CA2066674A1 - Rheumatoid factor activity encoded by variant immunoglobulin variable region - Google Patents

Abstract

Diagnosis and therapy of inflammatory diseases is provided by detecting the presence of mutations in the framework regions of light chains of immunoglobulins. Particularly, a multiplicity of mutations in the framework region (3) at conserved sites is found to be associated with erosive arthritis. By detecting the presence of mutations in the nucleic acid or resulting encoded light chain, propensity for inflammatory diseases and prophylactic and therapeutic treatments is provided.

Description

~O91/os2ss æ~r~ Pcr/Vs90/0~311 S R~EWATOID FACTOR ACq~ NCODED BY
VARIA2~ IMM~NOGLO~ULIN VA~ I.E R~GION
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INTRODUCTION ~:

Technical Field .
~ he ~echnical field of ~he subject invention is the diagnosi~ and therapy oF erosive arthritis.

;~ Erosive ~rthriti~ i~ a chronic de~tructive inflam~atory joint di~ea~e, which inc1udes rheumatoid arthr~ ~i5 as one type of the disease. ~heuma~old ~actors are antiglobulin antlbodies that bind the F~
portion of IgG immunoglobulin and are imp1icated in the pathogeneQis of rheumatold arthritis. Rheumatoid factor~ in patlents with erosive arthritis, however, may be d~ffer~nt ~ro~ rheumato$d factor ~n individuals without rheumatoid arthriti~, as indicated by ~tudie~
o rheumatoid fa~tor specificity and idiotype. In contrast to rheum~toid factor in p~tients without ero~ive arthritis, the genetlc and molecular basis for rheumatoid factor autoantibodies ~n patients with erosive arthritl~ is comp1ete1y unknown.
There is substantial lnterest in understandlng the ba~is for the oc~urrenc~ of ero~lve arthrltis. Tne under~tanding of the characterlstics o~ the compo~itions in the immune syste~ ~hlch lead to erosiYe ; arthritis could provide means for dlagnosis and - 35 treatment o~ the disease. ;~

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R e_e_n t_L l~a t u r e Waaler, Acta Pathol. Microbiol. Scand. 17:1721B8 S ll940); Rose et al., _ roc. soc~ Ex~ Biol. Med. 68:1-6 ~1948); and Natvig et al., Clin. Exp. Immunol. 12:177-183 ~1972) report that rheumatoid factor~ are antiglobulin antibodies that bond the Fc portion of IgG immuno-globulins and are implicated in the pathogenesis of rheumatoid arthritis. Allen and Kunkel, Arthritis _aqum.
9 758-768 (1966) and Fong et al., J. Immunol. 137:122-128 (1986) suggest that rheumatoid factor~ in patient~ with rheumatoid arthritis may be different from these factors in individuals without rheumatoid arthriti~ as indicated by studies of rheumatoid factor specificity and idiotype.
See also Jirik et al., Proc. Natl. Acad. Sci. ~SA
83:2195-2199 (1986). ~he human lymphoblastoid cell line hRF-l i~ reported by Weisbart et al., J. Immunol.
139:2925-2g28 (1987).

SUMMARY OF THE INVENTION
Methods and compositions are provided for the diagnosi~ and treatment of rheumatoid arthriti~, where peptides of conserved framework regions encoded by germline gene~ for antibody light chains are found to be diagnostic of rheumatoid factor.

This invention provides a method of diagnosing susceptibility to erosive arthritis comprising:
determining the presence o a human kappa II light chain having amino acid substitutions at sites 62 and 65 in the third framework region of said human kappa II
light chain, as indicative of said susceptibi1ity.

This invention a1so provides a method of diagnosing susceptibility to erosive arthritis comprising:
determining the presence of a human kappa II light chain having a third framework region having a sequence associated with susceptibility to erosive arthritis, wherein the presence of said human kappa II light chain is indicative of said susceptibility. `~

