WO1994010188A1 - Activation antigen cd69 - Google Patents

Abstract

DNA encoding a cell surface antigen designated CD69 is isolated, and the nucleotide and encoded amino acid sequences determined. cDNA encoding the CD69 protein is inserted into an expression vector for production of the protein via recombinant DNA technology. Soluble forms of CD69 (comprising the extracellular domain), dimeric forms of the protein, and fusion proteins comprising CD69 as one component are prepared. CD69 is associated with activation of a number of cell types.

Description

ACTIVATION ANTIGEN CD69

BACKGROUND During the activation process, T cells acquire several new cell-surface glycoproteins which in turn are involved in further aspects of cellular activation. The earliest of these newly-synthesized cell-surface activation molecules is CD69. CD69 expression is seen within 60 minutes of T cell stimulation, but is absent in resting cells (Hara et al., J. Exp. Med. 764:1988, 1986; Cosulich et al., Proc. Natl. Acad. Sci. USA 84:4205, 1987; Cebrian et al., J. Exp. Med. 765:1621, 1988). CD69 appears to be involved in cellular . activation in that cross-linking with antibodies in the presence of phorbol ester is capable of inducing T-cell proliferation (Cosulich, supra; Cebrian, supra; Nakamura et al., J. Exp. Med. 169:611, 1989; Risso et al., Eur. J. Immunol. 19:323, 1989; Testi et al., J. Immunol. 143:1123, 1989; Testi et al., J. Immunol. 742:1854, 1989). In addition to activated T cells, CD69 is constitutively expressed on a subset of thymocytes and on platelets (Testi et al., J. Immunol. 141 :2557, 1988 and Testi et al, J. Exp. Med. 772:701 , 1990) and is inducible on K cells, B cells and neutrophils (Risso, supra; Lanier et al., J. Exp. Med. 767:1572, 198s; and Gavioli et al., Cell. Immunol. 742:186, 1992). In each case, binding of the CD69 molecule with a specific antibody is capable of activating the expressing cell (Testi, 1988, supra; Testi, 1990, supra; Lanier, supra; Gavioli, supra; Moretta et al., J. Exp. Med. 174:1393, 1991). CD69 also has been detected on platelets and is reported to mediate platelet activation and aggregation (Testi et al., 1990, supra). Biochemical characterization of the CD69 antigen has revealed it to be a homo- dimer of differentially-processed 32 Kd and 28 Kd subunits (Hara, supra; and Cosulich, supra). Activation of certain cell types (e.g., peripheral blood T-cells) results in phosphorylation of CD69, and CD69 expressed in thymocytes is constitutively phosphorylated (Risso, supra; Testi et al., J. Immunol. 742:1854, 1989; and Testi, 1988, supra). While there is evidence that activation of T cells through CD69 results in the association of CD69 with a GTP-binding protein (Risso et al., J. Immunol. 746:4105, 1991), the nature of the signal transduced by CD69 has not yet been determined.

SUMMARY OF INVENTION The present invention provides isolated DNA encoding an activation antigen designated CD69, recombinant expression vectors containing the isolated DNA, and host cells transformed with the recombinant vector. CD69 proteins encoded by the isolated DNA, including soluble CD69 proteins and fusion proteins comprising CD69, are also disclosed herein. Also provided is a method for producing CD69 protein by cultivating the transformed host cells under conditions that promote expression of CD69, and recovering the expressed CD69 protein. The nucleotide sequence of cloned CD69 cDNA is provided, along with the amino acid sequence encoded thereby.

DETAILED DESCRIPTION OF INVENTION CD69 is a cell surface antigen associated with activation of certain cell types. The present invention provides isolated DNA encoding CD69, recombinant expression vectors containing the isolated DNA, and host cells transformed with the recombinant vector. A method for producing recombinant CD69 protein involves cultivating the transformed host cells under conditions that promote expression of CD69, and recovering the expressed CD69 protein from the cell culture. DNA sequences encoding fusion proteins comprising CD69 as one component are also disclosed herein, as described in more detail below.

Homodimers of CD69 result when interchain disulfϊde bonds form. The disulfide bonds may form between cysteine residues on two CD69 pόlypeptide chains, or between cysteine residues on a heterologous polypeptide (e.g., an antibody-derived Fc polypeptide) fused to each of two CD69 polypeptides, as described below for one embodiment of the present invention. The two CD69 polypeptides of a homodimer may be differentially glycosylated, while having the same amino acid sequence.

Human CD69 is within the scope of the present invention, as are CD69 proteins derived from other mammalian species. As used herein, the term "CD69" includes membrane-bound proteins (comprising a cytoplasmic domain, a transmembrane region, and an extracellular domain) as well as truncated proteins that retain the desired biological properties. Such truncated proteins include, for example, soluble CD69 comprising only the extracellular (ligand binding) domain.

As described in example 1, human CD69 cDNA (designated clone #33) was isolated from a cDNA library prepared from an alloreactive human T-cell clone. A second human CD69-encoding cDNA (designated HCD69- 11) was isolated from a cDNA library derived from stimulated human peripheral blood T cells. Both clones comprise the same coding region (presented as SEQ ID NO: 1) but clone HCD69-11 comprises additional non- coding sequence. The entire human CD69 cDNA insert of clone #33 was excised and inserted into pBLUESCRIPT® SK, a cloning vector available from Stratagene Cloning Systems, San Diego. The resulting recombinant plasmid in E. coli strain DH5α was deposited with the American Type Culture collection on October 23, 1992 and was given accession number ATCC 69100. The strain deposit was made under the terms of the Budapest Treaty.

The DNA sequence of the coding region of the two human CD69 cDNA clones described above is shown in SEQ ID NO.l, along with the amino acid sequence encoded thereby. This amino acid sequence is also shown in SEQ ID NO:2.

When the encoded amino acid sequence was examined, several interesting features were apparent. One open reading frame of 199 amino acids was present. The sequence lacks a signal peptide and contains an internal segment of hydrophobic amino acids believed to constitute a transmembrane region (residues 35-78 of SEQ ID NO:l). These features are characteristic of type II integral membrane proteins (Singer et al., Proc. Natl. Acad. Sci. USA 84:1960, 1987). Amino acids 1-34 constitute an intracellular (cytoplasmic) domain believed to function in signal transduction. Amino acids 79-199 constitute the extracellular domain, available for binding of a putative ligand.

There are single N-linked glycosylation sites in both the putative cytoplasmic domain and the extracellular domain (at amino acids 11-13 and 166-168, as described further below). The finding of only one N-linked glycosylation site in the extracellular domain of CD69 was quite surprising. CD69 is expressed as a homo-dimer of different sized subunits (Bjorndahl et al., J. Immunol. 747:4094, 1988). As described in Example 2, when the CD69 cDNA of clone #33 was expressed in COS-7 cells, a di er was immunoprecipitated from the cells. Others have shown that each subunit can be converted to a 24 Kd polypeptide using N-glycanase (Bjorndahl, supra), suggesting that the difference in size was due to differential glycosylation. The finding that CD69 contains only one N-linked glycosylation site in its extracellular domain indicates that the size difference is likely to be attributable to differential processing of a single glycosylation site. CD69 is constitutively expressed as a phosphoprotein in human thymocytes, and is phosphorylated upon activation of PBTs (Risso, supra; Testi et al., J. Immunol. 742:1854, 1989; and Testi, 1988, supra). One report showed that this phosphorylation was on tyrosine residues (Cosulich et al., Leukocyte Typing IV, B. Knapp et al., editors, Springer-Verlag, 432, 1989). The cytoplasmic domains of clones #33 and HCD69-11 contain no tyrosine residues, however. While the explanation for the discrepancy is unclear, it suggests the possibility of multiple forms of CD69, or that the CD69 protein is phosphorylated on its extracellular domain, where there are several tyrosine residues.

