CA2144055C - Cloning and sequencing of allergens of dermatophagoides (house dust mite) - Google Patents

Abstract

The present invention features isolated DNA encoding allergens of Dermatophagoides (house dust mites) particularly of the species Dermatophagoides farinae and Dermatophagoides pteronyssinus, which are protein allergens or peptides which include at least one epitope of the protein allergen. In particular, the invention provides DNA encoding the major D. farinae allergens, Der f I and Der f II and DNA encoding the major D. pteronyssinus allergens, Der p I and Der p II. The present invention further relates to proteins and peptides encoded by the isolated D. farinae and D. pteronyssinus DNA, including proteins containing sequence polymorphisms. In addition, the proteins or peptides encoded by the isolated DNA, their use as diagnostic and therapeutic reagents and methods of diagnosing and treating sensitivity to house dust mite allergens, are disclosed.

Description

~''LONING AND SEQUENCING OF ALLERGENS
OF DERMATOP AGOIDES (HOUSE DUST MITEI
Descri tn ion Recent reports have documented the importance of responses to the Group I and Group II allergens in house dust mite allergy. For example, it has io been documented that over 60% of patients have at least 50% of their anti-mite antibodies directed towards these proteins (Lind, P. ~L, IIerQV, X2:259-274 ( 1984); van der Zee, J.S. gala, ~. AllerQV Clin. Immunol., $1:884-896 ( 1988)). It is possible that children show a greater degree of reactivity (Thompson, P.J.
, ImmunoloQV x:311-314 (1988)). Allergy to mites of the genus is Dermatophag~ides (p.) is associated with conditions such as asthma, rhinitis and ectopic dermatitis. Two species, D. pteron, ss~ and ~~, predominate and, as a result, considerable effort has been expended in trying to identify the allergens produced by these two species. D. pteron" ssy inus mites are the most common Dermatonhagoides species in house dust in Western Europe and 2o Australia. The species j~farinae predominates in other countries, such as North America and Japan (Wharton, G.W., J. Medical Entom, x:577-621 (1976)). It has long been recognized that allergy to mites of this genus is associated with diseases such as asthma, rhinitis and atopic dermatitis. It is still not clear what allergens produced by these mites are responsible for the allergic response and 2s associated conditions.
The present invention relates to isolated DNA which encodes a protein allergen of Dermatoph~goides ((p.) house dust mite) or a peptide which 3o includes at least one epitope of a protein allergen of a house dust mite of the genus Dermatophago, ides. It particularly relates to DNA encoding major allergens of the species D.D. farinae, designated Der f I and Der f II, or portions of these major allergens (i.e., peptides which include at least one epitope of Der f I or of Der f II).
It also particularly relates to DNA encoding maj or allergens of D. pteron, ssinus, 3s designated ~ I and j2~ II, or portions of these major allergens (i.e., peptides which include at least one epitope of Der n I or of p~2 II).

la The invention provides a protein allergen of Der n II comprising the amino acid sequence:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Xaal Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Xaa2 Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Xaa3 Arg Asp where Xaal is selected from the group consisting of Thr and Ser;
where Xaa2 is selected from the group consisting of Asp and Asn; and where Xaa3 is selected from the group consisting of Ile and Lue, except for the amino acid sequence where Xaal is Thr, Xaa2 is Asp and Xaa3 is Ile.
The invention also provides a protein allergen of Der f II comprising the amino acid sequence:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn Xaal Glu Ile Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Xaa2 Glu Xaa3 Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Xaa4 Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp XaaS Lys Tyr lb Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Xaa6 Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp where Xaal is selected from the group consisting of Asn and Ser;
where Xaa2 is selected from the group consisting of Thr and Ile;
where Xaa3 is selected from the group consisting of Ile and Thr;
where Xaa4 is selected from the group consisting of Met and Val;
where XaaS is selected from the group consisting of Ala and Ile; and where Xaa6 is selected from the group consisting of Val and Ile, with the proviso that when Xaal is Asn, then Xaa3 is Thr; and when Xaa3 is Ile, then Xaal is Ser.

2144p~~

The present invention further relates to proteins and peptides encoded by the isolated Dermato h~agoides (e.g., D i , D. pteronyssinus) DNA including proteins containing sequence polymorphisms. Several nucleotide and resulting amino acid sequence polymorphisms have been discovered in the Der n I, per n II and Der f II allergens. All such nucleotide variations and proteins, or portions thereof, containing a sequence polymorphism are within the scope of the invention.
Peptides of the present invention include at least one epitope of a D. farinae allergen (e.g., at least one epitope of Der I or Der f II) or at least one epitope of a D. ~teron, ss.~' inus allergen (e.g., at least one epitope of Der n I or of D_ er n II). It also relates to antibodies specific for D. ri proteins or peptides and to antibodies specific for D. tn eronyssinus proteins or peptides.
Dermatonhagoides DNA, proteins and peptides of the present invention are useful for diagnostic and therapeutic purposes. For example, isolated D.
ina proteins or peptides can be used to detect sensitivity in an individual to house dust mites and can be used to treat sensitivity (reduce sensitivity or desensitize) in an individual, to whom therapeutically effective quantities of the D. farinae protein or peptide is administered. For example, isolated D. farinae protein allergen, such as Der f I or Der f II, can be administered periodically, using standard techniques, to an individual in order to desensitize the individual. Alternatively, a peptide which includes at least one epitope of Der f I or of Der f II can be administered for this purpose. Isolated D. tp eronvssinus protein allergen, such as Der n I or 1II, can be administered as described for Der f I or Der f II. Similarly, a peptide which includes at least one D r~ I epitope or at least one Der n II epitope can be administered for this purpose. A combination of these proteins or peptides (e.g., Der ff I and Der f II; Dery I and Der n II; or a mixture of both Der f and p~ proteins) can also be administered.
The use of such isolated proteins or peptides provides a means of desensitizing individuals to important house dust mite allergens.
Brief Descr~ion of the Drawing Figures lA and 1B show the nucleotide and predicted amino acid sequence of cDNA ~,gt 11 p 1 ( 13T) (SEQ ID NOS: 1 and 2, respectively).
Numbers to the right are nucleotide positions whereas numbers above the sequence are amino acid positions. Positive amino acid residue numbers correspond to the sequence of the mature excreted Der n I beginning with threonine. Negative sequence numbers refer to the proposed transient pre- and preproenzyme forms of Der n I. The arrows indicate the beginning of the proposed proenzyme sequence and the mature Der n I, respectively. Residues -15 to -13 enclosed by an open box make AMEWED SHEET
IPEA/EP

2~.4~0~~

up the proposed cleavage for the proenzyme formation, and the dashed residues ~2-54 represent a potential N-glycosylation site. The termination TAA codon and the adjacent polyadenylation signal are underlined. Amino acid residues 1-41, 79-95, 11 I -142, and 162-179 correspond to known tryptic peptide sequences determined by conventional amino acid sequencing analysis.
Fi r shows the restriction map of the cDNA insert of clone ~,gt 11 p 1 ( 13T) and the strategy of DNA sequencing. Arrows indicate directions in which sequences were read.
Fi r is a comparison of N-terminal sequences of D~ I and D r I. The amino acid sequence for Der n I is equivalent to amino acids 1-20 in Figures lA and 1B; the Der f I sequence is from reference (12).
F. igu~e 4 shows the reactivity of ~,gt 11 p 1 ( 13T) with anti-Der n I.
Lysates from Y1089 lysogens induced for phage were reacted by dot-blot with rabbit anti-Der nn I (Der nn I) or normal rabbit serum (Nrs). Dots (2m1) were made in triplicate from lysates of bacteria infected with ~,gt 11 p 1 ( 13T) (a) or ~,gt 11 (b).
When developed with 125I_protein A and autoradiography only the reaction between ~,gt 11 p 1 ( I 3T) lysate and the anti-Der n I showed reactivity.
Figure 5 shows reaction of clone pGEX-p 1 ( 13T) with IgE in allergic serum. Overnight cultures of pGEX or pGEX-p 1 were diluted 1 / 10 in broth and grown for 2 hours at 37°C. They were induced with IPTG and grown for 2 hours at 37°C. The bacteria were pelleted and resuspended in PBS to 1/10 the volume of culture media. The bacteria were lysed by freeze/thaw and sonication. A
radioimmune dot-blot was performed with 2m1 of these lysates using mite-allergic or non-allergic serum. The dots in row 1 were from ~, s~~i containing pGEX and row 2-4 from different cultures of ~ ~ infected with pGEX-p 1 ( 13T).
Reactivity to pGEX-p 1 ( 13T) was found with IgE in allergic but not non-allergic serum. No reactivity to the vector control or with non-allergic serum was found.
Fi shows seroreactivity of cDNA clones coding for Der p II in plaque radioimmune assay. Segments of nitrocellulose filters from plaque lifts were taken from clones 1, 3, A, B and the vector control Ampl. These were reached by immunoassay for human IgE against allergic serum (AM) in row 1, non-allergic serum (WT) in row 2 and by protein A immunoassay for Der n I with rabbit antiserum in row 3. The clones 1, 3 and B reacted strongly with allergic serum but not non-allergic or vector control. (Clone B and vector control were not tested with non-allergic serum).
Al~fEf~~E~ ~I-~EET
IPEAIEi~' _.._ Figures 7A and 7B show the nucleotide and predicted amino acid sequences of cDNA of ~,gtl l p II (C1) (SEQ ID NOS: 3 and 4, respectively).
Numbers to the right are nucleotide positions and numbers above are amino acid positions. Positive numbers for amino acids begin at the known N-terminal of Der p II and match the known sequence of the first 40 residues. Residues -1 to -16 resemble a typical leader sequence with a hydrophobic core.
Fi ur shows the N-terminal amino acid homology of Der p II and Der f II. (Der f II sequence from reference 30).
Fi is a restriction map of the cDNA insert of clone 7~gt11 fl, including a schematic representation of the strategy of DNA sequencing. Arrows indicate directions in which sequences were read.
Figures l0A and lOB are the nucleotide sequence and the predicted amino acid sequence of cDNA ~,gtl 1 fl (SEQ ID NOS: 5 and 6, respectively).
Numbers above are nucleotide positions; numbers to the left are amino acid positions.
1 S Positive amino acid residue numbers correspond to the sequence of the mature excreted D~f I beginning with threonine. Negative sequence numbers refer to the signal peptide and the proenzyme regions of Der f I. The arrows indicate the beginning of the proenzyme sequence and the mature Der f I, respectively. The underlined residues -81 to -78 make up the proposed cleavage site for the proenzyme formation, while the underlined residues 53-55 represent a potential N-glycosylation site. The termination TGA codon and the adjacent polyadenylation signal are also underlined. Amino acid residues 1-28 correspond to a known tryptic peptide sequence determined by conventional amino acid sequencing analysis.
Fi I is a composite alignment of the amino acid sequences of the mature Der n I (SEQ ID NO: 11 ) and Der f I proteins. The numbering above the sequence refers to Der n I. The asterisk denotes the gap that was introduced for maximal alignment. The symbol (.) is used to indicate that the amino acid residue of Der f I at that position is identical to the corresponding amino acid residue of Der n I.
The arrows indicate those xesidues making up the active site of Der n I and Der I.
Figures 12A and 12 B are a comparison of the amino acid sequence in the pre- and pro-peptide regions of Der f I with those of rat cathepsin H, rat cathepsin L, papain, aleurain, CP 1, CP2, rat cathepsin B, CTLA-2, MCP, I. ~~. I and actinidin.
Gaps, denoted by dashes, were added for maximal alignment. Double asterisks denote conserved amino acid residues which are shared by greater than 80% of the 3 5 proenzymes; single asterisks show residues which are conserved in greater than 5 5 of the sequences. The symbol (.) is used to denote semiconserved equivalent amino acids which are shared by greater than 90% of the proenzyme regions.
AME(~DED SHEET
IPEA/EP

