CA2136973A1 - Recombinant timothy grass pollen allergen ph1 pii - Google Patents
Recombinant timothy grass pollen allergen ph1 piiInfo
- Publication number
- CA2136973A1 CA2136973A1 CA002136973A CA2136973A CA2136973A1 CA 2136973 A1 CA2136973 A1 CA 2136973A1 CA 002136973 A CA002136973 A CA 002136973A CA 2136973 A CA2136973 A CA 2136973A CA 2136973 A1 CA2136973 A1 CA 2136973A1
- Authority
- CA
- Canada
- Prior art keywords
- recombinant
- phi
- polypeptide
- allergen
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
(54) Title: RECOMBINANT TIMOTHY GRASS POLLEN ALLERGEN Phi p II
(57) Abstract:
A recombinant DNA molecule codes for a peptide with the antigenicity of timothy grass pollen allergens Phi p II. The amino acid sequence (5) and the most important B coil and T cell epitopes of the molecule are derived therefrom. The recombinant Phi p II allergies is expressed in Escherichia coli and binds serum IgE of more than 60 % of all those allergic to grass pollen and many therefore be used in the same way as the naturally occurring Phi p II for processes based on antigen-antibody interaction, mediator release and T cell reactivity.
Phi p II
(57) Abstract:
A recombinant DNA molecule codes for a peptide with the antigenicity of timothy grass pollen allergens Phi p II. The amino acid sequence (5) and the most important B coil and T cell epitopes of the molecule are derived therefrom. The recombinant Phi p II allergies is expressed in Escherichia coli and binds serum IgE of more than 60 % of all those allergic to grass pollen and many therefore be used in the same way as the naturally occurring Phi p II for processes based on antigen-antibody interaction, mediator release and T cell reactivity.
Phi p II
Description
SENT BY:PENNIE ~ EOMONDS ~ 2e-94 ; 3'45PM ; PENNIE 8 EDMCNDS~ PER!EY-ROBERTSON;# 3 .. .. . .. . . . . . .. .. . ... . .. . . . . . . ..
~ 2.~.30~97~
WO 9~ J23035 P~/A'r94f O0039~ :
~bln~nt ~y~
ara~ ~9 th~ t ~ear~t ~ t llorols~
o~currlng durln~ th~ ~u.. . ~ thul 20~ o~ lv~ 'co l~ort~t or~ n ~lorgen- Lnclud- Oro~ I t1~, ~up V ~2~, Cro~p dl-oo~d to ~ ~r-rs pollo gu~ ~7~ 3)- ~
allorg r~o ~$~ nd ~o l~ologLe-lly or ~truo~ ,ly olo~ly rol-tot ~ poll~ o~ a~f-r-At gr~ ~lo~, ~d r-l~t-t ~11~ o~ o~o group ~h~w oro~- rewtlv~ty wlth ~tl~ 50 ~r, ~1~ D~ th~ .~ ~ ~ L~ tod b~r r~bl~t ~hnl~
~n~ ps~ ooll ~9~. ~ny o~ t~ ~llor~n~ ~pr-~ ln E oolJ how ~ro~ r to th~- o~ tu~ rot L~, ~na c~
th-~nlor- b us d ~Dr ~ho dl~o-L~ u~d th-r~y Or llor~l~ dL~
(10, 11, 11). Jl~, do~crl~d ~os t~ ~ t tl~ 1- ~ol oul~
ohnru~ elon o~ o~plolt oD~A rhloh aod for Pbl p I~, ~nd th lon o~ ot-Ln ln ~. ooll. ~nt~l no~ t- ~ca~bln~nt ~rou~ rI~III or~u ~ ll~n q~ lhbl~ d, ~o o n ~ ~:
o_n ~rom th ~pl~u, Lt o~n ~ u~ llho t~ n~tu~ ro~ln ~or : -b--~ on ~ ntl~o~y ll~t~r~tlol~, uoh ~e ~ u--t ~o~ :
o ~lol~ ~OO-U~ UJ- .11 S-~ll ~Lto~o r ~nt ln tl~
s~ln-nt ~o~oaul-, ln th- n tur-1 ~l-cul- rurt~or ~ th r~o~blsu~t ~1~1 p 11 1~ eult~1~ for proo ~ whloh s uult ln ~ur bl- ~dL-to~ r-l--~e. ~ th~s-~tlo u~- Or r~o~ bl p I~, b-od on ~n d~ ~t on L~u~or gul~tory ~roo ~ , ~e~n-~At~body lnt-r-ctlon, S-o-11 r~tlvlty ~d ~odl~tor re1-~u~, L~ th con-4uon4-Or t~ ot~otur~ u~d blolo~lo~l o~l1 rlty ~tw n th- ~tur-1 ~d th-r-oolobln~nt ~-~ tr-~e~t l~ Atlon ~1~- ~ ca~pl~ oD~la ~hloh cod-- rOr ~ bl~t ~hl p II ~ll~q~ 01~ th b l- or tho ~no ~ald ~u~ ~duo d fro~ eh~ cD~ c~ d ~c-ll ~lto~ o~
tb- ~hl p II nll-rg~- ~ dot ~l~d Th r-oo bl~nt ~1 p ~S
~llor~n n- pr~d Ln ~ ~oll ~ h~- ~rop rtl~ r to tho~- o~
~urnl Oroup ~ S~ gr~ oll n 110r~no ro~ ~hl- lt ~ollow~ th-t ~ho r~ombln~nt ~hl p lI ~ rg~n, ~Llko th- n~tus-l Group II/III
ll~n-, o~n b- u--q ~oo pr~ o- ba~d on nt~n--ntl~o~y 13to~ot~on, ntlg~n~ nt T~-11 ~Lon or ~tLg~n~nd ~t -~
- ~: .
SENT BY:PENNIE ~ EDMONDS ~ 28-94 , 3:5~PM ; PENNIE ~ EDMONDS` PERLEY-ROBERTSON:# 7 . .
. _ .
Z1!~ 7~
m~l~or rdo~-e, wh~r~, ~ow~vor~ the r~oa~lbLn~nt Ll~r~n~ ha~ th~
~tdltlonal aav~nta~ of g~nt~r ~urlty and hlqhor ~ lol'cy.
4toril-1 ~4 thod~ ~
: ~
~rl~othy qr4~1~ pl~ar, ~All~rgon ~ ~nq~lhol~, Bw~n), whloh h~,d ~e~n ln ~lgated ~or E~urity ~y ~ n~ o~ llght ~loro~co~y n~ l ctro~
~ro~o~y, ~aa ~ood ~or tho l~olat~n of ~oly d-nyl~tod lINA ~13). ODI~
~ynthanl~ ua- oarr ~ d out w~tb oll~ nd ~ ri ~ r~; tho end~ o~ ~ -th oD~A ~ro dl~nntod wlth ~4 poly~4r~- and provldad wlth ~ooXI
l~n~or~ Th~ l~nb r~d oD~A wa- llg t-d lnto do~ho-~horylatod l~bd~
gtll r~b nd ~ d A oDNA g~a lLbrary o~ ~DO~OOO indYp~n4~t clono~ w~- obtaln-d ~
: . ~ -: ' na~y~l-~ ,~
Iot ~r~-n~n~ of tho tl~othy ~ra~- Fol~on ~D~A o-n lLbrary w o carsid out ~ d-ocr~b d ~y ~rolt-n-t-r t al ~14) S~-bin413g olo~-~ w~r-~rl~ d and gha~a-DNa w~ ~r-p r-d ~rc~ th-~- clon-~ ) 3y ~oanr of ~pn I ~n~ ~o I cut- t~o D~A ~r 4m~t~ u~r- obtaln-~ whloh oont-lned hoth ~ortlon~ o~ th oo~pl t- ~h~ ~ IS OD~A ~d ~I-nkln~ lambd- Ctll ~-~u Do-o~ Th r--~ltlng ~p I/~-c~ nd B~cl D~A ~r~gm nt- w-ro -~
rubclo~ d lnto th pl~-mld p~C 18~ ~nd tr~n~ormed ~nto ~ coli ~
~lu~ 9ult-bl- clonn~ w~r ld ~tlfled by re~trlotlon ~n~lyc$~ ~nd bcth ~tr~nd~ wYr- r-qy-nc-d by mo~n- o~ l~mbG~ ~t11 forw~rd oqu nclng prlm r ~2~ r) 4n~ l rbd- gtll r~v~r~ qu~ncln4 prlm r ~22-m~r~, Clo~t-ch ~bor-to~o~, P~lo Alto~ ~A, ~nd by m~ nG o~ H13 ~Cl~ forw rd ~nd ro~ r~ rl~ r-, ~o-hrlngor-~n~h-im, ~ rm~ny, ~ccordLng to ~n4~r 16~. -' ' ' ~ RNA_L~o~th4~ blot~
10 ocO o~ tot~ A fro~ poll-~ ol tl~othy yr~-~ rPhl-un pr ton~eJ nd ry~ g~ r~ollUS P-r-n~-) w~ p~r~t-d by den~turln~ gel elec~o-~hor~ nd blotte~ onto nlt~oc-11ulo~e ~17~ ~o l~ol~t- the ~DNA
codlng ~or ~bl p II, the oorse~pon~lng D~A ln--rt w~ u~plifl d by ~CR, ~tnrtlnq ~rom r-oo~bln nt ph y~ 5 pioo~ol-- o~ primer ~ bd- gtll ;~
~osw ¢d -quonciAq ~ri~ r l~2-~er~ ~na la~b~4 ~tll r~ o ~eqY-noln~
S ' ~' ., ; .~
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SENT BY:PENNIE ~ EDM~NDS ;11-23-94: 3:51PM; PENNIE ~ EDMONDS~ PERLE~'-R3~ERTS~N;# ~
-- ~ .