A comparison of the predicted amino acid sequence of CD69 with the GENBANK data base demonstrated that CD69 was a member of the C-type lectin family which includes the asialoglycoprotein receptor and the low affinity IgE receptor, CD23 (Yokoyama et al., J. Immunol. 743:1379, 1989). Most closely related to CD69 were two groups of NK cell activation antigens, NKG2 (also known as NKRPl) (Houchins et al., J. Exp. Med. 773:1017, 1991) and Ly-49 (Yokoyama, supra). The amino acid sequence of the extracellular domain of the CD69 protein of the present invention is 26.4% identical to the amino acid sequence of the extracellular domain of human NKR-P1 (129 amino acid overlap). The amino acid sequences of the extracellular domains of human CD69 and murine Ly-49 (human Ly-49 sequence unknown) are 23.6% identical (123 amino acid overlap). The percent identity was determined using the GAP computer program described below. The intracellular domains of CD69, NKR-P1 and Ly-49 exhibit no significant homology.

NKR-P1 and Ly-49 are homo-dimers, and have been shown to be involved in NK cell functional activation and possibly NK cell recognition (Giorda and Trucco, J.

Immunol. 747:1701, 1991; Giorda et al., Science 249:1298, 1990; and Karlhofer et al., Nature (London) 358:66, 1992). Crosslinking of NKR-P1 with specific antibodies can cause phosphoinositide turnover and changes in intracellular calcium levels in NK cells (Ryan et al., J. Immunol. 747:3244, 1991), and, as is the case with CD69, antibodies against NKR-P1 can redirect NK-mediated lysis to Fc receptor-bearing target cells (Moretta, supra and Ryan, supra). Thus, in addition to its likely role in lymphocyte activation, CD69 may be involved in aspects of NK cell activation. In contrast to NKR-P1 and Ly-49, however, CD69 is expressed in an inducible manner on a wide variety of hematopoietic cells. CD69 is unique within this gene family in its wide expression pattern. While its function on NK cells seems to be similar to that of NKR-P1, CD69 is involved in the co- stimulation of T cell, B cell and thymocyte proliferation, as well as the activation of neutrophils and platelets. This broad spectrum of biological activities suggests that CD69 (presumably in conjunction with a ligand that binds thereto) plays crucial roles in the regulation and function of the immune system.

Isolation of CD69 cDNA and determination of the nucleotide and encoded amino acid sequences as disclosed herein affords a number of advantages in characterizing CD69 and investigating the roles played by this protein in activation and other cellular processes. The amino acid sequence contained certain features that were not as expected, as discussed above. Isolation of CD69 DNA affords the opportunity to alter the nucleotide sequence, e.g., to inactivate N-glycosylation sites to study the apparent differential glycosylation of CD69 polypeptides in dimers. Fragments of the DNA may be isolated for production of desired portions of the CD69 protein. The sequence information provided herein, including identification of the extracellular domain, enables one to subclone DNA encoding the extracellular domain for production of soluble CD69 proteins, as discussed below. The cloned DNA of the present invention further enables one to produce fusion proteins comprising CD69 as one component, via recombinant DNA technology. Quantities of CD69 may be produced in recombinant expression systems, and simplified purification procedures employed. Soluble CD69 is secreted into the culture medium, thus eliminating the need to purify CD69 from cellular proteins that would be present in cell lysates. Peptides that facilitate purification may be fused to CD69, as discussed below. cDNA encoding a CD69 polypeptide may be isolated from other mammalian species by procedures analogous to those employed in isolating the human CD69 clone. For example, a cDNA library derived from another mammalian species may be substituted for the human cDNA libraries that were screened in example 1 below. An antibody directed against murine CD69 may be substituted for the antibody employed in example 1. Alternatively, the human CD69 cDNAs isolated in example 1 are labeled and used as probes to screen mammalian cDNA or genomic libraries using cross-species hybridization techniques. The probe preferably is derived from the coding region of the above-described human CD69 cDNAs. Cell types from which cDNA and genomic libraries may be prepared include those from which CD69 cDNA was isolated in example 1. mRNAs isolated from various cell lines can be screened by Northern hybridization to determine additional suitable sources of mammalian CD69 mRNA for use in cloning an CD69 gene. Nucleic acid from mammalian sources that include but are not limited to murine, bovine, porcine, and primate, may be screened to identify CD69 genes.

A murine CD69 cDNA was identified by cross-species hybridization to human CD69 cDNA, as described in example 5. The amino acid sequence encoded by this murine cDNA is 58% identical to that of the human clone. The nucleotide and encoded amino acid sequences of this murine CD69 cDNA are presented in SEQ ID NO:3 and SEQ ID NO:4. In addition to the membrane-bound full length proteins depicted in SEQ ID NO:2 and SEQ ID NO:4, the present invention provides soluble forms of the CD69 protein. "Soluble CD69" as used in the context of the present invention refers to polypeptides that contain all or part of the extracellular domain of a CD69 protein and that, due to the absence of a transmembrane region that would cause retention of the polypeptide on a cell membrane, are secreted upon expression. Since the CD69 protein lacks a signal peptide, a heterologous signal peptide may be fused to the N-terminus of a soluble CD69 protein to promote secretion thereof, as described in more detail below. The signal peptide is cleaved from the CD69 protein upon secretion from the host cell. Fragments of the extracellular domain may be employed as long as the fragment possesses the desired biological activity (e.g., binding to an anti-CD69 antibody or to the ligand for CD69). Soluble CD69 may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble CD69 protein is capable of being secreted. Preferred soluble CD69 polypeptides include the entire extracellular domain (amino acids 79 to 199 of SEQ ID NO:2 or SEQ ID NO:4). The present invention thus provides DNA sequences encoding soluble CD69 polypeptides. Examples include isolated DNA comprising nucleotides 235-600 of SEQ ID NO:l or SEQ ID NO:3. Soluble CD69 polypeptides may be identified (and distinguished from their non- soluble membrane-bound counterparts) by separating intact cells which express the protein in question from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of CD69. The presence of CD69 in the medium indicates that the protein was secreted from the cells and thus is a soluble form. Soluble CD69 includes naturally-occurring forms of the protein, such as those resulting from alternative splicing events. Alternatively, soluble fragments of CD69 proteins may be produced by recombinant DNA technology or otherwise isolated, as described below.

The use of soluble forms of CD69 is advantageous for certain applications. Purification of the proteins from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. The smaller soluble fragments may be advantageous for use in certain in vitro assays. The soluble CD69 polypeptides may be employed to competitively bind the ligand in vivo, thus inhibiting signal transduction activity via endogenous cell surface bound CD69 proteins. Further, soluble proteins are generally more suitable for intravenous administration and may exert their desired effect (e.g., binding a ligand) in the bloodstream.

Truncated CD69 proteins, including soluble polypeptides, may be prepared by any of a number of conventional techniques. A desired DNA sequence may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein.

