CA2142203A1 - Basophil granule proteins - Google Patents

Abstract

Several natural polypeptides (basophil granule proteins, "BGP") derived from the cytoplasmic granules of human basophils, and modified forms thereof, are described. These polypeptides, the DNA
which encodes them and antibodies which recognize them, are useful as diagnostics for, and treatments for, pathologies involving inflammatory and IgE-mediated responses, parasitic and helminthic infections, hypersensitivity reactions and certain types of leukocytic leukemias.

Description

W094/06829 ~ 1 4 2 2 0 3 PCT/US93/08511 BASOPHIL GRANULE PROTEINS

Technical Field This invention i~ related to no~el proteins, pharmaceutical compositions containing such, therapeutics and human immunology. More specifically, it relates to proteins found in the cytoplasmic granules of human - basophils, to the gene~ which encode them, to the antibodies which recognize them, and to the use of these lS proteins, oligonucleotide~, and antibodies in the diagnosis and treatment of disease.

Back~round Art The~basophil,~along-with the mast cell, 20~ contains~cytoplasmic~ granules with an affinity for basic s~ The basophil~is produced by the bone~marrow and 9~ circulates~in the blood. 9asophils are associated with he~iminthic parasiti~c infections and allergic reactions and~they~possess a high affinity receptor for IgE
25~antibod~les.~ Little is known howe~er about the proteins which~comprise the~granùle,~ in part because, under normal co~ditlo~s, basophils constitute le8s than l~ of peripheral blood cells and it is therefore difficult to obtain~an adequate ~amount of purified material for study.
While some researchers have proposed that ba~ophils-areitheipre~ursors of mast cells, recent data suggests that basophils represent terminally differentiated leukocyte8, pos~ibly more closely related to eo8inophils~(Galli, S~J. and Lichtenstein, ~.M., in ., :
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WO94/06829 ~ O3 PCI/US93/01~5ll-~ ~

All~rqy:Princi~les and Practice, Middleton et al (Ed~.), -3rd Ed, Vol. 1 (1988), pp 106-134).
Ba~ophil~ appear to participate in many inflar~unatory, irr~nunological and pathological react~ions.
5 For a general review ~ee ~alli et al, Proq_Allergy (1984) 34:1. The most ~triking tissue infiltrates of ba~ophils occur in cutaneou~ ba~ophil hypersensitivity reaction~
(Galli and A~kenase, in The Reticuloendothelial Sy~tem:A
Com~rehensi~re Treatise, Abramof~ et al (Eds.) pg 321, 10 Plenum Press 19~6). Recent studies suggest that basophils are es~ential for expression of i~nunity to the feeding of larval Amblyo~ma americanum ticks. Here, basc)phils rn~y collaborate with eosinophils in the expression of inmunity by acting to attract eosinophils 15 into tissues where the eosinophils subsequently release toxic cationic proteins (Brown, S.J. et ai, J Immunol (1982) 129:790). ~asophils are also elevated during ` helminthic infections, suggesting that they might participate in host defense to these parasites (Ogilvie, 3.M. et al, I~nunol (19aO) 39:3~5; Lindor, h.J., Para~ite I~nuno} (1983) 4:13; Juhlin, h. and Michaelsson, G., Lance~ (1977) 1:1233). E~ridence also exists that basophils function in hypersensitivity reaction (Schwartz, L.B. and Austen, K.F. in Immunological ~; 25 Disease~, Samter et al (Eds) Little E~rown & Co 4th Ed, pg 157 (1988); Mitchell, E.B. Clin Re~ ~ller~ sa3) 427), and in IgE mediated cutaneous late phase reactions (Solley, G.O. et al, J Clin Invest (1976) ~1.:408; Charlesworth, E.N. et al, J Clin Invest (1989) ~.:1519).
Studies (of human basophil granule proteins ha~re been limited by the difficulty of obtaining sufficient - numbers of basophils because they constitute o~ly about 0.5~ of the total leukocyte population. Prior studies f proteins isolated from the basophil3 of guinea pig8 i~

W094/06B29 ~14 2 ~ 0 3 PCT/US93tO8511 reFeatedly immunized with sheep blood revealed a mixture of neutral esterases-pro~ea~es and both trypYin and chymotrypsin-like serine hydrolase~, Dvorak, H.F. et al, J Immunol ~1974) ll3:l69; J Im~munQl (1977) 119:38J~
Studies of the glycosaminoglycans (GAG) of these proteins showed a mixture of GAGs including chondroitin sulfate, dermatin sulfate, and small amount~ of heparin sulfate (Orenstein, N.S. et al, J Immunol (1978) l2l:586).
Several proteins have been localized to the human ba30phil granule including the eosinophil major basic protein (Ackerman, S.J. et al, J Ex~ Med (1983) 158:946) and the Charcot-Leyden crystal protein (Ackerman, S.J. et al, J Ex~ Med (1982) l55:l597). In addition, mast cell tryptase can be identified in human ba~ophils at about 40 pg/cell, a level roughly 500-fold lower than in human mast cellq (Castells, M.C. et al, J
Imm~aQl (1987) l38:2l84). In addition, bradykinin ~generating aceivity has been ascribed to basophils by virtuè;of the release of this enzyme from peripheral ~whlte~blood cel1s by IgE dependent stimulation (Newball, H.H. et al, J Clin Invest (1979) 64:466), The present invention was facilitated by a patient that presented with basophilic leukemia.
~ Leukocyte counts were over 105 cell/~l and contained 78%
r,~ 25 basophils. On two~occasions this patient underwent ~ cytophoresis for removal of leukocytes and a total of -~ l.SxlOll basophi1s were obtained. Examination of the ~-~ granule proteins of these ba~ophils have revealed a number of novel pro~eins with unique N-tenminal amino acid sequences.

DisclQ~ure of the Invention Several newly identified polypeptides (basophil ;~
granule proteins, nBGP9 n ) are de8cribed which constitute somè of the proteins found in the cytopla~mic gra~ules of ' ~
:~
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W094/06829 PCT/US93/08~

2 ~ 03 - 4-human basophils. These polypeptides, the DNA which encodes them and antibodies which recognize them, are critical for diagnostics for, and treatments for, pathologies involving inflammatory and IgE-mediated responses, parasitic and.helminthic infections, hypersensitivity reactions and certain types of leukocytic leukemias.
One aspect of the invention is directed to BGPs, which include proteins found in the granules of basophils, and fragments, mutations and modifications of these natural proteins which retain their respective BGP
biological characteristics. The polypeptides can be recombinantly produced by cell~ in culture, produced by chemical synthesis or isolated and purified from basophils.
Other aspects of the invention'are an : : expression system comprising DNA~ which encode these . BGPs; host cells transfonmed with these expression systems; and methods to produce BGPs~which utilize host ~ 20 cells transformed with said expression systems.
;~ ~ Sti}l other aspects include antibodies, both monoclonal and polyclonal, which are specific for BGPs.
Additional aspects include methods of diagnosis and treatment of diseases characterized by abnormal .25 expression or re}ea~e of BGPs by basophils or other cells, or by genetic abnormalities within genes encoding BGPs. Further aspects include methods of treating `~: disease by the~ administration of the BGPs, antibodies, and DNA described herein.
Brief Descript~onlof!the Drawin~s Figure 1 shows a HPLC chromatogram of human ~ basophil granule proteins obtained per Example l;

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~, , W094/06X29 ~ 1 4 2 2 ~ 3 PCT/US93~08511 Figure 2 include~ HPLC chromatograms 2-1 to 2-8 of human basophil granule protein~ obtained per Example 2; and Figure 3 include~ HPLC chromatograms 3-l to 3-3 of human basophil granule proteins obtained per Example 3.

