WO1993004695A1 - T cell receptor-based therapy for rheumatoid arthritis - Google Patents

T cell receptor-based therapy for rheumatoid arthritis Download PDF

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Publication number
WO1993004695A1
WO1993004695A1 PCT/US1992/007289 US9207289W WO9304695A1 WO 1993004695 A1 WO1993004695 A1 WO 1993004695A1 US 9207289 W US9207289 W US 9207289W WO 9304695 A1 WO9304695 A1 WO 9304695A1
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Prior art keywords
cell receptor
variable regions
mammal
seq
receptor variable
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PCT/US1992/007289
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French (fr)
Inventor
William V. Williams
David B. Weiner
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The Wistar Institute
The Trustees Of The University Of Pennsylvania
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Application filed by The Wistar Institute, The Trustees Of The University Of Pennsylvania filed Critical The Wistar Institute
Priority to EP92919946A priority Critical patent/EP0602178A4/en
Priority to JP5505301A priority patent/JPH07504399A/en
Publication of WO1993004695A1 publication Critical patent/WO1993004695A1/en

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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
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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 relates to the field of mammalian therapeutics. More particularly, methods of treating rheumatoid arthritis and methods for immunizing against, rheumatoid arthritis are provided.
  • Rheumatoid arthritis is a systemic polyarthropathy characterized pathologically by proliferation of synovial fibroblast-like and macrophage-like cells and infiltration of the synovium with lymphocytes, predominately T cells of the helper (CD4+) phenotype (1,2).
  • CD4+ T cells are typically activated by an antigenic peptide complexed with Class II MHC molecules (HLA-DR/DP/DQ).
  • Immunogenetic analysis reveals that RA is associated with HLA- DR4, and more specifically with glutamine/lysine residues at amino acids 70/71 of the HLA-DR ⁇ chain (3-8).
  • T cells are involved in the development of rheumatoid joint disease. This includes the presence of lymphocytic infiltrates composed primarily of CD4+ T cells in the synovium (2, 21-23) the linkage of RA to HLA-DR4 which comprises a ligand for CD4+ T cell antigen receptors (3-8), and experimental models of arthritis and related autoimmune diseases which can be transferred by T cell lines (10, 13, 24- 33).
  • anti-T cell reagents can be of therapeutic efficacy (11, 25, 34-40). However, if these reagents are non-specific and delete too large a portion of the T cell repertoire, immunodeficiency (such as seen in acquired immune deficiency syndrome or AIDS) may result.
  • a better therapeutic alternative is to delete only those T cells involved in the autoimmune response. Since these comprise only a small portion of the total T cell repertoire, eliminating these T cells should not result in significant generalized immunosuppression.
  • the method comprises the steps of obtaining a sample of synovium from the mammal; identifying in said sample T cell receptor variable regions; and administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof.
  • the invention further provides a novel method of treating rheumatoid arthritis in a mammal comprising the steps of administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 14, V ⁇ 17 and V ⁇ 7 and antigenic fragments thereof.
  • the invention further comprises a novel method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis or to treat ongoing rheumatoid arthritis.
  • the method comprises the steps of administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 14, V ⁇ 17, V ⁇ 7 and antigenic fragments thereof.
  • Kits useful in the methods of the present invention comprising mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 14, V ⁇ 17 and V ⁇ 7 and antigenic fragments thereof or antibodies to said variable regions are also provided by the invention.
  • RA Rheumatoid arthritis
  • MHC major histocompatility
  • oligonucleotide primers specific for each of the major TCR subfamilies - one set for the TCR alpha chains and one for the TCR beta chains were used. These were utilized to amplify cDNA derived from whole synovium or synovial tissue T cell lines in a family specific manner. Amplified cDNA was sequenced to determine the corresponding amino acid sequences. Detection of amplified DNA was facilitated by utilizing oligonucleotide probes derived from the constant regions of the TCRs. Synovial T cell lines were developed by stimulation with phytohemagglutinin followed by maintenance in IL-2.
  • TCR repertoire present in these cell lines was quite heterogeneous, with an average of 15 alpha chains and 15.8 beta chains detected.
  • synovial tissue was analyzed, the predominant TCR subfamilies detected tended to be more restricted, with an average of 4.2 alpha chains and 9.7 beta chains detected.
  • predominance of one subfamily was apparent.
  • TCR transcripts in RA synovial T cell lines were developed by initial culture in PHA for 3-5 days, then maintained in IL-2 at 10 U/ml. Following 1-3 weeks of passage, the cells were frozen, and RNA later extracted for analysis of TCR expression as outlined in Materials and Methods. The sample designations are shown on the left, with the corresponding TCR alpha and beta family-specific primers used indicated above each lane.
  • FIG. 1 TCR transcripts in RA synovium. RNA was extracted and cDNA synthesized form 10 rheumatoid synovial tissues obtained at the time of joint surgery. These were analyzed for TCR expression as noted above. The sample designations are shown on the left, with the corresponding TCR alpha and beta family-specific primers used indicated above each lane.
  • Figure 4. Graphic representation of the frequency of occurrence of individual alpha chain variable regions in rheumatoid synovial tissue and T cell lines;
  • FIG. 1 T cell receptor PCR primers.
  • the asterisk denotes antisense primer.
  • C ⁇ 1 and C ⁇ 2 primers were used mixed together in equimolar concentrations.
  • Figure 6. T cell receptor ⁇ chain expression in ten rheumatoid synovia, The asterisk denotes > 2 standard errors from the mean.
  • a method of treating rheumatoid arthritis in a mammal comprises obtaining a sample of synovium from the mammal; identifying in said sample T cell receptor variable regions; and administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof.
  • synovium such as synovial tissue or fluid are obtained as is known to those in the art.
  • PCR polymerase chain reaction
  • Sequences of T cell receptors are generally available in the literature and in computer-based sequence data bases such as "Genbank” and "EMBL".
  • sequence of the family-specific oligonucleotide primer of interest can be matched against these data bases utilizing a variety of computer software tools (For example, the University of Wisconsin package. (49)) with programs such as "Word Search and Segments” or "Best Fit”.
  • the matched sequence are retrieved from the data base and translated from nucleic acid to protein sequence.
  • the T cell receptors of interest can be identified by in situ hybridization. Northern or Southern blot analysis of synovial fluid or tissue with family-specific probes or by immunohistochemistry or immunofluorescence with antibodies to the various T cell receptor variable regions, where available.
  • an effective amount of antibodies to at least one of the T cell receptor variable regions is then administered to the mammal.
  • antibodies to at least one of the T cell receptor variable regions is meant to denote antibodies which recognize T cell receptor variable regions and portions or fragments thereof.
  • An effective amount of antibodies is that amount which reduces the level of T cells bearing the corresponding receptor in the synovium or which results in clinical signs of improvement in the patient.
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of an antigen which can be recognized and bound by an antibody.
  • An antigen may have one or more than one epitope.
