BACKGROUND OF THE INVENTION PATHOGENS THAT ENTER MONONUCLEAR CELLS Pathogens may infect a patient via a number of mechanisms. One such mechanism is by infecting and subsequently parasitizing mononuclear cells such as monocytes or macrophages.

This mechanism has been observed in Leishmania (Berman et al., 1979, Infect. Immun. 26: 375- 379), Listeria, Mycobacterium tuberculosis (Schlesinger et al., 1990, J. Immunol. 144: 2771- 2780), and human immune deficiency virus (Hegde, 1995, Medical Hypotheses 45: 443-440).

Attempts have been made to prevent the entry of these pathogens into monocytes or macrophages by treatment with cytokines such as granulocyte macrophage colony stimulating (GMCSF), interferon-gamma, interleukins such as IL-3 and IL-7, and tissue necrosis factor- alpha (Al-Zamel et al., 1996, Zbl. Bakt. 285: 92-105 and Zarniecki and Sonnenfeld, 1993, APMIS 101: 1-17). However these treatments have only been of limited success.

It is therefore an object of the invention to provide a more effective method for inhibiting entry of a pathogen into a monocyte or macrophage.

HEPARIN-BINDING PROTEIN The covalent structure of two closely related proteins isolated from peripheral neutrophil leukocytes of human and porcine origin have recently been determined (cf. H. Flodgaard et al., Eur. J. Biochem. 197,1991, pp. 535-547; J. Pohl et al., FEBS Lett. 272,1990, p. 200 ff.). Both proteins show a high similarity to neutrophil elastase, but owing to selective mutations of the active serine 195 and histidine 57 (chymotrypsin numbering (B. S. Hartley,"Homologies in Serine Proteinases", Phil. Trans. Roy. Soc. Series 257,1970, p. 77 ff.)) the proteins lack protease activity. The proteins have been named human heparin-binding protein (hHBP) and porcine

heparin-binding protein (pHBP), respectively, owing to their high affinity for heparin; Schafer et al. (W. M. Schafer et al., Infect. Immun. 53,1986, p. 651 ff.) have named the protein cationic antimicrobial protein (CAP37) due to its antimicrobial activity. The protein has been found to regulate monocyte/macrophage functions such as chemotaxis, increased survival, and differentiation (reviewed in Pereira, 1995, J. Leuk. Biol. 57 : 805-812, also see U. S. Patent Nos.

5,458,874 and 5,484,885). However, there has been no disclosure as to whether it would inhibit infection of monocytes or macrophages by a pathogen.

Furthermore, HBP has been shown to mediate detachment and contraction of endothelial cells and fibroblasts when added to such cells grown in monolayer culture. HBP also stimulates monocyte survival and thrombospondin secretion (E. Ostergaard and H. Flodgaard, J. Leukocyte Biol. 51,1992, p 316 ff).

From the azurophil granules, a protein with the first 20 N-terminal amino acid residues identical to those of hHBP and CAP37 called azurocidin has also been isolated (J. E. Gabay et al., Proc. Natl. Acad. Sci. USA 86,1989, p. 5610 ff.; C. G. Wilde et al., J. Biol. Chem. 265, 1990, p. 2038 ff.) and its antimicrobial properties have been reported (D. Campanelli et al., J.

Clin. Invest. 85,1990, p. 904 ff.).

The presence of hHBP in the neutrophil leucokytes and the fact that 89% of CAP37 (which is identical to hHBP) is released when the leukocytes are phagocytosing Staph. aureus (H. A. Pereira et al., op. cit.) indicate that a function of hHBP could be its involvement in the inflammatory process since the protein is apparently released from activated neutrophils. Pereira et al., op. cit., suggested a function of CAP37 to be at the site of inflammation where it could specifically attract monocytes and thus be one of the factors responsible for the influx of mono- cytes in the second wave of inflammation. Ostergaard and Flodgaard, op. cit., suggest that, in addition to being important for the recruitment of monocytes, HBP might play a key role in the mechanism of neutrophil as well as monocyte extravasation.

The structure of HBP appears from WO 89/08666 and H. Flodgaard et al., op. cit. HBP has otherwise been termed CAP37 (cf. WO 91/00907, U. S. Patent Nos. 5,458,874 and 5,484,885) and azurocidin (cf. C. G. Wilde et al., J. Biol. Chem. 265,1990, p. 2038).

SUMMARY OF THE INVENTION It has surprisingly been found that a composition comprising heparin-binding protein

and an effective carrier or diluent can inhibit the entry of pathogens into mononuclear cells of a patient. Thus, the invention is directed to a method for inhibiting entry of a pathogen into mononuclear cells of a patient comprising administering a pharmaceutical composition comprising: (a) a mammalian heparin-binding protein which (i) in glycosylated form, has a molecular weight of about 28 kD as determined by SDS-PAGE under reducing conditions; (ii) is produced in the azurophil granules of polymorphonuclear leukocytes; and (iii) is a chemoattractant for monocytes; and (b) a pharmaceutically acceptable carrier or diluent in an amount effective to inhibit entry of said pathogens in said mononuclear cells. The composition may further comprise a cytokine.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A and 1 B respectively show the constructs used, pcDNA3-HBP and pcDNA pro.

Figure 2 shows Coomassie-stained SDS-PAGE (8-15%) comparing different HBP forms. Lane 1: recombinant HBP obtained from RBL-lcells. Lane 2: recombinant HBP obtained from insect cells.

Figure 3 shows a peptide map of human HBP (top), RBL-1 HBP (middle) and insect cell HBP (bottom).

Figure 4 shows a carbohydrate map of the three HBP samples after release and 2-AB labelling of the carbohydrates. CarboPac PA-100 column, sodium acetate gradient at high pH, fluorescence detection.

Figure 5 shows a carbohydrate map of the three HBP samples after release, 2-AB labelling and desialylation of the carbohydrates. Conditions as in Figure 4, except the gradient is modified.

Figure 6 shows size-exclusion chromatography (SEC) of the three HBP samples after release, 2-AB labelling and desialylation of the carbohydrates. Top panel: Human HBP; Middle panel: RBL-1 HBP; Bottom panel: Insect cell HBP.

Figure 7 shows MALDI-MS of the three HBP samples. Top panel: Human HBP; Middle panel: RBL-1 HBP; Bottom panel: Insect cell HBP.

Figure 8 shows CE of HBP samples in 25 mM ammonium phosphate, 0.05% polyvinyl alcohol, pH 2.5. Capillary: fused silica with Z-cell (LC Packings), ID 75 Am, L 45

cm, I 37.5 cm. Inj.: 10 sec. Voltage: 11 kV-31 pA. Detection: 214 nm.

Figure 9 shows CE of HBP samples in 25 mM ammonium phosphate, 0.005% polyarginine, pH 6.5. Capillary: fused silica with Z-cell (LC Packings), ID 75 Am, L 45 cm, 137.5 cm. Inj.: 20 sec. Voltage: 11 kV 23 A. Reversed polarity. Detection: 214 nm.

Figure 10 shows cation-exchange HPLC of the three HBP samples. Column: MonoS HR 5/5, NaCI gradient in 20 mM Ches, pH 9.0, UV detection 280 nm.

Figure 11 GP-HPLC of the three HBP samples. Column, Waters Protein-Pak 125,7.8 x 300 mm, 0.2 M ammonium sulfate, 5% isopropanol, pH 5.0, UV detection 215 nm.

DETAILED DESCRIPTION OF THE INVENTION The HBP may suitably be of mammalian, in particular human or porcine, origin. In particular, the HBP is a mature human HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO: 1, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% (hereinafter"homologous polypeptides"), which qualitative retain the activity of said heparin-binding protein, or a fragment thereof which inhibits the entry of a pathogen into a mononuclear (e. g., monocyte or macrophage) cells of a patient. Alternatively, the HBP is a mature porcine HBP which has at least about an 80% identity with the amino acid sequence set forth in SEQ ID NO : 2, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%, which qualitative retain the activity of said heparin-binding protein, or a fragment thereof which inhibits the entry of a pathogen into a mononuclear (e. g., monocyte or macrophage) cells of a patient.

In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NOS: 1 or 2. The degree of identity between two or more amino acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443-453). For purposes of determining the degree of identity between two amino acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

The amino acid sequences of the homologous polypeptides differ from the amino acid sequence set forth in SEQ ID NOS: 1 or 2 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is, conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine) and small amino acids (such as glycine, alanine, serine, threonine and methionine).

Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, e. g., by H. Neurath and R. L. Hill, 1979, in, The Proteins, Academic Press, New York. The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse.

