CA2116526A1 - T cell receptor-based therapy for rheumatoid arthritis - Google Patents

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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17 and V.beta.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.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17, V.beta.7 and antigenic fragments thereof. Kits comprising mammalian T cell receptor variable regions selected from the group consisting of V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17 and V.beta.7 and antigenic fragments thereof or antibodies to said variable regions are also provided by the invention.

Description

W093/0~95 P~T/US92/07289 2116~2S

T C~LL RECEPTOR-Ba8ED T~E~PY FOR RHE~MATOID ART~RITI~

Cro~ Re~erenc~ to ~elat~ ~pplicatio~
This application is a continuation-in-part of UOS.
Serial No. 750,913 entitled "T Cell Receptor Based Therapy ~or Rheumatoid Arthritis" filed in the U.S. Patent and Trademark Office on August 28, 1991 which is incorporated by reference herein.

Fiela of the I~Y~ntio~
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.

Governme~t Rights The work presented herein was supported in part by National Institute of Health grant lR-~AI-28503-01. The United States Governmsnt has certain rights in the invention.

Backgrou~d of the I~Ye~tion Rh~umatoid 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-W093/0469s PCT/US~2tO7289 21~6.~26 2 -DR4, and more specifically with glutamine/lysine residues at amino acids 70/71 of the HLA-DR~ 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 rhe~matoid 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 arthriti~ 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). Howe~er, 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 t~ese T cells should not result in significant generalized immunosuppression.

8ummary of the Inventio~
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 ~aria~le regions selected from the group consisting of V~17, V~l, V~12, V~14, V~17 and V~7 and antigenic fragments thereof.

WO 93/0469~ PCI /US92~07289 21i6~21~

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 ~roup 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~l, V~12, V~14, V~17 and V~7 and antigenic fragments therec: f 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 inf iltration of the tissue with CD4+ T lymphocytes, and a genetic linkage to the major historompatility (MHC) antigen HLA-DR4. Since T cells are restricted by Class II MHC molecules such as DR4, this suggests a d.irect 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 (I'CR). To investigate the TCR us,age 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 determin2 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 W093/04695 PCT/US92~072~9 2116~26 - 4 - ~
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 su~family was apparent. These results suggest that while a polyclonal population of T cells is pre~ent in RA
synovium, the predominant patterns of TCR transcript expression may be~somewhat more restricted. This suggests that TCR ~ased therapy of RA is possible.

Brief DeQcription of the Dr~wi~g~
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 ~or 3-5 days, t~en maintained in IL-2 at lO U/ml. Following 1-3 weeks of passagé, the cells were frozen, and RNA later extracted for analysis of TCR expression as outlined in Materials and Methods. ThP 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 Pxpression 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. C~1 and C~2 primers were used mixed together in equimolar concentrations.

W093/04695 2 1 1 ~ ~ 2 ~ PCT/~S92/07289 Figure 6. T cell receptor ~ chain expression in ten rheumatoid synovia. The asterisk denotes > 2 standard errors from the mean.
Figure 7. T cell receptor ~ chain expression in ten rheumatoid synovia. The asterisk denotes > 2 standard errors from the mean.

-'' Detaile~ Description of the Inve~tion 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 sampla 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 al so e . g . U . S .
patent 4,386,202 issued to Mullis which patent is incorporated by reference as if fully set forth hereini By utilizing oligonucleotide primers specific for the different T cell receptor variable region families, family specific amplification is possible (14-16). This techni~ue 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 comput~r-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 si~u 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 2116~2~ ~

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 re~cting 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 fragment~ 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 circulakion, 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. Antibodie~ useful in the present invention include antibodies to the T cell receptor ~ariable 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, calls 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 W093/04695 1 6 - ~ 6 - 8 - PCT/US~2/07289 may range in size from about 25 to about soo 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 i5 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 "&en Bank" and "EMBL". Additional sequences of interest may be W093/04695 2 1 :~ G ~ `2 6 PCT/US~2/07289 ~ `
_ g _ .
determined by cloning and sequencing cDNA clones of T cell receptors isolated from synovial tissue or fluid (48).
In particular sequen~es of T cell receptor variable regions V~14, V~17, V~l 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. 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.