  • An "antigen” is a substance capable of inducing an animal to produce antibodies capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the antigen will immunoreact, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example. Fab and F(ab') 2 fragments) which are capable of binding an antigen. Fab and F(ab' 2 ) fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding.
  • the antibodies useful in the present invention may be prepared by any of a variety of methods.
  • Antibodies useful in the present invention include antibodies to the T cell receptor variable region as well as antibodies to antigenic fragments thereof. Methods for the production of such antibodies are well known and described fully in the literature. (19) For example, cells expressing the peptide, synthetic peptides or an antigenic fragment thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the peptide.
  • Peptides useful in the present invention may range in size from about 25 to about 500 amino acids in length. In some embodiments of the present invention peptides may be from about 50 to about 300 amino acids in length.
  • peptides may be from about 50 to about 200 amino acids in length.
  • a peptide fragment is prepared and purified to render it substantially free of natural contaminants or a peptide fragment is synthesized, according to means known in the art. Either the purified fragment or the synthesized fragment or a combination of purified natural fragments and/or synthesized fragment may be introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • Monoclonal antibodies can be prepared using known hybridoma technology.
  • such procedures involve immunizing an animal with a peptide antigen, which includes the T cell receptor variable region and antigenic fragments thereof.
  • the splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention.
  • the resulting hybridoma cells are selectively maintained in a suitable medium and then cloned by limiting dilution.
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the peptide antigen.
  • hybridoma cells will produce antibodies capable of binding to the peptide (other hybridoma cells will produce antibody capable of binding to the peptide contaminants). Thus, it may be necessary to screen among the hybridoma cells for those which are capable of secreting an antibody which is capable of binding to the peptide. Once such a hybridoma cell has been identified, it may be clonally propagated by means known in the art in order to produce the peptide-specific monoclonal antibody.
  • T cell receptor variable regions are known and are available in data bases such as "Gen Bank” and "EMBL”. Additional sequences of interest may be determined by cloning and sequencing cDNA clones of T cell receptors isolated from synovial tissue or fluid (48).
  • sequences of T cell receptor variable regions V ⁇ 14, V ⁇ 17, V ⁇ 1 and V ⁇ 17 are preferred.
  • Preferred DNA sequences and corresponding amino acid sequences of these regions are set forth in Table 1.
  • Table 1 sets forth preferred sequences of rheumatoid synovial T cell receptor ⁇ and ⁇ chain variable regions derived from human synovial tissue. Such sequences and portions of said sequences are useful for the development of antibodies useful in the present invention. It should be understood by those skilled in the art that, in some embodiments of the present invention nucleic acid analogs may be substituted for naturally occuring nucleic acids.
  • nucleic acid sequences may range from about 75 to about 1500 nucleic acid bases in length based upon the portion of the T cell receptor variable region being coded and the size of a particular T cell receptor variable region. In other preferred embodiments nucleic acid sequences may range in length from about 150 to about 900 nucleic acid bases. In yet other embodiments of the present invention from about 150 to about 600 nucleic acids may code for a selected T cell receptor variable region or portion thereof.
  • Antibodies may be developed against the T cell receptors or against amino acid sequences and portions thereof, corresponding to said T cell receptor variable regions such as those set forth in Table 1 for commercial purposes by developing monoclonal antibodies as indicated herein and known in the art. These murine or rat or other species monoclonals could be administered directly. Alternatively, to reduce xenogeneic responses to the monoclonals, these antibodies can be "humanized” by grafting a human constant region onto the non-human variable region, or by transplanting the non-human hypervariable regions onto a human antibody. (50, 51) Polyclonal antibodies can also be employed, particularly if they are from a species which exhibits little immunogenicity in humans such as pigs.
  • Antigenic fragments may be derived from family-specific sequences such as those contained in the variable region primers or from hypervariable regions as defined in Jones et al. (52)
  • a method of treating rheumatoid arthritis in a mammal comprising administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 14, V ⁇ 17 and V ⁇ 7 and antigenic fragments thereof.
  • antibodies to amino acid set forth in Table 1 and portions thereof are preferred.
  • Antibodies to mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 14, V ⁇ 17 and V ⁇ 7 and antigenic fragments thereof can be prepared as described above.
  • An effective amount of antibodies to at least one of the T cell receptor variable regions described above is then administered to the mammal.
  • An effective amount of antibodies is that amount which reduces the level of T cells bearing the corresponding receptor in the synovium or which results in clinical signs of improvement in the patient.
  • the therapy of the invention could be administered at any point in the course of rheumatoid arthritis.
  • a method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis or to ameliorate active disease comprises administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of V ⁇ 17, V ⁇ 1, V ⁇ 12, V ⁇ 17, V ⁇ 7 and antigenic fragments thereof.
  • Amino acid sequences as set forth in Table 1, and portions thereof, are preferred for some embodiments of the invention.
  • Mammals could be immunized by using the T cell receptor variable regions described above and antigenic fragments thereof, with or without agents known to those in the art attached thereto to increase the antigenic potential of the antigen.
  • the antigen or protein can be dissolved at between about l ⁇ g/ml to about lg/ml in sterile saline or saline with 0.4 mg aluminum hydroxide per ml as a vehicle.
  • 0.5 to 1.0 ml of the protein solution is injected intramuscularly and then followed by booster injections at one and 6-12 months after the initial immunization.
  • An effective amount is that amount of antigen sufficient to raise antibodies to the antigen in the animal.
  • Kits with the antibodies described herein useful in the treatment of rheumatoid arthritis or kits with antigens for immunization are also within the scope of this invention.
  • Tissue was obtained at the time of joint surgery, and was handled sterily at all times. The tissue was rinsed in sterile phosphate buffered saline (PBS), placed in a petri dish, the superficial layer snipped off with scissors and minced with a sterile scalpel. The minced tissue was placed in 20 mis PBS with 5% HEPES buffer, 0.4 g hyaluronidase (type 1-S), 0.04 g DNA-ase 1 (type II from bovine pancrease) and 1.2 g collagenase (Type Z) (all from Sigma, St. Louis, MO) with 1% fetal calf serum (FCS), and stirred continuously for 90 minutes at 37°C.
  • PBS sterile phosphate buffered saline
  • FCS fetal calf serum
  • the large chunks of tissue were decanted, and the cells centrifuged and washed twice in culture media (RPMI 1640 with pen/step, L-glutamine, sodium pyruvate, non-essential amino acids, HEPES buffer 5X10- 5 M ⁇ -mercaptoethanol (all from Gibco, Gaithersburg MD), and 10% FCS (Hyclone).
  • the T cells were purified by standard nylon wool chromatography (17), cultured overnight at 1x10 6 /ml in culture media, and the non-adherent cells separated, centrifuged, and maintained in culture. Stimulation of the cells was with either phytohemagglutinin (1% solution, from Sigma), interleukin-2 (Amgen Biologicals, Thousand Oaks, CA), or media alone. Cells were stimulated for 3-5 days, and then maintained for varying periods of time in 10 U/ml IL-2 prior to analysis.