Modes of Carryinq Out the In~ention Unless defined otherwi~e, all technical and scientific terms used herein have the same meaning as - commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equi~alent to those described herein may be used in the practice or testing of the present in~ention, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference for the purpose;of disclosing and~describing the~specific aspects of the invention to whih those publications relate.
~ As used herein, "basophil granule protein" or "BGPn;refers~to purified forms of any and/or all of the novel proteins that may be purified from basophil ~; granules as disclosed here as well as fragments and variantq thereof, which retain a useful biological cha~acteristic of natural BGP~which BGP may be obtained by extraction, synthetically produced or produced using recombinant DNA methodology. It is recognized that basophil granules contain certain proteins that have been demonstrated from other 30urces. The in~ention allow~
the description of additional novel proteins that are discoverable f~omlbasophil granules.
A fragment of a BGP is a polypeptide having a primary amino acid se~uence identical to any part of a naturally occurring BGP and retaining a u9eful biological characteristic of the BGP.
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A ~ariant of a ~GP i9 any naturally occurring (allelic variant), recom~inantly engineered or chemically synthesized peptide or protein resulting from changes in the primary amino acid ~equence or posttranslatio~nal p.ocessing of the BGP described, but retaining useful ~iological characteristic~ of the BGP isolated from the cytoplasmic granule of human basophils. The variant forms of natural BGP include tho~e wherein one or more instances of amino acid deletions, substitutions or ;0 insertions occur. The variant forms of natural BGP also include those wherein aItered patterns of glycosylation or lipidation occur. Variants also include ~GP made ~ynthetically wherein substitutions by amino acids which are not encoded by the gene are made. Examples of such amino acids include but are not limited to norleucine, citrulline, ornithine, hydroxyproline, and cysteic acid.
- The biological l'characteri~tics" refer to the structural and/or biochemical properties of a BGP and include i~ts specific antigenicity or immunogenicity and its abl}ity to mediate inflammatory and immunological - ~ ~ respon9e9 n vL~Q. ~;
A "mutated" protein i8 a protein with an altered primary structure (relative to the commonly : : :
- - occurring protein) resulting from changes in the nucleotide-sequence of the DNA which encodes it. These mutations~include allelic variant~. A "modified" protein differ~s from the commonly occurring protein a9 a result ~ ; of post-translational e~ent~ which change the - glycosylation or lipidation pattern, or the primary, 3econdary, or tertiary structure of the protein. Changes in ~the9primaryl'structure!of a protein can also result from deletions, additions or sub~titutions. A "deletionl~
-~ - i9 defined~as a polypeptide in which one or more internal ~-- amino acid residues are absent. An ~addition" i9 defined as a polypeptide which has one or more additional . , , , W094/068~9 ~1~ 2 2 0 3 PCT/US93/08~11 internal amino acid residue~ as compared to the wild type. A '~sub~titution" result~ from the replacement of one or more amino acid residue~ by other residues. A
protein "fragment" i~ a polypeptide consisting of a primary amino acid sequen~e which i~ identical to a portion of the primary sequence of the protein to which the polypeptide is related.
Preferred altered forms of "natural" BGP
described above are those which have at least 80~
homology with natural BGP. At least 90~ homology is more preferred, especially those including conservati~e substitution~.
Homology is calculated by standard methods which involve aligning two sequences to be compared 90 that maximum matching occurs; and then calculating the percentage of matches. The altered forms of natural ~GP
include those wherein one or more of the residues of the native 3eque~ce i8 deIeted, substituted for, or inserted by a different amino acid or acids.
~ Preferred substitutions are those which are ; conservati~e, i.e., wherein a residue i8 replaced by another of the same general type. As is well understood, naturally occurring amino acids can be subclassified a~
~ acidic, basic, neutral and polar, or neutral and - ~ 25 nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generaIly preferred that peptides ~- ~ differing from the natural BGP contain substitutions , which are from the same group as that of the amino acid ~-1 replaced. Thus, in general, the basic amlno acids ~y9, Arg, and Hi~ are interchangeable; the acidic amlno acids aspartic and gl~tamic'are interchangeable; the neut~ai ;~ polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, Ile, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely . ~ ~

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W094/06829 214 2 2 0 3 PCT/US93/~8~

related and Val, Ile and Leu are more clo~ely related), and the aromatic amino acids Phe, Trp, and Tyr are interchangeable. While proline i8 a nonpolar neutral amino acid, it presents difficulties becau~e of ~ts effects on confonmation, and substitutions by or for proline are not preferred~ except when the same or similar conformational result~ can be obtained. Polar amino acid~ which represent conservative charge include Ser, Thr, Gln, Asn; and to a le~ser extent, Met. In addition, althoùgh classified in different categories, Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids. Some substitutions by amino acid~ from different classes may also be useful to produce altered 3GP.
It should further be noted that if the BGP i~
- made synthetically, substitutions by amino acids which cannot be encoded by genes~may also be made. Alternative ~residues include, for example, the omega amino acids of the fonmula N2N(CH2)nCOOH wherein n i9 2-6. These are neutral, nonpolar amino acids, as are sarco3ine ~Sar, t-butyl alanine (t-BuA), t-butyl glycine (t-BuG), N-methyl Ile (N-MeIle), and norleucine (Nle)~ Phenyl glycine, for example, can be substituted for Trp, Tyr, or Phe as aromatic neutral amino acids; citrulline (Cit) and - . methionine sulfoxide (MSO) are polar but neutral, .-~, .
;;~; cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic ~- acid ~(Cya) is~acid~;c, and ornithine (Orn) i9 basic. The ~ confonmation conferring properties of the pxoline ; 30 residues may be retained if one or more of these i9 substituted by hydroxyproline (Hyp).
It should be further noted that if the ~GP is " ~ ..
produced recombinantly as an intracellular protein, an N-terminal methionine residue may be retained in the fini~hed product. Cleavage of the N-terminal methionine ' , ' ~;

~ ; WO 94t06829 2 i 4 2 ~ ~ 3 PCT/US93/08~11 to liberate the native sequence may or may not be complete.
The biological "characteristic~ll of a protein refer to the structural or biochemical function of the protein in the normal biological processes of the organism in which the protein naturally occurs. Examples of biological characteristics of a BGP include its specific antigenicity or immunogenicity, its anti-helminthic activity when this is associated with a particular protein, and/or it~ ability to mediate inflammatory and immunological respon~eR in vivo.
A host cell "expresse~" a gene or DN~ when the gene or DNA i~ transcribed. A protein or polypeptide is "expressed" when the protein or polypeptide has been produced.
"Recombinant host cell n means a procaryotic or eucaryotic~cell which contains an expression vector comprising heterologous structural DNA and is capable of expressing~the polypeptides encoded by the heterologou~
^ ~ - 20 DNA.
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-~ A. Isolation of Baso~hil G~anules ClinicaI hematology laboratories are monitored to identify patients with chronic myelogenous leukemias ~with~greater ~han~2-3x104 leukocytes/~l of blood and lO-20~ basophils. The basophils are purified by , ~
,7',` centrifugation over a cushion of Ficoll-Hypaque from ~- ~ which 95% are recovered from the inter~ace with greater than 90~ purity.
30 ~ , Purified basophi1s are lysed u8ing modifications of the procedures de8cribed by Dvorak et al, J Immunol (1977) 119:38 (5~ ), for purification of ~-~ guinea pig basophil granule proteins. Briefly, and in a -;~ typical and illustrative procedure, purified basophils ~ 35 are washed with PBS and contaminating erythrocyte~ are -;

