CA2181142A1 - Cross-reactive allergen - Google Patents

Abstract

Screening of a Kentucky Bluegrass pollen-.lambda.gt11 library with sera from patients allergic to grass pollen led to the identification of a partial cDNA clone, KBG51. Nucleotide sequence analysis of KBG51 indicated that the polypeptide encoded by this cDNA is different from that of the known recombinant grass pollen allergens. Using murine antiserum to a GST-KBG51 fusion protein, produced with aid of pGEX-2T-1 expression system, two polypeptides of about 30 and 59 KDa in size, were detectable in SDS-PAGE immunoblot analysis of KBG pollen proteins. The reactivity of this antiserum, with a number of polypeptides, which ranged in size from 29.5 to 115 kDa, from pollen extracts of several grasses, birch, ragweed and parietaria and the hybridization of RT-PCR products from various pollens with ?diolabelled KBG51-cDNA,demonstrated the cross-reactivity (CR) of this AL with other pollen ALs. Because of the broad CR, the protein(s) corresponding to KBG51 has been designated as CRAL51. Analysis by ELISA using sera of about 1000 individuals worldwide who were allergic to pollens demonstrated that individuals from a variety of geographical areas possessed IgE antibodies that recognized the GST-KBG51 fusion protein. On the basis of these findings, CRAL51 represents a member of a family of highly cross-reactive ALs in plant pollens.

Description

WO9~119437 2 ~ 8 ~ 1 r~ ~ PCT/CA95/00021 TITLE QF 1~ l~V~ UN
CROSS-~F''"~'~VE aT.TT~T~
FIELD OF lNv~ LloN
The present invention is related to the f ield of molecular genetics and is particularly rnnr~7nP~ with the cloning of a cro 3s-reactive allergen .
K~ :N~; TO RRT.2-~T~n APPLICATION
This appli~cation is a rnnt;n11~tion-in-part of rnp~-n~;n~ United States patent ~rPl ir~t;n~ Serial No.
08/181,383 filed January 14, 1994.
BAr_~r~ KUUNL~ TO THE INVEN-TION
Pollen allergens are multidet~rrnin int proteins or glycoproteins capable of eliciting IgE-r '; ~t~orl allergic diseases such as hayfever and asthma in approximately 17%
of the pop1~1~ti~n who are g~n~tjr~l1y predisposed to develop allergies (ref. l - Throughout this srer;~;r~t;nn, various references are referred to in par~nthP~; ~ to more fully describe the state of the art to which this il1vention pertains. Full bibliographic infnrr-t;nn for each citation is found at the end of the disclosure. The disclosure of these references are hereby incorporated by ref erence into the present disclosure) . In contrast to some other allergens (e . g ., cat or houfie dust mite allergens), the global distribution of pollens of a large variety of monocotyledonous (gras5es) and dicotyledonous plants (trees and weeds) preclude any realistic pnss;h;1;ties that individuals allergic to pollen allergens can avoid these aero-allergens. The current treatment for hayfever consists primarily in symptomatic relief. Sufferers take drugs, such as an~i-histamines and steroids, which do not suppress the formation of IgE ~nt;horl;~ and often have harmful side effe:cts.
Attempts to ~ yulate the IgE immune r~pnn~ of allergic subject13 by the "time-h~ ,ul,=d~' immunotherapy consist of a series of inj ections of increasing amounts W0 95/l9437 ~ PC}/CA95/OOa21 of the allergenic extracts of the ~y~ U~Liate pollen or pollen-mixtures over prolonged periods lasting usually 3 to 5 years. Most of the pollen extracts used th_L~ r~l l y are crude mixtures of a multiplicity of 5 rhPm;r~l constituents, some of these , ~ bearing no relation whatsoever to the few allergenic ~ 'L""" "~ Q
which are act~ally r~RrrnQ;hle for a given patient's lly~eL~e~lsitivi~y. Because some of the proteins present in these extracts may not be al}ergeng, gtandar-l; 7At; nn 10 of allergenic ~Yt~A~rtQ based on total protein content is an unreliable guide for rl~t~rm;n;n? the potency of an extract. :- ~ oveL, large (up to lOOx) variations in allergen conte:nt occur in the prPr~t;nnC used for immunotherapy IDecause of the different methods used for 15 (i) pollen collection and storage, which lead to variations in ~aw --tor;Al R from lot to lot and from year to year, and ( ii) the extraction ~Lu~duLe8 . 1l~ euveL ~
although diffe~-ent r~t;Pnt~ may be allergic to different constituents of a given pollen, all r~A~t; Pnt Q receive 20 injections of the "same" complex mixture cnnt~;n;n~ all the constituents of different pollens, i.e., they receive even ~ ts to which they may not be allergic. It i5, therefore, not surprising that treatment with an ill-defined pollen extract may lead to the ;n~llrtion Of 25 additional IgE ;lnt;hori;~c~ i.e., to sensitiZ~t;nn of the patients to nei,r ~ t~ (refs. 2 to 6).
While up to 809~ of patient8 gain rl ;n;r~Al ;, uv~ from this immunotherapy (refs. 7, 8), the risk of side effects, the lengthy course of therapy, the 30 inconvenience to the patient of the mode and fre~uency of administration, and thê r ;nrJ costs of this t~.-limit the ut ility of the current immunotherapy.Although, local and systemic rP~rt;nnQ may occur as a result of this therapy, they may be managed by a 35 physician Rrer;Al;7~d in allergy. However, ocrARinnS~lly this mode of treatment is aQsor; ~tP~ with the risk of wo 95/19437 2 ~ ~ t 1 4 2 PCT/cAss/00021 severe ~nt` ~;r or anaphylactic reArr;nnQ~ which can result in deat~1 ~refa. 9, lO).
To ~1 ;m;nAIte some of the above di6advantages of the allergenic ~ .L.~Lions currently used for; ~hP--apy, 5 one of the major objectives in allergy research ha6 been the ; Rn] At j nn _nd rharArt~r; 7At i nn of the individual allergens of t.he complex repertoire of allergens of a given pollen by phyQi co~ h~m; ~-~1 and i ~ Al methods. Several lAhnr~Ator;~Q have iRn7Ated some of the lO allergens from the crude aqueous extracts of grass pollens by the use of rls~QQ;rAl physiorh~m;cAl methods and reverse i - - R consisting of I ~; 1; 7~d murine I --~nnAl Ant;horl;P-Q to the pollen constituents (refs. ll, 12). Although such; ~ 1 methods 15 appear to be yL, R;nrJ for the characterization of individual allergens, the main drawback of these ~LL~ ly labour intenQive pur;f;~-At;nn methods is the minute yield of allergens. ~Lt:UVe~, these methods do not ensure AhAol-lte purity of the allergenic constituents 20 and, therefore, the ~l~tPrm;nAt;nn of their amino acid sequences is difficult, if not ;~roQ~;hle. As a corollary, the dev~l.), of new theL~,ue:~Lic derivativeQ
of grass polle1l allergens and of reliable diagnostic procedures for pollen allergies are severely restricted 25 by the u_e of allergens ; RO1 At~cl by the existing PLUCe~ULe8~ Holiever, recent innovations in re~ ' nant DNA (rDNA) ter-hnr~ Qy have paved the way for the synthesis of allergenic proteins, and of their epitopic LL _ R r~RF~nQ;hle for their activation of the 30 d~LUyLiate B a:Dd T cells leading interactively to IgE
f n~ t; nn, on a;l industrial scale and in a consistently pure state.
Upon initial ~05UL~ to allergenic constituents, they are ;nt-o nAl;- ~ by antigen-pr~R~nt;n~J cells ~APC), 35 which include nnnAntirJPn-Rrer;f;c phagocytic cells or specific B cell,s, and ~re "~L~,~es~d" by these cells.