Figures 13A and 13B are a hydrophilicity plot of the p~ I mature protein and a hvdrophilicity plot of the Der f I mature protein produced using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven ) using a 6 residue window. Positive values indicate relative hydrophilicitv and negative values indicating relative hydrophobicity.
Figure is the nucleotide sequence and the predicted amino acid sequence of ~7er f II cDNA (SEQ ID NOS: 7 and 8, respectively). Numbers to the right are nucleotide positions and numbers above are amino acid residues. The stop (TAA) signal is underlined. The first 8 nucleotides are from the oligonucleotide to primer used to generate the cDNA, based on the j~g~ II sequence.
Figure 1 S is a restriction map of Der f II cDNA, which was generated by computer from the sequence data. A map of p~ II similarly generated is shown for comparison. There are few common restriction enzyme sites conserved. Sites marked with an asterisk were introduced by cloning procedures.
is figures 16A. 16B. and 16C show the alignment of Der f II and , er n II
cDNA sequences. Numbers to the right are nucleotide position and numbers above are amino acid residues. The top line gives I~ II nucleotide sequence and the second the Der n II amino acid residues. The next two lines show differences of Der f II to these sequences.
20 ~lgyres 17A and 17B are hydrophilicity plots of Der f II and p~ II
using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven) using a 6-residue window.
FiQU~ 18 is a composite alignment of the amino acid sequences of five ~ I clones (a)-(e) which illustrates polymorphism in the p~ I protein (SEQ ID
2s NO: 11 ). The numbering refers to the sequence of the p~ 1(a) clone. The symbol (-is used to indicate that the amino acid residue of a ~ I clone is identical to the corresponding amino acid residue of Der n I(a) at that position. The amino acid sequences of these clones indicate that there may be significant variation in Der n I, with five polvmorphic amino acid residues found in the five sequences.
3o F' 1 is a composite alignment of the amino acid sequences of three Der p II clones (c ). ( 1 ) and (2) which illustrates polymorphism in the ~ II
protein.
The numbering refers to the sequence of the II(c) clone. The symbol (.) is used to indicate that the amino acid residue of a , er D II clone is identical to the correspondng amino acid residue of Der D II (c) at that position.
3s ~i~ure 20 is a composite alignment of the amino acid sequences of six Der f II clones (i.e., pFL 1, pFL2, MT3, MTS, MT18 and MT16) which illustrates polymorphism in the her f II protein (SEQ ID NO: 13). The numbering ~r ~.
. -.. j ( _. ,~..
SU:3~T1"~'UTE ~fr~EET

2144~5~

refers to the sequences of the D r II pFL 1 clone. The symbol (.) is used to indicate that the amino acid residue of a Der f II clone is identical to the corresponding amino acid residue of Der f II pFL 1 at that position.
Figures 21A) 21B. and 21C are the nucleotide and predicted amino acid sequences of cDNA ~.gtl l pl(13T) (SEQ ID NOS: 9 and 10, respectively), including the full length of the preproenzyme form of ~r I. Negative sequence numbers refer to the proposed pre- and preproenzyme forms of II.
Detailed Description of the Invention The present invention relates to a nucleotide sequence coding for an allergen from the house dust mite Dermato~hagoides and to the encoded Dermatonhaggides protein or peptide which includes at least one epitope of the Dermatophagoides allergen. It particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of D.
farinae, such as Der f I or Der f II, or of a peptide which includes at least one epitope of Der f I
or of Der f II. It also particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of D. pteronvssinus, such as I or Der p II, or of a peptide which includes at least one epitope of Der n I
or of I. )~ II. The Dermato a~oides nucleotide sequence is useful as a probe for identifying additional nucleotide sequences which hybridize to it and encode other mite allergens, particularly D.D. farinae or D. pteronvssinus allergens.
Further, the present invention relates to nucleotide sequences which hybridize to a D.D.
farinae protein-encoding nucleotide sequence or a D. tp eronyssinus protein-encoding nucleotide sequence but which encode a protein from another species or type of house dust mite, such as D. microceras (e.g., Der mm I and Der m II).
The encoded Dermato~, h_a~oides mite allergen or peptide which includes at least one Dermatophagoides (Der f I or Der f II; I or I~r II) epitope can be used for diagnostic purposes (e.g., as an antigen) and for therapeutic purposes (e.g., to desensitize an individual). Alternatively, the encoded house dust mite allergen can be a protein or peptide, such as a ~ microceras protein or peptide, which displays the antigenicity of or is cross-reacitve with a Der f or a Der n allergen; generally, these have a high degree of amino acid homology.
Accordingly, the present invention also relates to compositions which include a Dermatophago,_.ides allergen (e.g., Der f I allergen, Der f II allergen; Der n I or Der n II
allergen or other D. allergen cross-reactive therewith) or a peptide which includes at least one epitope of a Dermato~a_gQides allergen (her f I, Der f II, Der n I, Der n II or other ~ allergen cross-reactive therewith) individually or in combination, and which can be used for therapeutic applications A~IEi~DtD SHEET
I PEA/~P

2~.4~~3~
_7_ (e.g., desensitization). As is described below, DNA coding for major allergens from house dust mites have been isolated and sequenced. In particular, and as is described in greater detail in the Examples. cDNA clones coding for the ~ I, ~ II, Der f I
and Der f II allergens have been isolated and sequenced. The nucleotide sequence of each of these clones has been compared with that of the homologous allergen from the related mite species (i.e., p~ I and Der f I; ~ II and Der f II), as has the predicted amino acid sequence of each.
The following is a description of isolation and sequencing of the two cDNA clones coding for leer f allergens and their comparison with the corresponding to D. ,pteronyssinus allergen and a description of use of the nucleotide sequences and encoded products in a diagnostic or a therapeutic context.
lfsolation and Sea_yence Analysis of Der f I
A cDNA clone coding for Der f I, a major allergen from the house dust is mite D.D. farinae, has been isolated and sequenced. A restriction map of the cDNA
insert of the clone is represented in Figure 9, as is the strategy of DNA
sequencing.
This Der f I cDNA clone contains a 1.1-kb cDNA insert encoding a typical signal peptide, a proenzyme region and the mature Der f I protein. The product is 321 amino acid residues; a putative 18 residue signal peptide, an 80 residue proenzyme (pro-2o peptide) region, and a 223 residue mature enzyme region. The derived molecular weight is 25,191. The nucleotide sequence and the predicted amino acid sequence of the Der f I cDNA are represented in Figures l0A and IOB. The deduced amino acid sequence shows significant homology to other cysteine proteases in the pro-region, as well as in the mature protein. Sequence alignment of the mature Der f I
protein with 25 the homologous allergen p~ I from the related mite D.~teronv, sinus (Figure 11 ) revealed a high degree of homology (81 %) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues comprising the active site of these enzymes were conserved and a potential N-glycosylation site was present at equivalent positions in both mite allergens.
3o Conserved cysteine residue pairs (31, 71) and (65, 103), where the numbering refers to Der n I. are apparently involved in disulphide bond formation on the basis of the assumed similarity of the three dimensional structure of ; er n I and er f I to that of papain and actinidin, which also have an additional disulphide bridge. The fifth and final cysteine residue for which there is a homologous cysteine 35 residue in papain and actinidin is the active site cysteine (residue 35 in Der f I). It is not unlikely that the two extra cysteine residues present in I2~~ I and Der f I may be involved in forming a third disulphide bridge.
y ...- r, 1 'e SU~~'4r'i'~'t~~~ S~°tEET ~.~~.~.~ ~.;~

_g_ The potential N-glycosylation site in Der,~ I is also present at the equivalent position in Der f I, with conservation of the crucial first and last residues of the tripeptide site. The degree of of er f I and Der p. I has yet to be determined. Carbohydrates, including mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine, have been reported in purified preparations of these mite allergens (Chapman, M.D., J. Immunol., x:587-592 ( 1980); Wolden, S. gL~l., Int. Arch. Aller~ppl. Immunol., x$:144-151 ( 1982)).
Given the degree of homology over the first thirty N-terminal amino acid residues between mature p~ I and Der m I (70%) and mature Der f I and Der m I
to (97%) with the Der m I residues determined by conventional amino acid sequencing (Platts-Mills TAE et al., In: Mite Aller,~. a World-Wide Problem, 27-29 ( 1988);
Lind, P. and N. Hom, In: Mite Aller~v. a World-Wide Problem, 30-34 ( 1988)), it is probable that the full mature Der m I sequence will confirm an overall 70-80%
homology between the Group I mite allergens. Der m I is an allergen from I~
is microceras. High homology between the proenzyme moieties of p~ I and Der f I
(91 %) over the residues -23 to -1 and the structural analysis of Der f I
suggests that the Group I allergens are likely to have N-terminal extension peptides of the mature protein of homologous structure and, at least for the pro-peptide, composition.
Studies on the fine structure of the design of signal sequences have 2o identified three structurally dissimilar regions so far: a positively charged N-terminal (n) region, a central hydrophobic (h) region and a more polar C-terminal (c) region that seems to define the cleavage site (Von Heijne, G., EMBO J., x:2315-2323 ( 1984);
Eur. J. Biochem.,1~:17-21 ( 1983 ); J. Mol. Biol., x:99-1 OS ( 1985 )).
Analysis of the signal peptide of Der f I revealed that it, too, contained these regions (Figures 12A
2s 'and 12B). The n-region is extremely variable in length and composition, but its net charge does not vary appreciably with the overall length, and has a mean value of about + 1.7. The n-region of the Der f I signal peptide, with a length of two residues, has a net charge of +2 contributed by the initiator methionine (which is unformylated and hence positively charged in eukaryotes) and the adjacent lysine (Lys) residue.
3o The h-region of Der f I is enriched with hydrophobic residues, the characteristic feature of this region, with only one hydrophilic residue serine (Ser) present which can be tolerated. The overall amino acid composition of the Der f I c-region is more polar than that of the h-region as is found in signal sequences with the h/c boundary located between residues -6 and -~, which is its mean position in eukaryotes. Thus, the Der f I
3s pre-peptide sequence appears to fulfill the requirements to which a functional signal sequence must conform.
SUSSTI ~ UTE SHEET (RULE 2fi) 2144U5~