  • FACS media 1% bovine serum albumin in PBS with 0.1% sodium azide
  • Primary antibody was added for 20-40 minutes on ice. After an additional two washings, the cells were subjected to second antibody (fluorescein isothiocyanate-conjugated goat anti-mouse Ig (Sigma); at 1:100 dilution), then washed twice again. The cells were then analyzed at the University of Pennsylvania Cancer Center FACS facility. Per cent positive was determined by comparing the samples to a no primary antibody control.
  • Antibodies used were OKT3 anti-CD3 (Ortho Diagnostics, Raritan, NJ), Leu3a anti-CD4 (Becton-Dickinson provide location), and OKT8 (Ortho), at the dilutions suggested by the suppliers.
  • RNA Extraction and cDNA Synthesis Tissue was homogenized in guanidinum isothiocyanate (GITC) solution, or cells resuspended in GITC solution, and vortexed for 30 seconds. 0.1 ml 2 M sodium acetate pH 4 was added, the solution vortexed, followed by 1.0 ml diethylpyrocarbonate (DEP) -water-saturated phenol, the sample mixed, then 0.2 ml phenylisoamyl alcohol, thorough vortexing, and the solution transferred to sterile EPPENDORF tubes.
  • GITC guanidinum isothiocyanate
  • DEP diethylpyrocarbonate
  • RNA precipitated with 2.5 volumes of 100% ethanol and 1/10 volume 1M sodium acetate pH 5.5 in dry ice/ethanol for 30 minutes.
  • the solutions were microfuged for 15 minutes, the supernatant decanted, the pellets washed in 70% ethanol and rotary evaporated.
  • the dried pellets were resuspended in 50 ⁇ l DEP-water and RNA quantitated spectrophotometrically.
  • RNA in 10 ⁇ l was utilized to synthesize cDNA primed with random hexamers in the following reaction mixture: 3 ⁇ l Maloney Murine Leukemia
  • Virus reverse transcriptase with 6 ⁇ l 5x reverse transcriptase buffer, 1.5 ⁇ l RNAse inhibitor, and 3 ⁇ l 0.1 M dithiothreitol
  • cDNA was amplified utilizing the primers listed in Figure 5 with Va/ ⁇ n and Ca/ ⁇ mid at 0.2 nM concentrations. cDNA was amplified utilizing Thermus aguaticus DNA polymerase (Tag polymerase) and standard reaction conditions suggested by the manufacturer (Perkin-Elmer Cetus Corp., Norwalk, CT).
  • the reaction mixture contained 10 ⁇ l of 10X reaction buffer, 16 ⁇ l 1.25 nM dNTPs (final concentration 200 ⁇ M in each dNTP), 5 ⁇ l of each oligonucleotide primer at 20 ⁇ M (final 1 ⁇ M in each primer), 5 ⁇ l of DNA, 0.5 ⁇ l of DNA, 0.5 ⁇ l Tag polymerase, and 58.5 ml distilled/deionized water. Primers were synthesized by the Wistar Institute oligonucleotide synthesis facility.
  • the program utilized 5 initial low temperature cycles for low stringency (95°C for 1 min., 37°C for 2 min., 52°C for 2 min.), followed by higher stringency for 40 cycles (95°C for 1 min., 52°C for 2 min., 72°C for 2 min), and a final 5 minute 72°C elongation phase.
  • the initial 20 cycles, described above was used followed by additional increments of 5 higher stringency cycles (95°C for 1 min., 52°C for 2 min., 72°C for 2 min), with PCR product removed following each increment of 5 cycles for analysis. Products were analyzed by electrophoresis on 2-3% agarose gels stained with ethidium bromide.
  • PCR products were cloned into the TA cloning vector (InVitrogen, San Diego, CA) according to kit instructions. Plasmid DNA was isolated from the clones as described by Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY) and Sambrook, et al., Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) incorporated by reference in their entireties.
  • Oligonucleotide labeling employed 100 ng DNA, 75 ⁇ C f 32P-ATP, 2.5 ⁇ l 10 x kinase buffer (500 mM Tris HCL pH 7.6, 100 mM MgCL 2 , 50 mM dithiothreitol, 1 mM spermadine, 1 mM EDTA), 10 U T4 DNA kinase adjusted to a final volume of 25 ⁇ l with distilled water. Labelling was carried out by incubation at 37°C for 30 minutes prior to use.
  • Blots were prehybridized in 5x SSC, 5x Denhardt's solution, 0.1% SDS for 1-1.5 hours at 55°C in sealable polyethylene bags, most of the solution poured off, 32 P-labelled oligonucleotide added (75 ⁇ Ci) and hybridized for 2-3 hours at 42°C or overnight at 4°C, the blots washed 1x in 2x SSC, 0.1% SDS for 20 minutes at 45°C, then 3x in 5x SSC, 0.1% SDS for 20 minutes at 45°C, an exposed to Kodak XRP film at -70°C for 2-72 hours.
  • Primers derived from the human TCR alpha and beta constant regions were utilized in conjunction with primers specific for individual variable region families. (14-16). The primers utilized in these studies are listed in Fig. 5, and their relative positions on the coding strand of cDNA indicated in Figure 1.
  • the constant region primers were designed as antisense primers to allow their use to prime both PCR reactions as well as probes for blotting.
  • Variable region primers were designed to act in a family specific manner as has been previously reported (14-16).
  • the PCR program used in these studies employed a low stringency initial 5 cycles, followed by 40 cycles at higher stringency. The rationale for using this program was twofold. As these studies were designed to investigate the range of T cell receptors expressed in RA synovium, and all TCR V regions have not yet been sequenced, related TCR families which have sequences related to the primers used here may also be amplified in the initial low stringency cycles. 40 cycles of amplification were then used to amplify even low frequency transcripts. This should help overcome the potential problem of sampling error, which is possible from surgical specimens. Thus, if local accumulations of specific TCR bearing T cells are present, and such a local accumulation is missed in the surgical specimen, their presence still may be detected if they are also present at lower frequency in the surgical specimen examined.
  • Synovial T Cells RNA was extracted and cDNA synthesized from both whole synovium and PHA stimulated/IL-2-maintained synovial T cell lines. Synovial T cell lines derived in this manner have been previously described (17), and early on represent a phenotypically mixed population, including CD8+ and CD4+ cells (17). FACS analysis was available for 4 of these cell lines at the time of analysis, and the data is shown in Table 3. In 3 of these, CD4+ cells predominated, while in the other, CD8+ cells were more prevalent.
  • T cell receptor transcripts were amplified from cDNA derived from rheumatoid synovial T cell lines. All rheumatoid synovia were obtained at the time of joint surgery, and thus represented late disease.