The heparin binding protein may be encoded by a nucleic acid sequence having at least about an 80% identity with the nucleic acid sequence set forth in SEQ ID NO: 3 (which encodes mature human HBP depicted in SEQ ID NO: 1), SEQ ID NO: 5 (which encodes a human HBP which includes the pro sequence and sequence of the mature protein, depicted in SEQ ID NO: 6), SEQ ID NO: 7 (which encodes human HBP which includes the signal sequence, the pro sequence and sequence of the mature protein, depicted in SEQ ID NO: 8) or SEQ ID NO: 4 (which encodes porcine HBP depicted in SEQ ID NO: 2), SEQ ID NO: 9 (which encodes a porcine HBP which includes the pro sequence and sequence of the mature protein depicted in SEQ ID NO: 10), SEQ ID NO: 11 (which encodes porcine HBP which includes the signal sequence, the pro sequence and sequence of the mature protein depicted in SEQ ID NO: 12), more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%, as determined by agarose gel electrophoresis. The nucleic acid

sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVSTVVLGA Y DLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLPLQNATV EAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNNVCTGVLTRRGGICNGD GGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGPGPA* (SEQ IDNO: 1) IVGG RRAQPQEFPF LASIQKQGRP FCAGALVHPR FVLTAASCFR GKNSGSASVV LGAYDLRQQE QSRQTFSIRS ISQNGYDPRQ NLNDVLLLQL DREARLTPSV ALVPLPPQNA TVEAGTNCQVEAGWGTQRLRR LFSRFPRVLN VTVTSNPCLP RDMCIGVFSR RGRISQGDRG TPLVCNGLAQ GVASFLRRRF RRSSGFFTRV ALFRNWIDSV LNNPPA* (SEQ ID NO: 2) ATCGTTGGCGGC CGGAAGGCGA GGCCCCGCCA GTTCCCGTTC CTGGCCTCCA TTCAGAATCA AGGCAGGCAC TTCTGCGGGG GTGCCCTGAT CCATGCCCGCTTCGTGATGA CCGCGGCCAG CTGCTTCCAA AGCCAGAACC CCGGGGTTAG CACCGTGGTG CTGGGTGCCT ATGACCTGAG GCGGCGGGAG AGGCAGTCCC GCCAGACGTT TTCCATCAGCAGCATGAGCG AGAATGGCTA CGACCCCCAG CAGAACCTGA ACGACCTGAT GCTGCTTCAG CTGGACCGTG AGGCCAACCT CACCAGCAGC GTGACGATAC TGCCACTGCC TCTGCAGAACGCCACGGTGG AAGCCGGCAC CAGATGCCAG GTGGCCGGCT GGGGGAGCCA GCGCAGTGGG GGGCGTCTCT CCCGTTTTCC CAGGTTCGTC AACGTGACTG TGACCCCCGA GGACCAGTGTCGCCCCAACA ACGTGTGCAC CGGTGTGCTC ACCCGCCGCG GTGGCATCTG CAATGGGGAC GGGGGCACCC CCCTCGTCTG CGAGGGCCTG GCCCACGGCG TGGCCTCCTT TTCCCTGGGGCCCTGTGGCC GAGGCCCTGA CTTCTTCACC CGAGTGGCGC TCTTCCGAGA CTGGATCGAT GGCGTTTTAA ACAATCCGGG ACCGGGGCCA GCCTAG * (SEQ ID NO: 3)

AT TGTGGGCGGC AGGAGGGCCC AGCCGCAGGA GTTCCCGTTT CTGGCCTCCA TTCAGAAACA AGGGAGGCCC TTTtGCGCCG GAGCCCTGGT CCATCCCCGC TTCGTCCTGA CAGCGGCCAG CTGCTTCCGT GGCAAGAACA GCGGAAGTGC CTCTGTGGTG CTGGGGGCCT ATGACCTGAG GCAGCAGGAG CAGTCCCGGC AGACATTCTC CATCAGGAGC ATCAGCCAGA ACGGCTATGA YCCCCGGCAG AATCTGAACG ATGTGCTGCT GCTGCAGCTG GACCGTGAGG CCAGACTCAC CCCCAGTGTG GCCCTGGTAC CGCTGCCCCC GCAGAATGCC ACAGTGGAAG CTGGCACCAA CTGCCAAGTTGCGGGCTGGG GGACCCAGCG GCTTAGGAGG CTTTTCTCCC GCTTCCCAAG GGTGCTCAAT GTCACCGTGA CCTCAAACCC GTGTCTCCCC AGAGACATGT GCATTGGTGT CTTCAGCCGC CGGGGCCGCA TCAGCCAGGG AGACAGAGGC ACCCCCCTCG TCTGCAACGG CCTGGCGCAG GGCGTGGCCT CCTTCCTCCG GAGGCGTTTC CGCAGGAGCT CCGGCTTCTT CACCCGCGTG GCGCTCTTCA GAAATTGGAT TGATTCAGTT CTCAACAACC CGCCGGCCTGA* (SEQ ID N0: 4) GGCTCCAGCCCCC TTTTGGAC ATCGTTGGCGGC CGGAAGGCGA GGCCCCGCCA GTTCCCGTTC CTGGCCTCCA TTCAGAATCA AGGCAGGCAC TTCTGCGGGG GTGCCCTGAT CCATGCCCGCTTCGTGATGA CCGCGGCCAG CTGCTTCCAA AGCCAGAACC CCGGGGTTAG CACCGTGGTG CTGGGTGCCT ATGACCTGAG GCGGCGGGAG AGGCAGTCCC GCCAGACGTT TTCCATCAGCAGCATGAGCG AGAATGGCTA CGACCCCCAG CAGAACCTGA ACGACCTGAT GCTGCTTCAG CTGGACCGTG AGGCCAACCT CACCAGCAGC GTGACGATAC TGCCACTGCC TCTGCAGAACGCCACGGTGG AAGCCGGCAC CAGATGCCAG GTGGCCGGCT GGGGGAGCCA GCGCAGTGGG GGGCGTCTCT CCCGTTTTCC CAGGTTCGTC AACGTGACTG TGACCCCCGA GGACCAGTGTCGCCCCAACA ACGTGTGCAC CGGTGTGCTC ACCCGCCGCG GTGGCATCTG CAATGGGGAC GGGGGCACCC CCCTCGTCTG CGAGGGCCTG GCCCACGGCG TGGCCTCCTT TTCCCTGGGGCCCTGTGGCC GAGGCCCTGA CTTCTTCACC CGAGTGGCGC TCTTCCGAGA CTGGATCGAT GGCGTTTTAA ACAATCCGGG ACCGGGGCCA GCCTAG * (SEQ ID NO:5)

GSSPLLDIVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVST VVLGAYDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLP LQNATVEAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNNVCTGVLTRRG GICNGDGGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGPGPA* ( SEQ ID NO: 6) ATGACCCGGC TGACAGTCCT GGCCCTGCTG GCTGGTCTGC TGGCGTCCTC GAGGGCC GGCTCCAGCCCCC TTTTGGAC ATCGTTGGCGGC CGGAAGGCGA GGCCCCGCCA GTTCCCGTTC CTGGCCTCCA TTCAGAATCA AGGCAGGCAC TTCTGCGGGG GTGCCCTGAT CCATGCCCGCTTCGTGATGA CCGCGGCCAG CTGCTTCCAA AGCCAGAACC CCGGGGTTAG CACCGTGGTG CTGGGTGCCT ATGACCTGAG GCGGCGGGAG AGGCAGTCCC GCCAGACGTT TTCCATCAGCAGCATGAGCG AGAATGGCTA CGACCCCCAG CAGAACCTGA ACGACCTGAT GCTGCTTCAG CTGGACCGTG AGGCCAACCT CACCAGCAGC GTGACGATAC TGCCACTGCC TCTGCAGAACGCCACGGTGG AAGCCGGCAC CAGATGCCAG GTGGCCGGCT GGGGGAGCCA GCGCAGTGGG GGGCGTCTCT CCCGTTTTCC CAGGTTCGTC AACGTGACTG TGACCCCCGA GGACCAGTGTCGCCCCAACA ACGTGTGCAC CGGTGTGCTC ACCCGCCGCG GTGGCATCTG CAATGGGGAC GGGGGCACCC CCCTCGTCTG CGAGGGCCTG GCCCACGGCG TGGCCTCCTT TTCCCTGGGGCCCTGTGGCC GAGGCCCTGA CTTCTTCACC CGAGTGGCGC TCTTCCGAGA CTGGATCGAT GGCGTTTTAA ACAATCCGGG ACCGGGGCCA GCCTAG * (SEQ ID NO: 7) IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVSTVVLGA YDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLPLQNA TVEAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNNVCTGVLTRRGGIC NGDGGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGPGPA* (SEQ ID NO: 8) ATGCCAGCAC TCAGATTCCT GGCCCTGCTG GCCAGCCTGC TGGCAACCTC CAGGGTT AT TGTGGGCGGC AGGAGGGCCC AGCCGCAGGA GTTCCCGTTT

