CA2188182A1 - Conserved t-cell receptor sequences - Google Patents

Abstract

Four unique transcripts have been isolated from the .beta. chain of the T-cell receptor in T-cells in the synovial tissue of a patient with rheumatoid arthritis. Two of these transcripts were isolated from fresh synovial tissue and two were isolated from a T-cell line derived from the synovial tissue The sequences of the four transcripts are highly homologous, with a conserved amino acid sequence of IGO N in the highly diverse V-D junction. The .alpha. chain and the antigenic specificity of the T-cell line derived transcripts has also been characterized.

Description

WO95/28481 21881~2 P~l/u~ s~o,~
CUN:jl!ilCY~:lJ T-CEL~ ~u~i~lOR b~;yu~iN~:~:S
This application is a continuation-in-part of Serial No. 07/766,751 filed September 27, 1991. This work was 5 supported by grants from the National Institutes of Health.
BACKGRO~IND OF TH~ INVENTION
T lymphocytes recognizes antigens through the T cell antigen receptor (TCR) complex. The TCR is a clone-specific 10 heterodimer on T cells, which recognizes its target antigen in association with a major histocompatibility antigen. Moreover, the TCR is highly polymorphic in dif ferent T cells .
Approximately 9 0 percent of peripheral blood T cells express a TCR consisting of an ~ polypeptide and a ,~ polypeptide and 15 a small percentage of T cells express a TCR consisting of a polypeptide and a ~ polypeptide. See Davis and Bjorkman, 1988, Nature 334:395-402; Marrack and Kappler, 1986, Sci. Azner.
254:36; Meuer et al., 1984, Arn. Rev. Irr~nunol. 2:23-50; Brenner et al., 1986, Nature 322:145-159; Krangel et al., 1987, Science 20 237:1051-1055; Hata et al., 1987, Science 238:678-682;
Hochstenbach et al., 1988, J. Exp. Med. 168:761-776).
The chains of the T cell antigen receptor of a T cell clone are each composed of a unique combination of domains designated variable (V), diversity (D), joining (J), and 25 constant (C) (Siu et al., 1984, Cell 37:393; Yanagi et al., 1985 Proc. Natl. Acad. Sci. USA 82:3430) . Hypervariable regions also have been identified (Patten et al., 1984, Nature 312:40; Becker et al., 1985, Nature 317:430) . In each T cell clone, the combination of V, D and J domains of both the alpha 3 0 and t~e beta chains or both the delta and gamma chains and defines a uni~ue antigen binding site in each T-cell clone.

WO95/28481 ~B~8~ lo~

In contrast, the C domain does not par~icipate in antigen binding .
TCR genes, like immunoglobulin genea, consist of regions which arrange during T cell ontogeny (Chien et al., 1984, Nature 312:31-35; ~Iedrick et al., 1984, Nature 308:149-153; Yanagi et al., 1984, Nature 308:145-149) . In genomic DNA, each TCR gene has V, J, and C regions; TCR ~ and ~ polypeptides also have D regions. The V, D, J and C regions are Yeparated f rom one another by spacer regions in the DNA . There are usually many variable region segments and somewhat fewer diversity, junctional, and co~stant regions segments. As a lymphocyte matures, these various segments are spliced together to create a cnnt ; nllnus gene sequence consisting of one V, (D), J, and C regions. TCR diversity, and thus T cell specificity, derives from several sources, (Barth et al,, 1985, Nature 316:517-523; Fink et al., 1986, Na~ure 321:219-225) including:
a multiplicity of germline gene se~ments (Chien et al, 1984, Nature 309:322-326; Malissen et al., 1984, Cell 37:1101-lllO;
Gascoigne et al., 1984, Nature 310:387-391; Kavaler et al., 1984, Nature 310:421-423; Siu et al., 1984, Nature 311:344-349;
Patten et al., 1984, Nature312:40-46), combinatorial diversity through the assembly of different V, D, .J, and C segments (Siu et al., 1984, Cell 37:393-401; Goverman et al., 1985, Cell 40:859-867), and junctional flexibility, N-region diversity and the use of either multiple D regions or any of the three translational reading frames for D~ segments. As a result of these me-~n; cmC~ TCRs are generated which differ at their N-terminal (called variable, or V regions, constructed from combinations of V, D, and J gene segments) but are the same elsewhere, including their C-terminal (called constant regions) . Therefore, an infinite number of TCRs can be established. ~:
The V~ gene of the TCR appears to resemble most closely the immunoglobulin V gene in that it has three gene segments, V,B, D~, and J~, which rearrange to form a contiguous V~ gene (Siu et al., 1984, Cell 37:393-401). The ,B locus has been well characterized in mice, where it spans 700-800 kilobases of DNA and is comprised of two nearly identical C

WO 95/28481 218 8 18 2 F~1/lJ., C'Q4 regions tandemly arranged with one D element and a cluster of 5-6 J elements 5' to each (Kronenberg et al., 1986, Ann Rev.
Imn?unol. 3:537-560) . Approximately twenty to thirtyV,B regions are located upstream (5' ) to the D, J, and C elements (Behlke 5 et al., 1985, Science, 229:566-570) although Vl~ genes may also be located 3 ' to the murine C~ genes (Malissen et al ., 1986, Nature 319 :28) . Study of the structure and diversity of the human TCR ~-chain variable region genes has led to the grouping of genes into district V~ subfamilies (Tillinghast et al., 1986, Science233:B79-883i r~nr~Ann~n et al., 1986, Proc. Natl., Acad. Sci. USA 83:6598-6602; Borst et al., 1987, J. Immunol.
139: 1952-1959) .
The ~TCR gene was identified, first in mice (Saito et al., 1984, Nature 309:757-762; Kranz et al., 1985, Nature 313:762-755; Hayday et al., 1985, Cell 40:259-269) and then in humans (Lefranc et al., 1985, Nature 316:464-466; Murre et al., 1985, Nature 316:549-552) . The human -yTCR locus appears to consist of between five and ten variable, five joining, and two constant region genes (Dialynas et al., 1986, Proc. Natl. Acad.
Sci. USA 83:2619) .
The TCR ~ and ~ locus are adjacent to one another on human chromosome 14. Many TCR ~ coding segments are located entirely within the ~Y gene locus (Satyanarayana et al., 1988, Proc. Natl. Acad. Sci. USA 85:8166-8170 Chien et al., 1987, Nature 330:722-727; Elliot et al., 1988, Nature 331:627-631).
It is estimated that there are a minimum of 45-50 V~ regions ~Becker et al., Nature 317:430-434) whereas there are only approximately 10 V~ regions (Chien et al., 1987, supra).
Nucleic acid ses~uences of TCR ~ genes have been reported (Sim et al., 1984, Nature 312:771-775; Yanagi et al., 1985, Proc.
Natl. Acad. Sci. USA 82:3430-3434; Berkout et al., 1988, Nucl.
Acids Res. 16:5208).
Rheumatoid arthritis (RA) is a chronic, recurrent, ln~l: tory disease primarily involving joints, affecting 1-396 of North Americans. Three time6 the number of women are afflicted with RA than men. Severe RA patients tend to exhibit extra-articular manifestations including vasculitis, muscle atrophy, subcutaneous nodules, lymrhA-l~n~pathy, splenomegaly _ _ _ _ _ _ _ _ _ _ _ _ , .. . . .. . . .. . . .. _ _ . . . _ .. ...

WO 95/28481 ~ 1 8 8 18 2 r~ ,o l~
and leukopenia. Spontaneous r~mission may occur; other patients have brief episodeæ of acute arthritis with longer periods of low-grade activity; still others progress to æevere deformity of joints. It iæ eætimated that about 1596 of RA
5 patients become completely incapcitated ~ " Primer on the Rheumatic Diseases, " 8th edition, 1983, Rodman, G P. &
Schumacher, H.R. Eds., Zvaifler, N.J., Aææoc. Ed., Arthritiæ
Foundations, Atlanta, Ga. ) .
The antigenic stimulus initiating the immune response 10 and consequent inflammation is unknown. Certain H~A types (DR4, Dw4, Dw14 and DRl) have an increased prevalence of RA, perhaps leading to a genetic susceptibility to an lln~ nr;fied factor which initiates the disease process. Relationships between Epstein Barr ~irus and RA have bee~ suggested.
Many cell types, notably macrophages, synoviocytes and polymorphonuclear leukocytes, participate in the complex ln~li tory response~which effects joint destruction in R.A.
However, a central role for T lymphocytes is suggested by: 1) the rich infiltration of activated T cells at the primary site of RA disease, the synovial tiæsue (van Boxel, J.A., et al., 1975; N. Engl. J. Med., 293:517; Panayi, J.S. et al., 1992, Arthritis Rheum. 35:729); 2) genetic ætudieæ ~inking R~ disease susceptibility to a def ined amino acid sequence in the third hypervariable region of the DR~ chain of the major hiætocompatibilty complex (MHC) class II molecule (P.
Gregersen, J. Sl~ver, R. J. Winchester, 1987, Arthritis Rheum.
30:1205); 3) animal modelæ of chronic arthritis in which antigen-specific T c~lls are capable of transferring disease to naive recipients (R. ~ hl, L. Klareskog, K. Rubin, E.
~arsson, H. Wigzell, 1985, Scand. .J. I~ unol. 22:295; W. van Eden et al., 1985, Proc. Natl. Acad. Sci. USA, 1985, 82:5117);
and 4 ) amelioration of arthritis, both in murine models of aut~immune diæeaæe and in patients with RA, by administration of monoclonal antibody (m~b) reactive with the CD4'T cell subset (G. E. Rangers, S. Sriram, S. M. Cooper, 1985, J. Exp.
Med. 162:11104; G. Horneff, G. R. Burmester, F. Emmrich, J. R.
Ralden, 1991, Arthritis Rheum. 34:129).

