WO1998021240A1 - Recombinant by55 and nucleic acids encoding same - Google Patents

Abstract

The present invention provides isolated nucleic acids that encode the human BY55 and BY55 fragments and derivatives capable of hybridizing to such BY55-encoding nucleic acids. cDNA (SEQ ID NO:1) encoding human BY55 has been isolated and expressed in eukaryotic cells, resulting in recombinant BY55 having an apparent molecular weight of about 80kD. The protein appears to contain multiple chains as a result of interchain disulfide bonds formed by the sic cysteines present in the mature form. Thus, we can otain a single chain recombinant of about 30kD by the present method. We can also replace any of the cysteines by a constitutive amino acid such as serine to prevent undesired disulfide bonding.

Description

RECOMBINANT BY55 AND NUCLEIC ACIDS ENCODING SAME

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to human BY55, including recombinant BY55 and fragments and derivatives thereof and isolated nucleic acids encoding BY55 and fragments and derivatives. In preferred aspects, assays for identifying compounds that can modulate activity associated with BY55⁺ cells are provided, particularly assays to identify a pharmacological agent useful in the diagnosis or treatment of disorders associated with BY55⁺ cells.

2. Background Natural killer (NK) cells are large granular lymphocytes (LGLs) comprising 2- 15% of peripheral blood mononu clear cells in healthy individuals. There are a number of cellular markers that are used to differentiate cells from each other such as whether a cell is CD3⁺ or CD3 > whether CD56 is expressed or not. These markers are frequently cell surface proteins such as receptors and often play a role in the activation of a particular cell. One marker was found using a monoclonal antibody called BY55 antibody. The antibody was raised by immunization with YT2C2 of a human Leukemia cell line displaying NK activity (Maiza et al 1993). This antibody bound to only a subset of 10-25% of E⁺ PBLs, which contain a protein, designated BY55. Among the lymphocytes that BY55 recognized, a sizeable proportion is CD3- and TCR γ/δ⁺ lymphocytes and a minor subset of CD8⁺ TCR α/β⁺ T lymphocytes. Although only the BY55⁺ lymphocyte population possessed cytolytic activity. Although not all NK cells display BY55, those NK cells that do have high cytolytic activity. Additionally, while only a small subset of circulatory lymphocytes display BY55, almost all human intestinal intraepithelial lymphocytes (IELs) express BY55. In addition, IELs and BY55⁺ circulatory lymphocytes show expansion in number in response to disease conditions. For example, BY55⁺CD8⁺ T lymphocytes show a four fold increase in HIV infected individuals as compared to uninfected people (Bensussan, et al., 1993). Similarly, IELs having BY55 increase in numbers in diseases of mucosal delayed type hypersensitivity, allograft rejection, graft-vs-host disease, parasite infections, enteral challenge after immunization (13), Crohn's disease (17, 18) and Celiac disease (19, 20). This indicates that BY55 activity is closely associated with immune activation. However, although an antibody identifying the protein was known, the isolated and purified protein had not been obtained. It would be useful to have the isolated protein to enhance our ability to modulate immunity. Similarly, obtaining nucleic acid encoding the protein would better facilitate our ability to manipulate the protein, where it is expressed, and to prepare derivative form thereof.

None of the activated PBL and TCR α/β or γ/δ T cell clones expressed the BY55 molecule. Moreover, BY55 expression is reduced immediately after NK activation. Upon activation with PMA E⁺ PBLs lost BY55 protein from their surface within 1 h and BY55 expression did not reappear even after 3 d of culture. Both MHC-unrestricted (NK activity) and MHC-restricted ( as measured by CD3-redirected CD8⁺ T cell activity) where BY55 is not present there is little cytolytic function (Bensussan et al 1993; Maiza, et al. 1993). Thus, BY55 is an important marker for immune modulation and cytolytic activation.

It thus would be desirable to have means to identify agents which can modulate cytolytic activity associated with BY55⁺ cells. Moreover, methods for identifying pharmacological agents of interest by automated, high throughput drug screening have become increasingly relied upon in a variety of pharmaceutical and biotechnology drug development programs. Unfortunately, however, requisite reagents for such high throughput screening assays to identify agents potentially useful in treatment of disorders associated with BY55⁺ cell are not readily available. For example, heretofore it was not possible to clone and isolate this transmembrane protein believed to be a receptor.

It thus would be desirable to have agents that can modulate BY55 activity and activities associated with BY55⁺ cells. It would also be desirable to have effective assays for identifying compounds that have the potential to modulate activity associated with BY55 and BY55⁺ cells or to diagnose or treat disorders relating to inappropriate or undesired BY55⁺ expression.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acids that encode the human BY55 and BY55 fragments and derivatives capable of hybridizing to such BY55-encoding nucleic acids. cDNA (SEQ ID NO: l) encoding human BY55 has been isolated and expressed in eukaryotic cells, resulting in recombinant BY55 having an apparent molecular weight of about 80 kD. The protein appears to contain multiple chains as a result of interchain disulflde bonds formed by the six cysteines present in the mature form. Thus, we can obtain a single chain recombinant of about 30 kD by the present method. We can also replace any of the cysteines by a constitutive amino acid such as serine to prevent undesired disulfide bonding.

The invention further provides isolated and pure recombinant human BY55 and BY55 fragments and derivatives.

The invention also provides novel assays for identifying compounds useful in the diagnosis or treatment of disorders related to the cytolytic activity associated with BY55⁺ cells. Preferred compounds identified through assays of the invention can modulate, preferably inhibit, BY55 activity and thus can be used to control the immune activation and cytolytic activity associated with BY55⁺ cells.

As used herein, "BY55 activity" refers to the particular activity displayed by a construct having the wild type nucleic acid sequence that when it is expressed results in a protein that has the native conformation .

A variety of such assays are provided including, e.g., direct binding assays and cleavage assays. Agents identified through assays of the invention will have potential for use in a number of therapeutic applications, especially to modulate, particularly inhibit, expression or activity of BY55 in particular cells. For example, binding assays can be used to identify agents that bind to BY55 and prevent cleavage and thus may be used to inhibit BY55⁺ cell activation. Cleavage assays can be used to identify agents that cleave or potentiate cleavage of BY55 and thus may be used to activate BY55⁺ cells. Specific areas that could be treated by administration of pharmacological agents identified through assays of the invention include those associated with undesired immune activation, e.g., graft-versus-host disease.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that BY55 antibody binds to COS cells transfected with BY55 cDNA. COS cells were transiently transfected with pCDM8 alone (Vec) or with pCDM8 encoding BY55 cDNA (BY55). Transfectants were stained with either BY55 antibody (BY55) or with an isotype matched control antibody (control IgM) and PE conjugated anti-antibody. Ab binding was detected by indirect immunofluorescence and flow cytometric analysis.

Figure 2A shows the nucleotide sequence (SEQ ID NO: 1) and predicted amino acid sequence of BY55 (SEQ ID NO: 2). The amino acids predicted to form the signal sequence are singly underlined and those for GPI anchorage are doubly underlined. The two N-linked glycosylation motifs are marked with an asterisk over the asparagines. The cysteines are in bold face. The extra nucleotides found in the 5' untranslated region of one size of BY55 cDNA are shown in lower case and are spliced out in the other size of BY55 cDNA. The polyadenylation signal sequence is underlined with a dotted line.

Figure 2B shows the basic-acidic characteristics and Chou-Fasman hydrophobicity index of BY55 polypeptide.

Figure 3A and 3B show BY55 mRNA expression in human tissues. Expression of BY55 in multiple human tissue Northern blots (CLONTECH) containing (3A) spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood lymphocytes and (3B) heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Each lane contains 2μg of poly (A)⁺ RNA and was probed with the 1.3 kb BY55 cDNA. The positions of RNA molecular weight markers are given on the left in kb. The mRNA expression of a house keeping gene G6PD is shown at the bottom of each panel.

Figure 4 shows BY55 mRNA expression in T, B, NK, and myeloid cell lines. Each lane was loaded with 20 μg of total RNA from the indicated cell lines and probed with the 1.3 kb BY55 cDNA. Shown at the bottom of the panel are the mRNA expression of G6PD and 18S ribosomal RNA (ethidium bromide staining) respectively. Figure 5 shows the results of a Southern blot analysis. Human genomic DNA (10 μg) was digested with Pst I, Eco RV, Eco RI or Xba I restriction enzymes and resolved in a 0.7% agarose gel and probed with BY55 cDNA.

Figure 6 shows immunoprecipitation analysis of BY55 protein. Cells were biotin labeled and proteins were immunoprecipitated with BY55 mAb or with an isotype matched control antibody. Immunoprecipitates were resolved in a 10% SDS-PAGE gel, western blotted, and the blot developed with ECL detection reagents. Lanes 1-2: Immunoprecipitates from NKL, a BY55 expressing NK-like cell line with BY55 mAb and control IgM, respectively. Lane 3: Immunoprecipitate with BY55 from untransfected 300.19, a murine pre-B cell line. Lanes 4-5: Immunoprecipitates with control IgM and BY55 mAb from 300.19 cells stably transfected with BY55 cDNA. Lane 1 : Immunoprecipitates were electrophoresed in the presence of the reducing reagent β-mercaptoethanol. Lanes 2&6. Immunoprecipitates were reduced with DTT and carboxyamidomethylated with iodoacetamide and then electrophoresed.

