MXPA98010619A - Materials and methods for the production of quitin - Google Patents

Abstract

The present invention relates to purified and isolated polynucleotide sequences encoding human chitinase. Materials and methods for the recombinant production of human chitinase products are also provided, which are expected to be useful as products for the treatment of fungal infections and for the development of products useful for the treatment of same.

Description

MATERIALS AND METHODS FOR THE PRODUCTION OF QUITINASE This is a continuation in part of the United States application series No. 08 / 663,618 filed on June 14, 1996.

FIELD OF THE INVENTION The present invention relates, in general, to the human chitinase enzyme, and more specifically to the novel used and isolated polynucleotides encoding human chitinase, to the chitinase products encoded by the polynucleotides, to the materials and methods for the recombinant production of chitinase products and specific antibody substances for chitinase.

Background Chitin is a linear homopolymer of N-acetylglucose residues ina ß- (1, 4) -linked, this polysaccharide is the second after cellulose as the most abundant organic substance. The exoskeleton of arthropods is composed of chitin. In addition, fungi and other parasites contain chitin in their outer cell walls where it plays important structural and protective functions. The breaking of the cell wall and the membranes of fungi has been a useful therapeutic strategy against fungi and parasites. For example, amphotericin B and fluconazole exert their antifungal activity affecting membrane steroids. Despite the existence of antifungal therapy, fungal infections in humans is increasingly responsible for life threatening disorders. See, Georgopapadakou et al., Trends Microbiol., 3: 98-104 (1995). The species of fungi and parasites responsible for these disorders are mainly Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in individuals with problems in the immune system, such as patients with AIDS, patients undergoing chemotherapy and immunosuppressed patients, with organ transplants. Chitin can be degraded by the enzyme chitinase. Chitinase enzymes are found in plants, microorganisms and animals. Bacterial chitinase helps provide a source of. carbon for bacterial development. The insects produce chitinase to direct their cuticle in each moult. It is considered that in plants, chitinase provides a protective function against fungal parasites. Chitinases have been cloned from different bacteria [for example, Serratia marcescens, Jones et al. , EMBO J., 5: 467-473 (1986)], in plants [eg, tubaceous, Heitz et al., Mol. Gen. Genet. 245: 246-254 (1994)], and insects [eg, wasps, Krishnan et al., J. Biol. Chem. 269: 20971-20976 (1994)] species. Several proteins with little homology for insect and plant bacterial chitinases (less than 40% amino acid identity) have been identified in mammals, such as the human cartilage gp-39 (C-gp39) [Hakala et al., J Biol. Chem., 268: 255803-25810 (1993)], the human glycoprotein YKL-40 [Johansen et al., Eur. J. Cancer, 31A: 1437-1442 (1995)], estrogen-induced, oviduct-specific protein from sheep [DeSouza et al., Endocrinology, 136: 2485-2946 (1995)], cows and humans; and a secretory protein from activated mouse macrophages [Chang et al., Genbank M94584]. However, the degrading activity of chitin has not been reported for these proteins. The function of these proteins is not known, but they have been postulated to be included in tissue remodeling. Hakala et al., Supra, reports that C-gp39 is detectable in synovial and cartilage samples from patients with rheumatoid arthritis, but not from normal humans. Recklies et al., Arthri tis Rheumatism 36 (9SUPPL.): S190 (1993) report the location of the C-gp39 protein for a different population of cells in the superficial layer of cartilage. Johansen et al., Supra, report that measurements of serum levels of YKL-40 are of value as a potential marker for predicting the degree of metastatic disease and survival of patients with recurrent breast cancer. Escott et al.I infected. Iwmun , 63: 4770-4773 (1995) demonstrated the enzymatic activity of chitinase in human leukocytes and in human serum. Overdik et al., Glycobiology, 4: 797-803 (1994) described the isolation of a chitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase) from human serum and rat liver. Renkema et al., J. Biol. Chem., 270: 2198-2202 (February 1995) prepared a human chytrotriosidase from the spleen of a patient with Gaucher's disease. This preparation showed chitinase activity and the article reports a small amount of amino acid sequence of the protein component of the preparation (22 amino terminal residues and 21 residues of a triptych fragment). The function of human chitinase is also unknown, but in the article a relationship with the pathophysiology of Gaucher disease is proposed. A later publication of the same group [Boot et al., J. Biol. Chem., 270 (44): 26252-25256 (November 1995)] describes the cloning of human macrophage cDNA encoding a product exhibiting chitinase activity. The partial amino acid sequence reported by the group in its February 1995 article coincides with portions of the deduced amino acid sequence of the human macrophage cDNA product. See also the publication of the international patent No. WO 96/40940. In view of the increasing incidence of life-threatening fungal infection in individuals with immune system problems, there is a need in the art to identify and isolate polynucleotide sequences encoding human chitinases, for the development of materials and methods useful for production recombinant of the enzyme, and to generate reagents for the detection of chitinase in plasma.

SUMMARY OF THE INVENTION The present invention provides novel purified and isolated polynucleotides (ie, DNA and RNA, both sense and antisense strands) that encode human chitinase or fragments and analogs thereof; methods for the recombinant production of chitinase polypeptides, fragments and analogs thereof; purified and isolated chitinase polypeptide fragments and analogs; antibodies to these polypeptides, fragments and analogues; and pharmaceutical compositions containing these polypeptides, fragments, analogs, or antibodies. Specifically provided are: purified, isolated polynucleotides encoding the amino acid sequence of human chitinase of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof, the DNAs containing the protein encoding the nucleotides of SEQ ID US: 1 6 3, particularly nucleotides 65 to 1402 of SEQ ID NO: nucleotides 90 to 1427 of SEQ ID NO: 3; the purified, isolated polynucleotides comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 14 or 15; purified, isolated polynucleotides encoding human chitinase selected from the group consisting of: (a) double-stranded DNA comprising the protein encoding portions of the sequence set forth in SEQ ID NO: l or SEQ ID NO: 3, (b) a DNA that hybridizes under severe conditions to a non-coding strand of the DNA of (a), and (c) a DNA that, except for the redundancy of the genetic code, would hybridize under severe conditions to a non-coding strand of the DNA sequence of (a) or (b); vectors containing these DNAs, particularly expression vectors wherein the DNA is operably linked to an expression control DNA sequence; host cells stably transformed or transfected with these DNAs in a form that allows the expression of human chitinase in the host cell; a method for producing human chitinase which consists in: cultivating these host cells in a nutrient medium and isolating human chitinase from the host cell or from the nutrient medium; purified, isolated polypeptides produced by this method; the purified, isolated polypeptides containing the amino acid sequence of human chitinase of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof; fragments of human chitinase lacking 1 to about 72 C-terminal amino acid residues of mature human chitinase, particularly the human chitinase fragment of SEQ ID NO: 14; analogous human chitinase of SEQ ID NO: 15; the hybridoma cell lines that produce a monoclonal antibody that is specifically reactive with one of the polypeptides described above; and monoclonal antibodies produced by these hybridomas. Preferred DNA sequences of the invention include cDNA and enomic sequences as well as fully or partially chemically synthesized DNA sequences. The nucleotide sequence of 2 human cDNAs encoding the putative allelic variants of human chitinase, and including the 5 'and 3' non-coding sequences, are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. These sequences are DNA and DNA sequences that hybridize to the non-coding strand thereof under normal severe conditions or that would hybridize but for the redundancy of the genetic code, are contemplated by the invention. Preferred DNAs of the present invention comprise the coding region for human chitinase (corresponding to nucleotides 2a 1402 of SEQ ID NO: lo nucleotides 27 to 1427 of SEQ ID NO: 3), and the putative coding sequence of the protein mature human chitinase, secreted without its signal sequence (nucleotides 65 to 1402 of SEQ ID NO: 1, or nucleotides 90 to 1427 of SEQ ID NO: 3). Examples of the conditions of severe hybridization are as follows: hybridization at 42 ° C in 50% formamide and washing at 60 ° C in 0.1 x SSC, 0.1% SDS. Those skilled in the art will understand that under these conditions variations may occur based on the length and base content of the GC nucleotide of the sequences to be hybridized. The formulas standard in the techniques are suitable to determine the exact conditions of the hybridization. See, Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Two amino acid sequences for human chitinase (s) are set forth in SEQ ID NOS: 2 and 4. The sequence SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 1, and the SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 3. Preferred polynucleotides of the present invention include, in addition to the polynucleotides described above, polynucleotides encoding amino acids -21 to 445 of SEQ ID NO: 2 or of SEQ ID NO: 4, and which differ from the polynucleotides described in the preceding paragraphs only due to the well-known degeneracy of the genetic code. In the same way, since 21 amino acids (positions -21 to -1) of SEQ ID NOS: 2 and 4 consist of a signal peptide that is split to produce mature human chitinase protein, preferred polynucleotides include those coding polypeptides that contain amino acids 1 to 445 of SEQ ID NO: 2 or SEQ ID NO: 4. Among the uses of the polynucleotides of the present invention is use as a hybridization probe to identify and isolate genomic DNA encoding human chitinase; to identify and isolate non-human genes that encode homologous proteins for human chitinase; to identify human and non-human proteins that are similar to human chitinase (including those that may be involved in tissue remodeling); and to identify those cells that express human chitinase and the biological conditions under which this protein is expressed. In another aspect, the invention includes biological replicates (ie, copies of isolated DNA sequences prepared in vivo or in vi tro) of the DNA sequences of the invention. Self-replicating recombinant constructs are provided, such as the plasmid and viral DNA vectors incorporating chitinase polynucleotides, including any of the DNAs described above. Preferred vectors include expression vectors in which the cDNA encoding chitinase incorporates is operably linked to an endogenous or heterologous expression control sequence and a transcription terminator. These expression vectors may additionally include DNA sequences encoding polypeptides operably linked to the DNA sequences encoding chitinase, which vectors can be expressed to produce a fusion protein comprising the polypeptide of interest. According to another aspect of the invention, the prokaryotic or eukaryotic host cells are stably transformed or transfected with DNA sequences of the invention in a form that allows the desired chitinase product to be expressed therein. The host cells expressing the chitinase products can serve a variety of useful purposes. These cells constitute a valuable source of immunogens for the development of antibodies specifically immunoreactive with chitinase. The host cells of the invention are useful in methods for the large-scale production of chitinase wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated, for example, by immunoaffinity purification, from the cells or from the medium in which they develop. The chitinase products can be obtained as isolates from natural cell sources or can be chemically synthesized, but are preferably produced by recombinant methods that include prokaryotic or eukaryotic host cells of the invention. The chitinase products of the invention can be full-length polypeptides, fragments or analogs thereof. Chitinase products having part or all of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 are contemplated. A preferred fragment lacking the 72 C-terminal amino acid residues of the mature protein is established in the SEQ ID NO: 14. It has been determined that these 72 C-terminal residues are not crucial for the enzymatic activity of chitinase. Example 5 illustrates the production of this C-terminal fragment; the introduction of a nonsense codon after the codon for amino acid 373 resulted in a fragment of approximately 39 kDa recombinant chitinase which retained the similar specific activity when compared to full length recombinant human chitinase.