7~
prl~r ~2~r~, Clont~h ~oro,tor~o-, ~3A~, w~o uond ln oaoh cao~.
Th~ PCR pra~lat WB~ l~ to 14 6~7Qro~ ~ol ~.nd th~ b-nd~ w~r nlute~d ~y ~an~ of ~ DJAII loll ~ch~ngor p~r ~ . T2~ D~IA oil~taln~d w~ ~
l~l-d by r~ndara ~ 13). Prohybrldi~lon an~ h~brldl~-'clon woro carrlod o~t l~y ot~d ~ho~o ~15). ~h~ blot~ ~re .t~.hea ~Lth 3.0 ~ 88C ~20 x ~sC ~ 3 11 ~aCl, O.g M ~ altrato, p~ 7.0), O.lt 5D8 (~odl~u~ dod-ayl oul~llto~ 1.5 ssc, 0.1~ t nd ~ lly wlth 0.75~ sW~
0,10 8D~ at 50'~, nd th~ utor~ r pb~d t~f~ ~, A~rd~
L~don, m~ .
l~y ~Qn~ o~ Igll ~or~nq ~ t-- oD~ oloo-- ~-o c~t~ w~ch ~or ~ Phl ,1~ llo~n. Sho 1Y~Og~A1C J!. coll l~t;ral~ Y 108q var ~l~td w1th r~coo~l~nt l~bd~ g~ll ~d t~o p-pl-Gtoeld~e ~u~
prot~ln w~.~ obt~lno~ fro~ tho ~ch ~ao~. Tho b~tch w~ rat~
oSrop~o~n~ y ln n 7.5~ yu:rylu~d~ and blott~ onto ~rocollulooe ~Z2). Th- ~u~lon ~r~tolD w~ toot~ by ~s o,~
Igl~ o~ lont- ~llorgLo to ~ n ~ n l,odL~ l d r~lt antlhu~n Ig8 ne~body ~Ph~ol~, ~aala, 8~).
Th- att-c2~ Flgur ~ ~orvo to lllwtr-t- thO u-~billty o~ tl~
r~a~ AZ p rl all~ ~ lor ~ro~-~o~ d or~ ~tlgon-~t~
lnt-ra~tLon, ~,ntl~n-d4~n~nt m~tor rQlo~, ~d nt~n-~ndant c-~.lul~ r~ot~vL~y. ~9. 1 ~ th~ A o~ t~o r ~rb~nt Phl p rI .
llg rl ~d tho l~tt-r'~ cld so~ duood ~r~ ~ld DNlL
Th- oa~lot- cD~ ~u~- o~ tl~O PAl p II tl~ lo oho~ b~--nonoodln~ o~-nc- -o ~ound ~t th 5~ ~nd Of th ~DNa, whloh 1-~ollo~d ~y 7~-bn~- lo~,dor ~ na-, ~hloh oodoo ~or th~ lgnnl ~tld undll~d L~ tho rl~ ~23). Sh ~lno ~c~d ooquonoo of th~
~tur proto~ ~ d duc-d rroD ba~ ~8 on, ~ b gln- ~lth v~l~. ~ ~
Th~ atop oodo~ ~AO, whlob b~-~lu Or~ tbo codlns~ t tho 3~ ond, ~ ~;
~ot~ t~lc~c. Th 3 ncodlng ~-~o~ -nd- lr tho ~oly A
rLg. 2 oho~- th~ ty o~ tl~ ~lno ~cl~ o- o~ th- . -~conbln~nt Phl p 1~ ~llorg~n to oth-r Oroup II/IIT cr~o~ n ;~
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SENT BY:PENNIE J ED~ONDS ~ 28-94 ; 3:51PM ; PENNIE ~ EDMONDS~ PERLEY-ROBERTSON;# 3 2~ ,g7~ ~
~ . 3 preo~tE th- d~-criptl~n o~ ll ~ltope9 of tho rooombl~ t PAI p 1~ ollor~-n~ ~d Flg. 4 th~t o~ th~ S-CQ11 opltOp~-~
Illg. 5 ~ tho ro~ot~rlty o~ tho r~oaablnu~t Phl ,p Il ~ rg n with ~orum ~g~ o~ ~ti~ nll~ to ~ ~oo~ln~t Pbl p I~ ~
r~ c~ ~ ~ l~to~ u~lon ~Iro~ 2f~o~nlo J. ool~ - .
Y Ioa9 by ~h~g ~ootl~ ~ Im~u~lo~ Ylth l~TO. ~h~ oll ~rotolno; ~vhlo~ oo~t~ln t~ r~o~ PhZ p Tl, w ~ el-~tro~
~hor-tlcnllr ~-~r~ted ~d tr~ ~rd o~to nJtroucll~loro ~oul 1 w ~ I~ o~ ~ O~Ou~ SI~S21-r- otl~e patl~tl ~llo ~ ~
2 ana 3 th d t~l w~- ~on- ulth I~ o~ all rglc ~tLent- ~rho ~ o r-~tlon wLth Cro~ II/~lI ~11~9~ ~ 4 ~ ~ co~trol ~r~nt wltb ~ o~ ~ non ll~lo ~ont~o~ ~r-o~.
F~g 6 ~re~nt- ~ t bl- whloh ll~to ~ ~c~t4- ~ gr~- ~ollon~
~llorglo ~t~-nt~ wlth ~g~ rowtl~lty wlth ~clfle ~r ~o ~ollor~
~llo~no ~ fr~u~y of r~tl~ltY of gr~s~ ~ollen ~ rglo pa~clonto wlth ~ r nt 5~r~ rg~ ~ ~lwwn Th~ flg~r r~-~t pprox~t- valu~a obt~ d by t-ct~ w~ tur~
r o~ln~nt gsar~ r pr ~nt-tlv- ~ ol ~-tl~t~
~1~. 7 ~rov~ th t r~lnnnt Phl p II o~rr1~ th- ~a I0 ~lt~
tl- tho o- wlth~ n~tur~l arOup IS/TI~ r~/Ig~ hlbltlon~ ~ho of ~ ~tLont wltll gr-o~ pllon ~11 rgy, who ~hh~ Ig~ r--otl~lty ~1~ orou~ ISJ~ ll~n~ /10~ u~ Oroup S ~ rgon~ ~t ubout 30 Im~ w-o ~r~ t-d ~ r~a-bln~nt Pbl p II t~n- 2),-o~bln~nt Phl p S tJ~ 3~. ~bln nt ~t v I tLu~ 4) or J ooll p~
1) Pr-lnou~-tlon wlth r oo~bln~t ~ p II s~r th Ig~ blndlng ~o r~t~u~ol JthJ, p II ~hllo-t ca~--t--ly dl~p~ r i~ th 12 kD ~ glon, wh-r~ ch- r~otlrlty ~lth Phl p I at 30 l~ not ~f-ot~
~oqllbln nt P*l p ~ r~uc-~ o~ly th hlndln~ to n~tur~l Plll p T, bu~ h~
~ rr-ct on th- ~ b~lng to 1~1 p ~ pr lnoub-tlon llth oontrol p~ot-ln~, r-o411bln nt ~-t v s ~ '. coll ~rot-;lnr ~oo not l~r th~ Ig~ bln~ to s~tur~ nd Ph~ p Ir~ ~o~ thlr lt rollowu ~ut r oom~n~nt ~ I h~ ~lto~- that ~ ~ r o~
ld ntl~-l to tho-- of n~tur~ PhJ p II, ~ut ehoro 1- ~o untl~nlc r-lut-dnu~- to natur~l or ~ollbln~nt aro~p ~ ll-rg Flg. B h4W th hybrldl~-t~on o~ th~ o~hA oo~ or 7hl p ~ wlth ~rom tlsothy gr ~ ry~ ot~l ~A w~ ol-tod ~ron~ rn~
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- , . : .. .-:: : - . . - . . .. . . - -5ENT BY:PENNIE ~ E~MONDS ;11-28-94; 3:52PM, PENNIE ~ EDM~NDSl PERLEY-RBBERTSDN;#l~
2~ 97~
nd tl~y gr~ ~na ~). IO ~ag wa~
ln denAtusing ~roae 9~ d blott~d ~to nitsoo~llulo~e. S~ 3~P-l~l-d c~A, cotl~ fos P~l p II~ hybr~dLz-l~ wlth botll ry gr~
arld tlmothy gra~d Rrla ~t aE~l~t~ly th l~al of la9 ~ t ~ven dl~tL~ly ~lo~ th~, ~t ~ ts~o~lpt ~ o~ ~bollt 60D b-s~
cro~ hy~rldlratlon ~how~ ~uctur~ il-rlty o~ th- ~orL~t~
vblA~h co~ ~or Oroup 21/III allganl- ln dlLf~rent 0s~-~ al~-a1o~-P~ur~ A ~d 9 ~ 1~ th- ra~Lvlty o~ r~o~bLnant PA~ p IT
~11~ wth ~ ~ntibody ~oi~io to a-o~ II~lTl ~ .8~ pollen A~ tll ~o~ uhloh ~ro~ tl~otlly ~ oll r~ ~llorg~8 ~d tho ~irclp~ ~iroh t~ ,ll~g~ ot ~ Co~
n~r~bl~nt ~ t-~ted 1~ tha dot blot ~rooe-~ Uit~l ~nt~o~ ~olflo ~co gs -~ poll~ ll~e~. 4~ to.oro~p Y
llor~n-, R4 d t~- a~p ~ rpDs~ ~nd ~5 l~tlLlo~
~o~p II/llI ~ll-rg ~-: , ~
1~ Th- dl~ lllu-tr t~ th- cl~ne d--Lg~4tlon ~ln nt PhJ p 1~ 1~ ~rc~ y clot~ A.