CTGGCCTCCA TTCAGAAACA AGGGAGGCCC TTTTGCGCCG GAGCCCTGGT CCATCCCCGC TTCGTCCTGA CAGCGGCCAG CTGCTTCCGT GGCAAGAACA GCGGAAGTGC CTCTGTGGTG CTGGGGGCCT ATGACCTGAG GCAGCAGGAG CAGTCCCGGC AGACATTCTC CATCAGGAGC ATCAGCCAGA ACGGCTATGA YCCCCGGCAG AATCTGAACG ATGTGCTGCT GCTGCAGCTG GACCGTGAGG CCAGACTCAC CCCCAGTGTG GCCCTGGTAC CGCTGCCCCC GCAGAATGCC ACAGTGGAAG CTGGCACCAA CTGCCAAGTTGCGGGCTGGG GGACCCAGCG GCTTAGGAGG CTTTTCTCCC GCTTCCCAAG GGTGCTCAAT GTCACCGTGA CCTCAAACCC GTGTCTCCCC AGAGACATGT GCATTGGTGT CTTCAGCCGC CGGGGCCGCA TCAGCCAGGG AGACAGAGGC ACCCCCCTCG TCTGCAACGG CCTGGCGCAG GGCGTGGCCT CCTTCCTCCG GAGGCGTTTC CGCAGGAGCT CCGGCTTCTT CACCCGCGTG GCGCTCTTCA GAAATTGGAT TGATTCAGTT CTCAACAACC CGCCGGCCTGA* (SEQ ID NO: 9) MPALRFLALL ASLLATSRV IVGG RRAQPQEFPF LASIQKQGRP FCAGALVHPR FVLTAASCFR GKNSGSASVV LGAYDLRQQE QSRQTFSIRS ISQNGYDPRQ NLNDVLLLQL DREARLTPSV ALVPLPPQNA TVEAGTNCQV AGWGTQRLRR LFSRFPRVLN VTVTSNPCLP RDMCIGVFSR RGRISQGDRG TPLVCNGLAQ GVASFLRRRF RRSSGFFTRV ALFRNWIDSV LNNPPA* (SEQ ID NO: 10) ATG CCAGCAC TCAGATTCCT GGCCCTGCTG GCCAGCCTGC TGGCAACCTC CAGG GTT GGC TTG GCC ACC CTG GCA GAC ATT GTGGGCGGC AGGAGGGCCC AGCCGCAGGA GTTCCCGTTT CTGGCCTCCA TTCAGAAACA AGGGAGGCCC TTTtGCGCCG GAGCCCTGGT CCATCCCCGC TTCGTCCTGA CAGCGGCCAG CTGCTTCCGT GGCAAGAACA GCGGAAGTGC CTCTGTGGTG CTGGGGGCCT ATGACCTGAG GCAGCAGGAG CAGTCCCGGC AGACATTCTC CATCAGGAGC ATCAGCCAGA ACGGCTATGA CCCCCGGCAG AATCTGAACG ATGTGCTGCT GCTGCAGCTG GACCGTGAGG CCAGACTCAC CCCCAGTGTG GCCCTGGTAC CGCTGCCCCC GCAGAATGCC ACAGTGGAAG CTGGCACCAA CTGCCAAGTTGCGGGCTGGG GGACCCAGCG GCTTAGGAGG CTTTTCTCCC

GCTTCCCAAG GGTGCTCAAT GTCACCGTGA CCTCAAACCC GTGTCTCCCC AGAGACATGT GCATTGGTGT CTTCAGCCGC CGGGGCCGCA TCAGCCAGGG AGACAGAGGC ACCCCCCTCG TCTGCAACGG CCTGGCGCAG GGCGTGGCCT CCTTCCTCCG GAGGCGTTTC CGCAGGAGCT CCGGCTTCTT CACCCGCGTG GCGCTCTTCA GAAATTGGAT TGATTCAGTT CTCAACAACC CGCCGGCCTGA* (SEQ ID NO: 11) MPALRFLALL ASLLATSRV GLATLAD IVGG RRAQPQEFPF LASIQKQGRP FCAGALVHPR FVLTAASCFR GKNSGSASVV LGAYDLRQQE QSRQTFSIRS ISQNGYDPRQ NLNDVLLLQL DREARLTPSV ALVPLPPQNA TVEAGTNCQV AGWGTQRLRR LFSRFPRVLN VTVTSNPCLP RDMCIGVFSR RGRISQGDRG TPLVCNGLAQ GVASFLRRRF RRSSGFFTRV ALFRNWIDSV LNNPPA* (SEQ ID NO: 12) The degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443-453). For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0. 3.

Modification of the nucleic acid sequence encoding the HBP may be necessary for the synthesis of polypeptide sequences substantially similar to the HBP. The term"substantially similar"to the HBP refers to non-naturally occurring forms of the HBP. These polypeptide sequences may differ in some engineered way from the HBP isolated from its native source. For example, it may be of interest to synthesize variants of the HBP where the variants differ in specific activity, thermostability, pH optimum, or the like using, e. g., site-directed mutagenesis.

The analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the HBP encoding part of SEQ ID NOS: 1,2,6,8,10, or 12, e. g., a sub-sequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the HBP encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general

description of nucleotide substitution, see, e. g., Ford et al., 1991, in Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide sequence. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e. g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for HBP activity to identify amino acid residues that are critical to the activity of the molecule.

PREPARATION OF HBP A nucleic acid sequence encoding HBP may be prepared synthetically by established standard methods, e. g., the phosphoamidite method described by S. L. Beaucage and M. H.

Caruthers, Tetrahedron Letters 22,1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3,1984, pp. 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e. g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

The techniques used to isolate or clone a nucleic acid sequence encoding the heparin binding protein used in the method of the present invention are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e. g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e. g., Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.

The nucleic acid sequence is then inserted into a recombinant expression vector which may be any vector which may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the

vector may be an autonomously replicating vector, i. e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e. g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.

In the vector, the nucleic acid sequence encoding HBP should be operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the nucleic acid sequence encoding HBP in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. 1,1981, pp. 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222,1983, pp. 809-814) or the adenovirus 2 major late promoter, a Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter (Boshart et al., 1981, Cell 41: 521-530) and a bovine papilloma virus promoter (BPV). A suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., FEBS Lett. 311,1992, pp. 7-11).

Examples of suitable promoters for directing the transcription of the nucleic acid sequence encoding HBP, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha- amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21- 25). Further promoters are described in"Useful proteins from recombinant bacteria"in Scientific American, 1980,242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid sequence encoding HBP in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,

Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U. S. Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral a-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.

In a yeast host, useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et aL, 1992, Yeast 8: 423-488.

The nucleic acid sequences encoding SEQ ID NOS: 1 and 2, e. g., SEQ ID NOS: 3 and 9 and may be operably linked to a nucleic acid encoding a heterologous pro sequence. The nucleic acid encoding SEQ ID NOS: 6,8,10, and 12, e. g., SEQ ID NOS: 5,7,9, and 11 and may be operably linked to a nucleic acid sequence encoding a heterologous signal sequence and/or pro sequence.

The nucleic acid sequence encoding HBP may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The vector may further comprise elements such as polyadenylation signals (e. g. from SV 40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e. g. the SV 40 enhancer) and translational enhancer sequences (e. g. the ones encoding adenovirus VA RNAs).

Furthermore, preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,

Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such a sequence (when the host cell is a mammalian cell) is the SV 40 or polyoma origin of replication. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM131. Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS 1. The origin of replication may be one having a mutation to make its function temperature-sensitive in the host cell (see, e. g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

The vector may also comprise a selectable marker, e. g, a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e. g., neomycin, geneticin, ampicillin, or hygromycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e. g, as described in WO 91/17243, where the selectable marker is on a separate vector.

The procedures used to ligate the nucleic acid sequences coding for HBP, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op. cit.).

The host cell into which the expression vector is introduced may be any cell which is capable of producing HBP and is preferably a eukaryotic cell, such as invertebrate (insect) cells

or vertebrate cells, e. g., Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are the COS (e. g., ATCC CRL 1650), BHK (e. g., ATCC CRL 1632, ATCC CCL 10) or CHO (e. g., ATCC CCL 61) cell lines.

The host cell may be a mammalian basophilic cell or mammalian hybrid cell. The mammalian basophilic cell may be human, guinea pig, rabbit or rat basophilic cells. In a specific embodiment, the mammalian basophilic cell is a rat basophilic cell. In a most specific embodiment, the rat basophilic cell may be an RBL-1 cell having the identifying characteristics of ATCC CRL-1378 or RBL-2H3 cell having the identifying characteristics of ATCC CRL- 2256.

Methods for transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e. g., Kaufman and Sharp, 1982, J. Mol. Biol. 159: 601-621; Southern and Berg, 1982, J. Mol. Appl. Genet. 1: 327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14: 725; Corsaro and Pearson, 1981, Somatic Cell Genetics 7: 603, Graham and van der Eb, 1973, Virology 52: 456; Fraley et al., 1980, JBC 225: 10431; Capecchi, 1980, Cell 22: 479; Wiberg et al., 1983, NAR 11: 7287; and Neumann et al., 1982, EMBO J. 1: 841-845. In a specific embodiment, the mammalian basophilic cell is transfected with DNA encoding HBP using an electroporation apparatus.