Analogs may consist of the chitinase analogs wherein one or more of the specified amino acids (i.e., naturally encoded) are deleted or substituted, or where one or more of the unspecified amino acids is added: (1) without loss of one or more of the enzymatic activities or specific immunological characteristics of chitinase; or (2) with specific disqualification of a specific biological activity of chitinase. Example 3 illustrates the production of this analog (SEQ ID NO: 15), in which the proline at position 370 is substituted with serine, and in which the 72 C-terminal amino acid residues have been deleted. It is also expected that the use of mammalian host cells will provide post translational or post-translational modifications (eg, myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be necessary to confer biological activity optimal in the recombinant expression products of the invention. Proteins or other molecules that bind to chitinase can be used to modulate their activity. Also encompassed by the present invention are antibody substances (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other specific binding proteins for chitinase. Proteins or other molecules (eg, small molecules) that specifically bind to chitinase can be identified using isolated plasma chitinase, recombinant chitinase, chitinase variants or cells expressing these products. The binding proteins are useful, in turn, in compositions for immunization, as well as for purifying chitinase, and are also useful for the detection or quantification of chitinase in fluid and tissue samples by known immunological methods. Anti-idiotypic antibodies specific for chitinase-specific antibody substances are also highly contemplated. The specific value of the information with which it is contributed through the descriptions of the DNA and amino acid sequences of the present invention is evident. As a series of examples, knowledge of the cDNA sequence for chitinase makes it possible to isolate by DNA / DNA hybridization the genomic DNA sequences encoding the chitinase and the regulatory sequences of the control of chitinase expression as promoters, operators and Similar. The DNA / DNA hybridization procedures carried out with the DNA sequences of the invention under conditions of standard severity in the art in the same manner are expected to allow the isolation of DNAs encoding human allelic variants of chitinase, other human proteins structurally related proteins that share one or more of the biochemical and / or immunological properties of chitinase, and proteins of non-human species homologous to chitinase. The information of the DNA sequence provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, for example, Kapecchi, Science, 244.- 1288-1292 (1989)], of rodents that ^ 10 stop expressing a functional chitinase enzyme, over express the enzyme chitinase or express a variant chitinase enzyme. The polynucleotides of the invention, when properly labeled, are useful in hybridization assays to detect the ability of cells to synthesize chitinase. The polynucleotides of the invention may also be the basis for the diagnostic methods useful to identify a genetic alteration (s) in the chitinase locus that underlies a disease state or states. Also by the present invention antisense polynucleotides relevant to regulate the expression of chitinase by those cells that commonly express the same are put at the disposal. The administration of the chitinase separations of the invention to mammalian individuals, especially humans, For the purpose of reducing the disease states caused by parasites containing chitin as fungi, it is contemplated by the invention. Mycotic infections (mycosis) such as candidiasis, aspergillosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis, chromoblastomycosis, sporotrichosis, mucormycosis, and dermatophytosis may manifest as acute or chronic diseases. Pathogenic fungi cause series and often fatal diseases in hosts with problems in the immune system. Cancer patients who are undergoing chemotherapy, immunosuppressed individuals, and individuals infected with HIV are susceptible to mycosis caused by Candida, Aspergillus, Pneumicystis carinni, and other fungi, amphotericin B and fluconazole are useful therapeutic agents for fungal infections, but the toxicity associated with these drugs causes serious adverse side effects that limit their usefulness. The mortality of systemic candidiasis is greater than 50% despite treatment with amphotericin B. Therefore, there is a need for agents that inhibit fungal development in vivo; and these products can be used as individual agents or in combination with conventional antifungal compounds, currently tested. Because the growth of fungi requires the synthesis of chitin for survival, inhibition by recombinant human chitinase may be useful in limiting fungal infections in vivo. The animal models for fungal infection are illustrated below in Examples 8 to 14 and have been described in the art. Specifically contemplated by the invention are chitinase compositions for use in methods for the treatment of a mammal susceptible or suffering from fungal infections, which consists in the administration of chitinase to the mammal in an amount sufficient to complement the endogenous activity of chitinase. It is contemplated that chitinase may be administered with other conventional antifungal agents, including amphotericin B and structurally related compounds nystatin and pimaricin; 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and thioconazole; allylamines-thiocarbamates, such as tolnaftate,. naftifine and terbinafine; griseofulvin; ciclopiroxolamine; haloprogin; undecylenic acid; and benzoic acid. [See, for example, Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill NY (1996).] Chitinase may improve the effectiveness of these conventional antifungal agents, perhaps making yeast more susceptible to its action, even in situations where chitinase alone is not effective in inhibiting the growth of fungi. By reducing the amount of conventional fungal agent necessary to exert the desired therapeutic effect, chitinase may allow drugs to be used at less toxic levels. For example, Davies and Pope, Nature 273: 235-236 (1978) reported that the administration of mycolases (enzymes that degrade the cell wall of fungi) together with a normally infectious dose of medically anti-icotic mice infected with Aspergillus provided a synergistically effective treatment. The combination of fungal chitinase and laminarinase was observed to be more effective in attacking the fungal cell wall than the enzyme alone. In this manner, the invention contemplates the use of chitinase in the preparation of a medicament for the prophylactic or therapeutic treatment of fungal infections, and further contemplates the use of chitinase in the preparation of a medicament for co-administration with another antifungal agent. Therapeutic / pharmaceutical compositions contemplated by the invention include chitinase and a physiologically acceptable diluent or carrier, and may also include other antifungal agents. The amounts of the indicated dosage would be sufficient to complement the endogenous activity of chitinase. For general considerations on dosage see Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, PA (1995). The dosages will vary between about 1 μg / kg to 100 mg / kg of body weight, and preferably between about 0.1 to about 20 mg of chitinase / kg of body weight. Therapeutic compositions of the invention may be administered by different days depending on the infection treated, including subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intratracheal, topical, oral or suppository administration. The invention also contemplates that the overexpression of chitinase in Gaucher disease or at sites of inflammation (as can be rheumatoid arthritis) may have deleterious effects on the extracellular matrix and in these parameters disease, inhibitors Chitinase activity may provide therapeutic benefit, for example, by reducing the remodeling or destruction of the extracellular matrix. The human chitinase cDNA of the present invention was isolated from a macrophage cDNA library. Macrophages are known to be closely associated with rheumatoid arthritis lesions [Feldman et al, Cell., 85: 307-310 (1996)], and macrophage products such as TNF-a are implicated in disease progression. A protein with homology to human chitinase, C-gp39, has been detected in the synovium and cartilage of patients with rheumatoid arthritis. Although the natural substrate for human chitinase is probably chitin from pathogenic organisms, the enzyme may also exhibit activity on endogenous macro molecules that form the natural extracellular matrix. For example, it has been suggested that hyaluronic acid, a major component of the extracellular matrix, contains a nucleus of chitin oligomers. [Semino et al., Proc. Nat 'l Acad. Sci. , 93: 4548-4553 (1996); Varki, Proc. Nat 'l. Acad. Sci. 93: 4523-4525 (1996)]. Chitinase, therefore, may be involved in the degradation of the extracellular matrix in diseases such as rheumatoid arthritis. The function of chitinase can be determined by measuring chitinase levels and / or the effects of chitinase administration or the inhibition of chitinase in synovial fluid isolated from arthritic joints. The levels of endogenous chitinase can be measured by enzymatic assay or with an antibody. The viscosity of the synovial fluid can be measured before and after the chitinase treatment; a decrease in viscosity associated with chitinase would be consistent with an endogenous chitinase substrate. The modulation of chitinase activity, therefore, could modulate the progress of joint destruction in rheumatoid arthritis.

Also contemplated by the invention are methods for the screening of inhibitors of chitinase activity, which may be useful in the manner described in the previous paragraph. A method for screening samples to identify agents that inhibit chitinase is reported in, for example, WO 95/34678 published December 21, 1995. In addition, methods for measuring endogenous levels of chitinase are contemplated, for example, for diagnosing the disease of Gaucher. Hollak et al., J. Clin. Invest., 93: 1288-1292 (1994), report that plasma chitinase levels are a diagnostic marker for Gaucher's disease. It is expected that the recombinant proteins of this invention will be more useful than preparations purified from humans, which have associated problems of performance and contamination with other impurities or infectious agents.

DETAILED DESCRIPTION Other aspects and advantages of the present invention will be understood taking into consideration the following illustrative examples. Example 1 describes the isolation of cDNAs from human chitinase from a human macrophage cDNA library. Example 2 shows the pattern of the genetic expression of chitinase in different human tissues. Example 3 describes the recombinant expression of the human chitinase gene in prokaryotic cells and the purification of the resulting enzyme. Example 4 provides a protocol for the recombinant production of human chitinase in yeast. Example 5 describes the recombinant expression of the human chitinase gene in mammalian cells and the purification of the resulting protein. Example 6 describes the production of analogs of the human chitinase polypeptide by the methods of peptide synthesis or recombinant production. Example 7 provides a protocol for the generation of monoclonal antibodies that are specifically immunoreactive with human chitinase. Example 8 describes an assay for the measurement of the catalytic activity of chitinase. Example 9 relates to the determination of the antifungal activity of human chitinase in vi tro. Example 10 relates to the determination of the antifungal activity of human chitinase in vivo in a mouse model, and examples 11 to 14 relate to rabbit models of invasive aspergillosis, disseminated candidiasis, Candida ophthalmitis, and endocarditis. for Candida.

EXAMPLE 1 Isolation of cDNA from chitinase A cDNA library was prepared from macrophages obtained from peripheral blood monocytes as described in Tjoelker et al., Nature, 374: 549-552 (1995). The library was chosen randomly and the plasmid DNA was purified from the individual genes. The approximately 300 to 500 base sequence of the DNA end of each clone was determined in an automated sequencer (model 373, Applied Biosystems, Foster City, CA) using the JHSP6 primer, which hybridizes to the plasmid vector pRc / CMV (Invitrogen, San Diego, CA) adjacent to the cDNA cloning site: JHSP6 5'GACACTATAGAATAGGGC-3 '(SEQ ID NO: 5) The nucleotide and deduced amino acid sequence of these cDNAs were compared with the sequences in the nucleotide and the databases of the peptide sequences to determine the similarity with the known genes. Sequence comparisons were performed by BLAST Network Service of the National Center for Biotechnology Information using the alignment algorithm of Altschul et al., J. Mol. Biol. 215: 403-410 (1990). The MO-911 clone presented significant homology with several different sequences, including the YM-1 precursor of the secretory protein of the mouse macrophage (Genbank Access No. M94584), the human cartilage gp-39 (Hakala et al., Supra), the sheep, cow and human oviduct glycoprotein (DeSouza et al., supra), and parasite chitinases (Oncocerca, Genbank access No. U14639), wasp (Chelonus, Genbank access No. U10422), plant (Nicotiana, Genbank access) NO.X77111), and bacteria (Serratia, Genbank access No. Z36295); its highest homology observed was with mammalian genes that encode proteins with chitinase homology but chitinase activity was not demonstrated. Another analysis of the sequence of MO-911 suggests that it contains a portion of the coding region for a homology of human chitinase. The DNA sequence of clone pMO-218 (deposited on June 7, 1996 under the terms of the Budapest treaty with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, under accession No. 98077) set forth in SEQ ID NO: 1, and the encoded acid sequence is set forth in SEQ ID NO: 2. It appears that MO-218 includes the entire coding region of human chitinase cDNA (nucleotides 2 to 1402 of the SEQ ID NO: 1), which comprises a putative signal sequence of 21 amino acids followed by 445 encoded amino acids (residues 1 to 445 of SEQ ID NO: 2). The 22 amino acids following the sequence of the putative signal exactly match the amino terminal sequence of the purified human chitotriosidase reported in Renkema et al., Supra. Renkema et al. They also described a sequence of 21 amino acids from a triptych fragment of human chitotriosidase that corresponds exactly to residues 157 to 177 of MO-218 (SEQ ID NO: 2). Boot et al., Supra, report the cloning of human chitotriosidase cDNA containing a coding sequence essentially identical to that of MO-218. The sequence of MO-218 differs from Boot et al., By 14 additional nucleotides at the 5 'end and by a change of nucleotides at nucleotide 330 in the coding region. To confirm that MO-218 actually contained the entire coding region of the cDNA, a 3G pT labeled TGGGATCATCAGCAGGACCATGAAACCTGCCCAGGCCACAGACCGCACCAT, SEQ ID NO: 6) probe was prepared which corresponded to the complement of nucleotides 2 to 52 of MO-218 (SEQ ID NO: 1) The Pl probe was designed to hybridize with samples that are at least as long as MO-218 at the 5 'end.The probe was hybridized with a portion (approximately 30,000 sections) of the library. CDNA of the human macrophage described above, in 40% formamide and buffer for hybridization (5 x SSPE, 10 x Denhardt, 100 μg / ml denatured salmon sperm DNA and 2% SDS) at 42 ° C overnight The filters were washed and 3 sections that hybridized were chosen for sequence / analysis.The longest clone was designated pMO-13B (deposited on June 7, 1996 under the terms of the Budapest Treaty with the American Type Culture Collection, 12301 Parklawn Driv e, Rockville, MD 20852, USA. Under access no. 98078). The DNA sequence of pMO-13B is set forth in SEQ ID NO: 3 and the encoded amino acid sequence is set forth in SEQ ID NO: 4. This clone contains 25 additional nucleotides at the 5 'end compared to the MO- 218; in addition, MO-13B (SEQ ID NO: 3) contains a nucleotide substitution at nucleotide 330 (corresponding to nucleotide 305 of MO-218, SEQ ID NO: 1) that changes the amino acid encoded at position 80 of the mature protein from a glycine (in SEQ ID NO: 2) to a serine, (in SEQ ID NO: 4).