2 ~ 4 ~ PCTICA95/00021 The net effect of thil3 ~Lc,~ ;n~ i8 the breakdown of the Slnt;~nR into ]?~rt;A;c A~t~rm;n~ntR which, in turn, are re-~L~8J~d in aR~nr;~t;nn with class I or class II
~ec~ Q of t]~e major hist~ - ;h;lity complex ~C) 5 on the surfsce of the APC. S ~ u- l ly, the binary peptide~ C ~ Y~ inter~ct with the corrF~cponA;-r~
Rrer;f;~r receptor5 ~TCR) of, respectively, (i) Th cells or (ii) CTLs or Ts cells, and the r~Rlllt;n~ triads ,l~t~rminF. the up- or down-re~-l~tinn of the ~-y~Lu~Liate 10 B cells (ref. 13).
On the basis of their l~, k;n~- secretion patterns, the Th cell s~hFop~l~r;nn may be further subdivided into three subsets, i.e., ThO, Thl and Th2 cells ~ref. 14).
In mice and man, the Th2 cells have been shown to produce 15 IL-4, IL-~ and IL-6, and IL-4 has been shown to activate B cells leading to the proAl~rt;nn of IgE ~nt;hoA;-~g. In contrast, the 1'hl cells produce I~r, which blocks the proA~rt;nn of IL-4. The r -` ; (8) involved in the suppression of antibody responses by Ts cells is still 20 not fully understood. It has been suggested that the suppression of Antibody prnA-~rtinn is due to inactivation of Rrer;f;C Th cells as a result of non-proff~R~Rk--~l APC, i.e. a T-cell (ref. 15).
The primar~ reaction of the IgE Ant;hQA;~ secreted 25 from the B~ cells is their binding to sp~r;f;~ IgE
receptors on the surface of mast cells, h~Fh;lR and eoR;nnrh;lR. On re-e~.~ODuLe of the patient to the specific multivalent allergen, the cell-fixed IgE
~nt;ho~ R react with and are cro~sl ;nk~d by the 30 allergenic molecules. This process leads to the release from these cells of rh~m;r~ tors of anaphylaYis.
In turn, these '; ~tr~rs act rapidly on the smooth muscles of di~i-erent target organs, resulting in the ;nfll tory mallifestations characteristic of; ';:~t~
35 type hy~eLDeusitivity.

- WO 9!ill943? 2 1 8 t 1 4 ~ PCT/~5,00021 .--The devel~, of th. Al~ - 1 ;r strategies ~ref. 16) that may ;nf I~P~re the fr~-tj nn of IgE ~ntiho~; e~
requires ~rtsl'i 1 P~ knowledge of (i) the DL-u~:Lu~ ~5 of individual allergens and, in particular, of their B cell 5 epitopes (whi~h are r~co3n;7-d by IgE/IgG :Int;horl;PQ) ~
lii) DL~u-_Lu ~3 r-ro3n; ' by M~C l~r~lP (i.e., Ia epitope), and (iii) the DLL~.Lu ~8 rerorJn; e~ by T cell receptors of l'h or Ts cells, known as T cell epitopes.
Therefore, ~C~t~;n~tir~n of primary s~ nr~Q of 10 individual pollen allergens of a complex repertoire of allergenQ of a give3l pollen by ; ' CR 1 and physiorhPm;r~l methods is of central importance, and has been a major objective in allergy research for a long time. ~owever, using these rl~QQir~l methods, the 15 progress in; Qr~l ~t; rn and characterization of allergens has been slow. For example, for ragweed pollen, which is one of the main allergenic polle:ls in North America, in spite of intensive studies of over 50 years only six allergens (i.e., A:nb a I, Amb a II, Amb a III, Amb a V, 20 Amo a VI and Amb t V) have been purified and characterized ~refs. 17 to 19). Similarly among grass pollens, one may cite pollens of Ryegrass, Timothy grass and Kentucky B~uegrass, and many others, which have been used for the isolation and characterization of their 25 protein allergens (refs. 20 to 25).
Similar to other protein antigens, the ep; tor~Q of allergenic proteins are either ~r,nt;nllr--Q or ~l;Rrrnt;nllrllQ. Generally, ~nnt;nllrllQ antige~ic epitopes can be lor ~ d to 3~_ Q ~ of amino acid 30 residues in a linear 8e~lu ~ e, whereag ~;Qrr,nt;nl~rllQ
(confor~--t; nnzll ) epitopes comprise residues which appear adjacent to on~. another on the protein surface, but are widely separated in their primary sequence. The latter epitopes depend on the native ~ u~ ation of the 35 protein. Conv~nt;rn~l methods for ;~t;f;r~tirn of B
cell epitopes consist of probing a polyspe~;f;~ antiserum Wo95/19437 2 ~ 8 1 ~ 4 ~ PCTICA95/00021 or a set of --lnnAl Ant;ho~;eq produced against the intact antigen with cleavage E _ c of antigen or synthetic pDrt~ DC, which may yield ;nfc~ t;nn on cnnt;nllnuq ,or;t-op~c (reEs. 26 to 27). Thus, on the basis of amino acid ~e~ nre data for ~3~ a III, Atassi and his AQqOr;At~q 8ynthD~; ' ten ovD~lArr;n~ p~nt~ r~L. l-Lides which L~L~ LCd the entire ~ rl-l e (ref .
28) . These pepti~C served to ~orAl ~e four antigenic sites in Amh _ III that were recogn;7Qd by IgG Antiho~ c in human, rahbit and murine antisera. It is noted that the same regions were also rec c~sn; 7ed by human IgE
Ant; ho~l; DC (reE . 29) .
Furthermo:re, by c~o~rl; n~ partially-purified ragweed pollen allergens to m hnYypolyethylene glycol lmPEG), it was shown that the resulting conjugates were (i) not only d~void of allergenicity and; ,_; city of the original ragweed pollen constituents in pAti~ntc and mice, respectively, (ii) but were also capable of ;n~l-c-;n~ a lon~-lasting suppression of IgE Ant;horl;~c in mice (ref. 30). It was also reported that conjugates of poly- (N-vinyl pyrrolidone) with Timothy grass pollen allergens suppressed the estAhl; Ch~.l IgE rDcpnnq~c to these allergens in mice (ref. 30).
Because of the limitation of the above r~ ACC; ~Al purif ication m~thods which yield only minute amounts of pure allergens, recently some investigators have used the rDNA methods fc~r the study of allergens. Thus, allergens present in dust mite (refs. 31 to 33), hornet venom (ref.
34), birch pollen (ref. 35) and grass pollens (ref. 36) have been cloned and the respective allergens yLu-luced by the Arpl;r~tinn of rDNA terhn;cr~Dc.
On the l~asis of secrlF-nre homology and cross-reactivities, the grass allergens cloned to-date may be r~Acc;f;e~ to two main groups, one group includes allergens Of 1l1 and 35 IcDa in size (ref. 13) and the other group ; nrl .1~3Dc allergens of 28 to 34 lcDa in size .

W095119437 2181 1 4~ pcrlcAsslnon2l pl~hl;nhPc~ A;s~n pate~t ~rrl; ~t;~n No. 2,066,801, in which we are named as coi.~ LuLd (O~ILL~ lin~ to .,~pPnrlin~ United Stateg patent ~rpl;r~t;on~ Serial No.
filed , the ~;crlosl~~e of 5 which is ir...~L~L~sted herein by reference) discloses the cloning of the cDNAs of three ; ~OAl l P--gen6 of Kentucky Blue Grass ~Q~L ~ratensis ~BG) pollen and ;-;Pnt;fied -lo~ir:llly; _ Lc~-L r~giong within thege latter group of allergens. These allergens are present in some lO other grasses and are thus u6eful for ~; r~nnsi ~ of and ~i~i7in~ for gra6g-sperif;c allergie~i.
As ~Yr1~;nPd above, pollens of a wide variety of grasses and other plants produce aeroallergens.
Ii~eLcfo~ e~ it would be ~l~R; rahl e to provide a cross-15 reactive allergen that is present in many yrasses andother plant poLlens. It would also be useful to provide the cDNA clone of such a cross-reactive allergen.
S~MMARY OF lN Vs!.w .L lUN
The present invention is directed toward 20 j uvc r~ in allergen-sper;fic immunotherapy by providing the means for ~veL~ 'n~ the lack of pure allergens or peptides corrP~pnn~ to portions thereof, as referred to above, by the ~i~t~rm;nat;nn of DNA
sequences codi~lg for a novel group of proteins of plant 25 pollens. This group of allergens, though constituting major allergenic _ ~-ltR of plant pollens, r~ inc~
nnl nn~d prior to this invention.
Accordingly, the present invention provides, in one aspect, a purified and ;~ol~ted nucleic acid i l~c-lle 30 comprising at least a portion coding for an allergenic protein which is present in pollens from both - LylP~nrml~ and dicotyledonous plants.
The m ~ Lyl~nnnuFl plants are ~n~ l l y tho6e selected from the family ~r---;nf-~P. particularly grasse6

