Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W~D 93~12814 1 2 l 2 ~ fi 8 ~; PCI'lUS92/11159
VACCINATIC)N AND METHODS AGAINST DISEASES RESULTING
F:ROM }?AT~IOGENIC RESPC:)NSES
BY SPECIFIC T CELL POPULATIONS
BACXGROUND OF T~E INYENTION
This inv~ntion relates to the ~ une system
and, mor~ ~pecifica11y, to methods of modifying ~:
pathological Lmmune respon~es.
Higher organi~m~ are ~haracterized by an L~mune
: ~ystem which protects th~m against inva ion by
- 10 pote~tia1ly de1et2rious ~ubstances ox m1~roorgani~s.
When a sub~t~ance,:termed an antig~n, e~ters the body, a~d
: ~ is recogni2ed~as foreign, the Lmm~ne system mou~ts bo~h
~an antibodyomediated response and a ce11Omediated
respon~e~ Ce11s of the Lmmune system termed B
lymphocytes, or B ce1ls, produce antibodies that
spe~ifically recogni~e and bind to the forei~n sub~tanc~.
Other lymphocytes: te ~ed T l yphocytes, or T ce11s, both
e~ect and reyulate the~cel1-mediated r~spo~se resu~tin~
eventually i~ the el~imInation of the a~ti~en.
20: : ~ A variety:of T~cells are involved in the cell-
mediated response.~ S~me induce particu1ar B cell clones
to:pro1iferat:e~and:produce antibodies specific for the
antigen. Others~recognize and~destroy cells pre~enting
ore1g~ antig ns~on their surfaces. Certai~ 11s
25;~:regu1~ate~:the~response ~y either~stimu1ating or
suppre~siny other cells. ~ ~ :
While the norma1 Lmmune system is clo~ly
~: regulated, aberrations in Lmmune response are not
unco~mon. :In ~ome inska~ces, the immun~ ~y~em functions
30 ~inappropriately~;and~reacts to a;oomponent o~ th~ host a~
if~it:wer ,:in fact, foreign. S~ch~a r~spo~se re~ults:in
: an autoi,.~un~ di~sease, in which:the ho~t's immune ~y~em
attac~s :the host's own ti~sùe,~ T c~11s, as the prLmary
regulators of the Lmmune system, dire~tly or indirect1y
; :
: :
2 2 1 17 ~ fi 8 r3
e_ ec_ suc~ ~m~u;le ?~--o 1 oc~ es .
~;~.e_-~s c~ se2ses z~e 3e 1 eve~ .~ ~2se~
;ne-;-r~ s;i~.s. ~-_~ner._ ~c. =hese ~=~
_. e~~sic z~
SVS ~2T"~ ?US e~,~=:~e~ sus,
_ _ = _ _ _ e s c -- s
___~ _s, - ~?e ~ c~ zDe~es, ,.~ zstheni2 c=~vis
cnc _e~_r.~ ~~s ~ 52-- s. ~u.^ ~-~r.e ~ seases z_~
.s c ~c ti d~21s wo= ~ 7ice -~ c. _..e c~st c these
~~ seases, . ~e--s o_ zclu2 =r e~ e-.= eY?enc~ s a~
_os. ~=_c~c_ -;- =-v, ~ s mezsu=ec in ~ ~: ^ .s c_ c~_' crs
:O -n-.u-~ ~ v. '._ _=esen_, .he~e z_e n_ k~.c~ . ef ec_~ve
~=ez~=en_a ^= suc:i zu~-i~u:~e r~ c c~- es. Usu2~ ~v,
C;l'~r =. e sv~ s c2n De ~~ea.e~, wh le =:le c se2se
-_n.-.. ~es _~ -~^c_ess, c~^~ar~. -es~;l=- ns _ seve~ e
-e~i 1 =a ~' cn C= ~e ~lh~
. .
_n c ~e~ s .2r.ces, ly~r.c_ ~-_es re~ 1~ c~_e
n2~ 2-e ~ v ~nd w' thou_ corl-=ol . ~^UC;~l .e 1~ ~a__3:
resui_a i:~ z c -~ce-::us c:ond~i Gn knowr. eS ë l~ cr~cm2.
Wne--e =:le ~,,r-gulzted ~lV'mD;nOC~'CeS ~ _ o .he ~ cell ty~e,
the t~ors 2re .er:~ed T c ~ rmphom2s . ~s w~ th G.her
20 mal~ nzncles, ~ cell lvmDhomas are cl__~ c~lt to l_eat
e_~ec t ~ vel~.
hus, a long-felt need exists for an effective means
of~curing or ameliorating T celI mediated pathologies. Such a
treatment should idéally control the inappropriate T cell
response~ rather than~merely reducing the symptoms. Attempts to
overcome this defa~lt:are disclosed in WO 90/11294, ~0 91/15225
and WO 91/~1133. ~hese documents relate to the use of T cell
receptor peptides as therapeutic agents for the treatment of
autoimmune~ dise~ses. ~ :
WO 90J11294 speciically relates to a ~accine com- :
prising an immunogenic:ally effecti~Te amount of a T cell rec:eptor
-- .
` ' '
AMENDD SHEE~
.. .~ .. .. ..
2 12 ~ fi~ ~
- 2a -
or fraoment thereof, especially the v~17 region.
WO 91/15225 specifically relates to an.immunogen
comprising a purified peptide containing at least a portion of
the T cell receptor, especially the V~12 or V~17 portion.
WO 91/01133 specific211y relates to peptides com-
prising a T cell receptor sequence or a derivative thereof,
especially the V region, the VDJ region and the CDR region of
tne T oell receptor.
It has, however, surprisingly been found according
to t~e presen~ invention, that the selection of specific sequen-
ces such as the V~3, the V~14, immunogenic fra~ments thereof,
and the CDR2 regions of V~3! V~14 and V~17 leads to much better
results in treating diseases resulting rom pathogenic responses
by specific T cell populations. The present in~ention therefore
satisfies the above mentioned need and pxo~ides related ad~an-
~tages as well.
:~ :
:
:.
: : ~ . .
: ~ . :
.,
'''~
AMENDED SHEE~ ~
`:
W~93/12814 PCT/US92/11159
21266``~
receptor (TCR) or an Lmmunogenic fra~ment thereof
corre~ponding to a TCR pre~ent on the surface of T cell~
mediating the pathology. The vaccine fragment can be a
peptide corresponding to se~uences of TCRs characteristic
of the T cells mediating said pathology.
~ he invention additionally provide~ specific ~-
chain variable re~ions and their ;m~unogenic ~egments,
a~d in particular three T cell re~eptors, designated VB3,
V~l4 and V~17, which are associated with the pathogenesis
of autoimmune di~ea es, for example rheumatoid arthritis
(R~) and multiple 6clerosis tMS). Additional VDJ
~u~ctional (CDR3) regions aææociated with other
: : autoimmune di3ea~es are also provided. The present
invention further relates to means for detPcting,
prev~nting a~d treatîng R~, MS and other autoLmmune
~: di~ea~es~
The inven ion:further provid~s method~ of
: pre~enting or treating T:~ell mediated pathologies,
; ;including RA and MS, ~y gene therapy. In these meth~ds,
20: :pure DNA~or R~A encoding for a TCR, an Lmmunogenic
ragment thereof or an anti-idiotype antibody having an
: internal ~ age of:~a~TCR:or an immunogenic fragment is
;administered~to~an:~individual. Vec~ors containing the
DN ~or RNA and compositions containing such; vectors are
2:5 also pro~ided~:for uB~e in these methods.
Brief~Description of the Fi~ures ~;
Figure 1~shows the variable region sequence~ of
3, V~14 and V~17.:~ The; boxed ~egments depict the CDRl,
: CDR2 and CDR4 hyper~ar~iable regions of each V~ chain.
The sequences~ between the CDR2 and CDR4 region~ repre.ent
an ove}lap b2tween;these two hypervariable regions.
Figu~e 2~A) shows the location of prLmers used :~
. .
: :
W093/128l~ J PCT/US92/11159
ln polymera~e chain reaction amplification of T cell
receptor ~-chain genes, and 2(B) shows prLmer sequences
used in polymerase chain reaction.
Figure 3 shows the location and ~equence of
5 prLmers used in polymerase chain reaction ampliication
of ~LA-DR Bl genes. Also shown are HLA-DR allele ~pecific
oligonucleotides.
Detailed Des~ription of the Invention
The invention generally relates to vac~ines and
theix u~e for pre~enting, ameliorating or treating T
: cell-mediated pathologies, such as autoLmmune di~ea~es
and T cell lymphomas. Vaccination provides a ~pecifi~
a~d ~ustained tr~atm~nt which avoids problems associaked
with othsr potential ~venues o~ therapy.
.
~ 15 As u ed herein, the term "T cell-mediated
:~ : : pat~ology1l r~fe~s to~any condition in which an
~ inappropriate T cell response is a componen~ of the
`~ pathology. The:term is intended to:encompass both T cell ,~
:mediat~d autoLmmune~ diGeaseG and disea~es resulting from ::
20~ unregu}ated:clonal T ~cell re~lication. In addition, the
t~rm is in~ended to~include both disea~es dire~tly
mediated by~T :cel:ls~and those, such as myasthenia gravis,
which are~characterized prlmarily by damage resulting
from antibody binding:, a~d also diseas~s in whi~h an
~: 25 inappropriate T cell:reGponse contribut~s to the
produ tlon of those antibodies.
; ~ As u~d herein; "~ubstantially the ~mino acid
:: 3~quen~e,'i or "~ubsta~tially the sequenee" wh~n referri~g
to an amino acid sequence, m~ans the~described sequence
or other ~equence ~:having any additions, deletions or
substitutio~s that do ~ot -~ub~tantially effect the
ability of the sequence to elicit an immune re3ponse
.
WO93~12814 2 ~ 2 6 ~3t PCT/US92/11l59
against the desired T cell receptor sequenoe. Such
sequences ~ommonly have many other sequences adjacent to
the d~scribed se~uence. A por~ion or segment of the
described immunizing sequence can be used so long as it
is sufficiently characteristic of the desired T cel~
receptor or ~ragment thereof ~o cauRe an effective Lmmune
respon~e against desired T cell receptors, but not
against undesired ~ cell receptors. Such variations in
the sequence can ea~ily be made, for examp~e by
synthe~izing an alternative sequence. The alternate
sequence can then be teited, for example by Lmmunizing a
vertebrate, to determlne its effectiveness.
As used;~herein, the term "fragment" mea~s an
immunogenically effe~tive subset of the amino acid
~equence that compri~e a TCR. The term is intended to
.
include ~uch fragments in conjunction with or combined
with add~tional sequence8 or moieties, as for example
where~the peptide is coupled to other amino a~id
se~uences or o a~carrier. The terms "fragm~t" and
"peptide" can, therefore,~be;used interchangeably since a
peptide will~be the~most common fra~ment of the T cell
receptor. Each fragment of the invention c~an have an
altered seguence,~ as described above ~or the term
"substantially~the~ equence~
25~Reference~;herein to;a ~'fragment,"~ "portio~" or
"segment"~of a~T c~ell receptor~does not mean that the
omposition~must~be~derived from intact T cell reeeptors.
Such "fragments," portions" or "segments" can be produ~ed
by various means~well-known to those skilled in the art, -~
3G such as, ~or exa~ple, manual or automatic peptide
ynthesis, various~methods of cloning or enzymatic
treatment of a whole~CR.
As used~herein when referring to the
relation~hip~between peptide frag~ent~ of the invention
.
WOg3/1281~ PCT/US92/11159
~ S6 6
and sequences of TCRs, ~corresponding to~ means that the
peptide fragment has an ~ no acid sequence which is
sufficiently homologous to a TCR sequence or fragment
thereof to stLmulate an effective regulatory response in
the indi~idual. The ~equence, however, need not be
identical to the TCR sequence as shown, for instance, in
Examples II and III~
By " ~ unogenically effective-l is meant an
amount of the T cell receptor or fragment thereof which ::
0 i5 effectively ~lioit~ an immune respo~e to prevent or
: treat a T cell:mediated pathology or an unregulated T
cell clonal replication in an individual. Such amounts
: : will vary between;~species and individuals depending on
many factors for which one skilled in the art can
:: : 15 determine.
As~ used herein, "binding partner"~means a
c:ompound which~is:reaotive~with a TCR. Generally, t~is
compound will be;:a~Major Histocompatibility Anti~en (MHC)
: but~can be:any compound~capable of directly or indi.rectly
20~ stimu~ating T cell~activation~or~proliferation when bound
to:the TCR. ~Such~oompounds~:can also, ~or ex2mple, be a
superant~igen:that;~binds:to a superantigen binding site on
the~TCR.; : - ~
As used:here~in,~ "individual" means any
25~vertebrater~including~humans~,~ capable of having a T cell
mediated pathology~or unregulated clonal ~ cell
: replication and is us~ed~interchangeably with
vertebrate."
~ ~ ,
As used~herein,:"ligand"~means any molecule
30~ ~that reacts with ~another molecule to form a complex.
:
:
As u:sed herein, "selectively binds" means that
a molecule binds to~ one type of molecule or related group
W0~3/12814 2 l 2 ~ ; PCT/US92/11159
of molecules, but not substantially to other types of
molecules. In relation to V~s, "selective binding"
indicates binding to TCRs or fragments thereof co~taining
a specific V~ without substantial cross-reactivity with
other TCRs that lack the speeific V~.
The Lmmune system is the prLmary biol~gical
defense of the ho~t (self) against potentially perniciou~
agents ~non-~elf). ~hese pernicious agents may be
pathogens, such a~ bacteria or viruses, as well as ~:
:lO modified sel~ cells, including viru~-infected cell~
tumor cells or other abnormal cells of the host.
Collecti~ely~ the~e targets of the immune 8y8tem are
referred to as antigens. The r~cognition of antigen by
the immune ~ystem rapidly mobilizes L~mune mechanisms to
destroy that antigen, thus preserving the sanctity of th~
host environment~ :
The principal manifestation~ of an antigen-
specific Lmmune respon~e are h~moral immunity ~antibody
mediat~d)~and cellular immunity~(cell mediated). ~ach of :~:
the~ae:lmmunological mechanisms are:~initiated through the
:activation of~:helper: (CD4+) T Cells~ These CD4+ T cells
in turn:stimulate~B Gells~ prLmed for antibody ~ynthesis
by:antig~n binding,~ to prolifsrate;and ~ecrete anti~ody. -:
This secreted~antibody binds to t~e an~igen and
: 2~5~:fac~ilitates i~ destructio~ by other ~ une mechanisms.
Similarly~,-:CD4+~T:cells provide ætLmulatory sig~al~ to
cytotoxic ~CD8+) T cells that recognize and destroy
cellular targets (~or ex ~ple, virus inf~ted cells of
the host). Thus,~the:activation of ~D~ T: cells is the
30 ~ proxLmal event in the stimulation of an Lmmun~ respon~e.
Therefore, elaboration of the mechani~ms underlying
antigen;~peci~ic activation o~ CD4+ T ~ells is crucial in
:any attempt to ~lectively D dify ~immunological function.
T cells owe their antigen specificity to the T
.
W093/12814 PCT/US92/11159
~ ~ 66R 6 8
cell receptor (TCR) which is expressed on the cell
surface. The TCR is a heterodimeric glycoprotein,
composed of two polypeptide chains, each with a molecular
weight of approxLmately 45 kD. Two forms of the TCR have
been identified. One is composed of an alpha chain and a
beta chain, while the ~econd consists of a gamma chain
and a delta chain. Each of these four TCR polypeptide
chains is encodéd by a di~tinct genetic locus containing
multiple discontinuous gene segments. The~e include
~: }O variable (V) region gene ~egments, joining (J) region
: gene ~egments and constant (C) region gene ~e$ments.