EXAMPLE 2 Genetic expression pattern of chitinase in human tissues The Northen blot analysis was performed to identify tissues in which human chitinase is expressed. A multiple human tissue Northen blot (Clontch, Palo Alto CA) was hybridized with the entire coding region of MO-218 under standard severe conditions (according to the Northen laboratory manual). The largest hybridization was observed for lung (+++) and ovarian tissue (+++), with much smaller (+) levels in thymus and placenta. The size of the hybridizing mRNA was 2.0 kb for lung, ovary and thymus, which corresponds well with the size of the cloned cDNA (1.6 kb, or approximately 1.8 kb including the poly A tail). The size of the hybridizing placenta mRNA was considerably smaller, at 1.3 kb. Hybridization of chitinase was not observed in spleen, prostate, testes, small intestine, colon, peripheral blood leukocytes, heart, brain, skeletal muscle, kidney or pancreas. The expression of chitinase in lung is consistent with a protective function against pathogenic organisms that contain chitin, since the lung represents the primary entry route for pathogenic fungi.

Example 3 Production of recombinant human chitinase in bacterial cells The mature coding region of MO-218 was designed for expression in E. coli as a truncated analog at the C-terminus. The PCR was used to generate a DNA fragment for expression using a primer corresponding to nucleotides 65 to 88 of the chitinase cDNA of MO-218 preceded by. a methionine initiation codon and a restriction endonuclease site Xbal (5'-TACATCTAGAATTATGGCAAAACTGGTCTGCTACTTCACC-3 ', SEQ ID NO: 7), and a primer in the transcription direction encoding nucleotides 1163 to 1183 of the MO-218 followed by a nonsense codon and a HindIII site (5 'AGATCTAACCTTAGGTGCCTGAAGACAAGTATGG-3, SEQ ID NO: 8). The primer in the direction of transcription contained an adenine in base 25, while the sequence of MO-218 contains a guanine in the corresponding position of the nucleotide. Accordingly, the resulting DNA fragment contains a thymine in place of a cytosine at the position corresponding to nucleotide 1172 of the M0-218 sequence, and the encoded chitinase fragment, set forth in SEQ ID NO: 15, is also a analog containing a serine at position 370 of the mature amino acid in place of the proline encoded by MO-218. The resulting DNA fragment was digested with Xbal and HindIII and cloned into the pAraBAD plasmid (which is also known by the designation pAraCB). Plasmid pAraCB was prepared as follows. Plasmid pUC19 was modified to include a arabinose promoter and subsequently to include AKAP coding sequences 79. The arabinic promoter [Wilcpx et al., Gene, 34: 123-128 (1985); Wilcox, et al., Gene 18: 157-163 (1982)] and the araC gene were amplified by PCR from the BAD arabinose operon of Salmonella typhimurium as an EcoRI / Xbal fragment with the araC-2 primers (SEQ ID NO: 9) and arab-1 (SEQ ID NO: 10): araC-2 TACAGAATTCTTATTCACATCCGGCCCTG SEQ ID NO: 9 arab-1 TACATCTAGACTCCATCCAGAAAAACAGGTATGG SEQ ID NO: 10 The araC-2 primer encodes an EcoRI site (underlined) and • a stop codon (italics) for the araC gene product. The araC-1 primer encodes a putative ribosome that is unites the domain (italics) and a restriction site Xbal (underlined). PCR with these produced a 1.2 kb fragment that was digested with EcoRI and XBal and subcloned into pUC19 (New England Biolabs, Beverly, MA) previously digested with the same two enzymes. The resulting plasmid was named ? t 10 araCB and contained a polylinker region (SEQ ID NO: 11) flanked at the 5 'end with a Xbal restriction site (underlined) and the 3' end with a HindIII site (italics).

AraCB polliynker TCTAGAGTCGACCTGCAGGCATGCAAGCTT SEQ ID NO: 11 Transformants containing the resulting expression plasmid (pAraM0218) were induced with arabinose and developed at 37 ° C. These transformants produced inclusion bodies that contained a 39 kDa protein which was a truncated form of chitinase (designed to contain 373 instead of 445 amino acids). This fragment of chitinase contains 4 cysteine residues, while full-length chitinase. contains 10 cysteine residues. The inclusion antibodies were separated from the E. coli culture and subjected to electrophoresis on SDS-PAGE. The 39 kDa band was transferred to a PVDF membrane and sequenced at its amino termini. The major part (approximately 2 thirds) of the material contained a sequence corresponding to the amino terminus of human chitinase. The remaining material corresponded with a contaminating E. coli protein, porin. This preparation of recombinant chitinase from E. coli was useful to produce polyclonal and monoclonal antibodies (described below in example 7). When the transformants containing the aRA-chitinase expression plasmid were developed at 25 ° C, the inclusion antibodies were not observed and the expression of the recombinant product was decreased from about 10% of the cellular protein. total to approximately 1%. However, this material produced at 25 ° C presented catalytic activity of chitinase.

Example 4 Production of recombinant human chitinase in yeast cells Exemplary protocols for the recombinant expression of human chitinase in yeast and for the purification of the resulting recombinant protein are as follows. The coding region of human chitinase is designed into vectors for expression in Saccharomyces cerevisiae using PCR or linker oligonucleotides designed to encode a fusion polypeptide containing a secretion-mediating leader for the coding region for the corresponding human chitinase with the amino terminal of the natural molecule. Signal peptides from secretion include, for example, SUC2 or equivalent leaders with a functional signal peptidase cleavage site or pre-pro-alpha factor or other complete complex leader of a pre, or signal peptide, and a pro, or region spacer, which has a KEX2 cleavage site, the DNA encoding the signal sequence can be obtained by synthesis of oligonucleotides or by PCR. The DNA encoding the pre-alpha leader factor is obtained by PCR using primers containing nucleotides 1 to 20 of the alpha coupling factor gene and a primer complementary to the nucleotides 255 to 235 of this gene [Kurjan and Herskowitz, Cell , 30: 933-943 (1982)]. The fragments of the coding sequence of the pre-pro-alpha leader and of the coding sequence of human chitinase are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, so that the promoter directs expression to a protein of fusion. As Rose and Broach taught, [Meth. Enz. 185: 234-219, D. Goeddel, ed. , Academic Press, Inc., San Diego, CA (1990)] ,. The vector further includes an ADH2 transcription terminator downstream or in the direction of transcription of the cloning site, the "2-μ" origin of replication of yeast, a selectable marker, eg, TRP1, CUP1 or LEU2 (or LEU2-d) or another equivalent gene, the REP1 and REP2 genes of yeast, the beta-lactamase gene of E. coli, and the origin of replication of E. coli. the beta-lactamase and TRP1 genes provide selection in bacteria and yeast, respectively. The genes for REP1 and REP2 encode proteins included in the replication of the copy number of the plasmid. Otherwise, other melting points within the coding region of chitinase can be chosen. The truncates of the coding region can be used to increase the homogeneity of the product, increase the specific activity or modify the substrate specificity. The DNA constructs described in the previous paragraphs are transformed into yeast cells using a known method, for example, the treatment with lithium acetate [Stearns et al., Meth. Enz. supra, pp 280-297] or by equivalent methods. The ADH2 promoter is induced with glucose depletion in the growth medium [Price et al., Gene, 55: 287 (1987)]. The pre-pro-alpha sequence or other leader sequence effects the secretion of the fusion protein by releasing the mature human chitinase peptide from the cells. The signal peptide leader is processed by the signal peptidase or, in the case of the elimination of pre-pro-alpha from the pro region, by the KEX2 protease [Bitter et al., Proc. Nati Acad. Sic. USA, 81: 5330-5334 (1984)]. Chitinase contains mature amino acid sequence 2 dibasic sequences at positions 107-108 (Lys-Arg) and 2 '09-210 (Arg-Lys) that can be proteolytically trimmed by the KEX2 protease during secretion. To stabilize and / or increase the level of the secreted product of the cells, these sequences can be mutated to eliminate the potential sites for proteolysis as shown by Gillis et al. [Behring Inst. My tt. , No. 83: 1-1 (1988)] or by the expression of chitinase without dibasic modifications in a host that is deficient in KEX2. This host can be obtained by scanning for the non-KEX2 protease containing mutants or by manipulation of the genomic KEX2 locus by genetic replacement / genetic disruption techniques [Orr-Weaver et al., Proc. Nati Acad. Sci, USA, 78: 6354-6358 (1981)]. Recombinant chitinase can be secreted from Saccharomyces cerevisiae using similar vectors containing alternative promoters PRBI, GAL4, TPI, or other adequately strong promoters carrying fragments or by fusion to a variety of leader sequences [Sleep et al. , Bio / Technol. , 8: 42-46 (1990)].

Other suitable expression hosts other than Saccharomyces cerevisiae include Kluyveromyces Lactis, Schizosaccharomyces pom.be, Pichia pastoris and members of the genus Hansenula or Aspergillus. Analogous recombinant expression systems for these fungi include the organism and its suitable autonomous replication vectors [eg, Falcone et al. , Plasmid, 15: 248-252 (1988)] or expression cassettes with multiple integrations. These systems also depend on signal sequences or leaders of the type described above to mediate the secretion in the medium. Secreted, recombinant human chitinase is purified from the yeast growth medium, for example, by the methods used to purify chitinase from supernatants of bacterial and mammalian cells (see example 3 above and example 5 below). Otherwise, the mature form of the recombinant chitinase product can be expressed in the cytoplasms of Saccharomyces cerevisiae or analogous hosts, and can be purified from host cells used. It is possible to re-double the protein during the act of purification to obtain the appropriate levels of specific activity.