3 5 including Berm~da grass, Kentucky Blue grass, Red Top grass, Reed Canary grass, Rye grass, Timothy grass, Brome Wo 95119437 ~ P(:T/CA95/00021 B
grass, Orchard grass and cultivated cor~. The dicoty~eArnn~ plants include trees, for example, birch, and weeds, for example, ragweed, particularly fihort ragweed, and pllrietaria.
The portil~n of the nucleic acid molecule coding for the allergenic protein provided herein generally is selected from:
(a) the DNA ler~1le inrl11~l;nrJ the DNA se~u~ set out in Figure 1 or its . ~ ~ry strand;
(b) a D~l~ i lecl~ nroA;nrJ a protein ;nrlllA;nrJ the amino acid 3e ~ re set f orth in Figure 2; and (c) DNA ~ which hybridize under stringent conditions to the DNA 8~-~r~ nr~ defined in (a) or (b), and is conserved among the r - ~ _ Lyledonous and dicotyl~Annn~ plants. Preferably, the DNA seo,uences defined in (c) have at least about 75~ se~"u~..ce identity with the 8~ defined in ~a) or (b).
In anothi~r aspect of the invention, the prese~t invention pro~ides a re n~n~ pla8mid adopted for 2 0 transf ormation of a host, comprising a plasmid vector into which has been inserted a DNA segment comprising at least a 15 bp E _ ~ of a DNA r 1 Pr~l e as provided herein. The present invention also i nrl l-A~ an expression vector adapted for transformation of a host, 25 comprising at least a DNA segment comprising at least a 15 bp C~ _ of a DNA molecule as provided herein and expression means operatively coupled to the DNA segment for expression thereof in a host. In particular, the DNA
segment encodes an allergenic protein ;nrluA;nrJ the amino 30 acid ser~ nre ~of Figure 2.
The DNA segment rnnt~;n~'Cl in the expression vector may further comprise a nucleic acid s~S~u~Ce ~nro~l;n J a carrier protei~, for example, glut~th;nn~-S-transferase (GST), ~-s~l~rtn~ e or protein A, for expression of a 35 carrier-allergen fusion. One particular expression vector provided herein is plasmid pNKl, deposited with .

wo g5/19437 2 1 8 1 1 4 2 PCT/CA9~0002l the r ~r~-n Type Culture Collection, Rockville, Mary-land, l:~. S .~., Qn January lO , 1994 and having ATCC
;r,n number 75,634.
An additil~nal afipect of the invention provides a 5 L~ ~ n~nt protein ~L~,-luc~d by e~L~ ion in the host of the DNA rL__ rnn~ain~d in the e..~,L~ssion vector provided hereill or a fl~nrt; rn~l analog of the protein.
In this Arpl i~atirn, a first protein is a "fl7nrt;rnal analog" of a ~econd protein if the f irst protein is ; -lo~; rAl l~ related to and/or has the same function as the second protein. The fllnrt;~7n~l analog may be, for example, a LL _ of the protein or a substitution addition or ~l~l ~t; nn mutant thereof . The present invention also ;nrl-~ synthetic allergenic or Ant;gen;r peptides corr~prn~;n~ in amino acid sequence to portions of the re: ' rl~nt protein or allergen.
The present invention provides, in a further aspect, a composition Eor protect;n~ allergic individuals from developing an allergic reaction, comprising at least one active ~ t E~lert~ from at least one nucleic acid molecule and at: least one L~_ ' n:~nt protein, provided in acc~L~ce with aspects of the invention, and a pharmaceutically-acceptable carrier therefor, particularly f~LI 1 ~t~1 for n vivo administration.
In such; -,_- ic compositions, the composition may comprise at least one re. ' in~nt protein conjugated to a non-; ,_L~iC substrate, which may be a polymeric material, for example, a ~L~ y -~hyl rolllll ~rse, a -hr~ypolyethylene glycol and a polyvinyl alcohol.
The non-; , ;r ~uL_LL~Le may comprise beads for targeted uptakel of the at least one re~ ' nant protein by antigen-pr~nt; n~ cells .
The composition may be foL, l ~ted as a microparticle, capsule or l i, - preparation and may be 35 provided a - ' ;n~tirn with a targeting l~c~ for - Wo 95/l9437 2 l ~ ~ t ~ 2 PCTICA95/00021 delivery to ~p,-ri f; c cell6 of the immune system or to mucosal surf ac~s .
The ~ t i nn provided in this aspect of the invention may }~e nF-cl with at least one additionsl 5 desensitizing agent, which may be s~ rted from Poa }2 IX
allergen, ~Ql }2 I allergen, Bet y I allergen, }~k a I
alleryen and ~ a II allergen. In Rri~itinn, the ,-~itir~n ma~ comprise at least one ~ _ ' having anti-histamine activity and/or at least one C
10 having anti-;nfl: nry activity and/or at least one ' which is i - ~ ~ssive. The composition also may comprise an a lj uv~...L .
In an adclitional aspect, the present invention provides a method for desensitizing an allergic 15 individual, particularly a human, by administering an effective amount of the composition provided herein, par~ir~lRrly on~ rnntRin;n~ the at least one additional desensitizing agent.
The presen~: invention provides, in a further aspect, 20 a method of depleting allergen-~pr~rif;r Ant;hQr~;t,s, from an individual, particularly a human, by rrntRrt;n~ the Rnt; hr~lr~ r~ with the composition provided herein to f orm a complex, and removing the complex frQm the individual.
In another aspect, the present invention provides a 25 method of anergizing allergen-specific antibody-producing cells, particular in a human, by contacting the cells with the compos:ition provided herein.
The present invention also provides, in an additional aspect, an antiserum sp~r;f;~ for a 3 0 r~ nRnt prol:ein as provided herein.
R~ r~ DESCRIPTION OF DRAWINGS
Figure 1 sllows the nucleotide s~ r~nre (SEQ ID NO:
1~ and the deduced amino acid cr~ e (SEQ ID NO: 2) of a portion of a cDlU~ clone (K~3G 51~ r~ht~;n~d by screening 35 a Kentucky Blueyrass pollen - Agtll library;

WO 95/19g3~ PCT/CA95100021 21~t 1~2 Figure 2 rnntA;n~ Northern blot analysis of RLG
pollen mRNA using KBG 30 as lane 1, K~3G 51 as lane 2 and K3G 60 as lane 3 insert DNAs as probes;
Figure 3 illustrates ~lPtprti c~n of homology in mRNA
5 in a ~umoer of pollen by RT-PrR followed by SollthPrn analysis using K~3G 51 cDNA as the probe;
Figure 4 illustrates vector plasmid pNHl and the cloning site for K~3G 51;
Figure 5 illustrates high-level expression of KsG 51 10 in the pGEX-2T-1 system; and Figure 6 crnt:~;n~ Western blots of a variety of pollen extracts ~lt;l;7;n~ antiserum to GST-K3G 51 fusion protei~ .
r.~N~R~T, DESCRIPTION OF INVENTION
It is clearly c,~a~e-lL to one skilled in the art, that the vario~s ~ of the present invention have many appl:ications in the fields of diagrLosis and therapy of allergic diseases, such as allergic rhinitis, asthma, f ood allergies and atopic eczema . In a 20 diagnostic: ' '; , the demonstrated cross-reactivity of the allergen is particularly useful. Thus, as described above, the usual method of determining to which aeroallergens an individual is allergic involves an intra-dermal e~O2~ULe to many crude extracts of allerger, 25 extracts (for example, mixtures of grasses, weeds and trees). This ~LLIceduLe involves the use and administration of many allergens. The cross-reactive allergen of th~! present invention (~rer;firAlly CRA~51) now allows for the demonstration of IgE An~;ho~l;e~
3 0 against a whole range of allergens . An allergic individual who has CR~L51 specific IgE/An~;hs~;es can now be immediately excluded from being allergic to this whole range of allergens. A further non-limiting discussion of such uses i8 f~lrther presented below.