Heta and de1ta chains contain an additional:element
termed the diversity~(D)~gene segment. Since D s2gments
and elements~are f~ound:in only some of the TCR qenetic
loci, and polypeptides, further references herein to D
egments and elements~will be in parentheses to indicate ~-
the inclusion~:of:the~se regions: only ~in the:appropriate ~'
TCR~chains.~ Thus,~ V(~DjJ:refers either to VDJ se~uences ,,
of chains which,-ha,ve~a,D region:or refers to VJ 3equ~nces
of~chains lacking D~:regions.~
Witb~r-spect to~the~beta chain of the variable
region referr~ed~to~as a V~, the~nomenclature used herein
to~identify~specific V~5 ~ follows~that~of KLmura et al.,
Eur. J. Immuno.~ 7::37:5-~83 (1987):, with ~he exception
25;~:that::the ~14~herein~corresponds~to V~3~.3 of KLmura et
During~:lymphocyte~maturation,: single V, ~rD) and
J gene segments~;are~ rearranged to form a functional gene
that determines~:the~;amino a~id~sequence:of the TCR
30~expressed~-by~that~cell.~ ~Sincé~the~pool of V, (~) and J
genes: which may~be~:;rearranged:is multi-membered and since
'individual ~e~bers~:~of~the~ poo~'s may be rearranged in
vir~tua11y any~oomoination,~he oomp1~t~ TCR repertoire is
hi~hly:diverse and~capable of specif~cally recognizing
35~: and binding the vast~ array of binding partners to which
.
:::~:: : : : : : :
.r .. :.r; ~ ... &S~.;,. J .; s~ r ~ r~ ~ r. ~ J. . ~
W093/12814 2 1 2 ~j ~j X ,. PC~/~S
an organi~m may be expo~ed. ~owever, a particular T cell
will have only one TCR molecule and that TCR molecule, to
a large degree if not singly, determines the specificity
of that T cell for its binding partner.
~:
~nLmal models have contributed significantly to
the under~tanding of the immunological mechan~sms of
autoLmmune di~a~e. One such anLmal model, experLmental :
allexgic encephalomyelitis (EAE), i~ an autoLmmune
di~ea~e of the ce~tral nervous system that can be induced
in mice and rats by immunization with myelin basic
: protein ~BP). The di~ea~e i~ characteriæed clini~ally
by paralysis and mild wasting and histologically by a
perivascular:mononuclear ~ell infiltr2tion of the central
nervous sy~tem par~chyma. The disea~e pathogenesis îs
~mediated by T:cells having ~peci~icity for MBP. Multiple :~
clo~es of MBP-~pecific T cells ha~e been isolat d from
animals suf~ering from~EAE and have been propagated in
continuous aulture.~ After ln vitro stLmulation with MBP,
20~: these T cell c~lones rapidly induce E~E when adoptively
ra~sferred to~healthy hosts. Importan~ly, these E~E-
indu~ing T cells are specific not only for the same
: antigen (MBPJ, but usually also ~or a single epitope on
;: that~antigen~ These: observations indicate that discrete
;2~ populations;of~autoaggressive~T cells are responsible for
the pathogenesis of:~EA~
A~alysis of the TCRs of EAE-induci~g T cells
ha~ revealed re tricted~heteroye~eity in the structure of ::
these di~eaæe-as ociated receptors. In one analysis of
33 MBP-reactive T cells, only two alpha chain V region
: ge~e ~egment~ and a single alpha chai~ J region ~ne
: se$ment were found~. Similar restriction of beta chain
TC:R:gene u~age was a I80 observed in this T cell
'~ population.: Only two beta chain ~ regio~ ~egments and
: 5 two J regio~ gen~ 3egment~ were ~ound. More Lmportantly,
approxLmately eighty percent of the~T cell clones had
W093/12814 6 PCT/U~92~11159
~ 7~ o
identical amino acid sequences across the region of beta
chain V-D-J joining. These findings confirm the notion
of common TCR structure among T cells with s~milar
antigen specificities and i~dic~te that the TCR is an
effective target for Lmmunotherapeutic strategies aLmed
at eliminating the pathogene~is of EAE.
An alterna~ive mechanism for T cell aativation
has been suggested in which endogenous and exoge~ous
superantig~ns have been shown to mediate ~-cell
~tLmulation as degcribed~ for example, in White ~t al,
S~ll 56:27-35 (1989:~ and Ja~eway, Cell 63:659-661 (1990).
:,
: A~ used ~herein, "superantigens" means antig ~s
or fragments thereof that bind preferentially to T cells
at ~pecific sites o~ the ~ chain of a TCR and stimulate T
cells:at very~high ~reguency rate. Such superantigen~
::can be endogenous~or exogenous, ~Frequency" r~fer~ to
~he proportion of T cells respo~ding to antigens and
:ranges from about 1~5:to 1/100 in response to
superantigens. ~Thus,~superantigens are d~stinguishable
20 ~from conve~ntio~al antigens, which have a ~uch lower T
ceLl response~fre~uency:rate ranging from about ltlO4 ~o
/ 106 - Superantigens activate T cells by binding to
speci~ic:V~s. The superantigen binding ~ites of ~arious
TCRs~:have been distinquished~from the conventional
hypervariable~regions ~DRs) of TCRs. These CDRs
represent the~regions of TCRs thought to be responsible
for~bindi~g conventi~onal antigens ~that are complexed to
M~C .
The~pre~ent inventio~ pxovides an effective
~ 30 method of Lm~unotherapy for T cell mediated pathologies,
: : : including autoimmune diseases, which avoids many of the
:problems:associated with previously suggested methods of
txeatment. By vaccinating, rather than pa~ively
admini~tering heterologous antibodies, the host's own
W093/12814 21~ B ~ PCT/US92/11159
11 '
immune system is mobilized to suppress the autoaggressive
T cells. Thus, the suppression is persistent and may
involve any or all immunological mechanisms in effecting :
that suppression. This multi-faceted response is more -:~
S effective than the uni-dLmensional suppression achieved
by passive administra~ion of monoclonal antibodies or ex
vlvo-derived regulatory T cell clones which requires a
highly individualized therapeutic approach becau~e of MHC
non-identity among humans in order to avoid graft versus
host reaction~ ~The methods of the present invention are
also more effective than vaccination with attenuated
disease-induc~ng T cells that lack speaificity for the
protective antigen~on the surface of a particular T cell
as-well a the:variable induction of Lmmunity to that ~:
15 antigen~. In addition, vaccination with attenuated T :~
; cells is plagued;by the:same labor intensiveAe~s and need
for:~:individualized therapies as noted above for ex vivo
derived regulstory~T cell clones.
As~they relate to:autoi~mune dioease, the
-20~:vaccine pep~ides of the:present invention compri~e TCRs
or~ ~ unogen:ic~fragments~thereof~ from specific T cells
t~hat mediate autoimmune diseases.~ The vaccines can be
wholé~TCRs substantially purified~from T cell ~lones,
individual T~;cell~receptor~chains:(for example, alpha,
25~ be~a:, etc.) or~:portions of such chains, either alone or
in~combination. ~The:vaccine can be;homogenous, f~r
example,~:a single;;peptide,~or~:can be composed of more
than:one:type~of:~peptide, each:of~which corresponds to a
dif~ferent portion:of the T~R. ~urther, these peptides
can be from diff~erent TCRs that contribute~to the T cell
mediated~pathology~ These vac~ine~peptides can be of
variabIe~length~:s~o~long~as they can elicit a regulatory
response.~Preferably, such peptides are betw~en about 5 -
lOO~ami~o àcids~in~length,~and more preferably between
about~6 - 25 amino~:acids in length.
:
:: : : :~ :
: ~ :
WO'33/12814 PCT/US92/11159
~2
~ a specific embodiment, the immunizing
peptide can ha~e the amino acid sequence of a ~-chain VDJ
region when the 3ubject has MS or RA. Any immunogenic
portion of these peptides can be effective, particularly ~:
a portion having substantially the ~eque~ce 5GDQGGNE or
CAIGSNTE of V~4 a~d V~12, respectively, for MS or
substantially the ~equence A5SLGGAVSYN, ASSLG&~ETQYF,
ASSLGGFETQYF or ASSLGGTEAFF or RA. Thus, amino acid
:~ ; substitutio~s can be made which do not destroy the
immuno~enicity of the peptide. Additionally, this
peptide can be linked to a carrier to further increase
its ~mmun~genicity~ Alternatively, whole T cell
receptors or TCR~fragments that include the~e sequence~
can be u~ed to vacci~ate directly. :;
:~
; 15 In a further specific embodLment, T cell
receptors, whole: T cells or fra~ments of TCRs that
contain V~17, ~Al4: or ~3 can be u~ed to Lmmunize an
individual having:a T:cell mediated patholo~y to treat or
prevent the~di ea8e.~ In a ~specific embodiment,
20 :rheumatoid arthritis can be so treated~ The Lmmune
response generated~in the individual;can neutra1ize or
: kill T ce1ls having V~17, V~14 or`V~3 and, thus, prevent
or treat the~deleterious effects of such VB-bearing T
:cell~. :Moreover,~to~:the extent that V~17, ~14 or Y~3 is
;25 common to T a:ell~receptors:on pathogenic T cells
mediating other::autoimmune diseases or autoLmmune
: :diseases~;in general,;~such vaccines~can aleo be effective
in ame1iorating such other autoimmune dis~ases.
:: , .
As used~herein, ~ 17" refers to a ~pecifi~
human ~-chain varia~le region of three T cell re~eptors O
: VB17 has the :following~amino acid~equence: MSNQVLCCVVLC
FLGA~VDGGITQSPKYLPRK~GQNVTLSCEQN~ ~DAMYWYRQDPGQG~RhIYYSQ
~: : IYNDFQ~GDIAEGY5VSREXKESFPLTVTSAQKNPT~FYLC~SS.
~V~14" ~refers to a speci ic human ~-chain
:
:
W093/12814 21 2 ~ ~ ) ~ PCT/USg2/11159
. ~3~3
13
variable regions of another TCR. V~14 has the following
amino acid ~equence: MGPQLLGY W LCLLGAGPLEAQVTQNPRYLITV
TGKXLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDYPEGYXVSRKEK
RNFPLILESPSPNQTSLYFCA5S.
"V~3~l refers to a fzmily of specific human B-
chain variable region. Two members of the V~3 family
have been identîfied as V~3~1 and VB3.2. V~3.1 ha~ the
foll~wing amino acid:s~equence: MGIRLLCR~AFCFLAVGLVDV~V
TQSSRYLV~RT OE KVFLECVQDMD~ENMFWYRQDPGLGLRLIYFSYD~RMKEKGDIP
EGYSVSREKK~RFSLILESASTNQTSMYLCASS. V~3 . 2 has the
followi~g amino~acid sequence: MGIRLLCR~AFC
r
FLAVGLVDVKVTQSSRY~VKRTGEKVFLECVDMD~ENMFWYQRQDPGLGLRLIYFSY
DYXMKEKGDIPEGYSVS~EgXERFSLILESASTNQTSMYLCASS.
,
:: The~hypervariable or iunctional regions are
15~ useful for the:vaccines:of~the~ pre ent:invention.
Hypervariable;~:regions~useful~in~the pre~ent inYention
include CDRl,::~CDR2,~CDR3~and CDR4.~ The amino acid
sequences of~the:CDR1, CDR2 and CDR4~:hyperYariable
regions:for~VB3,~V~14 and YB17~are shown in Figure 1.
20 ~ The~ CDR3,~ als~ known~as:~the V(D)J region, is
useful~as a~vacc:ine~of:~;;the~present invention~since T cell
immunity ;e;licited~ ~by ~ peptides~ cor}esponding to ~this
region~is~:expécted~to~be~:~highly::speDific for~a particul~r
;an~tigén~.~ Due~t~a;~the~recombination:of:the~V,:~D and J
2~5~ region genes~prior~to~maturation~ the amino acid equence
;:across~:these~règions is~virtually unique t~o eaoh T cell
:and its clones. ~:
owever,~as a germ-line~element, the CDR2
region:is~also;~u~eful~in~human~::disea~es æuch as MS and in
30~pa}ticular:RA.~ In~RA~studies,~ the~results indicate a
limited~number~of~V~s~among the~wtivated~ cells
:infiltrating the synoYial target ~ti~su~ with only a few
incidences:~f~ equenc~e homology in:the ~VDJ region.
:: :
: ~
,
WO93J12814 ~ PCT/US92/11159
14
~hus, peptides cvrresponding to the CDR2 region are
viable al~rnatives for use as vaccines of the present
invention. For example, the CDR2 region of V~3,
DPGLGLRLIYFSYDVK~KEKG , of VB14, DPGLGLRQIYYSMNYEVTDKG, or
of V~17 ~ DPGQGLRLIYYSQIYNKFQKG, can be us~d.
~ odifications in these ~eque~ces that do ~ot
affect the ability of the re~eptor or an Lmmunogeni~
fragment ~hereo~ to ac~ a~ an Lmmunogen to ~tLmulate the
desired .ummune respon~e are contemplated and ar~ included . 10 in the:definition of TCR fra~ment. The variable region
can:~be joined:with any D and J ~egm~nt of the TCR.
Further, Lmmunogenically repre~entative fragm~nts of ~3,
V~14 and V~17:are~al~o included in the de~inition of
"V~3," "VB14" and -V~17," respectively.
~ By~'lsubstantially pure," it is mea~t that the
TCR iB substantially~free o~ other bi~chemical moieties
with which;it~is normally a~ociated in nature. Such
sub~tantial:ly~pure ~CRs or fxagments;thereof, for
;instanoe:, can be~synthesized, produced recombinantly by
2~0 ~means known:~to~those~skilled in the~art. In addition,
whole TCRs~an be~enzymatically treated to produce such
fragment~
In~another~embodiment, vaccine peptides ~an
correspond~to~the~Y~ regions that~contain ~equences of
25~high:homology~which~are conserved among pathogeni~ TCR5r
The3:e~ region~ of~conserved homology include the
conven~ional CDRs,~such as CDRl and CDR2, which are
~: common to T cells bearing the same V~, and also the
superantigen binding site, which can be co~mo~ to
pathogenic T~Rs:~bearing:different VBs. The superantigen
binding~site i~:also known to:be in or around the CDR4
hypervariable~region. : ~:
The vac~ines of the present invention compri~e
: ~ :
W093/12814 15 2 i ~ ~3~
peptides of varying lengths corresponding to the TCR or
Lmmunogenic fragments thereof. The vaccine peptides can
correspond to region~ of the TCR which distingui~h that
TCR from other nonpathogenic TCRs. Such specific regions
can, for example, be located within the various region(s)
of the respective TCR polypeptide chains~ for example, a
short sequence ~panning the V(D)J junction, thu~
:~: restricting the immu~e reBpon~e solely to those T cells
bearing this single determinant.
. 10 T~e vaccine~ are admini~tered to a host
: ~ exhibiting or at risk of exhibiting an autoLmmune
: ~ respon~e~ Definite clinical diagnosis of a particular
autoimmuné disea~e~warrants the administration of the
relevant di~ea~e-sp~cific TCR vaccines. Prophylactic
applications are warranted in diseases where the
autoim~une mech~nism6 precede the onset of overt cli~ical
di~ea~e (for example, Type I Diabetes). Thus,
indi~idual with~:f ~ lial:history of disease and
predicted to ~e at ri~k by:reliable prognostic indicator~
2~0 could be treated prophylactically~to i~terdict autoLmmune
mechanisms prior to their onset.
TCR~:peptides:can be administered in many
:: possible formulationG, including:pharmaceutically
accept:able mediums.~In~the case o~ a short peptide~ the
25~ peptide can ~e conjugat d to a ~arrier, such as X~, in
order to~increase its~Lmmunogenicity. Th~ vaccine can
clude or be a ~ nistered in conjunction with an
: adjuvant, of which several are known to those 6killed in
the art. Arter;initial~immunization with the ~accine,
:furthgr:booster~ aan be provided~. T~ vaccines are
administered by conventional methods, in dosag~s which
are ~u~fici~nt to:elicit an Lmmunological respon~e. Such
dosages can be easily determined~y tho~ skilled in the
art.
