RELATED APPLICATIONS This application is a continuation-in-part of Tedder, U.S. Patent Application Serial No. 07/983,606, filed November 30, 1992, which is a continuation under 37 CFR 1.62 of Serial Nos. 07/730,503, filed July 8,1991, and 07/313 , 109, filed February 21, 1989, both now abandoned. This application also is a continuation-in-part of Tedder, U.S. Patent Application Serial No. 07/700,773, filed May 15, 1991; of Tedder, U.S. Patent Application Serial No. 07/737,092, filed July 29, 1991; of Tedder et al., U.S. Patent Application Serial No. 07/770,608, filed October 3, 1991; and of Tedder et al., U.S. Patent Application Serial No. 07/862,483, filed April 2, 1992, the whole of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION This invention relates to the selectin family of receptor adhesion molecules and particularly to agents interfering with selectin function.

GOVERNMENT RIGHTS Part of the work leading to this invention was made with United States Government funds. Therefore, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The ability of leukocytes to leave the circulation and to migrate into tissues is a critical feature of the immune response. Normally, the infiltrating leukocytes phagocytize invading organisms or dead or damaged cells. However, in pathologic inflammation, infiltrating leukocytes can cause

serious and sometimes deadly damage. Leukocyte-mediated inflammation is implicated in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury. The selectin family of receptor adhesion molecules mediates the initial interactions of leukocytes with endothelium (Springer, Nature 346:425-434 (1990); Lasky, Science 258:964-969 (1992)). L-selectin (also known as LAM- 1) is expressed on the surface of most classes of leukocytes (Griffin et al., J. Immunol. 14_5:576-584 (1990); Tedder et al., J. Immunol. 144 :532-540 (1990)) and mediates the binding of lymphocytes to high endothelial venules (HEV) of lymph nodes and to activated endothelium. P-selectin (also called PADGEM, CD62 or GMP-140) is expressed by activated platelets and endothelial cells (Johnston et al., Cell 5 >:1033 (1989); Hsu-Lin et al., J. Biol. Chem. 259:9121 (1984)) and mediates adhesion between myeloid cells and activated endothelium or activated platelets (Gallatin et al, Nature 304:30-34 (1983); Spertini et al, J. Immunol. 147:2565-2573 (1991) ; Larsen et al, Cell 5_9:305-312 (1989); Geng et al, Nature 343:757-760 (1990) ) . Another member of the family E-selectin (also known as ELAM-1) is expressed by activated endothelial cells (Bevilacgua et al., Science 243:1160 (1989) ; Bevilacqua et al., Proc. Nat'1 Acad. Sci. USA 84.:9238 (1987)) and partially mediates the binding of neutrophils to endothelium at sites of inflammation (Bevilacqua et al., Proc. Nat'l Acad. Sci. USA J4:9238 (1987)). All selectins are derived from evolutionarily related genes (Collins et al., J. Biol. Chem. 266:2466-2478 (1991); Johnston et al., J. Biol. Chem. 34 _. :21381-21385 (1990); Ord et al., J. Biol. Chem. 265:7760-

7767 (1990) ; Watson et al., J. Exp. Med. 172:263-272 (1990)), and are characterized by an NH.-terminal, Ca ⁺ -dependent lectin domain, an epidermal growth factor (EGF) -like domain followed by multiple short consensus repeat (SCR) domains, a transmembrane region, and a cytoplasmic tail. Although the lectin domains are critical for the binding of specific

carbohydrate ligands (Springer, Nature 346:425-434 (1990) ; Lasky, Science 258:964-969 (1992)) , the role of the conserved EGF-like domains is unknown.

It has been proposed that the treatment of a patient suffering from pathologic inflammation with an antagonist to adhesion receptor function can result in the reduction of leukocyte migration to a level manageable by the target endothelial cells and the subsequent dramatic recovery of the patient. Local administration of therapeutic agents can block competitively the adhesive interactions between leukocytes and the endothelium adjacent to an inflamed region. Therapeutic agents can also be administered on a systemic level for the treatment of a patient suffering from disseminated inflammation (Harlan and Liu, eds., Adhesion: Its Role in Inflammatory Disease, W. H. Freeman (in press)).

SUMMARY OF THE INVENTION We report here that the EGF-like domains of P-selectin and of E-selectin can participate directly in cell adhesion, having ligand binding sites distinct fron those of their respective lectin domains. Cell adhesion mediated at least by the P and E-selectins may be complex, involving interactions between the lectin domain and a carbohydrate ligand, and separately, between the EGF-like domain and one or more proteins or other ligands. Our discovery of a difference in at least one of the ligands recognized by the lectin and EGF domains of a given selectin has permitted the preparation of chimeric polypeptides that combine within one molecule the ability to target two or more different selectin ligands and the use of these agents in therapy or in the preparation of additional classes of antagonists to selectin function.

Thus, the invention generally features chimeric peptides or polypeptides that combine ligand binding portions from within the lectin and EGF domains of two different selectins. The peptides or polypeptides can be composed solely of the

indicated portions of lectin or EGF domains or they can include portions of any of the remaining domains (SCR, transmembrane or cytoplasmic) , or the entire extracellular portion, of a generic selectin molecule. The peptides or polypeptides also can be joined to a carrier protein (e.g., a soluble portion of an immunoglobulin molecule) to increase the serum half-life of the therapeutic agent. In another aspect, the invention features nucleic acid encoding the chimeric polypeptides. The chimeric polypeptides can be used as therapeutic agents to antagonize selectin function. They are also useful for screening for agents that are simultaneously antagonists of the function of lectin and EGF domains of different selectins. As used herein, the term "polypeptide" is intended to include shorter molecules, or "peptides." The term "essentially purified" refers to a polypeptide sequence that has been separated or isolated from the environment in which it was prepared or in which it naturally occurs.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, taken in conjunction with the accompanying drawings in which: Fig. 1 shows the structure and expression of

L-selectin/P-selectin chimeric proteins (Lectin, lectin domain; EGF, epidermal growth factor-like domain; SCR, short concensus repeat; TM, transmembrane domain; C, cytoplasmic domain) ; Fig. 2a shows binding of HL-60 cells to COS cells transfected with natural and chimeric selectins (Original magnification, 200x) ;

Fig. 2b shows mapping of the domain(s) of L2P responsible for adhesion of HL-60 cells (bars represent the

means + SD, and are representative of at least six experiments) ;

Fig. 3a shows neuraminidase sensitivity for adhesion mediated by the lectin and EGF domains of P-selectin; Fig. 3b shows protease sensitivity and Ca ²⁺ requirement for adhesion mediated by the lectin and EGF domains of P-selectin;

Fig. 3c shows an analysis of the binding of the chimera L2P3L; Figs. 4a, 4b, and 4c show the cDNA nucleotide sequence

(SEQ ID NO: l) encoding L-selectin and also show the amino acid sequence of L-selectin (SEQ ID NO: 2) ;

Fig. 5 shows the restriction map and the -exon-intron organization of the lyam-1 gene; Figs. 6a, 6b, and 6c show the nucleotide sequence of exons II through X of the lyam-1 gene; and

Fig. 7 shows the ho ology among the lectin and EGF domains of L-, P- and E-selectin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS We have examined the role of the lectin and EGF-like domains of the L-, P-, and E-selectins in cell adhesion and have determined that the EGF-like domains of P- and E-selectin play a direct role in ligand recognition and leukocyte adhesion mediated by the respective selectin. This discovery has permitted the preparation of chimeric polypeptides that combine within one therapeutic agent the ability to target two or more different selectin ligands and the use of these agents in therapy or in the preparation of additional classes of antagonists to selectin function.

Determination of the binding specificity of selectin lectin and EGF domains

The functional activities of the lectin and EGF-like domains of L-, P-, and E-selectin in the adhesive events described above were examined using a panel of

L-selectin/P-selectin and L-selectin/E-selectin chimeric molecules. Referring to Fig. 1, these hybrid polypeptides were created by exchange of cDNA encoding the lectin, EGF, or both lectin and EGF domains of the desired selectin and expression of the chimeric nucleic acid, as described in the Experimental Procedures section. Many other methods familiar to those of skill in the art can be employed to prepare similar products.

The chimeras conserve the overall polypeptide backbone structure of the selectins, and resulted in minimal changes in the junctions between domains. The functional characterization of these chimeric selectins therefore offered a powerful approach to determining the molecular basis of cell adhesion mediated by these molecules and to preparing diagnostic and therapeutic agents incorporating properties of individual selectins into the same polypeptide.