,~,, W094/06829 PCT/US93/08~11r--` 21~2203 `

ly~ed by e~posure to Tris-ammonium chloride for 5 minutes. The cell suspension is centrifuged at about 400g, washed with Hank's ~SA-EDTA and su~pended in cold 0.25M sucrose containing DNAase and heparin using~a volume of l5 ml for 8x108 basophils. The cell suspension i~ next centrifuged at 400g for lO minutes and the sediment i5 again suspended in 0.25M ~ucrose containing 2mg DNAase per 15ml cell suspen~ion. After 1-2 minutes, heparin (20 IU) dissolved in 2ml 0.24M sucrose i5 added and the preparation is subjected to a shearing force by repeated passage (15 times) through a 20 gauge needle.
The suspen~ion is centri~uged a~ 400g to remo~e any remaining intact cells, and the granules are then purified by centrifugation through a cushion of 40~
sucrose Finally, the proteins of the isolated basophil granules are solubilized by expo~ure to O.C5M borate buffer at pH 9 in the presence of 5mM
diisopropylfluorophosphate, lx10-7M pepstatin A, and lOmM
~ .
~ EDTA~to inhibit protease acti~ity.
~ - .
~- 20 B. Protein Fractionation The solubilized proteins of human basophilic granu1es, in the same sol~ent described su~ra, are separated by reverse phase HPLC using a Brownlee BU-300 25~ C4 column. ~The mobile phase i9 0.1~ trifluoroacetic acid ~TFA) containing 0-70~ acetonitrile. Fractions are collect~ed across the acetonitrile gradient as shown in Figure l, where absorbance at 214 nm is shown on the ordinate. The relative homogeneity of each fraction i8 determined by SDS-PAGE electrophoresis.
~ ~ Although re~erse phase HPLC is an extremely ;~ powerful technique, not all human basophil gra~ule - protein~ can be purified by thi technique alone. Thus, ~ ize exclusion chromatography can also be employed as a -~ 35 ~-'''"~:' ~ :- W094/06829 2 ~ 4 2 2 ~ 3 PCT/US93/08511 preliminary fractionation (e.g. Bio Sel TSK 125 in 50mM
phosphate pH 6.9) prior to HP~C.
If additional purification of size exclusion chromatography fraction~ i~ necessa~y prior to HPLC, ion exchange chromatography can also be employed. A Mono-Q
column (Pharmacia) is used under conditions as would be understood in the art whereby mo~t typical proteins would bind to the column (e.g. 20 mM Tris, pH9.0). The protein~ are then eluted in a gradient from 0 to 2 M
NaCl.
Purified proteins recovered from re~erse phase HPLC are sequenced by subjecting up to 100 pmole~
(estimated ~rom chromatographic peak height and ~taining intensity on acrylamide gels) to automated Edman degradation.
For some granule proteins N-terminal sequencing may not be adequate to support efforts to clone the -~ cDNAs. ~For instance, some granule proteins~may be blocked~or modified at the N-terminal or alternatively, the~-N-~terminal sequences may not show fa~orable regions for generation of oligonucleotide probes. Such proteins are~digested with trypsin and the tryptic peptides are purified and sequenced in order to generate additional information. The protein i9 concentrated to a 5~1 volume by vacuum centrifugation, and i9 then digested by i~cu~atian (4 hours, 37 C) with 1/40 (w:w) TPC~-trypsin in~1 ml: of 50mM ammonium bicarbonate, pH 8Ø Tryptic fragments are puri~fied for sequencing by reverse phase -~ -HP~C using a ~rownlee RP 18 narrow bore column and an Applied Biosystems 130A liquid chromatograph or other suitable de~ice for HPLC - designed specifically for ~; ~ purification of pmole samples.
-~ Sequence data thus obtained are compared to krown protein ~equences by computerized searches of the Protein Identification Resource of the N~RF, and/or of , ,, ~

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~, W094/06829 PCT/US93/08511f 2142~03 i ` -12-th~ Swi~9 protein databa~e, in order to determine their novelty or relationship to other protein sequences.

C. Protein Sequencing.
Protein sequen~ing i5 performed by automated Edman degradation utilizing a Applied ~i 09y8 t ems 477A
Protein Sequenator. Those skilled in the art will recognize that sequence assignments based on a ~ingle analysi~ are subject to a degree of uncertainty.
[Hunkapiller, M.W. et al. in Method~ of Protein Microcharacterization ed. Shively, J.E. (19~6) The Humana Press, Clifton, N.J., pp. 223-247; Hunkapiller M.W., Applied Biosystems Protein Sequence~ Users Bulletin #14 (1985)]. Particularly, when assignments at a given cycle lS are based on analysis of low quantities of the deri~atized amino acid~, a certain le~el of error i~
expected. These error~ are most frequently misidentification~ of amino acids which are more difficult to identify, namely Serj Thr, His, Arg and Trp.
~ Po~itive identification of Cys is a special problem. Without prior modification of the pxotein~, Cys is not detectable but its presence may be inferred by the a~aence of an appropriate signal in that cycle.
Lag i~ a well known phenomenon in sequencing, where signal deri~ing from cycle n i9 also present in ~ cycle n~+l. Lag present3 a special problem when Cys is - ~ present at cycle n+1 since the amino acid present at ~ cycle n may mistakenly be as3igned at cycle n+1 due to -~ the lag phenomenon.
Becau3e of such characterigtic uncertainties, data from an analysis may support more than one intexpretation. Nevertheless, examination of the ~- sequence data allows one skilled in the art to judge whether two qequences derive from the same protein, e~en when ~ome discrepancies exist.

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In accordance with the considerations, the sequences determined are submitted as a description rather than a definition of the proteins which have been isolated.
D. Screenin~ of cDNA Libraries and the Molecular Cloninq of Unique Basophil Granule Protein Encodinq PNA
Basophils mature in culture~ of human umbilical cord blood cell~. Thus these cultures can be used to prepare a cDNA library which is then screened for particular DNA sequences that encode proteins unique to human basophil granules (BGP) (Saito, H. et al, Proc Nat Acad Sci (1988) 85:2288).
Other candidate cDNA libraries which may express BGP~ include unstimulated HL-60 cells, or Hh-60 cells driven to basophilic differentiation by culturing in a;protein free medium (Muroi, K. et a}, heukemia Res (1989) l3:l~57) or in the pre3ence of sodium butyrate (~ut~t-~Taylor, S.R. et al., ~l~Ç~ (1988) 71:209).
20 ~ Although~basophils and mast cel}s appear to be distinct n~eheir lineageq, granules of both cells contain mast cell~tryptase (Castells, M.C. et al, ~_Immunol (1987) 13~:21843 and these cells may therefore share other common pxoteins. Therefore rDNA libraries made from human mast cel}s (~e.g. HMC-l) are another source of BGP
encoding~sequences.~The preparation of these cDNA
libraries i9 described in detail in Maniatis, T. et al, Molecular~ClQ~lng, (1982) CSHL Press, and i8 well known to those skilled in the art. A convenient approach is ~ 30~ the insertion of cDNA fragments into a lambda phage : vector e.g. lambda gtiO or lambda gtll as`described by M~niatiq, .su~ra.
i- Methods of screening cDNA libraries are also - well known to those skilled in the art. The amino acid sequence of the BGPs is analyzed for example utilizing i, , ,:: -, , ''' -~ ' W094/06829 ~ PCT/US93/08511;