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W093/04695 PCT/US92/07289 ~
21 16~26 28 -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 ~pecies 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 r.estriction 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 (1~ ). 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 embodimant 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 V~17, V~1, V~12, V~14, ~9 _ 2116~2~
V~17 and V~7 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 recep or 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 aboYe is then administered to the mammal. An effective amount of antibodies is that amount which reduces the level of T cells bearing the rorresponding 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 ma~malian T cell receptor variable regions selected from the group consisting o~ V~17, V~l, V~12, V~17, ~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. Generally 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. Generally O.S 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 W O 93/04695 21i6-Z6 PC~r/~S92/07289 ~

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.
Rits with the antibodies described herein useful in the treatment of rheumatoid arthritis or kits with antigen~
for immunization are also within the scope of this invention.

W093/04695 ~ 1 1 6 5 2 ~ PCT/US92/07289 ~teriAlR ~nd Meth~d~
8y~ovial ti~ue ~a C~ll Lins3: 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 mls PBS with 5% HEPES buffer, 0.4 g hyaluronidase (type l-S~, 0.04 g DNA-ase 1 (type II from bovine pancrease) and l.Z g collagenase (Type Z) (all from Sigma, St. Louis, MO) with 1% fetal calf serum (FCS), and ~stirred continuously for 90 minutes at 37C. 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 5Xl0 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 lx106/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 Oaksj 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.

Fluore~c~nce-~cti~ate~ Cell ~orter (FACS) A~aly~
Following culture, cells were centrifuged, washed and resuspended in FACS media (1% bovine serum albumin in PBS with 0.1% sodium azide), at lx106 cells per 100~1. 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 ~: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, W093/~95 P~T/US92/07289 211 6 3 2 6 - 32 - ~
Raritan, NJ), Leu3a anti-CD4 (Becton-Dickinson provid~
location), and OKT8 (Ortho), at the dilutions ~uggested by the suppliers.

RNA ~xtr~ctio~ and cDNA 8ynthesis: 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 lM sodium acetate pH 5.5 in dry ice/ethanol for 30 minutes. The solutions were microfuged for 15 minutes, the supernatant decantedl the pellets washed in 70% ethanol and rotary evaporated. The dried pellets were resuspended in 50 ~1 DEP-water and RNA quantitated spectrophotometrically~
For reverse transcription, 1-20 ~g of RNA in 10~1 was utilized to synthesize cDNA primed with random hexamers in the following reaction mixture: 3~1 Maloney Murine Leukemia Virus reverse transcriptase with 6 ~l 5x reverse transcriptase buffer, 1.5 ~l RNAse inhibitor, and 3 ~l O.l M dithiothreitol ~all from GIBCO/BRL, Gaithersburg, MD), 3 ~l random hexamers (from Pharmacia LKB Biotechnolo~y, Piscataway, NJ), and either 1 or 3 ~l 100 mM dNTPs (25 mM in each dNTP, from Boehringer Mannheim, GmbH W. Germany). FO11QWing a 10 minute preincubation at 25C, the reaction was carried out for l hour at 42C, then 95C for 5 minutes followed by storage at -20C
until use.

PCR Amplification T ~all R~ceptor Vari~ble Regio~: cDNA was amplified utilizing the primers listed in.Figure 5 with Va/~n and Ca/~mjd at O.2 nM concentrations. cDNA was amplified utilizing ~hermus aquaticus DNA polymerase (Taq polymerase) and standard reaction conditions suggested by the manufacturer W093~0~95 2 ~ PCT/US92/07289 (Perkin-Elmer Cetus Corp., Norwalk, CT). The reaction mixture contained 10 ~l of lOX reaction buffer, 16 ~l 1.25 nM dNTPs (final concen~ration 200 ~M in each dNTP), 5 ~l of each oligonucleotide primer at 20 uM (final 1 uM in each primer), 5 ~l of DNA, 0.5 ~l of DNA, 0.5 ~l Taq polymerase, and 58.5 ml distilled/deionized water. Primers were synthesized by the Wistar Institute oligonucleotide synthesis facility. The program utilized 5 initial low temperatur~ cycles for low stringency (9SC for 1 min., 37C for 2 min., 52C for 2 min.), followed by higher stringency for 40 cycles (95C for 1 min., 52C for 2 min., 72C for 2 min), and a final 5 minute 72C elongation phase. For some experiments r the initial 20 cycles, described above, was used followed by additional increments of 5 higher stringency cycles (95C for 1 mîn., 52C for 2 min., 72C for 2 min), with PC~ product removed following each increment of 5 cycles for analysis. Products were analyzed by electrophoresis on 2-3% agarose gels stained with ethidium bromide. :~