Expression of the chimeric proteins in COS cells was verified and quantitated using panels of P-, E- and

L-selectin domain-specific monoclonal antibodies (mAb) in direct adhesion assays. The chimeric cDNA were subcloned into the pMT-2 vector and used to transiently transfect COS cells as described under Experimental Procedures. Twenty-four hours before analysis, 2.5 x 10 ⁴ transfected COS cells were replated into 96 well plates. Expression of domain-specific epitopes by chimeric selectins was assessed using mAb reactive with specific domains and a surface immunofluorescence assay as described (Luscinskas et al, J. Immunol. 149:2163-2171 (1992)) .

Referring again to Fig. 1, the individual constructs, containing the indicated domains of P-selectin and L- selectin, reacts only with anti-L-selectin and anti-P- selectin monoclonal antibodies recognizing the specific, indicated domains. These results demonstrate that the chimeric cDNA encoded the expected proteins and that each protein was expressed and readily detected on the surface of COS cells. Note that the LAMl-1 mAb fails to recognize

either the L2P or P2L proteins, and therefore defines an epitope composed of residues in both the lectin and EGF domains. Values given are means of absolute optical density (OD) units from which OD units obtained with a control non-binding mAb were subtracted. Experimental values less than the control values are represented as zero. The values presented are typical of those obtained in three experiments. The standard deviations for these fluorescence measurements were less than 15%.

Characterization of the adhesion reactions mediated by the selectin lectin and EGF domains

The HL-60 myelomonocytic cell line expresses ligands for P-selectin (Larsen et al. Cell 5_9_:305-312 (1989) ; Geng et al. Nature 343:757-760 (1990)) and E-selectin (Bevilacqua et al. , Science 243:1160 (1989); Bevilacqua et al. , Proc. Nat'1 Acad. Sci. USA 84.:9238 (1987)) and was therefore used to assess P-selectin and E-selectin function. Referring to Fig. 2a, HL-60 cells bound at high levels to COS cells expressing P-selectin, but did not bind to COS cells expressing L- selectin. HL-60 cells also bound to COS cells expressing the chimeric protein P2L, in which the lectin domain from P-selectin was substituted for the lectin domain of L-selectin. Therefore, the lectin domain of P-selectin alone, when attached to the EGF-like and other domains from L-selectin, was sufficient to mediate high levels of HL-60 cell adhesion. However, the lectin domain of P-selectin alone was not solely responsible for all HL-60 binding, because HL-60 cells also bound to COS cells expressing L2P, in which the P-selectin lectin domain was replaced with that of L-selectin.

The domain(s) within L2P responsible for HL-60 adhesion were mapped using additional chimeric selectins. Referring to Fig. 2b, HL-60 cells bound to COS cells expressing L2P3L, which contains only the EGF domain from P-selectin, but did not bind to COS cells expressing L3P, which lacks both the

lectin and EGF-like domains of P-selectin but contains the P-selectin SCR domains. Furthermore, binding of HL-60 cells to P3L, which contains both the lectin and EGF domains from P-selectin, was approximately equivalent to that of native P-selectin and significantly (P < 0.01; Student's T test) higher than binding of cells to P2L, even though expression of P2L and P3L were approximately equal. These data indicate that both the lectin and EGF-like domains of P-selectin contain ligand binding sites capable of independently mediating HL-60 cell adhesion.

For study of ligand binding sites in E-selectin, four L-selectin/E-selectin chimeric molecules were constructed. L2E has the lectin of L-selectin and the remainder of E-selectin; E2L is the precise converse. L5E has the amino terminal 88 amino acids of L-selectin (i.e., 74% of the lectin domain) and E5L is the precise converse. Binding of HL-60 cells to COS cells expressing these chimeras was assessed as before. The constructs containing only a portion of a specific lectin domain exhibited zero binding, while the binding to chimeras containing lectin and EGF domains from different selectins was reduced by about a half. Therefore, the overall pattern of binding was similar to the results with L-selectin/P-selectin chimeras.

Determination of the influence of EGF-domains on the adhesive activity or specificity of the lectin domains

Adhesion mediated by P-selectin involves sialic acid-bearing carbohydrates, possibly including the sLe ^x and/or related structures, as well as protein determinants (Larsen et al, Cell 63 _. :467-474 (1990) ; Zhou et al, J. Cell Biol. 115:557-564 (1991); Moore et al, J. Cell. Biol. 118:445-456 (1992); Moore et al, J. Cell Biol. 112:491-499 (1991); Polley et al, Proc. Natl. Acad. Sci. USA :6224-6228 (1991); Larsen et al, J. Biol. Chem. 267:11104-11110 (1992)). Therefore, the effects of neuraminidase and protease

treatment of HL-60 cells on binding to COS cells expressing either P-selectin, P3L, or P2L was examined.

Removal of sialic acid residues from HL-60 cells significantly (P< 0.01) but incompletely reduced adhesion of HL-60 cells to COS cells expressing native P-selectin or P3L (Fig. 3A) . In contrast, removal of sialic acid residues from HL-60 cells nearly completely eliminated adhesion of HL-60 cells to P2L (Fig. 3A) . In addition, binding to the E- selectin/L-selectin construct E2L was partially inhibited by pretreatment of the HL-60 cells with CSLEX1 mAb directed against sLex. Similarly, pretreatment of COS cells with mAb directed against the lectin domain of P-selectin significantly (P < 0.005), but only partially, inhibited the adhesion of HL-60 cells to P-selectin or P3L, but abolished adhesion to P2L (Fig. 3A) .

Treatment of HL-60 cells with either chymotrypsin or papain eliminated >90% of binding to COS cells expressing P-selectin, P3L, or P2L (Fig. 3B) . In addition, 2.5 mM EGTA abolished adhesion (Fig. 3B) . These results confirm the importance of the lectin domain in adhesion, and offer independent evidence that the EGF domain of P-selectin also plays a direct role in adhesion, recognizing a protein ligand.

A similar analysis was performed on L2P3L to characterize adhesion mediated by the EGF-like domain of P-selectin. Adhesion of HL-60 cells to COS cells expressing L2P3L was also reduced, but not eliminated, by neuraminidase treatment of the HL-60 cells (Fig. 3C) . MAb directed against the L-selectin lectin domain present within L2P3L also significantly (P < 0.01) inhibited adhesion. These results suggest that recognition of specific carbohydrate structures on HL-60 cells by the lectin domain of L-selectin, which alone was not sufficient for cell adhesion, contributes to leukocyte adhesion in this system when this interaction is supported by the adhesive activity mediated by the EGF domain of P-selectin. Consistent with this hypothesis, rare

(~1 plate) rosettes of HL-60 cells were occasionally observed on COS cells transfected with L-selectin (data not shown) , and a soluble L-selectin/IgG fusion protein has a measurable affinity for purified, immobilized sLe ^x (Foxall et al, J. Cell Biol. lT7:895-902 (1992)). Adhesion of HL-60 cells to L2P3L was eliminated by EGTA, indicating that adhesion mediated by the EGF-like domain is also Ca ⁺ -dependent, and was significantly (P < 0.01) reduced by protease treatment (Fig. 3C) .

Determination of the influence of EGF domains on the adhesive activity or specificity of the lectin domains

In order to determine if the EGF-like domains of selectins have any influence on the adhesive activity or specificity of the lectin domain, stable transfectants of the 300.19 murine pre-B cell line expressing L-selectin, P-selectin, L2P or L2P3L were generated. These cells were tested for binding to lymph node HEV using the standard in vitro frozen section assay (Stamper Jr. et al, J. Exp. Med. 144.:828-833 (1976) ) . Transfectants expressing P-selectin did not bind to HEV, whereas transfectants expressing L-selectin, L2P or L2P3L bound to HEV equivalently and at high levels (Table I) .

Table I. Adhesion of cell lines to lymph node HEV is mediated by the lectin domain of L-selectin.

cDNA expt 3

none <0.01

<0.01 ND ND

L-selectin

L2P

L2P3L

P-selectin <0.01 <0.01 ND

The lectin domain of L-selectin alone was therefore capable of mediating binding to HEV when attached to the EGF-like and other domains of P-selectin. The L2P and L2P3L chimeric selectins can therefore bind to different types of cells which express ligand(s) for either L- or P-selectin, reflecting the specificities of both of the parent selectins from which they are constructed. This acquisition of novel adhesive properties as a result of the exchange of lectin or EGF-like domains between selectins reinforces our hypothesis that the EGF-like domain of P-selectin can mediate cell adhesion. In addition, these data directly demonstrate the functional independence of the lectin and EGF-like domains, and therefore argue against a role for the EGF-like domain in determining the carbohydrate specificity of the lectin domain.