While the signal sequence of Der f I and other cysteine proteases share structural homology, all being composed of the n,h and c-regions, they are highly variable with respect to overall length and amino acid sequence, as is clear in Figures 12A and 12B. However, significant sequence homology has been shown between the s pro-regions of cysteine protease precursors (Ishidoh, K. ~j,,,, FEBS
Letters, xø:33-37 ( 1987)). Alignment of the proenzyme regions of Der f I and a number of other cysteine proteases (Figures 12A and 12B) indicated that these proregions share a number of very conserved residues as well as semi-conserved residues which were present in over half of the sequences. This homology was increased if conservative io amino acids such as valine (Val), isoleucine (Ile) and leucine (Leu) (small hydrophobic residues) or arginine (Arg) and Lys (positively charged residues) were regarded as identical. The per f I proregion possessed six out of seven highly conserved amino acids and all the residues at sites of conservative changes.
The homology at less conserved sites was lower. Homology in the pro-peptide, in is particular the highly conserved residues, may be important when considering the function of the pro-peptide in the processing of these enzymes, since it indicates that these sequences probably have structural and functional similarities.
Highly cross-reactive B cell epitopes on Der f I and , er n I have been demonstrated using antibodies present in mouse, rabbit and human sera (Heymann, 2o P.W. gt~., J. Immunol.1~Z:2841-2847 (1986); Platts-Mills, TAE ~L, J. Allergy Clin. Immunol. x:398-407 ( 1986)). However, species-specific epitopes have also been defined in these systems. Murine monoclonal antibodies bound predominantly to species-specific determinants (Platts-Mills TAE gels, J. Aller~r Clin. Immunol. x:1479-1484 ( 1987)). Some 40% of rabbit anti-pg~ I
2s reactivity was accounted for by epitopes unique to ~ I (Platts-Mills, TAE , Z
Alle,r~ Clin. Immunol. 7:398-407 (1986)), and some species-specific binding of antibodies from allergic humans was observed, although the majority bind to cross-reactive epitopes (Platts-Mills TAE ~, ~. Immunol. x:1479-1484 (1987)).
The recombinant DNA strategy of gene fragmentation and expression was used (Greene, W.K. g~L, Im~nunol. ( 1990)) to define five antigenic regions of recombinant ] er n I which contained B cell epitopes recognized by a rabbit anti-Der n I antiserum. Using the technique of immunoabsorption, three of these putative epitopes were shown to be shared with Der f I (located on regions containing amino acid residues 34-47, 60-72 and 166-194) while two appeared to be specific for p~ I
3s (regions 82-99 and 112-140). Differences in the reactivity of these peptides to rabbit anti-D.D. farinae supported the above division into cross-reactive and species-specific epitopes. The sequence differences shown between SUBSTIT°UTE SHEET (RULE 26~

WO 94/05790 r PCT/US93/08518 the er I and the Der f I proteins are primarily located in the N and C
terminal regions, as well as in an extended surface loop (residues 85-136) linking the two domains of the enzyme that includes helix D (residues 127-136), as predicted from the secondary and tertiary structures of papain and actinidin (Baker, E.N. and J.
Drenth, s ~: Biological Macromolecules and Assemblies, Vol. 3, pp. 314-368, John Wiley and Sons, NY ( 1987)). The surface location of these residues is supported by the hydrophilicity plots of I and Der f I in Figures 13A and 13B, which illustrate the predominantly hydrophilic nature of this region that predicts surface exposure. This region also contains the two species-specific B cell epitopes recognized by the rabbit to anti-pg~~ I serum (see above). Analysis of the sequences in the regions containing the cross-reactive epitopes (located in regions 34-47 and 60-72) are completely conserved between ~ I and Der f I, while the majority of residues in a third cross-reactive epitope-containing region (residues region 166-194) were conserved.
Expression of cDNA encoding Der f I results in production of pre-is pro-D r I protein in ~,. ~, a recombinant protein of greater solubility, stability and antigenicity than that of recombinant 1~ I. Protein encoded by Der f I
cDNA has been expressed using a pGEX vector and has been shown by radioimmune assay to react with rabbit anti-D.D. farinae antibodies. The availability of high yields of soluble Der f I allergen and antigenic derivatives will facilitate 2o the development of diagnostic and therapeutic agents and the mapping of B
and T
cell antigenic determinants.
With the availability of the complete amino acid sequence of recombinant Der f I, mapping of the epitopes recognized by both the B and T
cell compartments of the immune system can be carried out. The use of techniques 2s such as the screening of overlapping synthetic peptides, the use of monoclonal antibodies and gene fragmentation and expression should enable the identification of both the continuous and topographical epitopes of Der f I. It will be particularly useful to determine whether allergenic (IgE-binding) determinants have common features and are intrinsically different firom antigenic (IgG-binding) 3o determinants and whether T cells recognize unique epitopes different from those recognized by B cells. Studies to identify the Der f I epitopes reactive with mite allergic human IgE antibodies and the division of these into determinants cross-reactive with p~ I and determinants unique to Der f I can also be carried out.
B
cell (and T cell) epitopes specific for either species can be used to provide useful 3s diagnostic reagents for determining reactivity to the different mite species, while cross-reacting epitopes are candidates for a common immunotherapeutic agent.
SUB.ST~'~UTE SHEET (R~JLE ?6) As described in detail in the Examples, a cDNA clone coding for Der n I which contained a 0.8-kb cDNA insert has been isolated. Sequence analysis revealed that the 222 amino acid residue mature recombinant p~ I
protein showed significant homology with a group of cysteine proteases, including actinidin, papain. cathepsin H and cathepsin B.
A cDNA clone coding for Der f II, a major allergen from the house dust mite D.D. farinae, has been isolated and sequenced, as described in the io Examples. The nucleotide sequence and the predicted amino acid sequence of the Der f II cDNA are represented in Figure 14. A restriction map of the cDNA
insert of a clone coding for Der f II is represented in Figure 15.
Figures 16A, 16B, and 16C show the alignment of Der f II and per n II cDNA sequences. The homology of the sequence of Der f II with ~ II
i5 (88%) is higher than the 81 % homology found with ~ I and Der f I, which is significantly different (p<0.05) using the chit distribution. The reason for this may simply be that the Group I allergens are larger and each residue may be less critical for the structure and function of the molecule. It is known, for example, that assuming they adopt a similar conformation to other cysteine proteases, many 20 of the amino acid differences in p~ I and Der f I lie in residues linking the two domain structures of the molecules. The 6 cysteine molecules are conserved between the group II allergens, suggesting a similar disulphide bonding, although this may be expected, given the high overall homology. Another indication of the conservation of these proteins is that 34/55 of the nucleotide changes of the 25 coding sequence are in the third base of a codon, which usually does not change the amino acid. Residues that may be of importance in the function of the molecule are Ser 57 where all three bases are changed but the amino acid is conserved. A similar phenomenon exists at residue 88, where a complete codon change has conserved a small aliphatic residue. Again, like j~ II, the Der f II
3o cDNA clone does not have a poly A tail, although the 3' non-coding region is rich in adenosine and has two possible polyadenylation signals ATAA. The nucleotides encoding the first four residues are from the PCR primer which was designed from the known homology of p~ II and Der f II from N-terminal amino acid sequencing. A primer based on the C-terminal sequence can now be 35 used to determine these bases, as well as the signal sequence.
~U~3~T6TvTE ~~ErT »i~~c ~!'~~