.
:: :
2 ~
. 16
o~=~2te ~e?t aes to De usea c-
~m~r. -2_' 0~. C~ e ce~e ~nea as _ollows. D~sease-
~ Uc - ~ c~ s~_nes ~e2c_~ve ~-ith .:ne .arge~ æn~=~ cens
c~- isc~elec ~_om 2 _ec~ea i~dividuals. Suc;n m cells 2r e
- ob.~ned Dre~e~a~iv --om the site of 2CI~ ve
au.cacc=essive 2CI' Vily SUC;~ 2S 2 lesion i~ tne c2se or
~em~hic-.~s vulga~ls, the cer.~ral nervous sys~em ~CNS) in
the case cr mul_~-l- scle~osis or the sv~o~-i21 _~ or
tissue ~ the c~se of rhe~ told ar~ is.
10 ~lt~r~2=~ vely, suc:~ T ~e~ls can be obtained l-om ~150d 0~
z---ected in~iv-id~zis. ~he ~C~ genes _rcm the~e
auloaag-essive ~ celis are t~en sequenced. ~olyDe~lides
c-r_es~-nding t~ TC~s or ?or~lons thereo~ ~hzt are
~ seiec=_-~ely re~-esen.ed 2mong dise2se ~ aucing T ce~s
: l_ (re~at~ve to non-oathogenic ~ cells) can then be se~ec~ea
as ~2CC' ~es 2n~ made cnd used as desc=~ ~ed above. ~
alternatlYe~metnod Lor isol~ting pathoaenic T cells is
ro~ide- by Al er~ in PCT Publlcat~on No. WO88J10314,
:publisned on Decembe~ 29, 1~88.
:: :
Alter~atlvely, the vacclnes can comprlse znti-
idlotypic antibodles whic~zre i~ternal Lmages of the
; peDtides descri~ed abo~e. Methods of making, selecting
and admi~isteri~nq~such anti-~dlotype vacc~nes are well
known Ln the art.~See, for examD~e, Eichmann, et al.,
25 C~C~Critical~Revlews in Immunoloov 7:193-227 ~1987)5
inc~p~or2'e~ ~erein ~y-~ete~e~ee.
In a further aspect of the present invention,
me~hods or pre~enting the proliferation of T cells
associated wLth a T cell mediated pathology are also
30~ contempl2ted.:~ `5uch methods include det~nmlning a T cell
: receptor binding~partner accordi~g to the a~ove methods
and:administeri~ an effective amount ox such binding
partner ~ an appropriate form:to:prev~nt the
pr~liîerztion of the T cells. The methods can be usPd,
35~ for example, to build a tolerance to self antigens 25 in
,~E~lDED S~E
2 ~ ~ 3~
.hQ c2se G - ar. Gu~c~mune _~sease.
The ~_~se~ ~ven_icn aisc =eie.es .o o~er
~e~hocs c_ J~ever.~nc or ~~eztins 2 ~ cel'~ oat~olocy DV
lnn' Di=:n5 ~he ~ir.c~ng of 2 T cell -ecQ~tor ~o its TC~
_ ~inaln~ ~artne- n o_der to orevent t;~e ~=oli era_ion or
T cells ~ssoc ateG with the T cell 02~hology. Licznàs
tha~ a=e -eac._ve with the m cell rece~cr or i_s -~ncinc
par_ne- z~ binà~ng sites t;~a~ inhiDit ~he T ce7.l -ece~tor
at.ac.~en~ to .:~.e bindina oar~ner can be usea~ Suc;,
_0 1~ sanas czn be, ~o- examole, antiDodies havinc
spec~ y-~~r .he T cell ~ece~lor o~ ~_s binair.
pa_~ne-.
: ~
The Lnven~lon zlso provides 2 me~hod o~
preven.:nc o~ t-eating a T cell mediated ~2.hoiogy in an
ndlvlcu~l comDr~ sLna; cy~ctoxically or cyl~s~aticaily
trealLn~ 2 V~-containing T-cells, par~culzrly V~3, V~14
a~nd V~17, in the~ina~vidual. The ~-con~aining T ceLls
are tre2ted with 2~cviotoxic or cytos~atic ageni thzt
select1vely~binds~lo the~VB region of 2 T cell receptor
at meaiates~a pa~hology,~such as RA or ~S for exam~le.
The agent ca~be~:~an~antibody attached to a radioactive or
chemotheraDeutlc~molety. Sucn~attachment znd~effective
agents~are well~kn~wn~in the:art.~ Seet Isr examDl ,
Earl~ow~ . a~d ~Lane,~ ~ntibodies,~eal,
Cold Spr~ng :E~àr~or Laboratory ~1988) 1~ ~5
Rheo~a ~ is
Rheumato~id arthritis~RAj is a T cell m2diated
autoLmmune disease.~The in~ention~describes c~lonal
3~0~ infiltrates of~act1vated V~3, ~l4~a~d VB17 T cells in
the synoY.ium~of rheumatoid arthAtis patients. The
presence:of these T~cells in the diseased ~issue of most
of patie~ts ~examined,~their clonal`ity,: and the~cytotoxic
~ ,
~ :
:~: : , :
AMENDE~S~EE~
:
~6 18 P~r/U~92~11159
acti~ity of one such T cell for synovial adherent cells,
demonstrate a central role for T cells bearin~ thexe ~s
in the pathogenesis o~ RA.
Activated T cell populations in the synovial
tissue of RA patients have been examined by a~alyæing T
cell recept~r (TCR~ mRNAs igolated from L-2 r~ceptor
positive (IL-2R+) synovial T cells. As de~cribed in
~xample X~C~, TCR mRNAs were amplified using a polymerase
chain react~on (PCR) protocol designed to amplify human
TCR ~-chain genes containing virtually any de~ired VB
gene element. In this analysis, clonal V~l7
rearrang~ment~ were found to b~ e~riched in the IL2-R+
population, indicati~g that ~17 T cel~s are likely
involved in the pathogeneRis of RA. A CD4~, V~17 bearing
T cell cl~ne has been isolated from one of the synovi~l
: tissue specLmens a~d its ln vitro ~ytotoxicity for
: ~ynovia1 adherent ce ls ~upports the direct involvement
o~ Y~17 T cells in R~. ~
: Additional s~udies were conducted to en~ure
that the prevalence of V~17 T cells in the initlal
: : studies did not result from an amplification bia~ for the
V~ consensus primer, and to examine the involvement of
other TCR V~ ~ene families:in RA~ As described in
Example XI,~RNAs from activated tIL2-R~) synovial T cells
2~ were:analyzed by PCR-amplifi~ation with a panel of V~-
specific PCR primers. In this analysis, V~17 tran~crip~s
;~ ~ were found in four of the five:patients testedr
:
confirming the a~sociation of V~317 with RA and validatirlg
the ~utIlity of t~e Vl~ con~ensus primer. In addition,
30 ~ Y1314 was found~ in four of the fi~re R~ pati~t samples and
33 and V139 were detectable iD three of five patients.
he uenc~ of the~e Yariou v~ polypeptides
: wer~ exami~ed for homology to V~17. The resul~s are
- r~ported i~ Table l.
WO 93/12~3~4 P~US92/l 1 159
2 ~ 2 6 ~
19
TABLE 1
Relative Homoloqies of TCR 1~ Chain PolypePtides
With Human V~3 17
hV~3 100% ( 94aa)
mV~36 69.1% t94aa)
hVf~3 58.5% t94aa)
hVJ31201 53.2g6 (94aa)
hVJ314 52 .1% ( 94aa)
mVJ37 5I.796 (89aa)
h~J39 33 . 0% ( 94aa)
hVJ3 - ~ human~ ~ ~
m~J3 = mou e VB
:: `
As shown in Table 1, mou~e VJ~6 is most closely
15 homologous, followed by hV~3, hVJ312.1, h~71314, aIld mVJ37.
Three of l;he~ human VJ3s,~ VJ33, VBl4 and ~J317, were detected
n the~ ~;yIlovium of ~RA patients. : V~12.1 was n~gli~ible in
the synovium despite corlsiderable o~erall ho~logy with
VB17. In co~tra8t, V13;9 w s found in three of :Eive
2~ synovial samples,: yet: is only weakly homologous to V1317~
A surpris:ing ~discovery is~ the greater homology
foum:l among all of: the VJ3s detected~ in the synovia of RA
: ;; ;; patients, ~ except VJ39, ln a contiguous stretch of 15 amino
`:acids ~located ~carboxy to the CDR2 ~egion~.: The 15 am~no
acid: segue`nces of; :the~e VJ3s ~s well as other humarl and
mou~e V~s are ~ how~ in Table 2. Within the ~-chain, thi~
region of conservation corresponds positionally to that
previously shown~to contain ~upera~tigen bi~ding ~it~s.
: ~ .` ~ ! .
`:
:~
2~2~
?~r~?ose~ S~e-- .tiae?. ~ :lC' lla C- . e is.
~ ~ssoc a~ed V~
J 9S~l
`nVB i 7 EGYSVS~E~ --S~_ ~
hV~3~YSVS~ U?SL 8 6 . 7
:~VJ31~ ~GY~VS~ ~L 66.
hV1312.: DGYSVSRS~D~II 66.
hV~4 NRFSP~CSPD~iI~L <30
: ~.
._ ~.. _ .... _. . ._ .. . _ .. _. ... .__ ~ .
1_ :nV.~6 2GYDASi~E:R~SS~S-, 73 . 3
_ _ .
mVB7 KGYRVS~ FS~ 64 . 3
20 mYB8 . 2a~ DG~S~ SQEN~SL
mVJ~ 8 . 2 c ....... ~ É
~: ~
hV1313 . 2 DGYNVSRL~QN~GI~: -
:~ : 2 5 %
i = % ~homolo~y comoared with V1317:
The exoyenous superanti;gen, ~ SEC2, st~mulates
human V1313.2~T cells as :a result ~of bi~ding to a site on
V:B l3 . 2 ~as described in Choi et al .:, Na'cure 34 6: 4 71-4 7 3
30 ; :~( l990 )~ ~. The
se~uence o:E thi~s~ binding: site is shown in T:~le 2 a~3 the
Last lI am~no ac:ids for VJ31~.2.
A bindin~g site~ for ~ls-la, an endogenous
;~ ; superan:tigerl, has also been mapped to this region as
35 aesc:ribed in Pullen et al., Cel~ 1365-137:4 ~1990 )
IdeIl~ification :of this regio~ as the Mls binding site
invol~ed the study of V138.2a, the VJS8.2 isofo~ common to
: :~
AMENDED SHEET
W093/~2~14 PCT/US9~/11159
21~G~i8 6
21
laboratory mice, and V~8.2c, a ~-chain found in wild
mice. ~hese B-chain polypeptides are distinguished
functionally by their differential reactivities with Mlq-
la and structurally by a differen~e of five amino acids.
Of particular importance are the residues at position 70
and 71. The responder ~-chain, VB8.2c, has lysine and
glutamic acid, respectively, at the~e po~itions.
Specific mutagenesis of the non-responder gene to encode
a lysi~e-glutamlc acid pair at posikions 70 and 71
rendered that non-responder ~-chain Mls-la-reactive.
This confirms ~he region as one of superantigen binding.
;Thus, both the e exogenous and endoge~ous superantigens
ca~ bind in the vicinity of the 15 amino acid ~equence
homoIogy identified in Table 2. In addition, the lysine-
glut~mic acid pair charge motif is L~plicated in Mls-la
reactivity~ Involvement of this charge motif in ~ls-la
reactivity is confirmed by its pre~e~cq in mou~e V~6 and
V~7~, two other Uls-la reactive murine ~-chainsO ~he
V~8.2c, VB6~and:~V~7 charge motif~of a lysine or arginine
followed by a glut ~ c acid residue is underlined in
Table 2~ ~Thus~, the s~per~ntigen binding site for Mls-la
~:~: is~characterized~by this: charge motif contained within
: the region~of~ local homology.
The present invention is directed to the
25~ unexpe~ted disoovery that human~:V~3, V~14 and Y~17 have a
regioh~ that~corresponds to~the Mls la binding site~
E ~These three~human;VB ~display~;a significant degree of
overall homology within~the~entirè 94 amino acid se~uence
:: and of local homology within the 15 am1no acid ~equence
with mV~6 and~ mV~7. Each of these Y~s pos~es~ a lysine
:or arginine~glutamic acid pair, which are underlined in
Table 2 and repre6eDt what is m~ant by the term "charge
mo~if. U VB12.1, while di~playing a high degree of
::~overall and lo~al:homology with Vfl3, ~14 and V~17, lack~
:~:: 35 the charge motif, perhaps acco~nting for its presence in
~::the ~ynovium of only one of five RA patients. V~9 shows
W093/12814 PCT/USg2/11~59
no~ ~eral1 or local homology to V~s 3, 14 or 17 and lacks
the charge motif.
The presence of V~3, V~14 and VBl7-bearing T
cells has been demonstrated among the activated synovial
T cells in RAo These three ~-chain polypeptides, in
contrast to other known V~s, po~ess overall and local
sequence homolo,~y and an apparent ~uperantigen binding
charge motif. These re~ults indicate that ~-specific T
ce~l activation by superantigen play3 a role in RA.
lOV~3, V~14 and V~17 are the only know~ human VB
chains to possess this apparent supexantigen binding site
charact~rized by this local sequence homology and the
identified charge tif. ~owever, it is possible that
other Y~s may ~ecome known that contain such binding .
sites. Thus, a substantially pure V~3, V~l4 or V~17
cequenae containiny the charge moti can be used as an
; immunogen in the vaccines of the pre~ent invention. ~or
example, the &equences or fragme~ts thereof shown in
Table 2 for V~3,~VB:l4 and V~17 can be used. Yaccines
~ ZO ~ontai~ing~any~combination of these three V.~ sequences,
;: : includi~g all three ~equences, can be used effectiYely to
ameliorate T cell associated diseases.
~:
In~ addition, other common V(D)J seque~ces of
the ~-chain~observed in RA patients are listed in Table
3. :The::results taken from two different RA studies show
: :
: sequence homologies in the ~VDJs ~rom four different
clones,~ which indicates the u~efulne~s of peptide~
corresponding to the CDR3 region:as appropriate ~accine
candidates.:
: ` ~
: ~
,
W~93/12814 PCT/~92/1115g
2 ~
23
T~BLE 3
_-Chain VDJ Sequences Found
in Com~on in RA Patients
Patient V~VDJ Sequence J~
1012 V~14 A S S L G G A V S - Y N J~2O1
1013 ~3 A S S L G G E E T Q Y F J~2.5
C V~3 A S 5 L G G F E T Q Y F J~2.5
A V~3 A S S ~ G G T E A - F F J~l.l
As noted r the invention provide~ the di~icovery
that specific variable regions of the ~-chains of three
TCRs, designated V~3~ V~14, and V~17, are clo~ely
associated with T ~ell mediated pathologies, e~pecially
rh~umatoid arthritis in humsn s~bjects. This disco~ery
; allows for the dotection, prevention and treatment o~
rh~umatoid arthritis using tbe m~thodolo~y ~et out in
his invention.~ S ~ Iar therapeutic approaches ~et out
: above for EAE can be applied to rheumatoid arthritis by
those skilied In the art.