Figure 7 shows that the BY55 protein is anchored to the cell surface via a glycosylphosphatidyl inositol (GPI) linkage. Top panels: BY55-CHO cells transfectants. Bottom panels: NKL cell line that expresses BY55. Cells were either treated with glycosylphosphatidylmositol phospholipase C or untreated, stained with either BY55 mAb or an isotype matched control antibody and phycoerythrin-conjugated goat anti-mouse IgM secondary antibody as indicated. Data are presented as single color fluorescence histograms plotted with cell number vs log scale of fluorescence intensity.

Figure 8 illustrates the expression of BY55 by human intra-epithelial lymphocytes (IELs). Freshly isolated human small intestinal IELs or human peripheral blood mononuclear cells (PBMC) were incubated with phycoerythrin-conjugated CD3 mAb (OKT3), TCR α/β mAb (BMA031), TCR γ/δ mAb (IMMU510), or CD56 mAb (NKH 1), and with unconjugated BY55 mAb and FITC- conjugated goat anti-mouse IgM secondary antibody and analyzed by flow cytometry. Cells incubated with isotype matched irrelevant antibodies served as negative controls to set up quadrant regions. Data are presented as double color fluorescence histograms with log scale of fluorescence intensity. Percentage of cells positive in each quadrant is marked within each quadrant.

Figures 9A and 9B illustrate BY55 expression in ilEL and LPL. Freshly isolated (9A) human small intestinal IELs or (9B) lamina propria lymphocytes (LPL) were incubated with FITC-conjugated CD3, CD4, CD8, TCR α/β or CD56 mAbs mAb or with unconjugated CD45RO, ^αEβ7, CD 101, or CD 1 lb mAbs followed by FITC-conjugated goat anti- mouse IgG secondary antibody. Cells were then stained with unconjugated BY55 mAb and phycoerythrin-conjugated goat anti-mouse IgM secondary antibody and analyzed by flow cytometry. Cells incubated with isotype matched irrelevant antibodies served as negative controls to set up quadrant regions. Data are presented as double color fluorescence histograms with log scale of fluorescence intensity. Percentage of cells positive in each quadrant is indicated.

Figure 10 illustrates BY55 is expressed on CD8⁺CD28^" PBL. PBMC enriched for CD8⁺CD28^" cells were prepared as described in Methods and stained with the indicated mAbs.

Figure 11 illustrates BY55 Expression in NK cell subsets. PBMC enriched for NK cells were prepared as described in Methods and stained with PE-conjugated NKH- 1 (CD56) and either FITC-conjugated CD 16 or unconjugated BY55, followed by secondary staining with FITC-labeled goat anti-mouse IgM. Logarithm of PE fluorescence is displayed on the abscissa and logarithm of FITC fluorescence on jthe ordinate. The data shown are representative of 3 separate experiments.

Figure 12 shows that that BY55 does not costimulate NK cell proliferation. Sorted CD56^brig^ht or CD56^dim NK cells were incubated with IL-2 and the indicated mAbs and proliferation was determined as described in Methods.

DETAILED DESCRIPTION OF THE INVENTION

We have now isolated a cDNA encoding human BY55 . This cDNA is represented by SEQ ID NO: 1 and encodes a protein that when expressed in a eukaryotic cell such as COS cells has an apparent molecular weight of 80 kD as determined by polyacrylamide gel electrophoresis. The cDNA encodes a pep tide that contains six cysteine residues and would be expected to have a nonprocessed molecular weight of 24 kD. We have found a band at 30 kD after reduction and carboxyamido-methylation. Thus, we believe the 80 kD protein represents a multimeric form. Accordingly, in one embodiment we disclose an isolated and purified single chain version of BY55. In a preferred embodiment of the single chain form, one to six of the cysteine residues have been changed to a conservative amino acid such as serine. The recombinant protein is bound by the BY55 monoclonal antibody. BY55 messenger RNA is expressed only in selected human tissues, spleen, PBLs and small intestine. Although it is expressed only on a small subset of circulating lymphocytes that typically have cytolytic activity such as certain NK cells, it is present on most human small intestinal intraepithelial lymphocytes (IELs).

As discussed above, the ability to regulate the cytolytic activity associated with such BY55⁺ cells in a particular environment can be very important. BY55⁺ cells are also important as modulators of immune reaction, as mediators of, e.g., disease. For example, IELs have been implicated with a number of transplant related problems, especially graft-versus-host disease and solid organ and allogenic bone marrow transplant rejection. Other BY55⁺ cells seem to proliferate in response to certain diseases. For example, HIV-infected individuals have 4x more BY55⁺ cells than uninfected individuals.

As discussed above, the invention provides methods to modulate, particularly to inhibit, expression or activity of BY55 in particular cells. For example, one can use a BY55 nucleic acid segment operably linked to a BY55 promoter to selectively direct it to desired cells. As another example, one can administer a BY55 protein or fragment or derivative to modulate BY55 activity. For example an inactive soluble form can be used to compete with BY55 activators. As a further example, a BY55 expressing cells may be activated by contacting the cell with an agent that selectively cleaves the glycophosphatidylinsitol (GPI) linkage. Such agents include, for example, phosphatedylinositol-specific phospholipase C. Activation of BY55 expressing cells may be inhibited by contacting the cell with an agent that blocks cleavage at the BY55 GPI linkage. Such agents include agents that bind to the GPI linkage, e.g., monoclonal antibodies.

The invention further provides an isolated and purified BY55 having an amino acid sequence represented by SEQ ID NO:2, as well as fragments or derivatives thereof.

The term "fragment" when referring to an BY55 protein means proteins or polypeptides which retain in one designated area essentially the same biological function or activity as the protein of SEQ ID NO:2. For example, for that function, e.g., immune modulation, the BY55 fragments or derivatives of the present invention maintain at least about 50% of the activity of the protein of SEQ ID NO:2, preferably at least 75%, more preferably at least about 95% of the activity of the protein of SEQ ID NO:2.

Derivatives can include agonists and antagonists derived from SEQ ID NO:2. For example by using deletion analysis to remove an undesired domain.

Fragments or derivatives as the term is used herein can include competitors of the native BY55 with respect to a particular BY55 domain activity. However, the fragment or derivative shows an overall similarity to BY55 in other areas as explained herein.

An BY55 fragment or derivative of the invention may be (i) a peptide in which one or more of the amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) a peptide in which one or more of the amino acid residues includes a substituent group, or (iii) a peptide in which the mature protein is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). For example, BY55 may be used to form a fusion protein with, e.g., an immunoglobulin. In one preferred embodiment cysteine residues are removed to prevent undesired disulflde bonding. Thus, a BY55 fragment or derivative includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The protein fragments and derivatives of the invention are of a sufficient length to uniquely identify a region of BY55. BY55 fragments and derivatives thus preferably comprise at least 8 amino acids, usually at least about 12 amino acids, more usually at least about 15 amino acids. Preferred BY55 fragments or derivatives of the invention include those that have at least about 55 percent homology (sequence identity) to the protein of SEQ ID NO:2, more preferably about 65 percent or more homology to the protein of SEQ ID NO:2, still more preferably about 75, even more preferably 90 percent or more homology to the protein of SEQ ID NO:2. Thus, uniqueness can be determined by comparison to known databases as of the filing date.

BY55 and fragments and derivatives thereof of the invention are "isolated", meaning the protein or peptide constitutes at least about 70%, preferably at least about 85%, more preferably at least about 90% and still more preferably at least about 95% by weight of the total protein in a given sample. A protein or peptide of the invention preferably is also at least 70% free of immunoglobulin contaminants, more preferably at least 85% free, still more preferably at least 90% free and even more preferably at least 95% free of immunoglobulin contaminants. The BY55 fragments and derivatives may be present in a free state or bound to other components, e.g. blocking groups to chemically insulate reactive groups (e.g. amines, carboxyls, etc.) of the peptide, or fusion peptides or polypeptides (i.e. the peptide may be present as a portion of a larger polypeptide) .

BY55 can exist in a number of forms in addition to its multimeric form, which is preferred. For example, we discussed above single chain forms and soluble or truncated forms. A truncated form is one where the molecule has been cleaved at the GPI linkage. Soluble forms include the truncated form and any other form which has been deleted of amino acid residues that bind the protein to the cell membrane.

As discussed above, BY55 nucleic acid fragments and derivatives are also provided. Those fragments and derivatives are of a length sufficient to bind to the sequence of SEQ ID NO: 1 under the following moderately stringent conditions (referred to herein as "normal stringency" conditions): use of a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.