~,v-le ~ th~ Phl n 11 ~
~he D~ no~ o~ the Phl p ~J, Uler~n w~l~ det~lrl d ~y ~oqu-noln~ ;
th oD~ ocord~g to th~ ~thod Or 8~gor ~16~ rlg~ u t~o dotermln~d D~A -qu-no- ~nd th~ o ~cld e~ t duc-d t~rdro~
A 1CD~1 ~ ptSd in rrcnt o~ th- s~tur- prok-Ln~ ~bl4h poctLda how~
~lgnl~lo nt ho~olooy ~lth oth r uk~syot~o ign~l }-~tldo~
uno~ui~oa~lly provo- th~t Oroup Il/rI~ xga~o of dlotlnct g~c~
~r~g~t~ ~rn ood d ~nd do t ro~ult rro~ Osoup ~ ~ll~rg-~ throu~h prot-olytlo d oo~po-ltlon Th~ hlgh d-~r-~ of -qu-n~- ho3Dlogy ~-~out ~ .
70~ ~quenco ld ntlty) of th- r~comblnu~t ~hl p Il wlth thD ~o~olo~o~
~rot-ln~ ~ro~ r~ gr~ oI p IT ~nd Lol p ~ r~. 2~ ohow~ th-oloro ~tr~otur41 rd ~e~d~uo~ of tho~- protolna ~d th~rQby provlde- th mol oul~r b~ or th ~munolo~ic~l rel~t-dno~ Or Oroup II/III
~ller~ ac Or ~ ront p ol~
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SENT aY:PENN]E 8 EDMONDS ,11-2B-~4 : 3'52PM : PEN~IE 8 EDMONDSl PERLEY-RC3ERTSCN:#ll .. . . . ..... . . . . . .. . .
2~ '.~ ~
D-terr~ of ~-o~ ~ T-cell~ ~
0~1 tha b~ o~ t~o dodYco~ ~no cld n~ o ~ tbo P~J1 p ~I ~llqr~-n tb~ ~ell ~d ~-ccll ~topo~ ln d wlth ~1 ~d o~
ao~tcr ~r~- 124, 25)- Th- ~-lor~t ~oll uld ~-cell ~pLtopo~ r~ bwnp~l d Ln F~ 3 ~d ~- ~Iynthe~l~ pept~ whlCb oorr-~pon4 to ~ plto~c, b~Lnd Igll ol c pOlloG-cllorglC
~tl-rlt~, Ybor~ ynth~t~o ~ptl,~l c~ ~1ch oo~apond to ~-oell ~lt;opo~ ot~t- ~-~llo o~ gr~o~ polloA~ o ~tLen~- to prolLf-r~to~ u~d r ~ult Ln an l~r-~od ~-ehylD14in~ UpW~
~o~ln~nt ph~o- oon~lnl~ th cDN1~ Sor P~ .o Tl wore ~ ~-d for l~-otlon ~Lth ly~og~lo t, ooll Y 1009. ~obl~nt ,O-gal~to-~da~
fu~ protoln w~ obt~ 4 by l~tlon ~1th IP~O lS~o;arapyl-~
th~o l~oto~ uld oulturo ~0)~ ~rho control pro~ln p- --ctw~ ho~ Io8 ~ludl~ ltl t~ ~-t~rn blot ~-~t~ wh-r-u~
th- r ~ln~t Phl p rI ~u~ pro'c-ln bo~ dl-t~ot Iq~ ~lndln~
~ol~lo~lly to ~ D Ip ol o tL~t- who r aot-d wlth Oroup ''fIlI
~11~A~ 0~ d~S-r, nt Cr~ pol~
~g~b~nd~o o~o ol~v S~ r ~P~ 12 IL A~r~
8-rum obt-~d rrO~ ~ gr~- pllon~ lo p~tllt Wh~ r-~ct~ w~th n-tur~l ~roUp II~ r~, W~ pr-lncu~to~ wlth r~ bln~nt ~Al p II, wh-r~ lsy arOu~ rT/rIl--p ol~lo Ig- w~- ~o~d rlg. 6 show- th-t th~ , blrldl~ to n~tt~r~l oroup I~J~ sgen~ ~oot co~pl-toly nnull-d by th- prolnoub~tLon, ~ro-~ th~ blndln~ to Oroup I 4t Group V
llo~n~ w~ not .~r~t~. Thl- ~ h~t th~ lto~o~ o~ ~ur~
croup Il~III ~lorg ~ - blo~}od by ~omb~nt ~ TI, ~d th~t .
th r lu h~rtly ~ r l~ r~iv~ty b~w~A Crou~ I, Croup v : ;
~4 Oraup SI/IIS ~
-:, - -: ;~ -:
Q~o~- hvb~d~
~ot-l ~A rro~ rgo ~r~- ~a tl~othy ~r~-- P11-A W~ ol~
~t-d ln d~ urln~7 ~ ro~ ~1 ~d tr~r~ r~ onto nlt~-llulo~e - .: ~`
m~br no~. ~ho ~r~ hrbrldl~-d wlth ~ pl t- oDllA whlch ~o4~
9 '' ~
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SENT DY:PENNIE ~ EDMONDS ;11-2~-94 ; 3:53PM ; PENNIE ~ EDM~NDS- PERLEY-RO~ERTSON:#l~
21L~
For Pn~ p II. 2ybridlzlng blu~d~ ~r3 ~llw~y~ fo~nd ~t thN lo~el o~ th 1f~8 r~o~om~l F~A ~nd ~l~o ~onsid-r~ly balow th~t. ~t~ hybrldl~tlo~
1~ cl-~ly ~r- ln't-n~l /e wlth tlr~othy gr-o~ F~liA t~ Lt~ rye gs~-~~, Although, ~oco~dl~ ~o the g 1 ~t~ ~g wlth ethldlun~ bro~
bollt ~ha o~ ~ou~t~ o~ to~ lll uor ~d ~e~rthe~ roo~
~rldl~tlos~. ce~uld b- utt~ a und~r ~tr~go~t ao~ld$tlon~ d~r o~rt-ln c~-tu~ the l~-r tc~ orlp~ r~roo~nto L~rcl-r ~
~tlll ~turo ~ 04 th~ hybrldl,-atlorl with~t~d~ ~n 3trln~nt wa-hl~. ~ lo ~t~d~ to do~t tho ~o109~ o~ t~o Oroul7 ll/I~r g~ao~ llorgon~ o~ dlff~nt ~ole-.
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"~.,'-' ~''."'', ::~;:
::: ~.~ .- -SENT BY:PENNIE & EDMOHDS .11-2~-94 ; ~'53PM; PEI~INIE ~ EOM~NDSl PERLEY-RCBE~T30N;#13 .. ... . .. .. . . . . ~ .. .. . .. . . .. .