. -14-programs from DNAstar (Madi30n WI) in order to identify optimal regions for construction of oligonucleotide probe~. Redundant oligonucleotide probes are Qynthe~ized with a DNA synthesizer (380A: Applied Bio~ystem~ Inc.
Fo~ter City CA) by the phosphoramidite method.
Oligonucleotides are purified on Sephadex G-50 columns and ~tored at -20C. The redundant probe~ are 5'-labeled with T- [32P)ATP (E.I. du Pont de Nemours & Co., Inc., Boston, MA) using T4 polynucleotide kinaqe. ~ibraries are ~creened u~ing up to 1o6 individual plaques per library, with the redundant oligonucleotide probes.
Duplicate nylon membranes containing phage are prepared and prehybridized in 5x SSPE ~0.9M Nail, 50mM NH2P04, 5mM
- EDTA, pH7.4), 0.2~ SDS, and 0.005% denatured salmon ~perm DNA for 2 hours at 50C with 8 filters per 50 ml prehybridization fluid per bag. Membranes are hybridized with approximately 1 ng of labeled probe per ml, in fresh hybridization fluid, overnight at the appropriate temperature for the redundant probe mixture. Membranes are then wa~hed at room temperature for 45 minutes in 1 liter of 5x SSPE per 40 filters, followed by a 1 minute wash in fre~h buffer at 50C, slightly air-dried, a~d exposed to Rodak XAR-5 film, with intensifying screens, for 72 hours at -70C.
.~ .
After analysis, filters are stripped of hy~ridized label by incubation in 5x SSPB at 70C for 10 minutes and sub~equently hybridized with a second probe -~ under the same conditions. This procedure is repeated , .~
for each probe. Recombinant clones which hybridize ~ith probes will be ~elected from the library and plaque pur~fied.
Recombinant phage DNA i9 then purified and digested with an appropriate reqtriction endonuclea~e to -~ yield the amplified cDNA insert. Inserts are then ~ 35 ligated into M13mp series phage and sequenced u~ing the , ~ W094/~6829 ~1422a3 PCT/~S93/0851i di~eoxy method described by Sanger ~igginl M.D. et al, Proc Nat Acad Scl ~l983) 80:3963). Depending on the ~ize of the cDNA, it may be necessary to restrict the clone, and subclone the fragments into ~13. If the cDNA clones are not complete, a repeat ~creen of the library with the partial cDNA would be required. The complete ~equence of the ~GP cDNA i then compared again~t known sequences in the GenBank databa~e. DNAstar is used for nucleotide and polypeptide analy~es and sequence comparison~.
Selected cDNA insert~ which encode a BGP can then be incorporated into an expression ~ystem. The cDNA
is operably linked to heterologous control sequences to form an expression ~ector. The control sequences are chosen to be functionally compatible with the recombinant host cell into which the expressio~ vector is introduced.
These procedures are known to tho9e skilled in the art and described in Maniatis, supra.
Expression can be in procaryotic or eucaryotic systems. ~Procaryotes most frequently are represented by varioùs~strains of E~coli. Howe~er, other microbial strains~ay also be used, ~uch a9 bacilli (e.g. Bacillus subtilis), various species of Pseudomonas, or other bacterial strains. In such procaryotic systems, pla3mid " ~
~ectors which contain replication sites and control 25~ sequences~derived from a species compatible with the host are~;us~ed.~ For example, E coli is typically transformed using deri~atives of pBR322, a pla~mid derived from an E.
li species by Bolivar et al., Gene (l977~ 2:95.
Commonly used procaryotic control sequences, which are defined herein to include operons with promoters for transcriptional i1nitiatlon, optionally w~th an operator, :~:
along with ribosome binding site sequences, include such commonly used promoter9 as the beta-lactamase (penicillina9e) promoter, lacto9e ~lac) promoter 9y9tems (Chang et al., Nature (1977) l98:1056), the tryptophan ,,".~
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WO 94/06829 2 1 ~ 2 2 0 3 PCT/US93/0851~

, (trp) promoter system (Goeddel et al., Nucleic Acids Res (1980) 8:4057), the lambda-deri~red PL promoter and N-gene ribosom~ binding site (Shimatake et al., Nature (1981) 292:128). Any available promoter sy~tem compatiple with S procaryotes can be u~ed.
The expression systems useful in eucar}rotic hosts comprise promoters derived from appropriate ~`
eucaryotic genes. A class of promoters useful in yea~t, for example, includes promoters for synthesis of 10 glycolytic enzyme~, including tho~e for 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem (1980) 255:207). Other promoters include those from the enolase gene (Holland, M.J., et al. J Biol Chçm (1981) ~56:1385) or the ~eu2 gene obtained from YEpl3 (Broacht, J.~ et al., ~aÇ (1978) ~:121).
Suitable ma~lian promoters include metallothionein, the early and late promoter~ from SV40 -~ (Fiers et al.~, Nature (1978) 273:113), or other Yiral promote~rs~such a those derived from polyoma, adeno~rirus ;20 II, bo~ine papilloma virus or retroviruses. Suitable - ~;
`In~ Yiral and~mammalian enhancers may also be used. In the event~plant cells are used as an expression system, the nopaline synthesis promoter is appropriate (Depicker, A., : ~ ~ et al~., J Mol A~?l Gen (1982) 1:561).
~ ~ The expreqsion system is constr~cted from the foresoing control elements~ which are operably linked to the BGP sequences by employing standard ligation and restriction techniques which are well understood in the ~- ~ art. Isolated pla mid~, DNA sequences, or synthe~ized ~; 30 oligonucleotides are cleaved, tailored, and relegated in the fo`rn~s desi~ed.

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' ; w094/06829 ~ 2 2 0 ~ PCT/US93/08511 E. Productlon_of AntisenSe~ and Trlple Helix 01iqonucleo~cldes Antisense oligonucleo~ides can be designed and produced ba~ed upon the nucleotide sequence of cDN~A
encoding a BGP. The anti$ense oligonucleotide can be designed and used to regulate the translation within the cell of the specific mRNA to which it i9 complementary according to methods known in the art (Green et al., Ann Rev Biochem (1986) 55:569; Rossi et al., Pharmacol Ther (1991) 50:245).
The antisense reagent is made complementary to some portion of the mRNA encoding the BGP, preferably including a portion of the mRNA at or near the translation initiation site in the 5' region. In one approach, the antisense reagent may be ~NA or preferably a modified RNA wherein the sugar phosphatq backbone has been~modified to increase resistance to RNase acti~ity or otherwise impro~e pharmacokinetic or pharmacodynamic properties. In another approach the antisense oligonucleotide may be a DNA, which has been ligated to a promoter in an antisense orientation, such that transcription of the DNA produces a mRNA complementary to the mRNA encoding a BGP. The antisense DNA may be incorporated into an expression ~ector for introduction into the body.
-~ Triple helix oligonucleotides can also be de~igned and prod~ced based upon the nucleotide sequence of cDNA encoding a BGP. The triple helix oligonucleotide can be used to-regulate the transcription within the cell 30 of the specific DNA to which it is taryeted and -~
particularly to'inhibit expression of a specific gene in individuals having diseases associated with expres~ion of the gene. ~omopurine and homopyrimidine sequenceq which are appropriate for triple helix formation can be designed by methods k~nown in the art (Griffen et al.