Structure of the L-selectin gene

As disclosed in U.S. Patent Application Ser. No. 07/983,606, cDNA encoding L-selectin has been cloned and the sequence determined, as shown in Figs. 4a, 4b and 4c (SEQ ID NO: 1) . The cDNA encodes a protein of 372 amino acids (SEQ ID NO: 2) .

The structure of the lyam-1 gene, which encodes the LAM- 1 protein, was determined by isolating overlapping genomic DNA clones that hybridized with a LAM-1 cDNA probe. The lyam-1 gene spans greater than 30 kb of DNA and is composed of at least 10 exons. The 5' end of the LAM-1 mRNA was mapped by primer extension analysis, revealing a single initiation region for transcription. Exons II through X contain translated sequences; exon II (SEQ ID NO: 3) encodes the translation initiation codon, residue 14 shown in Fig. 2; exon III (SEQ ID NO: 4) encodes the leader peptide domain, residues 15-41; exon IV (SEQ ID NO: 5) encodes the lectin- like domain, residues 42-170; exon V (SEQ ID NO: 6) encodes the epidermal growth factor-like domain, residues 171-206; exons VI (SEQ ID NO: 7) and VII (SEQ ID NO: 8) encode the

short consensus repeat unit domains, residues 207-269 and 270-331; exon VIII (SEQ ID NO: 9) encodes the transmembrane region, residues 332-373; exon IX (SEQ ID NO: 10) encodes seven amino acids containing a potential phosphorylation site, residues 374-380; and exon X (SEQ ID NO: 11) encodes the five remaining amino acids of the cytoplasmic tail and the long 3' untranslated region.

The pLAM-1 cDNA was labeled with ³² P and used as a probe to isolate hybridizing DNAs from a human leukocyte genomic DNA library. Approximately 1 X 10 ⁶ plaques were screened, and 13 plaques that hybridized with the cDNA probe were identified and isolated. Seven of these clones were found to contain inserts with unique restriction enzyme maps representing overlapping genomic fragments spanning at least 30 kb. These inserts, LAMG-17, -19, -20, -28, -35, -37, and -47,* were further digested and subcloned into plasmids. Detailed restriction maps of these subclones were made and compared to those of intact inserts to determine their correct locations (Figs. 4A and 4B) . The correctness of the restriction map was verified with

Southern blot analysis. DNA isolated from two B cell lines, BL and BJAB, and one T cell line, HSB-2, was digested to completion with Bam HI, Bal II, or Pvu II, size-fractionated, and transferred onto nitrocellulose. This filter was probed with the LAM-1 cDNA clone, pLAM-1. All genomic fragments derived from endonuclease digested DNA hybridized with cDNA probe to generate hybridizing bands of the appropriate size.

The pLAM-1 cDNA clone encodes an 85-bp 5' untranslated region. An oligonucleotide homologous with the 5' sequence of the pLAM-1 cDNA was used as a probe for primer extension analysis. This oligonucleotide was hybridized with poly (A ⁺ ) RNA isolated from the human B cell line RAJI, the LAM-1 negative human B cell line Namalwa, the mouse pre-B cell line A20, and yeast tRNA as a control. Complementary DNA was synthesized by extending the primer with reverse transcriptase. The major primer extension product obtained

using the human LAM-1 positive B cell line RNA was extended 126 nucleotides beyond the translation initiation site. There was a single cluster of transcription initiation sites for the lyam-1 gene apparent in the reaction with RAJI RNA that was not found with the LAM-1 negative Namalwa RNA. Several primer extension products of size similar to those of the human B cell line RNA were obtained with mouse B cell RNA. Therefore, murine B cells may express an RNA species that cross-hybridizes with the oligonucleotide probe used. No primer extension products were obtained in the yeast tRNA control reactions.

The relationship of the primer extension results to the cloned LAM-1 cDNAs and the most 5' exon of lyam-1 isolated was used to determine the nucleotide sequence of the exon that encodes the translation initiation AUG codon. This exon ends immediately after the site that encodes the translation initiation codon (Fig. 5) and overlaps precisely with the pLAM-1 cDNA sequence. The length of the cDNA clone obtained by Bowen et al., J. Cell Biol. 109:421-427 (1989) agrees precisely with the primer extension results except for two nucleotides. However, 15 nucleotides before the 5' end of the cDNA the sequence diverges from the genomic sequence at a site homologous with the 3' splice acceptor site consensus sequence. Therefore, it is most likely that this 15-bp region is derived from the exon upstream of this potential splice site. Thus, the primer extension results indicate that exon I would most likely be composed of 15 or fewer base pairs.

A 15-bp oligonucleotide homologous with the 5 ' nucleotides present in the cDNA clone of Bowen et al., supra but not encoded by exon II, was used to probe the 10 kb of cloned DNAs 5' of exon II; however, specific hybridization was not detected by Southern blot analysis. Under the conditions necessary for hybridization of this oligonucleotide, significant cross-hybridization occurred

with λ-DNA, making it difficult to use this oligonucleotide to isolate the first exon from a λ-based genomic library.

These results suggest that the exon which encodes the translation initiation site is the second exon of the lyam-1 gene (Fig. 4C) . Consistent with this, the 900 bp upstream of exon II did not contain any apparent "TATA" or "CCAAT" sequences frequently found in promoter regions of eukaryotic genes (Fig. 5) . Therefore, it is likely that the transcription initiation region and exon I are further than 10 kb upstream from exon II of the lyam-1 gene. SI nuclease protection analysis was carried out using the 5' region of exon II as a labeled probe for hybridization with poly (A ⁺ ) RNA from RAJI, Namalwa, and A20 cells. Two mRNA-species were protected in the RAJI mRNA, while no SI protection was provided by the other RNAs. The length of these fragments was consistent with differential splicing at the two potential CAG/N splice sites located within the potential splice acceptor site in exon II. It is therefore likely that the transcription initiation region has not been identified. The majority of the exons were localized by comparison of the restriction enzyme maps of the genomic clones and the pLAM-1 cDNA. In cases where this method did not provide definitive results, subcloned DNA fragments were digested with selected restriction enzymes, electrophoresed through agarose gels, and transferred to nitrocellulose. Fragments containing exons were identified by Southern blot analysis using labeled cDNA or oligonucleotide probes. The exon that encodes the 3 untranslated region of the LAM-1 cDNA was not contained within the 30 kb of isolated DNA fragments. Therefore, a labeled 0.9-kb Dra I fragment containing most of the 3' untranslated region of the pLAM-1 cDNA was used as a probe to identify a homologous 3.2-kb fragment generated by complete Eco RI digestion of genomic DNA. Eco RI-digested genomic DNA fragments of this size were used to make a partial λ-gtll genomic library from which the 3.2-kb Eco RI

fragment was cloned. This 3.2-kb fragment did not overlap with the previously isolated genomic DNAs.

The exact boundaries of the exons were determined by nucleotide sequence analysis. From this analysis, nine exons were identified which make up the entire pLAM-1 cDNA. Exon II encodes the translation initiation codon, and exon III encodes the leader domain of the LAM-1 protein (Fig. 5) . Each of the lectin-like, epidermal growth factor¬ like, transmembrane, and short consensus repeat domains was encoded by a separate exon. The smallest exon, IX, is 19 bp in length and may encode a carboxyl-terminal phosphorylation cassette. The last 5 amino acids of the LAM-1 protein and the 3' untranslated region which includes the poly(A) attachment site, AATAAA, are encoded by exon X as shown in Fig. 5. The nine exons which encode pLAM-1 were split inside codons in all cases, except the junction between exons II and III. In each instance, the consensus sequences of 5' donor splice sites and 3' acceptor splice sites were adhered to. Nucleotide sequence polymorphisms within the coding region were observed between the genomic clones containing exon V that encoded SCR I and the pLAM-1 clone at cDNA nucleotide positions 741 and 747 (A to G) , leading to a coding change from Asn to Ser in both cases, and at position 816 (A to G) changing the Glu to a Gly.