DESCRIPTION O~ SPECIFIC 13MBODIMENTS

Methods and compositions are provided for the diagnosis and treatment o~ erosive arthritis, which includes about 70% of patients with rheumatoid arthritis and 26% of patients with psoriasis and polyarthritis (Waaler, Acta Pathol. Microbiol. Scand. 17:172-188 -~
(1940); Rose et al., Proc. Soc. Exp. Biol. Med. 68:1-6 (1948); Natvig et al., Clin. Exp. Immunol. 12:177-183 ~1972); and Wright "Psoriatic Arthritis" in Textbook of Rheumatoloqy (EDS Kelley, W.; Harris, E.D.; Ruddy, S.;
Sledge, C.B. (1051) W. B. Saunders, Philadelphia, 1981).
The framework regions are the 1, 2 and 3 framework regions (FR~ 2, and -3) where the ~ites of intere~t ar~ particularly in the FR-3 region and more particuLarly at sites 62 and 65. Furthermore, the light chains are divided into kappa and lambda, where the sites are of particular interest in the kappa light chain, and where the kappa light chains are divided into subgroups I~IV, of particular interest is kappa II.

2D Conserved amino acid~ of the third framework region include the following sequence: ~-G-V-P-R-F-S-S-G-S-T-F-T-L-I-R-V-V-G-V-Y-Y-C
(57-88). For example, at residue 62, phenylalanine occurs in all four of the kappa subgroups and is present in 78/80 of reported kappa light chains; only one light chain (a VI ) contains a change to valine while the other chain was a different amino acid. Similarly, serine is present at residue 65 in 75/80 reported kappa chain sequences but only one light chain (a VIII) contained a change to arglnine (Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services) (1987). None of the 80 reported kappa light chain sequences had changes at both residue 62 and 65.

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l'he am1no acLd difference in the conserved region may be a cooservative or non~conservative change, that lS, it may be an e~change of one polar amino acid for anol:her polar amino acld, or a nonpolar amino acid for a polar amLno ac1d, an aliphatic amino acid or an aromatic am1no acicl, a charged polar amino acid for a non-charged . polar amino acid, v1ce versa, or the like. In one kappa - chain, at residue 62, phenylalanine was substi-tu-ted by valine and at residue 65, serine was substituted by arginine. Therefore, the substitutions may be conservative or non-conservative, where the following table indicates conservative substitutions, where the sa`me amino acids are found on the same line.

TABLE I
Amino~3~L5D~ cb~L

Aliphatic Non-polar G, A, P
L, I, V

Polar Neutral : C, M, S, T, N, Q
Charged Negative : D,E
Posikive ~,R :

30 . Aromatic F, H, W, Y

,~

, :: . . . :-: - . : : : - . . . .: .:- : : : ;:: :: ::; . ::: ::: :.,.. . .:, . .. : -: : . ., : . , . .:

Deslrablv, the sllbstltution may have the i~ame charge or neu~rality as the WLl(:t type a~ino acid.
In addltlon, o interest will be the particlllar J
chaln associated with the subject peptide. Of particular lnterest is the presence of a J5 segment associ~ted with the subject peptide light chain.
The mutations will normally be associated with the germline gene, so that the variable region e~on will include the mutation.
Oligopeptides may be provided which are of at least eight amino acids, and, furthermore, encompass the subject framework region. ~he oligopeptides will generally be fewer than about 60 amino acids, more usually fewer than about 36 amino acids, although large oligopeptides may be employed to inhibit binding of the subject framework region to other antibodies. In addition, usiing the procedure of PCT applications WO
84/03564, WO 84/03506 and WO 86/00991, oligopeptides may be prepared synthetically which are specific for the binding site of the subject antibody, so as to be able to bind to the subject antibody and prevent its binding to an homologous ligand other than the synthesized peptide.
These peptides can be readily prepared in large number and screened for binding affinity to the particular antibody. The peptides may be used individually or in combination, blocking binding of the subject antibody to other antibodies or proteins. If desired, the entire subject light chain may be employed, but since this is not necessary, it will be frequently convenient to use a fragment of the light chain. The entire light chain may be used in purified form, generally greater than about 90%, usually greater than about 95% pure, in place of the oligopeptide.

Besides using oligopeptide sequences which are cross-reactive with the subject framework region, one may . ' ' I'' ~.

use other compositions which will bind to the subject framework region. Of particular interest are antibodies in a monovalent form, such as Fab, Fab', or Fv or an S antiidiotype, where the variable region of the antiidiotype may compete with the subject framework region and the antiidiotype is monovalent. Thus, only a single variable region of the antiidiotype antibody may be employed to inhibit binding.