WO 95/28481 1 ~11 1,~ ' 'C I

Previous studies designed to correlate TCR structure with antigen-MHC molecular complex recognition have emphasized the importance of critical amino acid residues in each of the three polymorphic CDR regions of both ~ and ,B chains, with CDR3 playing a dominant role. In both the murine and human systems, T cells specific for a particular peptide--MHC complex often utilize a characteristic amino acid or sequence cluster in the CDR3 region (S.M. Hedrick et al., 1988, ~cience 239:1541) .
Recent studies demonstrate that the introduction of charge altering amino acids in a well defined antigenic peptide results in a T cell response characterized by antigen-specific TCR3 which have incorporated reciprocal charge changes in the CDR3 amino acid residues of both ~ and ,~ chains (J.L. Jorgensen et al., 1992, l~ature 355:224) . This result suggests that these TCR residues bind directly to the antigenic peptide. In a related study, it was found that the murine TCR repertoire re-~rrn;7;nr foreign peptides which are highly homologous to self is markedly constrained with respect to TCR V~ and V,B gene usage, CDR3 length, and the presence of canonical amino acid residues in the CDR3 domain (J.-L. Casanova et al., 1991, .J.Exp.Med. 174:1371). These data suggest that pathogenic T
cells mediating autoimmune disease will express TCR which share crucial structural characteristics.
Further support for this hypothesis is found in studies of other autoimmune diseases. Myelin basic protein (MBP) specific Th cells induce exp~r; t~1 allergic ~.nr~rh~lomyelitis (EAE) (S.S. Zamvil et al., 1988, J.Exp. Med.
167:1586; J.L. Urban at al., 1988, Cell 54:577; F.R. Burns et al., 1989, ~J.Exp.Med. 169:27). ~nr~orh~litogenic T cell clones 3 0 are strongly biased with respect to V~ and V~ gene usage as well as CDR3 region structure (D.P Gold et al, 1992, J.
I~nnlunol . 148 :1712 ) . Recently, it has been shown that TCR V,(3 transcripts isolated from ce~tral nervous system lesions of patients with multiple sclerosis (MS) exhibit sequence motifs in the CDR3 region homologous with those expressed by ~n~rh~l itogenic M~3P reactive murine T cell clones (R. Martin et al., 1991, ,;r Exp. Med. 173:19; J.R. Oksenberg et al., 1993, Nature 362:68) .

Wo95~28481 ~ g~ . '0 While the importance o~ T cells in RA appears clear, neither the antigen specificity nor the structure of the TCR
expressed by disease-inducing T cells has been determined. In an attempt to identify pathogenic T cells among the vast number present in the inf lamed j oint, investigators have applied molecular techniques to detect T cells which: 1) share TCR
structural features, i.e. restricted usage of particular TCR
variable gene elements, or 2) are "oligoclonal" with respect to the highly polymorphic antigen binding CDR3 region of the TCR, suggesting antigen-driven ex~?ansion at the site of pathology. To date, this approach has yielded conflicting results. Several laboratories have reported evidence of oligoclonality and over-usage of particular TCR V gene products among RA joint-derived T cells (M.D. Howell et al., 1991, Proc.
Natl. Acad. Sci. USA 88:10921; X. Paliard et al., 1991, Science 253:325; W. V. Williams et al., 1992, J. Clin. I~vest. 90:326) .
However, the TCR V gene families implicated vary from study to study and still other investigations find no evidence for TCR
skewing in RA (Y. Uematsu et al., 1991, Proc. Natl. Acad. Sci.
USA 88: 8534; J. M. van Laar et al., 1991, Clin. Exp. Immunol .
83 :353) .
SUMMARY OF INVENTION
Four unique V,(~ 17 transcripts encoding conserved CDR3 regions of the T cell receptor of RA patients has now been discovered. These unlque transcripts while not i~llontic~l, are highly homologous in an otherwise variable region of the TCR.
Also, within these transcripts is a highly conserved sequence IGQ_N (sequence I.D. No.13). These transcripts were isolated from the synovial tissue of a RA patient and a cell line ~n~ from synovial tissue T-cells.
It has also been discovered that two unique ~ chains with conserved CDR3 sequences are utilized by the uni~ue T cell clones .
Methods ~or diagnosing and treating RA with the peptides encoded by the transcripts and/or monoclonal antibodies specific for the peptides is also part of this invention .

WO9~128481 2 ~88ig2 r~ , DESCRIPTION OF THE FIGI~RES
Figure 1 depicts mnnnnllnl ear cells (MNC) from healthy subjects, patients with seropositive RA, patients with non-RA
;nfl~r~-tory arthritis, and patients with systemic lupus 5 erythematosus (SLE) tha~ were analyzed by indirect immunofluorescence for expression of V,(~ 17 TCR gene products.
Results for peripheal blood (PB) are shown in the top panel and for synovial fluid (SF) in the bottom panel and are expressed as 9~ of cells reactive with the anti-TCR V~ mAb/96 of cells 10 reactive with anti-CD3 mAb.
Figure 2 depicts MNC isolated from synovial fluid and analyzed by indirect immunofluorescence for expression of the T cell surface epitopes indicated. Fluorescence is demonstrated on the abscissa (~og scale) and cell number on the 15 ordinate (linear scale) of each cytofluorograph histogram.
Figure 3 depicts two color immunof luorescence analysis of SF T cells. SF T cells were stained with anti-VB
(left panel) or anti-V~ 17 (right panel) mAb.
Figure 4 depicts the CDR3 sequences of ~n--i n~nt V~17 20 transcripts identified among freshly isolated synovial tissue T cells (V~17seql and seq2) and culture 5 derived T cell clones P~n~ in vitro (V,B17seq3 and seq4). The conserved amino acid residues at the N-D-N area are presented in boldface.
Figure 5 depicts the nucleotide and deduced amino 25 acid sequences in the CDR3 regions for V~2.3 (A) and V~3.1 (B) .
The Va2.3, cnnt:1;n;n~ a J~(IGRJaO9) segment, ~Csnmr~nyS
V~17seq3 and the V~3.1, using a J~k segment, is linked to V~17seq4 expressing in culture 5-derived T cell clones.
Figure 6 shows the proliferation of synovial tissue 30 T ce~l clones induced by EBV-transformed B cell lines.
DETATT En DESCRIPTION OF THE INVENTION
The invention is directed to unique tY/,3 T cell receptor (TCR) sequences which are conserved in the synovial 35 tissue of persons afflicted with RA. These four sequences show sequence homology in the complementarity-determining-region (CDR3), which is normally a highly variable region of the TCR.