Example 5 Production of recombinant human chitinase in mammalian cells A. Expression in COS cells Clone MO-218 and clone M0-13B were isolated, both of which contain the 3 'full-length human chitinase cDNA to the CMV promoter of pRc / CMV. A third plasmid, which corresponded to the same C-terminal fragment expressed in bacterial cells in Example 3 above was prepared as follows. The plasmid MO-218 was amplified by PCR using the oligonucleotide primer 218-1 (CGCAACGTTGAGAGCTCCGTTCCGCCACATGGTGCGGTCTGTGGCCTGGG, SEQ ID NO: 12), which contains a HindIII site and nucleotides 2 to 23 of the chitinase cDNA for MO-218 of SEQ ID NO: 1, and with the complementary T-END primer in the sense of transcription (GACTCTAGACTAGGTGCCTGAAGGCAAGTATG, SEQ ID NO: 13), which contains nucleotides 1164 to 1183 of MO-218, a nonsense codon 1 and an Xbal site. The amplified DNA was purified by electrophoresis, digested with Xbal and HindIII and cloned into the pRc / CMV vector (Invitrogen, San Diego, CA) previously cut with the same restriction enzymes. The junctions of the resulting clone were sequenced in a model 373 (Applied Biosystems, Foster City CA) and encoded the designed, predicted protein sequence, as set forth in SEQ ID NO: 14. Three plasmids were transiently transfected into COS cells by the DEAE transfection method [see, for example, Sambrook et al., Molecular Cloning: a Laboratory Manual, 2d ed., Cold Sprring Harbor, New York: Cold Spring Harbor Laboratory (1989).) After 3 days 37 ° C, the cell medium was assayed for the chitinase activity in vi tro as described below in Example 8. Each culture produced significant activity of the chemtinase (600-800 U / ml / min), and similar amounts were observed. for each construction. Recombinant human chitinase was purified as follows. 5 days after transfection of COS cells with the pásmid pRc / CMV-MO-13B, conditioned medium from the culture was harvested and diluted with an equivalent volume of water. The diluted medium, conditioning was applied to a Q-Sepharose Fast Flow column (Pharmacia Biotech, Uppsala, Sweden) which was pre-equilibrated in 25 mM tris, 10 mM sodium chloride, 1 mM EDTA, at pH 8.0. Approximately 95% of the chitinase activity flowed and did not bind to the column. This flow through Q-Sepharose was adjusted with 1.2 M ammonium sulfate and applied to a Butyl-Sepharose 4 Fast Flow column (Pharmacia) which was pre-equilibrated in 25 mM tris, 1.2 M ammonium sulfate, 1 mM EDTA, at pH 8.0. The protein was eluted using inverse gradient of ammonium sulfate 1.2 m to 0 M in 25 mM tris; pH 8.0. A single absorbance peak at 280 mM corresponding to the peak of chitinase activity was eluted at low salt concentration. This material was greater than 85% pure as determined by SDS-PAGE and contained approximately 60% chitinase activity. The protein was then concentrated and the buffer solution exchanged in 20 mM tris; 150 mM sodium chloride; at pH 8.0 using a 10,000 MWCO membrane (Ultrafree 10K, Millipore Corp., Bedford, MA). This preparation was then tested for in vitro enzymatic and antifungal activity as described in Examples 8 and 9 below. The recombinant preparation had a chititriosidase activity of 90 nm / min per mg of protein.

B. Expression in CHO cells The chitinase gene was inserted into pDEFl (the construction of which is described in example 4 of US Application Serial No. 08 / 847,218, filed May 1, 1997, incorporated herein by reference). reference) cleaving the 1.77 kb HindIII / Xbal fragment containing the full-length chitinase gene from pEc / CMV / MO-13B and ligating the fragment with pEDF1 digested with HindIII / Xbal, to create the plasmid pHDEFI / CTN. l. The 1.7 kb HindIII / Xbal fragment containing the chitinase gene was also ligated into pHDEFl digested with HindIII / Xbal to create the plasmid. pHDEFl / CTN.l. The plasmid pHDEFl is the same as pDEFl except for differences: (1) in pHDEFl, an Ehel / Sall fragment of 2 kb from pCEP4. { Invitrogen Carlsbad) containing a hygromycin resistance gene replaced the 19 pb pmel / SalI fragment of pDEFl; (2) in pHDEFl, the expression of the dihydrofolate reductase (DHFR) gene is controlled by a shortened SV40 promoter contained in a 120 bp Nhel / Asp718 fragment that replaced the 212 bp Nhel / Asp718 fragment of pDEFl. This 120 bp Nhel / Asp718 fragment was prepared by first amplifying a 171 bp PCR fragment with the oligonucleotide primer 94-26 (5'-TGATACGGTACCGCCCCATGGCTGACTA-3 ', SEQ ID NO: 16) (containing a new Asp718 site), and the primer 94-27 (GCAAGTTTGGCGCGAAATCG-3M SEQ ID NO: 17), using as a template the DNA from the pDCl (described in example 4 of the US Application Serial No. 08 / 847,218 filed May 1, 1997) bearing the SV40-DHFR cassette , and then digesting this 171 bp RCP fragment with Nhel and Asp 718. The Chinese hamster ovary DG44 (CHO) cell line negative for DHFR was transfected with plasmid pDEF1 / CTN.l as described in example 5 of the application of the United States series no. 08 / 847,218 filed May 1, 1997. The CH44 DG44 cell line was also transfected with the plasmid pHDEF1 / CTN.1, followed by selection using the following modified procedure. The cells were first selected for hygromycin resistance only, in medium (DMEM / F-12 supplemented with 2-10% dialysed FBS) with a content of 800 mg / liter of hygromycin (Calbiochem, San Diego) and also with a hypoxanthine and thymidine content (which therefore made the medium non-selective for the HFR gene). After selecting the transfectants that were resistant to hygromycin, the cells were also selected for the expression of the DHFR gene by growing them in medium lacking hypoxanthine and thymidine. Next, hygromycin-resistant and DHFR-positive CHO cells were selected in medium containing first 10 nM, then 20 nM, and finally 50 nM methotrexate, which resulted in the selection of. cells that express higher levels of chitinase. The supernatant of the CHO cells transfected with pHDEF1 / CTN.l containing expressed recombinant human chitinase over (rH-chitinase) was purified as follows. In the preparation for anion exchange chromatography, the supernatant was diluted 1: 3 with 20 nM tris, pH 7.0 (Buffer A). An anion exchange column, packed with Q-Sepharose Fast Flow resin (Pharmacia Biotech Inc. Piscataway, NJ), was equilibrated with Buffer A and loaded with 15 1 of diluted supernatant per 1 1 of resin. The rH-chitinase was collected in the flow through the Q-Sepharose column and adjusted to 5% polyethylene glycol (PEG) 400 (MillincKrodt Baker, Inc., Phillipsburg, NJ), 30 mM sodium acetate, pH 4.3 in preparation for cation exchange chromatography. A cation exchange column packed with CM-Sepharose Fast Flow resin (Pharmacia Biotech Inc. Piscataway, NJ) was equilibrated with 30 mM sodium acetate, 5% (PEG) 400, pH 4.3 (Buffer B). The rH-chitinase sample was loaded on the CM-Sepharose column at 1 mg per ml of resin, and the rH-chitinase was eluted from the column with 40 mM tris, 5% PEG 400 pH 7.5 (Buffer C). The sample of rH-chitinase was then prepared for interaction chromatography, hydrophobic adding (NH4) 2S04 1.5 M. A column packed with Macro-Prep support Methyl Hlc, (Bio-Rad Laboratories, Hercules, CA) was equilibrated with tris 20 mM, 5% PEG 400 pH 7.0 (Buffer D). That contained 1.5 M of (NH4) 2S04. The rH-chitinase sample was loaded onto the Macro-Prep Methyl 1 mg column per ml of resin. The column was washed with Buffer D containing 1.1 M (NH4) 2S? 4, and the rH-chitinase was eluted with buffer D containing 0.2M (NH4) 2S04. The purified eluate was exchanged in NaCl, 150 mM, tris 20 mM, pH 7.0 (Buffer E) by membrane filtration.

Example 6 Production of analogs and fragments of human chitinase. Recombinant techniques such as those described in the above examples can be used to prepare the analogs and fragments of the human chitinase polypeptide. More specifically, the polypeptides encoding human chitinase are modified to encode the polypeptide analogs of interest using well-known techniques, for example, site-directed mutagenesis and polymerase chain reaction. C-terminal and N-terminal deletions can also be prepared by, for example, deleting the appropriate portion of the polynucleotide coding sequence. See, generally, Sambrook et al., Supra, chapter 15. The modified polynucleotides are expressed recombinantly, and the analogs or fragments of the recombinant polypeptide are purified as described in the previous examples. Crude residues are identified for human chitinase activity, for example, by homology with other chitinases or substituting alanine for the amino acid residues of native human chitinase. Cysteines are often crucial for the functional integrity of proteins due to their ability to form disulfide bonds and limit secondary structure. To determine if any of the cysteines in human chitinase are crucial for enzymatic activity, each cysteine is used individually in a serine. A truncated fragment at the C-terminus of 39 kDa of the mature human chitinase protein was prepared as already described in Examples 3 and 5 by introducing a nonsense codon after the codon for amino acid 373. This fragment of 39 kDa lacking 72 C-terminal amino acid residues of the mature protein, including 6 cysteines, I still retain similar specific enzymatic activity compared to full-length recombinant human chitinase. This result indicates that the 72 missing C-terminal residues, including the six cysteines, are not necessary for the enzymatic activity. It is possible to prepare other C-terminal deletions, for example, by carrying out the digestion of the 3 'end of the truncated human chitinase coding sequence described in Example 3 with hexonuclease III for different amounts of time and then ligating the shortened coding sequence for the plasmid DNA coding stop codons in all three reading frames. N-terminal deletions are prepared in a similar manner by digesting the 5 'end of the coding sequence and then ligating the digested fragments into a plasmid containing a promoter sequence and an initiating methionine immediately upstream of the promoter site. These analogs or fragments of the N-terminal deletion can also be expressed as fusion proteins. Otherwise, analogs of the human chitinase polypeptide can also be prepared by partial or complete chemical synthesis of peptides using known techniques. [See, for example, Synthesis of IL-8 in Clark-Lewis et al. J. Biol. Chem., 266: 23128-34 (1991); Synthesis of IL-3 in Clark-Lewis et al. Science, 231: 134-139 (1986) and synthesis by ligation in Dawson et al., Science 266: 116-119 (1994).] These synthetic methods also allow the selective introduction of unnatural, novel amino acids and other chemical modifications. The biological activities, including enzymatic, antifungal and remodeling activities of the extracellular matrix of the human chitinase polypeptide analogues are assayed by recognized techniques, such as those described in examples 8 to 14 below.