L0 A sequence of particular interest is an eight amino acid sequence coming within the sequence GVPDRVSGRGSGTDF, where additional amino acids from the framework region of the kappa light chain particularly the kappa II light chain may be present, but the eight or greater amino acid sequence includes the sequence VSG~.

`; Where oligo- or polypeptides are employed, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as the D- amino acid, at other than the tetrad indicated above, by functionalizing the amino or carboxy terminus, e.g., for the amino group, acylation or alkylation, and for the carboxy group, esterification or amidification, or the ~ike. Other methods of stabilization may include liposome formation, other forms of encapsulation, etc. ~-. , . ,.; - ~ .

::: : . : :: : .: ;: : : : . . : . : .

If necessary, one n~ay isolate antisera and identify the light chain and seqllence the light chain or portions thereof including the framework region of interest and determlne the presence of mutations.
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A wide variety of protocols are available for performing immunoassays, sequencing nucleic acids and --peptide sequences, and any or all of these may be employed, depending upon the particular situation.
10 Immunoassays include ELISA, EMIT, CEDIA, SLIFA, and the ~
like. A variety of patents have issued describing a -number of diagnostic procedures, which include ~.S.
Patent Nos 3,7~1,932; 3,817,837; 3,998,943, and references cited therein.

The subject peptides may also be used for therapy, in inhibiting rheumatoid factor activity of the subject framework region antibodies. These compositions may be ; administerea by any convenient way, preferably intravascularly or peritoneally, conveniently in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like#
Generally, the amount administered will be in the range of about 100 to 1000 ~g/kg of the recipient. A single bolus may be employed or repetitive administrations may 25 be employed, generally not more frequently than once ?~
weekly. The concentration of the peptide will generally be in the range of about 100 to 500 ~g/ml in the dose administered. Other additives may be included, such as stabilizers, bacteriocides, etc. These additives will be present in conventional amounts.
~';
The subject peptides may be prepared in a variety of ~-' ways. Peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers. Alternatively, DNA
35 sequences may be prepared encoding ~

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wo 91/052g8 2 ~ ~ ~r 7 ~ PCT/~S90/0~311 the de~ired peptide and inserted into an appropriate expres~ion vector for eYpre~sion ln a prokaryotic or eukaryotic host. A wide variety of expression vectors are available today and may be used in conventional way~ for trans~ormation of a competent host for expression and isolation. If desired, the open reading frame encoding the desired peptide may be joined to a signal sequence for secretion, so as to permit isolation from the culture medium. Method for preparing the desired ~equence, in~erting the sequence into an expression v~ctor, transforming a competent host, and growing the ho~t in culture for production of the product may be found in U.S. Pa~ent Nos. 4,710,473;
4,711,843 and 4,7l3,339.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES

Preparation of Immortalized Cell Line Secretinq IqG
Rheumatoid Pactcr ~uman ~ynovium from rheumatoid arthritis patients was obta~ned at the time of surgery for ~oint replacement. The inflammatory cell population conta~nin~ 107 to 108 lymphoid cells was freed from stroma ~y digesting synovial tissue with DNAse and collagenase. The B-lymphocytes in the cell population were t~ansformed with Epstein-3arr virus according to the method of Cole et al., Molecular and Cellular 8iochemistry ~l984) ~ 09. ~he culture sup*rnatant erom one of ten tran3formed lymphoblastoid synovi~l primary cell cultures, hRF-l, produced dete~tabl~
levels of IgG RF autoantibody by two weeks after E~V
tran~for~ation. The IgG RF was detected by an enzyme-linked immuno~orb~nt assay ~ELISA~ using purified human IgG Fc fragments bound to 96 well microtiter plate~
~Weisbart et al., J. Immunoloqv (l984~ 132:2909-7 ~ :
~'0 91/05258 PCT/US90/05311 2912~. ~ound IgG RF wa~ identified with peroYidaselabeled rabbit antibodies specific for the F(ab')2 of hu0an IgG. After repeated cloning, these hRF-l B- :
lymphoblastoid cel1s ~ecreted only IgG (1-2 ~g/ml), but :~
5 not IgA ~le~s than 20 mg/ml), or IgM (less than 10 :.
mg/ml).
Icotype testing with subclass ~pecific antiglobulins identified only human IgG4 ~ubclass gamma heavy chains. ~nti-light chain specific an~iglobulins detected only kappa, but not lambda, light chains.
Initial specificity o~ this IgG4 RF for human immùnoglobulin was demonstrated by its binding to purified Fc fragmant~ of human IgG, but not human ~erum albumin, bovine eollagen, histone 2~, or denatured DNA, as shown in the following table.