; : Spec~ically, tne inYention provides a method
:: : 20 ~of diagnosing or:predicting susceptibility to T cell
mediated pathologies in an individual comprisin~
: detecting T cells having the ~-chain variable region from
V~3, V~14 or V~1~7 in a sample from the individual, the
presence of abnonmal lev ls of such VB~contai~ing ~ cells
indicating the pathology or ~usceptibility to the
pathology, The V~-containin~ T cells can he
qualitatively~ or~uantitatively compared to that of
normal individuals. Such diagnosis can be performed, for
~: ~ example, by ~eteGting: a portion o~ the V~s that doe~ not
:: ~ 30 occur on non-rheumatoid arthritisi a~sociated ~ochain
variable region T-cell receptors. The V~s of the pre~ent
invention can be detected, for example, by contacting the
WO93/1~814 ~ PCT/US92/11159
~ ` 24
V~s with a detectable ligand capable of specifically
binding t~ the individual V~s. Many such detectable
ligands are known in the art, e.g. an enzyme linked
antibody. Alternatively, nucleotide probes,
complement~ry to the individual V~ of interest, encoding
nucleic acid sequences can be utilized to detect ~uch V~
containing T cells, as taught, for instance, in Examples
: X and XI.
The invention also provides a method of -
pre~enting or treating a T ceIl mediated pathology
: comprising preventi~g the attachment of a V~3-, V~l4- or
: : V~l7-containing T-cell receptor to its binding partner~
~: In on~ embodLment, attachment is pr2vented by binding a
ligand to V~3, V~14 or V~17. In an alternative
embodiment, attach~ent is prevented by binding a lig~nd
to the V~3, V~14 or Y~17 bindin~ partner. Attachment can
be pre~nted~by known methods, e.g. blnding an antibody
o the individual V~s or to its binding partner in order
to~physically~block attachment.
: 20 : ; :~: Mult:iplè Sclerosis
5~ oell~ cau~ative of multiple sclerosis ~MS)
have not pre~iously been~ identifi~d, though MBP reacti~e
T cells~havè~;been proposed~to play~a role due~to the
clinic~l and:histologic sLmilaritie~ between MS and ~AE.
25; In rat~and mouse models of EAE,~MBP-reactive,
encephalit~ogenic T cells show striking conservation of ~-
: chain V~D~J amlno: acid se~uence, despite k~own
~'; differences in MHC restriction and MBP-peptide antigen
specificit~.~ One~:embodLment o~ the i~vention is premis~d
on the observation that a~human myelin basic protein
: (MBP)-reactive:;T~ell line, derived from an MS patient,
: has a TCR ~-chain with a V(D~J amino acid sequ~nce of V~4
:
:~ ~ : homologous~with~:that of B-ehains~from MBP-reactive T
cells mediating~pathoge~esis in experimental allsrgic
: : : :
WO93/12814 2 ~ ~ 6~ PcT/us92/lll59
encephalomyelitis (EAE), an animal model of MS, as shown
in Table 4~ This finding demonstrates the involvement of
MBP-reactive T cells in the pathogenesi~ of MS and
demonstrates that TCR peptides sLmilar to those described
herein for the prevention of EAE can be appropriate in
treating MS. As shown in Table 4, a VDJ ~equence of
YBl2, CAIGSNTE and one of V~4, SGDQGGNE, have been
ob~erved in MS patients.
TABLE 4
lO~-Chain VDJ Sequences in MS_Patients
: HomoIoqous to Rat VDJ Sequences
: :-
V~ VDJ Sequence
RA~ V~8.2 S S D S S ~ T E
~: : :
RAT VP~B.2 S S D S G N T E
:::: : ~ :: :
~ HUMAN V~4 S G D:Q G G N E
UMAN V~12 C A I G S N T E
:: :The ac~ivated cells of one MS~ patient from the
: CSF~were~:analyzed~and found to be predominantly V~14 and
3. In both:cases, there was a::predominate clone of
20~ V~14~ and~a predominate clone of V~3. These findings
: indicate that the~results from the RA ~tudies r~lating to
: : the same V~s~can~be extended to other autoLmmune
: pathologies, including MSo
In this regard, ~he invention is directed to
the discovery that ~-chain VDJ fragme~ts homolog~us to
VDJ se~uences~found in rodent:~AE, such as SGDQGGNE and
C~IGSN~E, ar~ c1Osely associated with multiple sclerosis
in:human subjects. This discovery allows for the
detection, pr~vention and treatment of multiple sclero~i
usi~g the methodology set out in this i~v~ntio~. Similar
therapeutic approaches set out herein for EAE can be
applied to multiple sclerosis by those skilled in the
.
W0~3/12~14 PCT/USg2/11159
26
art.
Specifically, the invention provides a method
of diagnosing or predictin~ susceptibility to multiple
sclerosis in an individual comprising detecting T cells
having various V~s, such as V~4 or VBl~, and particularly
having ~ubsta~tially the sequence SGDQGGNE or CAIGS~TE,
in a sample from the individual, the prese~ce of th
~equence indiaa~i~g multiple sclerosis or ~u~ceptibility
to multiple sclerosi~. The sequences can be detected,
: . 10 for example, by contacting T cells or TCRs with a
detectable ligand. Many such ligands are known in the
:~ art, for example, an enzyme linkzd or otherwi~e labeled
antibody specific for the sequence. Alternatively,
nucleotide probes complementary to the nucleic acid
encoding the ~equence can be utilized as taught, for
instanae~ in ~xample IX.
The invention also provides a method of
pxeventing or:treating multiple sclerosis co~priging
prev~nting the:attachment of a T-cell receptor containi~g
~: 20: various V~s, including V~4, V~12 or fra ~ e~ts thereof,
such::as those having substantially the S~DQGGNE vr
CAIGSNTE ~equence to its binding partner. In one
embod~ment, attach~nent i5 prevented by binding a ligand
to the sequence`. In an alternative embod~ment,
attachment~ is:prevented by bindi~g a ligand ts the
binding par~ner~. Attachme~t can be prevented by known
metho~s, such~ as~binding an antibody to these V~s, and in
particular to the SGDQGGNE or CAIGSNTE se~u~nces, to
physically block attachment.
The~invention also provides a method of
preventing or treating multiple sclerosi~ in an
; : indi~iduaI c~mprisi~g cytotoxically or ~ytostatically
t~eating T ells containing various V~s, including V~4,
V~12 and fragments thereof, particularly those having
W~g3/128~4 PCT/US92/1115g
2 ~ 2 ~
27
substantially the SGDQGG~E or CAIGSNTE sequence in the
individual. In one embodiment, T cells are treated with
a cytotoxic ox cytostatic agent which ~Plectively binds
to these V~5 or their immunogenic fragment~. The agent
S can ~e, ~or example, an antibody attached to a
radioactive or chemotherapeutic moiety.
T~Cell Patholoqies_of Maliqnant ~tioloqy
To illu trate the utility of TCR
vacci~a~ion~ autoLmmun~ di~ease has been di3cu~edO
~owever, T cell lymphoma is another T cell p~tholo~y
which would be amenable to this type of treatmentO
Appllcation of this technology in the treatment of T
lymphoma would be conducted in virtua~ ly i~entical
fashio~. In one respect, however, this tech~ology i~
~: 15 more r~adily applied to T cell proliferative di ea8e
sinoe the isolation o the pathogenic T cells i~ more
easily accomplish~d. Once the ~lones are i~ol~t~d, the
technology i~ applied in the manner described herein.
: Sp~cifically, the TCR genes of the T lymphomas are
se~uenced, appropriat~ regions of those TCRs are
identified and used as vaccines. The vaccines can
comprise single~or multiple peptides, and can be
admini~tered in~pharmaceutically acceptable for~ulations,
with ~or ~ithout~adjuvants, by conventional means.
: ~ : : Gene Therapy
~ The present inv~ntion f~rther relates to an
alternative method of treating: or preventing a T ce
media~ed pa hology by gene therapy. In this method, a
nucleic acid encoding for a TCR or a~ Lmmunogenic
3û fragment th~reo~ i~ first inserted into an ~appropriat~
d~li~e:ry ~y~tem, for example a plasmi d. The mlcleic acid
can be D~7A- or RNA ~ncoding for TCRs, ~unogenic
: fragmerlts thereof or anti-idiotype antibodie~ that can be
~: :
2~2~66~ :
23
use~ zs vaccines ~n the ~_ese~ ve~= on Suc; 3NA c=
~NA c2~ De isol2.e~ bv s~anQ2-~ me.:~ocs ~nown i~ =ne z-
~~he ~sola_ed ~.ucle~ cci~. c2n ~;nen _e i ~.se~_ed ~n~o 2
sui_~ e vec=^- ~v knowm me~hods Suc;. me~hoas 2re
_ Gesc=i -e~, _sr ex2mDie, in Manizt~s e. al , Molec_l~r
Clon~n- ~ ~aborato~v M2nual (Ccl~ S~-ing ~arDo-
~a~0-2-0rV 1~82)~ c -_ ~cor~ h~
_-cf~A~ c
The vec~o_ is subseauenll~ zamin~ stered
cir~c=~v ~to t~ssue o cn indiviàuzl ~-ere_~nlv, ~ne
DNA c= ~NA-cont~ning vec_or is in3e~_~d i~to the
skele~z~ musc~e Gî the ~nài~icual ~o~ examDle, 2 i ~ cm
inc~s~on can be ~ade to exDose the ~uadrice~ musc-~es o~
. .he su~jec' . A 0.1 ml solution con~zi~,ing ~~om 10-100 ~g
15 of ~ 3NA o- ~NA plasmid and ~-20% suc_ose is i~je~_eà
o~Ter :1 mLnu~e~ in~o the exposed quaa~ice3 muscles 2L:~OU._
~- 0.2 cm deep The sKin is therearter closed The amour.t
G~ DN~ or ~NA p~asmid can range ~rom 10 to lon ~i 0-
~: : hypo~onic,: i50toniC ~ or hypertonic sucr~se solutions or
sucrose solutlons containing 2 mM CaCl3. The plasmlà
contzi~ng:solutions can also:be:a inistereà ove_ a
longer period:~of time, for examDle, 20~mlnutes, by
lnfuslon,: The ln ~ expression OI the des~red gene can
be tested~by~determining an increased Droduction of the
25~ enc~ded polypeptide by the su~ject accordins to methods
known in:the~art:or as described, for:example, in Wolff
et al., Sc~ence 247:146~-1468 (1990).
~ It;is believed that the treated cells will
,~ ~ res`pond tb the direct~injection of DNA or RNA by
expressing the~encoded polypept1de for at least about 60
days. Thu~, the desired TCR, Lm~uno~enic rragment or
anti-idiotype: antibody ca~ be e~fecti~ely expressed by
~: the cells of the iadi~idual as an alternative to
: ~accinating with such polypeptides~
` -
~:
:: :
AMENDED SHEET
W093~12814 2 1 2 6 ~ PCT/US92/11159
29
The present invention also relates to vectors
u~eful in the gene therapy methods and can be prepaxed by
methods known in the art. Compositions containi~g such
vectors and a pharmaceutically acceptable medium are al~o
provided. The phanmaceutically acceptable medium ~hould
not contain elements that would degrade the desired
nucleic acids.
'
The fol14wing example~ are intended to
: il1u~trate but not ILmit the invention.
XAMPLE I
RA~ MODEL OF 13AE
Female Lewis rats; (Charles River Laboratories,
Raleigh-Durham,~ NC~ were Lmmunized in each hind foot pad
with;50 yg~of guinea pig myelin basic protein em~l~ified
15 ; i~ complete Freun~d's adjuvant. The ~irst signs of
di~ea~e were typioally observed:9-ll days po~t-
: immuniza ion.~Di~ease everity is:s~ored on a three
p4int~scale as~follows: ~l=limp tail; 2-hind leg
: weakne~s;~3=hind~leg:paralysis. Following a disease
: 20~:: course of:approximately~four to six days, most rats
: spontaneously~rec~vered~and were refractory ~ subsequent
EAE induction.~
5ELEC~TION AND PREPAR~TION OF ~ACCI~ES
Vaccinations w~re conduct~d with a T cell
: receptor peptide::whose sequence was deduced from the DNA
sequen~e of~ T~ cel1~ receptor beta gene pr~dominating
: among EAE~ ucing T ce}ls~of B10.PL mice. The DNA
:seque~ce was that~reported by Urban, et a~,, supra, which
~ :30 is incorporated~herein by refere~e. A nin~ amino acid
;~ ~: :peptide, h~vi~g the~e~ue~ce of the YDJ ju~ction of the
~ ~ ~ TCR beta chain of the mouset :was synthesized by methods
: ~ :
W~93/12~14 PCT/U~92/11159
~ ' 30
known to those skilled in the art. The sequence of this
peptide i~: SGDAGGGYE. (Amino.acids are repre~ented by
the conventional single letter codes.) The equivalent
sequence in the rat ha~ been reported to be: SSDSSNTE
~Burns et al., J. Exp. Med. 169:27 39 ~l~89)). The
peptide was desalted by Sephadex G-25 (Pharmacia Fine
Chemlcals, Piscataway, ~J) column chromatography in O.l M
acetic acid and the solvent was ub~equently removed by
two cycles of lyophilization. A portion of the peptide
was conjugated to keyhole limpet hem~cyanin (~L~) with
glutaraldehyde at a ratio of 7.5 mgs of peptide per mg of
: : KLH. The r~sulting conjugate was dialyzed against
phosphate buffered saline (PBS).
XAMPLE III
V~CCINATION AGAIN5T ~AE
:
: Vaccines u~ed in these studies consisted of
ree~VDJ peptide a~d also of VDJ peptide conjugated to
LH~ These~were~ dis~olved in:PBS and wer~ emulsified
: ~ :with equal volume~ of:either`(l) incomplete Freund's
20::~adjuvant (~ FA) or:~(2) complete Freund's adjuvant lCFA)
made by suspending 10 mg/ml heat killed desi~cated
:~ycobacterium tuberculosis H37ra: (:Difco Labor~tories,
étroit,:MI) in~IFA. ~Emulsions were a~inistered to 8-12
we~ek old female Lewi~ rats in a:inal volume of lOO
25~ microliters:per~animal (50~l in each of the hind
~otpads).: 5~yg:of unconjugated V~J peptide were
administered:per~xat. RLH-VDJ conjugat~ was administered
at a dose eguivalent to lO yg of ~L~ per rat. Twenty-
nine d~ys later ~ach rat was challenged with 50 ~g of
30~: guinea:pig myelin;basic proteiD in complete Freund's
::adjuvant in the~ front ~ootpads. :AnLmals w~re monitored
: : daily begin~ing at day 9 for clinical s~gn of EAE and
were core~ as d~scribed above. Th~ re~ults are
presented in Table 5. As can be seen, not only was there
::
~ ~ 35 a r~duced incidence of the di~ase i~ the vacci~ated
:
wo g3,l28l4 2 ~ 2 ~ PCT~US92/11159
individuals, but in th~se which did contract the disease,
the severity of the di~ease was reduced and/or the on~et
was delayed. The extent of proteciion varied with the
vaccine formulation, those ? ncluding CFA as the ad~uv~t
5 demonstrating the greatest degree of protection.
TABLE 5
AnLmal Vacci~atio~ Days After Challenge
No. (Adjuvant) lO ll 12 13 14 15 16 17 18
lO 1 VDJ (IFA) - - 2 3 3 3 - - -
: 2 " - - l 3 3 3 2
" _ _ _ 3 3 3 2
4 VDJ (CFA) - - - - 1 1 1 _ _
: : : 15 6 i' - - - l 3 3 3 ~ -
7 KhH-VDJ (CFA) - - - 1 3 2 - _ _
g - __ _ _ _ _ _ _ _
KLH-~DJ (TFA) - ~ 3 3 2 2 1 - -
20 ll ~ 3 3 3 3 3 ~ -
: 12 i~ l 3 3 3 3 - -
: l3 :~ONE : l 3~ 3 3 3
14 " ~ l 3 3. 3
"~ : l 3 3 3 l - - - -
: Scoring: - :no signs ~
l) l ~p tail :~;
: 2:):hind leg weakne~s:
:3) hind:leg paralysis
30 ~ EXAMoeLE IV
yaccinatlon:~aaa~inse ~AE wlth Lewis Rat VDJ peptides
:The ~D3:~peptide uséd in the pre~ious exa~ples
was synthesized:~according to~the~sequence o TCR B chain
molecules fou~d on E~ -inducing T cells in BlO~PL mlce.