. -~

W094/06~29 ~1 A 2 2 ~ 3 PCTtUS93/08 ScIe ce (19a9~ 245:967). The effectivenes~ of triple helix oligonucleotide can be improved by synthe~izing the reagents using the unnatural ~-anomeric nucleotides to improve their nuclease resistance properties or b~
derivitizing the oligonucleotides with an intercalating agent such as ethidium bromide to 3tabilize the triple helix, once it has been formed.
Anti~ense molecules are introduced into the body in one method by injecting the oligonucleotide either alone, encapsulated in a liposome or incorporated into a viral particle.
~ .
F. Analysis of the Genomic Seq~nce of B~P DNA
~GP-encoding genes are obtained from the genomic library of human fetal liver DNA in Charon 4A
phage (ATCC 37333). The library contains 106 independent recombinants with an insert size of 15-20 kb and it is ~ screened wieh cDNA essentially as previously described.
-~ Phage are seguentially adsorbed onto duplicate 8x8 cm nylon membrane filters. Filters are prehybridized in 5x ~-- SSPE, 50~ formamide, 5x Denhardt's solution, 0.5% SDS and 0.005~ denatured salmon qperm DN~ for 2 hours at 42C
with 8 filters per 50 ml of prehybridization fluid.
Fi1ters are hybridized with approximately 1.0 ng of 25~ lab~eled ba ophil protein cDNA per ml of fre~h - prehybridization fluid, containing 10~ dextran sulphate and 2x Denhardt's solution, overnight at 42C. BGP cDN~
~- is labeléd with ~32p dCTP and purified by Sephadex G-50 chromatography. Filters are then washed twice at room temperature for 15 minutes in 1 liter 2x SSPE and 0.2~
SDS per!40 filt~rs', followed by two 15 minute 50C washes in O.lx SSPE and 0.2~ SDS, ~lightly air-dried,and expoQed to Kodak XAR-5 film, with intensifying screens, for 48 hour~ at -70C.
~ 35 '~:
~' :' ;~- W094/06829 ~1 ~ 2 2 0 3 PCT/US93/08511 Po~itive clone~ are selected from the library and plaque purified. Various probes derived from the cDNA are utilized to determine whether or not a complete copy of the gene is contained within the genomic clone.
Recombinant phage DNA is next extracted, purified, and subjected to restriction digestion - all processes which are well known to those skilled in the art. Soùthern blots of the restriction fragments are hybridized with BGP cDNA to identify fragments containin~ the BGP gene.
10 These f-agments are then isolated and sequenced. From "`
this inf-ormation a restriction map is con~tructed and the inerons of the gene are identified.
. .
G. Preparaeion of Antibodies to BGP3 `.
Two approaches are utilized to raise antibodies to BGP and both approaches can be used to generate either poly~clonal~or monoclonal antibodies. In ~ne approach, as `dena~ured~pro~ein from~ehe~reverse~phase HPLC separation is-~0=btàlned in~quantities up~to 75 ~g, this~denatured 20~ proeein~can~-be~used to immunlze mice;using standard protocols;~abQut 25 ~g is adequaee for immu~ization. For c~reening~hybridomas, the denatured protein, which is soluble in 0.1% TFA~and acetonitrile, can'be radioiodinated;and used to s~creen murine ~-cell 25~ hybrid~mas'~for those which produce antibody. This proc~dure~r~equire9 only small quantitie~ of protein such ehat 20~g would be~sufficient for labeling and screening of~several~thou:~and~clones.
~`-9~ In the sec~ond approach, the amino acid sequence of BGPs as deduced from the gene is analyzed to determine ' regions of high' i~mu~o'genicity. The corresponding I ' polypeptides are synthesized and are used in suitable immunizat~ion~prQtocols to rai9e antibodie9. Analy9is to se1ect appropriate~epitopes is described by, for example, Au-qubel,~ F.~M. et al, in ~rent Protocols in Molecular , ~ ~

W094/06X29 ~ 1 4 2 ~ 0 3 PCT/US93/08511 ~i~loqy, John Wiley ~ Sons, Vol. 2, Sec. IV, ppll.14.l, 1989). The optimal selections are usually the C
terminus, the N terminus and internal regions of the polypeptide, which are likely to be exposed to the external environment when the protein is in its natural conformation (this determination is based on the hydrophilicity of the sites). Typically, selected peptides, about 15 residues in length, are synthesized usi$g an Applied ~iosystems Peptide Synthesizer Model 431A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma) by reaction with m-maleimidobenzoyl-N-hydroxysuccinimide e~ter (MBS) (See Ausubel et al, supra at pp ll.15.l). A cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are lmmunized with the peptide-KLH complex in complete Freund' 9 adjuvant and the resulting antisera tested for antipeptide acti~ity, for example, by blnding the peptide tQ plastic, blocking with -~ 0.1%~ SA, reacting with antisera, washing and reacting w1th radioiodinated affinity purified specific goat antirabbit IgG.
; Hybridomas may be also be prepared and screened - using standard techniques. Hybrids are scrèened using radioiodinated 9GP to identify those producing monoclonal antibody. In a typical protocol, prongs of plates (FAST, 9ecton-Dickinson, Palo Alto, CA), are coated with - affinity purified specific rabbit-antimouse (or suitable anti species Ig) antibodies at lO ~g/ml. The coated prongs are blocked with O.l~ ~SA, washed and exposed to supernatants from hybridoma~. After incubation the prongslare exposed td radiolabeled protein, 1 ng/ml.
Clone~ producing antibodies will bind a quantity of radioactivity which is detectable abo~e background. Such clones are expanded and 3ubjected to 2 cycles of cloning at 0.3 cell/well. Cloned hybridomas are injected into ~":

"- ~
, ~, ~ W094/06829 ~1 ~ 2 2 0 3 Pcr/usg3/o85l, pristine treated mice ~o produce ascite~q, and monoclonal antibody i9 purified from the ascitic fluid by affinity chromatography on protein A.

H. Use of Anti-BGPs in Diagnosls Anti-~GPs are useful for the diagnoqis of prepathologic conditionq and aq well as chronic and acute di~ea~es which are characterized by abnormalities in the amount or distribution of BGPs. For example, the ~GPs disclosed herein can be used to generate polyclonal and preferably monoclonal antibodies. The3e antibodies can be used to detect the BGPs in a sample such as a blood sample and determine the presence of and level of the BGPs in the ~ample. The type and/or amount of BGPs detected can be compared to a known standard -- an average for healthy individuals. Readings out~ide of the standard would be useful information in diagnosing abnormalities such as basophilic leukemia.
A variety of protocols for the conduct of immunoassays, using either polyclonal or monoclonal antibodies specific for BGPs, known in the art and include competiti~e binding assays and immNnoradiometric assays. A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific BGP is preferred, but a competitive binding a~say can also be employed.
The~e assays are deYcribed in the following publications, --~ hereby incorporated by reference: Maddox, D.E. et al, J
Ex~ Med (1983) 1$8:1211; Gleich, G.J. et al, J Lab Clin ~ (1971) 77:690; Gleich, G.J. et al, J ~llergy Clin Immun (~974) ~ 15~; Gleich, G.~. et al, J Allergy Clin Immun (1977) 60:188; Dunnette, S.h. et al, J ImmunQl - (1977) 11~:1727; Wa~som, D.~. et al, J Clin Invest (19 67:651.

:

W094/06829 ~ 2 ~ O ~ PCT/US93/08511 Immunoassay procedures are utilized to mea~ure several major parameters in immunopathologic and prepathologic condition~ which are characterized by ~GP
abnormalitie~ - e.g. the increased or decrea~ed production of BGPs by basophils, the aberrant production o~ BGPs by cells other than basophils, and the change in intracellular or extracellular distribution o~ BGPs during the genesis of disea~e. In order to determine the normal distribution of ~GP in leukocytes, peripheral blood mononuclear cells from normal individual~ are prepared and analy~ed a~ described by Ackerman et al for the localization of eosinophil granules ~3P and Charcot-Leyden crystal protein to human basophils. (J Exp Med (19~3) 158:946; J Exp Med (1982) 155:1597). To determine the ~uantity of ~GPs in basophils, freeze-thawed detergent extracts of cell ~uspensions enriched for basophils are analyzed by immunoassay, and the slope of the b~nding curves are then compared to comparable binding curves generated by the purified protein.
2~
I. Pharmaceutical Compositions BGPs are also useful to remedy deficiencies in these proteins or to amplify immune-responses which are stimulated by these proteins. BGPs can be administered to subjects exhibiting such conditions using standard formulations such as those set forth in Reminaton's ~ Pharmaceutical Sciences, Mac~ Publishing Co., Easton PA, -~ Latest Ed.
Thus the present in~ention also provides compositions containing an effective amount of compounds of the;present invent;lon,`including the nontoxic add!ition f salts, amides and esters thereof, which may alone ser~e to pro~ide the abo~e-recited therapeutic benefits.
.
Such compositions can also be pro~ided together with :

.