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing mammalian cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e. g. in catalogues of the American Type Culture Collection). The cells are then screened for antibiotic resistance. Subsequently, the selected clones are subsequently assayed for HBP activity using assays known in the art such as a chemotaxis assay and assaying for cytokine release from monocytes (see, for example, Rasmussen et al., 1996, FEBS Lett. 390: 109-112).

Alternatively, the host cell may be a hybrid mammalian cell. A myeloma line e. g., mouse, rat, human) is transfected with DNA encoding HBP using the procedures described above. It may be subsequently be fused with a mammalian cell expressing an acidic proteoglycan such as a mammalian basophilic cell or mast cell using the following procedures.

In one embodiment the parental cells are mixed in culture media such as RPMI-1640 and exposed to a chemical fusion agent such a polyethylene glycol (see, for example, Gefter et al.,

1997, Somat. Cell Genet. 3: 231-236). The fusion agent is subsequently diluted out and the cells are incubated in media and HAT. Selected clones are subsequently assayed for HBP activity as described above.

Alternatively, two parental cells may be fused by electrofusion. Membrane contact between cells are achieved by a non-uniform alternating field that leads to dielectrophoresis and cell chain formation. Fusion is then triggered by the injection of a field pulse that is strong enough to induce reversible breakdown in the membrane contact zone (see, for example, Okada et al., 1984, Biomed. Res. 5: 511-566). Alternatively cell fusion may be induced by Sendai virus (see, for example, Wainberg et al., 1973, J. Cell Biol. 57: 388-396).

The host cell may be a unicellular pathogen, e. g., a prokaryote, or a non-unicellular pathogen, e. g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e. g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e. g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. The transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e. g, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e. g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnar and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e. g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e. g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a fungal cell."Fungi"as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

Representative groups of Ascomycota include, e. g., Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed above. Examples

of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e. g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.

Representative groups of Oomycota include, e. g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include, e. g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell."Yeast"as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e. g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e. g., genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e. g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e. g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980). The biology of yeast and manipulation of yeast genetics are well known in the art (see, e. g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

In a more preferred embodiment, the yeast host cell is a cell of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia. In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

The medium used to culture the cells may be any conventional medium suitable for

growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e. g,. in catalogues of the American Type Culture Collection).

The HBP produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e. g., ammonium sulphate, purification by a variety of chromatographic procedures, e. g., ion exchange chromatography, affinity chromatography, or the like. The recombinant host cells may also produce an acid proteoglycan such as heparin sulfate. To obtain active HBP, the acid proteoglycan will need to be removed. This may be accomplished using a series of separation methods, ie., precipitation or column chromatography, such as reverse phase HPLC, HIC, SEC, IEC and affinity based techniques. The separation method may be combined with other treatments like increasing salt concentration, by change in the pH and by other means that reduce interactions between the acidic proteoglycan and HBP.

The recombinant host cells may also produce an acid proteoglycan such as heparin sulfate. To obtain active HBP, the acid proteoglycan will need to be removed.

COMPOSITIONS In the pharmaceutical composition used in the method of the present invention, the HBP may be formulated by any of the established methods of formulating pharmaceutical compositions, e. g. as described in Remington's Pharmaceutical Sciences, 1985. The composition may typically be in a form suited for local or systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution. The compositions may be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The concentration of HBP may vary widely, i. e. from less than

about 0.5%, such as from 1%, to as much as 15-20% by weight. A unit dosage of the composition may typically contain from about 10 mg to about 1 g of HBP.

The pharmaceutical composition of the invention is contemplated to be advantageous to use to prevent entry of a pathogen into mononuclear cells (e. g., monocytes or macrophages).

Such pathogens include bacteria, which include but are not limited to Listera e. g., Listeria mycogenes, and Mycobacterium, specifically, e. g., Mycobacterium tuberulosis, protozoa, e. g., Leishmania, and a virus, e. g., HIV. A daily dosage of HBP of 0.1-100 mg/kg body weight is at present contemplated to be suitable, dependent on the severity of the condition to be treated and the patient's condition.

The composition of the invention may additionally comprise at least one cytokine.

Examples of such cytokines include but are not limited to interferon-gamma, TNF-alpha, IL-3, IL-7, GM-CSF, G-CSF, and M-CSF.

EXAMPLES Example 1: Production of HBP Materials The vectors pBlueBacIII and pcDNA3 are obtained from Invitrogen. All primers and oligos are synthesized on an Applied Biosystems Model 394 DNA synthesizer. Restriction enzymes are obtained from New England Biolabs. Pfu polymerase, used in PCR reactions is obtained from Stratagene. RBL-1 cells (ATCC CRL-1378) and RBL-2H3 cells (ATCC CRL- 2256) are obtained from American Type Culture Collection (ATCC) in Rockville, MD. Cells are grown as recommended by the supplier or in RPMI 1640 culture medium (Gibco, Life Technologies) supplemented with 10% heat inactivated gamma-irradiated FCS (origin: NZ, Gibco, Life Technologies) or fetal calf serum (FCS) North American origin from HyClone or BioWhittaker. Cells are grown in 5% Cor art 37 C in an 80% humidified atmosphere.

Exponentially growing cells are used in all experiments.

Construction of Expression Vectors A 770 bp BamHI-HindIII fragment is constructed using PCR technology from a human bone marrow DNA library (Clontech) based on the human HBP amino acid sequence (Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547) and the CAP 37/azurocidin DNA

sequence (Morgan et al., 1991, J. Immunol. 147: 3210-3214 and Almeida et al., 1991, Biochem. Biophys. Res. Commun. 177: 688-695). This fragment contains the entire coding region of HBP, including a 19-residue signal peptide, a 7 amino acid pro-peptide, a mature part of 22 amino acids, and a 3 amino acid C-terminal extension. The fragment is inserted into pBlue-BacIII resulting in the plasmid pSX556. For deletion of the pro-region, an oligonucleotide linker of 99 bp, covering the signal peptide and the first 4 amino acids of mature HBP (from BamHI to EagI) is substituted for the original BamHI-EagI fragment in pSX556 giving rise to pSX559.

For expression of these two cDNA sequences in RBL-1 cells, pSX556 and pSX559 described above are used as templates in PCR reactions using the primers PBRa 246 (5'-CCGGGGATCCAACTAGGCTGGCCCCGGTCCCGG-3') (SEQ ID NO: 13) PBRa247 (5'-CCGGGGATCCGATGACCCGGCTGACAGTCCTGG-3') (SEQ ID NO: 14) with a Pfu polymerase according to manufacturer's instructions (Stratagene).

After BamHI cleavage of the PCR reaction products, the fragments are ligated in correct orientation into the mammalian expression vector pcDNA3 (Invitrogen), linearized with BamHI, resulting in two plasmids, pcDNA3-HBP (Figure 1 A) and pcDNA3-HBP pro (Figure 1B).

Transfection Procedures Transfection is performed according to the following procedures. 25 ug of pcDNA3- HBP or pcDNA3-HBP pro is transfected into RBL-1 cells or RBL-2H3 cells (8 x 106 cells are transfected using a BioRad Electroporation Apparatus with electric settings 960 uF and 300V as described by Gullberg et al., 1994, J. Biol. Chem. 269: 25219-25225 and Garwicz et al., 1995, J. Biol. Chem. 270: 28413-28418, or are transfected using LipofecAmine (Gibco, Life Technologies) or Superfect (Qiagen) transfection reagents as recommended by the suppliers.

Cells are grown in RPMI 1640 medium supplemented with 10% heat inactivated gamma- irradiated FCS in 5% Cor art 37 C in an 80% humidified atmosphere. Geneticin (2 mg/ml) is added 48 hrs. post-transfection to select for recombinant clones.

Individual clones growing in the presence of geneticin are isolated and tested for HBP expression by ELISA. The HBP ELISA is a sandwich immunoassay using a monoclonal antibody as catcher and a polyclonal rabbit antibody conjugated to horseradish peroxidase as

detector. Antibodies are prepared according to standard procedures by immunizing mice and rabbits with HBP purified from human buffy coat cels (Flodgaard et al., 1991, Eur. J.

Biochem. 197: 535-547). Specifically, each well is coated with 0.5 pg monoclonal anti-hHBP dissolved in 100 pl of PBS overnight. The coated wells are washed three times with a solution of 5% lactose, 0.5% Byco A 0.05% Tween 20 and 0.024% thiomersal. After the last washing, the plates are left to dry at room temperature upside-down on a piece of cloth. The coated plates are rapped with staniol and can be stored up to three months.

Purified hHBP is used as reference preparation. A working dilution of 100 ng hHBP/ml is prepared in a BSA-EDTA buffer and stored in aliquots at-80°C for a maximum of two weeks. Serial dilution's containing 0; 0.3; 1 ; 4 and 12 ng hHBP/ml diluted in BSA- EDTA are made fresh and 100 1ll are added to each well. hHBP samples are also diluted in BSA-EDTA buffer and all the samples are in-cubated agitated for 1 hour at room temperature.