In additio~, peptides~were synth~sized and te~ted which
correspond:to~:~equences~found on encephalitog~ic T cells
in Lewis rats. ~Th~ese VDJ s.~quences are homologous wi~h
that of B10~.~PL micet but not identical~ l'he rat p~ptid~
were synthesize~ accordi~g to the; DNA eque~ces r~ported
: : 40 by Burns, ~t al. and Chluba, et al., Eur. 3. Immunol.
19:279-284 (1989).~ The sequences of these peptides
WO 93tl2~14 PCr/USg2/1 1 159
? ~ jS~ 3~
de~ignated IRl, 2, 3 and 9b are 3hown below, aligned with
the BlO.PL mou~e ~;equence u~ed in Examples I through III
(YDJ ):
VDJ S G D A G G Y E
S IRl C A S S D - S S N T E V F F G K
IR2 C A S S D - S G N T 1~ V F F G K
IR3 C A S S D - S G El - V L Y F G E G S R
IR5b A S S D - S S N T ~
The preparation, administration and e~aluation
1 Q of tht3s~ vacciTles were conducted as described in Examples
I through III with the following exceptionE;: 50 ~g of
the individual VDJ peptides were incorporated into
vaccine :Eormulatior~s containing CFA; neither vacc:inations
in IFA nor vaccinations with p~ptides conjugated to KLH
15 were conductedO Corltrol animals w3re un~reated prior to
BP challeIlge as in ~xample III or were vac:Y:inated with
: : emulsions of PBS and FR to as~ess th~ pro~ectiv~ ef f ect
of adjuvant alone. The results are shown in Table 6
: below.
~: :
W093~12814 2~ 2~ P~/USg2/lll59
TAB~E 6
AnLmal VacinationDays After Challenge
No. tAdjuYant)lO ll 12 13 14 15 16 17 18
l None - l 2 3 3 2 - - -
2 " l 3 3 3 2
3 " - 2 3 3 3
4 PBS~CFA l 2 3 3 3
" l 2 3 3 3 - - - -
6 " - 2 3 3 3 ~
: 7 IRl (50 yg) - - - 2 l - - -
8 l 3
9 " ~
: lOIR2 (50~g) ~ - l 3 3 3 - - -
ll " ~ 2 2 3 3
12
l3IR3 (50~g): l 3 3 3 2 - - - .-
: 14 " - - 2 3 3 ~
: 15 ~ - - _ _ _ _ _ _ _
20 16 IR9b (50 yg3
17 -
: 18 .. ~ -
1 9
Scoring: - no s~gns
lLmp~tai~
2~ hind leg weakness
3) hind leg paralysis
: As shown in Table 6, disease in unvaccinat~d
control anLma1s;was observed as early as day lO. Disease
was ch~racterized~by severe paralysis and wasting,
-: persis:ted f~r 4~to 6:days and~spontaneously r~mitted.
PBS-CFA vac~inated rats disp1ayed di~eaRe cour~es
virtua~lly~indistingui~hable from those of unvaccinated
35 ::contro1s. In~contrast, delays in on~e~were observed in
: some of the IR1~ 2:or 3 vaccinated anL~ls and others
:~ show~d:both delayed~onset as:well~as decrea~ed severity
:~ a~dfor duration of disease. Overall, however,
: v~cci~ations~with the rat VDJ~peptides:(IRl-3) were
slightly les~ effe~tive than ~hos~ with the mouse VDJ
peptide ~xample III). Vaccina~ion with IR9b, hvwever,
affortsd co~plete protection in all four anLmals in which
it was tested. Importantly, no histologic le~ions
c~aracteristic of disease were found in any of the fvur
W093/12814 PCT/US92/11159
2~
~ 34
anLmals vaccinated with IR9b indicating that sub-clinical
signs of di3ease were also abrogated.
E ~ L~ V
Vaccination with V reqion sPecific Peptides
A peptide specific for the V~8 gene family wa~
tested as a vaccine against E~E. VB8 i~ the mo~t common
chain gen~ f amily used by encephalitogenic T cells in
both rats and mice. ~ peptide was synthesized ba~ed on a
unique DNA ~equen~e found in the VB8 gene t and whi~h LS
~0 n~t f~und among oth~r rat V~ genes who~e ~equences w~re
reported by Morris, et a;l., Immunog~netics 27: 174 179
(1938~ o The sequence ~of this VJ38 peptide, designatet:l
IR7, is:
: ~ : IR7 ~DMGHGLRLIHYSYDVNST~K
;15 ~ The efficacy of this V~8 peptide wa~ te~ted in
:tbe Lewis rat:model of ~AE (Example I) ~s described in
Examples II and III.~ 5~ ~g of peptide were t~sted in
CFA. Vaccinations in I~A or with peptideDKL~ ~on jugat~s
:; w~re not co~ducted. :The result~ of the~e studies are
:: 20 shown in Table 7:~
:: TABLE 7~
Animal ~ ~accination ~ Days After Challenge
o:.~ Adjuva~t~ lO ll12 13 14 15 16 17 l8
~' 25l ~ I~7 (50 yg) :- - l 2 3 3 3 - -
Scoring: - ~ no ~ign~
1~ ~: limp; tail
2)~ hind leg :weakness
3) ~hind leg paralysi~
W~93/12814 2 1 ,~ 6 ~ PCT~US92/11159
EXAMPLE_VI
Comparison of VB8.2 Peptide Lenqths
The results of vaccinations conducted with the
rat VBB peptide are similar to those observed with the
mouse and rat IRl, 2 nd 3 peptides. Delayed onset a~
well as decreased severity and duration of di~ea~e was
observed in one anLmal. One animal was co~pletely
: protected.
It has been found that a correspondi~g 2l amino
acid sequence of ~8~2 (residues 39-59) provided le~
; : protection tha~ IR7 as shown in Table 8. The 2l amino
acid sequence of VB8.2 is DMG~G~RLIHYSYD~NS~BKG~
: ~ :
,,:
:.
: :
:
: :
:
WO 93/12~14 PCI/U$92/11159
36
c~7,~ J _
U~
I o ~r I
~'~ I ~
~-- ~
o
~ ~` ~D O
I
a~ a~ u
~ ~ U~
,~ _
G~
Q u~ :~t
_ ~
P~
~ O
C~ P; oo
.,.~
~o ~ ~ o o o
~ ~
.,~
~ Q~ ~ - U~ O O~
,~C ~ :~ E~ _l .
.
~H ~ ~ ~ ~
~: ,.;~ ~ O ~: 0 h . o o
: ~ ~ 5~ ~
U h ~
U~
Ql C : O _t _I
~: O
,~
O
h a~ ~ ~: : ~ c~ _
:: : ~ : :~ :
: a.~ :
Q ~
Q) :
,c ~ ~ o o o o
m
, ~ :
~ ~ O ~
: ~ ~c~ u
: -~ o ~ ~
c)
~ g ~ ~:
WO93/12814 PCT/USg2/1115
X, ~
37
Each peptide was used at l00 ~g doses and
dissolved in saline prior to being emulsified in an equal
~olume of complete Freund~s adjuvant (CFA). AnLmals were
challenged after 42 days with 50 yq of guinea pig myelin
basic protein in CF~. The CFA contained l0 mg/ml of
mycobacteria tuberculosis. Injections and evaluation of
cl inical signs and histology were performed as previously
described~ Other anLmals (five per group) were L~munized
with peptides in the same way and their splenocytes were
removed aft~r 14 days to test for lymphocyte
proliferation as described in Olee et al., J.
~ Neuroimmunol. 21:235-240 (1~89). The sequences of the 20
: amino acid p~ptide and the 21 amino acid peptide are
designated in Table 8 as V~8 . 22o and ~8.221 respectively.
: XA~PLE_VII
Vaccination with J renion peptides
,~:
A pep~ide was ~ynthesized which corresponds to
the 3 a gene egment, TA39, ~ound~amo~g both rat ~nd
mouse encephalitoge~ic ~:cell receptoræ. ~he 3equence of
this peptide,~designated IR5, is:
IR5 :~ RFGAGTRLTVK
:: : :
: : The:eff~icacy of the J3TA39 peptide was tested
in the Lewis rat model of EAE (ExampLe I~ as described in
Examples ~ II and I:II. ~50 ~g o~ peptide were t~sted in
25 CFA. Vac~inations in IFA or with :peptide~ conjugates
were nc: t conducted:. The results of these studies are
shown in Table 9.
:~: ~ :: :
WO 93/12~14 P~r/US92/1 1 15~
~f ~ 3 - 33
Animal Vaccination Days P,fter Challenge
No. (AdjuYant) lO ll12 13 1415 l6 17 18 l9 20
1 IR5 ( 5 0 ,ug ) - - - - - 2 1 1 1 1 -
3 _ _ _ _ _ _ _ _ _ _ _
Sc:oring: - no signs
1 ) l~mp tail
2~ hind leg weaknes~
3 ~ hind leg paraly~is
~ he results of vaccinations conducted with the
rat J a TA39 peptide are more effective than tho~e
~bæerved with the mou~e VDJ peptide or the V~8 peptide.
~wo~of three anLmals were totally protected and, in the
third, dis~a~e onset was markedly delayed~ Severity was
` al~o reduced in this anLmal though disea~e persisted for
a nonmal cour~e of 5~d~ysO Importantly, the twv anlmal~
`20~which were~completely protected howed no histologic
evidence of T cell infiltration of the CNS. This result
indicates that~:~vaecinating with the JaTA39 very
efficiently~induces a regulatory response directed at
encephalitogenic~T cells. :Even sub-clinical signs of
2~ disea~e were abrogated.
~ : EXAMPLE VIII
: Vaccination with mixtures of TCR peptides
:~- ~accina~ions were condu~ted with a mixture of
: TCR peptidess~ This mixture contained 50 yg of each of
30 ~the peptides I~l, 2, 3 and 5 (the three rat VDJ peptides
~ and the rat JaTA39 peptide),
,~
The ef f icacy of this peptide m~xture was tested
in the Lewis rat~ model (Example I) as described in
. .
WO 93/12814 ~ ~ 2 ~ 6 ~ ~ PCr/US92/11159
39
Examples II and III. Peptides were tested in CFA.
Vaccinations in IFA or with peptide-KL~ conjugates were
not conducted. The results of these studies are shown in
Table l O .
TABLE l O
AnLmal Vacaination Days After ~hallenge
No. (Adjuvant) lO 11 12 13 14 15 16 17 18
4 IR1, 2, 3, 5
~50 yg each)
: Scoring: - : no ~;igns
1 ) limp tail
2 ) hiIld leg weakn~3ss
3~ ~ hir~d leg paraly iR ~
The results of:vaccinati~ns co~ducted with th2
rat JaTA39 and~;three~VDJ peptides~were a~ eff~cti~e as
those described for IR9b~in Table 6. All three anLmals
20~:were totally~protected. In a W i~ion to the ab~en~e of
any clinical~signs~of ~ , two of these three anLmals
were:~complete~ly~;free of~histological evidence of T cell
infiltration~;into~the CNS while~the thlrd showed only two
~all foci~of~lymphocytic:in~iltration:at the base of the
25~:splnal:aort.:~
: Multiple Sclerosis Vacc:ine
:~ :
A. Human ~BP-reactive T cells
MBP-reactive T cell lines were e~tabli~hed from
30~ peripheral~;blood~ nonuolear cells~(PBMC3 of ni~e chronic
: progre~sive:~S~patients and two healthy control&~ Cells
were maintai~ed in culture by regular ~tLmulation with
purified huma~ MBP and irradiated-autologous PBMC for
W093/12~14 PCT/US92/11159
' 40
three days followed by four days in IL-2 containing
mediu~.
B. _ PCR Ampli~ication of TCR ~-chain qenes from MBP-
reacti~e T cell lines
T cell~ were harvested from log pha~e cultures
and RNA was prepared, amplified with the ~l6mer primer
and neste~ C~ prLmers for 55 cycles as described in
Example X.
C. TCR B-chain ~equ n ~s of human MBP-reactive ~ cell~
V~l6mer amplified TCR ~-chain genes from human
MBP-r~active T cell lines were ~equenced using the C~seq
prLmer. Ampllfication products w~re gel purified, ba~e
denatured and sequenced from the C~3eq prLmer. Readable
: DNA se~uence was~ obtai~ed from 5 of the~e line~,
indicating that :predominant ~ cell clones had be~n
; : :: se1ected by long term in vitro passage. One of the~e
sequences,:fr~m the MS-Re cell line (Table ll~, po~se~sed
a ~-chain VDJ amino acid sequence that shared five of the
irst six and~ix of nine total residues with the B-~hain
~DJ a~ino acid sequence conserved ~ ong MBP reactive,
encephalitogenic T cells in the Bl0.PL mouse mod~l of
AE.~This sequence was not~pre~ent among the predominant
TCR rearrangement~ found in the remaining f our human MBP
: reac~ive T ceIl ~lines.
To determine if sLmilar sequences were pre~nt
in the B-chain repextoire of the MBP-r~active T c ll
~: ~ lines from other ~S patients, ~CR ~mplification was
conducte~: with a degenerate (n=1024) 2l-nucleotide primer
(V~e) ~orr~sponding to se~en amino acid~ of thi~
: 30 se~uence. R~As were rever~ed transcribed and ~mplified
i~ 20 cycle stage I reactions with the ~l6mer and C~xt
primers. One ~l aliquots of these ~tage I reactions were
;
WO93/12~14 ~ ~ 2 ~ ~ PCT/U~92/11159
41
reamplified for 35 cycles with the V~Re and C~ int
prLmer~. One ~l aliquots of the~e reactions were
analyzed by Southern ~lot hybridization with a 32P-labeled
human CB probe. This analysis revealed the 300 bp
amp~ified product in the Re cell line a~d in one of the
other ~S patient lines, but not in MBP-reactive T cell~
from control ~ubjects or in non-MBP reactive human T c~ll
lines and clones. ~he presen~e o this ~equen~e in two
of the nine ~S patient lines te~ted is c~mpelling. Since
this ~e~u~n~e i~ known to be con~erved among
: encephalitogenic T cells in E~E, it~ detection among MBP-
rea~tive T cell~ from MS patients demon~trate~ a role for
T cells bearing thiS determlnant in the pathogenesis of
MS.
Immu~ogenic peptides haviny the ~e~u~nce -
: SGDQGGNE can be synthesized as ~hown i~ Example II and
u~d to L~unlze human subjects by method~ demon~trated
in ~xample III.~Sueh immunizatio~ can result in an
ef~ective im~une respon~e. ::
20 : ~ TABLE l1
A~:~Sample VB: ;_ : D~ _J~
: 2~ V~4~2 J~2.1
:MS-Re ~ ~ctc~gc agcggagaccagggcggc aatgagcagtt~ttc
S G :D : Q ~ G - N E Q ~ F
- : Bl0~P~ : S G D ~ G G G Y E
B~
: ~ A~ A A
:C C A C C C A
5' ~ G : G A C A G:G G G A A G A 3'
G T G G ~ T G
: T T T
::
WO93/12~14 P~T/US92/111
EXA~PLE X
Detection of Clonal Infiltrates of
Activated V~l7 T Cells in the Synovium of
Rheumatoid Arthritis Patient~
O T ~ell preparations from svnovial tissue
Synovial tissue ~pecLmens w~re obtained from
radiographically proven rheumatoid arthriti~ patients
: und~rgoing joint replacement therapy. Activated T cells
were ~elected using magnetic beads and antibodie~
: 10 reac i:ive with the human IL2 -R ( aIL2 -R ) as f ollows.
. Syno~rial tis~;ue was digested for 4 hrs at 37C i~ RPPqI +
10~ Fetal Bovirle Serum (FBS ) containing 4 mg/ml
col:lagena~e ( Worthington Biochemical, Freehold, NJ ) and
0.1~ mg/ml DNA3e~(Sigma, St. Loui~, MO~. Digests ware
lS pa~d through an 80-mesh scree~ a~d ~i~gle cella were
collected~by Fic~ll density gradi~nt ce~trifl1gation.