Uses of the subject allergenic proteins/peptides and DNA encoding same The materials resulting from the work described herein, as well as compositions containing these materials, can be used in methods of diagnosing, treating and preventing allergic responses to mite allergens, particularly to mites s of the genus Dermatophagoides; such as D. farinae and D,~teronvssinus. In addition, the cDNA (or the mRNA from which it was transcribed) can be used to identify other similar sequences. This can be carried out, for example, under conditions of low stringency and those sequences having sufficient homology (generally greater than 40%) can be selected for further assessment using the method described herein. Alternatively, high stringency conditions can be used.
In this manner, DNA of the present invention can be used to identify sequences coding for mite allergens having amino acid sequences similar to that of Der f I, Der f II, per n I or Der n II. Thus, the present invention includes not only D.D. farinae and D.~teron, ss~ inns allergens, but other mite allergens as well (e.g., is other mite allergens encoded by DNA which hybridizes to DNA of the present invention).
Proteins or peptides encoded by the cDNA of the present invention can be used, for example, as "purified" allergens. Such purified allergens are useful in the standardization of allergen extracts or preparations which can be 2o used as reagents for the diagnosis and treatment of allergy to house dust mites.
Through use of the peptides of the present invention, allergen preparations of consistent, well-defined composition and biological activity can be made and administered for therapeutic purposes (e.g., to modify the allergic response of a house dust mite-sensitive individual). Der f I or Der f II peptides or proteins (or 2s modified versions thereof, such as are described below) may, for example, modify B-cell response to Der f I or Der f II, T-cell response to Der f I and Der f II, or both responses. Similarly, pg~ I or pg~ II proteins or peptides may be used to modify B-cell and/or T-cell response to p.~l~ I or p~ II. Purified allergens can also be used to study the mechanism of immunotherapy of allergy to house dust 3o mites, particularly to Der f I, Der f II, Der nn I and Der n II, and to design modified derivatives or analogues which are more useful in immunotherapy than are the unmodified ("naturally-occurring") peptides.
In those instances in which there are epitopes which are cross-reactive, such as the three epitopes described herein which are shared by Der f I
3s and peg I, the areas) of the molecule which contain the cross-reactive epitopes can be used as common immunotherapeutic peptides to be administered in treating allergy to the two (or more) mite species which share the epitope. For example, the cross-reactive epitopes could be used to induce IgG blocking antibody against both allergens (e.g., Der f I and ~ I allergen). A peptide containing a WO 94/05790 ~ ,'~~ ~ PCT/US93/08518 univalent antibody epitope can be used, rather than the entire molecule, and may prove advantageous because the univalent antibody epitope cannot crosslink mast cells and cause adverse reactions during desensitizing treatments. It is also possible to attach a B cell epitope to a carrier molecule to direct T cell control of allergic responses.
Alternatively, it may be desirable or necessary to have peptides which are specific to a selected Dermatopha~o, ides allergen. As described herein, two epitopes which are apparently p~ I-specific have been identified. A similar approach can be used to identify other species-specific epitopes (e.g., p~ I
or II, Der f I or II). The presence in an individual of antibodies to the species-specific epitopes can be used as a quick serological test to determine which mite species is causing the allergic response. This would make it possible to specifically target therapy provided to an individual to the causative species and, thus, enhance the therapeutic effect.
is Work by others has shown that high doses of allergens generally produce the best results (i.e., best symptom relief). However, many people are unable to tolerate large doses of allergens because~of allergic reactions to the allergens. Modification of naturally-occurring allergens can be designed in such a manner that modified peptides or modified allergens which have the same or 2o enhanced therapeutic properties as the corresponding naturally-occurring allergen but have reduced side effects (especially anaphylactic reactions) can be produced.
These can be, for example, a peptide of the present invention (e.g., one having all or a portion of the amino acid sequence of Der f I or Der f II, ~ I or I~ II).
Alternatively, a combination of peptides can be administered. A modified peptide 2s or peptide analogue (e.g., a peptide in which the amino acid sequence has been altered to modify immunogenicity and/or reduce allergenicity or to which a component has been added for the same purpose) can be used for desensitization therapy.
Administration of the peptides of the present invention to an 3o individual to be desensitized can be carried out using known techniques. A
peptide or combination of different peptides can be administered to an individual in a composition which includes, for example, an appropriate buffer, a carrier and/or an adjuvant. Such compositions will generally be administered by injection, inhalation, transdermal application or rectal administration. Using the 3s information now available, it is possible to design a I~Lp I, Deb II, Der f I or Der f II peptide which, when administered to a sensitive individual in sufficient quantities, will modify the individual's allergic response to Der n I, p,~ II, Der f I and/or Der f II. This can be done, for example, by examining the structures of these allergens, producing peptides to be examined for their ability to influence B-WO 94/05790 ~ PCT/US93/08518 cell and/or T-cell responses in house dust mite-sensitive individuals and selecting appropriate epitopes recognized by the cells. Synthetic amino acid sequences which mimic those of the epitopes and which are capable of down regulating allergic response to D_ er o I, Der n II, Der f I or Der f II allergens can be made.
s Proteins, peptides or antibodies of the present invention can also be used, in known methods, for detecting and diagnosing allergic response to Der f I or Der f II. For example, this can be done by combining blood obtained from an individual to be assessed for sensitivity to one of these allergens with an isolated allergenic peptide of house dust mite, under conditions appropriate for binding of or stimulating components (e.g., antibodies, T cells, B cells) in the blood with the peptide and determining the extent to which such binding occurs. Der f and ~
proteins or peptides can be administered together to treat an individual sensitive to both allergen types.
It is now also possible to design an agent or a drug capable of is blocking or inhibiting the ability of p~ I, ~ II, Der f I or Der f II to induce an allergic reaction in house dust mite-sensitive individuals. Such agents could be designed, for example, in such a manner that they would bind to relevant anti-I2~I~ I, anti-pg~ II, anti-Der f I or anti-Der f II IgEs, thus preventing IgE-allergen binding and subsequent mast cell degranulation. Alternatively, such 2o agents could bind to cellular components of the immune system, resulting in suppression or desensitization of the allergic response to these allergens. A
non-restrictive example of this is the use of appropriate B- and T-cell epitope peptides, or modifications thereof, based on the cDNA/protein structures of the present invention to suppress the allergic response to these allergens. This can be carried 2s out by defining the structures of B- and T-cell epitope peptides which affect B-and T-cell function in u1 vitro studies with blood cells from house dust mite-sensitive individuals.
The cDNA encoding I2~ I, Der n II, Der f I or Der f II or a peptide including at least one epitope thereof can be used to produce additional peptides, 3o using known techniques such as gene cloning. A method of producing a protein or a peptide of the present invention can include, for example, culturing a host cell containing an expression vector which, in turn, contains DNA encoding all or a portion of a selected allergenic protein or peptide (e.g., Der nn I, ~ II, Der f I, Der f II or a peptide including at least one epitope). Cells are cultured under 3s conditions appropriate for expression of the DNA insert (production of the encoded protein or peptide). The expressed product is then recovered, using known techniques. Alternatively, the allergen or portion thereof can be synthesized using known mechanical or chemical techniques. As used herein, the 2i44p~~
-IS-term protein or peptide refers to proteins or peptides made by any of these techniques. The resulting peptide can, in turn, be used as described previously.
DNA to be used in any embodiment of this invention can be cDNA
obtained as described herein or, alternatively, can be any oligodeoxynucleotide sequence having all or a portion of the sequence represented in Figures 1 A
and 1 B, 7A and 7B, l0A and l OB, and 14 or their functional equivalent. Such oligodeoxynucleotide sequences can be produced chemically or mechanically, using known techniques. A functional equivalent of an oligonucleotide sequence is one which is capable of hybridizing to a complementary oligonucleotide sequence to which the sequence (or corresponding sequence portions) of Figures 1 A and 1 B, 7A and 7B, 1 OA and 1 OB, and 14 hybridizes and/or which encodes a product (e.g., a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) represented in these figures. Whether a functional equivalent must meet one or I S both criteria will depend on its use (e.g., if it is to be used only as an oligoprobe, it need meet only the first criterion and if it is to be used to produce house dust mite allergen, it need only meet the second criterion).
The structural information now available (e.g., DNA, protein/peptide sequences) can also be used to identify or define T cell epitope peptides and/or B
cell epitope peptides which are of importance in allergic reactions to house dust mite allergens and to elucidate the mediators or mechanisms (e.g., interleukin-2, interleukin-4, gamma interferon) by which these reactions occur. This knowledge should make it possible to design peptide-based house dust mite therapeutic agents or drugs which can be used to modulate these responses.
The present invention will now be further illustrated by the following Examples, which are not intended to be limiting in any way.
~'loning and Expression of Der ~ I cDNA.
Polyadenylated mRNA was isolated from the mite WPrmatgphagoide heron ss~ inus cultured by Commonwealth Serum Laboratories, Parkville, Australia, and cDNA was synthesized by the RNA-ase H method (5) using a kit (Amersham, International, Bucks). After the addition of EcoRI
linkers, 3 5 the cDNA was ligated into ~,gt 11 and plated in E ~ Y 1090 (r-) (Promega B
iotec, Madison, Wisconsin), to produce a library of Sx105 recombinants. Screening was performed by plaque radioimmune assay (6) using a rabbit anti-Der n I
antiserum (7). Reactivity was detected by hydrochloride in 0.1 AMEMDED SHEET
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M sodium acetate buffer pH 5.2 were then added and the mixture was homogenized and spun at 10,000 rpm for 30 min in a Sorval*SS34 rotor. The supernatant was collected and layered onto a CsCI pad (Sml of 4.8 M CsCI in 10 mM EDTA) and centrifuged at 37,000 rpm for 16h at 15°C in a SW41 TI
rotor (Beckman Instruments, Inc., Fullerton, CA). The DNA band at the interphase was collected and diluted 1:15 in IOmM Tris HCl/1 mM EDTA buffer, pH 8Ø
Banding of genomic DNA in CsCI was carried out by the standard method.
Isolation of DNA from ~,gt 11 p 1 cDNA Clone.
Phage DNA from ~,gtl l pl clone was prepared by a rapid isolation procedure. Clarified phage plate lysate ( 1 ml) was mixed with 2701 of 25%
wtlvol polyethylene glycol (PEG 6000) in 2.5 M NaCI and incubated at room temperature for 15 min. The mixture was then spun for ~ min in a microfuge (Eppendorf, Federal Republic of Germany), and the supernatant was removed.
The pellet was dissolved in 100 ~l of 10 mM Tris/HCI pH 8.0 containing 1 mM
EDTA and 100 mM NaCI. This DNA preparation was extracted 3 times with phenol/chloroform ( 1:1 ) and the DNA was precipitated by ethanol.
DNA HXbridization.
Nucleic acid was radiolabelled with 32P by nick translation ( 10).
DNA samples were digested with appropriate restriction enzymes using conditions recommended by the supplier. Southern blots were prepared using Zeta-Probe membranes (Bio-Rad Laboratories, Richmond, CA).
Prehybridization, hybridization, posthybridization washes were carried out according to the manufacturers recommendations (bulletin 1234, Bio-Rad Laboratories).
Clonine and DNA Se,~uencine To clone the 0.8-kb cDNA insert from clone ~.gtl 1 pl into plasmid pUCB, phage DNA was digested with EcoRI restriction enzyme and then ligated to EcoRI-digested pUC8 DNA and used to transform E,scherichia cue' JM83. The resulting recombinant plasmid was designated as pHDM 1.
To obtain clones for DNA sequence analysis, the cDNA insert was isolated from pHDM 1 and ligated to M 13-derived sequencing vectors mp 18 and 3 S mp 19 ( 16). Transformation was carried out using E,. Eli JM 107 and sequencing was performed by the dideoxynucleotide chain termination method ( 11 ).
Trade-mark RESULTS
Several phage clones reacted with the rabbit anti Der n I serum and hybridized with all 3 oligonucleotide probes. One of these, ~.gt 11 p 1 ( 13 T), was examined further. The nucleotide sequence of the cDNA insert from this clone, ~,gt 11 p l, was determined using the sequencing strategy shown in Fig. 2. The complete sequence was shown to be 857 bases long and included a 69-base-long 5' proximal end sequence, a coding region for the entire native Der ,p I protein of 222 amino acids with a derived molecular weight of 25,371, an 89-base-long 3' noncoding region and a poly (A) tail of 33 residues (Figures lA and 1B).
The assignment of a threonine residue at position 1 as the NH2-terminal amino acid of Der p I was based on data obtained by NH2-terminal amino acid sequencing of the pure protein isolated from mite excretions ( 17). The predicted amino acid sequence matched with data obtained by amino acid sequence analysis of the NH2-terminal region as well as with internal sequences derived from analyses of tryptic peptides (Figures lA and 1B). The complete mature protein is coded by a single open reading frame terminating at the TAA stop codon at nucleotide position 736-738. At present, it is not certain whether the first ATG
codon at nucleotide position 16-18 is the translation initiation site, since the immediate flanking sequence of this ATG codon (TTGATGA) showed no homology with the Kozak consenses sequence (ACCATGG) for the eukaryotic translation initiation sites ( 18). In addition, the 5' proximal end sequence does not code for a typical signal peptide sequence (see below).
The amino acid sequence predicted by nucleotide analysis is shown in Figures lA and 1B. A protein data-base search revealed that the Der n I amino acid sequence showed homology with a group of cysteine proteases. Previous cDNA studies have shown that lysosomal cathepsins B, a mouse macrophage protease and a cysteine protease from an amoeba have transient pre- and proform intermediates ( 19-21 ), and inspection of the amino acid sequence at the 5' proximal end of the ~,gt 11 p 1 cDNt~ clone suggests that Der n I may be similar.
First, the hydrophilicity plot (22) of the sequence preceding the mature protein sequence lacks the characteristic hydrophobic region of a signal peptide (23 ) and second, an Ala-X-Ala sequence, the most frequent sequence preceding the signal peptidase cleavage site (24,25 ), is present at positions -13, -14, -15 (Figures 1 A and 1 B).
Therefore, it is proposed that cleavage between pro-I sequence and the pre-Der n I sequence occurs between Ala (-13) and Phe (-12). Thus, pro- er I
sequence begins at residue Phe (-12) and ends at residue Glu (-1). The amino acid residues numbered -13 to -23 would then correspond to a partial signal peptide sequence. The full length of the I~ I preproenzyme sequence has been AMENDED SHEET
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2144~~5 -1g -determined and is shown in Figures 21A and 21B. The negative sequence numbers refer to the pre- and preproenzyme forms of Der n I.
When the 857-by cDNA insert was radiolabelled and hybridized against a Southern blot of EcoRI-digested genomic DNA from house dust mite, hybridization to bands of 1. 5, 0.5, and 0.3 5 kb was observed (data not shown). As shown in the restriction enzyme map of the cDNA insert (Figure 2), there was no internal EcoRI site and the multiple hybridization bands observed suggest that D_ er n I is coded by a noncontiguous gene. The results also showed little evidence of gene duplication since hybridization was restricted to fragments with a total length of 2.4 kb.
The N-terminal can be compared with the N-terminal of the equivalent protein from D. farinae (Der f I) ( 12). There is identity in 11 /20 positions of the sequences available for comparison (Fig. 3).
To examine the protein produced by ~,gt 11 p 1 ( 13 T), phage was lysogenized in Y1089 (r-) and the bacteria grown in broth culture at 30°C. Phage was induced by temperature switch and isopropyl thiogalactopyranoside (IPTG) (6) and the bacteria were suspended in PBS to 1/20 of the culture volume, and sonicated for an antigen preparation. When examined by 7.5% SDS-PAGE
electrophoresis, it was found that ~,gtl 1 p 1 ( 13T) did not produce a Mr 116k 13-galactosidase band but instead produced a 140K band consistent with a fusion protein with the D~ I contributing a 24kDa moiety (6). Rabbit anti-Deb I was shown to react with the lysate from ~,gtl 1 pl(13T) (Fig. 4).

Expression of Der.p I cDNA products reactive with IgE from allergic serum.
The DNA insert from ~,gt 11 p 1 ( 13T) which codes for Der n I was subcloned into the EcoRI site of the plasmid expression vector (pGEX)(26) where it could be expressed as a .fusion with a glutathione transferase molecule. ~, coli infected with this plasmid pGEX-p 1 ( 13T) or with the vector alone were grown to a log phase culture and harvested by centrifugation. The bacteria were suspended in PBS to 1/20 of their culture volume and lysed by freeze- thawing. The lysate was shown by sodium dodecylsulphate polyacrylamide electrophoresis to express a fusion protein in high concentration of the expected Mr 50,000. These lysates were then tested for their ability to react with IgE from allergic serum by radioimmune dot-blot conducted by the method described by Thomas and Rossi (27). The serum was taken from donors known to be mite-allergic or from non-allergic controls. Reactivity was developed by 125I_monoclonal anti-IgE and AMEWED SHEET
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2i4~0~~
autoradiography. Figure 5 shows the lysate from pGEX-p 1 ( 13T), but not the vector control reacted with IgE in allergic serum, but not non allergic serum.