The wells are emptied and washed three times with phosphate buffered saline followed by the addition of 100 ul/well diluted (1: 1000) Fab-peroxidase conjugated rab-bit anti-hHBP, and incubated agitated for 1 hour at room temperature. Peroxidase activity is measured using 100 ul/well TMB-perborate substrate solution, resulting in a color formation measurable photometrically at 450 nm. The reference curve is linear when the logarithm to the absorbance is plotted against the logarithm to the dose.

Clones with the most pronounced expression are chosen for further experiments, recloned and retested for expression levels. The highest HBP producers are selected and grown into mass culture or adapted to serum free or protein free medium.

Isolation of HBP The isolation of HBP from RBL-1 cells is carried out essentially as described by Rasmussen et al., 1996, FEBS Lett. 390: 109-112. The transfected and selected RBL-1 cells are initially filtered to remove any remaining cells and cell debris. The culture medium is subsequently applied to a CM-Sepharose cation-exchange column (Pharmacia and Upjohn), previously equilibrated with 50 mM sodium phosphate, pH 7.3. Unbound and loosely bound materials are eluted with equilibration buffer until baseline is achieved measured by on-line UV detection at 280 nm. The coulumn is then developed with a linear gradient from 0 to 1 M sodium chloride in equilibration buffer. HBP eluted with about 0.7 M sodium chloride and

fractions are combined based on UV absorption. Pooled fractions are diluted with two volumes of distilled water and applied on a new CM-Sepharose column. Following equilibration HBP is step-eluted with 1 M sodium chloride in equilibration buffer and fractions combined based on absorption at 280 nm. Highly concentrated and pure HBP is obtained by this procedure. Final purification is carried out on a Sephadex G-25 gel-filtration column (Pharmacia & Upjohn) equipped with a UV-flow cell and equilibrated and eluted with 0.02% trifluoroacetic acid. HBP is collected based on absorption at 280 nm. The gel filtration serves mainly as a buffer exchange step to produce a stable preparation of HBP that is kept at 4 C until use.

Characterization of Isolated HBP Recombinant HBP obtained from RBL-1 cells, hereinafter referred to as RBL-1 HBP, is analyzed by SDS-gel electrophoresis, peptide and carbohydrate mapping, mass spectrometry (MS), capillary electrophoresis (CE), and HPLC and compared to recombinant HBP obtained from insect cells (Rasmussen et al., 1996, FEBS Lett. 390: 109-112), hereinafter referred to as insect cell HBP, and native HBP obtained from polymorphonuclear leukocytes (Flodgaard et al., 1991, Eur. J. Biochem. 197: 535-547), hereinafter referred to as human HBP.

SDS Gel Electrophoresis RBL-1 HBP and insect cell HBP is tested on SDS-PAGE (see Figure 2). It appears that RBL-1 HBP has a molecular wieght slightly larger than insect cell HBP.

Amino Acid Sequence Analysis Amino acid sequence analysis is determined by automated Edman degradation using an Applied Biosystems Model 477 gas-phase sequencer described by the manufacturer.

Characterization shows that human HBP and RBL-1 HBP are homogenous with regard to their N-terminal but heterogenous with regard to their C-terminal. RBL-1 HBP contained the three peptide forms HBP 1-221, HBP 1-223 and HBP 1-225. Human HBP contains the two peptide forms HBP 1-221 and HBP 1-223, and possibly also the HBP 1-225 peptide. HBP from baculovirus infected insect cell culture (insect cell HBP) is used as reference. Insect cell HBP contains two peptide forms: HBP 1-225 peptide (80-90%) and HBP 5-225 peptide (10- 20%, tHBP).

Tryptic peptide map Samples of human, RBL-1 and insect cell HBP, respectively, are desalted into a 10 mM Mes buffer, pH 5.9 on NAPS columns. A volume containing about 50 p9 HBP is withdrawn each sample and concentrated to 10 1 in a Speedvac concentrator. Mes and CaC12 buffers, pH 5.9 are added to a final concentration of 100 mM Mes, 10 mM CaCl2 in 30 pi.

Following addition of 20 17 M ion-exchanged urea, the samples are allowed to denature for 10 min. before injection on a Poroszyme immobilized trypsin cartridge (Perceptive Biosystems, part nr. 2-3128-00), which has been previously equilibrated in 95 % A buffer (50 mM Mes, pH 5.9) and 5 % B buffer (100 % acetonitrile). The sample is washed onto the column by the same buffer for 3 min. and then allowed to incubate for 2 hrs. at 37'C. The peptides generated are eluted from the column by the equilibration buffer. 300 1 are collected manually from the column after the first 50 1 had been discarded. 50 I of each collected digest (about 8g HBP before digestion) are analysed on a RP-HPLC Vydac 218 TP52 (250 mm x 2.1 mm i. d.) column using an Applied Biosystemsl30 HPLC system and a linear gradient of 3-80 % B (0.055 % TFA, 70 % acetonitrile) over 65 min.

The tryptic peptide maps of human-, RBL-1-and insect cell HBP, respectively, obtained following digestion on a Poroszyme immobilised trypsin cartridge and separation on a Vydac C 18 RP-HPLC column are shown in Figure 3. It is seen that the peptide maps of human HBP and RBL-1 HBP are very similar and that they differ considerably from the peptide map of insect cell HBP.

Differences are observed in insect cell HBP compared to the other two HBP samples with respect to glycosylation at Asn (100) and Asn (114). The RBL-1 and human HBP seem to be more heterogeneous. Differences are also seen in the glycostructures at Asn (145) of insect cell HBP compared to the two others. The degree of glycosylation of the three glycosylation sites cannot be estimated at present. A major differnece is the extra peak eluting before peak 40 both in the RBL-1 HBP and in the human HBP chromatogram compared to insect cell HBP and the xtra double peak following peak 40. These extra peaks may represent C- terminal truncated forms. In addition, an extra peak elutes just after peak 50 in RBL-1 and human HBP, but is not seen in insect cell HBP.

Monosaccharide Composition After TFA hydrolysis, the monosaccharide composition is determined by AIE-HPLC with pulsed amperometric detection. The results (Table 1) are consistent with the results from both carbohydrate map and MALDI-MS (see below), is that the dominating structures are a fucosylated core-structure (Fuc-GIcNAc2-Man3) and truncated forms thereof. Larger complex structures are indicated in low amounts for human HBP by the galactose content and in moderate amounts for RBL-1 HBP by the galactose and N-acetylgalactosamine contents.

Table 1. Monosaccharide content determined in the human HBP and RBL-1 HBP.

Contents are given mol/mol. Numbers in brackets are relative contents with Nacetylglucosamine = 100. ND, not detected. The amount of glucose found is not above the expected background.

Carbohydrate Mapping The carbohydrates are released by hydrazinolysis on a GlycoPreplO00 unit using N+O mode (434-084) and labelled with 2-aminobenzemide (2-AB) using a labelling kit (K-404, Oxford GlycoSystems). The 2-AB carbohydrate pools are analysed by IE-HPLC (Figure 4) on a CarboPac PA-100 column eluted with a NaAc gradient in NaOH. The pools are desialylated by sialidase treatment (Sialidase X-5022, Oxford GlycoSystems, 37 C, 16 hrs) and analysed by IE-HPLC (Figure 4) and size exclusion chromatography (Figure 6) on a Glycan Sizing column eluted with water and calibrated in glucose units (g. u.). For both LC analyses, fluorescence detection is used (exe. 330 nm, emm. 420 nm).

The carbohydrate structures found in the three HBP forms can be divided in 3 major groups Figures 4-6): 1. Core-structures:

Structure A: Truncated core-structure or non-carbohydrate impurity.

Structure B: Core-structure with fucose, lacking one mannose, 4.8 g. u., 876.3 Da.

Structure C: Core-structure with fucose, 5.8 g. u., 1038.4 Da.

Structure D: Core-structure without fucose, 4.8 g. u., 892.3 Da.

The core-structures represent approx. 80% of the human HBP carbohydrates, approx. 50% of the RBL-1 HBP carbohydrates, and 100% of the insect cell HBP carbohydrates.

2. Sialylated structures: Stuctures E-F and neighbouring structures eluted as mono-and possibly disialylated structures (IE-HPLC, Figure 4). After sialidase treatment, the carbohydrates eluted as neutral mono-or biantenna complex structures, ie. structures from 9 to 13 g. u. (IE-HPLC, Figure 5, SEC, Figure 6). The sialylated structures represent approx. 20% of the human HBP carbohydrates and approx. 40% of the RBL-1 HBP carbohydrates.

3. Late-eluting structures: The late-eluting structures (Figure 4) eluted as highly sialylated structures, but the retention times were un-changed after sialidase treatment (not apparent in Figure 5 due to change in the gradient compared to Figure 4). The late-eluting structures represented approx. l0% of the RBL-1 HBP carbohydrates.