In another approach, enzymatic treatment (e.g., using Bal 31 exonuclease) may be employed to delete terminal nucleotides from a DNA fragment to obtain a fragment having a particular desired terminus. Among the commercially available linkers are those that can be ligated to the blunt ends produced by Bal 31 digestion, and which contain restriction endonuclease cleavage site(s). Alternatively, oligonucleotides that reconstruct the N- or C- terminus of a DNA fragment to a desired point may be synthesized. The oligonucleotide may contain a restriction endonuclease cleavage site upstream of the desired coding sequence and position an initiation codon (ATG) at the N-terminus of the coding sequence. The well known polymerase chain reaction procedure also may be employed to amplify a DNA sequence encoding a desired protein fragment. 3' and 5' oligonucleotide primers that anneal to the CD69 DNA at the termini of a desired fragment are employed in the PCR reaction which is conducted using any suitable procedure, such as those described in Saiki et al., Science 239:487 (1988); in Recombinant DNA Methodology, Wu et al., eds., Academic Press Inc., San Diego (1989), pp. 189-196; and in PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).

The present invention also provides antigenic fragments of CD69 that can act as im unogens to generate antibodies specific to the CD69 immunogens. The above- described procedures for producing CD69 fragments may be employed in producing CD69 fragments for use as immunogens.

Expression of Recombinant CD69 Proteins

The present invention provides recombinant expression vectors to express DNA encoding the CD69 proteins of the present invention. The inventive recombinant expression vectors are replicable DNA constructs which contain a synthetic or cDNA- derived DNA sequence encoding an CD69 protein, operably linked to suitable transcriptional or translational regulatory elements. Examples of genetic elements having a regulatory role in gene expression include transcriptional promoters, operators or enhancers, a sequence encoding suitable mRNA ribosomal binding sites, and appropriate transcription and translation initiation and termination sequences. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. The regulatory elements employed in the expression vectors are generally derived from mammalian, microbial, viral, or insect genes. Expression vectors derived from retroviruses also may be employed. . DNA regions are operably linked when they are functionally related to each other.

A DNA sequence encoding CD69 is said to be operably linked to one or more of the above- described regulatory elements when the CD69 DNA sequence is transcribed, or the resulting mRNA is translated, under the control of the regulatory element(s).

Transformed host cells are cells which have been transformed or transfected with foreign DNA using recombinant DNA techniques. In the context of the present invention, the foreign DNA includes a sequence encoding the inventive CD69 protein. Host cells may be transformed for purposes of cloning or amplifying the foreign DNA, or may be transformed with an expression vector for production of the fusion protein under the control of appropriate promoters. Suitable host cells include prokaryotes, yeast, or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual. Elsevier, New York, 1985), the relevant disclosures of which is hereby incorporated by reference. Cell-free translation systems could also be employed to produce fusion protein using RNAs derived from the DNA constructs of the present invention.

Prokaryotes include gram negative or gram positive organisms. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. Examples of suitable prokaryotic hosts for transformation include E. coli, bacilli such as Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (Bolivar et al., Gene 2:95, 1977; ATCC 37017). Such commercial vectors include, for example, pKK223-3

(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, providing simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4051, 1980; and EPA 36,776) and tac promoter

(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful bacterial expression system employs the phage λ P promoter and cI857ts thermoinducible repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

The recombinant CD69 protein may also be expressed in yeast hosts, preferably from Saccharomyces species, such as S. cerevisiae. Yeast of other genera such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2μm yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the CD69 protein, sequences for polyadenylation and transcription termination and a selection gene. Yeast vectors may include origins of replication and selectable markers permitting transformation of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and the S. cerevisiae trpl gene. The trp 1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2013, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 77:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Examples of suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli (Amp^r gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and α-factor secretion leader. The ADH2 promoter has been described by Russell et al. (_/. Biol. Chem.258:2614, 1982) and Beier et al., (Nature 300:124, 1982). Advantageously, a DNA segment encoding a leader sequence functional in yeast is operably linked to the 5' end of the DNA encoding the CD69 protein. The encoded leader peptide promotes secretion of the CD69 protein from the host cell and is generally cleaved from the CD69 protein upon secretion. As one example, the yeast α-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30:922, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Patent 4,546,082; and EP 324,274. The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill in the art. An exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, (1978), selecting for Trp⁺ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, lOμg/ml adenine and 20 μg/ml uracil. Host strains transformed by vectors comprising the above-described ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4°C prior to further purification.

Various mammalian or insect cell culture systems can be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651), described by Gluzman (Cell 23:115, 1981), CV-1 cells (ATCC CCL 70) also derived from monkey kidney, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a poly-adenylation site, splice donor and acceptor sites, and transcriptional termination sequences. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the S V40 viral origin or replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BgR site located in the viral origin of replication is included. Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol.5:280, 1983). A useful system for stable high level expression of mammalian receptor cDNAs in C 127 murine mammary epithelial cells can be constructed substantially as described by Cos an et al. (Mol. Immunol. 23:935, 1986). Other expression vectors for use in mammalian host cells are derived from retroviruses.

Producing and Purifying the CD69 Protein

Substantially homogeneous CD69 protein may be produced by recombinant expression systems as described above or purified from naturally occurring cells. The CD69 protein is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A process for producing the recombinant CD69 protein of the present invention comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes said CD69 protein under conditions that promote expression of the CD69 protein, which is then purified from culture media or cell extracts. Any suitable purification process may be employed, with the procedure of choice varying according to such factors as the type of host cells and whether or not the desired protein is secreted from the host cells. The fusion protein will be secreted into the culture medium when it is initially fused to a signal sequence or leader peptide operative in the host cells, or when the protein comprises soluble forms of the CD69 polypeptides. For example, supematants from expression systems which secrete recombinant protein into the culture medium can be first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, an immunoaffinity column comprising antibodies directed against CD69 and bound to a suitable support may be employed. Monoclonal antibodies that bind human or murine CD69 are described in examples 1 and 5, respectively. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. One or more reversed-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify CD69. Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant fusion proteins can disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express CD69 as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:\1\ , 1984), involving two sequential, reversed-phase HPLC steps for purification of a recombinant protein on a preparative HPLC column. Some or all of the foregoing purification steps, in various combinations, can be employed to provide an essentially homogeneous recombinant protein. Recombinant cell culture enables the production of the CD69 protein free of those contaminating proteins which may be normally associated with CD69 as it is found in nature, e.g., in cells, cell exudates or body fluids. The foregoing purification procedures are among those that may be employed to purify non-recombinant CD69 proteins of the present invention as well.

Variants and Derivatives of CD69

Variants and derivatives of native CD69 proteins that retain the desired biological activity are also within the scope of the present invention. Such variants and derivatives are considered to be equivalents of the CD69 proteins presented in SEQ ID NOS:2 and 4. One method of verifying that a CD69 protein, including variants and derivatives thereof, is biologically active is by using a fetal thymus culture development assay. One biological activity of CD69 is inhibiting differentiation of early thymocytes in culture, e.g., in fetal thymus organ cultures.

A CD69 variant, as referred to herein, is a polypeptide substantially homologous to a native CD69, but which has an amino acid sequence different from that of native CD69 (human, murine or other mammalian species) because of one or a plurality of deletions, insertions or substitutions. The variant amino acid sequence preferably is at least 80% identical to a native CD69 amino acid sequence, most preferably at least 90% identical. The degree of homology (percent identity) may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. For fragments derived from the CD69 protein of SEQ ID NOS:2 or 4, the homology is calculated based on that portion of the CD69 protein that is present in the fragment. Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein.

Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.

CD69 also may be modified to create CD69 derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of CD69 may be prepared by linking the chemical moieties to functional groups on CD69 amino acid side chains or at the N-terminus or C-terminus of an CD69 polypeptide or the extracellular domain thereof. Other derivatives of CD69 within the scope of this invention include covalent or aggregative conjugates of CD69 or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions.