Inhibition of IsE antibo~,~re~onses to Der p I by treatment with the product from a cDNA clone coding for Der n I.
E coli lysogenized by ~,gt 11 p 1 ( 13T) were grown and induced by temperature switch to produce a recombinant fusion protein which was consistent with a 24 kD Der I2 I moiety and a 116 kD 13-galactosidase moiety p 1 ( 13T) (28).
This protein was mostly insoluble and could be isolated to about 90% purity, judged by sodium didodecyl polyacrylamide electrophoresis, by differential centrifugation. A similar protein was produced from another gtl 1 cDNA mite clone ~,gt pX (2c). To test for the ability of the recombinant protein to modify IgE
antibody responses to Der n I, groups of 4-S CBA mice were injected intraperitoneally with 2 mg of the p 1 ( 13T) or pX (2c) fusion proteins and after 2 days given a subcutaneous injection of S~,g of native Der n I (from mite culture medium) in aluminium hydroxide gel. The IgE antibody titres were measured by passive cutaneous anaphylaxis (PCA) after 3 and 6 weeks. The methods and background data for these responses have been described by Stewart and Holt (29). For a specificity control, groups of mice injected with p 1 ( 13T) or pX
(2c) were also injected with 10~g of ovalbumin in alum. Responses were compared to mice without prior p 1 ( 13 T) or pX (2c) treatment (Table 1 ). After 3 weeks, mice either not given an injection of recombinant protein or injected with the control pX (2c) had detectable anti Der o I PCA titres (1/2 or greater). Only 1/5 of mice treated with recombinant p 1 ( 13T) had a detectable titre and this, at 1/4, was lower than all of the titres of both control groups. Titres of all groups at 6 weeks were low or absent (not shown). The PCA response to ovalbumin was not significantly affected by treatment with recombinant proteins. These data show the potential of the recombinant proteins to specifically decrease IgE responses as required for a desensitizing agent.
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21~4~55 TABLE 1 Inhibition of anti-Der n I IgE by preinjection with recombinant Der p I.

prei njection immunizing IgE (PCA y titres at d21 group -2 days injection (d0) (Sp.g/alum) responders titres 1 - Der p I 4/4 1 / 16-1 /64 2 pX(2C) Der n I 5/5 1/8-1/16 3 pl(13T) per n I 1/S* 1/4*

4 - ovalbumin 4/4 1 /64-1 /256 pX(2C) ovalbumin S/5 1/32-1/128 6 pl(13T) ovalbumin 5/5 1/64-1/256 Mice were given a preinjection on day -2 and then immunized with Der n I or 5 ovalbumin on day 0. Serum antibody titres were measured on day 21 and 42 by PCA in rat skin. Significant anti-I titres were not detected on day 42 (not shown). The PCA were measured to I for groups 1-3 and ovalbumin for groups 4-6. The anti-II titres were lower (p<0.001 )* when pretreated with recombinant Der p I p 1 ( 13T).
*Mann Whitney analysis.

F~~re~~;~~ of Der n I antigenic determinants by fraements of the cDNA from ~,~t~ll pl(,l3Tl The cDNA from ~,gtl l (13T) coding for Der n I was fragmented by sonication. The fragments (in varying size ranges) were isolated by electrophoresis, filled in by the Klenow reaction to create blunt ends. EcoRI
linkers were attached and the fragment libraries cloned in ~,gt 11 . The methods used for the fragments cloning were the same as that used for cDNA cloning (6).
Plaque immunoassay was used for screening with rabbit anti-Der n I. Three phage clones reacting with the antiserum were isolated and the oligonucleotide sequences of the cloned fragments obtained. Two of these were found to code for ~ I
amino acids 17-5 5 (see Figures 1 A and 1 B for numbering) and one for amino acids 70-100. Such fragments will eventually be useful for both diagnostic reagents to AMENDED SHEET
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~144~~~

determine epitope reactivity and for therapy where molecules of limited allergenicity may increase safety of desensitisation.

Cloning and expression of cDNA coding for the major mite allergen Der ~I_I.
The Dermato.~hag~ides eronXssinus cDNA library in ~,gtl l previously described was screened by plaque radioimmune assay using nitrocellulose lifts (6). Instead of using specific antisera, the sera used was from a person allergic to house dust mites. The serum (at 1/2 dilution) was absorbed with E coli. To detect reactivity an 1251 labelled monoclonal anti-IgE was used (at 30ng/ml with 2x106 cpm/ml (approx. 30% counting efficiency)). After 1 hour, the filters were washed and autoradiography performed. Using this procedure, 4 clones reacting with human IgE were isolated. It was found they were related by DNA hybridization and had an identical pattern of reactivity against a panel of allergic sera. Fig. 6 shows IgE reactivity in plaque radioimmunoassay against allergic serum (AM) (top row) or non allergic (WT j. Here, clones l, 3 and 8 react strongly, but only against allergic sera. The amp 1 segments (present in row 1 ) are a ~,gt 11 vector control. The bottom row is an immunoassay with rabbit anti-Der p I, developed by 1251 staphylococcus protein A which shows no significant reactivity. The clones were tested against a panel of sera. Serum from five patients without allergy to mite did not react, but serum from 14/ 17 people with mite allergy showed reactivity. The DNA insert from the clone ~,gtl l pII(C1) was subcloned into M 13 mpl8 and M 13 mp 19 and sequenced by the chain termination method. The nucleotide sequence (Figures 7A and 7B) showed this allergen was Dg~2 II by (a) the homology of the inferred amino acid sequence of residues 1-with that of the N-terminal amino acid of Der n II (30); and (b) the homology of this sequence with the equivalent Der f II allergen from Dermatonha~oides f ri (30).

Isolation and Characterization of cDNA Coding for Der f I
MATERIALS AND METHODS
Dermatonha~oides farinae culture Mites were purchased from Commonwealth Serum Laboratories, Parkville, Australia.
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21440~~

Construction of the D farinae cDNA ~,gtl 1 1 Polyadenylated mRNA was isolated from live D. farinae mites and cDNA was synthesized by the RNase H method (Gubler, V. and B.J. Hoffman, Gene x:263-269 ( 1983 )) using a kit (Amersham International, Bucks.). After the addition of EcoRI linkers (New England Biolabs, Beverly, MA) the cDNA was ligated to alkaline phosphatase treated ~,gt 11 arms (Promega, Madison, WI).
The ligated DNA was packaged and plated in E.E. coli Y1090 (r-) to produce a library of 2x104 recombinants.
Isolation of Der f I cDNA clones from the D. farinae cDNA ~,gt 11 library Screening of the library was performed by hybridization with two probes comprising the two D~ I cDNA BamHI fragments 1-348 and 349-857 generated by BamHI digestion of a derivative of the Der n I cDNA which has had two BamHI restriction sites inserted between amino acid residues -1 and 1 and between residues 116 and 117 by site-directed mutagenesis (Chua, K.Y. ~, .I. Exp Med. 17:175-182 ( 1988)). The probes were radiolabelled with 32P by nick translation. Phage were plated at 20,000 pfu per 150mm petri dish and plaques were lifted onto nitrocellulose (Schleicher and Schull, Dassel, FRG), denatured and baked (Maniatis, T. e~1" Molecular Cloning: Laboratory Manual, Cold Spring Harbor Laboratory Press ( 1982)). Prehybridizations were performed for 2 hours at 42°C in 50% formamide/5 x SSCE/1 x Denhardt's/poly C
(0.1 mg/ml)/poly U(O.lmg/ml) with hybridization overnight at 42°C at cpm/ml. Post hybridization washes consisted of 15 min washes at room temperature with 2 x sodium chloride citrate (SSC)/0.1 % sodium dodecylsulphate (SDS), 0.5 x SSC/0.1% SDS, 0.1 x SSC/0.1% SDS successively and a final wash at 50°C for 30 min in 0.1 x SSC/1% SDS.
Isolation of DNA from ~,;"" 11 fl cDNA clones Phage DNA from ~,gtl 1 fl clones was prepared by a rapid isolation procedure. Clarified phage plate lysate (1 ml) was mixed with 270 of 25%
wt/vol polyethylene glycol (PEG 6000) in 2.SM NaCI and incubated at room temperature for 15 min. The mixture was then spun for 5 min in a microfuge (Eppendorf, FRG), and the supernatant was removed. The pellet was dissolved in 100 mL of l OmM Tris/HC 1 pH8.0 containing 1 mM EDTA and 100 mM NaCI (TE). This 3 5 DNA preparation was extracted with phenol/TE, the phenol phase was washed with 100 p l TE, the pooled aqueous phases were then extracted another 2 times with phenol/TE, 2 times with Leder phenol (phenol/chloroform/isoamylalcohol;
25:24:1 ), once with chloroform and the DNA was precipitated by ethanol.
AMEWED SHEET
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DNA seauencine To obtain clones for DNA sequence analysis, the ~.gt 11 fl phage DNA was digested with EcoRI restriction enzyme (Pharmacia, Uppsala, Sweden) and the DNA insert was ligated to EcoRI-digested M 13-derived sequencing vectors mp 18 and mp 19 (Maniatis, T. te~l~, Molecular Cloning: A
I,aboratorv Manual, Cold Spring Harbor Laboratory Press (1982)).
Transformation was carried out using E.E. coli TG-1 and sequencing was performed by the dideoxynucleotide chain termination method (Sanger, F. t~j~, Proc. atl. Acad. Sci. USA, x:5463-5467 ( 1977)) using the Sequenase version 2.0 DNA sequencing kit (U.S.B., Cleveland, Ohio).
Polvmerase chain reaction f,PCRI
PCR was performed by the Taq DNA polymerase method (Saiki, R.K. etet al., ,~cienc~ ?x:487-491 ( 1988)) using the TaqPaq kit (Biotech International, Bentley, WA) and the conditions recommended by the supplier with l ong of target DNA and l Opmol of ~,gt l 1 primers (New England BioLabs, Beverly, MA).
RESULTS
Isolation of Der f I cDNA clones Two clones expressing the major mite allergen Der f I were isolated from the D. farinae cDNA ~.gt 11 library by their ability to hybridize with both of the per p I cDNA probes (nucleotides 1-348 and 349-857). This approach was adopted because amino acid sequencing had shown high homology (80%) between these two allergens (Thomas, W.R., te~l~, Advances in the Biosciences, 1_4:139-147 (1989)). Digestion of the 7~gt11 fl clone DNA with EcoRI
restriction enzyme to release the cDNA insert produced three Der f I cDNA EcoRI
fragments: one approximately 800 bases long and a doublet approximately 150 bases long. The er f I cDNA insert was also amplified from the phage DNA by the polymerase chain reaction (PCR) resulting in a PCR product of approximately 1.1-kb. Each Der f I cDNA fragment was cloned separately into the M13-derived sequencing vectors mp 18 and mp 19 and sequenced.
DNA seauence analysis The nucleotide sequence of , er f I cDNA was determined using the sequencing strategy shown in Figure 9. The complete sequence was shown to be bases long and included a 335-base long 5' proximal end sequence, a coding region for the entire native Der f I protein of 223 amino acids with a derived molecular weight of 25,191 and an 80-base long 3' noncoding region (Figures l0A and l OB).
Trade-mark 2144~~~