Cell at the interface were wa~hed and were inc~bat~d at
lO~ml fox 30 min at 0C with 5 yg/m~ control mou3e IgG
oul~er I ~ unology, Hialeah, ~L) in PBS containing 2%
20 ~FBS~(PBS-FB~ o ~Cells were washed three times and
incubated for 30:mln at O~C with magneti~ beads conjugated
to~goat~ an*i-mouse IgG (Advanced Magnetics, Cambridge,
MA)~. Beads were:magnetically:separated and wa~hed three
tLmes with PBS-FBS. This preselectioa with mou~e IgG
25~ !mIgG~ and~magnetic beads was used to control for non-
specific ad~orption~of T ells. The c~l1s re~ai~ing in
the initial su~pension:were further incubated 30 minut~
at 0C wi~h~;5 ~g/ml monoclonal mou~e IgG reacti~e with the
:human T cell:IL2-R:ICou~ter I~munolo~y,~Hial~ah, FL).
` 30 Cells:wer~ wa~hed and ~elected with mag~etic bead~ a~
above. Beads~from the IgG preadsorption and the IL20R
antibody ~el0ction w~re Lmmed3ately~re~uspended in
: acidified-guanidinium-phe~ol chloroform a~d R~A prepar~d
: as described in Chonezynski and~Sacchi, Anal. BiochemO
~: 35 162~156 (1987~,:which is incorporated h~rein by
:~
WO 93/12814 2 1 2 ~ ~ S ~ PCI/US92/1115~
43
ref erence . Since Rl~A5 were prepared without in vitro
culture of the cells and the accompanying bia~ that may
be induced, they are expected to accurately ref lect T
cell di~tributions in synovial tissue at the t~e of
5 urgical removal. Only half of the mIgG ~nd ~xIL2-R beads
from patient 1012 were immediately proce3~ed for ~NA.
The remainder were cultured for 5 days in RPMI 1640, 5%
FBS, 20% ~L-1 (Ventrex Laboratories Inc., Portland, ME),
25m~ ~EP~S, glutamine, antibiotics and 20% LAK
~ 10 supernatant (Allegretta et al., Science, 247:718 (1990)),
- , which is incorporated by reference herei~, as a source of
IL-2. R~A wa~ extracted from culture~ of the aI~2-R
. b~ads (1012IL2.d5), but not from the 1012mIgG sample as
no ~iable cells were pre3ent at the: end of the 5 day
culture.
A ~ cell cIone was derived from the Ficoll
pellet of patient 1008. The cel~1s in the pellet were
: :c~ultured at 2 x 106/ml: in m~dia without IL-2 for ~wo
we~ks.: Non-adherent cells from this culture were cloned
by 1Lmiting dilut.ion onto autologous synovial cell
: monolayers;.~ A~CD4+ T cell clone~1008.8 was obtained and
adapted to~cul~ure by regular stLmulation with autologous
synovia1 mono1ayers:for 3 day in media without IL-2
fo11Owed by a 4 day culturè in medium with LAR
25~ ~supernatant.
B. Lysis of ~novial Adherent Cel:ls by l008 . 8
1~ : Lysis of synovial adherent cell~ by 1008 . 8 wa~
dems:)nstrated as ~follow~. Synovia1 cell monolayers were
lab~led as described ir. Stedman: arld S~ampbell, J. Immunol.
:
~eth~ 119:~291 (1989), which i8: incorporated herein by
reference, with~35S for use ~s targets in C~L assays.
Cell~ were- ~ypsiniz~d, wash~d and plate~ at 2~00 c~lls
~:~ : per well of a~96-well rou~d bottom microtiter plate~
1008.8 cells, cu1tured for 3 days priox ~o the assay with
,~
WO93/1281~ PCT/US92/111~9
'3~ 44
synovial adherent cells and medium containing LAK
supernatant, were added to the targets at the indicated
effector:target ratios. Cultures were incubated
overnight at 37~C, centrifuged at 300xg for 2 minutes and
radioactivity in 50 yl of the supernatant quantified.
Per cent specifi~ lysis was calculated relative to
detergent-ly3ed targets by ~tandard formula~. This clone
is cytotoxic for ~ynovial adherent cell targets in CTL
assays (Table 12).
TABLE 12
.
ffector:TILEget Ratlo _ % SPecific LY~is
5:1 7
: lO:l 16
25:1 32
; 15 C. PCR AmpIification of TCR ~-chain qenes
TCR ~-chain genes were amplified with ~everal
combinations of the prLmers shown in Figure 2. The
v~l6mer p~imer is a degenerate V~ primer ~n=256) which is
; predicted to ~ind 85% of human TCR ~-chain genes at all
,
1:6 residues and 95% at 15 residue~. This prLmer has been
: used to amplify TCR B-chains from more than 25 different
human T cell~clone~, line~ or prLmary ti~sue
preparations.~A ~pectrum of V~ genes has be~n sequenced
: from these amplified DNAs, arguing again~t a ~ignificant
: ~ 25 bias of the prLmer for certain V~ familles. Thus, PGR
amplification~with the VBl6mer primer facilitates
analy s of T c~ll populatîons for which a Priori
: kn~wledge o V~ gene usage is unavailable.
30T cell receptor ~-chain genes were amplified in
two-stage amplification reactions with neæ~ed pairs of
: the prLmers shown in Figure 2. The primer sequences u~ed
W0~3/12814 2 1 2 ~ ; PCT/US92/11159
in the polymera~e chain reactions are listed in Tab~e 13.
T ~ LE 13
G AC CAAA
~cons 5' T TC TGGTA CA 3'
T TT TCGT
V~17 5' TCACAGATAGT~A~TGACTTTCAG 3'
VB8 5' TCTCCACTCTGAAGATCC 3'
V~12 5' GATTTCCTCCTC~CTCTG 3'
5'C~ 5' CAaGCTGTTCCCACCCGA 3'
10. C~xt5' CCAGAAGGTGGCCGAGAC 3'
: C~int5' GCGGCTGCTCAGGCAGTA 3'
~ ~ : C~seq .5' CG~CCTCGGGTGGGAACA 3'
: ~ :
~ s ~were~ :rever3e trar~cribed for 1 hour at 42C
with 4 0pmol of the CJ~ext primer in ~ a 12 7ul reaction using
1 5~ : condit~ons de~;c~ibed by Elart et al ., The Lancet , p . 5 9 6
( 1988 ), included by reference herein. Reactis~ns were
:diluted: with a :master mix con~aining 40 pmols of the
VJ3:16mer primer, nucleotides and reaction buffer as above
but without: MgCl:2 to give a final Mg~2 Gorlcentration of
: 2~ 3.6 mM. Samples were denatured~ for 1~ ~ILinutes at 95C,
unit of heat stable recombinant ~D~A polymerase (t::etus
Corpo~ation, ~3meryville, CA, Ampli-taq~) was added and 20
~ : cycles of P~R c~nducted. Each cycle con~i~sted of a 1 min
: denaturation at;95C,~a two minute annealing step a~d a
: 25 two mi~ute extension:at~72CC. The first ~wo cycles were
: an~ealed at 37~C and~45C, r specti~ely, and the remainder
: at 50C. One~microliter aliquots of these stage I
reac*ion~ were a~ded to l00 ~1 stage II ~mpli~icatio~
reactions (Cetus, Ge~e-Amp git~M) containing l00 pmols of
the Bint prLmer and l00:pmols of the V~8, Y~17 or 5'C~
prLmers ox 7QO pmols of the V~16mer prLmer. Stage II
ampli ications were conducted as above with a 50C
~ '
WO93/12~4 ~ PCT/US92/1l1~9
~S'^` 46
annealing temperature and without the 37C and 45C
ramping.
RNA samples from 1012IL2.d5 and 1008.8 cultures
were ~mplified with the V~16mer and CBext prLmers in
stage I reactions and with the VB16mer and the C~int
prLmer in 35 cycle stage II reactions. Reaction
products, purified from low melting agarofie gel slice~
with Gene Clean gla~s beads ~Bio 1~1, Sa~ Diego, CA),
were base denatur~d and ~equenced from the C~seq prLmer
with T7 poLymerase~(Sequenase, United States Bio~hem,
Cleveland,~OH). A;~pred~minate ~ sequence, corresponding
to a single V~17~ rearrangement (Table 14), was clearly
readable in the 10}2IL2.d5 sample. Other, less frequent
rearrangement~ were det~cted as faint, uninterpretable
background bands~in~the ~equencing gels. Culture of
;these 1012.IL2 ~ead~ in~IL2-oontaining medium without
added ~ccessory~cells or antigen~is~not expe~ted to
induce de ~yy~a~tivation of ~ cell~. ~hus, the
predominan~e of a~single~YB17 rearrangement in this
20; sample~re~l~ects~in vivo clonal expaDsion of V~17~ T cells
in~this~patient.~ DNA~s~quence;determination of TCR ~-
chain~DNA~amplified~from;the~cytotoxic T cell clone,
lQ08.8,~ als~o~revealed a V~17 rearrangeme~nt (Table 14).
The pre~ence~of~ l7~rearrangements in these two
25~different~types~of~synovial T cell~sample~,~derived from
two~separate RA~patients,~plicates V~17 bearing T cells
in the~pathogenesis~of~RA.
: ~ :
:: : : ~ :
..... ,, . .. ~ .. , ~ .. .,~ .,.
2~ 2 ~ fi ~ ~ /US92/111~
WO93/12814 P~T
47
TAB~E 14
Sample V~ D~ J~_ _
.
1012 Y L C A S K ~ P T V S Y G Y T F
day 5 tatctct ~ gccagt aaaa.tcccacggtctcc tatggctacaccttc
VB17 J~1.2 ;-.
: .
Y L C A S D N E S F F G Q G
1008.8 tatctctgtgccagt gacaacgagagtttctttggacaaggc
10 : : V~17 : ~ J~l.l .
:~ 1014 Y L C A S~ V R D R R N Y G Y
IL-2 tatctotgtgccagt gtgagggacagga~aaactatggctacacc
V~17:~ J~1.2
1015~ Y~ L :~C~:A ~ S:~ S~ S I D S S Y E Q Y
15;~ 2 ~tatctctgtgccagtagt agtatagactcc tcctacgagcag~c
: To`:determine-whether~or~not ~B17~:rearrang~ment~
were~pres~nt::in~:the other:magnetic bead:RNA preparations,
TCR B-~:hain~genes:~were~ampliied with ~a~V~17~-specific~
20~ prLmer~;in~the~;seco~d stage:ampl~ifiaation after ~n initial
:amplification~with~::;the~V~:16mer.~ V~17 TCR DNA could be
amplified~from~magnetic bead~samples derived~from the 4:
patiénts~::exam~ned~ thidium bromide stainin~ of
lectroph~res:ed~r~action~products revealed greater V~17
amplifi~ation i~ 50m~ of ~he IL-~R+ sa~ples than i~ the
rre~pondi~g~ ntrols. A~cordin~ly, the::relative
; æmounts:of~V~17~ T~Rs~in ea~h control and IL-2R~ æample
were qua~tified~ y~slot blot hybridi~ation analysi~ as
foll~w~
~ P~B; from magnetic bead preps were amplified in
the fir~t ~tage with the VJ316mer and C~ext primers and
: : : :
:: : :
WO93/12814 ~ PCT/US92/11159
6~ ;r~ 48
then reamplified for twenty cycles with the CBint primer
and each of the ~ 17, Bfl8 and 5'C~ primers.
Amplification reactions were 8erially dilutsd in 20X SSC,
denatured by boiling and chilled in an ice slurry.
Sampl~s were loaded onto nitrocellulo~e membranes,
hybridized to a human TCR ~-chain constant region probe
and washed with O.lX SSC, 0.1% SDS at 56~C. Bound
;~ radioactivity was quantified by li~uid scintillation
;~ spectroscopy and endpoint dilutions were tho~e samples
: 10 with fewer than 200 cpm bound. The amounts of product
produced by forty total cy les with each of the
re3pective prLmer combinations falls in the linear
. portion of a product versus cycle number quantification
curve.
- Amplifications with the 5'C~ and C~int primer
pair were u ed~to~e6tLmate the total ~-chain amplified
from each ~ample,:;proyiding benc ~ rks for normalizing
the~results of~V~17~and VB8 quantification in th~
respective IL-2R~+ and control:sampls pairs (Table 15) .
: 20~The~ ~uantity~of~V~17 DNA:ampli~ied was increa3ed in the
IL-2R+ ~samples,~relative to~the~control samples, in 3 of
the ~4 patients.: The magnitude; o~ ~the increase ranged
f~rom:5-fold~in~patient 1015 to:40-fold in patient 1014
(Table~15~. This~enriohment was not a product of the
25 ~isolation~procedure, since:the quantity of V~8 DNA
amplified was~increased in the IL-2R~ fraction only in
patient~1015.~
~ ,
~: ~
:
WO93/12814 2 ~ ` PCT/US92/11159
49
TABLE l5
Endpoint Dilution
Sample C~V~17V~8 V~17/C~ V~17IL-2RV~8/C~ V~8IL-2
mIgG mIgG
3 ~ 1253 ~ 125 625 1 0 . 2
23 ~ 125 125 625 0 . 04 0 . 2
315 ~ 625 25 ~2~ 0 . 001 0 . 04
0.12 0.04 `~
43 ~ 125 25~ 3, 12~; 0 . 008
515 ~ 625:625 125 0 . 04 t) . 008
0.04
63 ~125 ~ 5 625 0 . 001 O . ;2
715~,1;25 625 l5r625 0.04
878,1;~5 ~6~25 ~ 3,125 0.008 ~ 0.04
Sample :l = lOl2~IL-2R+, Sample 2 = :10~12 mIgG,
Sample 3 = ~1013 I~-2R+~ SampIe 4 = lOl3 mIgG,
Sample 5 o 1014 I~-7R+,:Sampl`e:6 =:1014 mIgG,
S~ample 7 - lOl5~:IL-~R~,: Sample 8 -:1015 mIgG.
*In~a follow-up~study in which~VB8 was:sequenced, it was
found that:the enrichment of this V~ was:an arti~act and
that the value is~:<:l. : : :~
:;:: YB17:~rearrangements from the IL-2R+~:R~As of the
three~patient~ howing enrichment were amplified with the
V~17 and~C~int prLmer pair and the reaction products
~equenced with the C~seq prLmer.: As was~how~ for ~ample
~: 1012 IL-2.d~ 14 and 1015 contain~d single ~quences
(Tabl~e 14):,~indicative of c1Onal expa~sion o~ V~17 T
cells in:vivo. :In~ c~ntra~t, direct ~e~uencing of the
rearrangements~ smpli~ied with th~ Y~8 specific prLmer was
: :35 ~not poY~ibl~ due~:to significant heterogeneity in ths ~-
chain pro~uct.;:::~
": : : :
: ~
:
2 1 ~ ~ ~3 ~ ri - ~
so
.- 3-_~ .hna~vs_s ~ he~m2.0ià ~ - .is __~e~lls
_~AY~R a~e~vsis ~ eum2lc~d 2r_~- _ s
?a~ s was ~e-~or~ed 2S -ollows. DN~ '~rom e2c:~ p2. ent
W2S ~=e~are~ _v Doil~n~ 105 svno~ia~ c~lls in 200 ~l d~.O.
Ten ~ were zm?li~ied for 3~ cycles i-. 2 100 ~l ~e2c~ion
(Celus, Gene ,~m~ Ki~-Y) cor..aining 100 ?mols of e~ o~
t.~e ~?~ ~CR p-~ne_s shown in ~zble lo 2s DR~l æ-,c DR~'.