. ~ , .
WO 94123035 2.~ T9410003g .
aibllog~aphy 1.1~reidl~off, ~.R., 3~hrlich ~tzky, E.,GI~lt, J~H., 1~syers, D.A~, ~nd Manh, D.~. (19~ AUergy Cl~n Immunol 7~, 1190-1201 a. Matthlesen, ~., Lowensteil~, H. Clin 13xp Allergy 21, 309-320.
~ 2.~.30~97~
WO 9~ J23035 P~/A'r94f O0039~ :
~bln~nt ~y~
ara~ ~9 th~ t ~ear~t ~ t llorols~
o~currlng durln~ th~ ~u.. . ~ thul 20~ o~ lv~ 'co l~ort~t or~ n ~lorgen- Lnclud- Oro~ I t1~, ~up V ~2~, Cro~p dl-oo~d to ~ ~r-rs pollo gu~ ~7~ 3)- ~
allorg r~o ~$~ nd ~o l~ologLe-lly or ~truo~ ,ly olo~ly rol-tot ~ poll~ o~ a~f-r-At gr~ ~lo~, ~d r-l~t-t ~11~ o~ o~o group ~h~w oro~- rewtlv~ty wlth ~tl~ 50 ~r, ~1~ D~ th~ .~ ~ ~ L~ tod b~r r~bl~t ~hnl~
~n~ ps~ ooll ~9~. ~ny o~ t~ ~llor~n~ ~pr-~ ln E oolJ how ~ro~ r to th~- o~ tu~ rot L~, ~na c~
th-~nlor- b us d ~Dr ~ho dl~o-L~ u~d th-r~y Or llor~l~ dL~
(10, 11, 11). Jl~, do~crl~d ~os t~ ~ t tl~ 1- ~ol oul~
ohnru~ elon o~ o~plolt oD~A rhloh aod for Pbl p I~, ~nd th lon o~ ot-Ln ln ~. ooll. ~nt~l no~ t- ~ca~bln~nt ~rou~ rI~III or~u ~ ll~n q~ lhbl~ d, ~o o n ~ ~:
o_n ~rom th ~pl~u, Lt o~n ~ u~ llho t~ n~tu~ ro~ln ~or : -b--~ on ~ ntl~o~y ll~t~r~tlol~, uoh ~e ~ u--t ~o~ :
o ~lol~ ~OO-U~ UJ- .11 S-~ll ~Lto~o r ~nt ln tl~
s~ln-nt ~o~oaul-, ln th- n tur-1 ~l-cul- rurt~or ~ th r~o~blsu~t ~1~1 p 11 1~ eult~1~ for proo ~ whloh s uult ln ~ur bl- ~dL-to~ r-l--~e. ~ th~s-~tlo u~- Or r~o~ bl p I~, b-od on ~n d~ ~t on L~u~or gul~tory ~roo ~ , ~e~n-~At~body lnt-r-ctlon, S-o-11 r~tlvlty ~d ~odl~tor re1-~u~, L~ th con-4uon4-Or t~ ot~otur~ u~d blolo~lo~l o~l1 rlty ~tw n th- ~tur-1 ~d th-r-oolobln~nt ~-~ tr-~e~t l~ Atlon ~1~- ~ ca~pl~ oD~la ~hloh cod-- rOr ~ bl~t ~hl p II ~ll~q~ 01~ th b l- or tho ~no ~ald ~u~ ~duo d fro~ eh~ cD~ c~ d ~c-ll ~lto~ o~
tb- ~hl p II nll-rg~- ~ dot ~l~d Th r-oo bl~nt ~1 p ~S
~llor~n n- pr~d Ln ~ ~oll ~ h~- ~rop rtl~ r to tho~- o~
~urnl Oroup ~ S~ gr~ oll n 110r~no ro~ ~hl- lt ~ollow~ th-t ~ho r~ombln~nt ~hl p lI ~ rg~n, ~Llko th- n~tus-l Group II/III
ll~n-, o~n b- u--q ~oo pr~ o- ba~d on nt~n--ntl~o~y 13to~ot~on, ntlg~n~ nt T~-11 ~Lon or ~tLg~n~nd ~t -~
- ~: .
SENT BY:PENNIE ~ EDMONDS ~ 28-94 , 3:5~PM ; PENNIE ~ EDMONDS` PERLEY-ROBERTSON:# 7 . .
. _ .
Z1!~ 7~
m~l~or rdo~-e, wh~r~, ~ow~vor~ the r~oa~lbLn~nt Ll~r~n~ ha~ th~
~tdltlonal aav~nta~ of g~nt~r ~urlty and hlqhor ~ lol'cy.
4toril-1 ~4 thod~ ~
: ~
~rl~othy qr4~1~ pl~ar, ~All~rgon ~ ~nq~lhol~, Bw~n), whloh h~,d ~e~n ln ~lgated ~or E~urity ~y ~ n~ o~ llght ~loro~co~y n~ l ctro~
~ro~o~y, ~aa ~ood ~or tho l~olat~n of ~oly d-nyl~tod lINA ~13). ODI~
~ynthanl~ ua- oarr ~ d out w~tb oll~ nd ~ ri ~ r~; tho end~ o~ ~ -th oD~A ~ro dl~nntod wlth ~4 poly~4r~- and provldad wlth ~ooXI
l~n~or~ Th~ l~nb r~d oD~A wa- llg t-d lnto do~ho-~horylatod l~bd~
gtll r~b nd ~ d A oDNA g~a lLbrary o~ ~DO~OOO indYp~n4~t clono~ w~- obtaln-d ~
: . ~ -: ' na~y~l-~ ,~
Iot ~r~-n~n~ of tho tl~othy ~ra~- Fol~on ~D~A o-n lLbrary w o carsid out ~ d-ocr~b d ~y ~rolt-n-t-r t al ~14) S~-bin413g olo~-~ w~r-~rl~ d and gha~a-DNa w~ ~r-p r-d ~rc~ th-~- clon-~ ) 3y ~oanr of ~pn I ~n~ ~o I cut- t~o D~A ~r 4m~t~ u~r- obtaln-~ whloh oont-lned hoth ~ortlon~ o~ th oo~pl t- ~h~ ~ IS OD~A ~d ~I-nkln~ lambd- Ctll ~-~u Do-o~ Th r--~ltlng ~p I/~-c~ nd B~cl D~A ~r~gm nt- w-ro -~
rubclo~ d lnto th pl~-mld p~C 18~ ~nd tr~n~ormed ~nto ~ coli ~
~lu~ 9ult-bl- clonn~ w~r ld ~tlfled by re~trlotlon ~n~lyc$~ ~nd bcth ~tr~nd~ wYr- r-qy-nc-d by mo~n- o~ l~mbG~ ~t11 forw~rd oqu nclng prlm r ~2~ r) 4n~ l rbd- gtll r~v~r~ qu~ncln4 prlm r ~22-m~r~, Clo~t-ch ~bor-to~o~, P~lo Alto~ ~A, ~nd by m~ nG o~ H13 ~Cl~ forw rd ~nd ro~ r~ rl~ r-, ~o-hrlngor-~n~h-im, ~ rm~ny, ~ccordLng to ~n4~r 16~. -' ' ' ~ RNA_L~o~th4~ blot~
10 ocO o~ tot~ A fro~ poll-~ ol tl~othy yr~-~ rPhl-un pr ton~eJ nd ry~ g~ r~ollUS P-r-n~-) w~ p~r~t-d by den~turln~ gel elec~o-~hor~ nd blotte~ onto nlt~oc-11ulo~e ~17~ ~o l~ol~t- the ~DNA
codlng ~or ~bl p II, the oorse~pon~lng D~A ln--rt w~ u~plifl d by ~CR, ~tnrtlnq ~rom r-oo~bln nt ph y~ 5 pioo~ol-- o~ primer ~ bd- gtll ;~
~osw ¢d -quonciAq ~ri~ r l~2-~er~ ~na la~b~4 ~tll r~ o ~eqY-noln~
S ' ~' ., ; .~
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SENT BY:PENNIE ~ EDM~NDS ;11-23-94: 3:51PM; PENNIE ~ EDMONDS~ PERLE~'-R3~ERTS~N;# ~
-- ~ .
7~
prl~r ~2~r~, Clont~h ~oro,tor~o-, ~3A~, w~o uond ln oaoh cao~.