  • cDNA was split into equal portions and amplified with the middle constant region primers (C ⁇ mid or C ⁇ mid ) in combination with each of the respective individual variable region primers noted in Fig. 5 (eg., C ⁇ mid +C ⁇ 1,
  • each TCR variable region was tabulated for synovial tissue in Figures 6 and 7. While the T cell receptor expression seen in the synovial T cell lines is quite heterogeneous, the expression in rheumatoid synovia was somewhat more limited. Specifically, V ⁇ 17 was present 7/10 synovia, and V ⁇ 1 was present in 5/10. V ⁇ 14 was seen in 9/10 samples, while V ⁇ 17 and V ⁇ 12 were present in 8/10 specimens and V ⁇ 7 was seen in 7/10. This suggests the presence of these variable regions in many rheumatoid synovia from many different patients. When analyzed statistically, the frequency of V ⁇ 12, 14 & 17 were >2 standard errors above the mean values for all TCR V ⁇ s detected, and V ⁇ 17 and >2 standard errors above the mean values for all TCR V ⁇ a detected.
  • Phenotypic characterization of lymphocytes infiltrating synovial tissue from patients with rheumatoid arthritis analysis of lymphocytes isolated from minced synovial tissue by dual immunofluorescent staining. J Rheumatol 17:142-148.
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Abstract

There is provided by this invention a novel method of treating rheumatoid arthritis in a mammal. The method comprises the steps of obtaining a sample of synovium from the mammal; identifying in said sample T cell receptor variable regions; and administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof. The invention further provides a novel method of treating rheumatoid arthritis in a mammal comprising the steps of administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof. The invention further comprises a novel method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis or to treat ongoing rheumatoid arthritis. The method comprises the steps of administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17, Vβ7 and antigenic fragments thereof. Kits comprising mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof or antibodies to said variable regions are also provided by the invention.

Description

T CELL RECEPTOR-BASED THERAPY FOR RHEUMATOID ARTHRITIS
Cross Reference to Related Application
This application is a continuation-in-part of U.S. Serial No. 750,913 entitled "T Cell Receptor-Based Therapy for Rheumatoid Arthritis" filed in the U.S. Patent and Trademark Office on August 28, 1991 which is incorporated by reference herein.
Field of the Invention
This invention relates to the field of mammalian therapeutics. More particularly, methods of treating rheumatoid arthritis and methods for immunizing against, rheumatoid arthritis are provided.
Government Rights
The work presented herein was supported in part by National Institute of Health grant 1R-29AI-28503-01. The United States Government has certain rights in the invention.
Background of the invention
Rheumatoid arthritis (RA) is a systemic polyarthropathy characterized pathologically by proliferation of synovial fibroblast-like and macrophage-like cells and infiltration of the synovium with lymphocytes, predominately T cells of the helper (CD4+) phenotype (1,2). Such CD4+ T cells are typically activated by an antigenic peptide complexed with Class II MHC molecules (HLA-DR/DP/DQ). Immunogenetic analysis reveals that RA is associated with HLA- DR4, and more specifically with glutamine/lysine residues at amino acids 70/71 of the HLA-DRβ chain (3-8).
Current therapy for rheumatoid arthritis is either poorly efficacious or toxic. Many lines of evidence indicate that T cells are involved in the development of rheumatoid joint disease. This includes the presence of lymphocytic infiltrates composed primarily of CD4+ T cells in the synovium (2, 21-23) the linkage of RA to HLA-DR4 which comprises a ligand for CD4+ T cell antigen receptors (3-8), and experimental models of arthritis and related autoimmune diseases which can be transferred by T cell lines (10, 13, 24- 33). Studies in both animal models and human rheumatoid arthritis indicate that anti-T cell reagents can be of therapeutic efficacy (11, 25, 34-40). However, if these reagents are non-specific and delete too large a portion of the T cell repertoire, immunodeficiency (such as seen in acquired immune deficiency syndrome or AIDS) may result.
A better therapeutic alternative is to delete only those T cells involved in the autoimmune response. Since these comprise only a small portion of the total T cell repertoire, eliminating these T cells should not result in significant generalized immunosuppression.
Summary of the invention
There is provided by this invention a novel method of treating rheumatoid arthritis in a mammal. The method comprises the steps of obtaining a sample of synovium from the mammal; identifying in said sample T cell receptor variable regions; and administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof.
The invention further provides a novel method of treating rheumatoid arthritis in a mammal comprising the steps of administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof. The invention further comprises a novel method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis or to treat ongoing rheumatoid arthritis. The method comprises the steps of administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17, Vβ7 and antigenic fragments thereof.
Kits useful in the methods of the present invention comprising mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof or antibodies to said variable regions are also provided by the invention.
Rheumatoid arthritis (RA) is characterized by massive proliferation of synovial tissue, elevated expression of HLA DR antigens, accompanying infiltration of the tissue with CD4+ T lymphocytes, and a genetic linkage to the major histocompatility (MHC) antigen HLA-DR4. Since T cells are restricted by Class II MHC molecules such as DR4, this suggests a direct role for these CD4+ cells in pathogenesis. One strategy for the development of novel therapies in T cell mediated autoimmunity is to specifically delete the autoreactive T cells. Such a strategy depends on understanding the molecular structure of autoreactive T cell receptors (TCR). To investigate the TCR usage in RA, oligonucleotide primers specific for each of the major TCR subfamilies - one set for the TCR alpha chains and one for the TCR beta chains were used. These were utilized to amplify cDNA derived from whole synovium or synovial tissue T cell lines in a family specific manner. Amplified cDNA was sequenced to determine the corresponding amino acid sequences. Detection of amplified DNA was facilitated by utilizing oligonucleotide probes derived from the constant regions of the TCRs. Synovial T cell lines were developed by stimulation with phytohemagglutinin followed by maintenance in IL-2. The TCR repertoire present in these cell lines was quite heterogeneous, with an average of 15 alpha chains and 15.8 beta chains detected. When synovial tissue was analyzed, the predominant TCR subfamilies detected tended to be more restricted, with an average of 4.2 alpha chains and 9.7 beta chains detected. In some synovial tissue samples predominance of one subfamily was apparent. These results suggest that while a polyclonal population of T cells is present in RA synovium, the predominant patterns of TCR transcript expression may be somewhat more restricted. This suggests that TCR based therapy of RA is possible.
Brief Description of the Drawings
Figure 1. T cell receptor specific oligonucleotides and their relative location.
Figure 2. TCR transcripts in RA synovial T cell lines. Rheumatoid synovial T cell lines were developed by initial culture in PHA for 3-5 days, then maintained in IL-2 at 10 U/ml. Following 1-3 weeks of passage, the cells were frozen, and RNA later extracted for analysis of TCR expression as outlined in Materials and Methods. The sample designations are shown on the left, with the corresponding TCR alpha and beta family-specific primers used indicated above each lane.
Figure 3. TCR transcripts in RA synovium. RNA was extracted and cDNA synthesized form 10 rheumatoid synovial tissues obtained at the time of joint surgery. These were analyzed for TCR expression as noted above. The sample designations are shown on the left, with the corresponding TCR alpha and beta family-specific primers used indicated above each lane.
Figure 4. (A) Graphic representation of the frequency of occurrence of individual alpha chain variable regions in rheumatoid synovial tissue and T cell lines;
(B) Graphic representation of the frequency of occurrence of individual beta chain variable regions in rheumatoid synovial tissue and T cell lines.