The assignment of the threonine residue at position 1 as the NH2-terminal amino acid of Der f I was based on data obtained by NH2-terminal amino acid sequencing of the native protein and the predicted amino acid sequence of recombinant Der n I
(Chug, K.Y. et al., J. Ex~. Med., x:175-182 (1988)). The predicted amino acid sequence of the Der f I cDNA in the NH2-terminal region matched completely with that determined at the protein level (Figures l0A and lOB).
The complete mature protein coded by a single open reading frame terminating at the TGA stop codon at nucleotide position 42-44 is presumed to be the translation initiation site since the subsequent sequence codes for a typical signal peptide sequence.
Amino Acid Seauence Analvsis The amino acid sequence of her f I predicted by nucleotide analysis is shown in Figures l0A and IOB. As shown in the composite alignment of the amino acid sequence of mature Deep I and Der f I (Figure 11 ), high homology was observed between the two proteins. Sequence homology analysis revealed that the Der f I
protein showed 81 % homology with the Der n I protein as predicted by previous conventional amino acid sequencing. In particular, the residues making up the active site of Der o I, based on those determined for papain, actinidin, cathepsin H, and cathepsin B, are also conserved in the Der f I protein. The residues are glutamine (residue 29), glycine, serine and cysteine (residues 33-35), histidine (residue 171) and asparagine, serine and tryptophan (residues 191-193) where the numbering refers to Der f I. The predicted mature Der f I amino acid sequence contains a potential N-glycosylation site (Asn-Thr-Ser) at position 53-55 which is also present as Asn-Gln-Ser at the equivalent position in Der n I.
Analysis of the predicted amino acid sequence of the entire Der f I cDNA
insert has shown that, as for other cysteine proteases (Figures 12A and 12B), the Der f I protein has pre- and proform intermediates. As previously mentioned, the methionine residue at posixion -98 is presumed to be the initiation methionine. This assumption is based on the fact that firstly, the 5' proximal end sequence from residues -98 to -81 is composed predominantly of hydrophobic amino acid residues (72%), which is the characteristic feature of signal peptides (Von Heijne, G., EMBO
J., x:2315-2323 ( 1984)). Secondly, the lengths of the presumptive pre- ( 18 amino acid residues) and pro-peptides (80 residues) are similar to those for other cysteine proteases (Figures 12A and 12B). Most cysteine proteases examined have about preproenzyme residues (of which an average of 19 residues form the signal peptide) with cathepsin B the smallest with 80 (Ishidoh, K. ~, FEB S Letters, ~:3 2-3 7 ( 1987)). Der f I falls within this range with a total of 98 preproenzyme residues.
AMEIt~ED ~~-IEET
IPEP~'EP

~. -25- ~1440~5 By following the method for predicting signal-sequence cleavage sites outlined in Von Heijne, it is proposed that cleavage from the pre-D r I
sequence for proenzyme formation occurs at the signal peptidase cleavage site lying between Ala (-81 ) and Arg (-80) (Von Heijne, G., Eur. J. Biochem., x:17-21 ( 1988) and J. Mol. Biol., _1$4_:99-105 ( 1985)). Thus, the sequence from residues -98 to -81 codes for the leader peptide while the proenzyme moiety of Der f I begins at residue Arg (-80) and ends at residue Glu (-1).

Isolation and Characterization of cDNA Coding for Der f II
MATERIALS AND METHODS
Amino acid seauence analvsis Preparation of ~ et l 1 D farinae cDNA ligat,_ ions D. ri was purchased from Commonwealth Serum Laboratories, Parkville, Australia, and used to prepare mRNA (polyadenylated RNA) as described (Stewart, G.A. and W.R. Thomas, Int Arch AllerQV A~pl Immunol., $x:384-389 (1987)). The mRNA was suspended at approximately 0.5~g/ml and 5 ~,g used to prepare cDNA by the RNase H method (Gubler, U. and Hoffman, B.J., ne, X5:263-269 (1983)) using a kit (Amersham International, Bucks). EcoRI
linkers (Amersham, GGAATTCC) were attached according to the method described by Huynh e~~., Constructing and screening cDNA libraries in gt 10 and gt 11, In:
Glover, ANA Cloning vol A practic pp~oach pp. 47-78 IRL Press, Oxford ( 1985)).
The DNA was then digested with EcoRI and recovered from an agarose gel purification by electrophoresis into a DEAF membrane (Schleicher and Schuell, Dassel, FRG, NA-45) according to protocol 6.24 of Sambrook , (Sambrook etet al., Molecular Cloning;. A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press ( 1989)), except 0.5M arginine base was used for elution. The cDNA was then ligated in ~,gtl0 and ~,gtl 1 at an arms to insert ratio of 2:1. Some was packaged for plaque libraries and an aliquot retained for isolating sequences by polymerase chain reaction as described below.
Isolation of Der f II cDNA bXPol~nerase Chain Reaction To isolate Der f II cDNA, an oligonucleotide primer based on the N-terminal sequence of Der o II was made because their amino acid residues are identical in these regions (Heymann, P.W. g~~l." J AllerQV Clin. Immunol., $~:1055-1087 ( 1989)). The primer GGATCCGATCAACTCGATGC-3' (SEQ ID N0:14) was used. The first GGATCC encodes a $~H1 site and the following sequence, GAT..., encodes the first four residues of Der n II. For the other primer, the ~,gtl l AMENDED SHEET
IPEA/EP

- 25.1 -TTGACACCAGACCAACTGGTAATG-3' (SEQ ID NO:15) reverse primer flanking the EcoRI
AMEWED SHEET
IPEAIEP

__ -26~~~~0~~
cloning site was used (New England Biolabs, Beverly, MA). The Der n II primer was designed to have approximately 50-60% G-C and to end on the first or second, rather than the third, base of a codon (Gould, S.J. etet al., Proc. Natl. Acad. Sci., 8~f :1934-1938 ( 1989); Summer, R. and D. Tautz, Nucleic Acid Res., 17:6749 ( 1989)).
The PCR reactions were carried out in a final reaction volume of 25.1 containing 67mM Tris-HCL (pH8.8 at 25°C), 16.6mM (NH4)2S04, 40~M dNTPs, SmM 2-mercaptoethanol, 6~M EDTA, 0.2mg/ml gelatin, 2mM MgCl2, lOpmoles of each primer and 2 units of Taq polymerase. Approximately 0.001 ~g of target DNA
was added and the contents of the tube were mixed and overlayed with paraffin oil.
The tubes were initially denatured at 95 °C for 6 minutes, then annealed at 5 5 °C for 1 minute and extended at 72°C for 2 minutes. Thereafter for 38 cycles, denaturing was carried out for 30 seconds and annealing and extension as before. In the final (40th) cycle, the extension reacton was increased to 10 minutes to ensure that all amplified products were full length. The annealing temperature was deliberately set slightly lower than the Tm of the oligonucleotide primers (determined by the formula Tm=69.3 + 0.41 (G+C%)-650/oligo length) to allow for mismatches in the N-terminal primer.
5 ~l of the reaction was then checked for amplified bands on a 1 agarose gel. The remainder of the reaction mixture was extracted with chloroform to remove all of the paraffin oil and ethanol precipitated prior to purification of the amplified product on a low melting point agarose gel (Bio-Rad, Richmond, CA).
Subcloning_of PCR Product The ends of the purified PCR product were filled in a reaction containing 10 mM Tris HC 1, 10 mM MgCl2, 50 mM NaCI, 0.025 mM dNTP and l~l of Klenow enzyme in a final volume of 100,1. The reaction was carried out at 37°C for 15 minutes and heat inactivated at 70°C for 10 minutes.
The mixture was Leder phenol extracted before ethanol precipitation. The resulting blunt ended DNA was ligated into M13mp118 digested with Sma I in a reaction containing O.SM ATP, 1 X ligase buffer and 1 unit of T4 ligase at 15°C
for 24 hrs and transformed into E.E. coli TG 1 made competent by the CaCl2 method. The transformed cells were plated out as a lawn on L + G plates and grown overnight at 37°C.
AMENDED SHEET
IPEA/EP

Preparation of Single-stranded DNA Template for Seauencing, Isolated white plaques were picked using an orange stick into 2.5 ml of an overnight culture of TG 1 cells diluted 1 in 100 in 2 X TY broth, and grown at 37°C for 6 hours. The cultures were pelleted and the supernatant removed to a fresh tube. To a lml aliquot of this supernatant 2701 of 20% polyethylene glycol, 2.SM NaCI was added and the tube was vortexed before allowing it to stand at room temperature (RT) for 15 minutes. This was then spun down again and all traces of the supernatant were removed from the tube. The pellet was then resuspended in 100p.1 of 1 X TE buffer. At least 2 phenol:TE extractions were done, followed by 1 Leder phenol extraction and a CHCP3 extraction. The DNA
was precipitated in ethanol and resuspended in a final volume of 20m1 of TE
buffer.
j2NA Analysis DNA sequencing was performed with the dideoxynucleotide chain termination (Sanger, F. g~.~l., Proc. Natl. Acad. Sci., L:5463-5467 ( 1977)) using DNA produced from M13 derived vectors mp 18 and mp 19 in E.E. coli TG 1 and T4 DNA polymerase (Sequenase version 2.0, USB Corp., Cleveland, Ohio;
Restriction endonucleases were from Toyobo, (Osaka, Japan). All general procedures were by standard techniques (Sambrook, J. gLal~, A Laboratory anual, 2d Ed. Cold Spring Harbor Laboratory Press ( 1989)). The sequence analysis was performed using the Mac Vector*Software (IBI, New Haven, CT).
RESULTS
D.D. farinae cDNA ligated in ~.gtl 1 was used to amplify a sequence using an oligonucleotide primer with homology to nucleotides coding for the 4 N-terminal residues of ~ II and a reverse primer for the ~.gt 1 I sequence flanking the coding site. Two major bands of about 500 by and 300 by were obtained when the product was gel electrophoresed. These were ligated into M 13 mp 18 and a number of clones containing the 500 by fragment were analyzed by DNA
sequencing. Three clones produced sequence data from the N-terminal primer end and one from the other orientation. Where the sequence data from the two directions overlapped, a complete match was found. One of the clones read from the N-terminal primer, contained a one-base deletion which shifted the reading frame. It was deduced to be a copying error, as the translated sequence from the other two clones matched the protein sequence for the first 20 amino acid residues of the allergen.
Trade-mark _2g- 2 1 4 4 0 5 5 The sequence of the clones showing consensus and producing a correct reading frame is shown in Figure 14, along with the inferred amino acid sequence. It coded for a 129 residue protein with no N-glycosylation site and a calculated molecular weight of 14,021 kD. No homology was found when compared to other proteins on the GenBanlc*data base (61.0 release). It did, however, show 88%
amino acid residue homology with Der R II shown in the alignment in Figures 16A, 16B, and 16C. Seven out of the 16 changes were conservative. The conserved residues also include all the cysteines present at positions 8, 21, 27, 73 and 119. There was also considerable nucleotide homology, although the restriction enzyme map generated from the sequence data for commonly used enzymes is different from ]Der p II
(Figure 15). The hydrophobicity plots of the translated sequence of L7er f II and peg II
shown in Figures 17A and 17B are almost identical.
Determination of Nucleotide Seauence Po~vmomhisms in the Der p I. Der p II and ;Der f It Allergens It was expected that there were sequence polymorphisms in the nucleic acid sequence coding for Der D I, ~ II, Der f I and j2er f II, due to natural allelic variation among individual mites. Several nucleotide and resulting amino acid sequence polymorphisms were discovered during the sequencing of different Der ~ I, Der p II and Der f II clones. The amino acid sequence polymorphisms are shown in Figures 18, 19 and 20.
The original Der n I ~,a 11 cDNA library was reprobed with cDNA
obtained from the ~,gt 11 p 1 ( 13T) clone to identify new clones. S imilarly, the 7~gt 11 cDNA library of I2~.~ II was reprobed with cDNA obtained from the ~.gt 11 pII(C 1 ) clone to identify additional Der ~ II clones. These clones were isolated, sequenced and found to contain nucleotide and resulting amino acid sequence polymorphisms (see Fig. 18 and 19).
Four ~ I clones, (b), (c), (d) and (e) were sequenced, as shown in Fig.
18. Clone ] er o I(d) was found to contain the following polymorphisms relative to the clone Der 12 I(a) sequence: ( 1 ) the codon for amino acid residue 136 was ACC
rather than AGC, which results in a predicted amino acid substitution of Thr for Ser;
(2) the codon for amino acid residue 149 had a silent mutation, GCT rather than GCA;
and (3) the codon for amino acid residue 215 was CAA rather than GAA; which results in a predicted amino acid substitution of Gln for Glu.
Trade-mark -29- ~1~~0 ~~
The Der n II clones, D~r.p II( 1 ) and Der n II(2) were sequenced as shown in Figure 19. Clone ~r II(2) was found to have the codon TCA, rather than ACA at amino acid residue 47, which results in a predicted amino acid substitution of Ser for Thr. This clone also was found to have the codon AAT
at amino acid residue 113 rather than GAT, which results in a predicted amino acid substitution of Asn for Asp. The codon for amino acid 127 of this clone was found to be CTC rather than ATC. This change in codon 127 results in a predicted amino acid substitution of Leu for Ile.
Additional Der f II cDNA clones containing nucleic acid and resulting amino acid sequence polymorphisms were obtained from PCR reactions using cDNA prepared with RNA isolated from D. farinae mites (Commonwealth Serum Laboratories, Parksville, Australia). cDNA was prepared and ligated in ~,gt 10 as previously described (Trudinger e~ ~ ( 1991 ) C lin. Exn. Allergy ?~:3 3-37). The clones described below were isolated following PCR of the ~,gtl0 library using a 5' primer, which had the sequence 5'-GGATCCGATCAAGTCGATGT-3' (SEQ ID N0:16). The nucleotides 5'-GGATCC-3' of the 5' primer correspond to a HI endonuclease site added for cloning purposes. The remaining nucleotides of the 5' primer, 5'-GATCAAGTCGATGT-3' (SEQ ID N0:17) correspond to the first 4 amino acids of Der n II (Chug .e~ ~ (1990) Int Arch Allergy Clin Immunol X1:118-123) as described in Trudinger ~ ~1_. (( 1991 ) Clin. Exp. Allergy ~ 1_:33-37). The 3' primer, which has the sequence 5'-TTGACACCAGACCAACTGGTAATG-3' (SEQ ID
NO: 15), corresponds to a sequence of the ~,gtl0 cloning vector (Trudinger ~
~.r.
a r ).
PCR was performed as described (Trudinger ~ ,~1~, supra) and four Der f II
clones, MT3, MTS, MT 16 and MT 18, were sequenced, as shown in Figure 20.
Three clones were sequenced that had potential polymorphisms relative to the published Der f II sequence (Trudinger ~ ~1,. supra). The codon for amino acid 52 of clone MT 18 was ATT rather than the published ACT (Trudinger ~ ~, s_uT). This change in codon 52 of clone MT18 would result in a predicted amino acid change from Thr to Ile. Clone MTS contained three changes from the published sequence (Trudinger e~
~1 su ): ( 1 ) the codon for amino acid 11 was AGC rather than the published AAC
(Trudinger e~ ~. ), which results in a predicted amino acid substitution of Ser for Asn; (2) the codon for amino acid 52 was ATT, rather than the published ACT
(Trudinger ~ ~ supra), which results in a predicted amino acid substitution of Ile for Thr; and (3) the codon for amino acid 88 was ATC rather than the published GCC
(Trudinger e~ ~1,. supra), which results in a predicted amino acid substitution of Ile for Ala. Clone MT16 had a AMEWED ~!--91=I=T
IP~RI~P