When initially synthesized, CD69 may comprise a heterologous signal or leader polypeptide sequence at the N-terminus. A leader peptide useful in yeast expression systems is the α-factor leader of Saccharomyces (described above). Examples of signal peptides useful in mammalian expression systems are the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleukin-2 receptor described in Cos an et al., Nature 372:768 (1984); the interleukin-4 signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin- 1 receptor signal peptide described in EP 460,846. Each of these references describing signal peptides is hereby incorporated by reference. The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site outside of the cell membrane or cell wall, and is cleaved from the CD69 protein. CD69-containing fusion proteins can comprise peptides added to facilitate purification or identification of CD69. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988, hereby incorporated by reference. One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK), which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli. A murine hybridoma designated 4Ε11 produces a monoclonal antibody that binds the peptide DYKDDDDK in the presence of certain divalent metal cations (as described in U.S. Patent 5,011 ,912) and has been deposited with the American Type Culture Collection under accession no HB 9259. The present invention further includes CD69 polypeptides with or without associated native-pattern glycosylation. CD69 expressed in yeast or mammalian expression systems (e.g., COS-7 cells) may be similar to or significantly different from a native CD69 polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of CD69 polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. In one embodiment of the present invention, N-glycosylation sites in the CD69 protein are modified to preclude glycosylation. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Carbohydrate residues attach to the Asn side chain. N-glycosylation sites are found at amino acids 11-13 and 166-168 of SEQ ID NO:2, and at amino acids 150-152, 166-168, and 180-182 of SEQ ID NO:4. Appropriate modification of the nucleotide sequence (to introduce substitutions, additions or deletions such that this triplet is no longer encoded) inactivates the N-glycosylation site. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Known procedures for inactivating N- glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846.

As discussed above, homodimeric forms of CD69 are believed to comprise two polypeptide chains that are identical in amino acid sequence but differ in their glycosylation patterns. The isolated CD69 cDNA of the present invention provides a means for studying this differential glycosylation. Homodimers comprising non-glycosylated CD69 (produced in bacteria or comprising inactivated N-glycosylation sites) may be compared with homodimers comprising differentially glycosylated chains. The ability to produce non- glycosylated CD69 also affords the opportunity to purify a more homogeneous product.

Other variants within the scope of the present invention include sequences in which Cys residues that are not essential for biological activity are altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. The resulting polypeptide will not dimerize and also finds use as a research reagent, e.g., in comparing the biological activities of the monomer and the dimer.

Other variants are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding, or substituting residues to alter Arg- Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. The resulting muteins are less susceptible to cleavage by the KEX2 protease at locations other than the yeast α-factor leader sequence, where cleavage upon secretion is intended. KEX2 p^* ^ease processing sites are found at amino acids 94-95, 103-104, and 133-134 of SEQ ID K J:2 and at amino acids 93-94 and 133-134 of SEQ ID NO:4.

Naturally occurring CD69 variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternative mRNA splicing events or from proteolytic cleavage of the CD69 protein, provided the desired biological activity (e.g., binding to an anti-CD69 antibody or to the ligand) is retained. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids (which may occur intracellularly or during purification). The N- terminal amino acid may, for example, be any of the amino acids at positions 1 to 5 of SEQ ID NO:2 or SEQ ID NO:4. The C-terminus may be truncated deliberately during expression vector construction (e.g., in constructing vectors encoding soluble proteins as described above) or as a result of differential processing which may remove up to about five C-terminal amino acids, for example. In certain host cells, post-translational processing will remove the methionine residue encoded by an initiation codon, whereas the methionine residue will remain at the N-terminus of proteins produced in other host cells. Due to the known degeneracy of the genetic code wherein more than one codon can encode the same amino acid, a DNA sequence may vary from that presented in SEQ ID NO: 1 or SEQ ID NO:3, and still encode a CD69 protein having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, respectively. Such variant DNA sequences may result from silent mutations (e.g., occurring during PCR amplification), and may be the product of deliberate mutagenesis of a native sequence.

Nucleic acid sequences within the scope of the present invention include isolated DNA and RNA sequences that hybridize to a native mammalian CD69 nucleotide sequence under conditions of moderate or severe stringency, and which encode biologically active CD69. Moderate stringency hybridization conditions refer to conditions described in, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989). Conditions of moderate stringency, as defined by Sambrook et al., include use of a prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55°C, 5 X SSC, overnight. Conditions of severe stringency include higher temperatures of hybridization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the probe.

The present invention thus provides isolated DNA sequences encoding biologically active CD69, selected from: (a) DNA comprising the human CD69 DNA sequence presented in SEQ ID NO: 1 or the murine CD69 DNA sequence of SEQ ID NO:3; (b) DNA capable of hybridizing under moderately stringent conditions to a DNA of (a) and which encodes biologically active CD69; and (c) DNA which is degenerate as a result of the genetic code to a DNA defined in (a) or (b) and which encodes biologically active CD69. CD69 proteins encoded by these isolated DNA sequences are encompassed by the present invention.

CD69 polypeptides in the form of oligomers are within the scope of the present invention. In one embodiment of the invention, a CD69 dimer is created by fusing CD69 to the Fc region of an antibody (IgGl ). The Fc polypeptide preferably is fused to the N- terminus of a soluble CD69 (comprising only the extracellular domain). A gene fusion encoding the CD69 fusion protein is inserted into an appropriate expression vector. The CD69 fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding divalent CD69. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a CD69 oligomer with as many as four CD69 extracellular regions. Alternatively, one can link two soluble CD69 domains with a peptide linker such as the Gly4SerGly5Ser linker sequence described in United States Patent 5,073,627. A fusion protein comprising two or more CD69 polypeptides (with or without peptide spacers) may be produced by recombinant DNA technology. Even though the CD69 protein is capable of forming homodimers without the Fc polypeptide, certain advantages are afforded by fusion of the Fc polypeptide to CD69.

Additional purification methods may be employed, e.g., affinity chromatography using

Protein A columns that bind the Fc portion of the fusion protein. When the CD69/Fc fusion proteins are employed in assay and screening procedures, reagents reactive with the

Fc portion, e.g., commercially available antibodies directed against human antibodies, may be used in the procedures.

In spite of these advantages, CD69 preferably is expressed without an Fc polypeptide fused thereto. Dimerization is achieved without the use of Fc moieties, as illustrated in example 2. Any possible disadvantage of using an Fc moiety (e.g., undesirable binding to Fc receptors in vivo or any possible adverse effect on biological activity of CD69) thus may be avoided.

The present invention further provides fragments of the CD69 nucleotide sequences presented herein. Such fragments desirably comprise at least about 14 nucleotides of the sequence presented in SEQ ID NOS:l or 3. DNA and RNA complements of said fragments are provided herein, along with both single- stranded and double-stranded forms of the CD69 DNA.

Among the uses of such CD69 nucleic acid fragments is use as a probe. Such probes may be employed in cross-species hybridization procedures to isolate CD69 DNA from additional mammalian species, as illustrated in example 5. The probes also find use in detecting the presence of CD69 nucleic acids in in vitro assays and in such procedures as

Northern and Southern blots. Cell types expressing CD69 can be identified, e.g., as described in example 2.

Other useful fragments of the CD69 nucleic acids are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target CD69 mRNA (sense) or CD69 DNA (antisense) sequences.

Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of CD69 cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given protein, is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block translation (RNA) or transcription (DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of CD69 proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629), wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties such as those described in WO 90/10448 or other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4- mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656). Alternatively, other promotor sequences may be used to express the oligonucleotide.

Sense or antisense oligonucleotides may also be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.

Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. Uses of CD69 Proteins

The CD69 protein of the present invention is useful for blocking thymocyte development in in vitro systems. CD69 may be used to enrich for early (immature) thymocytes in cell culture, for example. Without CD69, the early thymocytes would differentiate as they are carried in culture. CD69 thus finds use as a tissue culture reagent that aids investigators in studies of early thymocytes.

One example of this use of CD69 involves adding a soluble CD69 polypeptide to a fetal thymic organ culture. Such cultures are described in Ritter and Larche, Current Opinion in Immunology 1 :203, 1988, and F. Ramsdell, "Fetal thymus organ culture for T cell development studies", in Current Protocols in Immunology, J.E. Coligan et al., eds, Wiley and Sons, New York, 1991, p.3.18.1-3.18.10. A preferred concentration is 50-100 μg CD69 per ml of culture supernatant.

Another use of CD69 is as a research tool for investigating the existance of a ligand that binds thereto and studying the biological effects of ligand binding. The CD69 polypeptides of the present invention also may be employed in in vitro assays for detection of CD69 or its ligand or the interactions thereof.

CD69 also finds use in studies of the mechanisms of cellular activation. The role of CD69 in regulation and function of the immune system also can be investigated, as discussed above.

The CD69 polypeptides of the present invention can be used in a binding assay to detect cells expressing a ligand for CD69. For example, CD69 or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as ¹²⁵I. Radiolabeling with ¹²⁵I can be performed by any of several standard methodologies that yield a functional ¹²⁵I CD69 molecule labeled to high specific activity. Alternatively, another detectable moiety such as an enzyme that can catalyze a colorometric or fluorometric reaction, biotin or avidin may be used. Cells to be tested for CD69 ligand expression can be contacted with the labeled CD69. After incubation, unbound labeled CD69 is removed and binding is measured using the detectable moiety. Soluble CD69 polypeptides may be employed to competitively bind the ligand in vivo, thus inhibiting signal transduction activity via endogenous cell surface bound CD69 proteins. Further, soluble proteins are generally more suitable for intravenous administration and may exert their desired effect (e.g. binding a ligand) in the bloodstream.

EXAMPLE 1 : Cloning of Human CD69 DNA

Levels of CD69 expression were analyzed on a variety of cell lines by FACS analysis using a monoclonal antibody designated TP1/55.3.1 (available from AMAC Inc., Westbrook, ME), which is directed against human CD69. An allo-reactive CD4⁺ human T cell clone (clone 14), stimulated with anti-CD3 antibodies for about 90 minutes, displayed the highest levels of CD69. A cDNA library was constructed in the mammalian expression vector pDC302 using RNA isolated from the αCD3-stimulated clone 14 cells. The cDNA was cloned into the Bglll site of the mammalian expression vector pDC302 by an adaptor method similar to that described by Haymerle et al. (Nucl. Acids Res. 14:8615, 1986). The pDC302 expression vector employed in preparing this cDNA library has been described by Mosley et al. (Cell 59:335, 1989). pDC302 is an expression vector for use in mammalian host cells, but also replicates in E. coli. pDC302 was assembled from pDC201 (Sims et al., Science 241 :585, 1988), SV40 and cytomegalovirus DNA and comprises a multiple cloning site (MCS) containing sites for Xhol, Asp718, Smal, NotI and Bglll.

Plasmid DNA generated from pools of 2500 clones from the clone 14-derived library was transfected into CV-1 EBNA-1 cells (McMahan et al., EMBOJ. 70:2821, 1991). Transfected cells were screened for CD69 expression using the above-described anti-CD69 antibody and a slide autoradiography technique, essentially as described by Gearing et al. (EMBO J. 8:3661, 1989). Briefly, transfectants were plated in chambered slides (Lab-Tek) and cultured for 48-72 hours to permit protein expression. The cells were then washed with binding buffer (RPMI 1640 medium containing 10% BSA, 20mM

HEPES, pH 7.2, 2 mg/ml sodium azide, and 10% freeze-dried milk) and incubated with the TP1/55.3.1 antibody in binding buffer. The slides were then treated with an -^'~-\- labelled goat-αmouse IgG F(ab')2 (New England Nuclear), also in binding buffer. After washing to remove unbound antibody, the cells were fixed by incubating for 30 minutes at room temperature in 10% glutaraldehyde in PBS, pH 7.3, washed twice in PBS, and air dried. The slides were dipped in Kodak NTB-2 photographic emulsion (5x dilution in water) and exposed in the dark for 3 days in a light proof box. The slides were then developed in Kodak D19 developer (40 g/500 ml water), rinsed in water and fixed in Agfa G433C fixer. The slides were individually examined with a microscope at 25-40x magnification, and positive cells expressing CD69 were identified by the presence of autoradiographic silver grains against a light background.

Five pools yielded positive cells. A single clone was isolated from one pool, #33. Plasmid DNA was isolated from clone #33, and the cDNA inseπ was determined to be about 950 bp in length. The entire cDNA insert was excised and cloned into pBLUESCRIPT® SK (Stratagene Cloning Systems, San Diego). The resulting recombinant vector containing human CD69 cDNA was transfected into E. coli DH5α host cells and deposited with the American Type Culture Collection on October 23, 1992, under Accession No. 69100.

A second human CD69 clone was isolated by screening a different cDNA library using the cDNA insert of clone #33 as a probe. The cDNA library was derived from RNA extracted from peripheral blood T lymphocytes (purified by E-rosetting) which had been activated for 18 h with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA). The cDNA was packaged into λgtlO. The library was screened using standard hybridization techniques. One hybridizing clone, HCD69-1 containing a 1.7 Kb insert, was chosen for further analysis.

The cDNA inserts of clones #33 and HCD69-11 were isolated for sequencing. The clones were found to encompass the same coding region, with additional non-coding sequences being present in clone HCD69-11. The DNA sequence of the human CD69 coding region is presented in SEQ ID NO: 1, and the encoded amino acid sequence is presented in SEQ ID NO:l and SEQ ID NO:2.

The human CD69 protein of SEQ ID NO:2 comprises an N-terminal intracellular (signal-transducing) domain (amino acids 1 -34), followed by a transmembrane region comprising amino acids 35-78, and a C-terminal extracellular (ligand-binding) domain comprising amino acids 79-199.

EXAMPLE 2: Characterization of CD69 Gene Product To examine the protein encoded by clone #33, plasmid DNA was isolated from clone #33 and transfected into COS-7 cells (monkey kidney cell line, ATCC CRL 1651 ) using a standard DEAE-dextran technique. Two days later the cells were metabolically labelled with ³⁵S-Met/Cys by standard techniques, then detergent solubilized. The ³⁵S- labeled lysates were incubated with the above-described anti-CD69 antibody TP1/55.3.1. The immuno-precipitated protein was analyzed by electrophoresis on a reducing SDS/polyacrylamide gel to visualize monomers rather than disulfide-linked dimers. Two protein bands corresponding to molecular weights of 28 Kd and 32 Kd were visible on the gel. This result supports the supposition that CD69 consists of a homodimer of differentially-processed proteins, as discussed above. A protein band corresponding to the molecular weight expected for this homodimer was visualized on an SDS-PAGE (non- reducing) gel.

To examine the expression of the human CD69 gene, RNA was isolated from peripheral blood T cells (PBT) unstimulated or stimulated for three hours with PMA or PHA. The clone #33 cDNA insert hybridized to a single 1.7 Kb mRNA present in RNA from the stimulated PBT populations, but not found in RNA from unstimulated PBTs.