Wo 95/19437 PCT/C, 9Sl00021 7 ~

Pr-r- at;n~ and U~e o~ Composition for Prot~cting Allergic Indivi~ual~ for Developing an Allersic RRaction --;t;n~c, suitable to be used for protecting allergic indi~iduals from developing an allergic reaction, may be ~Le~eLL~d from CR~L51 allergen, analogs, ~_ _ c and,/or peptides as ~i; Rrl os~cl herein.
Compositions may be ~L~=paLed as inje~t~hl~c, as liSiuid 8Olllt;nnc or: lRinnC. The CRA~51 allergen f~
analogs or peptides may be mixed with rh~rr-reutically-acceptable ~Yr;ri~ntc which are - ;hle with the allergen proteins, LL _ analogs or peptides.
~Yr;ri~ntc may include, water, saline, dextrose, glycerol, ethanol, and ' ;n~t;ons thereof. The composition may further contain minor amounts of ~llY;l;~ry substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness t3lereof. Methods of achieving an adjuvant effect for the compositions includes the use of agents, such as aluminum hydroxide or 3?hnsrh~te (alum), commonly used as about 0.05 to about 0.1 percent snlllt;nn in rhnSph~te buffered saline. Compositions may be administered parenterally, by injection sllhcut~n~nusly or intr~r lcclll ~rly. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, f or example, polyalkylene gl}~cols or triglycerides. Oral for~ t;~nR
may include normally employed ;n~-;r;~ntc~ such as, for example, pharrriaceutical grades of saccharine, cellulose, ~ n~cillm r~rh^n~te and the like. These compositions take the form of solutions, sucr~nc;nnc, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 959~ of the allergen ~L _ analogs and/or 3?eptides.
3 5 The compositions are administered in a manner ~;hlP with the dosage form.ulation, and in such Wo 95ll9437 13 PCr/C~95/00021 amount as is theL~ l ;CA11Y effective to protect allergic individuals from developing an allergic reaction. The quantity to be administered depends on the subject to be treated, ;nrl~ ;nr,, for example, the 5 capacity of the individual~s immune system to synthPc;~e Ant;hori;PR. Precise amounts of allergen required to be administered clepends on the j~d!~ of the practitioner. However, suitable dosage ranges are readily ~PtPr~;nAh]~ by one skilled in the art and may be 10 of the order of n~--oyL.~...,, to mi- Luy ~ of the allergen, analog fragment and/or peptides. Suitable regimens for initial admini~;tration and booster does are al60 variable, but IDay include an initial administration followed by sub~sequent administrations. The dosage of 15 the compositior~ may also depend on the route of administration and will vary according to the size of the host .
As described above, the cross-reactive allergen may be conjugated to a non-; r~Pn;c substrate including 20 polymeric materials, such as ~ L~.y, thyl cP~ cpc/
monomethoxypolye~thylene glycols (mPEG) and polyvinyl alcohols, to rem~er it non-; -_, ;r and non-allergenic for protecting ,~llergic individuals from developing an allergic reaction. mPEG conjugates to a variety of 25 allergens have been tested in human clinical trials ~ref.
4s). The avA;lAh;l;ty of a cross-reactive allergen in a purif ied f orm in large amounts now makes it possible to syn~hPc; 7e well defined conjugates for these molecules .
The nucleic acid molecules ~nro~;nr~ the allergen of 3 o the present invention or portions thereof may also be used directly for; ;~:~t;r,n by admingtration of the DNA directly, i or example, by injection for genetic ; 7~t; rn or by constructing a live vector, such as ,CA1 ^llA, BCG, adenovirus, poxvirus, vaccinia or 35 poliovirus. A discussion of some live vectors that have been used to ca:rry heterologous Ant;~Pnc to the immune WO95/19437 2 1 ~ 2 PCTICA95/00021 system are ~i qc~lqs~l in, ~or example, O'~agan (1992 ref. 46) . P ~,c~s~ases for the direct injection of DNA into test subjects for genetic; ;7at;r,n are degcribed in, for example, Ulm!er et al., 1993 (ref. 47).
The use of E~eptides corr~cpnn~;n~ to portions of the cross-reactive allergen in Yivo may first require their rh~m;rAl r ~;f;~rAtinn, since the p~pt;~i"c themselves may not have a suf f iciently long serum and/or tissue half -life. Such rh~T;rA7ly, ';f;ed peptides are referred to - 10 herein as "peptide analogs~. The term "peptide analog"
extends to any functional rh~m; ~l equivalent of a peptide characterized by its inCreased stability and/or ef f icacy in vivo or in vitro in respect of the practice of the invention. The term "peptide analog" i8 also used herein to extenl to any amino acid derivative of the peptides as A~qr~; h~l herein. Peptide analogs rnntl l~ted herein are produced by procedures that include, but ar~ not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of cross-linkers and other methods which impose confor--t;c~n~l constraint on the peptides or their analogs .
33xamples of side chain r- ';f;r~tir~nC cnnt~mrlAted by the present in~ention include modif ication of amino groups, such as 3~y reductive alkylation by reaction with an aldehyde followed by reduction with NaB3~ m;~At;l n with methylAret;m;~9~te; acetylation with acetic anhydride; carbamylation of amino groups with cyanate;
3 o trinitrobenzylation of amino groups with 2, 4, 6, trinitrnhen7~n~0 13ulfonic acid (TNBS); alkylation of amino groups with 8--rr; n; c anhydride and tetrahydrorhthAl; c anhydride; and p~ridoxylation of lysine with pyridoxa-5' -phosphate followed by r~ ct;nn with NaBH,.
The ~lAn;~linn group of arginine residues may be , ~;fied by ~he formation of heterocyclic cnn~nCAt;nn W095/19437 2 1 ~ 1 1 4 2 PCT/C~95/00021 products with reagents, such as 2, 3-~utAn-~;nnF-, phenylglyoxal and glyoxal.
The carboxyl group may be, ~ifiPrl by r~rho~;im;~;~
activation via o-acylisour~a frr~-t;rn followed by 5 subsequent deriv~t;cat;~n, for example, to a L:ULL~ ; n~ amide .
Sulfhydryl groups may be ' f; ed by methods, such as caL~u.~ hylation with ; ~rrinAreti c acid or ir~ r~Pret;~ ; p~:LLoLllliC acid nY;~l~t;r~n to cysteic acid;
10 fr~rm~t;r,n of mixed ~;Clllrh;~Fc with other thiol -c; reaction with maleimide; maleic anhydride or other substitut~d maleimide; f ormation of mercurial derivatives lLsing 4-chluLI .uLibenzoate, 4-ChlULI - .uLiphenylsulfonicacid, phenylmercuryrhlorirl~, 15 2-chluL, ~ uLic-4-niLLu~he~lol and other mercurials;
carbamylation with cyanate at ~lk~1 ;n~ p~.
Tryptophan residues may be ';f;ed by, for example, rY;rl~t;rn with ~-bromosllrr;n;m;~ or alkylation of the indole ring with 2-hydroxy-5-niLLub~ ;yl kromide or 20 sulphonyl halides. Tryosine residues may be altered by nitration with tetraniLL ~h~n~ to form a 3-nitrotyrosine derivative.
;f;r~t;0n of the imidazole ring of a histidine residue may be a~ ,1 j Ch~d by alkylation with iodoacetic 25 acid derivatives or N-r~rhethrrylation with diethylpyrocArhrn~te .
Examples of incoL~uL..ting unnatural amino acids and derivatives durirLg peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-30 amino-3-hydroxy-5-phenylrPnt~ ic acid, 6-Am;nr~hF~Y~nric acid-, t-buty}glycine, norvaline, phenylglycine, ornithine, sarco6ine, 4-amino-3-hydroxy-6-methylh-~rt~nr;c acid, 2-thienyl alanine and/or D-isomers of amino acids.
The purif ied and isolated DNA molecules comprising 35 at least a portion coding for an allergenic protein which is present in pollens from both - Lyledo~Lous and Wo 95119437 PCT/CA95/00021 2~81 1~2 dicotyledonous plants typified by the ~"~o~; c described herein are adv~nt~eo 1c as:
- nucleic acid probes f or the specif ic nt;f;~:-t;vn ~f other allergens that contain a DNA
5 ~ re compriF~ing at least a portion coding f or the cross-reactive allergen CRAL51 or analogs thereof;
- the products encoded by the DNA molecules are useful as diagno3tic reagents to identify CRAL51 specific IgE Ant;h~i;Pc i~l an individual to demonstrate or exclude lO allergy to a whole range of allergens;
- peptides ~:~.ILL. C~ ;nrJ to portions of the allergen as typified by the : ' - '; c described herein are advantageous as diagnostic reagents, antigens f or the pro~rt;nn of allergen-specific antisera, for example, 15 f or the demonstration of an allergic reaction to a whole range of allerg~ns.
T -- v Sl Zly~
The cross-reactive allergen, analog, LL~ t and/or peptide of the present invention are useful as 20 ; ~ s, as a~ltigens in; --c8~yS including enzyme-linked; -- 7 assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or ~LUCe~UL.S known in the art for the detection of allergen specific IgE
;~nt;hor3;es. Ill ELISA assays, the allergen, analog, 25 fra; and/or peptide corr~c~nn~;n~ to portions of the allergen are ; ; l; zed onto a selected surface, for example, a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely ~Co~h~d allergen, analog, 30 fragment and/or peptide, a nr~ncrec;f;c protein, such as bovine serum albumin (BSA) or casein that is known to be antigenically neutral with regard to the test sample, may be bound to t~Le selected surface. This allows for blork;n~ of r~rncper;f;r adsorption sites on the 35 ; ,h;1;7;n~ surface and thus reduces the ba~.k~L-,u--d caused by n~ncper;f;c h;nrl;n~C of antisera onto the Wo 95/19437 PCT~CA~100021 ~ 218t 142 surface. Normally, the peptides are at least 12 residues in length and pref erably 14 to 3 0 residues . It i5 understood however, that a mixture of peptides may be used either as an; _ in a composition or as a 5 ~ nnst; c agent: .
The; ';'I;~;n~ 8urface is then rnntArted with a sample, such as rl; n; rA1 or biolo~; rAl materials to be tested in a manner conducive to immune complex (antigen/antibody) formation. This may include ~ t;ng lO the sample with diluents, such as solutions of BSA, bovine gamma ~lnh1-l;n ~BGG) and/or phosphate buffered saline ~P5S) /T~een. The sample is then allowed to inrllh;lte for from about 2 to about 4 hours, at temperatures, s1lch as of the order of about 25- to about 15 37 C. Following inr11h~t;nn, the sample-cnntArted surface is washed to remove non-; - lexed material. The washing procedure may include washing with a solution, such as PBS/Tween, or a borate buffer.
Following f ormation of specif ic ; - 1 exes 20 between the test sample and the bound allergen, analog, LL _ and/o~ peptide, and subsequent washing, the oc.uLLe..ce, and even amount, of; ~ Y formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first 25 antibody. If the test sample is of human origin, the second antibody would be an antibody having sper;f;cjty for human IgE or IgG Ant;hoA;~A. To provide detecting means, the second antibody may have an associated activity, such as an enzymatic activity that will 3 0 generate, f or example, a color dev~ upon ;nr1~hAt;nS with an ay~ ,Liate e1-L j ;c substrate.
Quantification may then achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer .

WO 95/19437 PCr/C~DOO2~
~ 21~ 142 UHel of 5~ - Z~ yh~i ~ Probo~l The nucleotide se~ r~ of the present invention, comprising the seguence of the cross-reactive allergenic protein, now allow for the ;~ntif;cation and cloning of 5 the allergenic protein genes from other sources.
The nucleotide sequences comprising the s~ nre of the allergenic protein of the present invention are useful for their ability to selectively fonm duplex molecules with , l~ ~ry stretches of other 10 allergenic protein genes. ~rpen~;n~ on the ~rPl ir:~t;nn, a variety of hybri~l;7~t;nn conditions may be employed to achieve varying degrees of selectivity of the probe toward the othe:r allergenic protein genes. For a high degree of selectivity, relatively stringent conditions 15 are used to f onrl the duplexes, such as low salt and/or high temperature conditions, such as provided by 0 . 02 M
to 0.15 M NaCl ilt t - eLLu-~=s of between about 50 C to 70 C. For some applicationg, lesg ~:trin~nt hybri~i; 7~tirn conditions are required such as 0 .15 M to 20 o.9 M salt, at temperatures ranging from between about 20 C to 55 C. EIybrjr~;7~tinn conditions can 8180 be rendered more ~tringent by the addition of increasing amounts of fnrr~~;~P, to dest~hil;7e the hybrid duplex.
Thus, particulal~ hybr;~i7~tirn conditions can be readily 2~ r-n;rulAted, and will generally be a method of choice l~r~n~;nS on the desired results.
A wide variety of appropriate indicator means are known in the art for detenmining hybri~;7~t;rn, ;nr~llA;n~
radioactive, ~,-zy tir or other ligands, such as 30 avidin/biotin, which are capable of providing a A~tect~hle signal. In some ~ '-'; ~, an en2yme tag such as urease, ;llk;-l ;n~ rhrDlh~t~e or peroxidase, instead of a radioactive tag may be used. In the case of enzyme tags, colorimetric ;n~;r~tnr substrates are known 35 which can be employed to provide a means visible to the human eye or spe-.LLu~hotometrically, to identify sp~c; f; r Wo 9S/19437 PCT1CA9S/00021 2~81 ~42 hybri~ tinn wi.th samples cnnt~;n;ng allergenic protein gene secr~nr~R
The nucleic acid ~ nr~R of allergenic protein genes of the present invention are useful as hybri~;7~t;nn p:robes in solution hybri~;7~t;nnR and in ~mho~l; R employing solid-phase PLUC~e1U~ eS. In emhO~ involving solid phase ~Luce~u~ eS, the test DNA (or RNA) from samples, is ~lRn~hed or otherwiae affixed to a s/~lected matrix or surface. The fixed, single-stranded nucleic acid is then subjected to sperifir hybr;tii7~t;nn with selected probes comprising the nucleic acid sec~uences of the allergenic protein genes or fragments thereof of the present invention under desired conditic)ns. The selected conditions will depend on the particular circumstances based on the particular criteria recluired A~r~n~l;n5 on, for example, on the G+C
rnnt~ntR, type c)f target nucleic acid, source of nucleic acid, size of hybr;~;7~tinn probe etc. Following washing of the hybritl;7~t;nn surface 80 as to remove non-specifically bound probe molecules, Rrer;f;r hybri~;7~t;nn i8 detected, or even crl~nt;f;~o~, by means of the label. 'rhe selected probe should be at least 18 bp and is preferably in the range of 30 bp to 9o bp long.
E~ression of t}le Allergenic Protein G~n~6 Plasmid vectors rnnt~inin~ replicon and control secluences which are derived f rom species - ~t; hl e with the host cell l~ay be used f or the expression of the allergenic protein genes in expression systems. The vector ordinarily carries a r~rl;r~t;nn site, as well as 3 o marking sec~uences which are capable of providing phenotypic selection in transformed cells. For e~cample, E~. coli may be transformed using p3R322 which C~nnt:~;nR
genes for ir;llin and tetracycline resistance and thus provides easy means for identifying tr~nRf -I cells.
The p~3R322 plasn1id, or other microbial plasmid or phage, must also contain, or be ';f;~cl to contain, promoters ~ WO95/19437 2 1 8 1 1 42 PCr/CA9~00021 which can be used by the microbial organi~m f or expression of its own proteins.
In addition, phage vectors rnntA;n;nr~ replicon and control se~ "r~A that are , ~ ;h~ ~ with the host S mi.;LuoLyCu~ism can be used as a transforming vector in rnnnf.rt; nn with these hosts . For example, the phage in lambda GEM~-ll may be ut; 1; 7--Cl in making rero--~inAnt phage vectors which can be used to transform host cells, such as E. coli LE392.
Promoters commonly used in re~ ' ;n:lnt DNA
con6truction include the ~ rt Ae (p~n;r;ll;n~e) and lactose ~L- t~r systems (Chang et al., 1978 (ref. 48):
Itakura et al., 1977 ~ref. 49); Goeddel et al., 1979 (ref. 50); Goedlel et al., 1980 ~ref. 51) ) and other 15 microbial promoters, such as the T7 promoter system.
Details rnnr~rn; nrj the nucleotide :ie~u~ s of promoters are known, ~nAhl;n~ a skilled worker to ligate them fl-nrt;nnAlly with plasmid vectors. The particular promoter used will generally be a matter of choice 20 ~r~n~;nS upon the desired results. Hosts that are a~L~Liate fo~ expression of the allergen genes, f~ q, analogs or variants thereof include E. coli, BacilluA species, fungi, yeast, higher eukaryotic cells, such as CE~O cells, or the baculovirus expression system 25 may be used.
In accordance with this invention, it is preferred to make the allergenic protein, ~L _ ' or analog by L~_ ' ;n ~nt met}lods. Particularly desirable hosts for expression include Gram positive bacteria which do not 30 have IJPS and are therefore ~n~tnY;n free. Such hosts include species of Bacillus and may be particularly useful for the production of allergenic protein, f ragments or analogs thereof .
The allergenic protein may also be produced as a 35 fusion protein with, for example, gll~tAth;nno-S-transferase, ,B-galactnsj~lAAe and protein A.

~ WO95/19437 2 ~ 8 1 1 42 pCI~/CA951D0021 R; ~lr,~J; r~ 1 Depos it~
A plasmid pNH1 ~pUM 94.1~ that rrnt~;nQ at least a portion coding for an allergenic protein that is described and referred to herein has been deposited with 5 the American Type Culture r~ rt i ~rn ~ATCC) located at Rockville, Maryland USA ~uL~ t to the Budapest Treaty on January lO, 1994 and has been accorded ;qrcF~ ;rn number 75, 634 . ~ further plasmid pUM 94 .2 ~KBG 15) also rrnt~;n;n~ at least a portion coding for an allergenic 10 protein has been deposited with ATCC on and has been accorded accession number . Samples of the deposited plasmids will become available to the public upon grarlt of a patent based upon this United States patent ap~lication. The invention described and 15 claimed herein is not to be limited in scope 3~y the plasmid deposite!d, since the deposited embodimlent is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as described in this application are 20 within the scope of the invention.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by ref erence to the f ollowing specif ic Examples . These Examples are described solely for purposes of 25 illustration and are not ; ntPn~ to limit the scope of the invention. Although specific terms have been employed herein, such terms are ;nt~nrl~-d in a descriptive sense and not for purposes of limitations. T ~ 'rjiCal and r~ ~ ;n~nt DNA methods may not be explicitly 3 0 described in thi.s disclosure but are well within the scope of those s3cilled in the art.
EXAMPLES
Examle This Example describes the cloning and 35 sequencing of a cDNA ~nr~;n~ a portion of the cross-reactive allergell (CRAL 51).