Example 7 Preparation of monoclonal antibodies to human chitinase The following two protocols (multiple-challenge or one-step immunizations) can be used to generate monoclonal antibodies to human chitinase. In the first protocol, a mouse is periodically injected with recombinant human chitinase (eg, 10-20 μg emulsified in complete Freund's adjuvant) obtained as described in any of examples 3 to 6. The mouse receives a final boost prior to the fusion of human chitinase in PBS, and 4 days later the mouse is sacrificed and the spleen removed. The spleen is placed in 10 ml of serum-free RPMI 1640, and a single cell suspension is formed by crushing the spleen between the frozen ends of two glass coverslips for microscopes immersed in serum-free RMPI 1640, supplemented with 2 mM L-glutamine. , 1 mM sodium pyruvate, 100 units / ml penicillin and 100 μg / ml streptomycin (RPMI) (Gibco, Canada). The cell suspension is filtered through a sterile 70 mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the package in 20 ml of free RMPI. serum. Splenocytes taken from 3 young Balb / c mice are prepared in a similar manner and used as a control. NS-1 myeloma cells, maintained in logarithmic phase in RPMI with 11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for 3 days before fusion, centrifuged at 200 g for 5 minutes and the package is washed twice as described in the previous paragraph. 1 x 108 spleen cells are combined with 2.0 x 10 7 NF-1 cells and centrifuged and the supernatant aspirated. The pack of C cells dislodged by tapping the tube, and 1 ml of PEG 1500 was added at 37 ° C (50% in Hepes, 75 mM, pH 8.0) (Boehringer Mannheim) with shaking for the course of 1 minute, followed by adding 7 ml of RPMI without serum for 7 minutes. 8 ml of additional RPMI is added and the cells are centrifuged at 200 g for 10 minutes. After discarding the supernatant, the package is resuspended in 200 ml of RPMI with a content of 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminophtine, 16 μM thymidine (HAT) (Gibco). 25 units / ml of IL-β (Boehringer Mannheim) and 1.5 x 105 splenocytes / ml and plated in 10 flat-bottomed 96-well tissue culture plates (Corning, Corning New York). On days 2, 4, 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusion is explored by ELISA, testing for the presence of mouse IgG that binds to human chitinase as follows. 4 Immulon plates (Dynatech, Cambridge, MA) are coated for 2 hours at 37 ° C with 100 ng / well of human chitinase diluted in 25 mM tris, pH 7.5. The coating solution is aspirated and 200 Ul / well of blocking solution [0.5% fish skin gelatin (sigma) diluted in CMF-PBS] are added and incubated for 30 minutes at 37 ° C. Plates are washed 3 times with PBS with 0.05% Tween 20 (PBST) and 50 μl of culture supernatant is added. After incubation at 37 ° C for 30 minutes and washing as in the previous case, 50 μl of horseradish peroxidase conjugated with goat anti-mouse IgG (fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted 1 : 3500 in PBST. The plates are incubated as above, washed four times with PBST, and 100 μl of substrate, with a content of 1 mg / ml of 0-phenylenediamine (sigma) and 0.1 μl / ml of 30% H202 is added. 100 mM citrate pH 4.5. The colorful reaction is interrupted after 5 minutes with the addition of 50 μl of 15% H2SO4. The A490 is read on a plate reader (Dynatech). The selected fusion wells are cloned twice by dilution in 96-well plates and a visual record of the number of colonies / well is made after 5 days. The isotype of the monoclonal antibodies produced by hybridomas is determined using the Isostrip system (Boehringer Mannheim, Indianapolis, IN). Otherwise, a second protocol that uses a single intrasplenic immunization can be performed, in general, according to Spitz, Methods Enzymol. , 121: 33-41 (1986). The spleen of the animal is exposed and injected with recombinant human chitinase (eg, 10-20 μg in PBS at a concentration of about 0.02% to 0.04%, with or without an aluminum adjuvant) obtained as described in any of the Examples 3 to 6, after which the spleen is returned to the peritoneal cavity and the animal is sutured. Three days later, the mouse is sacrificed and the spleen is removed. A suspension of spleen cells is prepared, washed twice with RMPI 1640 supplemented with 3% fetal bovine serum (FCS) and resuspended in 25 ml of the same medium. The myeloma cells (NS-O) are harvested in the logarithmic growth phase, washed 1 time and added to the suspension of spleen cells in a 50 ml tube, in a ratio of 3: 1 or 2: 1 ( splenic cells: myeloma cells). The mixture is packaged at approximately 450 g (1500 rpm), the supernatant is aspirated, and the package is separated by tapping the tube. The fusion is carried out at room temperature adding 1 ml of polyethylene glycol (PEG) 1500 for 1 minute, with constant agitation. The mixture is incubated for another minute, then 1 ml of warm RPMI (30 to 37 ° C) is added for 1 minute followed by 5 ml of RPMI for 3 minutes and another 10 ml of RPMI for another 3 minutes. The cell suspension is centrifuged and resuspended in approximately 200 ml of selective HAT medium consisting of RPMI 1640 supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 20% FCS, 100 mM hypoxanthine, 0.4 mM aminofterin and thymidine 16 mM. The cell suspension is distributed in 1 ml volumes in tissue culture plates and incubated at 37 ° C in a humid atmosphere with C02 5% -air 95% for 8 to 10 days. The supernatants are aspirated and the cells are fed with 1 ml of HAT medium per well, every 2 to 3 days according to cell growth. Supernatants from confluent wells are screened for specific antibodies and positive wells are cloned.

Example 8: Catalytic Activity of Recombinant Chitinase The activity of chitotriosidase (chitinase) was measured using the fluorogenic substrate 4-methylumbelliferyl-β-DN, N ', N "-triacetylchitotriose (4 MU-quitotrusside, Sigma Chemical, St. Louis, MO ), in buffer McILvain (Hollak et al., supra). Samples of 10 μl of the recombinant product were combined with 10 μl of bovine serum albumin (10 mg / ml), 15 ml of fluorogenic substrate (2.71 mM), and 65 μl of buffer (0.1 M citric acid, 0.2 M sodium phosphate) , pH 5.2) in a total volume of 100 μl. The reactions were incubated at 37 ° C for 15 minutes, then the reaction was interrupted with the addition of 2 ml of 0.3 M glycine / NAOH buffer (pH 10.6). The product of the fluorescent splitting, 4-methylumbelliferone, was monitored with a fluorimeter (SLM-AMINCO Instruments, Inc., Rochester, NY) at 450 nm. To obtain a standard curve, various substrate concentrations were combined with excess bacterial chitinase to ensure that the substrate was completely unfolded. The known quantity of 4-MU was then correlated with the fluorescence signal from the fluorometer and the linear regression was used to determine a standard curve. The signal produced with purified recombinant chitinase, diluted in the assay was then used to interpolate the amount in nm of the substrate cleaved by the enzyme during the reaction time. This number was then divided by the concentration of the protein to obtain nm / in per mg of protein (determined by A2so and the calculated molar extinction coefficient). The chitotriosidase activity of the recombinant human chitinase produced in COS cells as described in example 5A was determined 90 nm / min per mg protein.

Example 9 Antimycotic activity of recombinant chitinase in vi tro In a preliminary experiment, recombinant human chitinase was tested for the inhibition of fungal growth in vi tro. The two fungi Candida albicans and Aspergill us fumigatis are serious pathogens for patients with problems in the immune system. Candida and Aspergill were developed in RPMI growth medium at 37 ° C for approximately 10,000-50,000 colony forming units (CFU) per ml. Recombinant human chitinase (produced in COS cells as described in example 5A) was added to the cultures at 0, 2.8, 11.25 or 45 μg / ml. After 24 hours, the growth of fungi was assessed by the turbidity of the crops. Under these non-physiological conditions in this trial, all cultures appeared to grow at comparable rates, independent of the chitinase concentration. However, the concentration of fungi tested is much higher than the fungal load observed during fungal infection in vivo. Different results can be obtained under different conditions, for example, with a lower fungal load, or when human chitinase is tested in combination with other antifungal agents. Chitinase is also expected to be more effective in vivo under physiological conditions. In additional experiments, the antifungal activity of recombinant human chitinase (produced in COS cells as described in example 5A) was evaluated in an agar diffusion assay, in a test broth according to National Commitee on Clinical Laboratory Standards, and in an assay of inhibition of the cell wall according to Selitrennikoff, Antimicrob. Agents, Chemother. , 23: 757-765 (1983).

In the agar diffusion assay, approximately 1 x 106 cells / ml of Candida albincans (ATCC No. 90028) inoculated on 1.5% agar (RPMI 1640 medium) buffered with 2- (N-moformolin) propanesulfonic acid (MOPS), pH 7.0 A disk with a content of 50 μg of the sample (A: recombinant human chitinase, B: control buffer, C: control protein, B: used bacterial with chitinase activity, or a known antifungal agent) were placed on the agar, and the zone of growth inhibition was measured. The results are shown in table 1 below.

Table 1 Samples: A = rH-chitinase prepared from COS cells according to example 5A, in 150 mM NaCl, EDTA, 1 mM, 20 mM tris, pH 7.5; B = buffer (150 mM NaCl, 20 mM tris, pH 7.5); C = inactive PAF-AH protein (diluted from a standard solution of 2 mg / ml); D = Serratia marcescens lysate (SIGMA) # C-7809) with chitinase activity. Key to the growth record: (+) = normal growth, no inhibition of growth was observed; (-) = inhibited fungal growth, zone of inhibition was observed. Sample D stimulated the growth of Aspergillus fumigatus. In the broth assay (50 μg / ml sample - (A: recombinant human chitinase, B: control buffer, C: control protein, D: bacterial lysate with chitinase activity, or a known antifungal agent) were added with a Some concentration of fungal test organism in RPMI 1640 medium buffered with MOPS, pH 7.0 The samples were incubated at 35 ° C, with shaking at 120 rpm, for 48 hours, and then the growth was evaluated by measuring the turbidity of the suspension. The approximate concentrations of the test fungus were as follows: 2.5 x 10 cells / ml of Candida albicans (ATCC No. 90028), 5 x 10 4 cells / ml of Candida albicans polyene resistant (ATCC No. 38247), 1 x 10 4 cells / ml of Aspergillus fumigatus (ATCC No. 16424); 1 x 10 4 cells / ml of Neurospora crassa (ATCC No. 18889); 1 x 10 4 cells / ml of Saccharomyces cerevisiae (ATCC No. 26108) The results are shown in the table 2 next. • 53 Table 2 (continued) to samples: A = rH-chitinase prepared from COS cells according to example 5A, in 150 mM NaCl, EDTA, 1 mM, 20 mM tris, pH 7.5; B = buffer (150 mM NaCl, 20 mM tris, pH 7.5); C = inactive PAF-AH protein (diluted from a standard solution of 2 mg / ml); D = Serratia marcescens lysate (SIGMA) # C-7809) with chitinase activity. b key to the growth record: (0) = fungal growth; (1) = growth is 25% of control; 2 = the growth is 50% of the control; 3 = the growth is 75% of the control; 4 = the growth equivalent to the control; 5 = growth greater than the control c IC9o: the lowest concentration at which a compound inhibits the growth of an organism by at least 90%; equivalent to at least one record of 0. D IC 50: the lowest concentration at which a compound inhibits the growth of an organism by at least 50%; equivalent to at least a record of 2.

The os-1 whole cell assay, which identifies the inhibitors of fungal cell wall biosynthesis, was performed essentially according to Selitrenikoff, supra, using an inoculum of 1.5 x 105 protoplasts / ml embedded in agar (Vogel N medium, 7.5% sorbitol, 1.5% sucrose, 10 μg / ml nicotinamide and 1% agar) incubated at 25 ° C for 75 hours. The cultures were monitored for changes in growth and morphology after the disks contained 50 μg of the sample (A: recombinant human chitinase, B: control buffer, C: control protein, D: bacterial lysate with chitinase activity, or a known antifungal agent) were placed on the agar medium. The os-1 cell is a mutant strain of Neurospora crassa that grows on protoplasts without cell walls when incubated under certain conditions at 37 ° C, but regenerates a cell wall under the right conditions when the temperature changes to about 22 ° C. The samples that inhibit the growth are considered inhibitors of the fungal growth and the samples that avoid the regeneration of the cellular wall, but do not destroy the cells, are considered specific inhibitors of the cell wall. The results are shown in table 3 below.

Table 3 to samples: A = rH-chitinase prepared from COS cells according to example 5A, in 150 mM NaCl, EDTA; 1 mM, tris 20 mM, pH 7.5; B = buffer (150 mM NaCl, 20 mM tris, pH 7.5); C = inactive PAF-AH protein (diluted from a standard solution of 2 mg / ml); D = Serratia marcescens lysate (SIGMA) # C-7809) with chitinase activity. b Registry key for fungal growth: (+) = normal growth, no inhibition of growth was observed; (-) = inhibited fungal growth, zone of inhibition was observed. c Registry key for cell wall regeneration: (+) = regeneration of the normal cell wall; (-) = regeneration of the inhibited cell wall. d Radial measurements of the regeneration of the cell wall inhibited from the center of the disk. e Radial measurement of growth inhibited from the center of the disk. The results of the tests showed that the chitinase sample was a specific inhibitor of the cell wall in the os-1 whole cell assay and was moderately antifungal in the broth assay.

Example 10 Antimycotic Activity of Recombinant Chitinase in vivo in Mouse The far kinakinetics of recombinant human chitinase in mice was determined as follows. Balb / c female mice of 6 to 8 weeks were injected intravenously into the tail vein with 0.5 mg / kg, 5.0 mg / kg and 50 mg / kg of recombinant human chitinase. For each dose, the mice were bled at 0.01, 0.25 1, 8, and 24 hours after the injection (two animals were used per point of time per dose). The serum samples were then assayed for the activity and concentration of chitinase. The results are shown in table 4 below.