Th~ PCR pra~lat WB~ l~ to 14 6~7Qro~ ~ol ~.nd th~ b-nd~ w~r nlute~d ~y ~an~ of ~ DJAII loll ~ch~ngor p~r ~ . T2~ D~IA oil~taln~d w~ ~
l~l-d by r~ndara ~ 13). Prohybrldi~lon an~ h~brldl~-'clon woro carrlod o~t l~y ot~d ~ho~o ~15). ~h~ blot~ ~re .t~.hea ~Lth 3.0 ~ 88C ~20 x ~sC ~ 3 11 ~aCl, O.g M ~ altrato, p~ 7.0), O.lt 5D8 (~odl~u~ dod-ayl oul~llto~ 1.5 ssc, 0.1~ t nd ~ lly wlth 0.75~ sW~
0,10 8D~ at 50'~, nd th~ utor~ r pb~d t~f~ ~, A~rd~
L~don, m~ .
l~y ~Qn~ o~ Igll ~or~nq ~ t-- oD~ oloo-- ~-o c~t~ w~ch ~or ~ Phl ,1~ llo~n. Sho 1Y~Og~A1C J!. coll l~t;ral~ Y 108q var ~l~td w1th r~coo~l~nt l~bd~ g~ll ~d t~o p-pl-Gtoeld~e ~u~
prot~ln w~.~ obt~lno~ fro~ tho ~ch ~ao~. Tho b~tch w~ rat~
oSrop~o~n~ y ln n 7.5~ yu:rylu~d~ and blott~ onto ~rocollulooe ~Z2). Th- ~u~lon ~r~tolD w~ toot~ by ~s o,~
Igl~ o~ lont- ~llorgLo to ~ n ~ n l,odL~ l d r~lt antlhu~n Ig8 ne~body ~Ph~ol~, ~aala, 8~).
Th- att-c2~ Flgur ~ ~orvo to lllwtr-t- thO u-~billty o~ tl~
r~a~ AZ p rl all~ ~ lor ~ro~-~o~ d or~ ~tlgon-~t~
lnt-ra~tLon, ~,ntl~n-d4~n~nt m~tor rQlo~, ~d nt~n-~ndant c-~.lul~ r~ot~vL~y. ~9. 1 ~ th~ A o~ t~o r ~rb~nt Phl p rI .
llg rl ~d tho l~tt-r'~ cld so~ duood ~r~ ~ld DNlL
Th- oa~lot- cD~ ~u~- o~ tl~O PAl p II tl~ lo oho~ b~--nonoodln~ o~-nc- -o ~ound ~t th 5~ ~nd Of th ~DNa, whloh 1-~ollo~d ~y 7~-bn~- lo~,dor ~ na-, ~hloh oodoo ~or th~ lgnnl ~tld undll~d L~ tho rl~ ~23). Sh ~lno ~c~d ooquonoo of th~
~tur proto~ ~ d duc-d rroD ba~ ~8 on, ~ b gln- ~lth v~l~. ~ ~
Th~ atop oodo~ ~AO, whlob b~-~lu Or~ tbo codlns~ t tho 3~ ond, ~ ~;
~ot~ t~lc~c. Th 3 ncodlng ~-~o~ -nd- lr tho ~oly A
rLg. 2 oho~- th~ ty o~ tl~ ~lno ~cl~ o- o~ th- . -~conbln~nt Phl p 1~ ~llorg~n to oth-r Oroup II/IIT cr~o~ n ;~
: .:
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SENT BY:PENNIE J ED~ONDS ~ 28-94 ; 3:51PM ; PENNIE ~ EDMONDS~ PERLEY-ROBERTSON;# 3 2~ ,g7~ ~
~ . 3 preo~tE th- d~-criptl~n o~ ll ~ltope9 of tho rooombl~ t PAI p 1~ ollor~-n~ ~d Flg. 4 th~t o~ th~ S-CQ11 opltOp~-~
Illg. 5 ~ tho ro~ot~rlty o~ tho r~oaablnu~t Phl ,p Il ~ rg n with ~orum ~g~ o~ ~ti~ nll~ to ~ ~oo~ln~t Pbl p I~ ~
r~ c~ ~ ~ l~to~ u~lon ~Iro~ 2f~o~nlo J. ool~ - .
Y Ioa9 by ~h~g ~ootl~ ~ Im~u~lo~ Ylth l~TO. ~h~ oll ~rotolno; ~vhlo~ oo~t~ln t~ r~o~ PhZ p Tl, w ~ el-~tro~
~hor-tlcnllr ~-~r~ted ~d tr~ ~rd o~to nJtroucll~loro ~oul 1 w ~ I~ o~ ~ O~Ou~ SI~S21-r- otl~e patl~tl ~llo ~ ~
2 ana 3 th d t~l w~- ~on- ulth I~ o~ all rglc ~tLent- ~rho ~ o r-~tlon wLth Cro~ II/~lI ~11~9~ ~ 4 ~ ~ co~trol ~r~nt wltb ~ o~ ~ non ll~lo ~ont~o~ ~r-o~.
F~g 6 ~re~nt- ~ t bl- whloh ll~to ~ ~c~t4- ~ gr~- ~ollon~
~llorglo ~t~-nt~ wlth ~g~ rowtl~lty wlth ~clfle ~r ~o ~ollor~
~llo~no ~ fr~u~y of r~tl~ltY of gr~s~ ~ollen ~ rglo pa~clonto wlth ~ r nt 5~r~ rg~ ~ ~lwwn Th~ flg~r r~-~t pprox~t- valu~a obt~ d by t-ct~ w~ tur~
r o~ln~nt gsar~ r pr ~nt-tlv- ~ ol ~-tl~t~
~1~. 7 ~rov~ th t r~lnnnt Phl p II o~rr1~ th- ~a I0 ~lt~
tl- tho o- wlth~ n~tur~l arOup IS/TI~ r~/Ig~ hlbltlon~ ~ho of ~ ~tLont wltll gr-o~ pllon ~11 rgy, who ~hh~ Ig~ r--otl~lty ~1~ orou~ ISJ~ ll~n~ /10~ u~ Oroup S ~ rgon~ ~t ubout 30 Im~ w-o ~r~ t-d ~ r~a-bln~nt Pbl p II t~n- 2),-o~bln~nt Phl p S tJ~ 3~. ~bln nt ~t v I tLu~ 4) or J ooll p~
1) Pr-lnou~-tlon wlth r oo~bln~t ~ p II s~r th Ig~ blndlng ~o r~t~u~ol JthJ, p II ~hllo-t ca~--t--ly dl~p~ r i~ th 12 kD ~ glon, wh-r~ ch- r~otlrlty ~lth Phl p I at 30 l~ not ~f-ot~
~oqllbln nt P*l p ~ r~uc-~ o~ly th hlndln~ to n~tur~l Plll p T, bu~ h~
~ rr-ct on th- ~ b~lng to 1~1 p ~ pr lnoub-tlon llth oontrol p~ot-ln~, r-o411bln nt ~-t v s ~ '. coll ~rot-;lnr ~oo not l~r th~ Ig~ bln~ to s~tur~ nd Ph~ p Ir~ ~o~ thlr lt rollowu ~ut r oom~n~nt ~ I h~ ~lto~- that ~ ~ r o~
ld ntl~-l to tho-- of n~tur~ PhJ p II, ~ut ehoro 1- ~o untl~nlc r-lut-dnu~- to natur~l or ~ollbln~nt aro~p ~ ll-rg Flg. B h4W th hybrldl~-t~on o~ th~ o~hA oo~ or 7hl p ~ wlth ~rom tlsothy gr ~ ry~ ot~l ~A w~ ol-tod ~ron~ rn~
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- , . : .. .-:: : - . . - . . .. . . - -5ENT BY:PENNIE ~ E~MONDS ;11-28-94; 3:52PM, PENNIE ~ EDM~NDSl PERLEY-RBBERTSDN;#l~
2~ 97~
nd tl~y gr~ ~na ~). IO ~ag wa~
ln denAtusing ~roae 9~ d blott~d ~to nitsoo~llulo~e. S~ 3~P-l~l-d c~A, cotl~ fos P~l p II~ hybr~dLz-l~ wlth botll ry gr~
arld tlmothy gra~d Rrla ~t aE~l~t~ly th l~al of la9 ~ t ~ven dl~tL~ly ~lo~ th~, ~t ~ ts~o~lpt ~ o~ ~bollt 60D b-s~
cro~ hy~rldlratlon ~how~ ~uctur~ il-rlty o~ th- ~orL~t~
vblA~h co~ ~or Oroup 21/III allganl- ln dlLf~rent 0s~-~ al~-a1o~-P~ur~ A ~d 9 ~ 1~ th- ra~Lvlty o~ r~o~bLnant PA~ p IT
~11~ wth ~ ~ntibody ~oi~io to a-o~ II~lTl ~ .8~ pollen A~ tll ~o~ uhloh ~ro~ tl~otlly ~ oll r~ ~llorg~8 ~d tho ~irclp~ ~iroh t~ ,ll~g~ ot ~ Co~
n~r~bl~nt ~ t-~ted 1~ tha dot blot ~rooe-~ Uit~l ~nt~o~ ~olflo ~co gs -~ poll~ ll~e~. 4~ to.oro~p Y
llor~n-, R4 d t~- a~p ~ rpDs~ ~nd ~5 l~tlLlo~
~o~p II/llI ~ll-rg ~-: , ~
1~ Th- dl~ lllu-tr t~ th- cl~ne d--Lg~4tlon ~ln nt PhJ p 1~ 1~ ~rc~ y clot~ A.