9437 PCT/C~95/00021 WO95/1 2~ 8~ 1 42 Certain aspects of the cloning of allergen_~n~-o~; n~
cDNAs is described in the afvL ;nn~ n~ n patent pl;cPtinn No. 2,066,801 (and corr~crnn~;n~ USA
08/ ).
Total polle~l RNA was ~Ytr~ctecl from 35 g of mature KBG pollen (~ollister-Stier, Miles IJaboratories, Toronto) uaing the ~u~n;ti;n~ isothyocyanate-cesium chloride method (ref. 37). Sc~n~nin~ electron mi~;Lus~u~y confirmed that the pollen was that of ~Q~L pratensis and was free of any cnnt~m;n:-nt~ Poly(a) +RNA was prepared by affinity C}~l n~r~rhy 011 poly-lr Sepharose (Pharmacia, Toronto) .
Plaques (250, 000) from a ~gtll library (ref . 38) were screened; -]o~;C~lly for the expression of pollen allergenc using pooled sera of eight p~t;~nt~
allergic to KBG pollen. Patients were selected on the basis of case history, positive skin prick and radioallerosnrh~nt tests but had not u-ldeLyu--e any hyposensit; ~t;nn tr~P ~. Plaques were lifted onto nitroc~ 1 ose ~.ilters (Bio-Rad, Toronto) and washed twice for 5 and 30 minute intervals in 30 mls of buffer (50 mM sodium I)h~sph~te~ pH 7.5, o.59~ v/v Tween 20, 0.5~
bovine serum albumin (BSA), 0 . 05~ sodium azide w/v) per filter. The filters were then allowed to hybridize overnight at 4 'C under slight agitation in buf f er cnnt~;n;nS O.ol~ pooled sera. The filters were then washed at 4-C, for three 15 minute intervals in 25 ml of buffer per filter, and ;nAllh~ted overnight at 15-C with goat-anti-human IgE conjugated with ~1 k~l; nf~ rhnsrhz't:~Re .
The f ilters were re-washed as previously described and developed using 5-bromo-4-chloro-3-ind31ylrhn~Fh~te p-toluidine and nitroblue tetrazolium chloride. Positive clones were picked and re-screened, one of these clones, (KBG51) was further characterized.
DNA was prepared from plaque purif ied phage using the liquid lysate method (ref. 39). Inserts L~-uvel~d from Eco RI digestion were ligated into the multiple wo 95/19437 PCT/CA95/00021 2181 ~2 cloning site (MCS) of pBluescript and used to transform E. coli D3~5CY.
The seqllon~ of the cDNA clone 3~BG51 ~SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) are 5 shown in Figure 1. cDNA KBG51 is a partial cDNA clone with an open reading f rame . The deduced polypeptide comprises 145 amino acids with an estimated Mr of 16.636 kDa and pI of 12 . 02 . Comparison of the nucleotide and deduced amino acid s~ Pn~ q with the GenBank DNA and PIR
10 protein s~ nre Data Banks, respectively indicated that the sequences were unique and not ~ lcsollq to any other known allergen or non-allergen DNA or proteins in these Data Banks. T3lus, the cloned cDNA encodes a novel allergen which has not been ~oRr~; h~rl previously.
15 ~Y 1~1 e 2 This Example describes the demonstration of m3~NA
species in 3~BG pollen correqp~n~l;n~ to the 3~BG51 cDNA.
Total pollen and leaf RNA was electrophoresed in 1.5~6 agarose gels under dewatering conditions (ref. 40) 2 o and blotted onto nylon membrane .
Northern blot hybri~i7~tinn using radit~l~hP1l~
3~BG51 cDNA insert revealed that the corr~qrnnrling transcripts are present in KBG pollen but not in leaf tissue (Figure :'). Northern hybridization using cDNA
25 probes corr~oqrnn~; n T to allergens of two other cDNAs belonging to Poa 3~ IX used in this eYperiment as controls indicated that transcripts corr~qp~n~in~ to these cDNAs were --Y;~-11y 1.5 kh in length. The transcripts of this size has also not been reported previously for any grass 30 allergen cDNAs. On the basis of the size of this transcript it is concluded that these transcripts encode a group of alle~-gens, cDNAs of which have not yet been described .
The results of this analysis are shown in Figure 2 35 and 8 L- ate that m3~NA transcripts of about 2 . O kb and about 1.4 kh (lane 2 of Figure 3) were ~l~t~c~hle in WO 9S119437 PCT/CA9Sl00021 2t8t 142 pollen mRNA but not in leaf mRNA from E3G pollen.
Furthermore, these mRNA species differed from those ~PRr~;hP~ in ~'~n~ n patent ~rpl;rAt;nn No. 2,066,801 as shown in lanes 1 (KBG 31) and 3 (KBG 60).
Exam~le 3 This ExaTrlple demonstrates the presence of transcripts corr~Rpnn~ to KBG51 in a variety of other pollens .
Total RNA prepared from the pollens indicated in Figure 3 as ~Pl~r~ihed in Example 1 was subjected to reverse transcri ption reaction . Thus, the f irst strand cDNA was synthP~; 7ed in a 50 ILl final volume at 42 C for 1 h using a 5 ~g of total RNA, reverse transcriptase and oligo dT primer. The double strand cDNA was 5ynth~ci7~
using DNA polymerase and dNTP according to the manufacturers instructions (BRL, ~; RR; Rs~ a, ON) . The products nhtA;n~d were heat denatured and an aliquot of 5 ~l was ~It;l;7Pd in amplification reaction with specific primers (DNA Synthesis Laboratory, Univ. of Manitoba) for the RBG51 cDNA The primers are shown on the DNA
sequence of Figure 1 and had the ser~ nr~C KBG, forward 5' - TCT TGG CTT GAC CGA AGC (SEQ ID NO: 3) and KBG, reverse 5 ' - GAT ACA GCC CAT CAC CGC ( SEQ ID NO: 4 ) .
The PCR amplification (ref. 41) was carried out in a buffer rnnt~;nlnr 50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 0.15~ (w/v) gelatin, 200 ~LM dNTP, 2.5 Taq polymerase (Cetus Perkin Elmer) and 50 pmol of each primer. The reaction consisted of 30 cycles of A~ ir~t;n" followed by a 15 min. final l~ytpnc;r~n at 72-C. Each cycle included denaturation at 95 C for 30 sec, Ann~ l;nr at 65-C for 60 sec. and ~tPnR;nn at 72-C
for 2 min. The 2mplified products were then precipitated with ethanol, run on an agarose gel, transferred to nylon membranes and hybridized under stringent conditions with random-primed KLG51 cDNA insert. The results of this analysis are 8hown in Figure 3 and ; nll; r~te that mRNA

Wo 95/19437 2 ! 8 1 1 ~ 2 PCTICA95/00021 from all pol~.ens tested ~nntA;n~d a transcript corr~Rpnn~ to KBG51. This analysis demonstrated that the allergen e~lcoded by RBG51 is present in a wide variety of pla lt pollens and is therefore a cross-reactive allerge.n. Analysis of nucleotide sequences of the PCR E _ c ; n~; rAt~ that the s~riu~nr~a of the l;fied cDNAs were ;rl~nt;C:~l to that of clone 51.
r le 4 This Example describes the production of a GST-KBG51 fusion protein.
For ide~tification of the polyp~rti-l~a encoded by the KBG51 cDNA clone, the insert was cloned in the pGEX
2T-1 expression vector (ref. 42) to produce plasmid pNHl (Figure 4) which permitted high-level expression of the GST-KBG51 fusion protein ~Figure 6). The fusion protein was ~ tss~d as a soluble fusion protein of apprrY;r~t~ly 43 kDa in conjunction with the glutathione-S-transferase (GST), which upon cleavage yielded the GST
of about 26 kDa. The protein expression was estimated to be about 209~ of the total cell extract or about 2 mg/g wet wt of cells. Total crude proteins were recovered form the host cells and the fusion protein was purified by electrophoresis on 109~ SDS-PAGE followed by electroelution from the polyacrylamide gel to produce the GST-KBG51 fusion protein as shown in lane 3 of Figure 5.
Exam~le 5 This Example describes the generation of GST-KBG51 fusion protein sperlfic antiserum and its cros6 reactivity to proteins in other plant pollens.
The GST-K~3G51 fusion protein with dextran sulfate as adjuvant, was u~ed as immunogen to immunize 6 to 8 week old female BDFl mice ~Jackson Laboratories, Bar Harbour Michigan) . The mice were ; ; 7ed ~l-hcutAn~nual y and after 23 days the mice were boosted with the same antigen preparation. Bl~od was cr,~ ct~cl at day 30 after primary ;7~tion and, serum was ~JL~ L~