Preferred BY55 nucleic acid fragments and derivatives of the invention will bind to the sequence of SEQ ID NO: 1 under the following highly stringent conditions (referred to herein as "high stringency" conditions): use of a hybridization buffer comprising 20% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a temperature of 42°C and remaining bound when subject to washing twice with that SSC buffer at 42°C.

These nucleic acid fragments and derivatives preferably should comprise at least 15 base pairs, more preferably at least 20 base pairs, still more preferably comprise at least 30 base pairs. In some preferred embodiments, the nucleic acid fragment or derivative is bound to some moiety which permits ready identification such as a radionucleotide, fluorescent or other chemical identifier.

Isolated BY55 and peptide fragments or derivatives of the invention are preferably produced by recombinant methods. A wide variety of molecular and biochemical methods are available for generating and expressing the BY55 of the present invention; see e.g. the procedures disclosed in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, N.Y. 1992) or other procedures that are otherwise known in the art. For example, BY55 or fragments thereof may be obtained by chemical synthesis, expression in bacteria such as E. coli and eukaryotes such as yeast, baculovirus, or mammalian cell-based expression systems, etc., depending on the size, nature and quantity of the BY55 or fragment. The use of mammalian-based expression systems, particularly human, is particularly preferred where the peptide is to be used therapeutically.

Nucleic acids encoding the novel BY55 of the present invention and fragments and derivatives thereof may be part of BY55 expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with expression of a BY55), etc. Nucleic acids encoding BY55 containing proteins are isolated from eukaryotic cells, preferably human cells, by screening cDNA libraries with probes or PCR primers derived from the disclosed BY55 cDNAs.

The nucleic acids of the present invention are isolated, meaning the nucleic acids comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome and usually constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid present in a given fraction.

The nucleic acids of the present invention find a wide variety of applications including: use as translatable transcripts, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of BY55 genes and gene transcripts; use in detecting or amplifying nucleic acids encoding additional BY55 homologs and structural analogs; and use in gene therapy applications. To inhibit BY55 activity, nucleic acid encoding a competitor or an antagonist can be administered to a subject. One preferred embodiment employs nucleic acid encoding an BY55 derivative that acts as a competitor or an antagonist. For example, in transplants many immune reactions are undesirable, it is advantageous to inhibit those activities dependent upon BY55 activity. Accordingly, the BY55 domain responsible for that activity can be appropriately altered (e.g. deleted or mutated), whereby the protein will still display the desired activity, but will not exhibit the undesired activity. For example, by mutations that inhibit cleavage of the gpi membrane anchor sequence. Moreover, the altered protein can compete with the native BY55 to thereby inhibit the undesired activity.

Further, to reduce BY55 activity, nucleic acids capable of inhibiting translation of BY55 also may be administered. These nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed relevant BY55 fragment-encoding nucleic acid. Antisense modulation of the expression of a given BY55 fragment containing protein may employ BY55 fragment antisense nucleic acids operably linked to gene regulatory sequences. Cells are transfected with a vector comprising an BY55 fragment sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous BY55 fragment containing protein encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given BY55 fragment containing protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the BY55. The BY55 nucleic acids are introduced into the target cell by any method which will result in the uptake and expression of the nucleic acid by the target cells. These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93/04701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/ US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller, A.I. et al, J. Neurochem, 64: 487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995);

Geller, A.I. et al, Proc Natl. Acad. Sci.: U.S.A.:90 7603 (1993); Geller, A.I., et al, Proc Natl. Acad. Sci USA: 87: 1 149 (1990)], Adenovirus Vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al, Nat. Genet 3: 219 (1993); Yang, et al, J. Virol. 69: 2004 (1995)] and Adeno- associated Virus Vectors [Kaplitt, M.G., et al, Nat. Genet. 8: 148 ( 1994)].

Pox viral vectors introduce the gene into the cells cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaP0₄ precipitation, DEAE dextran, electroporation, protoplast fusion, lipofecton, cell microinjection, and viral vectors.

The vector can be employed to target essentially any desired target cell, such as a glioma. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell (Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration .

The invention provides efficient screening methods to identify pharmacological agents or lead compounds for agents which modulate, e.g. interfere with or increase an BY55 activity. The methods are amenable to automated, cost-effective high throughput drug screening and have immediate application in a broad range of pharmaceutical drug development programs.

A wide variety of assays for BY55 binding agents are provided including, e.g., labelled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, cell based assays such as one, two and three hybrid screens and expression assays.

An assay mixture of the invention comprises at least a portion of the BY55 protein. An assay mixture of the invention also comprises a candidate pharmacological agent. Generally a plurality of assay mixtures are run in parallel with different candidate agent concentrations to obtain a differential response to the various concentrations. Typically, one of these assay mixtures serves as a negative control, i.e. at zero concentration or below the limits of assay detection. Candidate agents encompass numerous chemical classes, though typically they are organic compounds and preferably small organic compounds. Small organic compounds generally have a molecular weight of more than about 50 yet less than about 2,500. Candidate agents comprise functional chemical groups necessary for structural interactions with proteins and/ or DNA, and may include at least one or two amine, carbonyl, hydroxyl or carboxyl groups.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.

Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. In addition, known pharmacological agents may be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidifϊcation, etc.

A variety of other reagents may also be included in the mixture. These include reagents such as salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal protein-protein and/ or protein-nucleic acid binding and/ or reduce non-specific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is then incubated under conditions whereby the candidate pharmacological agent and the BY55 or fragment or derivative thereof, if capable, bind. The mixture components can be added in any order that provides for the requisite bindings. Incubations may be performed at any temperature which facilitates optimal binding, typically between 4° and 40°C, more commonly between 15° and 40°C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high throughput screening, and are typically between 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.

After incubation, the binding is detected by any convenient way. For cell-free type assays, the BY55 may be bound to a solid substrate and the agent labelled, e.g., radiolabelled. A separation step can be used to separate the bound BY55 from unbound agent. The separation step may be accomplished in a variety of ways known in the art. The solid substrate may be made of a wide variety of materials and in a wide variety of shapes, e.g. microtiter plate, microbead, dipstick, resin particle, etc. The substrate is chosen to maximize signal to noise ratios, to minimize background binding, to facilitate washing and to minimize cost.

Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead (e.g. beads with iron cores may be readily isolated and washed using magnets), particle, chromatographic column or filter with a wash solution or solvent. Typically, the separation step will include an extended rinse or wash or a plurality of rinses or washes. For example, where the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific binding such as salts, buffer, detergent, nonspecific protein, etc. may exploit a polypeptide specific binding reagent such as an antibody or receptor specific to a ligand of the polypeptide.

As mentioned, detection may be effected in any convenient way, and for cell-free assays, one of the components usually comprises or is coupled to a label. Essentially any label can be used that provides for detection. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. The label may be appended to a reagent or incorporated into the peptide structure, e.g. in the case of a peptide reagent, a methionine residue comprising a radioactive isotope of sulfur.

A variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected. Labels may be directly detected through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. For example, in the case of radioactive labels, emissions may be detected directly, e.g. with particle counters or indirectly, e.g. with scintillation cocktails and counters. The assays of the invention are particularly suited to automated high throughput drug screening. In a particular embodiment, an automated mechanism, e.g. a mechanized arm, retrieves and transfers a microtiter plate to a liquid dispensing station where measured aliquots of each of an incubation buffer and a solution comprising one or more candidate agents are deposited into each designated well. The arm then retrieves and transfers to and deposits in designated wells a measured aliquot of a solution comprising a BY55 protein or fragment or derivative thereof as well as solutions of other reagents. Thereafter, the arm transfers the microtiter plate to an analysis station where the reaction mixture can be analyzed for the presence or absence of binding.

Antibodies also can be prepared that will bind to one or more particular domains of a peptide of the invention and can be used to modulate BY55 activity. The BY55 antibody that was used to initially identify BY55 was made using the whole cell. We can make BY55 fragments to prepare a range of antibodies including antibodies that will cross-link the bound protein, antibodies that will bind to the GPI domain and inhibit cleavage, and also antibodies that will promote cleavage.

For therapeutic applications, peptides and nucleic acids of the invention may be suitably administered to a subject such as a mammal, particularly a human, alone or as part of a pharmaceutical composition, comprising the peptide or nucleic acid together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well know in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences.

Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Application of the subject therapeutics often will be local, so as to be administered at the site of interest. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access. Where an organ or tissue is accessible because of removal from the patient, such organ or tissue may be bathed in a medium containing the subject compositions, the subject compositions may be painted onto the organ, or may be applied in any convenient way. Systemic administration of a nucleic acid using lipofection, liposomes with tissue targeting (e.g. antibody) may also be employed.