W094/06829 ~ 4 2 2 0 3 PCT/US93/~8511 phy~iologically tolerable liquid, gel o~ solid diluents, `~
adjuvant~ and excipients.
These compounds and composition~ can be administered tO mammal5 for veterinary use, such a5 with domestic animals, and cllnical use in humanc in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will range from about 0.01 to 10,000 mcg/kg, more u~ually 0.1 to 1000 mcg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion o~er an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
Typically, such compo~itions are prepared as injectibles, either as liquid solutions or suspensions;
solid form suitable for solution in, or suspended in, liquid prior to injection may also be prepared. The prepara~ion may al80 be emulsified. The active ingredient is often mixed with diluents or excipients ~-~ 20 which are physiologically tolerable and compatible with the acti~e ingredient. Suitable diluents or excipients -~ are/ for example, water, saline, dextrose, glycerol, or the like, and combinations thereof~ In addition, if desired, the compositions may contain minor amounts of auxiliary 3ubs~ances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
The compositions are conventionally -~ administered parenterally, by injec~ion, for example, either 3ubcutaneously or intra~enously. Additional formulations which are suitable for other modes of -~ - administrationiinclude 3uppositories, intranasal aeroso1s, and in some cases, oral formulations. For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or ;~ 35 triglycerides; such suppositories may be formed from :, W094/068~9 PCT/US93/085l1;~;

mi~ures containing the active ingredient in the range of 0.5~ to 10~, preferably 1%-2~. Oral formulations include such normally employed excipients a~, for example, phar~ceutical grades of mannitol, lactose, starch~, S magnesium stearate, sodiu~ saccharin, cellulose, magne~ium carbonate, and the like. These compo~itions take the fonm of solutions, suspensions, tablets, pills, capsule~, su~tained-release formulations, or powders, and contain 10~-95% of active ingredient, preferably 25~-70~.
The peptide compound~ may be formulated into the compositions as neutral or salt forms.
Pharmaceutically acceptable nontoxic salt~ include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or ~uch organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric 20~ hydroxides, and such organic bases a~ isopropylamine, ~; ~ trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
Based on the above, it can be seen that the core of the present in~ention relates to the novel ; 25 proteins which are present in the cytoplasmic granules of human~basophils, including a subset of those basophil granule proteins which are disclosed and described herein. In addition, the proteins can be applied to ~-~; various procedures for the diagnosis of disease3 related to the abnormal (e.g., o~er or under) expres~ion of the protein. The p~ot!eins can be formulated into pharmaceutical compositions of ~arious types and used for various types of treatments. In addition, the genetic material which encodes the proteins is useful in producing the proteins. The genetic material can be :
~ .
~ ' , , .

;; W094/06829 ~1 4 2 2 0 3 PCT/US93/08511 pl~ced in plasmids and the plasmid~ used to transfect ho~ts each of which are part of the present in~ention.
The proteins can be used to produce monoclonal and polyclonal antibodies and these antibodies can be~used in detection ~ssays of the t~pe described above.

EXAMPL~S

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how~to make the proteins of the invention~and are not inte~ded to limit the scope of what the inventors regard as their invention Efforts ha~e been made to ensure accuracy with respect to numbers used (e.g., amount~, temperature, etc.) but some experimental errors and deviation should -~ be accounted for. Unless indicated~otherwi~e, parts are parts~by~weight, molecule weight is weight average of molec~ular~weight, temperature is in degrees Centigrade, and~pressure~is~at or near atmo~pheric.
The following procedures are used to purify h ~ n ba~ophil3, isolate the basophil granules, extract basophil granule proteins, fractionate and purify those proteins and determine~their N-terminal amino acid ; 25 ~ sequence.

Pre~ara~ion~l~f Purifled Huma~ ~asophil Granules A patient with a form of chronic myelogenous leukemia (basophil~leukemia with leukocyte counts o~er 105 cell~/~l and 78% basophils) underwent two treatment3 of cyt~phorèsis f~om which 1.5xlO11 baqophils were ~:
~ - reco~ered. The basophils were purified by centrifugation -~ o~er a cu~hion of Ficoll-Hypaque from which 95% were ;, -recovered from the interface with greater than 90 ` 35 purity.

", ~ .

,~:

W094/068~9 ~ ~ g~ PCT/US93/~8511 Subsequently, these basophils were lysed using modifications of the procedures described by Dvorak et al. Purified basophil~ were washed with PBS
and erythrocytes were lysed by exposure to Tris-ammonium 5 chloride for 5 minutes. The cell su~penqion wa~ ;
centrifuged at 400 x g, washed with Hank'~ ~SS-EDTA and suspended in cold 0.25M sucrose containing DNAase and heparin u~ing a ~olume of 15 ml for 8 x 108 basophil~.
The cell suspension was centrifuged at 400 x g for 10 minute~ and the sediment was again ~uspended in 0.25M
sucrose containing DNAase (2mg/15ml solution). After 1-2 minutes, heparin (20 IU) dissolved in 2ml 0.24M sucrose - was added and the preparation was subjected to a shearing force by repeated passage ~15 times) through a 20 gauge needle. The suspension was centrifuged at 400 x g to remove intact cells and granules were purified by centrifugation through a cushion of 40~ sucrose. The granules were resuspended and stored in 30 aliquots at 70C.
, :
Exam~le 1 ~-~ Extraction of Baso~hil ~xanule Proteins.
Four consecutive freeze/thaw cycle~ were carried out on a single aliquot of purified ba30phil granules in;order to lyse the granule~. Proteins were extracted at 4C by addition of 1 ml of SOmM ~odium borate pH 9.0 to the lysed granules. The extraction mix was ~ortexed frequently over a 1 hour period to aid extraction. The insoluble material was removed by centrifugation for 15 minutes at 20000 x g, and the soluble extract was takén for chromatographic ! '`
~ fractionation.
,~
. ~
" .
~ 35 -W O 94/06829 ~ ~ /US93/08511 Re~rse Phase HPLC.
The borate extract was acidified by adjustment to 0.05-0.1~ trifluoroacetic acid and wa~ fractionated by reverse phase chromatography on a ~rownlee ~U300 column (2.1mm x 30mm) eluted with a 0-70~ gradient of acetonitrile in 0.1~ trifluoroacetic acid. Fractions judged to be appropriate for analysis were concentrated in a SpeedVac concentrator and sequenced by automated Edman degradation on the ABI 477 Protein Sequenator.
The results are shown in Figure 1 and de~cribed as follows:
Analyses of ba~ophil granule extracts by HPLC
chromatogram. Basophil granules were solubilized in 0.05M borate buffer, pH 9, and separated-by re~er~e phase HPLC using a Brownlee BU-300 C4 column. The mobile phase was 0.1~ trifluororacetic acid (TFA) conta-ning 0-70~
` ~ acetonitrile. The ~ B (0.1~ TFA and 70~ acetonitrile) is indlcated on the graph. The fractions are indicated on the absclssa. The ordinate shows absorbance at 214 nm.
TA~LE 1. N-terminal amino acid ~equences of proteins i ~ purified from basophil granule extract~ X" represents ,- ~
an unidentified residue.
Each of the sequences li3ted in Examples 1-3 i~
25~ given a number designation at the left referred to as the "Praction No. n which is provided only as a matter of re~erence convenience.

,~ ~

~, W094/06829 PCT/US93/0~511~;`

Fraction No.