One-~e~..h ul c~ .his -_ac.ion was rea~ iea i~ ls
c~r.~ ns onlv the DR~2 ~~imer ana 17 pmol ol ~322-dCTP :~
0 25 the sole sourc2 oî dCT~ fo~ 10 cvc'es. React-or.s were
s~ikec with 200 ,uM dC~ cnd chased o~ 2 cycles. The
r2su~=~ns nea2.i~e s~rand Drobes we_e ~ briài7ec to sio~
io.s containing~10 pmol o~ the HL~-3R ailele sDe~ c
oligcs (posi~ ve~ strands) usin~ condlt~ons previouslv
aesc=:~ed by~ ~ r el 21., J. ~mmunol. ~38:1947 (lg87),
whlc;- s lnco-?orated herein by refere~ce~ The siots
were washed twice for:20 minutes with
tetramethyla~oniumchloritie (Wood ei~ ai., Proc~ Natl.
Acad. ~ Sc . U51~ ~:82 ~1585; ( 1985 ) )~ d
20 ~ nc=c~ ~ : at ~ 65-68C and exposed to X-rav
; Each ~of ~the patlents in this study possessed at
Least one~ ailele ~ of: :the ~ DR genes, DR4w4, I:)R1, DR4w14
o:r DR4wlS~, that~are k~own to predis~ose~for RA (Table
25 ~16~. Also shown in Table 16:are ELA-DR allele sDecisic
oligonuc}eotldes.
~ . :
.
:::
AMENDEDSHE~
~:
WO93/12814 2 ~ 2 6 ~ PCT/US92/11159
TABLE l6
DR~l5' G A G T C T G G A A C A ~ C 3' ~:
C ,
A
DRB25' G T A G T T G T T C T G C A 3'
:~ G
~ HhA-DR ALLEL~-SPECIFIC OLIGONUCIEOTID~S
:
DR~l Gene~
DRl,DR4w1:4,DR4w15 5' CTC CTC GAG CAG AGG;CGG GCC GCG 3'
DR2* ~ 5' T~ G-C ~ -C ~ 3'
DR3 5' ~ A- ~ G- CG- 3'
DR4w4 5' ~ A~ 3'
DR4w13 :~: 5~ - -A 3~
15: DR~t DR6, DR4wl0 : 5' A~ A G-C G~ - 3'
DR7 5'~A~ :G~ G- CA- 3'
R8 : 5' T~ A~G-C ~ CT- 3'
DR9 ~ 5':T~ -G~ A- 3
DRB3~ &enes~
0~:~D~2 ~ 5' A~ - GC~ - 3'
:DR3~ : : 5' --~ AG 3'
DR7, DR9 ~ 5'~ - -A- 3'
; :; Patient __ _ _ ~LA-D~
1008~ w4
:~ 1012 l 3
1014 : : l,4w~
1015 ~ ~ 4w4,4w~
30~*:The dashes~refer tO~ a Lds 1~ com~on with DRl, DR4w14,
T:;cell receptors:containing ~17 or fragments
ther~of which:are~im~unog ic or can be made i~munog~ni~
: can~be u~ed to immu~ize human subjec~s by methods
; ~ 35 de~on~trated by ~xample VIII. 6uch Immunizations can
: resul~ in an effective Lmmune respon~e,
, ~
:: :
W093~12814 PCT/US92/11159
~7 ~ J EXAMPLE XI
Synovial tissue specLmens were obtained from
proven RA patients undergoing joint replacement surgery.
HLA DR analysis was conducted as de~cribed in Example
IX(D).
A Polymerase chain reaction ~PCR~ amplification of T
cell recept~r ~-chain gen~
T ~ell receptor ~-chain genes were amplified in
~ two-stage amplification reactions with nested pairs of
: ~ 10 HPLC-purlfied oligonucle~tide prLmers (Midla~d Certîfied
~ Reayents, Midland,~ TX.) shown in Figure 3. RNAs were
:~ reverse tran cribed (l hour, 42C) with the C~ext primer
: (40 pmol) in 12 ~l of reaction buffer (~art et al. t
Lancet ii:596-599 (1988)~ Reactions were diluted with a
15~ master mlx ~8 ~l) containing the V~cons primer ~40 pmol),
nucleotides and~reaotion buffer minus MgCl2 (final Mgl2
:c:oncentration~:3.6 mM~.: Samples were denatured (15
minutes, 95C) and:~20 cycles of:~PCR were conducted using
Taq~:polymeraYe:~(~l unit,: Cetus Ampli-taq). Eac~ cycl~
20`:~:consisted of a 1 minute denaturation at 95C, a two minute
;a~nnealing step and a two minute~extension at 72C. The
first two cycles~were~annealed at 37C nd 45C and the
remainder~at 50C:.~ One mlcroliter~ a1iquots of th~se Stage
I r~actions~:~were~added to l00 yl~Stage II amplification
reactions (C~etus-:Gene-Amp Rit) contai~ing the CBint
prLmer::(ln~O~pmol)~ and;either~the V~8, V~12, V~17, or 5'C~
prLmerY (lOO~pmol) or the v~cons~prLmer (l00-700 pmol).
Stage II amp~ifica~ions were co~ducted for the indicated
number of~cy~1eY~with a 50C ann~aling temp~rature and
: 30 ~without the 3~7C ~and~45C r2mping.;:
: ~ -
B.~ V~17 T_cells are cytotoxic for synovial adkerenk
cells : ::
Two parallel cultures were establi~hed from
: :
::
~2~-fi~
sir.c~e cell 5~s~e~.s~0r.s, _e~ve~ ~ e~~~Y~alic c-ces='on
~s c~sC=i~ec -e~l CW -rcm ~-~e svnovial t~ssue OL ~_~_' ent
coc . ~l~.e -~ -s~ c cu~L; c~ e c- ,~ s~nov~ 2' cells~
~as _~aLe~ a_ ^~ x 10~/mi ~ M.T 1640~ 5~ 20~ n~-
_ (Ve.~_ex Labo-2tc_ies, ~0-_12na, ME), 2~mM ~PE~,
~t2-tne and an.~_~o.ics. ~he cultu~e was gro~L. ~-
'~G` s~a-bed ,o_ two wee.~s -. t:~e aDsence of exoce~.ous
æn._ ~e~ o- g-ow-Lh _ac_~s. ~he secsn~, 2 synov a .ell
~ono -ver C't 1 t~e~ was ~n~=~ate~ as aDove and reg~alarly
~0 _ Y-~s: ized and ~ ssage~ g this ~-~o-wee.k pe~~od.
Monc~2ver cells we' e oiate~ at ~ x 10: cells/we~ r~at-
DO ~_~ed, 96-weil ~ic-o~ite- plates anc culture~
over-.:~ht. Non-adhere~l c-71s ~_om the lotal svr,cvi21
C5~ lture were then 21a_ed at 10 cells/well o.-~ the
monci_Yers. wells positive ror ~ ce;l g=owlh we;-
~eY~?anaed and za2D~ed to cul_ure by regular stLmu~a~ion
wi,_h autologous synovi21 monolaye_ s f or 3 davs i~ meàia
: witho~ 2 -ollowed bv 2 4 day cultu~e in medium
con_a~ning 20% .supernatant ,~om lymphokine activz.e~
20 killer (LA~) ~cells, a source of IL-2 (Allegretta er 21. ~
Sc~ence 247:~718-721~ gO)). Later passages were aaapted
to weekly sti~ulation with allogeneic P~l~;s a~d anti-CD3
antibody (Coulter Immunoloav, ~ialeah, ~IJ ) in place of
syno~ial cell~monolayers.
~Cytotoxicity assays-were conducted as described
Steoman et al.,~:Immu ol. Meth-.:119:291-294 (lg89~r
using ~5S-labeled
:: .synovial mo~olayer~cells or E~V trans~ormed B cell~ as
targets. ~Target cells were labeled o~ernight,
trypsinized (adherent cells only), washed and plated at
2000 cells per~ well in 96-well round bottom microtiter
plates. ~ cells~were activated~with allogenic PBLs and
- anti-CD3 antibody in medium containing LAK supernatant
: for 7 days~prior:~to the assay and added to the targets at
the indicated effector:~arget ratios. Cultures were
incubated overnight at 37C, centrifuged at 300xg for 2
AMENDED SHEE~
~2G~;~&
s 2nc _2C` C2C='~v~ V ' -. ~ C- =he suDe~..zl2nt
~U2~ -' ec.
T~e ~e~ _en~ s?ec _ _ lvs~s was calcuiz~ed
~ela_-ve to ce-e~ ent-lvsea ~arge~s as desc_i3ea ~n
Townsen~ et 2 ' ., Cell A 4 g~4_gç8 ~lg86)~ e-__c~
e-c~ ~r; ~e ~ .ee. ~. ef ec-o-:_2rge_ .ztios cf 1.O,
^~.5 c~- _.0, _~e pe~-e~.~ vsis o_ svnovial adhelenl cells
~v ~08 . 8 m c~ s was 2~D-ox:m2~eiv 4%, 14~ and 33~,
_es3ec_1ve~v. 3v comDz_-ison, 21 .he same e~feclo_:_arge~
lC ~~ _s, 1008.C ~ cells hzc ~o aemo~str~ted e~ . on EBV-
~ans ~~mea 3 cell targe.s. S~ ilarly, at the same
. e__ec~ .ar~e_ -Z=lOS, ~;~e ?ercen~ lvsis c~ svnovLa
~cne _n_ ce~ls ~y M53 cells was zD~roxlmately 0%, 0~ a~
3~ spec~_vely. :
T ce~ls were ~sol2tec '~rom the synovi2l tis~ue
;o~ Da.:e~t 1008 by co-cultivation:with svno~ial cPll
: monol~vers. Slnce the ant-gens recognized by ~athogenic
T cel 1 s in R~ 2re unknown, synovial ce11 monolayers were
used as sti~ulat~ors of~ in vitro syno~ial T cell growch.
2:D: ~ The: relevant targe~ cells we~e :assu~ed to be pxeseni in a
: bulk adherent~ cell culture from diseased synovium. Of
l92 mic-owell~co-cultures plated,: 7 were posLtive ~or T
cell: srowth~ wlthln :10-14 davs ~ T~cells~were expanded a~d
maint2i~ed in vitro by alternately stimulating :with
2~ ~ynovi~l ce11 mon~olayers and medium containing LAK
supernata~t~ Flow cyto~et~y re~ealed that each of these
: seven cultures was 100% CD4+. One culture designated
08.8, grew especially ~igorously and, as as~essed
microscopLcally~promoted the destruction of the mo~olayer
lls.~ C~ assays confirmed the cytotoxicity of 1008~2
~: for these monolayer targets as:`discussed abo~e.
Cytolysis was ~specific ~or syno~ial cell targets, as no
lysis of autologous Epstein~Barr ~iru~-transformed B
cells was demons~rable. Neither of the targets was lysed
: 35 by ? CD4~,~myelin basic protein-reacti~e human CTL clone,
~: :
:~:
AtllENûED SHEFI
2 1 ~ ~S ~
M53, exc~c -.c t~e ~ossi_ l~,v .~2. tne monoiaver -ells
were susce?.~_1e ~o lvsis DV 2n~ 2Ct' Vzrec ~ C~-' -' . In
-e?eZreG 2SS2VS Wit;~ iOOa . 8, s~ec --ic lvsis neve~
exceeaea 30~ , sugges. ~5 .;~at the -elevan~ svnovial
- _2rye_ cell c-mD-~ses aD~~oxl~ateiv t;~at ~rooo-_ on of
.he total monolave~ c~ltu=e, wnic:~ basea on ~or~;~oloov is
a mlx~ure OL mu1t~Dle cell tv~es.
~he m cell ~ece ~or (TC~) B-cnal~ cer.e c.
1008.8 was am~li_ied bv t;~e Dolvmerase cha~n reac=~on
(PCR)~ as ~esc=~bed in Mullis el 21., Me~h. ~-.z~m.
155~:~35-3~0 (1~87):, i Re~--or-~e~ he_e~ ~ and
: its DNA seauenc2 was dete ~lned. ~mpll icztion was
: accom~iished:using a consensus V~ primer (VBcons),
degenera~e 16 nuc~eotide ?rimer (n=2~6) wni~h is
15: homoiogous~at c11 16 residues wlth 78%, and at 1~ o. 16
residues:witn:~98~, or known hu~an TCR V~ gen~s, wh~ch
ha~e~been com~l1ed DV~Kimura et ~al., ~~.-T~mo7. 17:37~-383
; (1987). This prLmer~was aesi~ned to amolify ~--ha~n
rearrangements~;~containlng~2ny of~the k~own VB genes, thus
20~ Q11Owlng:~ana1ysls~of;T cell clones for which a ~r~or~
knowle~ge~of~VB gene:~usage is unavaila`ble. Se~uencinq or
the:V~con~s-ampliIied B-chain aene of 1008.8 re~ealed a
s~ingle VB17~-J~ rearrangemen.~;as shown in Table 16.
Su~se~uently,~ the remaining six Gultures derived from
2~5~ :pa~ie~t~100a~:were~ana1yzed~by;PCR amplification using a
VB17-specific:~primer shown~ in Table 13. TCR genes from
three ~f those six~were amD1i~iable, indicating that, of
the~7:~T: cell cultures obtained:`from the s~no~lum of this
. patient, 4 had~:rearra~ged and expressed the V~17 gene.
3~0 C._ V~7 ~ cells~are_enriched~amona a~tivated T cells in
~A svno~ium
: Synovial tissue~was digested with agitation for
4 hrs at 37C in~RPMI-1640 :and 10% fetal bo~ine serum
(FBS) containing 4 mg/ml collagen se (Worthingt~n
Biochemicals, Freehold, NJ~ and 0~15 mq~ml DNAse (Siqma
'
AMENr~F~) ~HF~T
WO93/12$14 PCT/US92/11159
?~C~J~ 3 ~6
Chemlcal, St. Louis, MO). Digests were passed through an
80-mesh screen and single cells were collected from the
interface of Ficoll density gradients, washed and
incubated at 106/ml for 30 min at 0C with 5 ~g/ml control :
mouYe IgG (Coulter) in PBS containing 2~ BSA. Cells were
washed 3X and incubated for 30 min at 0C with magnetic
beads con jugated to goat anti-mG~.lse IgG ( Advanced
Magnetics, Cambridge, MA). After ~agnetic removal of the
beads, the remaining cells were incubated 30 minutes at
0C with 5 ~g/ml mou e anti-human IL-2R (Coulter), washed
and selected with magnetic beads as above. Cell-coated
; ~ beads from the mIgG: preadsorption and the IL-2R antibody
selection were;washed 3X,~Lmmediately re~uspended in
~:M ~ :acidified-guanidinium-pheno~-chloroform and RNA was
.
: 15 prepared as~ described in Chomczyn ki et al., Anal.
~ Biochem.~162:156-159 ! l98? ) . :
: . RNAs~:from magnetic bea~ preparations were
;reverse transcribed~aAd ampli~ied for 20 cycles in stage
reactions with the~Y~cons~and CAext primers. One ~l of
~each reaction~was reamplified for 20 cycles in indi~idual
stage;II reactions containing the~int primer in
: ;:conj:unction with~the;VBl7, V~8,~Y~12 and 5'CB primers.
Aliquots~ of ~each~:~reaction were diluted i~ 20X SSC,
:denatured:~by~boiling:and chilled in an ice slurry.
2~5 Samples were~:loaded~onto nitrocellulose membranes,
hybridized to:~a~:human TCR ~-chain constant règion probe
: and washèd~with:0.1X:~SSC:,: 0.1%~SDS at 56C. Bound
: : radioacti~lty~was~:quantified by liquid scintillation
spectroscopy. The amounts~ of product produced by 40
: 30: total:~cycles:; with;~ each of: the respectiYe prLmer
combinations falls in the linear portion of a product
: versus cycle number~ quantification curve. Values shown
Table 17 b:elow reflect the relative increa~e or
: :decrease for:the:~specific ~Bs in:the IL2-R+ versus mIgG
: 35 controls Galaulated aocording to the formula:
:
WO 93~12~14 PCr/U~;92/1 1 159
2 ~ 2 S ~
~7 .