EXAMPLE 3: Expression Vector Encoding Soluble CD69/

Peptide Linker/Fc Fusion Protein

An expression vector encoding a soluble human CD69 protein is constructed as follows. The CD69 is initially expressed as a fusion protein comprising a heterologous N- terminal leader peptide (the interleukin-7 leader peptide), followed by a peptide of the sequence Asp-Tyr-Lys-(Asp)4-Lys to facilitate purification, followed by a polypeptide derived from the Fc region of a human IgGl antibody, followed by a Gly3SerGly3Ser peptide linker, followed by the entire extracellular domain (amino acids 79-199) of human CD69.

A DNA fragment comprising the extracellular domain of human CD69 is produced and amplified using the well known polymerase chain reaction (PCR) procedure. The 5' primer employed in the PCR reaction is a single-stranded oligonucleotide comprising a sequence identical to the 5' end (preferably about 15-20 nucleotides) of the CD69 extracellular domain. This primer additionally comprises a BspEl restriction site so that the amplified fragments comprise a BspEl site upstream of the CD69 DNA. The 3' primer is a single-stranded oligonucleotide comprising a sequence complementary to the 3' end (preferably about 15-20 nucleotides) of the extracellular domain. The 3' primer comprises an additional sequence that inserts a Not/ restriction site downstream of the CD69 sequence in the amplified fragments.

PCR is conducted according to conventional procedures, using the recombinant plasmid of CD30-L clone #33 as the template. An example of a suitable PCR procedure is as follows.^" All temperatures are in degrees centigrade. The following PCR reagents are added to a 0.5 ml Eppendorf microfuge tube: 10 μl of 10X PCR buffer (500 mM KC1, 100 mM Tris-HCl, pH 8.3 at 25°C, 25 mM MgCtø, and 1 mg/ml gelatin) (Perkin-Elmer Cetus, Νorwalk, CΝ), 8μl of a 2.5 mM solution containing each dΝTP (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 2 mM dTTP), 2.5 units (0.5 μl of standard 5000 units/ml solution) of Taq DΝA polymerase (Perkins-Elmer Cetus), 1 ng of template DΝA, 100 picomoles of each of the oligonucleotide primers, and water to a final volume of 100 μl. The final mixture is then overlaid with 100 μl parafin oil. PCR is carried out using a DΝA thermal cycler (Ericomp, San Diego, CA). The template is denatured at 94° for 5 minutes and PCR is carried out for 25 cycles of amplification using a step program (denaturation at 94°, 1.5 minutes; annealing at 60°, 1 minute; extension at 72°, 1 minute).

The amplified DΝA is recovered by phenolchloroform extraction, purified by spin column chromatography (e.g., using a G-50 column from Boehringer Mannheim), and digested with BspEl and NotI. The desired fragment is separated on, and recovered from, a low gelling temperature agarose gel. The purified fragment is inserted into the mammalian expression vector HAV-EO described by Dower et al. (J. Immunol. 742:4314, 1989).

Additional peptide-encoding sequences may be inserted into the expression vector such that a fusion protein comprising the following components (from Ν- to C- terminus) is encoded: interleukin-7 (IL-7) signal peptide/FLAG® peptide/Fc polypeptide/ Gly3SerGly3Ser/CD69. The IL-7 signal peptide promotes secretion of the fusion protein from the host cell, and is described in U. S. Patent 4,965,195. The FLAG® peptide facilitates purification and provides other advantages described above and in U. S. Patent 5,011,912. Bovine mucosal enterokinase cleaves this peptide at the residue immediately following the Asp-Lys pairing (i.e., cleaves the FLAG® peptide from the Fc polypeptide). The Fc polypeptide is derived from the Fc domain of a human IgGl antibody. The Fc polypeptide comprises at least the hinge region, and may extend from the hinge region to the C-terminus of the heavy chain. Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88: 10535, 1991) and Byrn et al. (Nature 344:611, 1990). Interchain disulfide bonds form between two Fc polypeptides (as occurs naturally in antibodies), thus creating a dimeric form of the fusion protein.

The Gly3SerGly3Ser octapeptide is a peptide linker (spacer) encoded by an oligonucleotide synthesized by conventional procedures. Other peptide linkers may be substituted, e.g., those described in U.S. Patent 5,073,627.

The HAV-EO expression vector comprising the above-identified gene fusion is transfected into CVl-EBNA cells by standard procedures. Alternatively, the transformed primary human embryonal kidney cell line known as 293 (ATCC CRL 1573) may be employed as host cells. The transfected host cells are cultured to express the fusion protein. The signal sequence is cleaved upon secretion of the remainder of the fusion protein. After purification of the fusion protein using a monoclonal antibody reactive with the FLAG® peptide (see U. S. Patent 5,011 ,912), the FLAG® peptide may be removed from the fusion protein by treatment with bovine mucosal enterokinase.

The purified fusion protein thus is a soluble protein comprising an Fc polypeptide joined to the human CD69 extracellular domain via the Gly3SerGly3Ser peptide linker. Dimers comprising two of these fusion proteins result from the disulfide bonds that form between the Fc portions of two fusion protein chains. Additional disulfide bonds may form between the CD69 components of two such fusion proteins.

EXAMPLE 4: Expression Vectors Encoding Soluble CD69 Protein a) Mammalian Expression Vector The Fc moiety described in example 3 is not required for dimerization of CD69, and

CD69 preferably is expressed without an Fc polypeptide fused thereto. An expression vector for use in expressing a soluble human CD69 protein in mammalian cells is constructed as follows.

A DNA fragment encoding the extracellular domain of human CD69 is isolated and amplified as described in example 3. The DNA fragment is inserted into a suitable expression vector such as HAV-EO (Dower et al., J. Immunol. 142:4314, 1989). DNA encoding the FLAG® peptide described in example 3 is fused to the 5' end of the CD69 DNA. Murine IL-7 signal peptide-encoding DNA (see U.S. Patent 4,965,195) is fused to the 5' end of the FLAG® peptide DNA.

Suitable mammalian cells such as CVl-EBNA-1 cells (McMahan et al., EMBO J. 10:2821, 1991) are transformed with the resulting recombinant expression vector and cultivated to express the soluble fusion protein. The signal peptide is cleaved during secretion of the fusion protein from the cell. The secreted FLAG®/soluble CD69 fusion protein is purified from the culture supernatant using a monoclonal antibody that binds the FLAG® peptide, and the FLAG® peptide is then cleaved from the soluble CD69 protein as described in example 3. b) Yeast Expression Vector

An expression vector useful for expressing soluble CD69 in yeast cells is constructed as follows. A DNA fragment encoding the extracellular domain of human CD69 is isolated and amplified by PCR as described in example 3. The DNA fragment is inserted into an expression vector suitable for use in yeast host cells. One such vector is similar to pIXY321, described in U.S. Patent 5,073,627

(hereby incorporated by reference). The pIXY321 vector comprises the yeast alcohol dehydrogenase II (ADH2) promoter, an Fl origin of replication and an origin of replication derived from pBR322, both functional in E. coli, and a 2u origin of replication functional in yeast. Selective markers include an ampicillin resistance gene (for selection in E. coli) and Trpl for selection in yeast. The yeast α-factor leader (pre pro) peptide fused to a GM- CSF/IL-3 fusion protein in pIXY321 is replaced by the signal (pre) peptide of α factor fused to the CD69-encoding DNA fragment isolated by PCR above. The yeast α-factor signal peptide is described in Waters et al. (7. Biol. Chem. 263:6209, 1988; see figure 2C; hereby incorporated by reference). Saccharomyces cerevisiae cells are transformed with the recombinant vector by standard techniques and cultivated to express the soluble CD69 protein. The CD69 is secreted into the culture medium.