-WO 95128481 218 8 1~ 2 PCT/1~595/04803 Both the o~ and ~ chains of these transcripts have been characterized, as well as their antigenic specificity.
The first step in isolating these transcripts was to identify the type of T cell which is pathogenic in RA and characterize the relevant antigens that maintain their chronic activation. This was done by utilizing a panel of murine mAb reactive with the products of particular TCR V3 gene families.
The results demonstrated the selective increase in the percentage of T celLs bearing the V,~17 TCR sequence in the peripheral blood (PB~ and synovial fluid (SF) from patients with RA.
In order to :evaluate the pathogenic potential o~ the oligoclonal V~17 synovial T cells, isolation of these cells in vi tro and characterization of the TCR ~/B chain structure and antigenicity had to be done. To accomplish this, one patient with "classic" RA was followed for eighteen months and then T
cells were explanted from his synovium. The uniriue transcripts were isolated from both the fresh synovium T cells and a cell line generated from the synovial tissue T cells.
Two of these transcripts, designated V~17seql ~amino acid sequence - seq . I . D . ~o . 1; nucleotide sequence - seq .
I.D. No.2) and V,~17seq2 (amino acid sequence - seq. I.D. No.
3 ; nucleotide sequence - seq. I.D. No. 4), were isolated from V317 cDNA clones derived from fresh synovial tissue. The other two of these sequences, designated V~17se~3 (amino acid sequence - seq . I . D . No . 5; nucleotide sequence - seq . I . D . No .
6) and V~17seq4 (amino acid sequence - seq. I.D. No. 7;
nucleotide sequence - seq . I . D . No . 8 ) were isolated f rom a cell line generated ~n vitro from synovial tissue T cells.
These four~transcripts, while not i~nt;r~l, contain highly homologous seriuences. The nucleotide and deduced amino acid sequences in the CDR3 region are shown in Figure 4.
Comparison of the sequences show that amino acid residue "I" at position 95 and "N" at position 99 is found in all four transcripts. It should be noted that residue N at position lY99 is encoded by the germline J~2.1 segment in V~17seq2, 3 and 4 but the same residue in V,B17seql is not germline encoded but results from the process of N region W095/28481 218~ 82 r~ o~
nucleotide addition which generates diversity in the antigen binding VDT region. See ~ieber, M.R. et. al., Proc. Nat'l.
Acad. Sci. USA, 1983, 85:8588.
Furthermore, homology of the sequences of transcripts 5 V~17seql and V,~17seq3, in the CDR3, region is 78 . 5% at the nucleotide level and 86.4% at the amino acid level. Also, 4 out of 5 amino acids , IGQ_N ( seq . I . D . No . 13 ), at residues #95-99, in the highly diverse V-D junction, are conserved in both transcripts.
Without being bound by any theory, it is believed that the four ~nm;n~nt transcripts recognize the same joint-localized antigen and that the conserved amino acids, " I " at position #95 and "N" at position #99 in the CDR3 region of the TCR~ chain may prove crucial for antigen recognition.
Because antigen recognition is a function of both TCR
Y and ~, the ~Y chain usage of the cell line derived T-cell clones characterized also ~Fig. 5). V~17seq3 expresses the Va2.3, and had an ~ rearrangement of V~2.3-J~ (IGRJaO9)-C~
(amino acid sequence - seq. I.D. No. 9 and nucleotide sequence 20 - seq. I.D. No. 10) . The expression of Vo~2.3 is of interest because recent reports have shown a selective increase in Vo~2.3 T cells in the synovial fluid of RA patients. See Pluschke, G. et al., ~3ur. ~. Immunol., 1991, 21:749; Broker, B.M. et al., Arthritis ~heum., 1993, 9:1234. V~17seq4 expressed Vol3.1-Jo~K-25 C~Y (amino acid sequence - seq. I.D. No. 11; nucleotide sequence - seq. I.D. No. 12) (Fig. 5).
Lastly, the antigen specificity of the culture derived T cell clones was determined. The synovial T cells expressing the conserved CDR3 sequences respond to the alleles 30 of the RA associated DR4 molecule.
These T cell clones which utilize the same V~ gene, are highly homologous in the antigen binding CDR3 region, and are reactive with or restricted by the HLA DR4 antigen probably have pathogenic potential in the rheumatoid process. This is 3s supported by: 1) m~b staining results in which only RA, but not other arthropathies, is characterized by an expansion of V~17+
T cells; 2) data from other laboratories demonstrating the selective representation of V~17, 14 and 3 among RA synovial WO 95/28481 ~ 1 8 8 ~ 8 2 P~ /01~^~

T cclls (Howell, M.D. et al, Proc. Nat'l. Acad sci ~SA, l991, 88:10921; Paliard et al, Science, 1991, 253:325;
Williams, W V., et al, .T. Clin. ~nvest., 1992, 90:326); and most compelling, 3) a CDR3 sequence highly homologous to that 5 in our V~17seq2 (nIQG_N") has been identified among the f~,r~n~l~.l, oligoclona1 V~14 TCR transcripts isolated from synovial fluid and tissue T cells o~ a DR4~, RF~ patient suffering from juvenile rheumatoid arthritis. See Grom et al., Proc. Nat'1. Acad. Sc1. USA, 1993, 90:11104.
The isolation and in Vitro growth of clones expressing TCR sequences homologous to the ~ln~; n~nt V,~17 sequences identified in fresh synovium have allowed el~ tinn of the complete structure of the o~ ,~ TCR expressed by potentially pathogenic RA T cells. These clones can also be 15 used to assess reactivity against a panel of potentially important self antigens, including joint-restricted antigens postulated to be targets of autoimmune attack, e.g. type II
collagen, proteoglycans, heat shock proteins, as well as an array of synthetic peptides cnnt~ln;n~ the sequence shared by 20 RA-associated DR molecules, QKRaA (seq. I.D No. 14).
These sequences could also be used as a confirmatory diagnostic tool for ~. A rii~gnnsi~ of RA may be made based upon clinical features and a in vitro assay using probes homologous to the conserved TCR sequences could confirm that 25 the patient is ; n-lP~rl suffering from RA, as opposed to other diseaees affecting the joints. Such a method would entail contacting the bodily fluid of a person suspected of suffering from RA with a probe homologous to the conserved TCR sequences.
Bodily f luid would include, but not ~e limited to, synovial 30 fluid. The presence or absence of the conserved TCR sequence determined by any method known to those skilled in the art.
Specific embodiments would include probes homologous to any one of the CDR3 sequences of V~seql, V,l317seq2, V,B17seq3 and V317seq4 or other -~ortions thereo~. Another preferred 35 embodiment would include probes homologous to the conserved CDR3 ~-chain sequences Yet another preferred embodiment would be a probe homologous to the nucleotide sequence ~ncn~ing the amino acid sequence IGQ_N