9 - Asp-Ile-Gly-Pro-Asp-Gln-His-Thr-Ser-Arg-Pro-Trp-Gly -Gln-Thr 11 - Asp-Val-Lys-Lys-Asp-Met-Glu-Val-Ser- Cy9 - Pro-Asp-Gly -Tyr-Thr 12 - Val-Met-X-Pro-Asp-Ala-Arg-Ser-X-Arg-Pro-Asp-Gly-X
-Thr 15 - Ala-Ile-Tyr-X-Arg-Ile-Pro-X-X-Ile-Ala-Gly-Glu-Phe -Arg-Tyr-Gly-Thr-Val-Tyr-Tyr-Gln-Gly-Ser-Leu 20 - Asp-Ile-Pro-Glu-Val-X-Val-X-Leu-Ala-Ala-A~p-Glu-Ser -Leu-Ala-Pro-Lys , 30 - Tyr-Pro-Gln-Leu-Al -Ile-Asn 43~- Ser-Ile-Gly-Phe-Val-Glu-Val-X-~eu-Val-Leu , ., ~

Exam~le 2 Extraction of Baso~hil Granule Proteins.
A~cocktail of protease inhibitors was prepared 25~ containing~;diisopropylfluorophosphate, e~th~ylenediaminetetraacetic acid and pepstatin A, and 0.~50 ml~of~the mix was added to each of 2 aliquots of ; frozen~granules. F~our consecutive freezejthaw cycles were`carried out in order to lyse the ~ranules. Protein~
~ 30 were extracted at 4C by addition of 3 ml of 50mM sodium -~ borate pH 9.0 to the pooled lysed granul~s. The ~ extraction mix was vortexed occa~ionally over 1 hour to q~ aid extraction. The insoluble material was removed by c-n~rlfugaelon for lS minute~ at 20000 x g, and the , ~

:.

-: .

` W094/06829 PCT/~S93/08511 -.

soP~ble extract was taken for chromatographic fractionation.

Size Exclusion HPLC.
~orate extract (4 ml) was concentrated to 0.6 ml and the pH was adjusted to neutral pH wi~h 0.5M
NaH2PO4. The neutralized extract was fractionated by size exclusion chromatography on a Bio-Sil TS~ 250 HPLC -~
column (7.5mm x 600mm). The A280 was monitored and the fractions containing protein~ were combined into 7 separate pools based on silver ~taining of Laemmli SDS
polyacrylamide gels.

Reverse Pha~e HPLC.
Pools of fractions from size exclusion HPLC
were acidified by adjustment to 0.05-O.l~ trifluoroacetic . acid and each pool was separately fractionated further by reverse~phase chromatography on a Brownlèe BU300 column (2.1mm x 30mm) eluted with a 0-70~ gradient of ~ 20 acetonitr1le in O.l~ tri~luoroacetic acid. Fractions :~ :judged to be appropriate for analysis were concentrated in a SpeedVAc and sequenced by automated Edman degradation on the A~I 477 Protein Sequenator.
The following se~uences were obtained:
25 l.18 Ala-Cys-Tyr-Cys-Arg-Ile-Pro-Ala- Cy9 -Ile-Ala-~ Gly-G1u-Arg-Arg-Tyr-Thr-Cys-Ile : 2.17b Ala-Pro-Ala-Leu-Thr-Ile-Ser-Asn-Gln 4.10a Asp-Ile-G1y-Pro-Asp-Gln-His-Thr-Ser-Xl-Pro-X2-Gly-Gln~Thr-Arg-X-Pro-Gln-Leu-Thr-Gly-Gly-Glu-Ala-X-Val . ~ " , where -l is Ser or Arg and X2 is Val or Trp 4.10b Arg-Asp-Val-Pro-Pro-Asp-X-Val-Val-Ser-X-Pro-Ser-Ser-Asp-Thr 4.12 Gly-Asp-Val-Lys-X-Asp-Met-Glu-Val-Ser-X-Pro-Asp-Gly-Tyr-Thr-X-X-Arg-Leu-Gln-Ser-Gly-Ala ' ~':

: ~ , ,~ .

, , W094/06829 PCT/US93/08511'~
~ ~14~2~ 30 A~p-Val-Lys-X-A9p-Met-Glu-Val-Ser-X-Pro-Asp- '~
Gly-Tyr-Thr-X-X-Arg-Leu-Gln-Ser~Gly-Ala 4.38 Gly-Pro-Pro-Thr-Ph2-A~n-Lys-Ile-Thr-Pro-~sn-Asp-Ala-Asp-Phe :~
It is pointed out that the above-listed sequence 1.18 is homologous to a family of peptides previously described (Selsted, M.E., Harwig, S.L., Ganæ, T., Schilling, J.W., and Lehrer, R.I. (1985) J. Clin.
Invest. 76,1436.), but differs from them by virtue of a - deletion of Gly found in position 17 of those peptides.
The above-listed sequence 4.12 is nearly identical to t~he ~equence BGP 11 of U.S. parent application serial number 07/55~,263 filed July 10, lg90, which i~ in turn very similar or identical to granulin A
described by Bateman et al. ~Bateman, A., Belcourt, D., Bennètt~ H., Lazure,~ C. and Solomon, S. (1990) Biochem.
Biopkys. Res;.~Comm~ 173, 1161), and bears sequence simi~l~a~ity~to~rat pept~ides termed epithelins, ;20~ par~ticularly~epithelin 1, described by Shoyab et al.
Shoyab~,~M.~ McDonald, V.L., 3yles, C., Todaro, G.J. and Pl~owman,~G.D. ~1990j Proc. Nat. Acad. Sci. 87, 7912).
Other members of a fami}y o~human and rat granulin~/epithelins have been described (Bhandari, V., ~Pal~f~ree,~ R.G.~E. and Bateman, A. ~1992j Proc. Nat. Acad.
S~ci.~89, ~1715~; Plowman, G.D., Green, J.M., Neubauer, M.G.~, Buckley, S.D., McDona}d! V.L., Todaro, G.J. and Shoyab~, M~. ~1992)~J. 3iol. Chem. 267, 13073.) Except for a Gly in~position l, the peptide of seguence 4.12 may be 3~0j ;identlcal to,peptlide i1~ of parent application 07/551,263.
The above-listed 4.38 i9 homologous to human antitrypsin (Long, G.L., Chandra, T., Woo, S.L.C., Davie,~.W.-and Kurachi, K. ~1984) ~iochemistry, ~l, 4828) and may represent an allelic variant of al-antitrypsin or may represent another member of a family , ."

, ~

:

W094/06829 ~14 2 ~ ~ 3 PCT/US93/0~511 of related molecule~, the serpins, of which ~1-antitrypsin is one.

Example 3 S Extraction of Basophil ~ranule Prot~ins.
A coc~tail of protease inhibitors was prepared containing diisopropylfluorophosphate, ,~
ethylenediaminetetraacetic acid and pepstatin A, and O.25 ml of the mix was added to each of 10 aliquots of frozen granules. Four consecutive freeze/thaw cycle~
were carried out in order to lyse the granules. Proteins were extracted at 4C by addition of 5 ml of 50mM sodium .
borate pH 9.0 to the pooled lysed granules. The extraction mix was vortexed occasionally over a 1 hour period to aid extraction. The insoluble material was removed by centrifugation for 15 minutes at 20000 x g, and~the soluble extract was~taken for chromatographic fractionation.

20~ Size~Exc~usion-~HPLC.
Borate extract ~7.2 ml) was concentrated to 1.6 ml and~was adjusted to neutral pH with 0.5M NaH~PQ4.
~r,,''~ The neutralized extract was fractionated by size exclusion chromatography in 3 identical runs on a Bio-Sil 25~ TSK~125 HP~C column (7~5mm~x 600mm). The A2~0 was nitored and the fractions containing proteins were combined~into 4 sepàrate pools based on sil~er staining of~àemmli SDS polyacrylamide gels.