silent mutation in the codon for amino acid 68 (ATC versus the published ATT
(Trudinger ~t al. a ra) that did not change the predicted amino acid at this residue. The following substitutes were also observed by Yuuki g~ ~
( n.J.Allerg.,ol. f :557-X61, 1990); Ile at residue 52, Ile at residue 54 and Ile at s residue 88.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments to of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

31- 2m~o~~
REFERENCES
1. Ford, A.W., Rawle, F.C., Lind, P., Spieksma, F.T.M., Lowenstein, H., Platts-Mills, T.A.E. (1985). Standardization of DermatophagQides pteronyssinus. Assessment of potency and allergen content in the coded extracts. Ice. Arch. All A~1_.
Immunol. 75:58-67.
2. Lind, P., Lowenstein, H. (1983). Identification of allergens in Dermato~hago~ides l2teronyssinus mite body extract by crossed radioimmunelectrophoresis with two different rabbit antibody pools.
S an . J_. Im ~Z:263-273.
3. Krilis, S., Baldo, B.A., Basten, A. ( 1984). Antigens and allergens 1 S from the common house dust mite Dermatop ' es pteronyssinus Part II. Identification of the major IgE binding antigens by crossed radioimmuno-electrophoresis. ~,, Allergy lir = Immunol. 74:142-146.
4. Tovey, E.R., Chapman, M.D., Platts-Mills, T.A E. (1981). Mite faeces are a major source of house dust allergens. Nature 2~:592-593.
5. Gubler, U., Hoffman, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Crene 25:263-269.
6. Huynh, T.V., Young, R.A., Davis, R.W. Constructing and screening cDNA libraries in g10 and ~,gtl l . p48-78 in DNA Cloning Vol 1 A
practical approach. Ed. D.M. Glover, IRL press.

7. Stewart, G.A., Thomas, W.R. (1987). It1 vitro translation of messenger RNA from the house mite Dermatop]L~o, ides pteronXssinus. ~. Arch. Allerw ~p~. Immunol. $x:384-389.