TAB~
Characterization and Binding Specificity of :
hR~-1 Monoclonal Rheumatoid Factor b~ ELISA

ale~a ~amma mu Isotype 0.00 0.60 0.00 IgG ~Y~l ~9G2 ~9~3 1q~4 ~:
subclass 0.06 OOOO 0.00 0.38 , 30 L$ght kaPpa lambda cha$n 0.47 0.00 Antigen Albumin Collaaen Bistone DNA IqG-Fc :;
binding 0.00 0.04 0.05 0.03 1.07 ~,:

.

The human monoclonal antibody hRF-l was bound to 96-well microtiter plastic plates at 1 ~9/100 ~L a~d characterized by ELISA using peroxidase conjugated antisera specific for human immunoglobulin lsotypes, S IgG subclasses, and light chains. The antigenic specificity of hRF-l monoclonal was measured with the : 5 antiqens bound to 96-well microtiter plates at 1 ~9/lOO ~L by adding hRF-l at 2 ~g/ml from culture `- media and detecting bound hR~-l with affinity . 10 purified peroxidase conjugated sheep antibodies speciflc for F~ab')2 o human IgG. Results were recorded as absorbdncy at 414 nm corrected for background values ùsing control E~ISA wells coated only with poly-L-lysine.

The h~F-l human monoclonal ~gG4 RF was tested - for binding to different speciesl IgGs and shown to bind to human, rabbit, mouse and guinea pig IgG more ;: strongly than to horse, goat, and sheep IgG. The 20 monoclonai I~G4 RF did not bind at all to chicken (nonmammalian) IgG. The observed binding pattern is . analogous to the species-specific binding of Staphylo-coccal protein-A ~SpA). SpA at concentrat~ons as low as l~g/ml in direct competetive binding assays with 25 hRF-l monoclonal IgG RF for the abillty to react with human IgG Fc resulted in a 30% inhibition of this low 3 concentration, whereas equimolar concentrations o~
ano~her Fc binding protein, complement factor Clq did not inhibit binding of the hRF-l monoclonal IgG to 30 human IgG ~c.

Purification of heavy an ~ ns o~ mAb hR~-l ~P-l ly~phoblast cell~ were grown in ~erum f~ee medium, and 2.0 mg of mAb hRF-l was purified from 35 40 liters o serum free supernatant by absorpt~on to 4 gm of Protein A Sepharose*CL-4B ~Pharmacla, Pisca~away, NJ), washed w~th 0.10 M phosphate bufer *Trademark co~taining 0.5 M NaCl, and eluted with o.io H glyclne, p~ 2.8 into tubes contalning carbonate buf~er to produce ~ final pH o 4Ø MAb hRF-l (1 m~/ml~ ~see U.S. applicat1on serial no. 562,781, filed Marc~ 30, 5 1987, whose disclosure is incorporated herein by :' re~erence), was ~educed in a nitrogen environment u~inq ~ mM dithioerythritol for 1 hr at room temperature, p~
8.3, and then alkylated with 22 mM iodoacetamide for 2 hr on ice., After dialysis a~aln6t loO M p~opion~ acid : , containing ~.5 M urea ~nd S0 m~ ~odium chlorlde ~S) ~or 1 h~, the immunoglobulin chains were 3eparated by ~-slze permeation chromatography on a W~ters*EP~C sy~tem using a Prote$n pak*SW300 17.5 X 300 mm) and SW125 ~7.8 ~' X 300 mm) column in series equilibrated ln Pns ' ~,~
bu~fer. ~he column ~ow rate wa~ 1.0 ml/mln, and 0.5 ml fractions were collected. The proteln fract~ons `~
were identifled spectrophotometrically (A280).