WO95/28481 ~ r~ ol~

The unique TCR sequences could also be used for immunotherapy for RA, for example by using them as "blocking"
antigenic peptides, activation of immunoregulatory cells, induction of an anti-TCR antibody or in mAb mediated deletion 5 of the pathogenic V gene expressing T cells. FL5YI ntq consisting of amino acids homologous to the unique TCR sequence could be administered to a patient directly. Such sequences would act by blocking the TCR of the T cells making the T cells unable to attack the antigens of the patient. The specific 10 peptides to be used in such a method would be homologous to the amino acid sequence of V~317seql, V~17seq2, V~17seq3 or V~17seq4. More specifically, a peptide homologous to the sequence IGQ_N could also be used.
In an alternative method of immunotherapy, mAb could 15 be produced, by conventional methods known in the art, which is directed to the unique TCR sequences. These mAb directed to the unique TCR sequences would target the unique TCR
sequences which are believed to be pathogenic.
Specific monoclonal antibodies to be used in such a 20 method would include mAb which recognize the CDR3 sequences of V,~seql, V~seq2, V~seq3 or V~17seq4 or portions thereof. A mAb directed to IGQ_N would also be useful.
Either the peptides or the mAb could be administered to an RA patient, for example in his synovial fluid, 25 systemically, or orally, in a suitable pharmaceutical carrier.
Exam~le In order to directly evaluate TCR V gene usage in the RA T cell repertoire, a panel of monoclonal antibodies specific 30 for human TCR V~3 gene products was utilized. Control subjects included 25 healthy volunteers (female/male = 2.4, mean age =
43.3) . Disease controls included patients with systemic lupus erythematosus (female/male = 10.0; mean age = 40) or 19 patients with non-RA ;n~li tory arthritis, including 35 osteoarthritis, gout, Reiter's syndrome and monoarticular arthritis; (female/male = 2.0; mean age = 59.5) . RA patients were defined using American Rheumatism Association criteria (Arnett et al., 1988, Arthritis Rheurn., 31:315-324) . Patients WO95/28481 2188182 F~ll~ c~ol~
were not selected with respect to medical therapy, which included aspirin, nonsteroidal ant;infl~r--tory drugs, corticosteroids, methotrexate, gold, hydroxychloroquine, or sulfasalazine. Serum samples from RA patients were assayed by latex fixation to determine if the patients were seropositive for RF. PB m~n~ ear cells (MNC) from some subjects were characterized for HI~A DR haplotype using standard serologic reagents .
Peripheral blood samples were obtained by venipuncture, and SF samples were obtained at the time of therapeutic arthrocentesis. ST specimens were obtained from the Department of ~athology at The Hospital for Special Surgery following therapeutic arthroscopic synovectomy, open synovecto-my, or total joint replacement. Synovial tissue was minced under sterile conditio~ls and incubated in 20 ml. of an enzyme preparation c~nt~;ning RPMI 1640 (GIBCO ~aboratories, Grand Island, NY), 20~ fetal calf serum (Whittaker Bioproducts, Inc., Walkersville, MD), 19~ penicillin and streptomycin, 1~ glutamine (GIBCO), 0.5 mg/ml collagenase, 0.15 mg/ml DNase, and 0.1 mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) at 37C., 59~
CO2 for 2-4 hours. Tissue was then mechanically disrupted using forceps and scalpel and pressed through a mesh sieve.
MNC were isolated from PB, SF, or ST digest on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient. In some cases, T cells were selectively enriched by rosetting of MNC
with sheep red blood cells (SRBC) followed by incubation at 4C
for 16 hours and subsequent fractionation of rosetted and unrosetted cells over Ficoll-Hypaque.
The T cells were stained with the following monoclonal antibodies : OKT3 (anti-CD3 , pan-T); OKT4 (anti-CD4 , helper/inducer subset); OKT8 (anti-CD8, suppressor/cytotoxic subset, American Type Culture (~llP~-ti~n, Rockville, MD); ,BV3 (reactive with TCR V33, T Cell Diagnostics, Cambridge, MA); C37 (reactive with TCR V~5.2/5.3, Wang et al., Hybridoma, 1986, 5:179); OT145 (reactive with TCR V,36.7a, ~i et al., J. Exp.
Med., 1990, 171:221); 16G8 (reactive with TCR V~8, T Cell Diagnostics); S511 (reactive with TCR V~12, Bigler et al., .J:
Exp. Med., 1983, 158:1000; Cl (reactive with TCR V~17, Freedman Wo95128481 13 21~8~ P~ C
et al., IJ. ~xp. I~ed., 1991, 174:891) and F1 (reactive with TCR
V~2.3, Janson et al., Cancer I~zununol. Immunother., 1989, 28:225) .
1-2 x 10; MNC or T cells were ;n~llh~tPrl with buffer 5 alone or a æaturating concentration of mAb at 4C for 30 minutes. Cells were then washed three times and incubated with a saturating concentration of fluorescein-labeled F (ab' ), fragments of goat anti-mouse IgG (Tago, Inc., Burlingame, CA) at 4C for 30 minutes. After 3 washes in buffer, the cells 10 were analyzed on a cytof luorograph . In some cases, two color immunofluorescence analysis was performed. The above procedure was followed by a blocking step, with cells incubated at 4C
for 30 minutes with an irrelevant murine mAb ~anti-trinitrophenol). After three washes, the cells were incubated 15 with a phycoerythrin-labeled murine mAb, washed, and prepared for analysis on a cytofluorograph. Cell fluoroscence was analyzed on an Ortho IIs cytofluorograph, gating on the small, nongranular lymphocyte population. The percentage of cells fluorescent with buffer or irrelevant control murine mAb and 20 fluorescein-labeled goat anti-mouse IgG alone was subtracted.
Cytofluorograph histograms of cells stained with anti-TCR mAb exhibited a peak of fluorescence distinct from the negative peak and with fluorescence intensity approximating that of cells stained with anti-CD3 m~b.
Exam~le 2 PB MNC from healthy subjects and from patients with seropositive RA, non-RA inf lammatory arthritis, or SLE were isolated and the percentage of CD3-positive cells expressing 30 the TCR V~3 gene product identified by a panel of anti-TCR mAb determined by indirect ;r--ln~lfluoresence analysis. The mean percentage of T cells reactive with mAb C3 7 (V~ 5 . 2 , 5 . 3 ), OT145 (V,B 6.7a), 16G8 (V~B 8), or S511 (V~ 12) is similar in each of the groups tested (Table 1), a result consistent with 35 previous studies of T cell repertoire in autoimmune disease which used these monoclonal reagents ( Posnett et al, .J.
Irnmunol., 1988, 141:1963; ~ lr-ln~ccon et al., Scand. J.
Irrununol, 1992, 36:681) . In contrast, analysis of CD3-positive WO 95/28481 21~ 8 ~ 8 2 r~ o ~ ~

Ps cells reactive with the more recen8:1y availa~le mAb C1, speci~ic for ~the V~ 17 TCR gene product, demonstrates a significant increase ~ the mean percentage o~ V,B 17-positive cells in RA patients when compared with the normal subjects or 5 control patients (p = 0.002) (Ta~le l and Figure 1) . No signi~icant increase in V~ 17-positive cells is observed in the non-RA arthritis or SLE patients when comr;~r,or~ with the normal controls. Taken together, these results demonstrate a selective expansion of V,(~ 17-positive T ce~ls in the PB o~ RA
10 patients.
T ble I
Sr~q Or T Ceit Pb~ f ItA ~d Co~l Sr bjeeta * OrcD3~. Cd~
T c.r~ ADt~8eD: CD4 CD8 IL.2BDSS V~3153 V~6.7~ V~g 11 (Monoclor~l ADtibody) (0~4) (OKTS) (AnD-Tac) (C37) (OT145) (16G8) (5511) PeriDh~ral i3100d Norraal Subjects 71.0 30.4 4.5 2.7 3.6 3.8 +8ga4) +11.3(14) +2.9(14) +1.7(15) +2.1(21) S~E 54.5 42.1 6.8 2.9 3,2 D
+11.8(13) +14.1(13) +8.0a3) +1.4(11) +1.6(25) ~ , Nor,BA Artbriti~ 54.9 41.4 12.6 3 5 3,1 11 +14.7(7) tl6.8(8) +19.2~7) +1.8~`7) +1.6(7) :!1 r~A 70.1 37.0 8.9 3.4 3.2-+l93a7) +18.7(27) +7,7(27) +1.8(22) +2.5(28) Norl-BA Artbrilis 6.24 313 9.5 3.1 3.6 4 +15.6~15) +l4.laS) +4.4(15) +1.9(17) +2.1(17) RA 53g 48.7 123 3.5 3,7 3 +16.8(46) +13.0(45) +6.6(45) +2.1(45) +2.4(48) ~1 Svnovial Tiuue ,DA 63.2 41.6 9.7 2.6 3,8 4 +15.2(19) +23.1(19) +7.0(20) +2 2(17) +2.7(21) . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ bnlated T r~os vle amly ed by ir~ect ' ~ a ~tolh~i~ The i~ge Or CD 3 ix~e ~lis exye~ eacb T cell ~face~v~se~LSilovD~tb~r~+~brddc~andDrr oerorir~akanaiv~i()roreacbsul~jea~roup ~tDdied.
~Ip ~ 0.002 I~p = O.Oi)l

2~182 W0 95/28481 P~ ~ riO I

Example 3 To characterize the T cell repertoire at the site of pathology in patients with RA, the synovial fluid, SF T cells were isolated from 49 r~ti Pnt~ with seropositive Rl~ and analyzed 5 for TCR V,B gene usage by indirect immunofluoresence staining ~Table 1 and Figure 1). The mean percentage of CD3-positive cells reactive with the V3 17-specific m~b C1 is significantly elevated (p = 0.001) in the RA patients (8.596 + 4.1) when compared with the percentage V,B 17-positive cells in the 19 SF
o specimens from p~tif~nt~ with non-RA infl t-lry arthritis (5.39 + 2.0) . Strikingly, 319~ (15/49) of the RA fluid samples and 0/19 of the control samples contain greater than 10~ V,B 17-positive T
cells (Figure 1). In contrast, no significant differences were noted between RA and control SF in the percentages of V,(~ 5 2/5.3, V,(~ 6 . 7a, V13 8, or V~ 12-positive T cells .
A representative study of SF T cells from an RA patient with an elevated percentage of V,~ 17-positive T cells i9 presented in Figure 2. Analysis of the TCR repertoire in this patient shows 17.3~ of cells expressing the V,~ 17 gene product, 20 but only 2.896, 2.4~, and 1.9~ expressing the V~ 5.2/5.3, V,B 6.7a, and V~ 12 products, respectively. Thus, of the five TCR V~ gene families studied, only V,~ 17 is si~n; f; ~-~ntl y increased in expression at the site of disease in the RA p~ti ~nt~. While we do not have ~A typing data on all of our subj ects, an increased 25 percentage of V~ 17-positive T cells in RA SF does not appear to correlate directly with expression of the DR4 RA susceptibility allele in thege p~ti~nt~ To date, 4 of the 15 r~ti,ont~ with ~10~ V3 17-positive SF T cells have been HI-A typed, and their DR
haplotypes are: DR 4, 7; DR2, 3; DRw13; and DR5, 7 .