~,v-le ~ th~ Phl n 11 ~
~he D~ no~ o~ the Phl p ~J, Uler~n w~l~ det~lrl d ~y ~oqu-noln~ ;
th oD~ ocord~g to th~ ~thod Or 8~gor ~16~ rlg~ u t~o dotermln~d D~A -qu-no- ~nd th~ o ~cld e~ t duc-d t~rdro~
A 1CD~1 ~ ptSd in rrcnt o~ th- s~tur- prok-Ln~ ~bl4h poctLda how~
~lgnl~lo nt ho~olooy ~lth oth r uk~syot~o ign~l }-~tldo~
uno~ui~oa~lly provo- th~t Oroup Il/rI~ xga~o of dlotlnct g~c~
~r~g~t~ ~rn ood d ~nd do t ro~ult rro~ Osoup ~ ~ll~rg-~ throu~h prot-olytlo d oo~po-ltlon Th~ hlgh d-~r-~ of -qu-n~- ho3Dlogy ~-~out ~ .
70~ ~quenco ld ntlty) of th- r~comblnu~t ~hl p Il wlth thD ~o~olo~o~
~rot-ln~ ~ro~ r~ gr~ oI p IT ~nd Lol p ~ r~. 2~ ohow~ th-oloro ~tr~otur41 rd ~e~d~uo~ of tho~- protolna ~d th~rQby provlde- th mol oul~r b~ or th ~munolo~ic~l rel~t-dno~ Or Oroup II/III
~ller~ ac Or ~ ront p ol~
: : :
SENT aY:PENN]E 8 EDMONDS ,11-2B-~4 : 3'52PM : PEN~IE 8 EDMONDSl PERLEY-RC3ERTSCN:#ll .. . . . ..... . . . . . .. . .
2~ '.~ ~
D-terr~ of ~-o~ ~ T-cell~ ~
0~1 tha b~ o~ t~o dodYco~ ~no cld n~ o ~ tbo P~J1 p ~I ~llqr~-n tb~ ~ell ~d ~-ccll ~topo~ ln d wlth ~1 ~d o~
ao~tcr ~r~- 124, 25)- Th- ~-lor~t ~oll uld ~-cell ~pLtopo~ r~ bwnp~l d Ln F~ 3 ~d ~- ~Iynthe~l~ pept~ whlCb oorr-~pon4 to ~ plto~c, b~Lnd Igll ol c pOlloG-cllorglC
~tl-rlt~, Ybor~ ynth~t~o ~ptl,~l c~ ~1ch oo~apond to ~-oell ~lt;opo~ ot~t- ~-~llo o~ gr~o~ polloA~ o ~tLen~- to prolLf-r~to~ u~d r ~ult Ln an l~r-~od ~-ehylD14in~ UpW~
~o~ln~nt ph~o- oon~lnl~ th cDN1~ Sor P~ .o Tl wore ~ ~-d for l~-otlon ~Lth ly~og~lo t, ooll Y 1009. ~obl~nt ,O-gal~to-~da~
fu~ protoln w~ obt~ 4 by l~tlon ~1th IP~O lS~o;arapyl-~
th~o l~oto~ uld oulturo ~0)~ ~rho control pro~ln p- --ctw~ ho~ Io8 ~ludl~ ltl t~ ~-t~rn blot ~-~t~ wh-r-u~
th- r ~ln~t Phl p rI ~u~ pro'c-ln bo~ dl-t~ot Iq~ ~lndln~
~ol~lo~lly to ~ D Ip ol o tL~t- who r aot-d wlth Oroup ''fIlI
~11~A~ 0~ d~S-r, nt Cr~ pol~
~g~b~nd~o o~o ol~v S~ r ~P~ 12 IL A~r~
8-rum obt-~d rrO~ ~ gr~- pllon~ lo p~tllt Wh~ r-~ct~ w~th n-tur~l ~roUp II~ r~, W~ pr-lncu~to~ wlth r~ bln~nt ~Al p II, wh-r~ lsy arOu~ rT/rIl--p ol~lo Ig- w~- ~o~d rlg. 6 show- th-t th~ , blrldl~ to n~tt~r~l oroup I~J~ sgen~ ~oot co~pl-toly nnull-d by th- prolnoub~tLon, ~ro-~ th~ blndln~ to Oroup I 4t Group V
llo~n~ w~ not .~r~t~. Thl- ~ h~t th~ lto~o~ o~ ~ur~
croup Il~III ~lorg ~ - blo~}od by ~omb~nt ~ TI, ~d th~t .
th r lu h~rtly ~ r l~ r~iv~ty b~w~A Crou~ I, Croup v : ;
~4 Oraup SI/IIS ~
-:, - -: ;~ -:
Q~o~- hvb~d~
~ot-l ~A rro~ rgo ~r~- ~a tl~othy ~r~-- P11-A W~ ol~
~t-d ln d~ urln~7 ~ ro~ ~1 ~d tr~r~ r~ onto nlt~-llulo~e - .: ~`
m~br no~. ~ho ~r~ hrbrldl~-d wlth ~ pl t- oDllA whlch ~o4~
9 '' ~
.-:: ~ .: ~
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:
SENT DY:PENNIE ~ EDMONDS ;11-2~-94 ; 3:53PM ; PENNIE ~ EDM~NDS- PERLEY-RO~ERTSON:#l~
21L~
For Pn~ p II. 2ybridlzlng blu~d~ ~r3 ~llw~y~ fo~nd ~t thN lo~el o~ th 1f~8 r~o~om~l F~A ~nd ~l~o ~onsid-r~ly balow th~t. ~t~ hybrldl~tlo~
1~ cl-~ly ~r- ln't-n~l /e wlth tlr~othy gr-o~ F~liA t~ Lt~ rye gs~-~~, Although, ~oco~dl~ ~o the g 1 ~t~ ~g wlth ethldlun~ bro~
bollt ~ha o~ ~ou~t~ o~ to~ lll uor ~d ~e~rthe~ roo~
~rldl~tlos~. ce~uld b- utt~ a und~r ~tr~go~t ao~ld$tlon~ d~r o~rt-ln c~-tu~ the l~-r tc~ orlp~ r~roo~nto L~rcl-r ~
~tlll ~turo ~ 04 th~ hybrldl,-atlorl with~t~d~ ~n 3trln~nt wa-hl~. ~ lo ~t~d~ to do~t tho ~o109~ o~ t~o Oroul7 ll/I~r g~ao~ llorgon~ o~ dlff~nt ~ole-.
: :::~::.-- :: : ~ : : : ~
"~.,'-' ~''."'', ::~;:
::: ~.~ .- -SENT BY:PENNIE & EDMOHDS .11-2~-94 ; ~'53PM; PEI~INIE ~ EOM~NDSl PERLEY-RCBE~T30N;#13 .. ... . .. .. . . . . ~ .. .. . .. . . .. .
. ~ , .
WO 94123035 2.~ T9410003g .
aibllog~aphy 1.1~reidl~off, ~.R., 3~hrlich ~tzky, E.,GI~lt, J~H., 1~syers, D.A~, ~nd Manh, D.~. (19~ AUergy Cl~n Immunol 7~, 1190-1201 a. Matthlesen, ~., Lowensteil~, H. Clin 13xp Allergy 21, 309-320.
3. ~ns~ .A.,Shen~amtl~thi, P.,and l~a~sh, ~.~. (198g) J. Biol. Chem, :
264, 11181-11185 4. Kisil, F. T., Ja~ . S., Lin, Z. W., Ekranwdullah, A, X. M. (19g9) in Sdwn ~ , D., Kunkcl, G. eds. Ep~topcs of Atopic Allcrgens, 22~
264, 11181-11185 4. Kisil, F. T., Ja~ . S., Lin, Z. W., Ekranwdullah, A, X. M. (19g9) in Sdwn ~ , D., Kunkcl, G. eds. Ep~topcs of Atopic Allcrgens, 22~
5. ~ei, K. S., ~kramDdullah, A. K. M., Kisil, P. T., ~zuba Pischcr, J. M. . ~ -;
M., Reclor, E. S., ~nt Sehon, A. El. (1989) J Allcrgy Clin Im~nuno183, 845-852.