EXAMPLE 5: Isolation of DNA Encoding Murine CD69 A cDNA library was prepared from the murine helper T-cell line designated 7B9

(Mosley et al., Cell 59:335, 1989), which was stimulated for 6 hours with 3 μg/ml Con A. 7B9 is a sheep red blood cell-specific helper T-cell line (TH0) derived by limiting dilution from primary antigen-induced cultures of murine C57BL/6 spleen cells.

7B9 was chosen as the nucleic acid source for this cloning attempt because the monoclonal antibody H1.2F3 (described by Yokoyama et al., 7. Immunol. 141:369, 1988) bound to an antigen on the surface of 7B9 cells. H1.2F3 was derived using a murine epidermal T-cell line as the immunogen. Yokoyama et al. noted that the antigen recognized by H1.2F3 had a number of properties in common with human CD69 (also known as EA-1), but stated that certain other evidence suggests that H1.2F3 does not recognize the murine homolog of EA-1. Certain functional and biochemical studies were said to suggest that the antigen recognized by H1.2F3 is the murine homolog of the human antigen known as CD28 (Yamasaki et al, supra, see abstract). Neveπheless, a cDNA library derived from stimulated 7B9 cells, in vector λgtlO, was employed in the cloning procedure of this example.

The cDNA library was screened using the cDNA insert of human CD69 clone #33 (see example 1) as the probe. One of the hybridizing clones, designated λM69-6, contained a 1.6 kbp insert and was chosen for further analysis. The DNA sequence was determined and is presented in SEQ ID NO:3. The amino acid sequence encoded by this cDNA is presented in SEQ ID NO:4 and is 58% identical to the human CD69 amino acid sequence in SEQ ID NO:2, with the homology extending the entire length of the proteins. The protein encoded by λM69-6 would also be predicted to be a type II membrane glycoprotein. The protein contains a cytoplasmic domain (amino acids 1-34), a transmembrane region (amino acids 35-78), and an extracellular domain (amino acids 79- 199 of SEQ ID NO:4).

EXAMPLE 6: Characterization of Murine CD69 To examine the expression of mouse CD69, RNA from a variety of murine cell lines was analyzed using the cDNA insert of clone M69-6 as a probe. The T cell lymphoma EL-40.5 (Armitage et al., Nature 357:80, 1992), the pro-B cell line HAFTL-1 (Davidson et al., 7. Exp. Med. 168:389, 1988), the IL-3-dependent myeloid leukemias NFS-60 and NFS-58 (Ihle et al., in G. Klein (Ed.) Advances in Viral Oncology, Raven Press, p. 95, 1984), and the myeloma MPC-1 all expressed a 1.7-kb mRNA that hybridized with the M69-6 probe.

To determine whether the protein encoded by the M69-6 clone contained the epitope against which MAb H1.2F3 is directed, CV-1/EBNA cells were transiently transfected with M69-6 cDNA in pDC302 (an expression vector described in example 1), or with pDC302 alone, and stained with the H1.2F3 antibody. Only those cells transfected with the pDC302/M69-6 construct showed staining with the H1.2F3 antibody. Also, H1.2F3 was able to specifically immunoprecipitate a doublet of bands of 30 kDa and 34 kDa from pDC302/M69-6 transfected CV-1/EBNA cells. SEQUENCE LISTING

(1) GENERAL INFORMATION:

( ) APPLICANT: Ziegler, Steven F.

H^errild, Kathryn A.

(ii) TITLE OF INVENTION: Activation Antigen CD69

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Immunex Corporation

(B) STREET: 51 University Street

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: USA

(F) ZIP: 98101

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Seese, Kathryn A.

(B) REGISTRATION NUMBER: 32,172

(C) REFERENCE/DOCKET NUMBER: 2610-WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 587-0430

(B) TELEFAX: (206) 233-0644

(2) INFORMATION FOR SEQ ID NO: 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 600 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..600 (ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 1..597

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

ATG AGC TCT GAA AAT TGT TTC GTA GCA GAG AAC AGC TCT TTG CAT CCG 48 Met Ser Ser Glu Asn Cys Phe Val Ala Glu Asn Ser Ser Leu His Pro 1 5 10 15

GAG AGT GGA CAA GAA AAT GAT GCC ACC AGT CCC CAT TTC TCA ACA CGT 96 Glu Ser Gly Gin Glu Asn Asp Ala Thr Ser Pro His Phe Ser Thr Arg 20 25 30

CAT GAA GGG TCC TTC CAA GTT CCT GTC CTG TGT GCT GTA ATG AAT GTG 144 His Glu Gly Ser Phe Gin Val Pro Val Leu Cys Ala Val Met Asn Val 35 40 45

GTC TTC ATC ACC ATT TTA ATC ATA GCT CTC ATT GCC TTA TCA GTG GGC 192 Val Phe He Thr He Leu He He Ala Leu He Ala Leu Ser Val Gly 50 55 60

CAA TAC AAT TGT CCA GGC CAA TAC ACA TTC TCA ATG CCA TCA GAC AGC 240 Gin Tyr Asn Cys Pro Gly Gin Tyr Thr Phe Ser Met Pro Ser Asp Ser 65 70 75 80

CAT GTT TCT TCA TGC TCT GAG GAC TGG GTT GGC TAC CAG AGG AAA TGC 288 His Val Ser Ser Cys Ser Glu Asp Trp Val Gly Tyr Gin Arg Lys Cys 85 90 95

TAC TTT ATT TCT ACT GTG AAG AGG AGC TGG ACT TCA GCC CAA AAT GCT 336 Tyr Phe He Ser Thr Val Lys Arg Ser Trp Thr Ser Ala Gin Asn Ala 100 105 110

TGT TCT GAA CAT GGT GCT ACT CTT GCT GTC ATT GAT TCT GAA AAG GAC 384 Cys Ser Glu His Gly Ala Thr Leu Ala Val He Asp Ser Glu Lys Asp 115 120 125

ATG AAC TTT CTA AAA CGA TAC GCA GGT AGA GAG GAA CAC TGG GTT GGA 432 Met Asn Phe Leu Lys Arg Tyr Ala Gly Arg Glu Glu His Trp Val Gly 130 135 140

CTG AAA AAG GAA CCT GGT CAC CCA TGG AAG TGG TCA AAT GGC AAA GAA 480 Leu Lys Lys Glu Pro Gly His Pro Trp Lys Trp Ser Asn Gly Lys Glu 145 150 155 160

TTT AAC AAC TGG TTC AAC GTT ACA GGG TCT GAC AAG TGT GTT TTT CTG 528 Phe Asn Asn Trp Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu 165 170 175

AAA AAC ACA GAG GTC AGC AGC ATG GAA TGT GAG AAG AAT TTA TAC TGG 576 Lys Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp 180 185 190

ATA TGT AAC AAA CCT TAC AAA TAA 600

He Cys Asn Lys Pro Tyr Lys

195 200 (2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 199 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Ser Glu Asn Cys Phe Val Ala Glu Asn Ser Ser Leu His Pro