Experimental Procedures

Construction of Chimeric Selectin cDNAs

Four new restriction endonuclease recognition sites were introduced separately into the pLAM-l cDNA by polymerase chain reaction (PCR)-based site-directed mutagenesis as follows. Oligonucleotides surrounding and including each new restriction sequence were synthesized in both the sense and antisense directions. PCR was carried out using the new restriction site sense oligonucleotide and an antisense oligonucleotide anchor located in the plasmid near the 3 end of the cDNA. In a separate reaction, the new restriction site antisense oligonucleotide plus a sense oligonucleotide

anchor from the plasmid 5' end were used to amplify the 5' end of the LAM-1 cDNA. The individual PCR products from these two reactions were gel purified, subcloned into the pSP65 vector (Promega Biotec, Madison, WI) , digested with the 5 appropriate restriction enzymes, and ligated together. The nucleotide changes were as follows: (a) GA ³⁶¹ ATCC to GGATCC in the lectin domain, creating a BamHI site, corresponding to amino acid number 53 in the mature protein (Fig. 2) ; (b) A ⁵⁸³ TGCAG to CTGCAG in the EGF domain, creating a PstI

10 site, corresponding to amino acid number 127; (c) GAGG ⁸⁸¹ C ⁸² C to GAGCTC in the first SCR domain, creating a Sad site, corresponding to amino acid number 164; and (d) GTCAAA ¹⁰²³ to GTCAAC in the second SCR domain, creating a Hindi site, corresponding to amino acid number 279. The fidelity of the

15 site-directed mutagenesis was confirmed by restriction mapping and sequence analysis of the cDNAs. Each of these sites is naturally present in the PADGEM cDNA (Larsen et al. , Cell .59:305-312 (1984)).

Domain Mapping of Binding Sites

20 The chimeric selectin cDNAs were subcloned into the pMT-2 expression vector (Kaufman et al, J. Mol. Cell. Biol.

9.:946-958 (1989)), and COS cells at -50% confluency were transiently transfected with 3 μg of the indicated cDNA by the DEAE dextran method. One day after transfection, the COS

25 cells were replated at ~50% confluency onto 35 mm Petri dishes (assay plates) (Becton-Dickinson, Lincoln Park, New

Jersey) . The following day, HL-60 cells were washed twice in cold RPMI 1640 media (Gibco, Grand Island, NY) , resuspended in RPMI 1640 at a final concentration of

^" 30 3.3 x 10 ⁶ cells ml, and 0.6 ml (2 x 10 ⁶ cells) were added to assay plates which had been washed twice with cold RPMI 1640 media. The plates were gently rocked for 20 min at 4°C, washed five times, and the number of HL-60 cells bound to individual COS cells were counted on a minimum of 100 COS

35 cells.

Assay for leukocyte attachment to HEV The cDNAs encoding native L-selectin, L2P and L2P3L were subcloned into the Bam HI site of the pZIPneoSV(X) vector (Cepko et al. Cell 12:1053-1062 (1984) ) , and were used to transfect the mouse pre-B cell line 300.19. Stable transfectants were selected in medium containing 0.5-1.0 mg/ml G418 (geneticin; Sigma), and cells expressing the proteins were selected by panning with the LAM1-3 mAb (Spertini et al, J. Immunol. i£7:942-949 (1991)). To generate P-selectin transfectants, the P-selectin cDNA in the pMT-2 vector was cotransfected with the pSV2neo vector containing the neomycin resistance marker. Rat lymph nodes were obtained from freshly euthanized Lewis rats, snap frozen in isopentane/liquid nitrogen, and stored at -70°C in isopentane until use. For the HEV assay, 5 x 10 ⁶ of each cell type were incubated on three 12 mm sections/ slide at 64 rpm for 25 min at ~4°C, the excess cells were gently removed, and the slides were placed vertically in ice-cold fixative (PBS/2.4% glutaraldehyde) overnight. The slides were then counterstained with Gill's hematoxylin, overlaid with glycerol gelatin, and cover slips were applied. Each slide was scored for the number of lymphocytes bound/HEV. Between 100-200 HEV were counted for each experiment. Data are presented as mean + standard deviation of lymphocytes bound/HEV. ND, not determined.

Determination of neuraminidase sensitivity, protease sensitivity and

Ca ²⁺ requirements for adhesion

COS cells were transfected with the indicated cDNA and replated as described above. HL-60 cells were incubated in

RPMI 1640 medium at 37°C for two hours with either 0.25 U/ml

Clostridium perfringens neuraminidase (Type VI, Sigma, St.

Louis, MO) , 50 mg/ml chymotrypsin or papain (Sigma) , washed twice, and the assay was performed as described in the Fig. 2 legend. Neuraminidase treatment of the HL-60 cells was effective in eliminating all reactivity with the CSLEXl

anti-sLe ^x mAb (data not shown) . In some groups, COS cells were preincubated with either 5 μg/ml Gl mAb (a gift of Dr. R.P. McEver) (Geng et al. Nature 343:757-760 (1990)), or ascites fluid containing the LAM1-3 mAb (Spertini et al, J. Immunol. 147:942-949 (1991)) diluted 1:200. To determine the effect of EGTA, both the HL-60 cells and the COS cells were washed in RPMI 1640 containing 2.5 mM EGTA, and this medium was also used for the assay. No detachment of COS cells occurred under these conditions. These results are representative of at least four experiments.

Use

The adhesion of leukocytes to the endothelial wall of blood vessels and their infiltration into the surrounding tissues contributes to inflammation of tissue at the cellular level. Normally, the infiltrating leukocytes phagocytize invading organisms or dead or damaged cells. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes deadly damage. Leukocyte-mediated inflammation is involved in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury.

Local administration of therapeutic agents antagonistic to the function of the selectins can block competitively the adhesive interactions between leukocytes and the endothelium adjacent to an inflamed region. On a systemic level, treatment of a patient in shock (e.g., from a serious injury) with an antagonist to selectin function can result in the reduction of leukocyte migration and adhesion and the subsequent dramatic recovery of the patient. Chimeric peptides or polypeptides combining ligand binding portions from within the lectin and EGF domains of two different selectins can be used for therapeutic treatment to interfere in the binding of leukocytes at the site of inflammation. Referring to Fig. 7, the close ho ology among the lectin and EGF domains of L-, P- and E-selectin can be

seen, and the intron/exon organization of the respective genes encoding P- and E-selectin (Collins et al., J. Biol.Chem. 266:2466 (1991); Johnston et al., J. Biol. Chem.265:21381 (1990)) is the same as that of the lyam-1 gene encoding L-selectin, disclosed herein. These chimeric agents will offer more efficient targeting than using a single function antagonist. Therapeutic agents of the invention can also be used to block platelet or platelet and mononuclear cell aggregation, thus preventing throbus formation. The therapeutic peptides or polypeptides can be composed solely of the indicated portions of lectin or EGF domains or they can include portions of any of the remaining domains (SCR, transmembrane or cytoplasmic) , or the entire extracellular portion of a generic selectin molecule. The peptides or polypeptides also can be joined to a carrier protein (e.g., a soluble portion of an immunoglobulin molecule) to increase the serum half-life of the therapeutic agent. Immunoglobulin chimera are easily purified through standard immunochemistry procedures, including IgG-binding protein A-Sepharose chromatography. The chimera have the ability to form an immunoglobulin-like dimer with the concomitant higher avidity and serum half-life. cDNA sequences encoding the chimeric polypeptides can be incorporated into replicable expression vectors and the vectors transfected into an appropriate host to express the encoded polypeptide. Stop codons can be incorporated into the DNA sequences or any other chain termination method can be used to prepare a soluble polypeptide. The expressed polypeptide can be purified by ordinary techniques well known to those of skill in the art, e.g., by affinity chromatograhic methods.

Other agents can also be joined to the therapeutic polypeptides to form a useful product. For example, the ligand binding portions of the lectin and EGF domains of two different selectins can be combined with the toxic portion of a cytotoxin to produce a fusion protein. In addition, the

selectin chimeric polypeptides may be coupled to a chemotherapeutic drug or drugs which could simultaneously bind to cells expressing their respective ligands, to administer the drug to a site of tissue damage or inflammation to treat, e.g., acute inflammation or vasculitus. Such drugs may include, anti-inflammatory agents or agents that provide regional relief from inflammatory distress. Syndromes, diseases, and conditions that could be treated by these agents would include, but not be limited to, treating inflammation, microbial/parasitic infections, post- reperfusion injury, leukemia, lymphoma, vasculitis, inhibition of the metastatic spread of tumor cells, organ transplantation, or graft rejection. As is well known to those of skill in the art, the fusion proteins can be transcribed from a hybrid cDNA molecule, or the agent may be covalently bonded to the chimeric polypeptide by routine procedures.