MALDI-MS MALDI-MS is performed on the three proteins using sinapinic acid as matrix. The main mass found by MALDI-MS of insect cell HBP (Figure 5) is 27236 Da. For RBL-1 HBP (Figure 7), the main mass of 26715.6 Da can be interpreted as the HBP 1-223 peptide with three core-structures with fucose and lacking one mannose (structure B), which has a theoretical mass of 26722 Da. Further, the mass spectrum shows a large amount of structures in the range 27-30 kDa, which can be assigned to glycoforms with one or more of the carbohydrate structure (s) B substituted with eg. sialylated structure (s) ; each substitution would add 600-1500 Da depending on the structure added. For human HBP (Figure 7), the main mass of 26681.9 Da can be the same structure, i. e., the HBP 1-223 peptide with three structures B.

Capillary Electrophoresis (CE) Initial studies have shown that CE of HBP is rather difficult. This is due primarily to a very strong ionic interaction between the positively-charged protein and the negatively charged fused silica capillary wall. However, HBP exhibits satisfactory behaviour in two different buffer systems. In both systems, the interaction of HBP with the fused silica wall is decreased through so called dynamic coating procedures.

One system is acidic, composed of 25 mM phosphoric acid with 0.05% polyvinylalcohol (PVA), titrated to pH 2.5 with ammonium hydroxide. It has been published that PVA interacts dynamically with the fused silica wall at pH below 5. In this way, the negative charge on the wall is shielded. The other system is a neutral buffer, 25 mM phosphoric acid with 0.005% poly-Arginine, titrated to pH 6.5 with ammonium hydroxide.

Poly-Arginine adsorbs to the fused silica wall through strong ionic interaction. The net charge of the wall becomes positive.

Analyses of capture eluate of insect cell HBP indicate that the two above-mentioned buffer systems have different selectivities. Therefore, the two test substances, RBL-1 HBP and human HBP, are analyzed in both buffer systems. Before analysis they are transferred to 10 mM MES pH 5.9 by gel filtration (NAP 5 column). Capture eluate of insect cell HBP is used as a reference.

Figures 8 and 9 show the separations of the three HBP samples in the two above mentioned buffer systems. Clearly, the figures show that the two buffers have different selectivities and that the microheterogeneity of the samples is highly different. However, there are also some similarities, which may be interpreted, takinn into consideration the data from other analyses. In the acid PVA-buffer (Figure 8), human HBP and RBL-1 HBP have a series of peaks eluting after the predominant peaks. In the neutral poly-Arg buffer (Figure 9) correspondingly peak complexes eluting prior to the main peak are observed. The intensity of the peaks is largest in RBL-1 HBP. At the same time insect cell HBP has virtually no peaks at the same positions.

The migration behaviour of these peaks seen in human HBP and RBL-1 HBP indicates that they are more negatively charged. The carbohydrate analyses have shown that human HBP as well as RBL-1 HBP, but not insect cell HBP, carry carbohydrate structures with one

or more sialic acid residues. Roughly 20% of the carbohydrate structures in human HBP are sialylated, whereas RBL-1 HBP contains twice as many.

Cation-exchange HPLC (CIE-HPLC) CIE-HPLC is performed on a MonoS HR 5/5 column eluted with a NaCI gradient in 20 mM Ches, pH 9.0 with UV detection at 280 nm. Samples are desalted to 10 mM MES, pH 5.9, before analysis. Human HBP elutes as two peaks, RBL-1 HBP eluted as three peaks and insect cell HBP elutes as one main peak with a front peak representing tHBP (Figure 10).

Although all the main-peaks for the three HBP forms are closely-eluting, none of them is co- eluting (Figure 10). These results may be due to the C-terminal heterogeneity.

Gel permeation chromatography (GP-HPLC) GP-HPLC is performed on a Waters Protein-Pak 125,7.8 mm x 300 mm, eluted with 0.2 M ammonium sulfate, 5% isopropanol, pH to 2.5 with phosphoric acid, then to pH 5.0 with triethylamine. UV detection at 215 nm. Samples are desalted to 10 mM MES, pH 5.9, before analysis. The GP-HPLC profiles of human HBP and of RBL-1 HBP show one broad peak (Figure 11). Human HBP elutes in a slightly higher elution volume compared to insectcell HBP and with a significant shoulder on the front (Figure 11). RBL-1 HBP elutes in the same elution volume as insect HBP but with a very large shoulder on the front (Figure 11).

These results could be due to the glycoform heterogeneity proposed from the carbohydrate mapping and MALDI-MS.

SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANTS: Novo Nordisk A/S (ii) TITLE OF THE INVENTION: Use Of Heparin-Binding Protein For Inhibiting Entry of Pathogens Into Mononuclear Cells (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Novo Nordisk A/S (B) STREET: Novo Allé (C) CITY: DK-2880 Bagsvaerd (D) STATE: (E) COUNTRY: Denmark (F) ZIP: (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 225 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ile Val Gly Gly Arg Lys Ala Arg Pro Arg Gln Phe Pro Phe Leu Ala 1 5 10 15 Ser Ile Gln Asn Gln Gly Arg His Phe Cys Gly Gly Ala Leu Ile His 20 25 30 Ala Arg Phe Val Met Thr Ala Ala Ser Cys Phe Gln Ser Gln Asn Pro 35 40 45 Gly Val Ser Thr Val Val Leu Gly Ala Tyr Asp Leu Arg Arg Arg Glu 50 55 60 Arg Gln Ser Arg Gln Thr Phe Ser Ile Ser Ser Met Ser Glu Asn Gly 65 70 75 80 Tyr Asp Pro Gln Gln Asn Leu Asn Asp Leu Met Leu Leu Gln Leu Asp 85 90 95 Arg Glu Ala Asn Leu Thr Ser Ser Val Thr Ile Leu Pro Leu Pro Leu 100 105 110 Gln Asn Ala Thr Val Glu Ala Gly Thr Arg Cys Gln Val Ala Gly Trp 115 120 125 Gly Ser Gln Arg Ser Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val 130 135 140 Asn Val Thr Val Thr Pro Glu Asp Gln Cys Arg Pro Asn Asn Val Cys 145 150 155 160 Thr Gly Val Leu Thr Arg Arg Gly Gly Ile Cys Asn Gly Asp Gly Gly 165 170 175 Thr Pro Leu Val Cys Glu Gly Leu Ala His Gly Val Ala Ser Phe Ser 180 185 190 Leu Gly Pro Cys Gly Arg Gly Pro Asp Phe Phe Thr Arg Val Ala Leu 195 200 205

Phe Arg Asp Trp Ile Asp Gly Val Leu Asn Asn Pro Gly Pro Gly Pro 210 215 220 Ala 225 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 221 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ile Val Gly Gly Arg Arg Ala Gln Pro Gln Glu Phe Pro Phe Leu Ala 1 5 10 15 Ser Ile Gln Lys Gln Gly Arg Pro Phe Cys Ala Gly Ala Leu Val His 20 25 30 Pro Arg Phe Val Leu Thr Ala Ala Ser Cys Phe Arg Gly Lys Asn Ser 35 40 45 Gly Ser Ala Ser Val Val Leu Gly Ala Tyr Asp Leu Arg Gln Gln Glu 50 55 60 Gln Ser Arg Gln Thr Phe Ser Ile Arg Ser Ile Ser Gln Asn Gly Tyr 65 70 75 80 Asp Pro Arg Gln Asn Leu Asn Asp Val Leu Leu Leu Gln Leu Asp Arg 85 90 95 Glu Ala Arg Leu Thr Pro Ser Val Ala Leu Val Pro Leu Pro Pro Gln 100 105 110 Asn Ala Thr Val Glu Ala Gly Thr Asn Cys Gln Val Glu Ala Gly Trp 115 120 125 Gly Thr Gln Arg Leu Arg Arg Leu Phe Ser Arg Phe Pro Arg Val Leu 130 135 140 Asn Val Thr Val Thr Ser Asn Pro Cys Leu Pro Arg Asp Met Cys Ile 145 150 155 160 Gly Val Phe Ser Arg Arg Gly Arg Ile Ser Gln Gly Asp Arg Gly Thr 165 170 175 Pro Leu Val Cys Asn Gly Leu Ala Gln Gly Val Ala Ser Phe Leu Arg 180 185 190 Arg Arg Phe Arg Arg Ser Ser Gly Phe Phe Thr Arg Val Ala Leu Phe 195 200 205 Arg Asn Trp Ile Asp Ser Val Leu Asn Asn Pro Pro Ala 210 215 220 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 678 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATCGTTGGCG GCCGGAAGGC GAGGCCCCGC CAGTTCCCGT TCCTGGCCTC CATTCAGAAT 60 CAAGGCAGGC ACTTCTGCGG GGGTGCCCTG ATCCATGCCC GCTTCGTGAT GACCGCGGCC 120 AGCTGCTTCC AAAGCCAGAA CCCCGGGGTT AGCACCGTGG TGCTGGGTGC CTATGACCTG 180 AGGCGGCGGG AGAGGCAGTC CCGCCAGACG TTTTCCATCA GCAGCATGAG CGAGAATGGC 240 TACGACCCCC AGCAGAACCT GAACGACCTG ATGCTGCTTC AGCTGGACCG TGAGGCCAAC 300 CTCACCAGCA GCGTGACGAT ACTGCCACTG CCTCTGCAGA ACGCCACGGT GGAAGCCGGC 360 ACCAGATGCC AGGTGGCCGG CTGGGGGAGC CAGCGCAGTG GGGGGCGTCT CTCCCGTTTT 420 CCCAGGTTCG TCAACGTGAC TGTGACCCCC GAGGACCAGT GTCGCCCCAA CAACGTGTGC 480 ACCGGTGTGC TCACCCGCCG CGGTGGCATC TGCAATGGGG ACGGGGGCAC CCCCCTCGTC 540