Figure 5. T cell receptor PCR primers. The asterisk denotes antisense primer. Cβ1 and Cβ2 primers were used mixed together in equimolar concentrations. Figure 6. T cell receptor β chain expression in ten rheumatoid synovia, The asterisk denotes > 2 standard errors from the mean.
Figure 7. T cell receptor α chain expression in ten rheumatoid synovia, The asterisk denotes > 2 standard errors from the mean.
Detailed Description of the Invention
In one aspect of the invention a method of treating rheumatoid arthritis in a mammal, such as a human, is provided. The method comprises obtaining a sample of synovium from the mammal; identifying in said sample T cell receptor variable regions; and administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof.
Samples of synovium such as synovial tissue or fluid are obtained as is known to those in the art.
Molecular characterization of human T cell receptors has been greatly aided recently through the application of the polymerase chain reaction (PCR). (19) See also e. g. U.S. patent 4,386,202 issued to Mullis which patent is incorporated by reference as if fully set forth herein. By utilizing oligonucleotide primers specific for the different T cell receptor variable region families, family specific amplification is possible (14-16). This technique can conveniently be applied to the identification of T cell receptors of interest.
Sequences of T cell receptors are generally available in the literature and in computer-based sequence data bases such as "Genbank" and "EMBL". Thus, the sequence of the family-specific oligonucleotide primer of interest can be matched against these data bases utilizing a variety of computer software tools (For example, the University of Wisconsin package. (49)) with programs such as "Word Search and Segments" or "Best Fit". The matched sequence are retrieved from the data base and translated from nucleic acid to protein sequence. Alternatively, the T cell receptors of interest can be identified by in situ hybridization. Northern or Southern blot analysis of synovial fluid or tissue with family-specific probes or by immunohistochemistry or immunofluorescence with antibodies to the various T cell receptor variable regions, where available.
An effective amount of antibodies to at least one of the T cell receptor variable regions is then administered to the mammal. It should be noted that "antibodies to at least one of the T cell receptor variable regions" is meant to denote antibodies which recognize T cell receptor variable regions and portions or fragments thereof. An effective amount of antibodies is that amount which reduces the level of T cells bearing the corresponding receptor in the synovium or which results in clinical signs of improvement in the patient.
An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of an antigen which can be recognized and bound by an antibody. An antigen may have one or more than one epitope. An "antigen" is a substance capable of inducing an animal to produce antibodies capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the antigen will immunoreact, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example. Fab and F(ab')2 fragments) which are capable of binding an antigen. Fab and F(ab'2) fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding.
The antibodies useful in the present invention may be prepared by any of a variety of methods. Antibodies useful in the present invention include antibodies to the T cell receptor variable region as well as antibodies to antigenic fragments thereof. Methods for the production of such antibodies are well known and described fully in the literature. (19) For example, cells expressing the peptide, synthetic peptides or an antigenic fragment thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the peptide. Peptides useful in the present invention may range in size from about 25 to about 500 amino acids in length. In some embodiments of the present invention peptides may be from about 50 to about 300 amino acids in length. In still other embodiments of the present invention peptides may be from about 50 to about 200 amino acids in length. Generally, a peptide fragment is prepared and purified to render it substantially free of natural contaminants or a peptide fragment is synthesized, according to means known in the art. Either the purified fragment or the synthesized fragment or a combination of purified natural fragments and/or synthesized fragment may be introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies can be prepared using known hybridoma technology. In general, such procedures involve immunizing an animal with a peptide antigen, which includes the T cell receptor variable region and antigenic fragments thereof. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained in a suitable medium and then cloned by limiting dilution. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the peptide antigen.
If the peptide source is impure, only some of the hybridoma cells will produce antibodies capable of binding to the peptide (other hybridoma cells will produce antibody capable of binding to the peptide contaminants). Thus, it may be necessary to screen among the hybridoma cells for those which are capable of secreting an antibody which is capable of binding to the peptide. Once such a hybridoma cell has been identified, it may be clonally propagated by means known in the art in order to produce the peptide-specific monoclonal antibody.
The sequence of many human T cell receptor variable regions are known and are available in data bases such as "Gen Bank" and "EMBL". Additional sequences of interest may be determined by cloning and sequencing cDNA clones of T cell receptors isolated from synovial tissue or fluid (48).
In particular sequences of T cell receptor variable regions Vβ14, Vβ17, Vα1 and Vα17 are preferred. Preferred DNA sequences and corresponding amino acid sequences of these regions are set forth in Table 1. Table 1 sets forth preferred sequences of rheumatoid synovial T cell receptor α and β chain variable regions derived from human synovial tissue. Such sequences and portions of said sequences are useful for the development of antibodies useful in the present invention. It should be understood by those skilled in the art that, in some embodiments of the present invention nucleic acid analogs may be substituted for naturally occuring nucleic acids. In preferred embodiments of the present invention nucleic acid sequences may range from about 75 to about 1500 nucleic acid bases in length based upon the portion of the T cell receptor variable region being coded and the size of a particular T cell receptor variable region. In other preferred embodiments nucleic acid sequences may range in length from about 150 to about 900 nucleic acid bases. In yet other embodiments of the present invention from about 150 to about 600 nucleic acids may code for a selected T cell receptor variable region or portion thereof.
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
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Figure imgf000020_0001
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Figure imgf000026_0001
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Figure imgf000028_0001
Figure imgf000029_0001
Antibodies may be developed against the T cell receptors or against amino acid sequences and portions thereof, corresponding to said T cell receptor variable regions such as those set forth in Table 1 for commercial purposes by developing monoclonal antibodies as indicated herein and known in the art. These murine or rat or other species monoclonals could be administered directly. Alternatively, to reduce xenogeneic responses to the monoclonals, these antibodies can be "humanized" by grafting a human constant region onto the non-human variable region, or by transplanting the non-human hypervariable regions onto a human antibody. (50, 51) Polyclonal antibodies can also be employed, particularly if they are from a species which exhibits little immunogenicity in humans such as pigs.
Antigenic fragments may be derived from family-specific sequences such as those contained in the variable region primers or from hypervariable regions as defined in Jones et al. (52)
The association of RA with HLA-is reminiscent of similar associations seen in experimental models of autoimmunity, such as experimental autoimmune encephalomyelitis, a model for multiple sclerosis triggered by autoreactive T cells reactive to myelin basic protein and specific MHC Class II antigens (9-12). The observation of a restriction to certain MHCs in such experimental systems correlates with a restricted repertoire of T cell antigen receptors which respond to that MHC + antigen (13). This has also been documented in multiple sclerosis T cell lines derived from humans (14, 15). In experimental systems, antibodies directed to the relevant T cell receptors, or immunization with peptides derived from these T cell receptors, is capable of ameliorating the disease (10, 11).
In another embodiment of the invention a method of treating rheumatoid arthritis in a mammal is provided which comprises administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof. In particular, antibodies to amino acid set forth in Table 1 and portions thereof are preferred.
Antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof can be prepared as described above.
An effective amount of antibodies to at least one of the T cell receptor variable regions described above is then administered to the mammal. An effective amount of antibodies is that amount which reduces the level of T cells bearing the corresponding receptor in the synovium or which results in clinical signs of improvement in the patient.
Of course the method of treating rheumatoid arthritis of the present invention may be combined with other traditional treatments for the disease where indicated.
It is believed the therapy of the invention could be administered at any point in the course of rheumatoid arthritis.
A method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis or to ameliorate active disease is also provided by the invention. The method comprises administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ17, Vβ7 and antigenic fragments thereof. Amino acid sequences as set forth in Table 1, and portions thereof, are preferred for some embodiments of the invention.
Mammals could be immunized by using the T cell receptor variable regions described above and antigenic fragments thereof, with or without agents known to those in the art attached thereto to increase the antigenic potential of the antigen. Generally the antigen or protein can be dissolved at between about lμg/ml to about lg/ml in sterile saline or saline with 0.4 mg aluminum hydroxide per ml as a vehicle. Generally 0.5 to 1.0 ml of the protein solution is injected intramuscularly and then followed by booster injections at one and 6-12 months after the initial immunization. An effective amount is that amount of antigen sufficient to raise antibodies to the antigen in the animal.
There is precedence for immunizing mammals with T cell receptor variable regions as protection against experimental autoimmune encephalomyelitis. (11, 12) It is believed that a patient to be immunized would either have clinical evidence of rheumatoid arthritis, have a strong family history of rheumatoid arthritis or have the genetic predisposition for rheumatoid arthritis described herein.
Kits with the antibodies described herein useful in the treatment of rheumatoid arthritis or kits with antigens for immunization are also within the scope of this invention.
Materials and Methods
Synovial tissue and Cell Lines: Tissue was obtained at the time of joint surgery, and was handled sterily at all times. The tissue was rinsed in sterile phosphate buffered saline (PBS), placed in a petri dish, the superficial layer snipped off with scissors and minced with a sterile scalpel. The minced tissue was placed in 20 mis PBS with 5% HEPES buffer, 0.4 g hyaluronidase (type 1-S), 0.04 g DNA-ase 1 (type II from bovine pancrease) and 1.2 g collagenase (Type Z) (all from Sigma, St. Louis, MO) with 1% fetal calf serum (FCS), and stirred continuously for 90 minutes at 37°C. The large chunks of tissue were decanted, and the cells centrifuged and washed twice in culture media (RPMI 1640 with pen/step, L-glutamine, sodium pyruvate, non-essential amino acids, HEPES buffer 5X10- 5 M β-mercaptoethanol (all from Gibco, Gaithersburg MD), and 10% FCS (Hyclone). The T cells were purified by standard nylon wool chromatography (17), cultured overnight at 1x106/ml in culture media, and the non-adherent cells separated, centrifuged, and maintained in culture. Stimulation of the cells was with either phytohemagglutinin (1% solution, from Sigma), interleukin-2 (Amgen Biologicals, Thousand Oaks, CA), or media alone. Cells were stimulated for 3-5 days, and then maintained for varying periods of time in 10 U/ml IL-2 prior to analysis.
Fluorescence-Activated Cell Sorter (FACS) Analysis:
Following culture, cells were centrifuged, washed and resuspended in FACS media (1% bovine serum albumin in PBS with 0.1% sodium azide), at 1x106 cells per 100μl. Primary antibody was added for 20-40 minutes on ice. After an additional two washings, the cells were subjected to second antibody (fluorescein isothiocyanate-conjugated goat anti-mouse Ig (Sigma); at 1:100 dilution), then washed twice again. The cells were then analyzed at the University of Pennsylvania Cancer Center FACS facility. Per cent positive was determined by comparing the samples to a no primary antibody control. Antibodies used were OKT3 anti-CD3 (Ortho Diagnostics, Raritan, NJ), Leu3a anti-CD4 (Becton-Dickinson provide location), and OKT8 (Ortho), at the dilutions suggested by the suppliers.
RNA Extraction and cDNA Synthesis: Tissue was homogenized in guanidinum isothiocyanate (GITC) solution, or cells resuspended in GITC solution, and vortexed for 30 seconds. 0.1 ml 2 M sodium acetate pH 4 was added, the solution vortexed, followed by 1.0 ml diethylpyrocarbonate (DEP) -water-saturated phenol, the sample mixed, then 0.2 ml phenylisoamyl alcohol, thorough vortexing, and the solution transferred to sterile EPPENDORF tubes. Each sample was then incubated on ice for 20 minutes, microfuged for 10 minutes, and the top layer recovered, RNA precipitated with 2.5 volumes of 100% ethanol and 1/10 volume 1M sodium acetate pH 5.5 in dry ice/ethanol for 30 minutes. The solutions were microfuged for 15 minutes, the supernatant decanted, the pellets washed in 70% ethanol and rotary evaporated. The dried pellets were resuspended in 50 μl DEP-water and RNA quantitated spectrophotometrically.
For reverse transcription, 1-20 μg of RNA in 10μl was utilized to synthesize cDNA primed with random hexamers in the following reaction mixture: 3μl Maloney Murine Leukemia
Virus reverse transcriptase with 6 μl 5x reverse transcriptase buffer, 1.5 μl RNAse inhibitor, and 3 μl 0.1 M dithiothreitol
(all from GIBCO/BRL, Gaithersburg, MD), 3 μl random hexamers
(from Pharmacia LKB Biotechnology, Piscataway, NJ), and either
1 or 3 μl 100 mM dNTPs (25 mM in each dNTP, from Boehringer
Mannheim, GmbH W. Germany). Following a 10 minute preincubation at 25°C, the reaction was carried out for 1 hour at 42°C, then 95°C for 5 minutes followed by storage at -20°C until use.
PCR Amplification T Cell Receptor Variable Regions: cDNA was amplified utilizing the primers listed in Figure 5 with Va/βn and Ca/βmid at 0.2 nM concentrations. cDNA was amplified utilizing Thermus aguaticus DNA polymerase (Tag polymerase) and standard reaction conditions suggested by the manufacturer (Perkin-Elmer Cetus Corp., Norwalk, CT). The reaction mixture contained 10 μl of 10X reaction buffer, 16 μl 1.25 nM dNTPs (final concentration 200 μM in each dNTP), 5 μl of each oligonucleotide primer at 20 μM (final 1 μM in each primer), 5 μl of DNA, 0.5 μl of DNA, 0.5 μl Tag polymerase, and 58.5 ml distilled/deionized water. Primers were synthesized by the Wistar Institute oligonucleotide synthesis facility. The program utilized 5 initial low temperature cycles for low stringency (95°C for 1 min., 37°C for 2 min., 52°C for 2 min.), followed by higher stringency for 40 cycles (95°C for 1 min., 52°C for 2 min., 72°C for 2 min), and a final 5 minute 72°C elongation phase. For some experiments, the initial 20 cycles, described above, was used followed by additional increments of 5 higher stringency cycles (95°C for 1 min., 52°C for 2 min., 72°C for 2 min), with PCR product removed following each increment of 5 cycles for analysis. Products were analyzed by electrophoresis on 2-3% agarose gels stained with ethidium bromide.