Determin~tion of 5eque~c~ of T Cell R~c~ptor V~ri~bl~
Region~: 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 de cribed by Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY~ and Sambrook, et al.~ Molecular Cloning . A La~oratory Manual ( Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) incorporated by referPnce in ~heir 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 e~tireties. 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 W093/04695 P~/US~2/07289 21~6~26 ~ ~

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W093/04695 PCT/US92/07289 ~
2116~26 35 and numbering of sequences of known T cell receptor regions.
Kabat, E.A.~ et al., Se~uences of Proteins of Immunological Interest~ 4th ed., U.S. Department of Health and Human Services, Public Health Service National Institute (1987) .

Trausfer and Probing Agarose gels were transferred to nylon fibers~Genescreen Plus, Du Pont New England Nuclear, Boston, MA) by capillary transfer overnight. ~ybridization was with --either Ca5' or C~5' primers noted in Figure 5.
Oligonucleotide labeling employed 100 ng DNA, 75 ~Cj 3ZP-ATP, 2.5 ~l 10 x kina~e buffer (500 mM Tris HCL pH 7.6, 100 mM
MgCL2, 50 ~M dithiothreitol, 1 m~ spermadine, 1 mM EDTA), 10 U T4 DNA kinase adjusted to a final volume of 25 ~1 with distilled water. Labelling was carried out by incubation at 37C for 30 minutes prior to use. Blots were prehybridized in 5x S5C, 5x Denhardt's solution, 0.1% SDS for 1-1.5 hours at 55C in sealable polyethylene bags, most of the solution poured off, 32P-labelled oligonucleotide added (75~Ci) and hybridized for 2-3 hours at 42C or overnight at 4C, the blots washed lx in 2x SSC, 0.1% SDS for 20 minutes at 45C, then 3x in 5x SSC, 0.1% SDS for 20 minutes at 45C, an exposed to Kodak XRP film at -70~C for 2-72 hours. -;.
8tati~tic~
The standard error of occurrence of each TCR V
region family was calculated by the formulae:

100 times the square root of (~r1-pl/n) where "n'l 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 i~ it was > 2 standard errors higher than the mean for all V regions of that type (a versus ~.

W093/04695 P~T/USg2/07289 :
- 3~ -~116i26 R~ult~
PC~ Pri~er~
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 pr-mers 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 two~
fold. As these studies were designed to investigate the range of T cell receptors expressed in RA synovium, and all TC~ V
regions have not yet been sequenced, related TCR families which have ~equences 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 V~16) 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 ((203 and data not shown).
~ynovi~l T C~lls ~NA 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 WO93/046g~ PCT/US92/07289 .

2116526 - 37 ~ ~
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.
TAB~E 3 PHENOTYPE OF 8YNOVIAL TISS~E T CELL LI~E8 PA~IE~T CO~rTROLCD3 t CD4+ CD8+
EP1 1% g0% 25% ~4%
NJ1 1% 80% ~1% 9%
MW1 1% 99% 77% 28%
HR~ 3% 98% 74% lS%
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 sur~ery, and thus represented late disease. cDNA was split into equal portions and amplified with the middle constant region primers (C~jd or C~mjd) in combination with each of the respective individual variable region primers noted in Fig. 5 (eg., C~mjd+C~
C~mjd+C~2, ., . C~mjd+C,l~20; C~m;d~C~ Crmid+C~2 ~ - - - CC~mid + C~18 ) -Following electrophoresis and transfer, these were probed with C~5' or C~5' r~spectively. The resultc 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. There~ore, similar analyses were performed on whole, unstimulated rheumatoid synovium.

Rheumatoia 8ynovium The results for the whole synovia or freshly isolated, unstimulated synovial T cells analyzed similarly are W093~04695 2 1 1 6 ~ 2 6 PCT/US92/07289 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 ~or 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~l 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 ~ifferent 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~z detected.
.