It will be appreciated that actual preferred amounts of a given peptide or nucleic acid of the invention used in a given therapy will vary to the particular active peptide or nucleic acid being utilized, the particular compositions formulated, the mode of application, the particular site of administration, the patient's weight, general health, 5 sex, etc., the particular indication being treated, etc. and other such factors that are recognized by those skilled in the art including the attendant physician or veterinarian. Optimal administration rates for a given protocol of administration can be readily determined by those skilled in the art using conventional dosage determination tests. In

10 general, a suitable effective dose of one or more the above-described compounds, particularly when using the more potent compound(s), will be in the range of from 0.01 to 100 milligrams per kilogram of bodyweight of recipient per day, preferably in the range of from 0.01 to 20 milligrams per kilogram bodyweight of recipient per day, more

15 preferably in the range of 0.05 to 4 milligrams per kilogram bodyweight of recipient per day. The desired dose is suitably administered once daily, or several sub-doses, e.g. 2 to 4 sub-doses, are administered at appropriate intervals through the day, or other appropriate schedule. Such sub-doses may be administered as unit dosage forms, e.g.,

20 containing from 0.05 to 10 milligrams of the above-described compound(s), per unit dosage, preferably from 0.2 to 2 milligrams per unit dosage.

The present invention further includes a kit for the in vivo 25 systemic introduction of a recombinant BY55 including fragments and derivatives thereof or nucleic acid encoding the same into a patient. Such a kit includes a carrier solution, protein or nucleic acid, and a means of delivery, e.g., a catheter or syringe. The kit may also include instructions for the administration of the preparation.

^'30

The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.

EXAMPLE 1 - Cloning and expression of BY55 cDNA

Materials and Methods.

The PE conjugated anti antibodies were purchased from Fisher Scientific, Pittsburgh, PA. TCR α/β specific antibody BMA031 and TCR γ/δ specific antibody IMMU510 were from Immunotech. All other antibodies were from Coulter (Hialeah, FL). Goat anti mouse IgM coupled Sepharose-4B was purchased from ZYMED laboratories Inc., South San Francisco, CA. Protease inhibitors, DTT, iodoacetamide and n-octyl-b-D-glucoside were from Sigma. Protein A- Sepharose 4B is from Repligen, Cambridge, MA. ECL western blot reagents were from Amersha . Monophosphatidylinositol specific phospholipase C is from Oxford Glyco System, Rosedale, NY. a-32P labeled dATP and dCTP nucleotides were from New England Nuclear, Boston, MA.

Isolation of BY55 cDNA.

NK cells from four different donors were isolated and poly (A)⁺ RNA prepared. A cDNA library was constructed using NK cell poly(A)⁺

RNA and the pCDM8 expression vector (Seed 1987, Aruffo et al 1987). Plasmid DNA from the cDNA library was prepared and used to transfect COS cells via the DEAE-dextran method (Seed et al 1987; Freeman et al 1989). Cells were harvested after 47 hrs and incubated for an hour with 10 μg/ml of the murine IgM antibody BY55 raised against YT2C2, a human cell line with NK functional characteristics (Maiza, et al. 1993). Antigen positive cells were isolated by panning on goat anti mouse IgM antibody coated plates (Seed, et al. 1987; Freeman, et al. 1989). Plasmid DNAs were isolated from COS cells in a Hirt supernatant (Seed, et al. 1987) and transformed into Escherichia coli DH10B/P3 by electroporation. Transformants were selected in 7.5 μg/ml tetracycline and 25 μg/ml ampicillin. The plasmids were introduced into COS cells by spheroplast fusion (Seed, et al. 1987). Antigen positive COS cells were isolated by panning as described above. After three rounds of selection, plasmid DNAs were isolated from individual clones and analyzed by restriction digest with Xba I. Nine out of ten plasmids showed cDNA inserts of two different sizes, 1.3 kb or 1.4 kb. COS cells tranfected with the miniprep plasmid DNA from eight of these clones bound BY55 but not control mouse IgM.

DNA sequence analysis. Both strands of a cDNA insert of one clone from the 1.3 kb and the 1.4 kb group were sequenced with synthetic oligonucleotide primers and dye-labelled terminator /Taq polymerase chemistry and analyzed on an automated fluorescent DNA sequencer (Applied Biosystems, Foster City, CA).

Northern and Southern blot analysis.

GCG blast search on the Combined nonreduntant data bases GenBank⁺EMBL⁺ DDBJ⁺PDB confirmed that the open reading frame is the same in both the 1.3 kb and the 1.4 kb cDNA inserts. The entire cDNA from the small size insert was used to generate a probe for hybridizations. Poly A⁺ RNAs were prepared as described in (Freeman et al 1992) from various cell lines and denatured with formaldehyde and electrophoresed on an agarose gel. The resolved RNAs were blotted onto nitrocellulose membranes (Scheicher & Schuell, Inc., Keene, NH). The BY55 probe was labeled by random oligonucleotide priming using a-32P labeled dCTP and dATP and Boeringher Mannheim random labeling kit following the protocol recommended by the supplier. Hybridization and washing and autoradiography were done as described previously (Freeman, et al. 1992). The blots were then stripped and hybridized with control glucose 6 phosphate dehydrogenase probe.

COS cell transient transfection. Transient transfection of COS cells was performed as described previously (Seed, et al. 1987). COS cells were transfected with BY55 plasmid DNAs or control vector DNA alone via the DEAE-dextran method. Transfected COS cells were harvested after 72 h for analysis.

Stable transfection and single cell cloning.

CHO, 300.19 and Jurkat cells were transfected with linearized BY55 plasmid DNA and the drug selection plasmid DNAs pGK hygromycin or pSV2 neomycin. Transfected cells expressing BY55 antigen were sorted twice with a FACS (Coulter) and single cells expressing BY55 were cloned by limiting dilution. All cell cultures were maintained mycoplasma free by treating with Ciprofloxacin and by frequent analysis by polymerase chain reaction for mycoplasma sequences (Stratagene).

Cell surface biotinylation and immunoprecipitation.

Cells were biotinylated by a sulfosuccinimidobiotin (Sulfo-NHS-biotin) (Pierce Chemical Co., Rockford, IL) procedure. Briefly, after three washes in PBS, cells were suspended at 10 x 10⁶/ml in PBS with 0. IM Hepes, pH 8.0 and 0.1 μg/ml of Sulfo-NHS-biotin.

After a 40-min incubation at room temperature with occasional shaking, cells were washed three times with RPMI 1640, 4°C. Cells were lyzed in a lysis buffer containing 50 mM Tris-HCl pH, 150 mM NaCl, 60 mM n-octyl-b-D-glucoside, 0.05% Triton X-100, in the presence of the protease inhibitors aprotinin (1 U/ml), benzamidine hydrochloride (10 μg/ml), Leupeptin (1 μg/ml), pepstatin (10 μg/ml), soybean trypsin inhibitor (10 μg/ml), and PMSF (1 mM) by rocking at 4°C for 45-min. After centrifugation, the lysate supernatant was precleared by incubating with protein A- Sepharose 4B beads and a rabbit polyclonal anti sera () plus protein A - Sepharose 4B. Immunoprecipitation was performed by an overnight incubation with BY55 or a control antibody plus protein A - Sepharose 4B. After five washes with a wash buffer containing 50 mM Tris-HCl pH 8.3, 0.5 M NaCl, 0.5% NP-40, protein was either eluted directly by boiling for 10-min in SDS sample buffer plus 5 mM β-mercaptoethanol or alternatively reduced with 10 mM DTT in wash buffer with a 1 h incubation at 60°C and after a brief spin and quick removal of the reducing buffer, carboxyamidomethylated with 100 mM idodo acetamide at 90°C and the protein was eluted as before. The immunoprecipitates were resolved by a 10% PAGE and blotted electrophoretically onto a membrane (Immobilon, Millipore, MA). The membrane was blocked overnight with 5% drymilk in PBST (PBS plus 0.05% Tween 20) and the protein bands were developed with horse radish peroxidase conjugated anti-antibody and ECL reagents.

Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment.

Cells were washed with chilled RPMI three times and incubated with phosphatidylinositol-specific phospholipase C (0.4 U/ml) plus 1 mM PMSF for lh at 37°C and washed again three times with chilled

RPMI. Control cells were also treated similarly but without PI-PLC.

Cells were then stained with either BY55 or a control antibody and a phycoerythrin (PE) conjugated anti-IgM antibody and analyzed by FACS.

Fluorescence activated cell sorting (FACS).

Cells were incubated with primary antibody (10 μg/ml) in RPMI plus 2% fetal calf serum for 30-min on ice and washed twice with the same media. In the cases where the primary antibodies are not directly conjugated, cells were again incubated with a FITC or PE conjugated secondary (anti) antibody as before and washed twice and either sorted or analyzed by FACS.

Isolation of Intra Epithelial Lymphocytes (IEL). Mucosa of surgically removed small intestinal specimen was separated from the submucosa by dissection. After washing several

2 times with RPMI, the mucosa was cut into 1 cm pieces. Tissues were washed three times with wash media (Hanks' Balanced Salt Solution without calcium or magnesium (HBSS-CMF) + 1 mM Dithithreitol (DTT)) by shaking in a water bath at 37 ∞C. ilELs were eluted three times from the tissue with 0.75 mM EDTA in 10 mM HEPES pH 7.4 buffer by incubating in a shaking water bath at 37 ∞C for 45 minutes each time. The eluted IELs were pooled and washed in culture media (RPMI + 10% Fetal Calf Serum) and kept at 4∞C overnight in culture media plus 20 U/ml collagenase and 0.1 mg/ml DNase. Pure ilELs were then isolated by percol gradient as described.