Comparison o~ rheumatoid factor bindi_q actlvity of 20 iQolated liqht and heavv chalns with lntact mAb hRF-l. ;
Purified ~Ab hR~-l and isolated light and : ' heavy chains t20 ~g/ml) were biotinylated in a molar ratio of 1:30 with N~S-LC-Biotin (~ierce Chemical Co., Rock~ord, IL) overnight at 4'C in N,N-dimethylformamite :, adjusted to p~ 8.5 with 5~ Na2C03. Biotinylated pept~des were dialyzed against P8S and as~ayed for '' binding human IgG and other mammalian IgG immunoglob- :
ulins by ELIS~. Binding was measured ~y adding streptavid$n conjugated with alkal~ne phosphat~se~ and bound phosphatase was measured by the conve~sion o p-nltrophenyl phosphate to p-nitrophenol (~405). The binding o~ mAb Gm607 to human IgG and other ma~allan IgG ~mmunoglobulins wa~ evaluated for comp~r~3cn and ' .
was measured by adding supernatant ~rom culturea of the Gm607 cell llne ~nd detecting ~Ab Gm607 ~Ig~) w~th ~ffin~ty purif~ed alkaline phosphatase labeled goat antlsera speciflc for human Ig~. The supernatant from *Trademarks ..
. :. .

0 ~/0~2~8 Z~ J~ 12 PCT/US90tO5311 Gm607 was ~hown to contain IgM u3ing the same preparation of goat antibodies to human IgM.

The amlno acid sequence of the variable region of mAb hRP-l licht chain Predicted from the nucleotide sequence The amino acid sequence is co~pared to Gm607, a prototype V~II light chain produced by a lymphobla~t cell l~ne deri~ed from a nor~al individua~ (Klobeck et àl., Nature 30g:73-76 ll984). The G~60~ amino aci~
sequence wa~ al~o predicted from the nucieotide sequence. Six oth~r reported V~I light chain ~re shown for compari~on. The amino acid ~e~uence o~ hR~-l is identical to Gm607 with the exception of two amino acid chan~e , eorre~pondins to a chanqe from phenyl-alanine to valine at residue 62 and a change from 3erine to arginine ~t re~idue 65. These amino acid changes occur in a relatively invariable region of F~3.

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., .

V~} ~IG8~ CaAI~S

FRl CDRl S
1 23abcdef 3 GM607CL DIVM~QSPLSLPVTPGEPASISC RSSQSL~BS ~GYNYLD
TEW ~ D-FD--N
MI~ ----------------------- ----N--Z -SBGB--- :
: 10 NIM ----------------------- -------WS-D--L--N
CUM ------~--- -- -------D-GD-N$--N
FR ----~--Q- ------VYR-8-BT--B
RPM16410CL -V--~ ---L-Q---~ ---YY--D-N~--N ~:
E~RFl , ~5 `~`

' GM607C~ WYLORPGQSPQLLIY ~GSNRAS
: 20 TEW -------- - - -----~IL ---Z---Z--Z---- ------- `;
i ~IM --------------- -------C~M -----A--------- TL-Y---FR ----------E---- -S-Y-D- .~:
RPM16410CL -FC-R-----RR--- KV---D- ~:
E[~l GM607CL GVPDRFSGSGSGTDFTLXISRVEAEDVGVYYC ~QALQTPOT
T ~ _______________________________H -Z---A~
MIL ____________-~--------Z-2~ H-NIM --~ -D-----I-----P-------- - A--S-P- ~CU~ ----------------------Q-Q------- --R-E~-Y- -35 PR ---~ D-----------T--Q--------- ---TZS-Y ~:
~PM16~10C~ -------------------------------- ff T~WSW-9RPl -----V--R----------------------- -------I- :
~;

: , ' The gene for the lmmunoglobu1ln light chain of hRF-1 cell3 ~as c10ned ~rom a cDNA library expressed in ~mbda Zap*(S~ratagene, La Jo11a, CA), and the cDNA
11brary was screened with a VJ chain olisonucleotide probe labeled wlth 32p. Lambda Zap-hR~-1 was converted to B1uescript~ from whlch double stranded and sing1e stranded DNA were isolated (Stratagene, ~a Jolla, CA), and dldeoxy ~equenc;ng wa3 performed us~ng an Applied ~ioSclence Sequenator*
Two new re~triction endonucIe~se cleava~e 3ite~ are produced as ~ result_of two nucleotide ~han~es ~n PR3 of hRF-l V,~II
The change from thymi~ine to guanine produced a TthIII1 site, a~d the chan~e from adenosine to rytldine produced a ~aeI~I ~ite in hR~-1 V~I compared ~:
to the prototyplc V~II o Gm607.