One method of imaging the sites of inflammation in a patient involves detecting the expression of the L-selectin ligand on the inflamed endothelium. The method includes administering to a patient a pharmaceutical composition consisting of a detectable amount of a labeled ligand binding fragment of L-selectin, alone or joined to a carrier protein, including a ligand binding portion of a different selectin receptor, in a pharmaceutically acceptable carrier. Sufficient time is allowed for the labeled polypeptide to localize at the site of L-selectin ligand expression, unbound polypeptide is permitted to clear from healthy tissue in the patient, and signal generated by the label is detected and converted into an image of the site of inflammation. The amount of labeled ligand binding fragment of L-selectin preferably would be from 1 pg/kg to 10 μg/kg although higher or lower doses are possible depending on the imaging agent label used and the sensitivity of the detection method. Some of the labels which can be detected externally from within the body of a human patient include radionuclides,

radiopaque labels, and paramagnetic isotopes. A radionuclide for in vivo diagnosis should have a half-life long enough that it is still detectable at the time of maximum uptake, but short enough that after diagnosis unwanted radiation does not remain in the patient. Coupling of radionuclides to antibodies or proteins is well known in the art (see, e.g., Daddona et al., U.S. Pat. No. 5,026,537, the teachings of which are incorporated by reference herein) and is often accomplished either directly or indirectly using an intermediary linking group. Examples of radioisotopes that could be used for in vivo diagnosis are ⁹⁹ Tc, ¹²³ I, ¹³¹ I, In, "Ru, "CU, ⁶⁷ Ga, ⁶⁸ Ga, ^As, ⁸⁹ Zr, and ²⁰¹ Ti. Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. Examples of elements that are particularly useful for use in magnetic resonance energy techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe. For radiopaque imaging, the LAM-1 protein or ligand binding fragment is coupled to an agent that produces an opaque field at the site of inflammation upon X-ray imaging. A ligand binding portion of the lectin or EGF-like domains of various selectins can be experimentally determined using this invention. In a typical procedure, fragments of the selectin cDNA can be fused with cDNA encoding a carrier protein, such as immunoglobulin heavy chain or another non- ligand binding portion of a different selectin. These chimeric proteins can be expressed on the cell surface, or as soluble molecules, and their ability to bind ligand assessed. Further segregated fragments of the lectin or EGF- like domain under study can be examined for their ability to bind ligands, as well as their ability to inhibit the binding of the parent selectin molecule to ligand.

Any of the peptides or polypeptides of the invention comprising lectin and EGF ligand binding portions from two different selectin molecules (including portions of any of the remaining domains (SCR, transmembrane or cytoplasic) of a generic selectin molecule or including a carrier protein)

can be used to screen for antagonists of lectin or EGF function individually or for agents that can simultaneously antagonize the functions of the lectin and EGF domain of different selectins. This invention will allow the development of phar acologic reagents composed of peptides, carbohydrate moieties, RNAs or other small molecules which may mimic the ligand of the lectin or EGF domains of the selectins or may mimic the ligand-binding epitopes of the respective selectin domains and thus be useful therapeutic agents.

In another therapeutic method, chimeric selectin polypeptides can be used in combination therapy with any other selectin or any other cell surface molecule, or soluble fragments thereof, involved in adhesion of leukocytes to endothelial surfaces. In addition, antagonists to any of the above receptors or receptor portions (or mAb reactive to the receptors or receptor portions) can be used in the above combinations or as independent antagonist combinations for treatment of a patient. Examples include ICAM-1, VCAM-1, VLA-4, CD18, CDlla, CDllb and CD31, and the mAb reactive with them.

The therapeutic agents may be administered orally, topically, or parenterally, (e.g., intranasally, subcutaneously, intramuscularly, intravenously, or intra- arterially) by routine methods in pharmaceutically acceptable inert carrier substances. Optimal dosage and modes of administration can readily be determined by conventional protocols.

The lyam-1 gene itself or portions thereof can be used to construct chimeric selectins. Thhe gene can also be used in genetic therapy. Individuals having a genetic defect in the lyam-1 gene would be unable to produce a fully active L- selectin leukocyte "homing" receptor and thus would be unable to mobilize sufficient leukocytes to a site of inflammation. Individuals suspected of having a congenital defect in the lyam-1 gene could be screened for this genetic disorder using

the sequence and structural information described. Treatment of affected individuals would then be possible using the lyam-1 gene or fragments thereof.

The normal regulation of the lyam-1 gene, as evidenced by the appearance and disappearance of L-selectin on the surface of a specific leukocyte sub-population can be monitored to test the effects of drugs or specific therapies that would alter gene expression.

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

(i) APPLICANT:

(A) NAME: Dana-Farber Cancer Institute, Inc. (B) STREET: 44 Binney Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 02115 (ii) TITLE OF INVENTION: CHIMERIC SELECTINS AS SIMULTANEOUS BLOCKING

AGENTS FOR COMPONENT SELECTIN FUNCTION

(iii) NUMBER OF SEQUENCES: 11

(iv) 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 (EPO)

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US94/00909

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/008,459

(B) FILING DATE: 25-JAN-1993

(vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/983,606

(B) FILING DATE: 30-NOV-1992

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/962,483

(B) FILING DATE: 02-APR-1992 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/770,608

(B) FILING DATE: 03-OCT-1991

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/737,092 (B) FILING DATE: 29-JUL-1991

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/730,503

(B) FILING DATE: 08-JUL-1991

(vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/700,773

(B) FILING DATE: 15-MAY-1991

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/313,109

(B) FILING DATE: 21-FEB-1989

2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2330 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D ) TOPOLOGY : linear ( ii ) MOLECULE TYPE : cDNA ( iii ) HYPOTHETICAL : NO ( iii ) ANTI-SENSE : NO

( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 53..1210

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

GAATTCCCTT TGGGCAAGGA CCTGAGACCC TTGTGCTAAG TCAAGAGGCT CA ATG 55

Met

1

GGC TGC AGA AGA ACT AGA GAA GGA CCA AGC AAA GCC ATG ATA TTT CCA 103 Gly Cys Arg Arg Thr Arg Glu Gly Pro Ser Lys Ala Met lie Phe Pro 5 10 15

TGG AAA TGT CAG AGC ACC CAG AGG GAC TTA TGG AAC ATC TTC AAG TTG 151 Trp Lys Cys Gin Ser Thr Gin Arg Asp Leu Trp Asn lie Phe Lys Leu 20 25 30

TGG GGG TGG ACA ATG CTC TGT TGT GAT TTC CTG GCA CAT CAT GGA ACC 199 Trp Gly Trp Thr Met Leu Cys Cys Asp Phe Leu Ala His His Gly Thr

35 40 45

GAC TGC TGG ACT TAC CAT TAT TCT GAA AAA CCC ATG AAC TGG CAA AGG 247 Asp Cys Trp Thr Tyr His Tyr Ser Glu Lys Pro Met Asn Trp Gin Arg 50 55 60 65 GCT AGA AGA TTC TGC CGA GAC AAT TAC ACA GAT TTA GTT GCC ATA CAA 295

Ala Arg Arg Phe Cys Arg Asp Asn Tyr Thr Asp Leu Val Ala lie Gin 70 75 80

AAC AAG GCG GAA ATT GAG TAT CTG GAG AAG ACT CTG CCT TTC AGT CGT 343 Asn Lys Ala Glu lie Glu Tyr Leu Glu Lys Thr Leu Pro Phe Ser Arg 85 90 95

TCT TAC TAC TGG ATA GGA ATC CGG AAG ATA GGA GGA ATA TGG ACG TGG 391 Ser Tyr Tyr Trp lie Gly lie Arg Lys lie Gly Gly lie Trp Thr Trp 100 105 110

GTG GGA ACC AAC AAA TCT CTC ACT GAA GAA GCA GAG AAC TGG GGA GAT 439 Val Gly Thr Asn Lys Ser Leu Thr Glu Glu Ala Glu Asn Trp Gly Asp

115 120 125

GGT GAG CCC AAC AAC AAG AAG AAC AAG GAG GAC TGC GTG GAG ATC TAT 487 Gly Glu Pro Asn Asn Lys Lys Asn Lys Glu Asp Cys Val Glu lie Tyr 130 135 140 145 ATC AAG AGA AAC AAA GAT GCA GGC AAA TGG AAC GAT GAC GCC TGC CAC 535 lie Lys Arg Asn Lys Asp Ala Gly Lys Trp Asn Asp Asp Ala Cys His 150 155 160

AAA CTA AAG GCA GCC CTC TGT TAC ACA GCT TCT TGC CAG CCC TGG TCA 583 Lys Leu Lys Ala Ala Leu Cys Tyr Thr Ala Ser Cys Gin Pro Trp Ser 165 170 175

TGC AGT GGC CAT GGA GAA TGT GTA GAA ATC ATC AAT AAT TAC ACC TGC 631 Cys Ser Gly His Gly Glu Cys Val Glu lie lie Asn Asn Tyr Thr Cys 180 185 190