Isolation of CD8+CD28^" PBL.

PBMC were isolated from fresh whole blood by centrifugation over Ficoll- hypaque. Monocytes were depleted by adherence to plastic flasks for 2 hrs to overnight at 37° C. Cells were incubated with antibodies specific for NK cells (anti CD 16 (3G8, IgGl) plus anti CD56 (3B8, IgGl)), B cells (anti CD19 (B4, IgGl) plus anti CD20 (Bl, IgG2a)), macrophages anti CD 14 (Mo2, IgM), CD4+ T cells (anti CD4,(T4, IgGl)) and CD28+ T cells (anti CD28 (9.3, IgG2a)) and NK cells, B cells, macrophages, CD4+ T cells and CD28+ T cells were depleted using goat anti-mouse IgG and IgM antibody coated magnetic beads (Perceptive Diagnostics,

Cambridge, MA).

Isolation and culture of human NK cells. PBMC were isolated by Ficoll- diatrizoate density gradient centrifugation from cytopheresis buffy coats obtained from normal volunteer donors. Adherent mononu clear cells were depleted by incubation on sterile scrubbed nylon wool columns for 60 minutes at 37 degrees C. Enriched NK cells were obtained as described previously [Robertson et al., (1992) JEM 175:779] by negative selection using T1/24T6G 12 (CD5), T3/RW2 (CD3), and MY4 (CD14) mAb together with immunomagnetic beads. Highly purified CD56^^rig^nt and CD56^^m NK subsets were isolated from populations of enriched NK cells by cell sorting as described [Caligiuri et al. (1990) JEM 171 : 1509]. Basal culture medium was RPMI- 1640 (Gibco, Grand Island, NY) supplemented with 2mM L- gl famine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml gentamicin and 15% heat inactivated FCS. Activation and in vitro expansion of NK cells using leukocyte conditioned medium (LCM) and ionomycin were performed as previously described [Robertson et al. (1993) J. Immunol. 150: 1705; Robertson et al. (1997) Immunity, in press]. Polyclonal NK cell cultures were maintained at cell concentrations of 1-2 x lO^/ml by addition of basal medium supplemented with 10- 15% LCM.

Proliferation Assays. Sorted NK cells were plated at 30,000 cells/well ( 1.5 x 10^ cells/ml) in 96 well microtiter plates (Flow Laboratories, McLean, VA) with the indicated cytokines and/or mAb. The final dilution of mAb containing ascites added to the cultures was 1 :500. In some experiments, sorted NK cells were cultured together with 5,000 irradiated (10,000 cGy) K562 stimulator cells [ ] at an NK:stimulator cell ratio of 5: 1. Cells were cultured for 96 hrs followed by a 16 hr pulse with 1 μCi ³H thymidine.

Results Molecular Cloning and Characterization of BY55.

A 1.3-kb and a 1.4-kb cDNAs were isolated by COS cell expression cloning using BY55 mAb and a cDNA library constructed from NK cells. On transient transfection into COS cells both the 1.3 and 1.4-kb cDNA inserts directed the expression of a protein molecule on the cell surface which was recognized by the BY55 mAb(fig.1) but not by an isotype matched control IgM.

The BY55 cDNA is comprised of 1449 nucleotides and another smaller transcript of 1343 nucleotides (Fig. 2 A). Sequence analysis of these two clones revealed an identical single long open reading frame.

Thus these two cDNA transcripts vary only in the 1.4 kb cDNA having an extra 106 nucleotides in the 5' untranslated region. This extra sequence is bounded by splice donor/ acceptor sites and is presumably spliced out to produce the smaller cDNA. The encoded polypeptide is 181 amino acids long. DNA and protein data base searches did not identify any BY55 homologous sequences. Thus, BY55 cDNA and polypeptides are unique and do not belong to any known family of genes or proteins.

B55 has two hydrophobic domains, one at the NH2 terminus and the other at the COOH terminus (Fig. 2 B). The NH2 terminus of the BY55 protein (amino acids 1-25) has characteristics of a secretory signal sequence with a predicted cleavage site after the glycine at position 25. The hydrophobic domain at the COOH terminus is of only 20 amino acids, a little smaller than expected for membrane anchorage and is not bounded by charged amino acids as is usual for transmembrane domains. The hydrophobic sequence at the COOH end has the characteristics of a glycophospatidylinositol (gpi) membrane anchor signal sequence. After the cleavage of the amino acids from 159 or 160 to 181, gpi molecule might be added to one of the serines at position 159 or 160. Thus the mature polypeptide have 135 or 136 amino acids. The polypeptide also has two potential sites for N-linked glycosylation. In addition to the two cysteines within the signal sequence, there are six more cysteines in the mature polypeptide. Thus this protein has ample possibility for forming intra and interchain protein disulflde bonds; however, the BY55 polypeptide does not have the conserved structural features of an Ig superfamily member. Similar to CC-chemokines there is a double cysteine in BY55, but the position of CC and also the encompassing amino acid sequences around CC are different in BY55 compared to CC-chemokines.

Expression of BY55 mRNA.

Two mRNA transcripts of 1.5 kb and 1.6 kb were identified by hybridization to the BY55 cDNA (Figures 3A &3B). The Northern blot analysis of T, B, NK, and various myeloid cell lines showed that BY55 is expressed only in human NK cells, NKL an NK-like cell line, thymocytes, splenocytes and resting T cells but not in any of the yeloid or B cell lines (Figure 3A & 3B). The level of BY55 mRNA expression is very high in NK and NKL cells compared to that in T cells, thymocytes and splenocytes. Although in NK and NKL cells both the small and large mRNA transcripts are expressed, in lymphocytes and splenocytes only the larger mRNA transcript was expressed. In human tissues BY55 mRNA is expressed only in spleen, small intestine and peripheral blood lymphocytes, but not in prostate, testis, ovary, heart, brain, placenta, lung, liver, skeletal muscle, kidney or pancreas (Figure 4A & 4B). From this Northern blot we are unable to ascertain BY55 mRNA expression in colon as the amount of RNA loaded in this lane seems very low as evidenced by the intensity of the band corresponding to the house keeping gene G6PD.

Immunoprecipitation Analysis.

The size of BY55 protein reported in an earlier report (-80 kD) was far larger than predicted from the mature polypeptide plus two N-linked glcosylations, approximately 24 kD. Since BY55 protein has six cysteines in the mature polypeptide, there is ample possibility for forming intra and interchain disulflde bonds which might increase the apparent molecular weight. We tested this by electrophoresing BY55 antibody immunoprecipitates from BY55 expressing NKL cells and BY55-300.19 cell transfectants in a polyacrylamide gel with β-mercaptoethanol in both the sample buffer and the electrophoresis running buffer (lane 1 in Figure 6). We saw a major band with molecular weight about ~80 kD, as reported earlier (Maiza, et al. 1993), but also a minor band with molecular weight about -30 kD. This 30 kD minor band became the major band when the immunoprecipitates were first reduced with DTT and cysteines were carboxyamidomethylated with iodoacetamide and then analyzed by PAGE (lanes 2 and 6 in Figure 6). Analysis of Surface Anchorage of BY55.

The hydrophobic domain at the COOH terminus of BY55 does not have the characteristics of a transmembrane domain. To test whether BY55 uses glycosylphosphatidylmositol mediated membrane anchorage, we incubated the BY55 expressing NKL cells and CHO cells stably transfected with BY55 cDNA with Phosphatidylinositol-specific phospholipase C (PIPLC) and then analyzed the cells for the surface expression of BY55. As shown in Figure 7, BY55 protein was cleaved from the cell surface by PIPLC, both in NKL cells (Figure 7, bottom panels) and in CHO cells transfected with BY55 cDNA (Figure 7, top panels), and also in Jurkat cells transfected with BY55 cDNA (data not shown). The level of cleavage is almost 100% as opposed to some other gpi-anchored proteins which are incompletely removed by PIPLC (Walter et al 1990; Solomon et al 1995).

BY55 is expressed on all intraepithelial and most lamina propria CD8⁺ intestinal lymphocytes.

As shown in figure 3, BY55 mRNA was detected in small intestine. As small intestine has abundant gut-associated lymphoid tissue, we stained freshly isolated human small intestinal intraepithelial (ilEL) and lamina propria lymphocytes (LPL) with BY55 mAb. Strikingly, BY55 was expressed on almost all ilEL (Figure 8). ilEL are a unique subpopulation of lymphocytes lining the gut and almost all are CD3⁺TCRα/β⁺CD8⁺CD56-CD28- (Figure 8, top panel). For comparison, we stained peripheral blood lymphocytes (PBL). Consistent with previous reports (Bensussan, et al. 1993; Maiza, et al. 1993), only 21% of peripheral blood CD3⁺ T cells expressed BY55. Most peripheral blood NK cells expressed BY55 with 59% of CD56⁺ PBL expressing BY55.