:
specif ic VB cpms t IL2-R+l / CB cpms ~ II.?-R~ )
specific VJ3 cpms tmIgG) / CB cpms (mIgG).
TABLE 1 7
Ratio I~-2R+
Sample mIaG _
ExDeriment # VB17 VJ3~ VJ312
: 1012
1~88 0.42 1.~5
2 -1.~0 0.39 0.72
; ~ 10 3 2.11 0.48 ~.64
X~ +S.D. 1.86 +0.25 0.43 ~0.04 0.900 ~0~38
~ : 1013
: ~ ~: 1 2.54 0~.49 2~
2 3~65 0.87 0,~7
1;5~ 3 4.29 2.07 ~.96
X-: ~S.D. 3.49 ~0.:8~ 1.14 0.82 0.91 ~0.06
~ ,
1 0 1 4
4 . 7 0 ~ 0 .. 1 7 N . D ,.
2 : 1 0 6 8 : ~ 0 . 1 0 1 . 5 0 .
: 3 ~ 1092 : 0.~9 0O44
4 : 2.34 0.07 0.5~
: X-- + S ., D . ~ 2 . 6 6 + 1 . 3 8 0 . l G ~ 0 . 0 4 ~0 . 8 3 0 . 5 8
1015
3 ~ 4 0 0 ~ 2 0 0 r 8 5
: 25 2 :2.85 0.47 1.82
X- +S.D,. 3.12 +0.50 0.38 +0.15 1~33 ~0.6B
Next,~ the~ presence of :~J317 T cells in the
ynovial ti~ue of other RA patient was: :det~rm~ned.
Sirlce the rh~umatoid:~ynovium ct)ntains a~mixture of
~ ,
:: 30 acti~ed a~d non-activated ~ cells, the: activated T
cells were identi~ied as the m~st rele~ant f or the
WO93/12~14 , PCT/US92/11159
~ 5 58
initiation and perpetuation of the disea~e pathogenesis.
Thus, activated T cells from single-cell suspension~ of
synovial tissue were selected using magnetic beads and
antibodies reactive with the human interleukin-2 receptor
5 ( IL-2R) . Cell suspensions from each patient were
pr~treated with an isotype-matchf~d mouse IgG ( mIgG ~ and
magnetic beads to control for non-specific ad~orption.
R~As were directly extracted from cells in the IL-2R+ and
contrsl samples without ln vitro culture and, therefore,
are expected to accurately reflect T cell distributions
- in synovial ti~sue at the tLme o~ surgical removal.
:The initial PCR amplifications of the~e
magnetic bead RNA preparations revealed greatex amounts
o~ V~17 PCR product in the IL2-R+ samples than in the
correspondîng mIgG Gontrols. The presence ~f TCR mR~A in
~; the mIgG s~mples indicates that T cells non pecifically
;~ adhered to the:magnetic beads. The apparent increa~e in
he IL2-R+ sample suggests~that th~ activated T cell
compartment contained m~re VBl7 T cells than the
20: unselected synovial T cell compartment. Thus, a
~ : quantitative PCR analysis was used to formally examine
; the relative:proportions of V~17 T cells in the IL-2R~
and mIgG control samples (Table l7~). Magnetic bead R~5
were reve~se transcribed, preamplified with V~con and
C~ext and reamplified in separate reactions with a
: constant region~prLmer (C~intl and each of the V~-
: specific prime}s~ 17, V~8 and V~12. The second stage
amplification was also performed with two CB primers
(5:'CB and C~int) in order to estLmate the t~tal ~-chain
30 present in each ~sample and to provide benchmarks for
normalizing the results of ~pecific VA guantification in
the respecti~e: IL-2R+ and control~ ~ample pairs. The
proportion o~ ~B17 DNA, relativ~ to tc~al C~3, waB
increa~ed iIl the IL-2R~ æamples o~er that f ound in the
~5 mIgG control ~amples for each of ~he 5 patiPnts examined.
This increa~e was observed in multiple analy3es and the
W093/12814 2 1 2 ~3fi !,3 PCT/US9Z/ I 1159
means are shown in Table 17. Enrichment was not a
product of the isolation procedure, since the quantity of
V~8 or V~12 DNA amplified was nG~ significantly increased
in the I~--2R+ fraction of any of the patients. Thus,
activated T cells in the rheumatoid synovium do not
repre~ent a cross sectio~ of all possible VB fami1ies,
but preferentia11y contain V~17 T cells, and po~sib1y
other V~ fami1ies which were ~ot quantified in this
ana1ysis.
D. Synovial_V~17 T cel1s disp1ay limited hetero~eneity
RNAs were revPrse transcribed and amplified
with the V~cons and C~ext prLmers in 20 cycle stage I
reactions and with the CBint and Y~cons (1008.8) or V~8,
V~12, and V~17 primers ~m~gnetic bead RNA preparations)
in 35 cycle stage II:reactionx. Doub1e stranded reaction
: : products were e1~ctrophoresed in 2% Nu-Siev~ agaro~e
;~ gels. After purification from gel ~1ices with Gene Clean
(Bio 101, San Diego, CA), samples were base denatured and
eith~ directly se~uenced or cloned int~ plasm1d for
sequencing of muItip1e indep~ndent rearrangement~ In
,
all ca~es, samp1es were sequenced from the C~eq primer
~Figure 2) with T7 polymerase tSe~uenasP, United States
Bio~hemicals, Cleveland, 08).
~17 rearrangements pxe~ent in the IL-2R~ RNAs
; 25 were amplified from the Vflcons and C~ext preamp1ification
with the Y~17 and CBint prLmer pair and the reaction
~ products se~ue~ced. PCR produ~ts from patients 1014 nd
,: ~ 1015 were directly sequenced and the result~ obtained
were consistent with the presence o~ a ~ingle V~17: - ; 30 r~arrangement in~each of the~e amplified ~amples (Table
17). The P~R product of patient 1~13 was cloned into
pla~mid and the thirteen isolate~ that were ~equ~nced
contai~ed identical VB17 r arrangements. Sequencing of
plasmid clones from patient 1012 revealed the presence of
two dominant rearrangements (5 i~olates of ea~h) and a
, ~ . . . _ .
2126~
si~cie ~soi2=e c_ 2no~e=. T~s, =ne ~-Bl/ -e~e~
.he ~ svnov ~ s G_ ~ e~ ;ne.e-~ ene~tv, i~c_ce_ ve
c_ ~`~n21 c_ ol ~__iz..21 eY._aIlsiOn C - V.r~17 ~ cells -7.
v~vc. T~is s s. cor..=2s= ._ _he ~-~78 ana V~12
-e?er-e~res, _~ hese se~e svnovial o~eDatzt-ons, wnic~
snowe~ sisn~_ -ar= hete_ocene~.y. None OI .he ~C~-
am~i~_ied V~8 zn_ V~12 szm?~es ar,zlvzec we~e di-ec_}v
secuence2ble 2nc -lasmic c onina c V~8 rea-_ance~e~._s
t~om ~a~ent '~12 -evealea 4 clf~e~en. seauences ~n _
iO clones analyze~.
V~17 --a-ranceme~=s in ~Ls f_om ~at en~ 012
also reveaie~ s=e2~e- c~ ve_si~y ~han t;n~t seen ~ he
svnovial T~ sampie. ~N~ l-om 2 3 aav c~lt~re o~
P~A/P.M~ slLmu~a_ec 1012 ~3_s was amDli~-lea with :~e V~17
; 15 or~me=, aâ ~or ~:~e synovia s2mDlet and the proa~c_s
cloned ~n~o plasm_d. Nine QiI rerent rezr~angemer.,s, ~one
of wh~ch corresponaed to t;~ose ~resent in the 1~12
syno~-um, were~^ound in the 10 clones that were
sequenced. Thus, .he res~r cted heterogeneity OI VB17
rearra~gements~in the~activated~syno~ial T cell
popuiatlon of ~a~l2nt 1017, as well as the other ~atients
examined,~ elv results, not lrom random T cell
tra~fic~ing, but f-om the selectlYe expansio~ of those
VB17-~earing~T cells ln the diseased tissue.
~ EXAMPLE XII
A. Detection o' ~ cr ~ ~ _novial T
cells us~n~ VB s~ecific ~CR-amDl _ication
T cell receptor r~-chain ~enes were amplified in
two-stage reactions with in~i~idual V~-specific ~rLmers
30 as described in WucherIpennig et al., Sclence, 248:1016-
1019 (1990)~ and nested
C~ prlmerg. RN~s were reverse txanscriked (1 hour, 42C)
~ with the CBext primer ~40 pmol) in 12 ~1 of reaction
,:
~IENDED StlEE~
WO93/12814 ~ 2 ~ ~ y ~ PCT/VS92/11159
61
buffer ~art et al., supra). Reactions were diluted with
a master mix (8 yl) containing nucleotides, reaction
buffer minus MgCl2 (final Mg+2 concentration = 3.6 mM) and
Taq polymerase, apportioned among l9 tubes containing the
individual V~ prLmers. Samples were denatured (15
minutes, 95C) and 20 cycles of PCR were conducted. ~ach
cycle con~isted of a one mlnute denaturation at g5C, a
two minut~ annealing step and a two mlnute extension at
72C. The first two cycles were annealed at 37C and
45C and the remainder at 50C. One microliter aliquots
of these Stag~ I reactions were added to l00 yl Stage II
amplification reactions (Cetus Gene-Amp Kit) co~taining
the CBint prLmer (l00 pmol) and l00 pmol of the Y~
prLmers u~ed in the corresponding preampli~ications.
Stage II amplifications were conducted for 20 cycles with
:a 50:C annealing temperature a~d without the 37~ ~nd
4SC rampi~g. Five:yl of each reaction wa
electrophoresed in:2%~agarose gels,~transferr~d to
nitrocellu1ORe:~and h~bridized to a 32P-labeled ~-chain
constant region probe. Blot~ were expo~ed to X-ray film
: and scored~ for the presence or absence of V~-specific
amplification~
B~. V~17, V~14~,~ V~9 and VB3 trans~:~ripts are common amonq
activated:synovial T cells
::: ~ ~ ;To ensure that the prevale~ce of V~17
rearrangements~ the;earlier:studies did ~ot result from
an amplification~bias of:the V~16mer pxLmer, and to
~ : assess:the:pre~ence of other V~ genes in the syno~iDl
,:~ TCR trans~ript :in~the IL2-R+ ~ampIes wer~ analyzed with
:~ ~ O a panel o~;l9~PCR~prLmers, specific for known human V~
gene~famil:ies~(~Table 18). The ~cons pr~mer of Example
: XI is eguivalent~to:~the VBl6mer prImer. The ~umbex of V~
genes detectabl:e in~hese samples was variablsr ra~ging
from two to twelve. V~17 was found in f~ur o~ the five
patients, confirming the previous analyses using the
V~16mer prLmer. V~14 transcripts also were found in four
:~ ~
.
~ : .
212t~'8
62
c_ ~:.e _ive ?2.ie~~s 2nG ~3 cnc v~9 _=ansc_~_s we~
ce.ec_~ble i~ ree c~ _he ve sam~es. m~5, ~ c~1~5
~e~ .- these _ vr, ~oi~?e~iaes mav 2 SO c~n.~ e
svnc~ a`~ ~n~~.~mæ~io~.
- m~3L~ 18
~n21vsis O~ _T- -2~ Svnovi2l ~ Cell s wi_ .
~nc~vicua~ s~e~- c ?-~e_s
V~ F~mllies
101 2 ~ ~ ~ 6 7 8 10 l1 '2 ~ 9 20
012
`
1 0 1
13i
101~ ~ T - - - - + - - - - - +
;1020 ~ T - - - + -
; : ; EX~LE XIII
Vac~ina~ ~aalnst EPE~with:~a CDR4 ~eotide o~ V~8.?
Rats were~Lmmunized with 100 ~g of a svnthetic
; 20 pe~ilde c~ntaini~g th~e seauence VPNGY~VS~PSQGDFFLTL found
n the~lourth~hypervaria~le or CDR4 region of the rat TCR
B chain id:entified as:V~8.2. The;peptide, dissol~ed in
: ; sallne~, was:~e~ lsi~ied in an e~ual volume of comp~ete
Freund's adjuvant (CFA);containing 10 mg~ul of
2~ mycobacterial~tuberculosis.::The~a~Lmals were challenged
30 days later with 50:yg of gui~ea pig myelin basic
:~ protein in CFA. The CDR4 peptide vacclnation resulted in
a ~Pàuced incidence of disease as::well as a reduced
se~erity as:~measured both clinically and histologically
30 as shown in Table 19. The histology was:p rformed
e3sentially ag~:described in ~ughes & Powell, J.
NeuroDath Ex~._Neurol. 43:154 (1984)~ W~O~-~5
_~eorDer~e~ he c~in~ r~*~4~co-
AMENDED SHEFr
.
W093/12814 `~ PCI'/US92/11~59
63
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WO93/12$14 PCT/U$92/1115s
p~E XIV
Cross-Specie~ Efficacv ~f Peptides
Synthetic peptides representing amino acid
sequences from the CDR4 region of both rat B~8.2 T cells
and human V~17 T cells can protect rats from the
development of EAE. The rat V~8.2 CDR4 peptide has the
~equence VPNGYKVSRPSQGDPFLTL (residues 61-79) and the
human V~l7 CDR4 peptide has the s quence ~RRES~PLTVT
(residu 68-79)c
~: :
0 Lewis rats were Lmmunized with emulsions
: containing equal volumes of pep~ide in saline and of IFA
intradermally in 0.1: ml doses divided equally between the
~ two hind footpads (O.05 ml/footpad). Each anLmal of
:~ ~ Groups A a~d B~received lOO~g of pPptide, Group C, 50 ~g
of pep~ide, G~oup D received IF~ alon~ and Group E, ~o
treatment. ~fter 30 days, each anLmal was challenged
:with 50 ~g guinea:~pig;myelin:basic protein ~GP-MBP) in
Baline emulsified with-an~equal volume of complete
Freund's~ adjuvant containi~g mycobacteria tuberculosis
20~ 0 mg/ml~.~ Each animal~received~O.l ml~in two dose~
divided ~equally~:between the~front~:footpads l0.05
~l/footpad)~:a~nd~observed for~:clinlcal signs of disease
graded:on a~:3:point Bcale con6isting of l) loss of motor
control of:the tail,::2) hind leg weakness and 3) hind leg
paralysis.:
Table 2 0 shows that animals receiving eikher
human CDR4 (~Group A) or rat CDR4 ( Group B ) had mild or
nonclinlcal s~i;gnB of disease a~ter ~hallenge whereas
anLmals:r~ceiving control peptide (C), IFA (D) or no
30: treatment~(E~ had a no ~al diseaBe~course. The resul~s
establish that :CD~4 peptides can prev2nt EAE and that a
peptide with the:se~uence ~XRESFPLTVT rom human VBl7 can
~; : protect f rom autoimmuDe disease in a different species
~: (rat).
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Ta~le 2l shows that animals receiving hu~an
: CDR4 peptides (Group A) from V~17 or V~l4 had mild or no
disease and 3 of 5 anLmals receiving a similar V~3
peptide had~no disease. In contrast, anLmals receiving
5 IFA ( Group D ) or no pretreatment (Group E) had a normal
~; disease cource.~ These resuIts establish that the human
~;: VBl4 CDR4 peptide having the sequence KEKRNFPLILE can
also protect from autoLmmune disease in a different
species (rat) and that a human V¢¢B3 CDR4 peptide having
10 the ~equ~nce ~EKKERFSLILE also has a protective effect.
. ~
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W093/12814 2 ~ 2 ~ 3 PCT/US92/11159
Although the invention has been described with
reference to the presently-preferred embodiment, it
should be understood that various modifications can be
made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the
following claims.
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