AAC TGT GAT GTG GGG TAC TAT GGG CCC CAG TGT CAG TTT GTG ATT CAG 679 Asn Cys Asp Val Gly Tyr Tyr Gly Pro Gin Cys Gin Phe Val lie Gin

195 200 205

TGT GAG CCT TTG GAG GCC CCA GAG CTG GGT ACC ATG GAC TGT ACT CAC 727 Cys Glu Pro Leu Glu Ala Pro Glu Leu Gly Thr Met Asp Cys Thr His 210 215 220 225 CCT TTG GGA AAC TTC AAC TTC AAC TCA CAG TGT GCC TTC AGC TGC TCT 775

Pro Leu Gly Asn Phe Asn Phe Asn Ser Gin Cys Ala Phe Ser Cys Ser 230 235 240

GAA GGA ACA AAC TTA ACT GGG ATT GAA GAA ACC ACC TGT GAA CCA TTT 823 Glu Gly Thr Asn Leu Thr Gly lie Glu Glu Thr Thr Cys Glu Pro Phe 245 250 255

GGA AAC TGG TCA TCT CCA GAA CCA ACC TGT CAA GTG ATT CAG TGT GAG 871 Gly Asn Trp Ser Ser Pro Glu Pro Thr Cys Gin Val lie Gin Cys Glu 260 265 270

CCT CTA TCA GCA CCA GAT TTG GGG ATC ATG AAC TGT AGC CAT CCC CTG 919 Pro Leu Ser Ala Pro Asp Leu Gly lie Met Asn Cys Ser His Pro Leu

275 280 285

GCC AGC TTC AGC TTT ACC TCT GCA TGT ACC TTC ATC TGC TCA GAA GGA 967 Ala Ser Phe Ser Phe Thr Ser Ala Cys Thr Phe lie Cys Ser Glu Gly 290 295 300 305 ACT GAG TTA ATT GGG AAG AAG AAA ACC ATT TGT GAA TCA TCT GGA ATC 1015

Thr Glu Leu lie Gly Lys Lys Lys Thr lie Cys Glu Ser Ser Gly lie 310 315 320

TGG TCA AAT CCT AGT CCA ATA TGT CAA AAA TTG GAC AAA AGT TTC TCA 1063 Trp Ser Asn Pro Ser Pro lie Cys Gin Lys Leu Asp Lys Ser Phe Ser 325 330 335

ATG ATT AAG GAG GGT GAT TAT AAC CCC CTC TTC ATT CCA GTG GCA GTC 1111 Met lie Lys Glu Gly Asp Tyr Asn Pro Leu Phe lie Pro Val Ala Val 340 345 350

ATG GTT ACT GCA TTC TCT GGG TTG GCA TTT ATC ATT TGG CTG GCA AGG 1159 Met Val Thr Ala Phe Ser Gly Leu Ala Phe lie lie Trp Leu Ala Arg

355 360 365

AGA TTA AAA AAA GGC AAG AAA TCC AAG AGA AGT ATG AAT GAC CCA TAT 1207 Arg Leu Lys Lys Gly Lys Lys Ser Lys Arg Ser Met Asn Asp Pro Tyr 370 375 380 385 TAAATCGCCC TTGGTGAAAG AAAATTCTTG GAATACTAAA AATCATGAGA TCCTTTAAAT 1267

CCTTCCATGA AACGTTTTGT GTGGTGGCAC CTCCTACGTC AAACATGAAG TGTGTTTCCT 1327

TCAGTGCATC TGGGAAGATT TCTACCTGAC CAACAGTTCC TTCAGCTTCC ATTTCACCCC 1387

TCATTTATCC CTCAACCCCC AGCCCACAGG TGTTTATACA GCTCAGCTTT TTGTCTTTTC 1447

TGAGGAGAAA CAAATAAGAC CATAAAGGGA AAGGATTCAT GTGGAATATA AAGATGGCTG 1507 ACTTTGCTCT TTCTTGACTC TTGTTTTCAG TTTCAATTCA GTGCTGTACT TGATGACAGA 1567

CACTTCTAAA TGAAGTGCAA ATTTGATACA TATGTGAATA TGGACTCAGT TTTCTTGCAG 1627

ATCAAATTTC GCGTCGTCTT CTGTATACGT GGAGGTACAC TCTATGAAGT CAAAAGTCTA 1687

CGCTCTCCTT TCTTTCTAAC TCCAGTGAAG TAATGGGGTC CTGCTCAAGT TGAAAGAGTC 1747

CTATTTGCAC TGTAGCCTCG CCGTCTGTGA ATTGGACCAT CCTATTTAAC TGGCTTCAGC 1807

CTCCCCACCT TCTTCAGCCA CCTCTCTTTT TCAGTTGGCT GACTTCCACA CCTAGCATCT 1867 CATGAGTGCC AAGCAAAAGG AGAGAAGAGA GAAATAGCCT GCGCTGTTTT TTAGTTTGGG 1927

GGTTTTGCTG TTTCCTTTTA TGAGACCCAT TCCTATTTCT TATAGTCAAT GTTTCTTTTA 1987

TCACGATATT ATTAGTAAGA AAACATCACT GAAATGCTAG CTGCAACTGA CATCTCTTTG 2047

ATGTCATATG GAAGAGTTAA AACAGGTGGA GAAATTCCTT GATTCACAAT GAAATGCTCT 2107

CCTTTCCCCT GCCCCCAGAC CTTTTATCCA CTTACCTAGA TTCTACATAT TCTTTAAATT 2167 TCATCTCAGG CCTCCCTCAA CCCCACCACT TCTTTTATAA CTAGTCCTTT ACTAATCCAA 2227

CCCATGATGA GCTCCTCTTC CTGGCTTCTT ACTGAAAGGT TACCCTGTAA CATGCAATTT 2287

TGCATTTGAA TAAAGCCTGC TTTTTAAGTG TTAAAAAGAA TTC 2330

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 385 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Gly Cys Arg Arg Thr Arg Glu Gly Pro Ser Lys Ala Met lie Phe

1 5 10 15

Pro Trp Lys Cys Gin Ser Thr Gin Arg Asp Leu Trp Asn lie Phe Lys 20 25 30

Leu Trp Gly Trp Thr Met Leu Cys Cys Asp Phe Leu Ala His His Gly 35 40 45

Thr Asp Cys Trp Thr Tyr His Tyr Ser Glu Lys Pro Met Asn Trp Gin 50 55 60

Arg Ala Arg Arg Phe Cys Arg Asp Asn Tyr Thr Asp Leu Val Ala lie 65 70 75 80 Gin Asn Lys Ala Glu lie Glu Tyr Leu Glu Lys Thr Leu Pro Phe Ser

85 90 95

Arg Ser Tyr Tyr Trp lie Gly lie Arg Lys lie Gly Gly lie Trp Thr 100 105 110

Trp Val Gly Thr Asn Lys Ser Leu Thr Glu Glu Ala Glu Asn Trp Gly 115 120 125

Asp Gly Glu Pro Asn Asn Lys Lys Asn Lys Glu Asp Cys Val Glu lie 130 135 140

Tyr lie Lys Arg Asn Lys Asp Ala Gly Lys Trp Asn Asp Asp Ala Cys 145 150 155 160

His Lys Leu Lys Ala Ala Leu Cys Tyr Thr Ala Ser Cys Gin Pro Trp 165 170 175

Ser Cys Ser Gly His Gly Glu Cys Val Glu lie lie Asn Asn Tyr Thr 180 185 190 Cys Asn Cys Asp Val Gly Tyr Tyr Gly Pro Gin Cys Gin Phe Val lie

195 200 205

Gin Cys Glu Pro Leu Glu Ala Pro Glu Leu Gly Thr Met Asp Cys Thr 210 215 220

His Pro Leu Gly Asn Phe Asn Phe Asn Ser Gin Cys Ala Phe Ser Cys 225 230 235 240

Ser Glu Gly Thr Asn Leu Thr Gly lie Glu Glu Thr Thr Cys Glu Pro 245 250 255

Phe Gly Asn Trp Ser Ser Pro Glu Pro Thr Cys Gin Val lie Gin Cys 260 265 270 Glu Pro Leu Ser Ala Pro Asp Leu Gly lie Met Asn Cys Ser His Pro

275 280 285

Leu Ala Ser Phe Ser Phe Thr Ser Ala Cys Thr Phe lie Cys Ser Glu 290 295 300

Gly Thr Glu Leu lie Gly Lys Lys Lys Thr lie Cys Glu Ser Ser Gly 305 310 315 320 lie Trp Ser Asn Pro Ser Pro lie Cys Gin Lys Leu Asp Lys Ser Phe 325 330 335