Example 4 Two color immunofluorescence analysis was performed on 5 RA SF samples and on 3 non-RA infl;~ tnry arthritis SF
samples, all of which ~nnt~ln~ less than 109~ V,~ 17-positive T
35 cells (Table 2). Th SF cells were stained with anti-V~ 3 or WOg5/~8481 ~88~2 ~ ~ ~, anti-V~ 17 m~b and FITC-goat anti-mouse IgG, followed by phycoerythrin anti~ 2 receptor antibody (anti-p55- TAC) and immunoflourescence was assessed on a cytofluL~Lu~L~L,h. The Tac-positive cells are found almost exclusively among the CD4-s positive SF T cells from the RA patients. ~loreover, Tac-positive T cells were enriched among the V3 17 -positive T cells f rom the RA SF, but not ~rom the non-RA arthritis SF. Cytofluorograph histograms demonstrating Tac expression on a high proportion of V,~ 17-positive T cells (apprn~lr-tPly 4536), but not V~ 3-positive T cells (less than l~), from an RA SF specimen are shown in Figure 3. Thus, even RA SF that do not contain a markedly P~ }`An-lPd V~ 17-positive T cell pO~ tinn show evidence for preferential activation of that T cell ,r~rt;nn, when compared with T cells expressing other V~ gene products.

;Imnl e 5 In order to assess the pathogenic potential of V~17+
gynovial tisgue T cellc~ an informative RA patient was analyzed.
This patient has "classic~ rhellm~toid factor-positive (RF+) 20 polyarticular, ~y r;r;ll joint ;nfl; tinn, expresses the RA
associated ~HC class II antigen DR4 and Pl-h;h; t~ an V~17+T cell population. Over an eighteen month period of study, this patient r-;nt~;npd a skewed peripheral blood T cell repertoire char~ctPr; 7Prl by persistently elevated percentages of 25 V,B17 IT cells, i.e. 13 .2-15.79~ as compared to an average normal value of 5 . 3 ~ .
Af ter the 18 month period, synovial tissue was explanted from the patient by an arthroscopic synovectomy and the synovial T cells were isolated to use in: 1) analysis of cell 30 surface antigen expression; 2) rnlPr~ r charartpr;7atinn of x/B
TCR reaLL~ c; and 3) in vitro prop~t;on and cloning of V,~17+ T cell clones.
In order ~ to analyze the cell surface antigen expression, synovial tissue T cells were washed in phosphate-35 buffered saline (PBS) and stained with a panel of monoclonal WO 95/28481 : 2 1 8 8 1 ~ 2 antibodies as described in Example 1. T cells were lnrllh~tPflwith buffer alone or a saturating c nn~pntrAtion of mAb at 4C for 30 minutes, washed three times with PBS, and ;nC1lhAt~.l with a saturating cnnrontration of flurescein-labeled F(ab')~ fragments s of goat anti-mouse IgG (Tago, Inc., Burlingname, CA) at 4cC for 30 minutes. After 3 washes in PBS, the cells were analyzed on a cytoflurograph. Two color i ~fluorescence analysis were performed using phycoerythrin-labeled anti-CD4 and anti-CD8 mAb obtained from Coulter Immunology (Hialeah, FI ) . The above procedure was followed by a hlocking step, with cells ;n~llhAt~
at 4C for 30 minutes with all ~rrelevant murine mAb (anti-trinitrophenol~. After three washes, the cells were ;ncllh~tpd with a phycoerythrin-labeled murine mAb, washed, and analyzed on an Ortho IIs cytofluorograph, gating on the small, nnn~rAnlllAr lymphocyte pOplll At; ~n . The percent:age of cells fluorescent with buffer or irrelevant control murine mAb and flurescein-labeled goat anti-mouse IgG alone was subtracted. The results are shown ill Table 3.

WO 95/28481 2 1 ~ ~ 1 8 ~ P~

~o N N o ~D Ul O
N U1 ,~
N

o r~ O
N
LO
~1 ~1 +
;3 ;3~

WO 95/28481 2 ~ 8 8 1 8 ~ PCI/US95/04803 s ., ~
z r _ i _ t q ~ ~ Y ~; -' I
-- 9 ~,~ -- 00 ,:, : 'e, f ' 2 ~ ~ q r ~ ~ q ~ 3 ~ .
, , Wo951284X1 ~ 8~ P ~ O1~

As shown in the table, the distribution o~ V,1~17+ T
cells in this patient' 8 peripheral blood ref lected the overall CD4/CD8 ratio (1/4), however, synovial tissue TCR repertoire analysis with m~bs demonstratQd relative ~h~lnrl~n~.e of V,B17+ T
5 cells ~nd their selective presentation in the CD4+ subset.
ExamT~le 6 In order to assess ol t ~Qrl~ln~l t ty of V,~117+ T cells at the side of pPthology O_ RA, sequences of the highly polymorphic 0 antigen binding CDR3 region in the V~17 transcripts were mtn~d The V,B17 transcripts were derived _rom the synovial tissue and peripheral blood o_ the RA patient described in Example 5. These transcripts were analyzed uslng reverse transcriptese-polymerase chain reactlon (RT-PCR) with total 5 r~ RNA as a template.
Total c~ r R~A3 were t ~ t~e~t _rom the peripheral blood or sy ovial tissue T cells by the g~pn~tn;llm/cesium chloride centrifugation method or an acidi_ied gll-n;rl;ntllm/phenol/chloroform method a8 degcribed in the manual 20 o_ ~R~azoln', TEL-TEST, INC., Texas. The _irst-strand cDNAs were reverse transcribed with cDNA synthesis kit (cDNA Cycle }~it, InYitrogen, San Diego, ~rAltforntP). TCR V,B gene 8-_ ta were ~mpli~ied by polymerDse chain reaction (PCR) with the 5' sense olig~n~ 7~1~tide primers sper; ftc _or V~17 (5' -ACAG~iL~ LCL~ ~G~AGA-25 3') (seq. I.D. No. 15), V,B6.7 (5'-~''Cr~7`"T~r~rr~a~r-3') (seq.
I.D. No. 16), V,~l (5'Gr~r~ L c~;c~ACTT-3') ~seq. I.D. No.
17), in connection with a ~ntt ~-~n~e primer ~ ry to TCR
constant region sequence (5'-GG~ G~AGA ~ ; -3') (seq.
I.D. No. 18) . The PCR products were s~lhrl~n~d into a T/A cloning

3 0 vector according to the instruction manual provided by Invltrogen, San Diego, CA. The 1 t ~a~-t ~n mixture was used to tr~n~lform competent D~50~ cells. The plasmia DNA samples were prepared and sub; ect to sequencing using a sequencing kit (Seguenase version 2.0, ~nited States Flto~h~m;rAl, O~).

W0 95128481 ~ ~ 8 ~ ~ 8 2 A total o~ twenty-nine ~29) V~17 cDNA clones from synovial tiasue T cells were seguenced. Twelve (12) of the twenty-nine (29) clones contain the jtlont~rAl seguence. Eleven (11) of the twenty-nine (29) clones contain a distinct but 5 structurally-related seq-~once. The two ' n~nt synovial tissue seguences are designated V~Bl7segl and V,Bl7seg2~ respectively.
The nucleotide and deduced amino acid seguences in CDR3 region are shown in Figure 4. Comparison o~ these seguences reveals identity in length as well as conservation of several amino acids 10 with the CDR3 region, ~nrl~ n~ ~ qolo~r~no (I) at position #95 and AqpA~ ne at position #99.
In contrast, V,1~17 transcripts ~rom the Ra patients peripheral blood were hetero~onoo~q. Twenty-eight cDNA clones were seguenced and twenty-two distinct patterns of CDR3 sPquonroq 15 were present. None of the peripheral blood cDNA cloneg rnn1~ no~1 the dominant synovial tissue V,817 se~rlonroq.
Exale 7 l~o~ ...J, tq of V,~6.7a and V~Bl gene 8--hrAm;lioq were 20 also assessed in order to ensure that the clonal ~' 'n~nre of the synovial RNA samples of Example 6 were not an aspect of the PCR
amplif ication .
As shown in Table 3, V~B6~7a+ T cells represent only 1.59~ of synovial T-cells, 6 distinct L~aLL~I~. I-q were obs~L~,cd 25 in the 8 clones sequenced. Three distinct ro~ tq in 9 V~Bl T cell clonea seq~onred were found. These re~LL~I~. ts were totally heterogeneous with respect to CDR3 8~ onro~ and the JB segment usage.
The results described in this Example, along with the 30 previous exaa~ples, demonstrate that V,~17+ T cells express structurally related CDR3 sequences are selectively 0~ r~ntlod in the synovial tissue of RA r~t;ont~.