1 5 10 15

Glu Ser Gly Gin Glu Asn Asp Ala Thr Ser Pro His Phe Ser Thr Arg 20 25 30

His Glu Gly Ser Phe Gin Val Pro Val Leu Cys Ala Val Met Asn Val

35 40 45

Val Phe He Thr He Leu He He Ala Leu He Ala Leu Ser Val Gly 50 55 60

Gin Tyr Asn Cys Pro Gly Gin Tyr Thr Phe Ser Met Pro Ser Asp Ser 65 70 75 80

His Val Ser Ser Cys Ser Glu Asp Trp Val Gly Tyr Gin Arg Lys Cys 85 90 95

Tyr Phe He Ser Thr Val Lys Arg Ser Trp Thr Ser Ala Gin Asn Ala 100 105 110

Cys Ser Glu His Gly Ala Thr Leu Ala Val He Asp Ser Glu Lys Asp 115 120 125

Met Asn Phe Leu Lys Arg Tyr Ala Gly Arg Glu Glu His Trp Val Gly 130 135 140

Leu Lys Lys Glu Pro Gly His Pro Trp Lys Trp Ser Asn Gly Lys Glu 145 150 155 160

Phe Asn Asn Trp Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu 165 ' 170 175

Lys Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp 180 185 190

He Cys Asn Lys Pro Tyr Lys 195

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 600 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 1..597

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..600

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATG GAT TCT GAA AAC TGT TCT ATA ACG GAA AAT AGC TCT TCA CAT CTG 48 Met Asp Ser Glu Asn Cys Ser He Thr Glu Asn Ser Ser Ser His Leu 1 5 10 15

GAG AGA GGG CAG AAG GAC CAT GGC ACC AGT ATA CAT TTT GAG AAG CAT 96 Glu Arg Gly Gin Lys Asp His Gly Thr Ser He His Phe Glu Lys His 20 25 30

CAT GAA GGA TCC ATT CAA GTT TCT ATC CCT TGG GCT GTG TTA ATA GTG 144 His Glu Gly Ser He Gin Val Ser He Pro Trp Ala Val Leu He Val 35 40 45

GTC CTC ATC ACG TCC TTA ATA ATA GCT CTC ATT GCC TTA AAT GTG GGC 192 Val Leu He Thr Ser Leu He He Ala Leu He Ala Leu Asn Val Gly 50 55 60

AAG TAC AAT TGC CCA GGC TTG TAC GAG AAG TTG GAA TCA TCT GAC CAC 240 Lys Tyr Asn Cys Pro Gly Leu Tyr Glu Lys Leu Glu Ser Ser Asp His 65 70 75 80

CAT GTT GCT ACC TGC AAG AAT GAG TGG ATT TCA TAC AAG AGG ACA TGT 288 His Val Ala Thr Cys Lys Asn Glu Trp He Ser Tyr Lys Arg Thr Cys 85 90 95

TAC TTC TTC TCC ACC ACA ACC AAG AGT TGG GCC TTG GCC CAA CGC TCT 336 Tyr Phe Phe Ser Thr Thr Thr Lys Ser Trp Ala Leu Ala Gin Arg Ser 100 105 110

TGT TCT GAA GAT GCT GCT ACT CTT GCT GTA ATT GAT TCA GAA AAG GAC 384 Cys Ser Glu Asp Ala Ala Thr Leu Ala Val He Asp Ser Glu Lys Asp 115 120 125

ATG ACG TTT CTG AAG CGA TAT TCT GGT GAA CTG GAA CAT TGG ATT GGG 432 Met Thr Phe Leu Lys Arg Tyr Ser Gly Glu Leu Glu His Trp He Gly 130 135 140

CTG AAA AAT GAA GCT AAT CAG ACA TGG AAA TGG GCA AAT GGC AAA GAA 480 Leu Lys Asn Glu Ala Asn Gin Thr Trp Lys Trp Ala Asn Gly Lys Glu 145 150 155 160

TTT AAC AGC TGG TTC AAC TTG ACG GGG TCT GGG AGG TGC GTG TCC GTG 528 Phe Asn Ser Trp Phe Asn Leu Thr Gly Ser Gly Arg Cys Val Ser Val 165 170 175 AAC CAC AAA AAT GTT ACC GCT GTG GAC TGT GAG GCA AAC TTC CAC TGG 576 Asn His Lys Asn Val Thr Ala Val Asp Cys Glu Ala Asn Phe His Trp 180 185 190

GTC TGC AGC AAG CCC TCC AGA TGA 600

Val Cys Ser Lys Pro Ser Arg

195 200

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 199 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Asp Ser Glu Asn Cys Ser He Thr Glu Asn Ser Ser Ser His Leu 1 5 10 15

Glu Arg Gly Gin Lys Asp His Gly Thr Ser He His Phe Glu Lys His 20 25 30

His Glu Gly Ser He Gin Val Ser He Pro Trp Ala Val Leu He Val 35 40 45

Val Leu He Thr Ser Leu He He Ala Leu He Ala Leu Asn Val Gly 50 55 60

Lys Tyr Asn Cys Pro Gly Leu Tyr Glu Lys Leu Glu Ser Ser Asp His 65 70 75 80

His Val Ala Thr Cys Lys Asn Glu Trp He Ser Tyr Lys Arg Thr Cys 85 90 95

Tyr Phe Phe Ser Thr Thr Thr Lys Ser Trp Ala Leu Ala Gin Arg Ser 100 105 110

Cys Ser Glu Asp Ala Ala Thr Leu Ala Val He Asp Ser Glu Lys Asp 115 120 125

Met Thr Phe Leu Lys Arg Tyr Ser Gly Glu Leu Glu His Trp He Gly 130 135 140

Leu Lys Asn Glu Ala Asn Gin Thr Trp Lys Trp Ala Asn Gly Lys Glu 145 150 155 160

Phe Asn Ser Trp Phe Asn Leu Thr Gly Ser Gly Arg Cys Val Ser Val 165 170 175

Asn His Lys Asn Val Thr Ala Val Asp Cys Glu Ala Asn Phe His Trp 180 185 190

Val Cys Ser Lys Pro Ser Arg 195

Claims

CLAIMSWhat is claimed is:

1. An isolated DNA comprising a DNA sequence encoding human CD69, wherein said CD69 comprises amino acids 1-199 of SEQ ID NO:l.

2. An isolated DNA comprising a DNA sequence encoding a soluble human CD69 polypeptide, wherein said CD69 comprises amino acids 79-199 of SEQ ID NO:l.

3. An isolated DNA according to claim 2, wherein said DNA additionally encodes a heterologous signal peptide fused to the N-terminus of said soluble human CD69 polypeptide.

4. An isolated DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 1-600 of SEQ ID NO: 1 and nucleotides 235-600 of SEQ ID NO:l.

5. An isolated DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 1 -600 of SEQ ID NO:3 and nucleotides 235-600 of SEQ ID

NO:3.

6. An isolated DNA encoding a biologically active CD69 polypeptide, wherein said CD69 comprises an amino acid sequence that is at least 80% identical to the sequence of amino acids 1-199 or 79-199 of SEQ ID NO:2.

7. A DNA according to claim 6, wherein said CD69 comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids 1-199 or 79-199 of SEQ ID NO:2.

8. An expression vector comprising a DNA sequence according to any one of claims 1, 2, 3, 4, 5, or 6.

9. A process for preparing a CD69 polypeptide, comprising culturing a host cell transformed with a vector according to claim 8 under conditions that promote expression of

CD69, and recovering the CD69 polypeptide.

10. A purified soluble human CD69 protein comprising an amino acid sequence that is at least 80% identical to the sequence of amino acids 79-199 of SEQ ID NO:2.

1 1. A purified soluble CD69 according to claim 10, wherein said CD69 comprises amino acids 79- 199 of SEQ ID NO:2.

12. A dimer comprising two soluble CD69 proteins according to claim 10, joined via disulfide bonds.

13. A dimer comprising two soluble CD69 proteins according to claim 11 , joined via disulfide bonds.

14. A nucleic acid molecule comprising a sequence of at least about 14 nucleotides of SEQ ID NO: 1 or the DNA or RNA complement therof.