More extensive phenotypic analysis of ilEL revealed other characteristic markers expressed on this T cell subset. In addition to being CD3⁺TCRα/β⁺CD4-CD8⁺CD56-CD28-> ilEL were almost all positive for gβ7 integrin (CD 103), the complement receptor type 3 CD l lb (data not shown), CD45RO, and CD 101 (Figure 9A). The quite uniform population of ilEL was compared to the more diverse population of lamina propria lymphocytes (LPL). Freshly isolated LPL were composed of various numbers of CD4⁺ and CD8⁺ T cells; however, BY55 expression was found in all donors in the CD8⁺ LPL with a similar phenotype as ilEL CD8+ cells (Figure 9B).

The subset of peripheral blood CD8⁺CD28^" T lymphocytes expressing BY55 differs from ilEL. The phenotype of ilEL, specifically, that they were CD8+CD28-

CD 1 lb⁺, is similar to a subset of peripheral blood T cells. We therefore examined expression of BY55 on CD8⁺CD28^" peripheral blood T cells. We have previously shown that in PBL the CD3⁺CD8⁺ subset which does not express CD28 is predominantly BY55⁺ (Schiavon, tissue antigen). We also reported that most of the CD 101⁺ T cells are CD28⁺.

In order to look for a subset of PBL with a similar phenotype as ilEL we performed further studies on PBL depleted of B cells , NK cells, CD4 T cells and CD28 T cells ( CD19-CD56-CD4-CD28^"). The resulting subset of PBL was enriched in cells expressing CD8, BY55 and CD 1 lb but lacked _Eβ7 (Figure 10) and CD 101 (not shown).

The subset of blood NK lymphocytes expressing BY55 expresses low levels of CD56 and is not proliferative.

CD56+ NK cells can be divided into CD 16⁺CD56^dim and CD 16^" CD56^brig^ht subsets. The CD 16⁺CD56^dim NK cell subset is highly cytolytic and does not proliferate in response to signals that costimulate NK cell proliferation. In contrast, the CD 16-CD56^brig^ht NK cell subset proliferates vigorously in response to signals that costimulate NK cells such as IL-2 and crosslinking of CD94 (); however, it shows little lytic activity (). We examined the expression of BY55 in these NK cell subsets and found that BY55 was expressed by the CD16⁺CD56^dim NK cell subset and was absent from the CD16-CD56^brig^nt NK cell subset (Figure 10). This is consistent with our previous reports showing the association of BY55 with cytolytic lymphocytes. Following in vitro activation and expansion with IL-2, sorted CD56^bng^nt cells remained BY55^". Sorted CD56dim cells were still 50% BY55+ after in vitro expansion and expression of BY55 paralleled expression of CD 16 (data not shown).

As expected, sorted BY55+ CD56^d^^m NK cells did not proliferate in response to IL-2 alone or in association with anti-CD94 mAb (figure 12). In contrast, sorted BY55^"CD56^brig^ht NK cells vigorously proliferated in response to increasing doses of IL-2 and this was augmented by costimulatory signals provided by anti-CD94 mAb.

BY55 mAb neither augmented nor inhibited the proliferative response of either CD56^brig^ht or CD56^dim NK cell subsets. In other experiments, inclusion of BY55 had no effect on the proliferation of CD56^dnτι NK cells in response to irradiated K562 cells (data not shown). Following in vitro activation and expansion, reculture of sorted NK cell subsets with IL-2 in the presence of BY55 had no significant effect on the proliferative response of these activated NK cell subsets (data not shown). Consistent with previous reports, the presence of BY55 did not augment or inhibit NK cell lysis (data not shown).

Taken together these results strongly strengthen our previous observation that BY55 is expressed only by NK cytotoxic cells which cannot be expanded in vitro with exogenous IL2.

As shown, BY55 mRNA expression is confined to only a selected subpopulation of human lymphocytes and tissues. To further explore expression of BY55 mRNA in small intestine, we stained freshly isolated human small intestinal intraepithelial lymphocytes and peripheral blood lymphocytes. In peripheral blood lymphocytes BY55 is expressed only in a unique subpopulation (Bensussan, et al. 1993; Maiza, et al. 1993). In contrast, in small intestinal IEL, BY55 was expressed in almost all lymphocytes (Figure 8). In PBMC only 20% of CD3⁺ T cells expressed BY55, whereas in IEL 97% of CD3⁺ T cells expressed BY55. In PBMC BY55 was expressed in 29% of TCR-α/β⁺ subpopulation as opposed to 91% of cells in IEL. In PBMC 59% of CD56⁺ lymphocytes expressed BY55 as opposed to 47% in IEL cells.

BY55 is a novel cytolytic T lymphocyte specific, cytolytic NK cell specific and small intestinal IEL specific cell surface molecule. This molecule is unique in its nucleotide and amino acid sequence and does not show homology with any of the gene families reported so far in molecular and immunological data bases. The BY55 cDNA has 1449 nucleotides and can code for a polypeptide of 181 amino acids. This molecule is anchored to the cell surface via gpi anchorage and when treated with the enzyme PIPLC, the protein was cleaved from the cell surface. Its gpi addition signal sequence is novel and hitherto not reported in the literature or protein data bases. This protein has two predicted N-linked glycosylation sites. With the cleavage of both the secretory signal sequence of about 25 amino acids at the NH2 terminus and a gpi anchor signal sequence of about 20 amino acids at the COOH terminus, the resultant mature polypeptide would be 135 amino acids. It has previously been reported that an 80 kDa protein was immunoprecipitated by BY55 mAb after cell surface iodination. We show here that BY55 mAb also immunoprecipitates an 80 kDa protein band from biotin-labeled NKL line and BY55 transfected cells. This molecular mass is far larger than predicted for the mature BY55 polypeptide with two N-linked glycosylations, approximately 24 kDa. The size of BY55 protein reported in an earlier report (-80 kD) was far larger than predicted from the mature polypeptide plus two N-linked glcosylations, approximately 24 kD. Since BY55 protein has six cysteines in the mature polypeptide, there is ample possibility for forming intra and interchain disulfide bonds which might increase the apparent molecular weight. Reduction and alkylation of the immunoprecipitated BY55 led to a major band of 30 kD whereas just reduction led to an 80 kD species suggesting that BY55 is a tightly disulfide linked homodimer. Moreover we could eliminate the possibility that the 80 kDa predominant band immunoprecipitated by BY55 mAb is an associated chain similar to what has been described for CD94 AP, as this 80kDa band was predominant in all immunoprecipitates including BY55 transfectants of CHO cells and murine B cells.

The messenger RNA expression of BY55 shows an interesting pattern. BY55 mRNA was expressed only in NK cells, NKL cells, thymocytes, spleen cells and T cells, but not in any T, B, or myeloid cell line tested. In human tissues BY55 mRNA expression showed a restricted pattern and was expressed only in spleen, small intestine and peripheral blood, but not in prostate, testis, ovary, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.

In an earlier report it was shown that 50% of TCR γ/δ⁺ T cells from peripheral blood expressed BY55 protein (Maiza, et al. 1993). In human the major source of TCR γ/δ⁺ T cells is small intestine. However the accumulation of a large body of data on the phenotypic differences between PBLs and IELs and controversies over thymic versus extra thymic development of IELs prompted us to further characterize the distribution of BY55 expressing on different phenotypic IELs. Surprisingly almost all of the IELs expressed BY55. This is in sharp contrast to what was seen in PBLs, where only 25-33% expressed BY55 (Figure 8 and data presented in (Maiza, et al. 1993)). Also in PBLs only 21% of TCR α/β⁺ T cells expressed BY55, but in IELs 98% of TCR α/β⁺ T cells expressed BY55. In BY55 expressing IELs only 2% expressed the NK phenotypic marker CD56, whereas in PBLs 40-50% of the BY55 expressing cells coexpressed CD56.

These ilELs do not or only weakly proliferate upon CD3/TCR engagement or with other mitogenic stimuli (1 1, 12). Interestingly, almost all of the ilELs, which are CD8⁺ T cells bearing α/β TCR, coexpressed BY55. In addition, as previously reported, all ilELs expressed CD 103 and CD 101 , while they lack the CD28 costimulatory molecule. It has been reported that most of the fresh ilELs have phenotypic characteristics of memory T cells. It was previously reported that CD 101 ⁺ cells represent a subset of blood CD8⁺CD28⁺ cells

(Gouttefangeas, Int. Immunol.). In addition, CD 101 expression, similarly to CD 103 and CD45RO, are induced during activation. In contrast, we showed that BY55 is downmodulated after activation in short or long term culture. Besides the difference in phenotype between the cells expressing BY55 in PB and ilEL, both cell populations are able to exert cytotoxic function. Previous report in mouse and man have indicated that IEL may function as cytolytic effector cells in vivo, as freshly isolated α/β IEL have lytic potential (13, 15 and Lundqvist, J. Immunol, 157: 1926- 1934, 1996). This is in agreement with our earlier studies showing that in fresh PBL, only sorted CD8⁺BY55⁺ cells exhibit CTL activity detected in a TCR mediated redirected lysis assay (Bensussan, PNAS 90:9427-9430 (1993)). Thus it appears that BY55 delineates cytotoxic CTL in both circulating blood and tissue.

