Note: Descriptions are shown in the official language in which they were submitted.
~O91/os2ss æ~r~ Pcr/Vs90/0~311
S R~EWATOID FACTOR ACq~ NCODED BY
VARIA2~ IMM~NOGLO~ULIN VA~ I.E R~GION
' ~
.~.
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. ;~
. .
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.
: ~ ?
^ ' '~ -~, 1., ~,
:~.,"~.~ :
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.
; ~
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$n conjugated with alkal~ne phosphat~se~ and
bound phosphatase was measured by the conve~sion o
p-nltrophenyl phosphate to p-nitrophenol (~405). The
binding o~ mAb Gm607 to human IgG and other ma~allan
IgG ~mmunoglobulins wa~ evaluated for comp~r~3cn and ' .
was measured by adding supernatant ~rom culturea of the
Gm607 cell llne ~nd detecting ~Ab Gm607 ~Ig~) w~th
~ffin~ty purif~ed alkaline phosphatase labeled goat
antlsera speciflc for human Ig~. The supernatant from
*Trademarks
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0 ~/0~2~8 Z~ J~ 12 PCT/US90tO5311
Gm607 was ~hown to contain IgM u3ing the same
preparation of goat antibodies to human IgM.
The amlno acid sequence of the variable region of mAb
hRP-l licht chain Predicted from the nucleotide
sequence
The amino acid sequence is co~pared to Gm607,
a prototype V~II light chain produced by a lymphobla~t
cell l~ne deri~ed from a nor~al individua~ (Klobeck et
àl., Nature 30g:73-76 ll984). The G~60~ amino aci~
sequence wa~ al~o predicted from the nucieotide
sequence. Six oth~r reported V~I light chain ~re
shown for compari~on. The amino acid ~e~uence o~ hR~-l
is identical to Gm607 with the exception of two amino
acid chan~e , eorre~pondins to a chanqe from phenyl-
alanine to valine at residue 62 and a change from
3erine to arginine ~t re~idue 65. These amino acid
changes occur in a relatively invariable region of F~3.
.,
. 25
.
.,
.
13
TABLE 3
V~} ~IG8~ CaAI~S
FRl CDRl
S
1 23abcdef 3
GM607CL DIVM~QSPLSLPVTPGEPASISC RSSQSL~BS ~GYNYLD
TEW ~ D-FD--N
MI~ ----------------------- ----N--Z -SBGB--- :
: 10 NIM ----------------------- -------WS-D--L--N
CUM ------~--- -- -------D-GD-N$--N
FR ----~--Q- ------VYR-8-BT--B
RPM16410CL -V--~ ---L-Q---~ ---YY--D-N~--N ~:
E~RFl ,
~5 `~`
PR2 CDR2
'
56
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
FR3 CDR3
57 89 97
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- :
~;
: , '
14
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.
' `:
~`::
. ... ,, . ~ . . . .. . ... . . .. .... .. . . . .. . . . .. . . . . .. . . . . .. .
: , . . , , .. , .,. ~ .. . .,. .. : . .. .. : :.: . ~ : . . . . .
.. .. ; . ., : - . ...... . . : . : . ~ , . .
16
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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