Identiflcatlon of the $thIIXI and ~aeIII endonuclea~
sltes ln hR~-1 cDNA bY PC~
A 156 bp fragment i9 ampliied in hR~-1 genomic DNA, hRP-1 c~NA, and norm~1 P~L DNA. The 156 bp frag~ent amp1ified from DNA from norma1 peripheral blood 1ymphocytes ~PBL) dd not contaln a TthIIII
site. In contrast, a TthIIII Qite wa~ present in hR~
cDNA. ~here 1~ one ~III 3$te in the 156 bp fragment amp1ified from hRP-1 genomic DNA, hRP-1 cDNA, and nor~21 PBL DNA which produces a fragment immediately bene~th the 156 bp f ragment. A second ~aeIII site i9 present in h~P-1 cDNA which produces an additlonal fragment.

?dentlflcat~on o~ the Tth~ nd ~aeIII endonuc1ease nites in hRF-1 qenomlc_DNA by PCR
A nucleotlde 8equence of 156 bp was ampllfled o110wing 25 ~ycle9 of PCR with h~F-1 genomic DNA, c~NA
prepared from hR~-1 poly~denylated mRMA by reYer~e *Trademarks .

.,~",~ ' .

transcriptase, and DNA isolated from normal PBL.
Oligonucleotide prim2rs ~100 ng) were used flanking the sequences in question. The primers used for PCR were TCTCCACAGCTCCTGATCT (sense) located at amino acids 43-48 and TTGTAGAGCTTGCATGCA ~antisense) located at amino acids B8-93 in the 5' to 3' orientation. The antisense primer was labeled with 1~32P]ATP. Reactions were performed in 2S mM tris-~Cl, pH 8.0, 5 mM MgC12, 50 mM NaCl, and 0.25 mM each o dPoxyadenosine ` 10 triphosphate, deoxycytidine triphosphate, deoxythy-~idine triphosphate, and deoxyguanosine triphosphate in a total volume of 25 ~1, cycling at 65 for 2 min and at 91b for t min. After amplification, 5 ~1 o~ the reaction were used for further analysis by 8 lS polyacrylamide gel electrophoresis, and the PCR
products were visualized by autoradiography after 2 and 12 hours of exposure.
The autoradiograph was overexposed to enhance the TthIIII and ~aeIII fragments in genomic D~ where the V~II of interest is diluted by multiple other V~
genes compared to amplification of the single hRF-l V~II in the hRF-l cDNA.
The autoradiograph showed the absence of bands in normal DNA rom PBL uncut or cut with ~ae~II
25 comparable to bands of hRF-l genomic DNA uncut or cut ~
by ~aeIII. In addition, both the cDNA and genomic DNA ~ ;
; cut with TthIlII showed a comparable band. ;
It is evident from the above results that one can diagnose and treat specific diseases associated with erosive arthritis, psoriasis and polyarthritis by detecting the presence of mutations of conserved amino acids in the Eramework reglons of immunoglobulin light chains. Thus, opportunities èxist to diagnose the propensity for these diseases and act prophylactically or therapeutically in the treatment of such diseases.

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.. .. ; . ., : - . ...... . . : . : . ~ , . .

Altho~gh the foregolng lnvention has been described in ~ome detail by way of lllustr~tion and example for purposes of clarlty of under3tandinq, it will be readlly apparent to those of ordinary skill in the ~rt in llght of the teachings of thl3 lnvention that certain changes ant ~odiflcatlons may be ~ade thereto without departing from the spirit or ~cope of the appended claims.

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Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS

1. A method of diagnosing susceptibility to erosive arthritis comprising:
determining the presence of a human kappa II light chain having amino acid substitutions at sites 62 and 65 in the third framework region of said human kappa II
light chain, as indicative of said susceptibility.

2. A method according to claim 1, wherein antibody specific for said amino acid substitutions is used for said determining.

3. A method of diagnosing susceptibility to erosive arthritis comprising:
determining the presence of a human kappa II light chain having a third framework region having a sequence associated with susceptibility to erosive arthritis, wherein the presence of said human kappa II light chain is indicative of said susceptibility.

4. A method according to claim 3, wherein said sequence comprises VSGR.

5. A method according to claim 3, wherein said sequence comprisesGVPDRVSGRGSGTDF.