BY55 is also found on CD56^dimCD 16⁺ PB lymphocytes exerting NK activity whereas it is not expressed on CD56^bl"ig^ntCD 16^~ cells which show a high proliferative response to IL2. Recently, several structures defined as receptors for HLA class I molecules have been identified on NK cells and a subset of CD8 TCR⁺ cells. These receptors have been shown to provide either inhibitory or activating signals following their ligation. Interestingly, the KIR/KAR can be coexpressed on the same NK cell, but they do not presently identify the whole circulating NK subset. It is clear, both at the molecular and functional level that the BY55 molecule is not a new member of any of the KIR family. We have previously reported that BY55 mAb does not inhibit or enhance allogeneic or NK cell lysis (Maiza et al, J. Exp. Med.178: 1 121- 1 126 (1993)). As BY55 does not show homology with any known family of cell surface molecules, the function of the molecule is based upon its association with particular cells. Further, gpi membrane anchorage of BY55 indicates that for triggering signal transduction, the BY55 molecule must be associated with another structure.

In T and NK cells BY55 is expressed only in the subpopulation of cells which are programmed for the final effector function, i.e., cytolysis. When considering all these observations it seems IELs and cytolytic BY55⁺/CD8⁺ PBT lymphocytes and BY55+ NK cells all share some common properties, viz., poor or null proliferative response, activated lymphocyte phenotype (1,2,9, 10, 16) and capacity for cytolytic activity ( 16,4). Another important characteristic shared by IELs and BY55⁺ lymphocytes is their expansion in certain disease conditions. For example, BY55⁺CD8⁺ T lymphocytes showed a four-fold increase in HIV infected people. Similarly, increased numbers of IELs have been reported in diseases of mucosal delayed-type hypersensivity, allograft rejection, graft-versus-host disease, parasite infections, enteral challenge after immunization (13) Crohn's disease (17, 18) and celiac disease (19,20).

The overwhelming expression of BY55 seen in all IELs and also in the cytolytic subpopulation of NK and T cells of PBLs is interesting as these two populations of immune cells defend the host either from constantly exposed intact microorganism, food antigens or from viral infection without requiring an activation signal. These findings indicates BY55 molecule plays an important role in the process of primary immune defense against viruses, other microorganisms and food derived antigens. The following references are cited throughout the specification. All documents mentioned herein are incorporated herein by reference.

1 Maiza, H., G. Leca, I-G. Mansur, V. Schiavon, L. Boumsell, and A. Bensussan. 1993. J. Exp. Med. 178:221.

2. Bensussan, A., C. Rabian, V. Schiavon, D. Bengouufa, G. Leca, and L. Boumsell. 1993. Significant enlargement of a specific subset of CD3⁺CD8⁺peripheral blood leukocytes mediating cytotoxic T-lymphocyte activity during human immunodeficiency virus infection. Proc. Natl. Acad. Sci. USA. 90:9427.

3. Aruffo, A., and B. Seed. 1987. Molecular cloning of the CD28 cDNA by a high-efficiency COS cell expression system. Proc. Natl. Acad. Sci. USA. 84:8573.

4 Seed, B., and A. Aruffo. 1987. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA. 84:3365.

5. Freeman, G.J., A. S. Freedman, J.M. Segil, G. Lee, J.F. Whitman, and L.M. Nadler. 1989. B7, a new member of the Ig super family with unique expression on activated and neoplastic B cells. J. immunol. 143:2714.

6. Freeman, G.J., D.B. Lombard, CD. Gimmi, S.A. Brod, K. Lee, J.C. Laning, D.A. Hafler, M.E. Dorf, G. Gray, H. Reiser, et al. 1992. CTLA4 and CD28 mRNAs are coexpressed in most activated T cells after activation: expression of CTLA4 and CD28 messenger RNA does not correlate with the pattern of lymphokine production. J. Immunol. 149:3795. 7. Walter, E.I., Roberts, W.L., Rosenberry, J.L., Ratnoff, W.D., and

Medof, M.E.1990. J. Immunol.144: 1030.

8. Solomon, K.R.,M. Chan, and R. Finberg. 1995. Expression of GPI-anchored complement regulatory proteins CD55 and CD56 differentiates two subpopulations of human CD56⁺CD3-lymphocytes (NK cells). Cellular Immunol. 165:294.

9. Sanders, M.E., M.W. Makgoba, aand S. Shaw. 1988. Immunol. Today. 9: 195.

10. Jarry, A., N.C. Bensussan, N. Brousse, F. Selz and D.G. Grand. 1990. Subsets of CD3⁺ (T cell receptor α/β or γ/δ) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in perpheral blood. Eur. J. Immunol. 20: 1097.1

1 1. Ebert, E.C. 1989. Proliferative responses of human intraepithelial lymphocytes to various T cell stimuli. Gastroentrology. 97: 1372.

12. Mosley, R.L., M. Whetsell, J.R. Klein. 1991. Proliferative properties of murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCRab or TCRgd are largely unresponsive to proliferation signals mediated via conventional stimulation of the CD3-TCR complex. Int. Immunol. 3:563.

13. Deusch, K., and K. Reich. 1994. Phenotypical features of human intestinal intraepithelial lymphocytes in health and disease. Mucosallmmunol. Update. 2: 1.4

14. Barrett, T.A., T.F. Gajewski, D. Danielpour, E.B. Chang, K.W. Beagley, J.A. Bluestone.1992. Differential function of intestinal intraepithelial lymphocyte subsets. J. Immunol. 149:224.

15. Deusch, K., F. Luling, K. Reich, M. Classen, H. Wagner, and K. Pheiffer. 1991. A major fraction of human intraepithelial lymphocytes simultaneously expresses the γ/δ T cell receptor, the CD8 accessary molecule and preferentially uses the VI 1 gene segment. Eur. J. Immunol. 21: 1053.

16. Sydora, B.C., P.F. Mixter, H.R. Holcombe, et al. 1993. Intestinal intraepithelial lymphocytes are activated and cytolytic but do not proliferate as well as other T cells in response to mitogenic signals. J.Immunol. 150:2179.

17. Cuvelier, N. De Wever, H. Mielants, M. De Vos, and H. Roels. 1992. T cell receptors in ileal mucosal biopsies of patients with Crohn disease and spondylarthopathy. Prog. Histochem. Cytochem. 26:21 1.

18. Kuner, I., S. Wilhelm, M. Classen, and K. Deusch. 1993. Inversion of phenotype and proliferative anergy of human Intestinal intraepithelial lymphocytes in inflammatory bowel disease. Gasteroenterology. 103:A874.

19. Halstensen, T.S.,and P. Brandtzaeg.1993. Activated T lymphocytes in the celiac lesion: non-proliferative activation (CD25) of CD4⁺α/β cells in the lamina propria but proliferation (Ki-67) of α/β and γ/δ cells in the epithelium. Eur. J. Immunol. 23:505.0

20. Lionetti, P., J. Spencer, E.J. Breese, S.H. Murch, J. Taylor, and T.T. MacDonald. 1993. Activation of mucosal Vb3⁺ T cells and tissue damage in the human small intestine by the bacterial superantigen, Staphylococcus aureus enterotoxin B.Eur. J. Immunol. 23:664. The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: DANA-FARBER CANCER INSTITUTE, ET AL.

(ii) TITLE OF THE INVENTION: RECOMBINANT BY55 AND NUCLEIC ACID ENCODING SAME

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DIKE, BRONSTEIN, ROBERTS & CUSMAN, LLP

(B) STREET: 130 Water Street

(C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 12-N0V-1997

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/7 7,853

(B) FILING DATE: 13-N0V-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Resnick, David S

(B) REGISTRATION NUMBER: 3 ,235

(C) REFERENCE/DOCKET NUMBER: 6964

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-523-3400

(B) TELEFAX: 617-523-6440

(C) TELEX:

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1361 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 216...758 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAGTCTGAGA ACAAGAAAGA AGAACTTCTG TCTCGAGGGT CTCACTGTCA ACCAGGCCAG 60 AGTGCAGTGA AGATCATACC TCACTACATC CGTGAACTCC CGGGCTCCTC CCACCTAAGT 120 CTCTTGAGTA GCTGGGACTT CAGGAGACTG AAGCCAAGGA TACCAGCAGA GCCAACATTT 180 GCTTCAAGTT CCTGGGCCTG CTGACAGCGT GCAGG ATG CTG TTG GAA CCC GGC 233 Met Leu Leu Glu Pro Gly 1 5

AGA GGC TGC TGT GCC CTG GCC ATC CTG CTG GCA ATT GTG GAC ATC CAG 281 Arg Gly Cys Cys Ala Leu Ala lie Leu Leu Ala lie Val Asp lie Gin 10 15 20