wog5n8481 2~8~182 r~ J.cQ4 F le 8 In order to examine the possible role played by T cells expressing these tlnm;n~nt V~17 sequences in the rhPllm~tni~l process, synovial tissue T cells were iEolated and P~r~lP-l ~r vi tro. Synovial tissue cells were incubated at 1 x 106/ml in RPMI 1640, 1096 fetal calf serum, 1~ pPn; r; 11; n and streptomycin, 1~ glutamine (culture medium) ~nnt~;n;ng V~Bl7 selective microbial superantigen ~¢Y~or7~l arthritidis mitogen (MaM, from Dr. B.
Cole, University of Utah School of MPr1; r;nP, Salt l~ake City, Utah) at a final concentration of 1/2000, or lO,ug/ml of anti-V~17 mAb C1.
As described in Priedman et al., J. Exp. Med. 174, 891 (1991), after 4 days of culture at 37C, purified interleukin 2 (IL-2) (srh;~rp~relli~ rnl i~, MD) was added to each culture at a final rnnrpntration of 1096. TCL were further P~r~n~Pfl by the weekly addition of x-;rr;~ tPfl sodium ppr;oll~tp-treated allogeneic peripheral blood non-T cells and I~-2.
While a large number of distinct V,317 TCR sequences are represented among the bulk T cell lines (TCLs) g~nPr~tPd, two V~17 transcripts from 1 TCL, designated culture 5, were analyzed using the method of RY~mple 6 . As shown in F; g . 4, the f irst transcript from culture 5, termed V~17seq3, utilizes V~17-D~2-J,1~2.1-C~2. This sequence is highly homologous to the .1l 'n~nt synovial tissue sequences, V~17seql and V,~17seq2.
The second ~J~17 transcript from culture 5, V317ses~4, Ehares the amino acid residue "I" at position #95 and ~'N" at position #99 with V,~17seql, 2, and 3 (Fig.4).
r le 9 ~ c_ain uEage by the culture 5-derived T cell clones expressing V~Bl7seq3 and 4 waE analyzed. The Culture 5 TCL cells were cloned by limited ~ lt;nn using 5xloJ x-;rr~ tpc~
periodate-treated allogeneic feeder cells and IL-2, then further P~rr~n~lPcl wltl~ epriodate- treated ~eeder cells and II--2 .

W0 95/28481 21~ 812 ~ r~

TCR ~Y reaLL~I~y. tq were analyzed using PCR and a panel of V~ specific primers from a TCR o~ constant region primer, (J. R. OkcPnh~rg et al., Nature 345: 344 (1990) ), OEcept primers for the VtY2 subfamily and for the TCR Cl chain constant region 5 sequence . The sense oligonucleotide primer f or V~Y2, 5lAGGTrr-~rr~TGATGA~ATccTTGAGAG-3l (seq. I.D. No. 19), is located at the 5' leader of VtY2 coding sequence and c--nt~lnc a Sal I site inside for further subcloning. The 3 ' antisense oligonucleotide complementary to TCR ~ constant region sequence is 5 ' -10 APTAGGTrr7~r~r~rTTGTCACTGG-3' (seq. I.D. No. 20) in which two nucleotides have been changed to create a Sal I site for future subcloning purpose.
This analysis revealed OEpression of only Va2.3 by T
cell clone expressing V~B17seq3. Formal sequencing was performed 15 and yielded a sequence consisting of V~2.3-J~Y(IGRJaO9)-CtY (Fig.
5). This V~Y designation is consistent with positive staining of this T cell clone by the Vc1!2.3 specific m~b F1 (Janson et al., 1989, C~ncer I~mlu~ol. Inmlunother. 28: 225) . A similar analysis of T cells OEpressing the V~B17seq4 has yield a TCR o~
20 rearrangement of V~Y3.1-Ja!-C~Y (Pig. 5). Thus, both TCR ~x and ~
chain of T cell clones OEpressing receptors homologous to the ;n~nt synovial tissue V,~17 transcripts have been characterized .
25 Exam~le 10 The culture 5 derived T cell clones were assayed for proliferative response against a panel of Epstein-Barr virus (EBV) transformed DR h-,...u,yyuus ly ~hnhl~ctoid B cell lines (BCL). As a control, an ~lnrlr~n~fl synovial tissue V~17+, CD4+ T
30 cell line designated culture lO was assayed simult~n~rl~-c~y (Fig.
6). 2 x 10~ T cell line cells were cultured, in triplicate, in 96 well round-bottom tissue cultures plate~ with medium alone or with 5 x lO~ EBV-transformed ~LA DR homozygous B cell line cells (from Dr. S. Y Yang, Sloan Xettering Institute, New York, NY) x-35 ; rr~ t~ with 4000 rads from a Cesium source. Cultures were Wo 95128481 2 ~ ~ 8 1~ ~ r~
suppleme~ted with 596 IL-2 and after 96 hours, 2 ,uCi 3[~I]-thymidine added to each culture and 16 hours later, the cultures were transferred to ~ilter paper using an ~llt~ tPrl cell harvester and counted i~ a beta counter. Clones expressing 5 Vcy2~3~v~Bl7seq3 showed poor growth rh~r~rtpriqtics and low levels of proliferation in response to all stimuli, inrlllfs;n~
~u~e ~ igens and anti-TCR mAb, ~lowever, as shown in the upper panel of Fig. 6, these cells pr~-l1fPr~te selectively to BCB cells expressing R~ associa~ed alleles o~ DR4, Dw4 and Dw14. T cells 10 expressing V~3.1/V,~17se~4 are highly responsive to DR4, DwlO
bearing BC~ cells (lower panel o~ Eig. 6) . This pr~l ;min~ry evidence o_ DR4 recognition ~y synovial T cells expressing the conserved CDR3 S~rl~lPnrPq is intriguing. It is not clear, however, if these clones recog~ize as yet undef i~ed antigenic 15 peptides in association with alleles o~ this ~C class II antigen or are speci_ic _or the DR4 alleles themselves.

WO 95/28481 2 1 8 8 1 ~ ~ P~ o l g -U~NlU~: LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLIQNT: Fried~an, Steven M
Crow, Mary K
Yi, y, Tumang, ~oseph Sun, Guang - Rong (ii) TITLE OF lNV~;N'l'l~JN: ConserVed T-Cell Receptor Sequences (iii) N~JMBER OF ~ u~;N~S: 21 ( iv) CORR~ NLI~N~:~; ADDRESS:
(Al ~nnT~Rc.sRR Darby ~ Darby PC
(Bl STREET: 805 Third Avenue ( C, CITY: New York (D STATE: New York ( E . COUN-TRY: US
(F, ZIP: 10022 (v) COMPUTER ~R~n~RT,T~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC nnTrlryt;hle (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Relea~e #1. 0, Version #1. 25 (vi) CURRENT APPLIQTION DATA:
(A) APP~ICATION N~JMBER:
( B ~ FILING DATE:
(C~ CLASSIFIQTION:
(viii~ ATTORNEY/AGENT INFORMATION:
(A~ NAME: Ludwig, S. Peter (B~ REGISTRATION NUMBER: 25, 351 (C~ K~ ;~;N~:~;/DOCKET NtlMBER: 59~3/09449 (ix~ TELECOMM~NIQTION INFORMATION:
(A~ TELEPHONE: 212-527-7700 ~B~ TELEFAX: 212-753-6237 (C) TELEX: 2366~7 (2) INFORMATION FOR SEQ ID NO:1:
UU~;N~ T~ TR~T~TICS:
(A) LENGTH: 23 amino acids ( B ) TYPE: amino acid (C~ ST~NnRnl\TR~,~ single (D~ TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide WO 95/28481 2 18 8 ~ 8 2 r~~ !O !~

(v~ FRAGMENT TYPE: interral (Vi) nRTr.TN,~T. SOURCE:
(A) Ol~r.~NT~M: HcTno sapiens ~vii) ~LMEDIATE SOD'RCE:
(B) C~ONE: v~17 seq 1-aa (Xi) ~ U~;N~:~; DESCRIPTION: SEQ ID NO:1:
Cy8 Ala Ser Ser Ile Gly Gln Glu Asn Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu mr Val Thr (2) INFORMATION FOR SEQ ID NO:2:
;UUl!;N~; rT-T1~T~rTRT~T.~TICS~
(A) LENGTH: 69 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MnT.RcTTT,R TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) nT~T~.'TN~T, SOURCE:
(A) nT~r7~NT~M Homo sapiens ( vii ) IMMEDIATE SOURCE:
(B) C~ONE: Vbl7-seq 1-nt (xi) ~ U~;N~:~; DESCRIPTION: SEQ ID NO:2:
TGTGCCAGTA GTATTGGTCA r.r.~.~rT~r GAGCAGTACT TCGGGCCGGG CACCAGGCTC 60 ( 2 ) INFORMATION FOR SEQ ID NO: 3:
U~ ; rT`Tl~T~Z~rTRT~T!:TIcS-(A) LENGT~: 22 amino acids (B) TYPE: amino acid (C) STT~NnT~.nNR.c.c single (D) TOPOI-OGY: liLear (ii) MO~ECU~E TYPE: peptide (v) FRAGMENT TYPE: in~rn;:

~0 95128481 2 ~ 8 8 ~ 8 2 ~ 5~Q `~

(Vi) ~RTc7TN~r, SOURCE:
(A) QRr~NT~h1- Homo sapiens (vii) IMMEDIATE SOURCE:
( B ) CLONE: vbl 7 - s eq2 - aa (xi) ~;UU~ ; DESCRIPTION: SEQ ID NO:3:
Cy8 Ala Ser Ser Ile Gln Gly Tyr Asn Glu Gln Phe Phe Gly Pro Gly hr Arg Leu Thr Val Leu ( 2 ) INFORMATION FOR SEQ ID NO: 4:
( i ) ~il~;UUl;N~; CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STR~NnP~nN~qS: single (D) TOPOLOGY: liIIear (ii) MOLECULE TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) ORIGINAL SOURCE:
(A) n~ NT.qM- Homo sapiens (vi i ) IMMEDTATE SOURCE:
(B) CLONE: vbl7 - seS~2 -nt (xi) ~l;uU~N~:~ DESCRIPTION: SEQ ID NO:4:
TGTGCCPLGTA GTATACAGGG GTACAATGAG r~lL, l1~ GGCCAGGGAC ACGGCTCACC 60 (2) INFORMATION FOR SEQ ID NO:5:
;QU~ ; rF~Z~R~rTRRTCTICS:
(A LENGTH: 22 amino acids (B TYPE: amino acid (C~ sTR~NnRnNR~s: single (D 1 TOPOLOGY: linear ( ii ) ~OLECU~E TYPE: peptide (v) FRAGMENT TYPE: interDal (vi) O~T(:TN~r~ SOURCE:
(A) nRr.~NT~M: Homo sapiens WO 95128481 218 8 ~ 8 2 r ~ o ~

(~ii) I~LMEDIATE SOrJRCE:
(B) CLONR: vbl7-seq3-aa (Xi) :j~;yU~N~ ; DESCRIP~ION: SEQ ID NO:5:
Cys Ala Ser Ser Ile Gly Gln Thr Asn Glu G1n Phe Phe Gly Pro Gly Thr Ar Leu Thr Val ~eu g 20 ( 2 ) INFOR~ATION FOR SEQ ID NO: 6:
:uu~;N~l; rr-T~RZ~rTRRT.qTICS
(A) LENGTH: 66 base pai~-s ~B) TYPE: nucleic acid (C) sTR~NnRnNR.q.q: sirLgle (D) TOPOLOGY: li~ear (ii) M~T,RCrrr,R TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) ORIGINAL sorJRcE
(A) oRr.~NT.qM- Homo sapiens (vii) IM~EDIATE SOrJRCE:
( B ) CLONE: vbl7 - seq3 ~ nt (xi) ~Uu~;Nu:~ DESCRIPTION: SEQ ID NO:6:
TGTGCQGTA GTATCGGGCA GACGAATGAG CA~LL~:ll~ r~-rr~r~Gr~r ACGGCTCACC 60 GTGCTA . _ 6 6 ( 2 ) INFORMATION FOR SEQ ID NO: 7:
;yu~ : rr~z~R~-TrJ~RT~sTIcs (A) ~ENGTH: 22 amino acids (}3) TYPE: amino acid (C) STR ~ .q.q: single ( D ) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: ; n (vi) ORTr.TN~r. SOURCE:
(A) ORGANISM: ~omo sapiens (vii) IM~!qEDIATE SOrJRCE:
(B) CLONE: vbl7-se~4-aa WO 95128481 2 9 2 1 8 8 1 8 ~ r . ~ . c c ~ Br (xi) ~iyu~;Ni_~; DESCRIPTION: SEQ ID NO:7:
Cys Ala Ser Ser Ile Pro Arg Ala Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg lieu Thr Val Leu ( 2 ) INFORMATION FOR SEQ ID NO: 8:
ilSyU~;NC~; rT~R~rTT~R T!: TICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STR~i~nRnNR.~.~ single ( D ) TOPOLOGY: l inear (ii) MnT.RCrTT,R TYPE: cDNA to mR~NA
( iv ) ANTI - SENSE: NO
(vi) r~RTr~TN~T~ SOURCE:
(A) r~Rr.~NT.~M. Homo sapiens (vii ) IMMEDIATE SOURCE:
(B ) CLONE: vbl7 - seq4 -nt (xi) ~uu~;N~:~; DESCRIPTION: SEQ ID NO:8:
TGTGCCAGTA GT~T~rCCrr. GGCCAATGAG CA~LLc~ GGrr~r~ r~r ACGGCTCACG 60 ( 2 ) INFORMATION FOR SEQ m NO: 9:
;yU~ rT~R7~rTRRT~C:TICS
(A:l LENGTH: 66 base pairs (B TYPE: nucleic acid (C STR~nRnNRqq: single (D TOPOLOGY: linear (ii) Mf~T,R~'ITT.R TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) nRTr.TN~T. SOTJRCE
(A) r,Rr.~NT.cM~ Homo sapiens (vi i ) IMMEDIATE SOURCE:
(B) CLONE: vbl7-seq~-nt (Xi) ~;yU~;N~:~; DESCRIPTION: SEQ ID NO:9:
., WO 95128481 ~0l 8 8 ~ ~ 2 r~ o I
TGTGCCAGTA GT~T7~rrrr~ GGCC~ATGAG CA~~ ~ GG~r~rr7r.~r ACGGCTCACG 60 ~2) INFORMATION FOR SEQ ID I;~O:l0:
(i) S~UU~;Nl~; rT~7~R,~rTRRT,5TICS
(A) LENGTH: 27 amino acids (B) TYPE: amino acid ( C ) S TR .~ N I ) ~ N ~: .4 C s i ng l e (D) TOPOLOGY: li~Lear (ii) MOLECULE TYPE: peptide (v) FRAC7MENT TYPE ; n~Prn~
(vi) r~RTr7TN7~T. SOUROE:
(A) oRr.~NT.qM- Homo sapiens (vii) IMMEDI~TE SOURCE:
~ B ) CLONE: Va2 . 3 - aa (xi) ~;UU~;N-:~; DESCRIPTION: SEQ ID NO:l0:
Cys Val Val Lys Gly Gly Gly Asn Lys Leu Val Phe Gly Ala Gly Thr 5 l0 15 Ile Leu Arg Val ~ys Ser Tyr Ile Gln Asn Pro ( 2 ) INFORWATION FOR SEQ ID NO: ll:
;UUb:N~; rFT,7lTI7'~l'r'RRT5TICS
(A) LENGTH: 81 base pairs (B) TYPE: ~ucleic acid (C) STR~NDRl~NE.55: single (D) TOPOLOGY: linear (ii) M ~r,R~T3r.R TYPE: cDNA to mRNA
(iv) ANTI-SENSE: NO
(Vi) (7RTr7Tr~T, SOURCE:
(A) ~Rr.~T5M- Homo sapiens (vii) IMMEDIATE SOURCE:
( ~3 ) CLONE: Va2 . 3 - nt (xi) ~;UU~:N~ DESCRIPTION: SEQ ID NO: ll:
tiA AGGGAGGGGG A~LCAaGCTG ~1cL1L~;CG r~rr.~rr~T TCTGAGAGTC 60 wo 95n848l 2 1 8 8 1 8 ~ F~ JL,.,'Q ,~