Table 4 ABC: area under the curve for infinity of time. Vee: volume of distribution in the steady state. eL: elimination. TEP: total average time of stay in the body. Cmax: maximum concentration in serum. Various models in animals have been developed to test the efficacy of antifungal compounds [see Louie et al., Infecí Immun. , 62: 2761-2772, 1994; Kinsman et al., Antimicrobial Agenis and Chemotherapy, 37: 1243-1246, 1993; Nakajima et al., Antimicrobial Agents and Chemotherapy, 39: 1517-1521 1995-; Tonneti et al., Eur. J. Immunol. , 25: 1559-1565 (1995); Denning and Stevens, Antimicrobial Agents and Chemother. , 35: 1329-1333 (1991) see also Stevens J. Mycol. Med., 6 (suppl. I): 1-10 (nineteen ninety six) ] . In short, the host animal is infected with the fungus, varying the doses of chitinase administered to the animals, and their survival is measured over time. The experiments are performed using chitinase as the sole therapeutic agent, or with a combination of conventional antifungal agents such as amphotericin B and fluconazole to determine if chitinase improves the efficacy of these compounds. Specifically, acute systemic candidiasis is obtained in mice by intraperitoneal or intravenous introduction of 10 x 106 CFU of Candida albicans. The therapeutic agents are administered before or at 1 to 5 hours after introduction, and the number of survivors is determined after 5 days. In addition, mice can be killed and the fungal load can be determined in specific organs such as brain, kidney, lung, liver and spleen. Otherwise, the mice are infected with lower doses of fungi, for example, Aspergillus (8-10 x 106 CFU) or Candida (1 x 106 CFU), in which case survival can be measured at more distant time points, for example, 45 days. The long-term fungicidal / fungistatic activity of chitinase alone or with another antifungal medicine can be evaluated by continuing the therapy for a week or more, for example, 11 days, and monitoring the animals for several weeks, for example, 18 days to a month. Effective antifungal agents promote the long-term survival of animals and reduce the fungal burden on blood and organs.

Example 11 Chitinase activity in vivo in a rabbit model of invasive aspergillosis The efficacy of chitinase, alone or in combination with other conventional antifungal agents, is evaluated in an immunosuppressed rabbit model of invasive Aspergillosis that has been used for 10 years to evaluate a variety of antifungal therapies. See, for example, Andriole et al., Clin. Infected Dis. , 14 (suppl.1); S134-S138 (1992) the study is generally performed according to Patterson et al., Antimicrob. Agents Chemother. , 37: 2307-2310 (1993) or George et al., J. Infect. Dis. 168: 692-698 (1993). In brief, on day 1 rabbits are administered with cyclophosphamide (200 mg) intravenously to make them leukopenic, followed by triamcinolone acetonide (10 mg) subcutaneously every day during the time of the experiment. On day 2, 24 hours after immunosuppression, the animals are infected intravenously with approximately 10d (lethal load) or approximately 105 (subletal load) of conidia A. Fumigatus. Antifungal therapy (chitinase alone, or in combination with other conventional antifungal agents amphotericin B, fluconazole, or 5-fluorosynthin) is initiated 24 hours after infection or 48 hours before (for prophylaxis) and is continued for 5 to 6 days or until death. Examples of conventional antifungal agent doses are 1.5 to 0.5 mg / kg / day of intravenous amphotericin B, 60 or 120 mg / kg / day of oral fluconazole and 100 mg / kg / day of oral 5-flukocitone. Control rabbits are not treated with any antifungal agent. At autopsy or death, semiquantitative fungal cultures and histopathological examination are performed on the liver, spleen, kidneys, lungs and brain. It is also possible to perform heart, urine and blood cultures. Blood samples are obtained at intervals and tested for leukocyte and circulating Aspergillus carbohydrate antigen counts using an ELISA assay. The effect of the treatment with the test drug is evaluated in three endpoints: reduction in the mortality rate, reduction in the number of Aspergillus organisms cultured from target organisms (fungal load), and reduction in the level of Aspergillus antigen circulating. Effective antifungal agents reduce mortality and / or fungal burden. Otherwise, pulmonary aspergillosis can be evaluated in this model generally according to Chilvers et al., Mycopathologia, 108: 163-71 (1989), in which immunosuppressed rabbits are infected with intratracheal instillation of conidia of Aspergillus fumigatus. , followed by bronchoalveolar lavage on days 1, 2, 4, 7 and 10 after infection; fungal culture, chitin assay, white cell count, and histopathology are performed in the wash fluids to determine the infective load within the lung. Effective fungal agents reduce the infective load or inflammation within the lung.

EXAMPLE 12 Chitinase activity in vivo in a mouse model of disseminated candidiasis The efficacy of chitinase, alone or in combination with other conventional antifungal agents, is evaluated in a rabbit model of disseminated candidiasis, generally in accordance with Rouse et al. Antimicrob. Agents Chemother. , 26: 56-58 (1992). White New Zealand rabbits are systemically infected with approximately 3 x 106 blastospores of Candida albicans. Antifungal therapy is started 48 hours after infection with Candida (or before infection for prophylaxis) and is continued for, for example, 4 days. The surviving animals are sacrificed, and the fungal cultures are performed in the aortic valve with attached vegetation, lung, kidney and spleen. Fungal cultures and histopathological examination can also be performed on these and other organs, such as liver, brain and heart. You can also make urine and blood cultures. The effect of antifungal therapy on mortality and fungal or circulating tissue load is determined. Bayer et al. Antimicrob. Agents Chemother., 19: 179-184 (1981), in which rabbits are inoculated intraperitoneally with approximately 5 x 108 CFU of Candida albicans. A saline peritoneal aspirate is obtained and cultured from each animal four days after intraperitoneal inoculation, and animals with a positive fungal culture aspirate are randomized to control and treatment groups. Antifungal treatment begins seven days after infection. The eyes of all rabbits are evaluated using indirect ophthalmoscopy, as disseminated candidiasis can result in endophthalmitis due to Candida. The animals are sacrificed at 7, 11 and 14 days after the initiation of therapy and their abdomens "are inspected for the evidence of • peritonitis and intra-abdominal abscess formation. The eyes are examined for macroscopic lesions. Tissue samples from peritoneal abscesses, all other visible accesses, kidneys, livers, spleens and ocular structures are weighed, homogenized in heart brain infusion broth, diluted in series and cultured to determine CFU per gram of tissue. Renal and peritoneal abscesses are also fixed in 10% neutral formaldehyde and histopathology is examined. The sections are stained with acid-Schiff reagent to determine the fungal charge and the morphology of the fungus. The effect of test drugs on improving survival and reducing fungal burden is evaluated.

EXAMPLE 13 Quiñitasa activity in vivo in a rabbit model of endophthalmitis fungal The efficacy of chitinase, alone or in combination with other conventional antifungal agents is evaluated in a rabbit model of endophthalmitis by Candida, generally according to Park et al., Antimicrob Agents Chemother. , 39: 958-963 (1995). In short, New Zealand white rabbits from 2 to 2.5 kg are infected with an intravitreal inoculation of approximately 1,000 CFU of Candida Albincans. The. Endophthalmitis is confirmed 5 days after inoculation by indirect ophthalmoscopy, and is defined as moderate to severe vitreous optical clarity with partial or complete obscuration greater than 50% of the retinal and choroidal vasculature. The vitreous turbidity is graduated on a scale, and the appearance of the background can be graduated and documented by background photography. The rabbits are then selected at random for the following treatment conditions: chitinase alone for 2 to 4 weeks, a combination of chitinase and another conventional antifungal agent (e.g. amphotericin B, fluconazole or 5-fluorocytosine) for 2 to 4 weeks, or without treatment (control). Exemplary doses of conventional anitmicotic agents are 80 mg / kg / day oral fluconazole and 100 mg / kg every 12 hours oral 5-fluorocytosine. The effect of the treatment is evaluated at 2 and 4 weeks after the therapy by indirect ophthalmoscopy, quantitative fungal culture and histopathology. For quantitative fungal culture, the eyes are dissected and weighed, and a heavy fraction of each sample is homogenized and cultured on 5% horse blood agar-brusela plates for 48 hours, at 35 ° C in 5 to 10% of C02 The homogenized sample can also be diluted 10 or 100 times with sterile saline against plating. The colonies are counted and the total CFU in the eye is calculated on the basis of the growth produced from the measured fractions of the sample. The effect of the treatment is evaluated in terms of a reduction in the total intraocular fungal load. For histopathology, the representative eyes are removed, fixed in formalin, embedded in plastic and cut into 5 μm sections. Sections are stained with hematoxylin-eosin or stained with Gomori methenamine silver and examined by light microscopy for inflammation, fibrous organization and fungal elements. The effect of antifungal agents on reducing mortality, reducing fungal burden or reducing inflammation associated with fungal infection is evaluated. Otherwise, a rabbit model of endophthalmitis by Aspergill can be used, generally in accordance with Jain et al., Doc. Opgthalmol., 69: 227-235 (1988). In short, New Zealand white rabbits are inoculated into one eye with approximately 40 spores of Aspergillus fumigatus. Their contralateral eyes (control) receive a similar but sterile inoculum. After treatment with the test drug (chitinase alone or in combination with another agent) the eyes of the rabbits can be evaluated for clinical appearance, electroretinogram waveforms, indirect ophthalmoscopy, quantitative fungal cultures and histopathology. Clinically evident endophthalmitis usually develops within 3 to 7 days after inoculation.