TCT GGT GGA TGC ATT AAC ATC ACC AGC TCA GCT TCC CAG GAA GGA ACG 329 Ser Gly Gly Cys lie Asn lie Thr Ser Ser Ala Ser Gin Glu Gly Thr 25 30 35

CGA CTA AAC TTA ATC TGT ACT GTA TGG CAT AAG AAA GAA GAG GCT GAG 377 Arg Leu Asn Leu He Cys Thr Val Trp His Lys Lys Glu Glu Ala Glu 40 45 50

GGG TTT GTA GTG TTT TTG TGC AAG GAC AGG TCT GGA GAC TGT TCT CCT 425 Gly Phe Val Val Phe Leu Cys Lys Asp Arg Ser Gly Asp Cys Ser Pro 55 60 65 70

GAG ACC AGT TTA AAA CAG CTG AGA CTT AAA AGG GAT CCT GGG ATA GAT 473 Glu Thr Ser Leu Lys Gin Leu Arg Leu Lys Arg Asp Pro Gly He Asp 75 80 85

GGT GTT GGT GAA ATA TCA TCT CAG TTG ATG TTC ACC ATA AGC CAA GTC 521 Gly Val Gly Glu He Ser Ser Gin Leu Met Phe Thr He Ser Gin Val 90 95 100

ACA CCG TTG CAC AGT GGG ACC TAC CAG TGT TGT GCC AGA AGC CAG AAG 569 Thr Pro Leu His Ser Gly Thr Tyr Gin Cys Cys Ala Arg Ser Gin Lys 105 110 115

TCA GGT ATC CGC CTT CAG GGC CAT TTT TTC TCC ATT CTA TTC ACA GAG 617 Ser Gly He Arg Leu Gin Gly His Phe Phe Ser He Leu Phe Thr Glu 120 125 130

ACA GGG AAC TAC ACA GTG ACG GGA TTG AAA CAA AGA CAA CAC CTT GAG 665 Thr Gly Asn Tyr Thr Val Thr Gly Leu Lys Gin Arg Gin His Leu Glu 135 140 145 150

TTC AGC CAT AAT GAA GGC ACT CTC AGT TCA GGC TTC CTA CAA GAA AAG 713 Phe Ser His Asn Glu Gly Thr Leu Ser Ser Gly Phe Leu Gin Glu Lys 155 160 165

GTC TGG GTA ATG CTG GTC ACC AGC CTT GTG GCC CTT CAA GCT TTG TAAGC 763 Val Trp Val Met Leu Val Thr Ser Leu Val Ala Leu Gin Ala Leu 170 175 180

CTTGTGCCAA AAGAAACTTT TAAAACAGCT ACAGCAAGAT GAGTCTGACT ATGGCTTAGT 823 ATCTTTCTCA TTACAATAGG CACAGAGAAG AATGCAACAG GGCACAGGGG AAGAGATGCT 883 AAATATACCA AGAATCTGTG GAAATATAAG CTGGGGCAAA TCAGTGTAAT CCTTGACTTT 943 GCTCCTCACC ATCAGGGCAA ACTTGCCTTC TTCCCTCCTA AGCTCCAGTA AATAAACAGA 1003 ACAGCTTTCA CCAAAGTTTC CTCCCTCTTC ATCAACATAG TAAAATAAGT CAAACAAAAT 1063 GAGAACACCA AATTTTGGGG GAATAAATTT TTATTTAACA CTGCAAAGGA AAGAGAGAGA 1123 AAACAAGCAA AGATAGGTAG GACAGAAAGG AAGACAGCCA GATCCAGTGA TTGACTTGGC 1183 ATGAAAATGA GAAAATGCAG ACAGACCTCA ACATTCAACA ACATCCATAC AGCACTGCTG 1243 GAGGAAGAGG AAGATTTGTG CAGACCAAGA GCACCACAGA CTACAACTGC CCAGCTTCAT 1303 CTAAATACTT GTTAACCTCT TTGGTCATTT CTCTTTTTAA ATAAATGCCC ATAGCAGT 1361

(2) INFORMATION FOR SEQ ID Nθ:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 181 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Met Leu Leu Glu Pro Gly Arg Gly Cys Cys Ala Leu Ala He Leu Leu

1 5 10 15

Ala He Val Asp He Gin Ser Gly Gly Cys He Asn He Thr Ser Ser

20 25 30

Ala Ser Gin Glu Gly Thr Arg Leu Asn Leu He Cys Thr Val Trp His

35 40 45

Lys Lys Glu Glu Ala Glu Gly Phe Val Val Phe Leu Cys Lys Asp Arg

50 55 60

Ser Gly Asp Cys Ser Pro Glu Thr Ser Leu Lys Gin Leu Arg Leu Lys 65 70 75 80

Arg Asp Pro Gly He Asp Gly Val Gly Glu He Ser Ser Gin Leu Met

85 90 95

Phe Thr He Ser Gin Val Thr Pro Leu His Ser Gly Thr Tyr Gin Cys

100 105 110

Cys Ala Arg Ser Gin Lys Ser Gly He Arg Leu Gin Gly His Phe Phe

115 120 125

Ser He Leu Phe Thr Glu Thr Gly Asn Tyr Thr Val Thr Gly Leu Lys

130 135 140

Gin Arg Gin His Leu Glu Phe Ser His Asn Glu Gly Thr Leu Ser Ser 145 150 155 160

Gly Phe Leu Gin Glu Lys Val Trp Val Met Leu Val Thr Ser Leu Val

165 170 175

Ala Leu Gin Ala Leu 180

Claims

What is claimed is:

1. An isolated nucleic acid encoding human BY55 or fragment thereof.

2. The nucleic acid of claim 1 where the nucleic acid comprises the sequence of SEQ ID NO: 1, or the complement thereto.

3. The nucleic acid of claim 1 where the nucleic acid codes for the human BY55 of SEQ ID NO:2.

4. The nucleic acid of claim 1 where the encoded human BY55, when expressed and processed, has a molecular weight of about 80 kD as determined by polyacrylamide gel electrophoresis.

5. The nucleic acid of claim 1 where the nucleic acid has at least about 55 percent sequence identity to SEQ ID NO: 1, or the complement thereto.

6. The nucleic acid of claim 1 where the nucleic acid has at least about 75 percent sequence identity to SEQ ID NO: 1 , or the complement thereto.

7. The nucleic acid of claim 1 where the nucleic acid hybridizes to the sequence of SEQ ID NO: 1 under normal stringency conditions.

8. The nucleic acid of claim 1 where the nucleic acid hybridizes to the sequence of SEQ ID NO: 1 under high stringency conditions.

9. The nucleic acid of claim 1 , wherein the nucleic acid is a unique fragment of huma BY55, where the unique nature is determined by comparison to public data bases as of this filing date.

10. The nucleic acid of claim 1 wherein the polynucleotide is cDNA.

1 1. The nucleic acid of claim 1 wherein the polynucleotide is RNA.

12. A recombinant vector comprising the nucleic acid of claim 1.

13. A host cell comprising the vector of claim 12.

14. A method of producing human BY55 comprising culturing a host cell of claim 13 under conditions suitable for expression of human BY55.

15. A method of identifying a compound useful in the diagnosis or treatment of a BY55 related disorder, comprising contacting a candidate pharmacological agent with human BY55 or fragment or derivative thereof and analyzing the mixture of the candidate agent and human BY55.

16. The method of claim 13 wherein the human BY55 has a sequence represented by SEQ ID NO:2.

17. An isolated human BY55 having an apparent molecular weight of about 80 kD as determined by polyacrylamide gel electrophoresis .

18. An isolated human BY55 of claim 17 comprising a sequence represented by SEQ ID NO:2.

19. A fusion protein comprising BY55 and an immunoglobulin.

20. An isolated mature human BY55.

21. A method for modulating BY55 activity comprising administering to human cells a modulation effective amount of a nucleic acid of claim 1 or fragment or derivative thereof.

22. A method for modulating BY55 activity comprising administering to human cells a modulation effective amount of an isolated human BY55 of claim 17 or fragment or derivative thereof.

23. A method for modifying an immune response comprising administering to a patient an effective amount of the fusion protein of claim 19.

24. A method for producing a truncated BY55 comprising contacting a cell expressing BY55 with an agent that selectively cleaves a glycophosphatidylinositol linkage.

25. The method of claim 24, wherein the agent is phosphatidylinositol specific phospholipase C and/ or the cell is selected from the group consisting of cytolytic T lymphocytes, cytolytic NK cells, and intra-epithelial lymphocytes.

26. The method of claim 24, wherein the cell is a host cell transformed with a DNA encoding BY55.

27. The method of claim 25, wherein the host cell is a COS cell.

28. A method of inhibiting cleavage of BY55 on a BY55 expressing cell comprising contacting the cell with an agent that blocks leavage of the BY55 GPI linkage.

29. The method of claim 26, wherein the agent blocks cleavage by binding to the GPI.

30. The method of claim 26, wherein the agent is a monoclonal Ab.