Determination of sequences of T Cell Receptor Variable Regions: PCR products were cloned into the TA cloning vector (InVitrogen, San Diego, CA) according to kit instructions. Plasmid DNA was isolated from the clones as described by Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY) and Sambrook, et al., Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) incorporated by reference in their entireties. A portion of the cloned cDNA was sequenced in accordance with methods provided by Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY) and Sambrook, et al., Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) incorporated by reference in their entireties. Amino acid sequences were determined as set forth in Table 1. Relative positions set forth in Table 2 were determined in relation to family specific variable region primers used and published data providing invariant residues
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
and numbering of sequences of known T cell receptor regions. Kabat, E.A., et al., Seguences of Proteins of Immunological Interest" 4th ed., U.S. Department of Health and Human Services, Public Health Service National Institute (1987) .
Transfer and Probing Agarose gels were transferred to nylon fibers(Genescreen Plus, Du Pont New England Nuclear, Boston, MA) by capillary transfer overnight. Hybridization was with either Cα5' or Cβ5' primers noted in Figure 5.
Oligonucleotide labeling employed 100 ng DNA, 75 μCf 32P-ATP, 2.5 μl 10 x kinase buffer (500 mM Tris HCL pH 7.6, 100 mM MgCL2, 50 mM dithiothreitol, 1 mM spermadine, 1 mM EDTA), 10 U T4 DNA kinase adjusted to a final volume of 25 μl with distilled water. Labelling was carried out by incubation at 37°C for 30 minutes prior to use. Blots were prehybridized in 5x SSC, 5x Denhardt's solution, 0.1% SDS for 1-1.5 hours at 55°C in sealable polyethylene bags, most of the solution poured off, 32P-labelled oligonucleotide added (75μCi) and hybridized for 2-3 hours at 42°C or overnight at 4°C, the blots washed 1x in 2x SSC, 0.1% SDS for 20 minutes at 45°C, then 3x in 5x SSC, 0.1% SDS for 20 minutes at 45°C, an exposed to Kodak XRP film at -70°C for 2-72 hours.
Statistics
The standard error of occurrence of each TCR V region family was calculated by the formulae:
100 times the square root of (p[1-p]/n) where "n" is the number of samples analyzed, and "p" is the number of positives. The frequency of occurrence of a particular TCR V region family was considered significantly increased if it was > 2 standard errors higher than the mean for all V regions of that type (α versus β). Results
PCR Primers
Primers derived from the human TCR alpha and beta constant regions were utilized in conjunction with primers specific for individual variable region families. (14-16). The primers utilized in these studies are listed in Fig. 5, and their relative positions on the coding strand of cDNA indicated in Figure 1. The constant region primers were designed as antisense primers to allow their use to prime both PCR reactions as well as probes for blotting. Variable region primers were designed to act in a family specific manner as has been previously reported (14-16).
The PCR program used in these studies employed a low stringency initial 5 cycles, followed by 40 cycles at higher stringency. The rationale for using this program was twofold. As these studies were designed to investigate the range of T cell receptors expressed in RA synovium, and all TCR V regions have not yet been sequenced, related TCR families which have sequences related to the primers used here may also be amplified in the initial low stringency cycles. 40 cycles of amplification were then used to amplify even low frequency transcripts. This should help overcome the potential problem of sampling error, which is possible from surgical specimens. Thus, if local accumulations of specific TCR bearing T cells are present, and such a local accumulation is missed in the surgical specimen, their presence still may be detected if they are also present at lower frequency in the surgical specimen examined. Preliminary experiments with these primers utilizing the program described in Materials and Methods revealed that all of them (except Vβ16) are effective in amplifying TCR V regions from PHA stimulated peripheral blood mononuclear cells, but that only the appropriate V region primers amplified Jurkat cell cDNA TCR ((20) and data not shown).
Synovial T Cells RNA was extracted and cDNA synthesized from both whole synovium and PHA stimulated/IL-2-maintained synovial T cell lines. Synovial T cell lines derived in this manner have been previously described (17), and early on represent a phenotypically mixed population, including CD8+ and CD4+ cells (17). FACS analysis was available for 4 of these cell lines at the time of analysis, and the data is shown in Table 3. In 3 of these, CD4+ cells predominated, while in the other, CD8+ cells were more prevalent.
Figure imgf000044_0001
T cell receptor transcripts were amplified from cDNA derived from rheumatoid synovial T cell lines. All rheumatoid synovia were obtained at the time of joint surgery, and thus represented late disease. cDNA was split into equal portions and amplified with the middle constant region primers (Cβmid or Cαmid) in combination with each of the respective individual variable region primers noted in Fig. 5 (eg., Cβmid+Cβ1,
Cβmid+Cβ2 , ... Cβmid+Cβ20; Cαmid+Cα1; Cαmid+Cα2, .....Cαmid + Cα18).
Following electrophoresis and transfer, these were probed with Cβ5' or Cα5' respectively. The results for the synovial T cell lines is shown in Figure 2. An average of 15 alpha chain and 15.8 beta chain families were detected in these cell lines. This suggests that a quite heterogeneous population of T cells is present in synovium. However, as these cell lines were initially expanded with PHA, it is possible that the proportion of the various TCR subsets alter during culture. In addition, the ability of PHA to activate resting T cells raises concern about the relative proportion of activated T cells following stimulation compared with prior to stimulation. Therefore, similar analyses were performed on whole, unstimulated rheumatoid synovium.
Rheumatoid synovium
The results for the whole synovia or freshly isolated, unstimulated synovial T cells analyzed similarly are shown in Figure 3. An average of 4.2 alpha chain and 9.7 beta chain families were detected by this technique. The intensity of the bands detected is quite variable in Figure 2 & 3. To further evaluate the technique, cDNA was pooled from 4 synovia, and amplified with these primers for increasing numbers of cycles (Figure 4). Note that the intensity of some bands which appeared in early cycles faded relative to the intensity of bands which arose at later cycles. Thus, the intensity of the bands cannot be taken as an indicator of their relative abundance.
The frequency of occurrence of each TCR variable region was tabulated for synovial tissue in Figures 6 and 7. While the T cell receptor expression seen in the synovial T cell lines is quite heterogeneous, the expression in rheumatoid synovia was somewhat more limited. Specifically, Vα17 was present 7/10 synovia, and Vα1 was present in 5/10. Vβ14 was seen in 9/10 samples, while Vβ17 and Vβ12 were present in 8/10 specimens and Vβ7 was seen in 7/10. This suggests the presence of these variable regions in many rheumatoid synovia from many different patients. When analyzed statistically, the frequency of Vβ12, 14 & 17 were >2 standard errors above the mean values for all TCR Vβs detected, and Vα17 and >2 standard errors above the mean values for all TCR Vαa detected.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Williams, William V.