5. v~n Rce, R., Dne~sen, ~. B. M., van Lc~uwen, W. A., Stapd, S. O., and .Aalbcrse, R. C. ~1992) Clin ~p t~iler~y.
7. Valenta, R" Puchene, M., Ebner, (:., Valent, P., Slllaber, C., Deviller, P., ~errdra, P., Tejkl, M., Edelmann, H" Klaft, P., and Scheiner, O. (199~) J
8xp Med 17S, 377-38$. . . -8. V~lenta, R., Dud~ene, M., Vrtala, S., Va~ent, P., Sillaber, C., Ferrdra, F., l~kl, M., Hirschwehrl R., Ebner, C., Ksaft, 1:~., and Sch~iner, O. (1993) In¢ ~: -Arch Allergy Appl Immunol 99, ~ 273.
9. Scheiner, O., Bohle, B., Broitenbach, M., Breitencd~r, H., Duch~ne, M., Ebner, C., Perrelra, F., Hirschwehr, ~, Hoffmann~ommergmber, K., Pettenburger, K., Rump~ld, H., Stciner, R., TejW;M., Valenh, R., and ~Craft, O. ~ `
tl992) in: Advanccs in Allergolo~y aod Clinical Immunology, Goda~t, P., ~ousquet, ~., and ~Iichel, P. 13. eds. The Par~henon Publishing Group. .
10. Valen~a, R., Duchene, M., Vrtah, S., Birkner, T., Ebner, C., Hirschw~hr, R, Brcitenbach, M., Rumpcld, E~., Scheiner, O., and Kraft, D. (l991) --~
J Aller~y Clln Immunol ~8, 88g-8g4. . ` ~ ~-11. Valenea, ~., Vrtala, S., Ebner, C., Kraft, D., and Schoinel, O. (1932) Int ~ ~ -Arch Al~ergy Appl Immunol g7, 287-294 .. _ .. ... . .. .. . .
. . . . .... ~:
.,.. , .
,... , .. ,.. . ~ - : . . :
.
- ~
. -, ~.:', . , ' :
S~NT BY:PENNIE ~ EDM~NDS ~ 2~-94; 3:54PM; P~NNIE ~ E~MONDS I PERL~Y-~BERTSON:#14 WO 941~30~5 2~ ~9~ 'rg4~00039 12. Valenla, R., Spur, W. R., Per~e~rA, F, Valeflt, P., Sillaber, C., Tejkl, M., Duch~nc, M., Ebner, C" Lechncr, ~., Kraft, D., and Scheincr, O. (1993). J
Allgy C1Jn ~mmunol 91, 88-91.
13. Vstal~, S, Spcrr, W. R., Ramittcr. I., Y~n~ec, R., Laffer, S., M~lbr, W.-D., V~ , P., I.ochncr, K.l Rumpold, H., Kraf~, D., Scheiner, O" and Y~Jenta, R. (1993) J Immunol (accepted proY~ded rwlsion), ~;
14. B~llened~r, H., Pcttenburser, K., Blto, A., Val~nta, ~.~ Kraft, 1)., Rumpold, II., Scheincr, O., and Brciten~aeh, M. ~198g) El~BO J 8, 1935-19~8 .
15. Ausubel, P. M. tl990) in Current protocols in molecular biology, Wiley, New York.
16. Sanger, ~., Ni~1den, S., Coulson, A. R, (lg77~ Proc Natl Acad Sci USA
74, 5463 5468.
t7. Vslenta, ~., Breilcneder, H., Pettenburger, K., Bre~tenbach, M., Rumpold, X., E~raft, D., and Schoiner, O. (l991) J Aller~y Clin Immunol 87, :~
677-682.
18. Sambrook,.~., Fritsch, E. P., Maniuis, T. (198g~ Molecular clonlng: a Iaboratosy manu~l. Cold Sprin~ Harbor Laboratory Press.
19, Fdn~cr~, A. P., ant Vog~stein, B, (1983) ~nal Bioohcm 132, 6-13.
20. Huynh, T. V., Youn~, R. A., Davis, R. W. (1985) in: cl:~N~ clonin~
O~ord, ~L Press, v~ A7g~
21. Laemml; ~. ~C. (1970) Naturc 227, 680-685.
22 Towbin, H., Staehdin, T., Gordon, 1. (19i9~ Proc ~atl Acad Sci IJSA 76, 435W3~4. . :~
23. ~3a~el, Y., and He~ne, G. tl9~0) FEBS Lctt 261, 455-45~
24. Kyte, J. and Doolittle, R. F. (1982) J Mol Biol 157, 105-132.
2S, Margaiit, H., Spouge, J. L., Comette, J. L" Cease, K E~., Delisi, C., and ~-~enofsky, J. A. (1987) J Immunol 138, 2213-2229.
- ~: : . ' . ' ' . . :
, :' . . : ' - ' ' , . .
M., Reclor, E. S., ~nt Sehon, A. El. (1989) J Allcrgy Clin Im~nuno183, 845-852.
5. v~n Rce, R., Dne~sen, ~. B. M., van Lc~uwen, W. A., Stapd, S. O., and .Aalbcrse, R. C. ~1992) Clin ~p t~iler~y.
7. Valenta, R" Puchene, M., Ebner, (:., Valent, P., Slllaber, C., Deviller, P., ~errdra, P., Tejkl, M., Edelmann, H" Klaft, P., and Scheiner, O. (199~) J
8xp Med 17S, 377-38$. . . -8. V~lenta, R., Dud~ene, M., Vrtala, S., Va~ent, P., Sillaber, C., Ferrdra, F., l~kl, M., Hirschwehrl R., Ebner, C., Ksaft, 1:~., and Sch~iner, O. (1993) In¢ ~: -Arch Allergy Appl Immunol 99, ~ 273.
9. Scheiner, O., Bohle, B., Broitenbach, M., Breitencd~r, H., Duch~ne, M., Ebner, C., Perrelra, F., Hirschwehr, ~, Hoffmann~ommergmber, K., Pettenburger, K., Rump~ld, H., Stciner, R., TejW;M., Valenh, R., and ~Craft, O. ~ `
tl992) in: Advanccs in Allergolo~y aod Clinical Immunology, Goda~t, P., ~ousquet, ~., and ~Iichel, P. 13. eds. The Par~henon Publishing Group. .
10. Valen~a, R., Duchene, M., Vrtah, S., Birkner, T., Ebner, C., Hirschw~hr, R, Brcitenbach, M., Rumpcld, E~., Scheiner, O., and Kraft, D. (l991) --~
J Aller~y Clln Immunol ~8, 88g-8g4. . ` ~ ~-11. Valenea, ~., Vrtala, S., Ebner, C., Kraft, D., and Schoinel, O. (1932) Int ~ ~ -Arch Al~ergy Appl Immunol g7, 287-294 .. _ .. ... . .. .. . .
. . . . .... ~:
.,.. , .
,... , .. ,.. . ~ - : . . :
.
- ~
. -, ~.:', . , ' :
S~NT BY:PENNIE ~ EDM~NDS ~ 2~-94; 3:54PM; P~NNIE ~ E~MONDS I PERL~Y-~BERTSON:#14 WO 941~30~5 2~ ~9~ 'rg4~00039 12. Valenla, R., Spur, W. R., Per~e~rA, F, Valeflt, P., Sillaber, C., Tejkl, M., Duch~nc, M., Ebner, C" Lechncr, ~., Kraft, D., and Scheincr, O. (1993). J
Allgy C1Jn ~mmunol 91, 88-91.
13. Vstal~, S, Spcrr, W. R., Ramittcr. I., Y~n~ec, R., Laffer, S., M~lbr, W.-D., V~ , P., I.ochncr, K.l Rumpold, H., Kraf~, D., Scheiner, O" and Y~Jenta, R. (1993) J Immunol (accepted proY~ded rwlsion), ~;
14. B~llened~r, H., Pcttenburser, K., Blto, A., Val~nta, ~.~ Kraft, 1)., Rumpold, II., Scheincr, O., and Brciten~aeh, M. ~198g) El~BO J 8, 1935-19~8 .
15. Ausubel, P. M. tl990) in Current protocols in molecular biology, Wiley, New York.
16. Sanger, ~., Ni~1den, S., Coulson, A. R, (lg77~ Proc Natl Acad Sci USA
74, 5463 5468.
t7. Vslenta, ~., Breilcneder, H., Pettenburger, K., Bre~tenbach, M., Rumpold, X., E~raft, D., and Schoiner, O. (l991) J Aller~y Clin Immunol 87, :~
677-682.
18. Sambrook,.~., Fritsch, E. P., Maniuis, T. (198g~ Molecular clonlng: a Iaboratosy manu~l. Cold Sprin~ Harbor Laboratory Press.