Ser Met lie Lys Glu Gly Asp Tyr Asn Pro Leu Phe lie Pro Val Ala 340 345 350 Val Met Val Thr Ala Phe Ser Gly Leu Ala Phe lie lie Trp Leu Ala

355 360 365

Arg Arg Leu Lys Lys Gly Lys Lys Ser Lys Arg Ser Met Asn Asp Pro 370 375 380

Tyr 385

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1192 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

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

AGAGAGCTGT TATAAAGATT AAACAATATA ATAAATATGG CGCGTGAGCT TCAGAGTTTT 60

TGTTGTTGTT ATTATTATTT TCCTAAAAAT GCAAATCTGA TTTGCATTTG ACTCATTGAC 120

TCACATCAGT GGGTCTTCCT TTTTATTGTC CTTCATCATA TGGGTCCTAA TTTCACATGC 180 AGTCTTATAA AACCATCTCA TTTTATAGTC CAAGAATATT AAAGGTACTT GTAGGCTCCC 240

AAACCTACAC GGTGAAAAGC TAGAGAGCAT GGGCTCTCTT CAGGGGTTAA CTTCAGGAAG 300

TGCCACTAAC AAGGACGTCC ACTAGGTGGT GAGCAAGGAA AGACGGAGGT GAAGGAACCG 360

AAACGAGTCA AGTCCACTGC TTAGCTCTAC TGAAGTTTTG CAAACATCAT AAATATGTCT 420

GAAATGCAGT TTTGATTTGT AGTATTTGCA ATTTCCAAGG GCCATTTACC ACAGGTAGCC 480 AAGAGTTAGT TTAGCATTTA TGAAAAAGAT AGGGGAGGGT GGTGGTTAAG AAGGAGGTGG 540

AGGAGAGAGT GAAGGAGGAA GAGGAGAACA AGAACCAAAC AAAAACAAGA ACAAGAACAA 600

GTAGAAGAAG AGGAGCAGGG AGGAAAAAGA AGAGGAAGAA GAACAGCAAC AACAATGAGT 660

GAAGGAGGAG GAGGGTAAGG AAAGATGCAT AGGAGAATGG AAGGAAGGAT AGAAAGGAGG 720

GAAGGAAGAG AGAATCTAGT CACATTACTT TCTGATCAGC AGTTCATTTT TGTCTCAGTG 780 GGAGGCAATA GAGGCCAGTC TAGGAAAGGG GTGGGGAAAG AGGAAAGAGA AGTGCAGGAG 840

GAAGGGGAGG CCCAAGGGGA GGAGGAGGAG GATGTGAGAC TGGGTTAGAG AAATGAAAGA 900

AAGCAAGGCT TTCTGTTGAC ATTCAGTGCA GTCTACCTGC AGCACAGCAC ACTCCCTTTG 960

GGCAAGGACC TGAGACCCTT GTGCTAAGTC AAGAGGCTCA ATGGGCTGCA GAAGAACTAG 1020

AGAAGGACCA AGCAAAGCCA TGGTGAGCCT TTCAGCCTAA AAGACGTTTA GATGCTCAGA 1080 TAGAAACTCT TGGGGTTGTA GAGGCAGGTG GCAAGGATAG GAATCACCCC ATTTCAATTC 1140

TGGTTTTAAA TAATATAGAA ACTAAACATT TTCTCAGACC CTCAAAAAAA GT 1192

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 363 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

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

CACTGAGACT AAGCGTAAAA TAAATAGAAC AAACAAACTG TGCATCAGTT CTGATGTAAA 60

TTTGAAGTAA TTTTCATCTA TGTCTGAGAA ACCTGTTACC TCAGACAGGG TTAGTAGACA 120

TATGTGTTTT ATTCTGATTA TTAAGAAAGT TGTAAGCACC ACCTCAAAGG CTATAAATGT 180

GTGGTTTAAG GGTATACATC TAAATATAAT TTTGTATTTC ATTTGCAGAT ATTTCCATGG 240

AAATGTCAGA GCACCCAGAG GGACTTATGG AACATCTTCA AGTTGTGGGG GTGGACAATG 300

CTCTGTTGTG GTATGTTATG ATATTTATAT ATCACTAAGT CTATTTTACT TATATTCATT 360 TTT 363

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 531 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CTGGAGTAGT GCTAGGTTCT TTTTAGCTGT AACATTATGT AAGTCTGCAT AGGTCACACT 60

GATGTCTTGC AGATTTCCTG GCACATCATG GAACCGACTG CTGGACTTAC CATTATTCTG 120

AAAAACCCAT GAACTGGCAA AGGGCTAGAA GATTCTGCCG AGACAATTAC ACAGATTTAG 180 TTGCCATACA AAACAAGGCG GAAATTGAGT ATCTGGAGAA GACTCTGCCT TTCAGTCGTT 240

CTTACTACTG GATAGGAATC CGGAAGATAG GAGGAATATG GACGTGGGTG GGAACCAACA 300

AATCTCTCAC TGAAGAAGCA GAGAACTGGG GAGATGGTGA GCCCAACAAC AAGAAGAACA 360

AGGAGGACTG CGTGGAGATC TATATCAAGA GAAACAAAGA TGCAGGCAAA TGGAACGATG 420

ACGCCTGCCA CAAACTAAAG GCAGCCCTCT GTTACACAGG TAGGGAGTGA CAAGACGGCT 480 ATGCTGCCTC AGACTCAGGA AGGGCCACGG TTAAGAGAAT ACTCAGATTT A 531 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 832 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

AAAATTTTAG CCATATGATT TTTATGCTAT GAATTTACCA AATAAACCTT TCCTGATTAT 60

TTAAATCATC TCAGACAAAA GGTTATCTAT GTCTAAAGAA ATGACTTTGA GTACTAAAAT 120 GTAATCACAT TAAAATATTT TTTTTCTGAC CTCCTTAAAG CTTCTTGCCA GCCCTGGTCA 180

TGCAGTGGCC ATGGAGAATG TGTAGAAATC ATCAATAATT ACACCTGCAA CTGTGATGTG 240

GGGTACTATG GGCCCCAGTG TCAGTTTGGT AAGTCTCTTT CCTTTCTTTG CTTCTTCTTA 300

GGTAAAGTCA CAGGAATCAT TATAGCTTAT CATGAAGCTG GTTGGAACAA AATGATACTA 360

GCCACTCTGA GAAATGGGAA GTTTTGATCA GAAAGCTCTG CTTTCACAAT ATTGTTACCT 420 TTCCGTAAAG ATTTCATAAG TCAGCATGAA GTTTCGATTC ACTTCTCAAC AAGTCTTTTT 480

GAGTACCACA AGAAGCACAG TGTTGGGATA AAGCTGTCAG GGTTACAATA AGGAATTAGC 540

ATGGTAGATT CCCGCTCTCA AGAAGCTCAC GATCTAATGA GCTTGTTAGA TTAATTAGAA 600

CTCTAAGGTC TGGAAGAAAC TATGCCATTT ATCATTAGGA GGCTGAGTTA CCCAGAAAGT 660

ATCTTGCTTT TTCCTTCTAG TAGTTCCTTT CCTTCTTGCA GTTCTCCACA CTTAACACAT 720 GTGCTCTGTA GCACACTGAC TTTGCTGGTG GCCTTCTCTC TCATTTTGCA CATGGCCAAA 780

AAACATGTCA TCTTTAAGAC ATTGTTCAAA GACAGTTTCT TCTAGGAAGC TT 832

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 712 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CTCTGATGTG ATAGTTATTT CCCGACTAAG CTGGTCATTC CCAGTTACAC CTATTTGGCT 60

TTAAGGATTC TCACTACAGA TAATACTGAA GATAATAATA TGAAGACTAG CTAATGTTTA 120 CTTAGAATTT CTGATGAGTC AGGCTTTGTT CTAACGTCCT TGACTTATGC TAATTGAATT 180