W093/04695 ~T/US92/07289 2116~26 - 39 ~
Ref~re~ce~
1. Firestein, G., and N. Zvaifler, 1987. The pathogenesis of rheumatoid arthritis. Immunology of rheumatic diseases 13:447-461.
2. Duke, O., G.S. Panayi, G. Janossy, and L. W. Poulter, 1982. An immunohistological analysis of lymphocyte subpopulations and their microenvironment in the synovial membranes of patients with rheumatoid arthritis using monoclonal antibodies. Clin Ex. Immunol 49:22-30.
3. Gao, X., E. Ball, L. Dombrausky, N. Olsen, T. ~incus, M.
Kahn, and F. Wolfe, 1988. Class II human leukocyte antigen genes and T cell receptor polymorphisms in patients with rheumatoid arthritis. Am J. Med 85~ 16.
4. Goronzy, J., C. Weyand, and C. Fathman, 1986, Shared T
cell recognition sites on human histocompatibility antigen class II molecules of patients with seropositive rheumatoid arthritis. J. Clin Invest 77: 1042-1049.
5. Gregerson, P., J. Silver, and R~ Winchester, 1988.
Genetic susceptibility to rheumatoid arthritis and human leukocyte antigen polymorphism. The role of shared conformational determinants. Am J Med 8S: 17-19.

6. Merryman, P., R. Crapper, S. Lee, P. Gregersen, and R.
Winchester. 1989. Class II major histocompatibility complex gane sequences in rheumatoid arthritis. Arthritis Rheum 3Z:251-~58.

7. Nepom, G., J. Hansen, and B. Nepom. 1987. The molecular basis for HLA class II association with rheumatoid arthritis.
J Clin Immunol 7:1-7.

8. Roudier, J., J. Petersen, G. Rhodes, J. Luke, and D.
Carson, 198~. Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR ~-1 chain and ~he Epstein-Barr virus glycoprotein gp 110. Proc Natl Acad sci USA 86: 5104-5108.

9. Saki, X., A.A. Sinha, D.J. Mitchell, S.S. Zamvil, J.B.
Rothbard, H.O. MCDevitt, and L. Steinman. 1988. Inv~lvement of distinct murine T-cell receptors in the autoimmune encephalitogenic responses tc nested epitopes of myelin basic protein. Proc Natl Acad sci USA 85: 8608-8612.

10. Hashim, G., A. Vandenbark, A. Galang, T. Diamanduros, E.
Carvalho, J. Srinivasan, R. Jones, M. Vainiene, W.
Morrisontand H. Offner. 1990. Antibodies specific for V~8 receptor peptide suppress experimental autoimmune encephalomyelitis. J Immunol 144:46Z1-4627.

W093/0469~ 2 1 1 6 ~ ~ 6 PCT/US92/07289 - ~0 - .

11. Vanden~ark, A.G. Hashim, and H. Offner. 1989.
Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis.
Nature 341:541-544.

12. Beraud, E., T. Resshef, A.A> Vandenbark, H.Offner, R.
Fritz, C.H.J. Chou, D. Brnard, and I.R. Cohen, 1986.
Experimental autoimmune encephalomyelitis mediated by T
lymphocyte lines: Genotype of antigen-presenting cells influences immunodominant epitope of basic protein. J Immunol 136:511-515.

13. Burns, F.R., X. Li, N. Shen, H. Offner, Y.K. Chou, A.A.
Vandenbark, and E. Heber-Katz. 1989. Both rat and mouse T
cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V~ and V~ chain genes even -though the major histocompatibility complex and encephalitogenic determinants being recognized ara different.
J Exp Med 169: 27-39 . ~:

14. Oksenberg, J.R., S. Stuart, A.B. Beegovich, R.B. Bell, ~
H.A. Erlich, L.Steinman, and C.C. A. Bernard, 1990. Limited ~ -heterogeneity of rearranged T~cell receptor V~ transcripts in brains of multiple sclerosis patients. 345:344-346.

15. Wucherpfennig, K.W., K. Ota, N.Endo, J.G. Seidman, A.
Rosenzweig, H.L. Weiner, and D.A. Hafler, 1990. Shared human T cell receptor V~ usage to immunodominant regions of myelin -basic protein, 248:1016-1019. -16. Choi. Y., B. Kotzin, L. Herron, J. Callahan, P. Marrack, and J. Kappler, 1989. Interaction of Staphylococcus aureus ~.
toxin" superantigens" with human T cells. Proc Natl Acad Sci USA 86:8941-8945.

17. Santoli, D., P. Phillips, T. Colt, and R. Zurier, 1990.
Suppression of interleukin-2 dependent human T cell growth in vitro by prostaglandin E ~PGE) and their precursor fatty acids in vitro. J Clin Invest 85:424-43~.