Weiner, David B.
(ii) TITLE OF INVENTION: T Cell Receptor-Based Therapy for
Rheumatoid Arthritis
(iii) NUMBER OF SEQUENCES: 79
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz and Norris
(B) STREET: One Liberty Place - 46th Floor
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19103
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(Vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(Viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Johnson, Philip S.
(B) REGISTRATION NUMBER: 27,200
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-568-3100
(B) TELEFAX: 215-568-3439
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(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
CTGAGGTGCA ACTACTCA
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GTGTTCCCAG AGGGAGCCAT TGCC
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GGTGAACAGT CAACAGGGAG A
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ACAAGCATTA CTGTACTCCT A
(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GGCCCTGAAC ATTCAGGA
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GTCACTTTCT AGCCTGCTGA
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
AGGAGCCATT GTCCAGATAA A
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GGAGAGAATG TGGAGCAGCA TC
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
ATCTCAGTGC TTGTGATAAT A
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: ACCCAGCTGG TGGAGCAGAG CCCT
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
AGAAAGCAAG GACCAAGTGT T
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: CAGAAGGTAA CTCAAGCGCA GACT
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
GCTTATGAGA ACACTGCGT
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GCAGCTTCCC TTCCAGCAAT
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: AGAACCTGAC TGCCCAGGAA
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CATCTCCATG GACTCATATG A
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: GACTATACTA ACAGCATGT
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
TGTCAGGCAA TGACAAGG 18
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
AATAGGTCGA GACACTTGTC ACTGGA
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
CTTGTCACTG GATTTAGATC TCTCAGCTG
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
GTACACGGCA GGGTCAGGGT TCTGGATATT
(2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
AAGAGAGAGC AAAAGGAAAC ATTCTTGAAC
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: GCTGCAAGGC CACATACGAG CAAGGCGTCG
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: AAAATGAAAG AAAAACCAGA TATTCCTGAG
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: CTGAGGCCAC ATATGAGAGT GGATTTGTCA
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
CAGAGAAACA AAGGAAACTT CCCTGGTCGA
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: GGGTGCGGCA GATGACTCAG GGCTGCCCAA
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: ATAAATGAAA GTGTGCCAAG TCGCTTCTCA
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: AACGTTCCGA TAGATGATTC AGGGATGCCC
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: CATTATAAAT GAAACAGTTC CAAATCGCTT
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
CTTATTCAGA AAGCAGAAAT AATCAATGAG
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
TCCACAGAGA AGGGAGATCT TTCCTCTGAG
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: GATACTGACA AAGGAGAAGT CTCAGATGGC
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: GTGACTGATA AGGGAGATGT TCCTGAAGGG
(2) INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
GATATAAACA AAGGAGAGAT CTCTGATGGA
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: CATGATAATC TTTATCGACG TGTTATGGGA
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: TTTCAGAAAG GAGATATAGC TGAAGGGTAC
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GATGAGTCAG GAATGCCAAA GGAACGATTT
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
CAAGAAACGG AGATGCACAA GAAGCGATTC
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
ACCGACAGGC TGCAGGCAGG GGCCTCCAGC
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
CCCTAGCAGG ATCTCATAGA GGATGGTGGC
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: CCCTAGCAAG ATCTCATAGA GGATGGTGGC
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: CTCTGCTTCT GATGGCTCAA ACACAGCGAC
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
CTCGGGTGGG AACACCTTGT TCAGGTCCTC
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
CTCGGGTGGG AACACGTTTT TCAGGTCCTC

Claims

Claims
1. A method of treating rheumatoid arthritis in a mammal comprising:
obtaining a sample of synovium from the mammal;
identifying in said sample T cell receptor variable regions; and
administering to said mammal an effective amount of antibodies to at least one of said T cell receptor variable regions or antigenic fragments thereof.
2. The method of claim 1 wherein said mammal is a human.
3. The method of claim 1 wherein said sample of synovium is synovial tissue or synovial fluid.
4. A method of treating rheumatoid arthritis in a mammal comprising:
administering to said mammal an effective amount of antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof.
5. The method of claim 4 wherein said antibody is specific for at least a portion of one or more peptides having amino acid sequences as set forth in Table 1.
6. The method of claim 4 wherein the mammal is human.
7. A method for immunizing a mammal to prevent the occurrence of rheumatoid arthritis comprising:
administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17, Vβ7 and antigenic fragments thereof.
8. The method of claim 7 wherein the mammal is a human and the mammalian T cell receptor variable regions are human T cell receptor variable regions.
9. The method of claim 7 wherein the mammal is a human and the mammalian T cell receptor variable regions comprise at least a portion of one of the amino acid sequences set forth in Table 1.
10. A method for immunizing a mammal to treat rheumatoid arthritis comprising:
administering to said mammal mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17, Vβ7 and antigenic fragments thereof.
11. The method of claim 10 wherein the mammal is a human and the mammalian T cell receptor variable regions are human T cell receptor variable regions.
12. The method of claim 10 wherein the mammal is a human and the mammalian T cell receptor variable regions comprise at least a portion of one of the amino acid sequences set forth in Table 1.
13. A kit comprising mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof.
14. The kit of claim 13 wherein the mammalian T cell receptor variable regions are human T cell receptor variable regions.
15. The kit of claim 14 wherein the mammalian T cell receptor variable regions comprise at least a portion of one of the amino acid sequences set forth in Table 1.
16. A kit comprising antibodies to mammalian T cell receptor variable regions selected from the group consisting of Vα17, Vα1, Vβ12, Vβ14, Vβ17 and Vβ7 and antigenic fragments thereof.
17. The kit of claim 16 wherein the mammalian T cell receptor variable regions are human T cell receptor variable regions.
18. The kit of claim 17 wherein the variable regions comprise at least a portion of one of the amino acid sequences set forth in Table 1.
PCT/US1992/007289 1991-08-28 1992-08-27 T cell receptor-based therapy for rheumatoid arthritis WO1993004695A1 (en)

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WO1994025489A1 (en) * 1993-04-26 1994-11-10 University Of Manitoba Downregulation of allergen-specific immune response
WO1995028481A1 (en) * 1994-04-18 1995-10-26 New York Society For The Ruptured And Crippled Maintaining The Hospital For Special Surgery Conserved t-cell receptor sequences
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AU2013206501B2 (en) * 2007-03-05 2016-05-19 International Institute Of Cancer Immunology, Inc. Cancer antigen-specific T-cell receptor gene, peptide encoded by the gene and use of them
WO2008125366A2 (en) * 2007-04-13 2008-10-23 Istituto Superiore Di Sanita Treatment of autoimmune diseases
WO2008125366A3 (en) * 2007-04-13 2009-07-16 Ist Superiore Sanita Treatment of autoimmune diseases
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CA2116526A1 (en) 1993-03-18
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EP0602178A1 (en) 1994-06-22

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