Example 14 Chitinase activity in vivo in a rabbit model of fungal endocarditis The efficacy of chitinase, alone or in combination with other conventional antifungal agents, is evaluated in a rabbit model of Candida endocarditis, in general according to Witt and Bayer, Antimicrob. Agents Chemother., 35: 2481-2485 (1991). See also Longman et al., Rev. Infect. Dis., 12 (Suppl 3): S294-298 (1990). Sterile thrombotic endocarditis occurs in New Zealand white rabbits by transaortic valvular placement of a sterile polyethylene catheter (internal diameter 0.86 mm), which remains in place during the course of the study. Infective endocarditis is then established 48 hours after catheterization by intravenous injection of approximately 2 x 10 'blastospores of C. albicans. Otherwise, C. parapsilosis can be used. Antifungal therapy (chitinase or chitinase in combination with another conventional antifungal agent) is started 48 hours before or 24 to 60 hours after fungal infection. The therapy is continued daily for 9 or 12 days. Exemplary doses of conventional antifungal agents are 1 mg / kg / day of intravenous amphotericin B, 50 mg / kg / day or 100 mg / kg / day of intravenous or intraperitoneal fluconazole. The control rabbits receive it antifungal agent. With the sacrifice, the hearts are removed and the position of the resident catheter is verified. The cardiac vegetations of each animal are removed, combined, weighed and homogenized in 1 ml of sterile saline. The homogenate is serially diluted and cultivated quantitatively on yeast potassium dextrose agar at 35 ° C for 48 hours. It is considered that the negative vegetations of the crop contain less than 2 logio of CFU / gram based on the average weight of the vegetation. Those skilled in the art will be able to make various modifications and variations to the invention described above. Therefore, only the limitations as they appear in the appended claims must be placed in the same LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: ICOS CORPORATION. (ii) TITLE OF THE INVENTION: Materials and methods for the preparation of chitinase (iii) NUMBER OF SEQUENCES: 17 (iv) POSTAL ADDRESS: (A) RECIPIENT: Marshall, O 'oóle, Gerstein, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South Wacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: USA (F) POSTAL CODE: 60606-6402 (v): LEGIBLE COMPUTATION FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IMB compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vi) APPLICATION DATA CURRENT: (A) NUMBER OF APPLICATION: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (vii) DATE OF PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 08 / 663,618 (B) DATE OF SUBMISSION: June 14 of 1996 (viii) INFORMATION OF THE ATTORNEY-LAWYER: (A) NAME: Rin-Laures, Li-Hsien (B) REGISTRATION NUMBER: 33,547 (C) REFERENCE NUMBER / RECORD: 27866/33994 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312 / 474-6300 (B) TELEFAX: 312 / 474-0448 (C) TELEX: 25-3856 (2) INFORMATION OF SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 1636 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY: (A) NAME / KEY: CDS (B) LOCATION: 2..1399 (ix) PECULIARITY: (A) NAME / KEY: mat_peptide (B) LOCATION: 65..1399 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: C ATG GTG CGG TCT GTG GCC TGG GCA GGT TTC ATG GTC CTG CTG ATG Met Val Arg Ser Val Wing Trp Wing Gly Phe Met Val Leu Leu Met -21 -20 -15 -10 ATC CCA TGG GGC TCT GCT GCA AAA CTG GTC TGC TAC TTC ACC AAC TGG 94 lie Pro Trp Gly Ser Wing Wing Lys Leu Val Cys Tyr Phe Thr Asn Trp -5 1 5 10 GCC CAG TAC AGA CAG GGG GAG GCT CGC TTC CTG CCC AAG GAC TTG GAC 142 Wing Gln Tyr Arg Gln Gly Glu Wing Arg Phe Leu Pro Lys Asp Leu Asp 15 20 25 CCC AGC CTT TGC ACC CAC CTC ATC TAC GCC TTC GCT GGC ATG ACC AAC 190 Pro Ser Leu Cys Thr His Leu He Tyr Wing Phe Wing Gly Met Thr Asn 30 35 40 CAC CAG CTG AGC ACC ACT GAG TGG AAT GAC GAG ACT CTC TAC CAG GAG 238 His Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Glp Glu 45 50 55 TTC AAT GGC CTG AAG AAG AAG ATG AAT CCC AAG CTG AAG ACC CTG TTA GCC 286 Phe Asn Gly Leu Lys Lye Met Asn Pro Lys Leu Lys Thr Leu Leu Wing GO 65 70 ATC GGA GGC TGG AAT TTC GGC ACT CAG AAG TTC ACA GAT ATG GTA GCC 334 He Gly Gly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Wing 75 80 85 90 ACG GCC AAC AAC CGT CAG ACC TTT GTC AAC TCG GCC ATC AGG TTT CTG 382 Thr Wing Aen Asn Arg Gln Thr Phe Val Asn Ser Wing He Arg Phe Leu 95 100 1 05 CGC AAA TAC AGC TTT GAC GGC CTT GAC CTG GAC TGG GAG TAC CCA GGA 430 Arg Lys Tyr Ser Phe Aep Gly Leu Asp Leu Aep Trp Glu Tyr Pro Gly 110 115 120 AGC CAG GGG AGC CCT GCC GTA GAC AAG GAG CGC TTC ACÁ ACC CTG GTA 478 Ser Gln Gly Ser Pro Wing Val Asp Lys Glu Arg Phe Thr Thr Leu Val 125 130 135 CAG GAC TTG GCC AAT GCC TTC CAG CAG GAA GCC CAG ACC TCA GGG AAG 526 Gln Asp Leu Wing Asn Wing Phe Gln Glp Glu Ala Gln Thr Ser Gly Lye 140 145 150 GAA CGC CTT CTG AGT GCA GCG GTT CCA GCT GGG CAG ACC TAT GTG 574 Glu Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val 155 160 165 170 GAT GCT GGA TAC GAG GTG GAC AAA ATC GCC CAG AAC CTG GAT TTT GTC 622 Asp Wing Gly Tyr Glu Val Aep Lys He Wing Gln Asn Leu Asp Phe Val 175 180 185 AAC CTT ATG GCC TAC GAC TTC CAT GGC TCT TGG GAG AAG GTC ACG GGA 670 Aen Leu Met Ala Tyr Asp Phe Hie Gly Ser Trp Glu Lys Val Thr Gly 190 195 200 CAT AAC AGC CCC CTC TAC AAG AGG CAA GAA GAG AGT GGT GCA GCC GCC 718 His Asn Ser Pro Leu Tyr Lye Arg Gln Glu Glu Ser G and Ala Ala Ala 205 210 215 AGC CTC AAC GTG GAT GCT GCT GTG CAG TGG CTG CAG AAG GGG ACC 766 Ser Leu Asn Val Asp Ala Ala Val Gln Gln Trp Leu Gln Lys Gly Thr 220 225 230 CCT GCC AGC AAG CTG ATC CTT GGC ATG CCT ACC TAC GGA CGC TCC TTC. 814 Pro Wing Ser Lys Leu He Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe 235 240 245 250 ACA CTG GCC TCC TCA TCA GAC ACC AGA GTG GGG GCC CCA GCC ACA GGG 862 Thr Leu Wing Being Ser Asp Thr Arg Val Gly Wing Pro Wing Thr Gly 255 260 265 TCT GGC ACT CCA GGC CCC TTC ACC AAG GAA GGA GGG ATG CTG GCC TAC 910 Ser Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Wing Tyr 270 275 280 TAT GAA GTC TGC TCC TGG AAG GGG GCC ACC AAA CAG AGA ATC CAG GAT 958 Tyr Glu Val Cye Ser Trp Lys Gly Ala Thr Lys Gln Arg He Gln Asp 235 290 295 CAG AAG GTG CCC TAC ATC TTC CGG GAC AAC CAG TGG GTG GGC TTT GAT 1006 Gln Lys Val Pro Tyr He Phe Arg Asp Asn Gln Trp Val Gly Phe Asp 300 305 310 GAT GTG GAG AGC TTC AAA ACC AAG GTC AGC TAT CTG AAG CAG AAG GGA 1054 Aep Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly 315 320 325 330 CTG GGC GGG GCC ATG GTC TGG GCA CTG GAC TTA GAT GAC TTT GCC GGC 1102 Leu Gly Gly Wing Met Val Trp Wing Leu Aep Leu Aep Asp Phe Wing Gly 335 340 345 TTC TGC AAC CAG GGC CGA TAC CCC CTC ATC CAG ACG CTA CGG CAG 1150 Phe Ser Cys Asn Gln Gly Arg Tyr Pro Leu He Gl n Thr Leu Arg Gln 350 355 360 GAA CTG AGT CTT CCA TAC TTG CCT TCA GGC ACC CCA GAG CTT GAA GTT 1198 Glu Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val 365 370 375 CCA AAA CCA GGT CAG CCC TCT GAA CCT GAG CAT GGC CCC AGC CCT GGA 1246 Pro Lys Pro Gly Gln Pro Ser Glu Pro Glu Hie Gly Pro Ser Pro Gly 380 385 390 CAA GAC ACG TTC TGC CAG GGC AAA GCT GAT GGG CTC TAT CCC AAT CCT 1294 Gln Asp Thr Phe Cye Gln Gly Lye Wing Aep Gly Leu Tyr Pro Aen Pro 395 400 405 410 CGG GAA CGG TCC AGC TTC TAC AGC TGT GCA GCG GGG CGG CTG TTC CAG 1342 Arg Glu Arg Ser Ser Phe Tyr Ser Cye Wing \ Wing Gly Arg Leu Phe Gln 415 420% 425 CAA AGC TGC CCG ACA GGC CTG GTG TTC AGC AAC TCC TGC AAA TGC TGC 1390 Gln Ser Cye Pro Thr Gly Leu Val Phe Ser Ain Ser Cys Lys Cye Cye 430 435 440 ACC TGG AAT TGAGTCGCTA AAGCCCCTCC AGTCCCAGCT TTGAGGCTGG 1439 Thr Trp Asn 445 GCCCAGGATC ACTCTACAGC CTGCCTCCTG GGTTTTTCCCCT GGGGGCCCCCA ATCTGGCTCC 1499 TGCAGGCCTT TCTGTGGTCT TCCTTTATCC AGGCTTTCTG CTCTCAGCCT TGCCTTCCTT 1559 TTTTCTGGGT CTCCTGGGCT GCCCCTTTCA CTTGCAAAAT AAATCTTTGG TTTGTGCCCC 1619 TCTTCCCAAA AAAAAAA 1636 INFORMATION OF SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 466 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met He -21 -20 -15 -10 Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala -5 1 5 10 Gln Tyr Arg Gln Gly Glu Wing Arg Phe Leu Pro Lys Asp Leu Asp Pro 15 20 25 Ser Leu Cys Thr His Leu He Tyr Wing Phe Wing Gly Met Thr Asn His 30 35 40 Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe 45 50 55 Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Wing He 60 65 70 75 Gly Gly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Wing Thr 80 85 90 Wing Asn Asn Arg Gln Thr Phe Val Asn Ser Wing Arg Phe Leu Arg 95 100 105 Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser 110 115 120 Gln Gly Ser Pro Wing Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln 125 130 135 Aep Leu Wing Asn Wing Phe Gln Gln Glu Wing Gln Thr Ser Gly Lys Glu 140 145 150 155 Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp 160 165 170 Wing Gly Tyr Glu Val Asp Lys He Wing Gln Asn Leu Asp Phe Val Asn 175 180 185 Leu Met Wing Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His 190 195 200 Asn Ser Pro Leu Tyr Lye Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser 205 210 '215 Leu Aen Val Asp Ala Ala Gl Gln Gln Trp Leu Gln Lys Gly Thr Pro 220 225 230 235 Wing Ser Lys Leu He Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr 240 245 250 Leu Wing Being Ser Asp Thr Arg Val Gly Wing Pro Wing Thr Gly Ser 255 260 265 Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Wing Tyr Tyr 270 275 280 Glu Val Cys Ser Trp Lys Gly Wing Thr Lys Gln Arg He Gln Asp Gln 285 290 295 Lys Val Pro Tyr He Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp 300 305 310 315 Val Glu Ser Phe Lye Thr Lye Val Ser Tyr Leu Lys Gln Lys Gly Leu 320 325 330 Gly Gly Wing Met Val Trp Wing Leu Asp Leu Asp Asp Phe Wing Gly Phe 335 340 345 Ser Cys Aap Gln Gly Arg tyr Pro Leu ll «Glp Thr Leu Aro ßln Glu 350 3SS 360 Leu be l Po Po Tyr Leu Pro Ser Gly Thr Peo Glu Leu Glu Val Pro 355 370 37S Lys Pro ßly Gin P? O be Glu Pro Glu Hi? Dly Pr Ser Pro Gly Glp 380 385 350 395 Aup Thr Phe Cyß Gln Gly Lys Ala. Asp Gly Leu Tyr Pro Asa Pro Arg 400 405 410 Glu Arg S & Ser Phe Tyr Ser Cyß Ala Ala Gly Arg Leu Phe Gln Gln 415 420 425 Se * Cys Pro Thr Gly Leu Val Phe Ser Aap Ser Cys lys Cys Cys Thr 435 4 0 Trp AB? 445 (2) INFORMATION OF SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1656 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear, ( ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY: (A) NAME / KEY: CDS (B) LOCATION: 27..1424 (ix) PECULIARITY: (A) NAME / KEY: mat_peptide (B) LOCATION: 90 .. 