A~GTCCTATA TCCAGAACCC T 81 (2) INFORMaTION FOR SEQ ID NO:12:
(i) S ~;QU~N1_:$ r~ 'T~ T~TICS:
(A) LENGTH: 2 8 amino acids - (B) TYPE: amino acid ( C ) ST~ ~NnRnN~.~ .C: 9 ingl e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: i 7ternal (Vi) nRTt'.TN~T, SOURCE:
(A) n~r.~NT.~M~ Homo sapiens (vii) IMMEDIATE SOURCE:
( B ) CLONE: ~Ja3 . 1- aa (xi) ~;uu~ ; DESCRIPTION: SEQ ID NO:12:
Cys Ala Thr Leu Gly Gly Ser Asn Tyr Lys Leu Thr Phe Gly Lys Gly Thr Leu Leu Thr Val Asn Pro Asn Ile Gln Asn Pro ( 2 ) INFORMa~TION FOR SEQ ID NO :13:
it5UUlSN~ rT~T~T~TIcs:
(A) LENGTEI: 84 base pairs (B) TYPE: nucleic acid ( C) ST~ ~Nn~nNl; CS: 8 ingle (D) TOPOLOGY: linear (ii) MnL~ClTT~ TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
( vi ) ~R T~'.T~T. S OIJRCE:
(A) O~ NTCM: Homo sapiens (Vii) TMMRnI~T~ SOURCE:
( B ) CLONE: Va3 .1- nt (xi) ~ U~ DESCRIPTION: SEQ ID NO:13:
TGTGCTACAC TGGGAGGTAG CAaCTATAAA CTGACATTTG GAAAAGGAAC TCTCTTAACC 60 GTGAATCCAa ATATCCAGAA CCCT 84 W0 95/28481 ~18 ~ r~ s o 2 ) INFORMATION FOR SEQ ID NO :14:
;Uu~;N~ ; rT-T~T~rTRT~T.qTICS:
(A) LENGTH 5 am~no acids (B) TYPE: amino acid ( C ) S TR ~NnEnNR .q .q: s i ng l e (D) TOPO~OGY: linear (ii) MO~ECT~E TYPE: peptide ( v ) FRAGMENT ~PE 1 n t Prn ;
(vi) tlRT(~TN~T. SOUROE:
(A) 9T~r-~NT.qM Homo sapie~s ( vi i ) TMMEn T ~TE S oTJRCE:
(B) C~ONE: V-D junction (xi) Y~ U~;NU~ DESCRIPIION: SEQ ID NO:14:
Ile Gly Gln Xaa Asn (2) INFORMATION FOR SEQ ID NO:15:
;(..?U~;N-:~; rT-T~T~rTET~T~qTIcs (A) ~ENGTH: 5 amino acids (B) TYPE: amino acid ( C ) STT~ ~NnT~nNR.q .c 5 iI~.gle ~ D ) TOPOLOGY: l~ear (ii) MOT,RC~:Tr.R TYPE: peptide (v) FRAGM~NT TYPE: ; nt- Prn:~
(vi ) o~Tr-TN~T~ SOURCE:
(A) OT~rDNTqM ~Iomo sapiens (vii) IMMEDIATE SOURCE:
(B) CBONE: RA-DR peptide (xi) ~ Ur...~ ~; DESCRIPTION: SEQ ID NO:15:
Gln Lys Arg Ala Ala S
(2) INFf~T~M~T~ON FOR SEQ ID NO:16:
U~ rT-T~T~ TR~T~qTIcs (A) ~ENGTH: 18 base pairs (B) TYPE: nucleic acid ( C) sTr~NnEnNR~qq: single 2~
WO 95/28481 r~ Q~

~D) TOPOLOGY: linear ( ii ) MOLECUI.E TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) n~Tt~,T~r, SOIJRCE:
(A) ORGANISM: Xomo sapiens (vii ) IMMEDIATE SOURCE:
( B ) CLONE: VB17 - 5 ' PRIMER
(Xi) ~ u~;N(.:l:; DESCRIPTION: SEQ ID NO:16:

(2) INFORMl~TION FOR SEQ ID NO:17:
U~;N1.~ rTR~T qTICS:
~A ~ENGTH: 19 base pairs ~B TYPE: nucleic acid ~C STT~1~NnRnl~R.C.C:: single ~D TOPO~OGY: linear ~ii) M(IT.RCTJT.R TYPE: cDNA to mRNA
( iv) ANTI - SENSE: NO
(vi) ORIGINAL SO~RCE:
~A) n~ ~TqM- Homo sapiens ~vi i ) IM~qEDL~TE SO~RCE:
B ) CLONE: VB 6 . 7 - 5 ' PRIMER
~Xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID NO:17:
AGGcAAcAGT Gr~r~ 19 ~2) INFORMATION FOR SEQ ID NO:18:
uU~N~ TRl~T C TICS:
~A) liENGTH: 21 base pairs ~B) TYPE: nucleic acid ~C) ST~2~T`TT)RnNR~qc: ~ingle ~ D) TOPOLOGY: linear rnr.RCNT,R TYPE: cDNA to mRNA
iv) ANTI - SENSE: NO
~vi) n~Tt~.T~T. SOURCE:
~A) n~ T~cM Homo sapiens WO95128481 ~ 182 r~l",~ c~

( vi i ) IMMED IATE S OURCE:
(B) CLONE: Vbl- 5' PRIMER
txi) ~;yU~;N~:l; DESCRIPTION: SEQ ID NO:l~:
G~ T ~ ACT T 21 ( 2 ) INFORMATION FOR SEQ ID NO: l9:
~i) ~.t!;~U~N(~ R~rTRRT.~TICS:
~A) LENGTH: 20 base pairs (B) TYPE: nuclei~ acid (C~) STRANn~nNR~ single (D) TOPOLOGY: linear (ii) MnT~CuT~ TYPE: cDNA to m~NA
( iv) ANTI - SENSE: YES
(vi ) ORIGINAL SOURCE:
(A) OR~NT~M: ~lomo sapiens ( vii ) IMMEDIATE SOUROE ::
(B) CLONE: TC~ B -AW'1'1~:N~;1;
(xi) ~;Uul;N~:l; DES~:KI~Ll~N: SEQ ID NO:19:
L~WA GATCTCTGCT 2 0 (2) INFORMATION FOR SEQ ID NO:20:
2ul;W~ R~-'TRRTCTICS:
(A) LENGTEI: 29 ~ase pairs (B) TYPE: nucleic acld ( C ) STR ~ND~nN~ ~: s ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNNA to mRNA
( iv) A-NTI - SENSE: NO
(vi) ~RT~'7TN~T. SO~RCE:
~A) nR-'~NTCM: E~omo sapiens (vii) IMMEDIATE SOURCE:
( B ) CLONE: Va2 5 ' PR~MER
(Xi) ~ Ul;N~:~!; DESCRIPTION: SEQ ID NO:20:
AGGTCGACGA ATGATGA~AT rt'TT~ ' 2 9 W0 9St28481 2 1 8 8 :~ 8 2 P~ 0 1 ~
~ 35 (2) INFORMATION FOR SEQ ID NO:21.
;UUlSNC~i r~ R~rTRRT~C:TICS
(A) LENGTX: 25 base pairs (B) TYPE: nucleic acid ( C ) S TR ~NnRnNR .~ S: s irlgl e (D) TOPOLOGY: linear ( i i ) ~qOLECULE TYPE: cDNA to mRNA
( iv) ANTI - SENSE: YES
(vi) i~RTf'.TN~T. SO~JRCE:
(A) ~Rr~NT.~M: Xomo sapiens (vii) IMMEDIATE SOUROE:
(B) CLONE: TCRa 3~n~,c~nce (Xi) ~;UU~;N~; DESCRIPTION: SEQ ID NO:21:
~ATAGGTCG~ C~GA TGTC ACTGG 25 '

Claims (15)

WHAT IS CLAIMED:

1) A purified and isolated DNA comprising a DNA sequence encoding the CDR3 region of the T cell receptor found in the synovial tissue of RA patients.

2) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.beta. region of the CDR3, V.beta.17seq1, as set forth in Figure 3.

3) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.beta. region of the CDR3, V.beta.17seq2, as set forth in Figure 3.

4) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.beta. region of the CDR3, V.beta.17seq3, as set forth in Figure 3.

5) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.beta. region of the CDR3, V.beta.17seq4, as set forth in Figure 3.

6) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.alpha. region of the CDR3, V.alpha.2.3-J.alpha.(IGRJa09)-C.alpha., as set forth in Figure 4.

7) A purified and isolated DNA of claim 1, comprising a DNA sequence encoding the V.alpha. region of the CDR3, V.alpha.3.1-J.alpha.k-C.alpha., as set forth in Figure 4.

8) A polypeptide comprising the CDR3 region of the T cell receptor found in the synovial tissue of RA patients.

37 g) A polypeptide of claim 8, comprising the V.beta. region of the CDR3, V.beta.17seq1 and having the amino acid sequence set forth in Figure 3.

10) A polypeptide of claim 8, comprising the V.beta. region of the CDR3, V.beta.17seq2 and having the amino acid sequence set forth in Figure 3.

11) A polypeptide of claim 8, comprising the V.beta. region of the CDR3, V.beta.17seq3 and having the amino acid sequence set forth in Figure 3.

12) A polypeptide of claim 8, comprising the V.beta. region of the CDR3, V.beta.17seq4 and having the amino acid sequence set forth in Figure 3.

13) A polypeptide of claim 8, comprising the V.beta. region of the CDR3, and having the amino acid sequence IGQ_N.

14) A polypeptide of claim 8, comprising the V.alpha. region of the CDR3, V.alpha.2.3-J.alpha.(IGRJa09)-C.alpha., and having the amino acid sequence set forth in Figure 4.

15) A polypeptide of claim 8, comprising the V.alpha. region of the CDR3, V.alpha.3.1-J.alpha.k-C.alpha., as set forth in Figure 4.