ACATTTAGTT TCCATATCAA TTTGATAAAG ATAACACAAT TTCATTATTC CTCTTATATA 240

GATGAAGAAA CTGAAGTTGG AGGGTTCAAG TAACCTTGTT TAAAGGCACA TGGTTATCAA 300

GTGGCAGGGC TAGGATTCAA ATCCAGGCGT CAGTTCCTCT TAACTCTTCC CCATACTGTT 360

TCTTTCCCTA TTGAAGTGAT TCAGTGTGAG CCTTTGGAGG CCCCAGAGCT GGGTACCATG 420

GACTGTACTC ACCCTTTGGG AAACTTCAGC TTCAGCTCAC AGTGTGCCTT CAGCTGCTCT 480

GAAGGAACAA ACTTAACTGG GATTGAAGAA ACCACCTGTG GACCATTTGG AAACTGGTCA 540

TCTCCAGAAC CAACCTGTCA AGGTGAGTAA CTTCAGACTA GAGGTTTTGT CATGCAATCC 600

TGGGCTTACA GTCAGAACAT TCAGTAGAAG TTTGCTGAGA AGTCAAACTT AGGATCCTAA 660 TTTAACCTAA CTTTTGTTTA ACCTACTGTG ATGTTTCTCA AAGGACTTAT TC 712

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 451 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GAGGGTCACC TTAGCTAGGG CAGCAGCCTG GAGTAGCTAC TCCTCTCCCC ACAGCTTTCA 60

ATGCTTCCTT GCCTTCATCT CTCATTCACC ACCCACCATC ATTCTCAAGA AAATAAAGCC 120

TGGAAGCAAT ATCACAAGTA ATGTAGTCAG GCAGCTTTGG CTAAAAATCC AAAGCTCAAG 180 GGAGGGTCTC TACTCAGAAA TACTGTTTTG TCTTTTTTTT TTTTTCTTTT TCATTGAAGT 240

GATTCAGTGT GAGCCTCTAT CAGCACCAGA TTTGGGGATC ATGAACTGTA GCCATCCCCT 300

GGCCAGCTTC AGCTTTACCT CTGCATGTAC CTTCATCTGC TCAGAAGGAA CTGAGTTAAT 360

TGGGAAGAAG AAAACCATTT GTGAATCATC TGGAATCTGG TCAAATCCTA GTCCAATATG 420

TCAAAGTGAG TAAGTTTGTC CTGGAACTGA A 451 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 544 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TATCAGAACT AAGAAAGCTT GGGCTGCAGG TCGACTCTAG GTGCATTTTC AGGAACTCTA 60

TGAACCACAA ATCTGGGCAT TGAGATTCTG TAGGCATTAG ACTAGCAAGG CTGGTCAGTC 120

TTTGCCTATG CTGTAGACTC ATCAGGGGCC TTCCCATGCC AGTTTCCTCA TCTGTCAAAT 180 GGCATCATTT GGGCTACTAC TGGGAGATGT AAGGAGGAAA AAAGTCAAAT ATCATGAGAT 240

AGACTAAGGA AATAATGCTG GTGGTCTCAT GCTATGTGCC TTACTGATTT CTCTTTCAGA 300

ATTGGACAAA AGTTTCTCAA TGATTAAGGA GGGTGATTAT AACCCCCTCT TCATTCCAGT 360

GGCAGTCATG GTTACTGCAT TCTCTGGGTT GGCATTTATC ATTTGGCTGG CAAGGAGATT 420

AAAAAAAGGT ATGTGAGTTT AACTTCACAT GAAAAGAACA CAACTTTAAA GTGAAAAAGA 480 AAAAAAAAAG AAACCCACAG GAAATTAAAT GTGATAGATT CAACACAAGC AGGATGCCAA 540

GCTT 544

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 524 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TAGTTTACAG TATTAGCAGC TGTCCCTCAA GGAAGAATCT GCAGGTAGAT GAGATGCAGA 60

TTGGGTGGGA TAAACACTTG AATGACATAT TGGGTCTTGC CACCAGGCAA TTTAGCAATT 120 CTGTCTTCTT GAGTAGCACG GAGATGGAAT GGAACCTCAG GAGGCATCTG CATCAACATG 180

TCTGTTCTGT ATTAGTGTCT ACCACTGTTT ATTAAGCCAG TTCCTCAAAT CTCCTTTGAC 240

ACAGATAGGG TCCACCTAAC AAATACCTAA TATACTTCAA AAGACAGTTT TGAGAGTGGG 300

AGTCTTCCTT CTCCCTTACT TGAAAAACTT TAAATTGTCT AATTTTTGCT AATGCCTTTT 360

TCTCTATTTT CTATTTCAGG CAAGAAATCC AAGAGAAGGT AAGTTTTATT AGTGGCGAGG 420 AGTTTCCACA TCTGCTGATT CATTCTCTAC TTCTTAAGTT ACTTCTGCTC TAGCTAGACA 480

CATACCCATA GTAGTTATTA CTGGGTCTAT CAATGACAGA TAGG 524

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1696 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ATAAGCATCA CTAAAGAGCT TGTTAGGGGT GCAGAATCTC AGGCTCCACT CAGACCTACT 60

GAATCAGAGT CTGCATTTTA ACACCATCTC TGAGTGGTAA GGACATGAAA ATCTGAGAAG 120

TGCTGCTACT AGGGTTTGCT TACATTTGTT CATCTTCAGA GGTTCCTAAA GCCTGGCCTC 180 TTGTCTGAGA TTTCCAGCTG AAAGCATTTC CTTGCTCCTC TTCTCATCTC TAATGAATAT 240

TTACCTTTAC TACTAACACT CCAAGTTTTG CAATTTTTAA ACTCTTATTA TCTTTTGTTT 300

TTCTTTCAGT ATGAATGACC CATATTAAAT CGCCCTTGGT GAAAGAAAAT TCTTGGAATA 360

CTAAAAATCA TGAGATCCTT TAAATCCTTC CATGAAACGT TTTGTGTGGT GGCACCTCCT 420

ACGTCAAACA TGAAGTGTGT TTCCTTCAGT GCATCTGGGA AGATTTCTAC CTGACCAACA 480 GTTCCTTCAG CTTCCATTTC ACCCCTCATT TATCCCTCAA CCCCCAGCCC ACAGGTGTTT 540

ATACAGCTCA GCTTTTTGTC TTTTCTGAGG AGAAACAAAT AAGACCATAA AGGGAAAGGA 600

TTCATGTGGA AT TAAAGAT GGCTGACTTT GCTCTTTCTT GACTCTTGTT TTCAGTTTCA 660

ATTCAGTGCT GTACTTGATG ACAGACACTT CTAAATGAAG TGCAAATTTG ATACATATGT 720

GAATATGGAC TCAGTTTTCT TGCAGATCAA ATTTCGCGTC GTCTTCTGTA TACGTGGAGG 780 TACACTCTAT GAAGTCAAAA GTCTACGCTC TCCTTTCTTT CTAACTCCAG TGAAGTAATG 840

GGGTCCTGCT CAAGTTGAAA GAGTCCTATT TGCACTGTAG CCTCGCCGTC TGTGAATTGG 900

ACCATCCTAT TTAACTGGCT TCAGCCTCCC CACCTTCTTC AGCCACCTCT CTTTTTCAGT 960

TGGCTGACTT CCACACCTAG CATCTCATGA GTGCCAAGCA AAAGGAGAGA AGAGAGAAAT 1020

AGCCTGCGCT GTTTTTTAGT TTGGGGGTTT TGCTGTTTCC TTTTATGAGA CCCATTCCTA 1080 TTTCTTATAG TCAATGTTTC TTTTATCACG ATATTATTAG TAAGAAAACA TCACTGAAAT 1140

GCTAGCTGCA ACTGACATCT CTTTGATGTC ATATGGAAGA GTTAAAACAG GTGGAGAAAT 1200

TCCTTGATTC ACAATGAAAT GCTCTCCTTT CCCCTGCCCC CAGACCTTTT ATCCACTTAC 1260

CTAGATTCTA CATATTCTTT AAATTTCATC TCAGGCCTCC CTCAACCCCA CCACTTCTTT 1320

TATAACTAGT CCTTTACTAA TCCAACCCAT GATGAGCTCC TCTTCCTGGC TTCTTACTGA 1380 AAGGTTACCC TGTAACATGC AATTTTGCAT TTGAATAAAG CCTGCTTTTT AAGTGTTAAC 1440

TAGTTTGCCT AGTTTGTTAT TTTGAAAATT GATCATATGT TTTGTTTTCT CCCCAGTGAG 1500

TTACATGCTC CTTCAGGGCA GAGTTTGTGT CAGATCCCTG GAGTATCTAG TGCATTACTT 1560

GACACTCAAT AAATGAATGT TCAAATAAAT CAGAAAGAGC ATACAGTGCA CTGCTGATAT 1620

AAGTTTCAGC ATCCCTCTTT CTCTATGGCA TCTGATGACC TGGGTCAGAT ATCACCTAAT 1680

GTCAACAGCT GAATTC 1696