wossll9437 2 1 8 1 1 4 2 PCT/CA95/0002l Proteins frt~m various pollens (as shown in Figure 6) were ;AolAted as previously ~cr~;hed (ref. 17) and electrophoresed on a 15~ polyacrylamide gel. Proteins were then stained with ~ csi~ slue or electroblotted 5 onto a nitrocellulose membrane. r~ L-l~es were blocked with 1% gelatin in TBS (lo mM Tris-HCl pH7.5, 150 mr~
NaCl ) and probed with the antiserum raised against GST-KBG51. T ~active bands were d~terted using an ;llk:ol;n~ rh~c,Fh~t~ce conjugated goat anti-mouse second 10 antibody.
A Western }~lot analysis (ref. 43) employing the polyclonal murine antiserum to the GST-KsG51 fusion protein led to the ;~nt;f;c~tion of two c Antc in the KBG extract, a major having an approximate 15 Mr of 59 kDa and a minor c~ n~nt having an approximate Mr- of 30 kDa (Fi.gure 6 lane 3 and Table 1 (the Tables appear at the end of the disclosure) ) . A Western blot analysis of a number of pollen extracts showed that a number of polypeptides in these extracts reacted with the 20 antiserum indicating the presence of cross-reactive allergens in these pollens. Table 1 summarizes the polypeptides recogn; 7~d by the antiserum in protein extracts ~L~ Lt:d from a number of different grasses, weeds and tree pollens. This antibody reco~nized two 25 high molecular weight polypeptides in the ryegrass pollen extract which has not been reported previously. From these studies it is c~n~ that the allergen encoded by the KBG51 cDNP~ comprises a very highly cross-reactive group of allergells in plant pollens.
3 0 ExamDle 6 This Example describes the percentage of the allergic population that have K}3G51 specif ic IgE
~nt; hof~; ~,c .
The sera of apprr~ t~l y lOOo individuals who were 35 allergic to a broad spectrum of allergens, ;nl ~ ;n5 WO95/19437 2 1 ~ 1 1 4 2 PCT/C~95/00021 pollen, were Aq~'R~ed for their ability to biIld GST-K~3G51 fusion protein in an ELISA assay Iref. 44) .
GST-K~3G51 protein (10 ~Lg/ml) was coated to wells of microtiterplatel3 (Nunc, T ,lAte~ Denmark) overnight.
5 The wells were further blocked with rhnsrhAte buffer t nntA;n;nq 1~ gelatin for 8 hrs. Subsequent to washing 5 times with rh~ h~e buffer cnntA;n;n~ 0.053~ Tween 20, the wells were ; nr~lhAted with human serum diluted with the washing buffer cnntA;nins 0.19~ gelatin overnight.
10 After washing, A'17~Al;nP Fhn8rhAtAqe-conjugated Se~nn~Ary :Int;h~ies (goat anti-human IgE (TAGO), diluted 300-fold) were added and the wells were incubated overnight.
Following washing with rhncFhAte buffer ~nntA;nlnS Tween 20, the color reaction was developed by adding the 15 substrate para-nitro phenyl rhnsphAte (1 mg/ml) in 0.1 M
Tris-HCl buffer cnntA;nln~ NaCl and MgCl~ and the OD was measured at 405 nm in a Titertek Multiscan ~e~L~.~hotometer ~Flow Laboratories, McLean, VA) .
The results of the ELISA assays are shown in Table 20 2 and indicate that individuals from a variety of geographical areas posRosRed IgE Ant; ho~l; 8 that recosn; 70d the GST-K~3G51 fusion protein. This indicates that the indiYiduals have been sensitized to CRAL51, portion6 or analogs thereof and suggests that CRAL51 25 allergen is a broadly cross reactive allergen present in many aeroallergens to which individuals are routinely exposed .
SUMMMY OF THE DISCLOSURE
In summary ~f this disclosure, the present invention 30 provides certain novel nucleic acid molecules which code for allergenic ~proteins present in a wide O~e.Llu.., of plant pollens. ModifirAtlnnq are posR;hle within the scope of this invention .

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~ r~ tD
. r ~t~ 0 O ~ ~ O ~r ~ _ WO95119437 2 1 8 1 1 4 2 PCT/C~ss/0002l Table 2 Human IgE binding of GST-K3G51 fusion protein using a panel of int~rn~t;nn~l ~era by ELISA
Laboratory Numbe~ Number of IgE positive sera Number of ~e~a with Sper; f; rj ty for (Country) ,Oy~m;ni d KBG extract GST-KBG51 fusion protein 5 (U.S.A. ) 9~ 16 16 466-03 (U.S.A. ) 20 7 1351-10 (U.S.A. ) 23 7 5 489-18 (U.S.A. ) 100 27 14 397-27 g 4 2 315-06 (Columbia) 89 17 26 13 (Japan) lO0 26 17 20 (Japan) 33 10 4 33 (Japan) 33 16 ll 36 (Japan) 7~. 25 8 1487-35 (Taiwan) l l' 0 2 l9 (Denmark) 25 14 0

4 (Sweden) 94 43 21 233-26 (Italy) 67 29 11 233-28 (Italy) 36 22 16 1488-34 (Bulgaria) 65 39 18 25 (Israel~ 3~) 8 6 O 951l9437 PCTICA9~00021 W 21g~2 1. Firedhoff LR. In: Genetic and EnYiL, ~1 factors in Clinical Allergy, l~arsh DG and Bl h:ll MN (eds . ) Univ-of Minnesota Press (1989).
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Immunol. Mounro-AshlTlan (ed. ), Elsevier pp. 394 (1974

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193 ( 1986 ) .

21. Johnson P, Marsh DG. Eur. Polymer J. 1: 63 (1965~.44.

22. Johnson P, Marsh DG T ~ try 3: 91 (1966~.

W095/19437 2 ~ 8 1 1 4 2 PCT~CA95100021

23. John50n P, Mar8h DG. T ` ~try 3: lO1 (1966).

24. Ansari AA. et al. J. Biol. Chem. 264: 11181 (1989) .

25. Ansari AA. et al. R;orh~m;Ç~try 28: 8665 (1989).

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27. Esch RE and Rlapper DG. Mol. Immunol. 26: 557 (1989) .

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29. Kurisake J. et al. Eur. J. Immunol. 16: 236 (1986).

30. Sehon A~I. Prog. Allergy 32: 161 (1982).

31. Chua RY. et al. J. Exp. Med 167: 175 (1988).

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35. Breitenader }~. et al. EMBO J. 8: 1935 (1989) .

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43. Zhang I. et al. Int:. Arch. Allergy Appl. Immunol. 96:28-34 ( 1991 )

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45. Dreborg S. and ~k~.rht~mh E., Lit. Review Ther. Drug Carrier Sept., CRC Press, ~ol. 6, p315-365 (1990) .

46. O'~lagan (1992) Clin. PhaL k;n~t. 22:1 (1992).

47. Ulmer et al., (1993) Curr. Opinion Invest. Drugs. 2 (9):
983 -989 .

48. Chang et al., (197E3) Nature 375:615.

~/0 95119437 ~ 1 8 1 1 ~ 2 PCT/CA95100021

49. Itakura et al., ~1977) Science 198:1056.

50. Goeddel et al., (1979) Nature 281:544.

51. Goeddel et al., ~1980) Nucl. Acids Res. 8:4057.

~VO 95119437 PCT/CA95/00021 SEQ~ENOE LISTING
( 1 ) GENERAL ~ r~
( i ) APP_}CANT:
A NAME: UhiverGity of Manitoba 3I STREET: 311 Ad~rini3tration Building C CITY: Winnipeg D 3TATE: Manitoba E COUNTRY: Canada FI POSTAL CODE (ZIP): R3T 2N2 G TELEP~IONE: (204) 474-8418 3~ TELEFAX: (204) 261-0325 ~A NAME: Shyam S. M~hAr~r~A
~B STREET: 364 T.;n-~n~And Drive East C CITY: Winnipeg D STATE: Maritoba E ~ COUNTRY: Canada F~ POSTAL CODE (ZIP): R3P 2~1 A) NAME: Alec ~I. Sehon B) STREET: 695 Academy Road C) CITY: Winnipeg D) STATE: Manitoba E) CODNTRY: Canada F) POSTAL CODE (ZIP): R3N OE8 (ii) TITLE OF lJ!I\/~ lU81: CROSS-REACTIVE ALLERGEN
(iii) NUMBER OF SEQUENCES: 2 iv) COM'UTER READA3LE FORM:
A MEDIUM TYPE: Floppy disk Bl COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
D SOFTWARE: PatentIr. Release #1.0, Version #1.30 (EPO) ( 2 ) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE ~T~ rT~TsTIcs:
A LENGT~}: 504 base pairs B TYPE: nucleic acid C~ ST~r~r'---c single D ~ TOPOLOGY: linear ix ) FEATURE:
~A) NAME/I~EY: CDS
~B) LOCATION: 7 . . 441 ~xi) SE0IJENCE ~ ~lrLl~: SEQ ID NO: 1:

Leu Arg Asp Val Leu Ala Ala Gln Cy8 Leu Asp Leu Leu Ala Val Asp Glu Pis Arg Arg Gly Arg Ile Leu Ser Arg Pro Arg Gln Ala WO 95119437 2 1 8 ~ 1 4 2 PCT/CA95100021 GAT GCC GAT ATT CGC CTG CTT CGA CTC GCC CGG ACC GTT G~C G~T ACA 144 Asp Ala Asp Ile Arg Leu Leu Arg Leu Ala Arg Thr Val Asp Asp Thr Ala E~is i5 Arg }~is Val ~is Leu Leu Asp Ala Arg Ile Leu Ile Ala Pro ~li5 Arg }lis Leu Arg Thr Gln Ile Gly Leu Asp Leu Arg Arg Gln 65 70 7s Phe Leu Glu Gln Cys Ala Gly Gly Ala Ala Thr Thr Arg Ala Arg A~p 80 85 go CAC CAT CGG CGT GAA CGA GCG CAG ACC CAT GGT CTG CAG GAT TTC CTG
E~is His Arg Arg Glu Arg Ala Gln Thr llis Gly Leu Gln Asp Phe Leu 336 Thr Asp Asp }lis Leu Ala Gly hla Arg Ala Ala Arg Phe Arg Arg Gln Arg Asp Ala Asp Arg Val Ala ~p Ala Leu Leu Gln Gl u Tyr Gly Gln CGC AGC GGT Tr~rr,rrDrr Ai~. ~.-~ Dnrrr~Trrr ~ ,,, 481 Arg Ser Gly D7,r~rr~r~T nr~rrnrr~ TTC 504 ~2) INFORMATION FOR SEQ ID NO 2 ( i ) SEQl~ENCE r7T~ L~ 'S -(A) LENGT~ 145 ami no acids IB) TYPE amino acid (D) TOPOLOGY line~r (ii) MOLECULE TYPE protein (xi) SEQUENOE LIL;~ L~ J~I: SEQ ID NO 2 Leu Arg Asp Val Leu Ala Ala Gln Cys Leu Asp Leu Leu Ala Val Asp lu EIis Arg Arg Gly Arg Ile Leu Ser Arg Pro Arg Gln Ala Asp Ala sp Ile Arg Leu Leu Arg Leu Ala Arg Thr Val A8p Asp Thr Ala EIis ~Iis Arg Eis Val ~lis Leu Leu A~p Ala Arg Ile Leu Ile Ala Pro ~lis Arg Elis Leu Arg Thr Gln Ile Gly Leu Asp Leu Arg Arg Gln Phe Leu Glu Gln Cys Ala Gly Gly Ala Ala Thr Thr Arg Ala Arg Asp ~is Elis Arg Arg Glu Arg Al~ Gln Thr Ilis Gly Leu Gln Asp Phe Leu Thr As W095/19437 2 ~ 8 1 1 42 Pcr/~95/0002l .

Asp Xis Leu Ala Gly Ala Arg Ala Ala Arg Phe Arg Arg Gln arg Asp Ala Asp Arg V~l Aln Asp Ala I.eu Leu Gl~ Gln Tyr Gly Gln Arg Ser Gl ( 2 ) 1~ TrN FOR SEO ID X'0: 3:
(i) SEQ~IOE rT~ rT~Tc~rTrc A I~GTX: 18 ba~e pairs B~ TYPE: nucleic acid C ::, : sillgle D TOPOI,OGY: linear (ii) MOLECULE TYPE: cDXA
(xi) SEQ~lENCE l,J~Kll:'~lL'rl: SEQ ID NO:3:
l ~ l l ~L l l i ACCGAAGC

(2) l~r~ --Tr,~ FOR SEQ ID X10:4:
( i ~ SEQI~EIIOE rT~
A LEI~GTX: 18 ~aso pairs 3I TYPE: nucleic ~cid C b : iingle D TOPOLOGY: linel~r (ii) MOLECULE TYPE: cDXlA
(xi) SEQIJENOE J~L$~I~llL'l~: SEQ ID X0:4:
naT~r~r.rrr ~lrcAcCGC 18

Claims (35)

What we claim is:

1. A purified and isolated nucleic acid molecule, the molecule comprising at least a portion coding for an allergenic protein which is present in pollens from both monocotyledonous and dicotyledonous plants, the portion coding for the allergenic protein being selected from:
(a) a DNA molecule including the DNA sequence set out in Figure 1 or its complementary strand;
(b) a DNA molecule encoding a protein including the amino acid sequence set out in Figure 2; and (c) a DNA sequence which hybridizes under stringent conditions to the DNA molecules defined in (a) or (b) and is conserved among said monocotyledonous and dicotyledonous plants.

2. The nucleic acid molecule of claim 1 wherein said monocotyledonous plants are selected from the family Gramineae.

3. The nucleic acid molecule of claim 2 wherein said monocotyledonous plants are selected from Bermuda grass, Kentucky Blue grass, Red Top grass, Reed Canary grass, Rye grass Timothy grass, Brome grass, Orchard grass and cultivated corn.

4. The nucleic acid molecule of claim 1 wherein said dicotyledonous plants are selected from trees and weeds.

5. The nucleic acid molecule of claim 4 wherein said tree is a Birch tree and said weeds are selected from ragweed and parietaria.

6. The nucleic acid molecule of claim 5 wherein said ragweed is short ragweed.

7. The nucleic acid molecule of claim 1 wherein said DNA sequences defined in (c) have at least about 75%
sequence identity with sequences defined in (a) or (b).

8. A recombinant plasmid adapted for transformation of a host, comprising a plasmid vector into which has been inserted a DNA segment comprising at least a 15 bp fragment of a nucleic acid molecule of claim 1.

9. An expression vector adapted for transformation of a host, comprising at least a DNA segment comprising at least a 15 bp fragment of a nucleic acid molecule of claim 1 and expression means operatively coupled to the DNA segment for expression thereof in the host.

10. The expression vector of claim 9 wherein said DNA
segment further comprises a nucleic acid sequence encoding a carrier protein for expressing a carrier-allergen fusion.

11. The expression vector of claim 10 wherein said carrier protein is selected from glutathione-S-transferase, .beta.-galactosidase and protein A.

12. The expression vector of claim 9 which is plasmid pNH1 having ATCC accession number 75, 634.

13. A recombinant protein produced by expression in said host of the DNA fragment contained in the expression vector of claim 9 or a functional analog of the protein.

14. A composition for protecting allergic individuals from developing an allergic reaction, comprising at least one active component selected from at least one nucleic acid molecule of claim 1 and at least one recombinant protein of claim 13, and a pharmaceutically-acceptable carrier therefor.

15. The composition of claim 14 formulated as a vaccine for in vivo administration.

16. The composition of claim 15 wherein said vaccine comprises said at least one recombinant protein conjugated to a non-immunogenic substrate.

17. The composition of claim 16 wherein said non-immunogenic substrate is selected from polymeric materials.

18. The composition of claim 17 wherein said polymeric material is selected from carboxymethyl celluloses, monomethoxypolyethylene glycols and polyvinyl alcohols.

19. The composition of claim 16 wherein said non-immunogenic substrate comprise beads for targeted uptake of said at least one recombinant protein by selected antigen-presenting cells.

20. The composition of claim 14 formulated as a microparticle, capsule or liposome preparation.

21. The composition of claim 14 in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces.

22. The composition of claim 15 comprising at least one additional desensitizing agent.

23. The composition of claim 22 wherein said at least one additional desensitizing agent is selected from Poa p IX allergen, Lol p I allergen, Bet v I allergen, Amb a I allergen and Amb a II antigen.

24. The composition of claim 15 further comprising at least one compound having anti-histamine activity, and/or at least one compound have anti-inflammatory activity and/or at least one compound which is immunosuppressive.

25. The composition of claim 14 further comprising an adjuvant.

26. The composition of claim 14 wherein said at least one nucleic acid molecule is contained in a vector.

27. The composition of claim 26 wherein the vector is selected from Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus.

28. A method for desensitizing an allergic individual, comprising administering to the individual an effective amount of the composition of claim 14.

29 . The method of claim 28 wherein the individual is a human.

30. The method of claim 29 wherein said immunogenic composition is that of claim 23.

31. A method of depleting allergen-specific antibodies from an individual, comprising contacting said antibodies with said composition of claim 14 to form a complex, and removing the complex from the individual.

32. The method of claim 31, wherein said individual is a human.

33. A method of anergizing allergen-specific antibody-producing cells, comprising contacting said cells with said composition of claim 16.

34. A method for diagnosing an allergic reaction to pollens from both monocotyledenous and dicotyledenous plants, which comprises administering to an individual with the recombinant protein of claim 13, and evaluating a response to said administration.

35. A method for diagnosing an allergic reaction to pollens from both monocotyledenous and dicotyledenous plants, which comprising contacting serum from an individual with the recombinant protein of claim 13, and determining the formation of a complex between said recombinant protein and pollen-specific IgE antibodies present in said serum.