Note: Descriptions are shown in the official language in which they were submitted.
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.
WO95/19437 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
11
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 ~
12
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
14
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
17
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
18
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
19
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
- 20
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
- 22
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
23
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
24
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
26
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 .
WO 95119437 2 1 ~ t 1 4 2 PCT/CA95100021
28
m .C
v E 'I r ~D N rO ~D INn
E tEa
r In ,~ ~
O O 1~ ~D 1~) IU r~ ~D
r~ rD In ~ ~ r' In
~ lJ
r r r m
I r t~ ~ m ~.
m .~ r~l m
.
r~ m
~D 1~1
~ m In In
~ ~ o 01 ~n tt; r;
ttl ~D ~D In N
,1 m m In
r E-~ D ~ ~
t~ ~
rl ~ ~ o
_~ ~ ~ tD ~D m r
E l .4 - In
o o
D ~ 1~)
O ~1
r ~ ~
r m
ta m rl r~ r~ r~ m
~1 ID ~D F)
~_ m
m
,n In ~D
115 ~1 ~D 'd~ m rD 1~) O
r' ~ O Q m
tn o ID
mm "
~ r~ tD
. r ~t~ 0
O ~ ~ O ~r ~ _
WO95119437 2 1 8 1 1 4 2 PCT/C~ss/0002l
29
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
1137 11 10
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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(eds.) pp. 285 (193).
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21. Johnson P, Marsh DG. Eur. Polymer J. 1: 63 (1965~.44.
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31
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~/0 95119437 ~ 1 8 1 1 ~ 2 PCT/CA95100021
32
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~VO 95119437 PCT/CA95/00021
2 1 8 1 1 42
33
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:
GAATTC TTG CGC GAT GTT CTC GCC GCG CAG TGT CTT GAC CTT CTC GCC 48
Leu Arg Asp Val Leu Ala Ala Gln Cy8 Leu Asp Leu Leu Ala
5 10
GTC GAT GAA CAC CGG CGC GGC CGG ATT CTC TCC CGA CCC CGG CAA GCT 96
Val Asp Glu Pis Arg Arg Gly Arg Ile Leu Ser Arg Pro Arg Gln Ala
15 20 25 30
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
35 40 45
GCC CAT CAC CGC CAC GTG CAT CTT CTC GAC GCC AGG ATA CTG ATC GCG 192
Ala E~is i5 Arg }~is Val ~is Leu Leu Asp Ala Arg Ile Leu Ile Ala
50 55 60
CCA CAC CGG CAT CTG CGT ACG CAG ATA GGT CTG GAT TTG CGA CGC CAG Z40
Pro ~li5 Arg }lis Leu Arg Thr Gln Ile Gly Leu Asp Leu Arg Arg Gln
65 70 7s
TTC CTG GAA CAG TGT GCT GGT GGT GCG GCC ACA ACC CGG GCA CGC GAT Z88
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
95 100 105 110
ACC GAC GAC CAC CTC GCC GGT GCG CGG GCC GCC CGG TTC CGG CGT CAG 384
Thr Asp Asp }lis Leu Ala Gly hla Arg Ala Ala Arg Phe Arg Arg Gln
115 120 125
CGA GAT GCG GAT CGT GTC GCC GAT GCC CTG CTG CAG CAA TAC GGA CAA 432
Arg Asp Ala Asp Arg Val Ala ~p Ala Leu Leu Gln Gl u Tyr Gly Gln
130 135 140
CGC AGC GGT Tr~rr,rrDrr Ai~. ~.-~ Dnrrr~Trrr ~ ,,, 481
Arg Ser Gly
145
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
20 25 30
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
50 55 60
Arg Elis Leu Arg Thr Gln Ile Gly Leu Asp Leu Arg Arg Gln Phe Leu
65 70 75 80
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
100 105 110
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
130 135 140
Gl
145
( 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
18
(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