19, Fdn~cr~, A. P., ant Vog~stein, B, (1983) ~nal Bioohcm 132, 6-13.
20. Huynh, T. V., Youn~, R. A., Davis, R. W. (1985) in: cl:~N~ clonin~
O~ord, ~L Press, v~ A7g~
21. Laemml; ~. ~C. (1970) Naturc 227, 680-685.
22 Towbin, H., Staehdin, T., Gordon, 1. (19i9~ Proc ~atl Acad Sci IJSA 76, 435W3~4. . :~
23. ~3a~el, Y., and He~ne, G. tl9~0) FEBS Lctt 261, 455-45~
24. Kyte, J. and Doolittle, R. F. (1982) J Mol Biol 157, 105-132.
2S, Margaiit, H., Spouge, J. L., Comette, J. L" Cease, K E~., Delisi, C., and ~-~enofsky, J. A. (1987) J Immunol 138, 2213-2229.
- ~: : . ' . ' ' . . :
, :' . . : ' - ' ' , . .
Claims (14)
1. Recombinant DNA molecule, characterized in that it has a nucleic acid sequence coding for a polypeptide which possesses the antigenicity of the allergen Phi p II (timothy grass pollen allergen), particularly of monocotyledonous plants, or for a peptide which has at least an epitope of this allergen, as well as a nucleic acid sequence which hydrodizes with said nucleic acid sequence under stringent conditions.
2. Recombinant DNA molecule according to Claim 1, characterized in that it has a nucleic acid sequence that is in homologous agreement with the total sequence shown in Fig. 1, or with partial regions thereof.
3. Recombinant DNA molecule according to Claim 1, characterized in that it has a nucleic acid sequence which is derivable through degeneration from the sequence shown i Fig. 1.
4. Recombinant DNA molecule according to Claim 1, characterized in that it has a nucleic acid sequence coding for a polypeptide which, as antigen, is cross-reactive with the Phi p II allergen, particularly of monocotyledonous plants, and has a high degree of homology thereto.
5. Recombinant DNA molecule according to on of Claims 1 to 4, characterized in that it is functionally combined with an expression control sequence to form an expression construct.
6. Host system, characterized in that it is transformed with a recombinant expression construct according to Claim 5.
7. Recombinant or synthetic protein or polypeptide derived from a DNA
molecule according to one of Claims 1 to 4, characterized in that it possesses the antigenicity of Phi p II or at least of an epitope thereof.
molecule according to one of Claims 1 to 4, characterized in that it possesses the antigenicity of Phi p II or at least of an epitope thereof.
8. Recombinant or synthetic protein or polypeptide according to Claim 7, characterised in that it has an amino acid sequence which fully or partially corresponds to the sequence shown in Fig. 1.
9. Recombinant or synthetic protein or polypeptide according to Claim 7 or 8, characterized in that it is a fusion product possessing the antigenicity of the Phi p II allergen of timothy grass or at least of an epitope thereof and has an additional polypeptide part, the whole fusion product being coded by the DNA
of an expression construct according to Claim 5.
of an expression construct according to Claim 5.
10. Recombinant or synthetic protein or polypeptide according to Claim 9, characterized in that said additional polypeptide part is .beta.-galactosidase or another polypeptide suitable for fusion.
11. Diagnostic or therapeutic reagent, characterized in that it contains a synthetic protein or polypeptide according to one of Claims 7 to 10.
12. Process for in vitro detection of a patient's allergy to the Phi p II allergen, characterized in that the reaction of the IgH
antibodies in the patient's serum is measured with a recombinant or synthetic protein or polypeptide according to one of Claims 7 to 10.
antibodies in the patient's serum is measured with a recombinant or synthetic protein or polypeptide according to one of Claims 7 to 10.
13. Process for in vitro detection of the cellular reaction to the Phi p II allergen, characterized by using a recombinant or synthetic protein or polypeptide according to one of Claims 7 to 10 for stimulation or inhibition of the cellular reaction.
14. Process for the treatment of a mammal suffering from a pollen allergy, characterized by the administration, to said mammal, of a recombinant or synthetic protein or polypeptide according to one of Claims 7 to 10.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0067293A AT401937B (en) | 1993-04-01 | 1993-04-01 | RECOMBINANT LYING GRASS POLLEN ALLERGEN PHL P II |
| ATA672/93 | 1993-04-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2136973A1 true CA2136973A1 (en) | 1994-10-13 |
Family
ID=3496784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002136973A Abandoned CA2136973A1 (en) | 1993-04-01 | 1994-03-31 | Recombinant timothy grass pollen allergen ph1 pii |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0643769B1 (en) |
| JP (1) | JP3657604B2 (en) |
| AT (2) | AT401937B (en) |
| AU (1) | AU684269B2 (en) |
| CA (1) | CA2136973A1 (en) |
| DE (1) | DE59406999D1 (en) |
| DK (1) | DK0643769T3 (en) |
| ES (1) | ES2124878T3 (en) |
| FI (1) | FI945648A (en) |
| NO (1) | NO944634L (en) |
| SI (1) | SI0643769T1 (en) |
| WO (1) | WO1994023035A2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2319437C (en) | 1998-01-31 | 2009-06-16 | University Of Arkansas | Methods and reagents for decreasing allergic reactions |
| DE19939982A1 (en) * | 1999-08-24 | 2001-03-01 | Merck Patent Gmbh | Process for the isolation and purification of grass pollen allergens |
| SE0200946D0 (en) * | 2002-03-27 | 2002-03-27 | Pharmacia Diagnostics Ab | Novel Allergen |
| US8709435B2 (en) | 2003-01-21 | 2014-04-29 | Biomay Ag | Hypallergenic mosaic antigens and methods of making same |
| DK1440979T3 (en) * | 2003-01-21 | 2005-12-19 | Biomay Prod & Handel | Process for the preparation of hypoallergenic mosaic proteins |
| AT503296B1 (en) * | 2006-05-03 | 2007-09-15 | Biomay Ag | Producing derivatives of wild-type protein allergen Phl p 1 with reduced allergenic activity by fragmenting wild-type protein allergen, the fragments having reduced allergenic activity, and rejoining the fragments |
| AT503690A1 (en) * | 2006-06-09 | 2007-12-15 | Biomay Ag | HYPOALLERGENIC MOLECULES |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1340729C (en) * | 1988-03-23 | 1999-09-07 | Mohan Bir Singh | Ryegrass pollen allergen |
-
1993
- 1993-04-01 AT AT0067293A patent/AT401937B/en not_active IP Right Cessation
-
1994
- 1994-03-31 JP JP52145094A patent/JP3657604B2/en not_active Expired - Lifetime
- 1994-03-31 SI SI9430214T patent/SI0643769T1/en unknown
- 1994-03-31 ES ES94910269T patent/ES2124878T3/en not_active Expired - Lifetime
- 1994-03-31 AT AT94910269T patent/ATE171725T1/en not_active IP Right Cessation
- 1994-03-31 CA CA002136973A patent/CA2136973A1/en not_active Abandoned
- 1994-03-31 AU AU62768/94A patent/AU684269B2/en not_active Ceased
- 1994-03-31 DK DK94910269T patent/DK0643769T3/en not_active Application Discontinuation
- 1994-03-31 DE DE59406999T patent/DE59406999D1/en not_active Expired - Lifetime
- 1994-03-31 WO PCT/AT1994/000039 patent/WO1994023035A2/en active IP Right Grant
- 1994-03-31 EP EP94910269A patent/EP0643769B1/en not_active Expired - Lifetime
- 1994-11-30 FI FI945648A patent/FI945648A/en unknown
- 1994-12-01 NO NO944634A patent/NO944634L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| SI0643769T1 (en) | 1999-04-30 |
| NO944634D0 (en) | 1994-12-01 |
| NO944634L (en) | 1995-01-23 |
| AU684269B2 (en) | 1997-12-11 |
| WO1994023035A3 (en) | 1994-11-24 |
| DE59406999D1 (en) | 1998-11-05 |
| ATE171725T1 (en) | 1998-10-15 |
| FI945648A0 (en) | 1994-11-30 |
| ATA67293A (en) | 1996-05-15 |
| WO1994023035A2 (en) | 1994-10-13 |
| JPH08500492A (en) | 1996-01-23 |
| EP0643769A1 (en) | 1995-03-22 |
| JP3657604B2 (en) | 2005-06-08 |
| AU6276894A (en) | 1994-10-24 |
| EP0643769B1 (en) | 1998-09-30 |
| ES2124878T3 (en) | 1999-02-16 |
| AT401937B (en) | 1996-12-27 |
| DK0643769T3 (en) | 1999-06-21 |
| FI945648A (en) | 1995-01-25 |
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