TGCGAGGGCC TGGCCCACGG CGTGGCCTCC TTTTCCCTGG GGCCCTGTGG CCGAGGCCCT 600 GACTTCTTCA CCCGAGTGGC GCTCTTCCGA GACTGGATCG ATGGCGTTTT AAACAATCCG 660 GGACCGGGGC CAGCCTAG 678 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 663 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATTGTGGGCG GCAGGAGGGC CCAGCCGCAG GAGTTCCCGT TTCTGGCCTC CATTCAGAAA 60 CAAGGGAGGC CCTTTTGCGC CGGAGCCCTG GTCCATCCCC GCTTCGTCCT GACAGCGGCC 120 AGCTGCTTCC GTGGCAAGAA CAGCGGAAGT GCCTCTGTGG TGCTGGGGGC CTATGACCTG 180 AGGCAGCAGG AGCAGTCCCG GCAGACATTC TCCATCAGGA GCATCAGCCA GAACGGCTAT 240 GAYCCCCGGC AGAATCTGAA CGATGTGCTG CTGCTGCAGC TGGACCGTGA GGCCAGACTC 300 ACCCCCAGTG TGGCCCTGGT ACCGCTGCCC CCGCAGAATG CCACAGTGGA AGCTGGCACC 360 AACTGCCAAG TTGCGGGCTG GGGGACCCAG CGGCTTAGGA GGCTTTTCTC CCGCTTCCCA 420 AGGGTGCTCA ATGTCACCGT GACCTCAAAC CCGTGTCTCC CCAGAGACAT GTGCATTGGT 480 GTCTTCAGCC GCCGGGGCCG CATCAGCCAG GGAGACAGAG GCACCCCCCT CGTCTGCAAC 540 GGCCTGGCGC AGGGCGTGGC CTCCTTCCTC CGGAGGCGTT TCCGCAGGAG CTCCGGCTTC 600 TTCACCCGCG TGGCGCTCTT CAGAAATTGG ATTGATTCAG TTCTCAACAA CCCGCCGGCC 660 TGA 663 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 699 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGCTCCAGCC CCCTTTTGGA CATCGTTGGC GGCCGGAAGG CGAGGCCCCG CCAGTTCCCG 60 TTCCTGGCCT CCATTCAGAA TCAAGGCAGG CACTTCTGCG GGGGTGCCCT GATCCATGCC 120 CGCTTCGTGA TGACCGCGGC CAGCTGCTTC CAAAGCCAGA ACCCCGGGGT TAGCACCGTG 180 GTGCTGGGTG CCTATGACCT GAGGCGGCGG GAGAGGCAGT CCCGCCAGAC GTTTTCCATC 240 AGCAGCATGA GCGAGAATGG CTACGACCCC CAGCAGAACC TGAACGACCT GATGCTGCTT 300 CAGCTGGACC GTGAGGCCAA CCTCACCAGC AGCGTGACGA TACTGCCACT GCCTCTGCAG 360 AACGCCACGG TGGAAGCCGG CACCAGATGC CAGGTGGCCG GCTGGGGGAG CCAGCGCAGT 420 GGGGGGCGTC TCTCCCGTTT TCCCAGGTTC GTCAACGTGA CTGTGACCCC CGAGGACCAG 480 TGTCGCCCCA ACAACGTGTG CACCGGTGTG CTCACCCGCC GCGGTGGCAT CTGCAATGGG 540 GACGGGGGCA CCCCCCTCGT CTGCGAGGGC CTGGCCCACG GCGTGGCCTC CTTTTCCCTG 600 GGGCCCTGTG GCCGAGGCCC TGACTTCTTC ACCCGAGTGG CGCTCTTCCG AGACTGGATC 660 GATGGCGTTT TAAACAATCC GGGACCGGGG CCAGCCTAG 699 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Ser Ser Pro Leu Leu Asp Ile Val Gly Gly Arg Lys Ala Arg Pro 1 5 10 15

Arg Gln Phe Pro Phe Leu Ala Ser Ile Gln Asn Gln Gly Arg His Phe 20 25 30 Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr Ala Ala Ser 35 40 45 Cys Phe Gln Ser Gln Asn Pro Gly Val Ser Thr Val Val Leu Gly Ala 50 55 60 Tyr Asp Leu Arg Arg Arg Glu Arg Gln Ser Arg Gln Thr Phe Ser Ile 65 70 75 80 Ser Ser Met Ser Glu Asn Gly Tyr Asp Pro Gln Gln Asn Leu Asn Asp 85 90 95 Leu Met Leu Leu Gln Leu Asp Arg Glu Ala Asn Leu Thr Ser Ser Val 100 105 110 Thr Ile Leu Pro Leu Pro Leu Gln Asn Ala Thr Val Glu Ala Gly Thr 115 120 125 Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg Ser Gly Gly Arg Leu 130 135 140 Ser Arg Phe Pro Arg Phe Val Asn Val Thr Val Thr Pro Glu Asp Gln 145 150 155 160 Cys Arg Pro Asn Asn Val Cys Thr Gly Val Leu Thr Arg Arg Gly Gly 165 170 175 Ile Cys Asn Gly Asp Gly Gly Thr Pro Leu Val Cys Glu Gly Leu Ala 180 185 190 His Gly Val Ala Ser Phe Ser Leu Gly Pro Cys Gly Arg Gly Pro Asp 195 200 205 Phe Phe Thr Arg Val Ala Leu Phe Arg Asp Trp Ile Asp Gly Val Leu 210 215 220 Asn Asn Pro Gly Pro Gly Pro Ala 225 230 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 756 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATGACCCGGC TGACAGTCCT GGCCCTGCTG GCTGGTCTGC TGGCGTCCTC GAGGGCCGGC 60 TCCAGCCCCC TTTTGGACAT CGTTGGCGGC CGGAAGGCGA GGCCCCGCCA GTTCCCGTTC 120 CTGGCCTCCA TTCAGAATCA AGGCAGGCAC TTCTGCGGGG GTGCCCTGAT CCATGCCCGC 180 TTCGTGATGA CCGCGGCCAG CTGCTTCCAA AGCCAGAACC CCGGGGTTAG CACCGTGGTG 240 CTGGGTGCCT ATGACCTGAG GCGGCGGGAG AGGCAGTCCC GCCAGACGTT TTCCATCAGC 300 AGCATGAGCG AGAATGGCTA CGACCCCCAG CAGAACCTGA ACGACCTGAT GCTGCTTCAG 360 CTGGACCGTG AGGCCAACCT CACCAGCAGC GTGACGATAC TGCCACTGCC TCTGCAGAAC 420 GCCACGGTGG AAGCCGGCAC CAGATGCCAG GTGGCCGGCT GGGGGAGCCA GCGCAGTGGG 480 GGGCGTCTCT CCCGTTTTCC CAGGTTCGTC AACGTGACTG TGACCCCCGA GGACCAGTGT 540 CGCCCCAACA ACGTGTGCAC CGGTGTGCTC ACCCGCCGCG GTGGCATCTG CAATGGGGAC 600 GGGGGCACCC CCCTCGTCTG CGAGGGCCTG GCCCACGGCG TGGCCTCCTT TTCCCTGGGG 660 CCCTGTGGCC GAGGCCCTGA CTTCTTCACC CGAGTGGCGC TCTTCCGAGA CTGGATCGAT 720 GGCGTTTTAA ACAATCCGGG ACCGGGGCCA GCCTAG 756 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 225 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ile Val Gly Gly Arg Lys Ala Arg Pro Arg Gln Phe Pro Phe Leu Ala