18. Ausubel, R., R. Brent, R. Kingston, D~ Moore, J. Seidman, J. Smith, and K. Struhl. 1989. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, New Yor~, N.Y.

19. Sambrook, J. E. Fritch, and T. Maniatis. 1989.
Molecular Cloning. A La~oratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

20. Williams, W.V., A. Sato, M. Rossman, Q. Fang, and D. B.
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24. Holmdahl, R., L. Klareskog, K. Rubin, E. larsson, and H.
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':

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Schattenkirchner, G. Riethmuller, and K. Kruger, 1991.
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51. Gorman, et al. "Reshaping a therapeutic CD4 antibody", ~
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W093/04695 PCT/US92/07289 ~.. .
21~6~2S - 83 -(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 tB) 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:

- 8~ _ 2~
(i) SEQUENCE C~ARACTERISTICS: .:
(A) LENGTH: 18 base pairs - .
(B) TYPE: nucleic acid ::
~D~ TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) ~ .
(xi) SEQUENCE DE5CRIPTION: SEQ ID NO:39:
GGCCCTGAAC ATTCAGGA
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CH~RACTERISTICS: ;~
(A) LENGTH: 2Q base pairs (B~ TYPE: nucleic acid -M
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40~
GTGACTTTCT 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 CHAR~CTERISTICS:
(A) LENGT~: ~2 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 WOg3/04695 PCT~US92~7289 2111i~2~ ~
~5 (B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) NOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
ATCTCAGTGC TTGTGAT~AT 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) INFORNATION FOR SEQ ID NO:45:
(i) SEQUENCE CHAR~CTERISTICS:
(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 C~ARACTERISTICS:
(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 CTCAA~CGCA GACT
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
tA) LEN5TH: 19 base pairs (B) TYPE: nucleic acid -~D) TOPOLOGY: unknown ~ `

W093/0469~ PCT/US92/07289 - ~:
862 1 i 6~ 2 6 ~ ~
(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) INFORM~TION FOR SEQ ID NO:49^
(i) SEQUENCE CXARACTERI5TICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: D~A (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
AGAACCTGAC TGCCCAGGAA
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic~
~xi) SEQUENCE DESCRIPTION: 5EQ ID NO:50: :
CATCTCCATG GACTCATATG A
(2) INFORM~TION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERI5TICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

W093J04695 2 1 ~ ~ 2 ~ PCT/US92/072~9 GACTATACTA ACAGCATGT
(2) INFORMATION FOR SEQ ID NO:S2:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: la base pairs (3) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CH~RA~TERISTICS:
(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 CHARACT~RISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucl~ic acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic~
(xi) SEQUENCE DESCRIPTION: 5EQ ID NO:54:
CTTGTCACTG GATTTAGATC TCTCAGCTG
(2) INFO~ATION 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:
G~ACACGGCA GG&TCAGGGT TCTGGATATT
(2) INFORMATION FOR SEQ ID NO: 56: ` -~

W093/0~5 2 ~ i 6 ~ 2 ~ PCT/US92/07289 - 8~ - :
~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) LENGTR: 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) INFORM~TION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 ~ase pairs (B) TYPE: nucleic acid (D) TOPOLOGY: unknown W0~3/04695 5 1 ~ ~ ` 2 PCT/U~92~07289 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
CAGAGAAACA AAGGAAACTT CCCTGGTCGA
t2) INFORMATION F~R 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:
&GGTGCGGCA GATGACTCAG GGCTGCCCAA
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS: ~~
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (~) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUEN OE 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 tii) MOLECULE TYPE: DNA (yenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
AACGTTCC&A TAGATGATTC AGGGATGCCC
(~) INFORMATION FOR SEQ ID NO:64:
(~) 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:

W93/04695 21 1 6~
Pcr~ls9~Jo7289 AAT GAAAcAG~Tc CAAATCG
ORMATIN FOR SEQ ID NO
(i) SEQuEMcE CHARAcTERIsTIcs (A) LENGrrH: 3 0 base pairS :
(BJ TYPE: nucleic acid (D) TOPOLOGy: unkrlown ~ OLECULE TYPE: DNA ~geno~ic) ~
(Xi) SEQuENcE DESCRIpTIoN SEQ ID NO 65 ~.
CAGA AAGcAGAaAT AATCAAT
( ) INFORMATIoN FOR SEQ ID N
(i) SEQUENCE CEARACTERI5TIC5 (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: unknown `. -:
) ~LECuLE TYpE DNA (yen (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
AGAGA AGGGAGATcT TTCCTCTGA
(2) INFoRMATIoN FOR SEQ ID NO 67 (i) SEQuENcE ClIaRAcTERIsTIcs (A) LENGI~ 3 o base pairs - :
(B) TYPE: nucleic acid (D) TOPOLOGY: unknown (ii) ~OLECULE TYPE: DNA (qenomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
GATACTGACA AAGGAGAAGT CTCAGATGGC
(~) INFOFMATI0N FOR SEQ ID NO:68:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) ~OPOLOGY: unknown (ii) ~OLECULE TYPE: DNA (genOmic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
GTGACTGATA AGGGAGATGT TCCTGAAGGG
~2) INPORMATION FOR SEQ ID NO:69:

pCT/I)S9t/01~89 ~i~6~26 ( i) S$QUENCE CG~RA30 base palrs ( ) TypE nucleic aCid ((~D~ ropOLOGY ~

MOLEcuLE ~ypE: DNA ( g ENCE DEscRIpTIoN SEQ

GG~GAGAT CTCTG~TGG
~TIoN FOR SEQ ID
(i) s}sQuE~rs E TYPE DNA ( gen~
E DESCRIpTION: S Q
TATCGACG TGTTATGGG

~A,~IoN FOR SEQ ID
( i) SEQUE~lc~E~cG~w%A3 0 ba5e palrS
~ TypE nuclelc ac (B ) TOpOgOGy unXnW

CPIPTION

~G G~G~T~rAGc TG
oR~,~ FOR SEQ ID
(i) SEQ(UA) ~ENG~rs ID) TOPO~OGY: U~knO
E TYPE DNA (genni ) GtrCAG G~rGCC~A G
FOP~5aTION ~~ SEQ
(i) 5EQUENCE C~A~RAC3TO~rs B) TypE: nUclelc ( D) ropoLoGy unkn ~O93/04695 21i652~ pCT/~sgtlo7289 _ 92 ~

(ii) ~OLECULE TYPE: DNA ~genomiC) 5EQUENCE DEscP~IPTION SEQ
CAAGAAACGG AGATGCACAA GAAGCGATTC
INFOP~ATION FOR SEQ ID No 74 (i) 5EQUENCE CHA~ACTERIsT
A) LENGTH 30 baSe pair B) TYP~ nucleic aCid (D) Top~LoGy: unknoWn ii) XoLEcuLE TypE: DNA (g CRIPTION SEQ
ACCGACAGGC TGcAGGcAGG GGCCTCCAGC

IoN FO~ SEQ ID No 75 UENCE C~ARAcTER~sTIcs-ENGTH 30 baSe palrS
B) TYPE nucleiC acid D) TopO~OGY unknown ii) ~OLECULE TYPE: DNA (genmiC) UENCE DEscRIpTIoN SEQ

TAGCAGG ATcTcATAGA GGAT
IoN FOR SEQ ID
UENCE CHA~AcTERIsTIcs A) LENGTH: 30 ba5e p B) TYPE nucleic aCid (D) TopOLOGY ~n~n~
i ~oLEcuLE TypE: DNA (g EQ~ENcE DEscRIp~IoN S~Q
TAGCAAG ATcTcATAGA GGAT
IoN FOR SEQ ID
i) SEQUENCE C~CTERI
~) LENGT~ 30 ba~e pai B) TYPE n ~D) ~oPOgO~
i) ~o~ECuLE TYPE DNA (9 EQUENCE DEscRIPTION SEQ

WO 93/04695 PCI~/U~92~072~g 2116~

CTCTGCTTCT GATGGCTCAA ACACAGCGAC
(2) INFORM~TION 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 CH~RACTERISTICS: :
(A) LENGTH: 30 base pairs -.
(B) TYPE: nucleic acid : ~:
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (g~nomic) ~ -(xi) SEQUENCE DESC~IPTION: SEQ ID No:7s:
CTCGGGTGGG AACACGTTTT TCAGGTCCTC

Claims (18)

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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17 and V.beta.7 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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17, V.beta.7 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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17, V.beta.7 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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17 and V.beta.7 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 V.alpha.17, V.alpha.1, V.beta.12, V.beta.14, V.beta.17 and V.beta.7 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.