8. Thomas, W.R., Rossi, A.A. (1986). Molecular cloning of DNA
coding for outer membrane proteins of Haemophilus influenzae type b. . fecti~l ~u.~ Immunity ,5:812-817.
AMENDED SHEET
IPEA/EP

~~~~a~~ -32-9. Simpson, R.J., Smith, J.A., Mortiz, R.L., O'Hare, M.J., Rudland. P.S., Morrison, J.R., Lloyd, C.J., Grego, B., Burgess, A.W. and Nice, E.L.
( 1985). Rat Epidermal Growth Factor: Complete amino acid sequence. ~ ~ Biochem. ~S :629-637.
s 10. Maniatis, T., Fritsch, E.F., Sambrook, J. ( 1982). Molecular cloning.
A Laboratory Manual, Cold Spring Harbor Laboratory.

11. Sanger, F., Nicklen, S., Coulson, A.R. ( 1977). DNA sequencing with io chain-terminating inhibitors. Proc. Acad. ~~j,, x:5463-5467.

12. Heyman, P.W., Chapman, M.D., Platts-Mills, T.A.E. (1986). Antigen Der f I from the house dust mite Dermato~ha oide farinae:
is Structural comparison with l~ I from Dermat~~o; ides nteronvssinus and epitope specificity of murine IgG and human IgE
antibodies. ~, j~,~, x:2841-2847.

13. Voorhorst, R., Spieksma-Boezeman, M.LA., and 2o Spieksma, F. Th.M. ( 1964). Is a mite ermat~ha oidet sp) the producer of the house dust allergen. Ah LQ:329.

14. Voorhorst, R., Spieksma, F.Th.M., Varekamp, H., Leupen, M.J. and Lyklema, A.W., (1967). The house dust mite ermat~hago. ides 25 nteronyssinus) and the allergens it produces. Identity with the house dust allergen. ~, All-er~~v. x:325.

15. Stewart, G.A. and Thomas, W.R. ( 1987). ~ vitro translation of messenger RNA from the house dust mite Dermat~hagoides so nteronvssinus. I~ ~ All ~ $x:384.

16. Messing, J. (1983). New M13 vectors for cloning. Methods Enzxm.QL 191:20.
3s 17. Stewart, G.A., Simpson, R.J., Thomas, W.R. and Turner, K.J. ( 1986).
The physiochemical characterization of a major protein allergen firom the house dust mite, EP. Asian per, ~, All j~, x:71.

WO 94/05790 4 ~ j .~] PCT/US93/08518 18. Kozak, M. ( 1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic. Acids ~ x:857.
s 19. San Segundo, B., Chain, S.J. and Steiner, p~ (1985). Identification of cDNA clones encoding a precursor of rat liver cathepsin B. Proc.
11~ Acad. ~ USA. $x:2320.
20. Portnoy, D.A., Erickson, A.H., Kochan, J., Ravetch, J.V. and to Unkeless, J.C. (1986). Cloning and characterization of a mouse cysteine proteinase. ~. ~ Chem. 21.:14697.
21. Williams, J.G., North, M.J. and Mahbubani, H. (1985). A
developmentally regulated cysteine proteinase in Dictvostelium ~5 discoideum. EMBO (~,, ~ ~.Q1,. Organ.) ~ 4:999.
22. Hopp, T.P. ( 1986). Protein surface analysis. Method for identifying antigenic determinants and other interaction sites. ~, j~, Methods. $$:1.
23. Von Heijne, G. ( 1984). Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications of protein export in prokaryotic and eukaryotic cells. ~Q (~ ~
Organ.) ~, x:2315.
24. Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J.
and Goodman, H.W. (1977). Rat insulin genes: Construction of plasmids containing th coding sequences. fence (Wash. DC) x:1313.
25. Carne, T. and Scheele, G. ( 1985). Cell Biology of the Secretory Process. M.Cantin, editor. S. Karger AG, Basel. 73.
26. Smith, D. and Johnson (1988), Gene (in press).
27. Thomas, W.R. and Rossi, A.A. (1986). Molecular cloning of DNA
coding for outer membrane proteins of ~mQph' Type b. Infection ~ Iml~i~ x:812-817.

28. Thomas, W.R., Stewart, G.A., Simpson, R.J., Chua, K.Y., Plozza, T.M., Dilworth, Dr. U., Nisbet, A. and Turner, K.J. ( 1987). Cloning and expression of DNA coding for the major house dust mite allergen Der n I in Escherichia ~. Ink, Arch. All 1. Immunol.
s $,x:127-129.
29. Stewart, G.A.. and Holt, P.G. ( 1987).
Immunogenicity and tolerogenicity of a major house dust mite allergen Der n I. 1~,, ~ All ~ x:44-51.
io 31. Chapman, M.D., Heymann, P.W. and Platts-Mills, T.A.E. (1987).
Mite allergens 1. Epitope mapping of major dust mite (permatop~4go, ides) allergens using monoclonal antibodies. Mite Allergy - A
is World Wide Problem. Ed. A.L. deWeck and A. Todt. The UCB
Institute of Allergy.
n . , , ~.~~~Q5~

SEQUENCE LISTING
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: IMMULOGIC PHARMACEUTICAL CORPORATION
(B) STREET: 610 LINCOLN STREET
(C) CITY: WALTHAM
lO (D) STATE: MASSACHUSETTS
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02154 (G) TELEPHONE: (617) 466-6000 (H) TELEFAX: (617) 466-6010 (ii) TITLE OF INVENTION: T CELL EPITOPES OF THE MAJOR ALLERGENS FROM
DERMATOPHAGOIDES
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD
(B) STREET: 60 STATE STREET, SUITE 510 (C) CITY: BOSTON
2S (D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02109 (v) COMPUTER READ:~BLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII TEXT
3S (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US 93/08518 (B) FILING DATE: 10-SEP-1993 (C) CLASSIFICATION:
AMEWE~ ~~EET
IPE/aIEP

i n WO 94/05790 ~ ~ ~ 4 PCT/US93/08518 (vii) PRIOR APPLICATION DATA:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NTJMBER: US 07/945,288 (B) FILING DATE': 10 SEPTEMBER 1992 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400 (B) TELEFAX: (617) 227-5941 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 834 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/FCEY: CDS
(B) LOCATION: 1..738 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn WO 94/05790 ~ ~ PCT/US93/08518 ._. -37-Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala 10 Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu A1a Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr ATT GAT CCA

Asp Gly Arg Thr Ile Gln Arg AsnGly Tyr Gln AsnTyr Ile Asp Pro GTT AGT GTC

His Ala Val Asn Ile Gly Tyr AsnAla Gln Gly AspTyr Val Ser Val AGT ACC AAT

Trp Ile Val Arg Asn Trp Asp AsnTrp Gly Asp GlyTyr Ser Thr Asn AAC TTG GAA

Gly Tyr Phe Ala Ala Ile Asp MetMet Ile Glu TyrPro Asn Leu Glu TAAACAAAAA
GACAATTTCT
TATATGATTG
TCACTAATTT

Tyr Val Val Ile Leu AAAATTTTTT A
AGAAAATGAA
TAAATTCATT

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
~ ,, Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly.Tyr Ser Asn Ala Gln Gly Val Asp Tyr 170 175 ' 180 185 Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 69..509 n . , , __ -41- 21~~a~~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Met Tyr Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala Val Ala Arg Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile CAT CGT GGT AAA CCA TTC CAA TTG GAA GCC GTT.TTC GAA GCC AAC CAA 254 His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Met Tyr Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala Val Ala Arg Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg WO 94/05790 ~ PCT/US93/08518 Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1072 base pairs (B) TYPE: nucleic acid --(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

~,~~~~~5 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 36..1001 (xi)SEQUENCE SEQID
DESCRIPTION: N0:5:

AAAAATTCAT
CAAAA

Met LysPheVal Leu Ala AGC

Ile Ala SerLeu LeuVal Leu ThrValTyr AlaArgPro AlaSer Ser AAA

Ile Lys ThrPhe GluGlu Phe LysAlaPhe'AsnLysAsn TyrAla Lys GCC

Thr Val GluGlu GluGlu Val ArgLysAsn PheLeuGlu SerLeu Ala GGT

25Lys Tyr ValGlu AlaAsn Lys AlaIleAsn HisLeuSer AspLeu Gly TCA TTG GATGAA TTCAAA AAC TATTTGATG AGTGCTGAA GCTTT'f 293 CGT

Ser Leu AspGlu PheLys Asn TyrLeuMet SerAlaGlu AlaPhe Arg GAT

Glu Gln LeuLys ThrGln Phe LeuAsnAla GluThrSer AlaCys Asp ~ ~ .

__ 21~~4~~~

Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Arg Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu ATT ATT AAA

Thr Gln ThrHis Thr Ala Ala Val Ile Gly Ile Asp Leu Ile Ile Lys GAT ATC CAT

Arg Ala PheGln His Tyr Gly Arg Thr Ile Gln Asp Asn Asp Ile His CAT ATT TAC

Gly Tyr GlnPro Asn Tyr Ala Val Asn Val Gly Gly Ser His Ile Tyr TGG AAC GAT

Thr Gln GlyAsp Asp Tyr Ile Val Arg Ser Trp Thr Thr Trp Asn Asp GGA GCC AAC

Trp Gly AspSer Gly Tyr Tyr Phe Gln Gly Asn Leu Met Gly Ala Asn TAT ATG AAATTGAATA

Met Ile GluGln Tyr Pro Val Val Ile Tyr Met TTTTCAAAAT CTCG
AAAAACAACT
ACTCTTGCGA

(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear r ~ . , , -4~- ~~~~Q~'S
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Lys Phe Val Leu Ala Ile Ala Ser Leu Leu Val Leu Ser Thr Val Tyr Ala Arg Pro Ala Ser Ile Lys Thr Phe Glu Glu Phe Lys Lys Ala Phe Asn Lys Asn Tyr Ala Thr Val Glu Glu Glu Glu Val Ala Arg Lys Asn Phe Leu Glu Ser Leu Lys Tyr Val Glu Ala Asn Lys Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Tyr Leu Met Ser Ala Glu Ala Phe Glu Gln Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu WO 94/05790 PCf/US93/08518 .f ~l~~t~~~,::~
Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu Gln Arg Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asp Val Lys Gln Ile Arg Glu Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Arg Ala Phe Gln His Tyr Asp Gly Arg Thr 145 150 ' 155 Ile Ile Gln His Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Gly Ser Thr Gln Gly Asp Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile Met (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..390 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lya Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Thr Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp AAT TTT AAA CCA

Val Pro GlyIle Asp Thr Ala CysHis Met Cys Leu Asn Phe Lys Pro GAT ACA AAT CCC

Val Lys GlyGln Gln Tyr Ala LysTyr Trp Val Lys Asp Thr Asn Pro AAC ACA AAA GTT

Ile Ala ProLys Ser Glu Val ValVal Val Leu Gly Asn Thr Lys Val TGC ACC GCT ATC

Asp Asn GlyVal Leu Ala Ala IleAla His Lys Arg Cys Thr Ala Ile GAT TF~AAAAAAAA AAATAAATAT 437 GAAAATTTTC
ACCAACATCG
AACAAAATTC

Asp ATTTGAATCA CTAC
AAAACGGAAT

(2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids (B) TYPE: amine acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein r WO 94/05790 ~ ~ ~ C~ PCT/US93/08518 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Thr Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg ~P

(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1172 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..738 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

Met Lys Ile Thr Leu Ala Ile Ala Ser Leu Leu Ala Leu Ser Ala Val Tyr Ala Arg Pro Ser Ser Ile Lys Thr Phe Glu Glu Tyr Lys Lys Ala Phe Asn Lys Ser Tyr Ala Thr Phe Glu Asp Glu Glu Ala Ala Arg Lys Asn Phe Leu WO 94/05790 O ~ ~ PCT/US93/08518 Glu Ser Val Lys Tyr Val Gln Ser Asn Gly Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val TGC AAT CGT

Ala Arg GluGln Ser Arg ArgPro AlaGln PheGly Ile Cys Asn Arg ATT AAT AAA

Ser Asn TyrCys Gln Tyr ProPro AlaAsn IleArg Glu Ile Asn Lys CAC GCC ATT

Ala Leu AlaGln Thr Ser AlaIle ValIle GlyIle Lys His Ala Ile CGT GGC ATC

Asp Leu AspAla Phe His TyrAsp ArgThr IleGln Arg Arg Gly Ile CCA GCT ATT

Asp Asn GlyTyr Gln Asn TyrHis ValAsn ValGly Tyr Pro Ala Ile GTC ATC AAC

Ser Asn AlaGln Gly Asp TyrTrp ValArg SerTrp Asp Val Ile Asn AAT TAT GCC

Thr Asn TrpGly Asp Gly TyrGly PheAla AsnIle Asp Asn Tyr Ala GAA GTT CTC

Leu Met MetIle Glu Tyr ProTyr ValIle Glu Val Leu TATATGATTG GAAAATGAAT
TCACTAATTT
ATTTAAAATC

~ , -5;
AAATTCATTC ACAAAAATTA AAAAAAAAAA F,~~AAAAAAAA F~~ AAAAAAAAAA 1139 P~AAAAAAAAA P~~AAAAAAAA AAAAAAAAAA AAA 1172 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 320 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Lys Ile Thr Leu Ala Ile Ala Ser Leu Leu~Ala Leu Ser Ala Val Tyr Ala Arg Pro Ser Ser Ile Lys Thr Phe Glu Glu Tyr Lys Lys Ala Phe Asn Lys Ser Tyr Ala Thr Phe Glu Asp Glu Glu Ala Ala Arg Lys Asn Phe Leu Glu Ser Val Lys Tyr Val Gln Ser Asn Gly Gly Ala Ile Asn His Leu Ser Asp Leu Ser Leu Asp Glu Phe Lys Asn Arg Phe Leu Met Ser Ala Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn WO 94/05790 ~, ~ PCT/US93/08518 '~1~~~~

Ala Glu Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile r WO 94/05790 ~'~ ~ PCT/US93/08518 Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 222 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 50 (D) OTFU;R INFORMATION: /label=Xaa is His or Tyr (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 81 (D) OTHER INFORMATION: /label=Xaa is Glu or Lys (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 124 (D) OTHER INFORMATION: /label=Xaa is Ala or Val A r (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 136 (D) OTHER INFORMATION: /label=Xaa is Ser or Thr (ix) FEATURE:
(A) NAME/K.EY: misc_feature (B) LOCATION: 215 (D) OTHER INFORMATION: /label=Xaa is Glu or Gln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly 25 30 ' 20 Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Xaa Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Xaa Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly r r 211~4~~~' Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Xaa Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Xaa Ala Ile Ala Val Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile 195 200 ~ 205 Asp Leu Met Met Ile Glu Xaa Tyr Pro Tyr Val Val Ile Leu (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 47 (D) OTHER INFORMATION: /Label=Xaa is Thr or Ser (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 114 (D) OTHER INFORMATION: /label=Xaa is Asp or Asn (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 127 (D) OTHER INFORMATION: /label=Xaa is Ile or Leu (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Xaa Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Asp Xaa Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Xaa Arg Asp (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 11 (D) OTHER INFORMATION: /label=Xaa is Asn or Ser (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 52 (D) OTHER INFORMATION: /label=Xaa is Thr or Ile (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 54 (D) OTHER INFORMATION: /label=Xaa is Ile or Thr 1~~~~~ -(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 76 (D) OTHER INFORMATION: /label=Xaa is Met or Val (ix) FEATURE:
(A) NAME/KEY: misc-feature (B) LOCATION: 88 (D) OTHER INFORMATION: /label=Xaa is Ala or Ile (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 111 (D) OTHER INFORMATION: /label=Xaa is Val or Ile (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn Xaa Glu Ile Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Xaa Glu Xaa Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Xaa Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Xaa Lys Tyr Thr Trp Asn Val Pro Lys 63_~I~40~5 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Xaa Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp IO (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid IS (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:

(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 3S (ii) MOLECULE TYPE: cDNA
AMENDED SHEET
IPEA/EP

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

S (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:

(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPEr cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

AME(~~ED S~iEET
IPEA/~P

Claims (5)

-64-

1. A protein allergen of Der p II comprising the amino acid sequence:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Xaa1 Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Xaa2 Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Xaa3 Arg Asp where Xaa1 is selected from the group consisting of Thr and Ser;
where Xaa2 is selected from the group consisting of Asp and Asn;
and where Xaa3 is selected from the group consisting of Ile and Leu, except for the amino acid sequence where Xaa1 is Thr, Xaa2 is Asp and Xaa3 is Ile.

2. A protein allergen of Der f II comprising the amino acid sequence:
Asp Gln Val Asp Val Lys Asp Cys Ala Asn Xaa1 Glu Ile Lys Lys Val Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Xaa2 Glu Xaa3 Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Xaa4 Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Xaa5 Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Xaa6 Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp where Xaa1 is selected from the group consisting of Asn and Ser;
where Xaa2 is selected from the group consisting of Thr and Ile;
where Xaa3 is selected from the group consisting of Ile and Thr;
where Xaa4 is selected from the group consisting of Met and Val;
where Xaa5 is selected from the group consisting of Ala and Ile; and where Xaa6 is selected from the group consisting of Val and Ile, with the proviso that, when Xaa1 is Asn, then Xaa3 is Thr; and when Xaa3 is Ile, then Xaa1 is Ser.

3. A therapeutic composition comprising a protein allergen of claim 1 and a pharmaceutically acceptable carrier or diluent.

4. A therapeutic composition comprising a protein allergen of claim 2 and a pharmaceutically acceptable carrier or diluent.

5. Use of the composition of claim 3 or claim 4 to treat sensitivity to house dust mite allergens.