1 5 10 15 Ser Ile Gln Asn Gln Gly Arg His Phe Cys Gly Gly Ala Leu Ile His 20 25 30 Ala Arg Phe Val Met Thr Ala Ala Ser Cys Phe Gln Ser Gln Asn Pro 35 40 45 Gly Val Ser Thr Val Val Leu Gly Ala Tyr Asp Leu Arg Arg Arg Glu 50 55 60 Arg Gln Ser Arg Gln Thr Phe Ser Ile Ser Ser Met Ser Glu Asn Gly 65 70 75 80 Tyr Asp Pro Gln Gln Asn Leu Asn Asp Leu Met Leu Leu Gln Leu Asp 85 90 95 Arg Glu Ala Asn Leu Thr Ser Ser Val Thr Ile Leu Pro Leu Pro Leu 100 105 110 Gln Asn Ala Thr Val Glu Ala Gly Thr Arg Cys Gln Val Ala Gly Trp 115 120 125 Gly Ser Gln Arg Ser Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val 130 135 140 Asn Val Thr Val Thr Pro Glu Asp Gln Cys Arg Pro Asn Asn Val Cys 145 150 155 160 Thr Gly Val Leu Thr Arg Arg Gly Gly Ile Cys Asn Gly Asp Gly Gly 165 170 175 Thr Pro Leu Val Cys Glu Gly Leu Ala His Gly Val Ala Ser Phe Ser 180 185 190 Leu Gly Pro Cys Gly Arg Gly Pro Asp Phe Phe Thr Arg Val Ala Leu 195 200 205 Phe Arg Asp Trp Ile Asp Gly Val Leu Asn Asn Pro Gly Pro Gly Pro 210 215 220 Ala 225 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 720 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGCCAGCAC TCAGATTCCT GGCCCTGCTG GCCAGCCTGC TGGCAACCTC CAGGGTTATT 60 GTGGGCGGCA GGAGGGCCCA GCCGCAGGAG TTCCCGTTTC TGGCCTCCAT TCAGAAACAA 120 GGGAGGCCCT TTTGCGCCGG AGCCCTGGTC CATCCCCGCT TCGTCCTGAC AGCGGCCAGC 180 TGCTTCCGTG GCAAGAACAG CGGAAGTGCC TCTGTGGTGC TGGGGGCCTA TGACCTGAGG 240 CAGCAGGAGC AGTCCCGGCA GACATTCTCC ATCAGGAGCA TCAGCCAGAA CGGCTATGAY 300 CCCCGGCAGA ATCTGAACGA TGTGCTGCTG CTGCAGCTGG ACCGTGAGGC CAGACTCACC 360 CCCAGTGTGG CCCTGGTACC GCTGCCCCCG CAGAATGCCA CAGTGGAAGC TGGCACCAAC 420 TGCCAAGTTG CGGGCTGGGG GACCCAGCGG CTTAGGAGGC TTTTCTCCCG CTTCCCAAGG 480 GTGCTCAATG TCACCGTGAC CTCAAACCCG TGTCTCCCCA GAGACATGTG CATTGGTGTC 540 TTCAGCCGCC GGGGCCGCAT CAGCCAGGGA GACAGAGGCA CCCCCCTCGT CTGCAACGGC 600 CTGGCGCAGG GCGTGGCCTC CTTCCTCCGG AGGCGTTTCC GCAGGAGCTC CGGCTTCTTC 660 ACCCGCGTGG CGCTCTTCAG AAATTGGATT GATTCAGTTC TCAACAACCC GCCGGCCTGA 720 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 239 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Pro Ala Leu Arg Phe Leu Ala Leu Leu Ala Ser Leu Leu Ala Thr

1 5 10 15 Ser Arg Val Ile Val Gly Gly Arg Arg Ala Gln Pro Gln Glu Phe Pro 20 25 30 Phe Leu Ala Ser Ile Gln Lys Gln Gly Arg Pro Phe Cys Ala Gly Ala 35 40 45 Leu Val His Pro Arg Phe Val Leu Thr Ala Ala Ser Cys Phe Arg Gly 50 55 60 Lys Asn Ser Gly Ser Ala Ser Val Val Leu Gly Ala Tyr Asp Leu Arg 65 70 75 80 Gln Gln Glu Gln Ser Arg Gln Thr Phe Ser Ile Arg Ser Ile Ser Gln 85 90 95 Asn Gly Tyr Asp Pro Arg Gln Asn Leu Asn Asp Val Leu Leu Leu Gln 100 105 110 Leu Asp Arg Glu Ala Arg Leu Thr Pro Ser Val Ala Leu Val Pro Leu 115 120 125 Pro Pro Gln Asn Ala Thr Val Glu Ala Gly Thr Asn Cys Gln Val Ala 130 135 140 Gly Trp Gly Thr Gln Arg Leu Arg Arg Leu Phe Ser Arg Phe Pro Arg 145 150 155 160 Val Leu Asn Val Thr Val Thr Ser Asn Pro Cys Leu Pro Arg Asp Met 165 170 175 Cys Ile Gly Val Phe Ser Arg Arg Gly Arg Ile Ser Gln Gly Asp Arg 180 185 190 Gly Thr Pro Leu Val Cys Asn Gly Leu Ala Gln Gly Val Ala Ser Phe 195 200 205 Leu Arg Arg Arg Phe Arg Arg Ser Ser Gly Phe Phe Thr Arg Val Ala 210 215 220 Leu Phe Arg Asn Trp Ile Asp Ser Val Leu Asn Asn Pro Pro Ala 225 230 235 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 741 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGCCAGCAC TCAGATTCCT GGCCCTGCTG GCCAGCCTGC TGGCAACCTC CAGGGTTGGC 60 TTGGCCACCC TGGCAGACAT TGTGGGCGGC AGGAGGGCCC AGCCGCAGGA GTTCCCGTTT 120 CTGGCCTCCA TTCAGAAACA AGGGAGGCCC TTTTGCGCCG GAGCCCTGGT CCATCCCCGC 180 TTCGTCCTGA CAGCGGCCAG CTGCTTCCGT GGCAAGAACA GCGGAAGTGC CTCTGTGGTG 240 CTGGGGGCCT ATGACCTGAG GCAGCAGGAG CAGTCCCGGC AGACATTCTC CATCAGGAGC 300 ATCAGCCAGA ACGGCTATGA CCCCCGGCAG AATCTGAACG ATGTGCTGCT GCTGCAGCTG 360 GACCGTGAGG CCAGACTCAC CCCCAGTGTG GCCCTGGTAC CGCTGCCCCC GCAGAATGCC 420 ACAGTGGAAG CTGGCACCAA CTGCCAAGTT GCGGGCTGGG GGACCCAGCG GCTTAGGAGG 480 CTTTTCTCCC GCTTCCCAAG GGTGCTCAAT GTCACCGTGA CCTCAAACCC GTGTCTCCCC 540 AGAGACATGT GCATTGGTGT CTTCAGCCGC CGGGGCCGCA TCAGCCAGGG AGACAGAGGC 600 ACCCCCCTCG TCTGCAACGG CCTGGCGCAG GGCGTGGCCT CCTTCCTCCG GAGGCGTTTC 660 CGCAGGAGCT CCGGCTTCTT CACCCGCGTG GCGCTCTTCA GAAATTGGAT TGATTCAGTT 720 CTCAACAACC CGCCGGCCTG A 741 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 246 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Pro Ala Leu Arg Phe Leu Ala Leu Leu Ala Ser Leu Leu Ala Thr 1 5 10 15 Ser Arg Val Gly Leu Ala Thr Leu Ala Asp Ile Val Gly Gly Arg Arg 20 25 30 Ala Gln Pro Gln Glu Phe Pro Phe Leu Ala Ser Ile Gln Lys Gln Gly 35 40 45 Arg Pro Phe Cys Ala Gly Ala Leu Val His Pro Arg Phe Val Leu Thr 50 55 60 Ala Ala Ser Cys Phe Arg Gly Lys Asn Ser Gly Ser Ala Ser Val Val 65 70 75 80 Leu Gly Ala Tyr Asp Leu Arg Gln Gln Glu Gln Ser Arg Gln Thr Phe 85 90 95 Ser Ile Arg Ser Ile Ser Gln Asn Gly Tyr Asp Pro Arg Gln Asn Leu 100 105 110 Asn Asp Val Leu Leu Leu Gln Leu Asp Arg Glu Ala Arg Leu Thr Pro 115 120 125 Ser Val Ala Leu Val Pro Leu Pro Pro Gln Asn Ala Thr Val Glu Ala 130 135 140 Gly Thr Asn Cys Gln Val Ala Gly Trp Gly Thr Gln Arg Leu Arg Arg 145 150 155 160 Leu Phe Ser Arg Phe Pro Arg Val Leu Asn Val Thr Val Thr Ser Asn 165 170 175 Pro Cys Leu Pro Arg Asp Met Cys Ile Gly Val Phe Ser Arg Arg Gly 180 185 190 Arg Ile Ser Gln Gly Asp Arg Gly Thr Pro Leu Val Cys Asn Gly Leu 195 200 205 Ala Gln Gly Val Ala Ser Phe Leu Arg Arg Arg Phe Arg Arg Ser Ser 210 215 220 Gly Phe Phe Thr Arg Val Ala Leu Phe Arg Asn Trp Ile Asp Ser Val 225 230 235 240 Leu Asn Asn Pro Pro Ala 245 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CCGGGGATCC AACTAGGCTG GCCCCGGTCC CGG 33 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CCGGGGATCC GATGACCCGG CTGACAGTCC TGG 33