3a Revers Phase HPLC.
e Pools of fractions from size exclusion HP~C
were acidi~ied by adjustment to 0.05-0.1~ trifluoroacetic ac~id~and ea~ch~pool was ~eparately fractionated further by reverse phase~chromatography on a Vydac C4 column ~- 35 ~(4.6mm x 25cm) eluted with a 0-100% gradient of ,,~,.
, i ~ .
" " ", W094/06829 PCT/USg3/08511~ .

~1~22~3 ace~onitrile in 0.1% trifluoroacetic acid. Fraction~ .
judged to be appropriate for analy~is were concentra~ed in a SpeedVac and sequenced by automated Edman degradation on the ABI 477 Protein Sequenator.
Proteins in ~ome fractions were further fractionated by an additional reverse phase HPLC step performed as described in Example 2. Proteins present in other reverse phase fractions were prepared for sequencing of component proteins by Laemmli SDS
polyacrylamide gel electrophoresis followed by electrophoretic transfer (2h at 500mA in lOmM CAPS
ph 11.0, 10% methanol, 0.05~ SDS) of proteins from the gel onto a polyvinylidineaifluoride membrane (ProBlott, :;
ABI). Such electroblotted bands were excised from the membrane and loaded directly onto the ABI 477 Protein Sequenator.
The following sequences were obtained:
19 Ala-~Ile-Gln-Cys-Pro-X3-Ser-Gln-Phe-X4-X5-Pro-X6-Phe-:~ Leu-Ala-Thr-Gly-Val-Met where 83 is Leu or Asp, _4 is Met or Glu, X5 is Lys, Ile, or Cys, and X6 i8 Pro or Leu.
26 Asp-Ile-Pro-Glu-Val-Cys-Phe-A~n 29 Asp-Pro-Gly-Glu-Val-Lys-Ala-Leu-Pro-Met-Gln Lys-Pro-Gln-Met-Phe-Thr-Ile-X-Gln-Asn-X-Ala-Thr-Trp-: ~ 25 Met 31 Lys-Ile-Gly-Gly-Phe-Glu-Val-Thr-Asp-Val-Phe-Ala-Pro-~ ` Val-Met-Ala - ~ ~ 31rp Ile-Leu-Gly-Val-Phe-X-Val-Glu-Gln-X-Phe-Ser-Phe-X-`~ Leu 37 Asp-Pro-Pro-Thr-Phe-Asn-Lys-Ile-Thr-Pro-Asn-Leu-Leu-Glu-Phe-Ala-Asp-Gly-Leu-Tyr-Lys-Gln-Glu ~- : 26bbl Ser-Glu-Leu-Thr-Lys-Met-Asn-Gln-Arg-Ser-Phe rp indicates that the sequence was obtained ~:~ 35 ~ollowing repurification of Fraction 31 of this example.
.
, , ~

~::

--- W0~4J06829 hl 4 2 Z 0 3 PCTIUS93/08511 bbl indicates that the sequence was o~tained following electroblotting of Fraction 26 of this example.

The above-listed sequence 37 has homology wit~
re~pect to human ~l-antitrypsin (Long, G.L., Chandra, T., Woo, S.L.C., Davie, E.W. and Kurachi, K. (1984) ;
Biochemi~try, 23,4828) and may represent an allelic variant of ~l-antitrypsin or may represent another member of a family of related molecules, the serpins, of which ~l-antitrypsin i~ one.
Each of the 29 remaining vials of basophil cell~
contains an estimated 200 ~g of extractable protein.
Individual proteins recovered had yields ranging from 250 pmoles for peak 21 down to 2S-50 pmoles for peak3 9 and 37 (Figure l). Since 25 pmoles i9 usually sufficient for sequencing 20 or more residues at the N-terminus, the expenditure of~more vials will enable rarer specieq of proteins~to be sequenced and will a1so e able more residues to be sequenced from all proteins.
The instant invention has been shown and described herein and was considered to be the most praatical, and preferred embodiments. It i9 recognized, however, that departures may be made therefrom which are within the scope of the in~entionj and that obvious modifications wilI occur to one ~killed in the art upon - ~ reading this disclosure.

, :
,. . ..

, ~

Claims (24)

What is claimed is:

1. A basophil granule protein (BGP) which can be extracted from human basophil granules by treatment at pH 9.0 or greater, wherein the BGP is in isolated and purified form, is produced synthetically or is recombinantly produced by cells in culture or in hosts of non-human origin.

2. The BGP of claim 1, wherein the BGP has an N-terminal sequence described by a sequence selected from the group consisting of:

where each X, X1, X2, X3, X4, X5 and X6 independently represents a variable amino acid residue.

3. The BGP of claim 2, wherein X1 is Ser or Arg and X2 is Val or Trp.

4. The BGP of claim 2, wherein X3 is Leu or Asp, X4 is Met or Glu, X5 is Lys, Ile or Cys, and X6 is Pro or Leu.

5. A polynucleotide, wherein the polynucleotide is in isolated and purified form or is recombinantly produced by cells in culture or in hosts of nonhuman origin which polynucleotide or its complement encodes any BGP as claimed in claim 1.

6. An oligonucleotide fragment of the polynucleotide of claim 5, wherein the oligonucleotide comprises at least 9 nucleotides.

7. The polynucleotide of claim 5, wherein the oligonucleotide is a cDNA in isolated and purified form, is synthetically produced, or is recombinantly produced by cells in culture or in hosts of nonhuman origin.

8. An expression vector comprising a cDNA as claimed in claim 7 operably linked to heterologous DNA
control sequences.

9. A mixture containing at least 10 polynucleotides as claimed in claim 5.

10. The mixture of claim 9, wherein the polynucleotides are each attached to a detectable label.

11. An antisense oligonucleotide capable of blocking expression of any BGP of claim 1.

12. A triple helix reagent capable of blocking expression of any BGP of claim 1.

13. An expression system comprising a vector of claim 8 contained within a recombinant host cell, wherein the transfected host cell is capable of producing said BGP.

14. A method to produce BGP which method comprises culturing the recombinant host cells containing the expression system of claim 13 under conditions permitting expression of said BGP encoding DNA so as to produce said BGP, and recovering the BGP produced thereby.

15. A composition of antibodies specific for the BGP of claim 1.

16. The composition of claim 15 in which said antibodies are monoclonal.

17. A method to diagnose diseases in which BGP
is over or underproduced or in which BGP is released from basophils, which method comprises contacting a biological sample from a subject to be diagnosed with the antibodies of claim 15 and detecting or quantifying the binding of said antibody to BGP, comparing the quantity of binding with a known standard and thereby diagnosing the over or under expression of BGP.

18. A method to treat diseases or conditions characterized by overproduction or release of BGP which method comprises administering to a subject in need of such treatment an effective amount of the antibodies of claim 15 or a pharmaceutical composition thereof.

19. A labeled polynucleotide probe which encodes a BGP of claim 1, which probe hybridizes to and is thereby effective to detect the presence or absence of BGP-encoding genes or its compliment.

20. A method for detecting sequence abnormalities in a gene encoding BGP which method comprises contacting a digest of the genomic DNA of a subject to be tested with the probe of claim 19, and characterizing the hybridized fragments.

21. A pharmaceutical composition which comprises the BGP of claim 1 as an active ingredient in admixture with a pharmaceutical excipient.

22. A method to treat diseases or conditions characterized by insufficient expression or granule release of physiologically active BGP which method comprises administering to a subject in need of such treatment an effective amount of the BGP of claim 1 or a pharmaceutical composition thereof.

23. A fragment of BGP of claim 1, wherein the fragment retains a biological characteristic of human BGP
and comprises at least seven amino acids.

24. A fragment of a polynucleotide of claim 5, wherein the fragment is of sufficient length to be used as a probe to find the entire polynucleotide.