1424 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: GCTGCAGCCT GCCGCTGAGC TGCATC ATG GTG CGG TCT GTG GCC TSG GCA GGT 53 Met Vel Arg Sex val Ins Trp Wing Gly - 21 -20 -IS TTC ATG GTC CTG TO ATG ATC CCA TGG GGC TCT GCT GCA A? A CTG GTC 101 F e Met val Leu Leu Mee lie Pro Trp Gly Sar Ala Wing Lyß Leu Val -10 -S 1 TGC TAC TTC ACC AAC TGG GCC CAG TAC AGA CAG GGG GAG GCT CGC TSC 149 Cys Tyr Phe Thr Aap Trp Wing Glp Tyr Arg Gln Gly Glu Wing Arg Phe 5 ío 15 20 CTG CCC AAG GAC TTG GAC CCC AGC CTT TGC ACC CAC CTC ATC TAC GCC 197 Leu Pro Lyß Asp Leu Aap Pro Ser Leu Cys Thr Hiß Leu ll * yr Wing 25 30 35 TTC GCT GGC ATG ACC AAC CAC CAG CTG AGC ACC ACT GAG TGG AftT GAC 245 Phe Ala Gly Met Thr Aβp His Gln Leu Ser Thr Thr Glu Trp Asp Aβp 40 45 50 GAG ACT CTC TAC CAG GAG TTC AAT GGC CTG AAG AAG ATG AAT CCC AAG 293 Glu Thr Leu Tyr Gln Glu Pha Aan Gly Leu Lyß Lys Ket Aan Pro Lye 55 60 S5 CTG AAG ACC CTG TTA GCC ATC GGA GGC TGG AAT TTC AGC ACT CAG AAG 341 Leu Lys Thr Leu Leu Wing He Gly Gly Trp Asn Phe Ser Thr Gln Lye 70 75 80 TTC ACA GAT ATG GTA GCC ACG GCC AAC AAC CGT CAG ACC TTT GTC AAC 389 Phe Thr Asp Met Val Wing Thr Wing Asn Asn Arg Gln Thr Phe Val Asn 85 90 95 100 TCG GCC ATC AGG TTT CTG CGC AAA TAC AGC TTT GAC GGC CTT GAC CTT 437 Ser Wing Arg Phe Leu Arg Lys Tyr Ser Phe Asp Gly Leu Asp Leu 105 110 115 GAC TGG GAG TAC CCA GGA AGC CAG GGG AGC CCT GCC GTA GAC AAG GAG 485 Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Wing Val Asp Lys Glu 120 125 130 CGC TTC ACÁ ACC CTG GTA CAG GAC TTG GCC AAT GCC TTC CAG CAG GAA 533 Arg Phe Thr Thu Leu Val Gln Asp Leu Wing Asn Wing Phe Gln Gln Glu 135 140 145 GCC CAG ACC TCA GGG AAG GAA CGC CTT CTT AGG GCA GCG GTT CCA 581 Wing Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser Wing Wing Val Pro 150 155 160 GCT GGG CAG ACC TAT GTG GAT GCT GGA TAC GAG GTG GAC AAA ATC GCC 629 Wing Gly Gln Thr Tyr Val Asp Wing Gly Tyr Glu Val Asp Lys He Wing 165 170 175 180 CAG AAC CTG GAT TTT GTC AAC CTT ATG GCC TAC GAC TTC CAT GGC TCT 677 Gln Asn Leu Asp Phe Val Asn Leu Mee Wing Tyr Aep Phe His Gly Ser 185 190 195 TGG GAG AAG GTC ACG GGA CAT AAC AGC CCC CTC TAC AAG AGG CAA GAA 725 Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr Lys Arg Gln Glu 200 205 - • • 210 GAG AGT GGT GCA GCC GCC AGC CTC AAC GTG GAT GCT GCT GTG CAA CAG 773 Glu Ser Gly Wing Wing Wing Leu Asn Val Asp Wing Wing Val Gln Gln 215 220 225 TGG CTG CAG AAG GGG ACC CCT GCC AGC AAG CTG ATC CTT GGC ATG CCT 821 Trp Leu Gln Lys Gly Thr Pro Wing Ser Lys * Leu He Leu Gly Mee Pro 230 235 240 ACC TGA GGA CGC TCC TTC TCC CTG TCC TCA TCA TAC GAC ACC AGA GTG 869 Thr Tyr Gly Arg Ser Phe Thr Leu Wing Being Ser Asp Thr Arg Val 245 250 255 260 GGG GCC CCA GCC ACA GGG TCT GGC ACT CCA GGC CCC TTC ACC AAG GAA 917 Gly Wing Pro Wing Thr Gly Ser Gly Thr Pro Gly Pro Phe Thr Lys Glu 265 270 275 GGA GGG ATG CTG GCC TAC TAT GAA GTC TGC TCC TGG AAG GGG GCC ACC 965 Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp Lys Gly Wing Thr 280 285 290 AAA CAG AGA ATC CAG GAT CAG AAG GTG CCC TAC ATC TTC CGG GAC AAC 1013 Lys Gln Arg He Gln Aep Gln Lys Val Pro Tyr He Phe Arg Asp Asn 295 300 305 CAG TGG GTG GGC TTT GAT GAT GTG GAG AGC TTC AAA ACC AAG GTC AGC. 1061 Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys Thr Lys Val Ser 310 315 320 TAT CTG AAG CAG AAG GGA CTG GGC GGG GCC ATG GTC TGG GCA CTG GAC 1109 Tyr Leu Lys Gln Lys Gly Leu Gly Gly Wing Met Val Trp Wing Leu Asp 325 330 335 340 TTA GAT GAC TTT GCC GBC TTC TGC AAC CAG GGC CGA TAC CCC CTC 1157 Leu Asp Asp Phe Wing Gly phe Ser Cys Asn Gis- Gly Arg Tyr Pro Leu 345 350 35S ATC CAG AOG CTA CGG CAG GAA CTG AGT CTT CC-TAC TTG CCT TCA GGC 1205 He Glp Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr Leu Pro Ser Gly 3fi0 365 370 ACC CCA GAG CTT GAA GTT CCA AAA CCA GGT CAG CCC TC5? GAA CCT GAG 1253 Thr Pro Glu Leu Glu Val Pro Lys Pro Gly Gln Pro Ser Glu Pro Glu 375 380 385 CAT GGC CCC AGC CCT GGA CAA GAC ACG TTC TGC CAG GGC AAA GCT GAT 1301 HÍB Gly ro be Pro Gly Gln? Sp Thr Phe Cys Gln Gly Lys Wing Asp 390 395 400 GGG CTC TAT CCC AAT CCT CGG GAA CGG TCC AGC TTC TAC AßC TGT GCA 1349 Gly Leu Tyr Pro Asa Pro Arg Glu Arg be Ser Phe Tyx Ser Cys Wing 435 10- 415 420 GCG GGG CGG CTG TTC CAG CAA AGC TGC CCG AC GGC CTG GTS TTC AGC 1397 Wing Gly Arg Leu Phe Gln Gln Ser Cyß Pro Thr Gly Leu Val Phe Ser 425 430 435 AAC TCC TGC AAA TGC TGC ACC TGG AAT TGAGTCOCTA AAGCCCCTCC 1444 Asn Ser Cye Lys Cys Cys Thr Trp Asn 440 445 AGTCCCAGCT TTOAGGCTGG GCCCAGGATC ACTCTACAGC CTGCCTCCTG GGTTTTCCCT X504 GGGGGGCCGCA ATCTGGCTCC TGCAGGCCTT TCTGTGGTCT TCCT? ATCC AGGCTTTCTG 1564 CTCTCAGCCT TGCCTTCCTT TTTTCTGGGT CTCCTGGGCT GCCCCTTTCA CTTGCAAAAT 1S24 AAATCTTTGG TTTGTGCCCC TCAAAAAAAA AA 1656 (2) INFORMATION OF SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 466 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu tia £ lie -31 -20 -15 -10 Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Aei n Trp Ala -5 1 S 10 Gln Tyr Arg Gln Gl? Glu Wing Arg Phe Leu Pro Lyß Aap Leu Asp Pro 15 2ü 25 Ser Leu Cys Thr His Leu laugh Tyr Wing Phe Wing Gly Mßt Thr Asp His 30 35 40 Glp Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe iS 50 55 Aan Gly Leu Lys Lys Mefc Aan Pro Lys Leu Lyß Thr Leu Leu Wing He 60 65 70 75 Gly Gly Trp Aßn phe Ser Thr Gln Lys Phe Thr Asp Met to Wing Thr SO 85 90 Wing Asn Asn Arg Gln Thr Phe Val Asn Ser Ala lie Arg Phe Leu Arg 95 100 105 Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser 110 115 120 Gln Gly Ser Pro Wing Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln 125 130 135 Asp Leu Wing Asn Wing Phe Gln Gln Glu Wing Gln Thr Ser Gly Lys Glu 140 145 150 155 Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp 160 165 170 Wing Gly Tyr Glu Val Asp Lys lie Wing Gln Asn Leu Asp Phe Val Asn 175 180 185 Leu Mee Wing Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His 190 195 200 Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser 205 210 215 Leu Asn Val Asp Ala Ala Gl Gln Gln Trp Leu Gln Lye Gly Thr Pro 220 225 230 235 Ala Ser Lys Leu Lie Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr 240 245 250 Leu Wing Being Ser Asp Thr Arg Val Gly Wing Pro Wing Thr Gly Ser 255 260 265 Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Wing Tyr Tyr 270 275 280 Glu Val Cys Ser Trp Lye Gly Wing Thr Lye Gln Arg - lie Gln Aep Gln 285 290 295 Lys Val Pro Tyr lie Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp 300 305 310 315 Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lye Gly Leu 320 325 330 Gly Gly Wing Met Val Trp Wing Leu Asp Leu Asp Aep Phe Wing Gly Phe 335 ^ 340 345 Ser Cys Asn Gln Gly Arg Tyr Pro Leu lie Gln Thr Leu Arg Gln Glu 350 355 360 Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro 365 370 375 Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly Gln 380 385 390 395 Asp Thr Phe Cys Gln Gly Lye Wing Asp Gly Leu Tyr Pro Asn Pro Arg 400 405 410 Glu Arg Being Ser Phe Tyr Being Cys Wing Wing Gly Arg Leu Phe Gln Gln 415 420 425 Ser Cye Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys Thr 430 435 440 Trp Asn (2) INFORMATION OF SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GACACTATAG AATAGGGC (2) INFORMATION OF SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TGGGATCATC AGCAGGACCA TGAAACCTGC CCAGGCCACA GACCGCACCA T (2) INFORMATION OF SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) 'LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7; TACATCTAGA ATTATGGCAA AACTGGTCTG CTACTTCACC (2) INFORMATION OF SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: AGATCTAACC TTAGGTGCCT GAAGACAAGT ATGG (2) INFORMATION OF SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: TACAGAATTC TTATTCACAT CCGGCCCTG (2) INFORMATION OF SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 pairs base (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10 TACATCTAGA CTCCATCCAG AAAAACAGGT ATGG (2) INFORMATION OF SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11 TCTAGAGTCG ACCTGCAGGC ATGCAAGCTT (2) INFORMATION OF SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: CGCAAGCTTG AGAGCTCCGT TCCGCCACAT GGTGCGGTCT GTGGCCTGGG (2) INFORMATION OF SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid - (C) HEBRA: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: GACTCTAGAC TAGGTGCCTG AAGGCAAGTA TG (2) 'INFORMATION OF SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 373 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Wing Lys Leu Val Cys Tyr Phe Thr Asn Trp Wing Gln Tyr Arg Gln Gly 1 5 10 15 Glu Wing Arg Phe Leu Pro Lye Asp Leu Asp Pro Ser Leu Cys Thr His 20 25 30 Leu lie Tyr Wing Phe Wing Gly Met Thr Asn His Gln Leu Ser Thr Thr 35 40 45 Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys 50 55 60 Met Asn Pro Lys Leu Lye Thr Leu Leu Ala lie Gly Gly Trp Asn Phe 65 70 75 80 Gly Thr G n Lys Phe Thr Asp Met Val Wing Thr Wing Asn Asn Arg Gln 85 90 95 Thr Phe Val Asn Ser Ala lie Arg Phe Leu Arg Lys Tyr Ser Phe Asp 100 105 110 Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Wing 115 120 125 Val Asp Lys Glu Arg Phe Thr Thu Leu Val Gln ABp Leu Ala Aen Wing 130 135 140 Phe Gln Gln Glu Wing Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser 145 150 155 160 Wing Wing Val Pro Wing Gly Gln Thr Tyr Val Asp Wing Gly Tyr Glu Val 165 170 175 Asp Lys lie Wing Gln Asn Leu Asp Phe Val Asn Leu Met Wing Tyr Aep 180 185 190 Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr 195 200 205 Lys Arg Gln Glu Glu Ser Gly Wing Wing Wing Leu Asn Val Aep Wing 210 215 220 Wing Val Gln Gln Trp Leu Gln Lye Gly Thr Pro Wing Ser Lys Leu lie 225 230 235 240 Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Wing Being Ser 245 250 255 Asp Thr Arg Val Gly Ala Pro Wing Thr Gly Ser Gly Thr Pro Gly Pro 260 265 270 Phe Thr Lys Glu Gly Gly Met Leu Wing Tyr Tyr Glu Val Cys Ser Trp 275 280 285 Lys Gly Wing Thr Lys Gln Arg He Gln Asp Gln Lys Val Pro Tyr He 290 295 300 Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys 305 310 315 320 Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly Gly Wing Met Val 325 330 335 Trp Wing Leu Asp Leu Asp Asp Phe Wing Gly Phe Ser Cys Asn Gln Gly 340 345 '350 Arg Tyr Pro Leu He Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr 355 360 365 Leu Pro Ser Gly Thr 370 (2) INFORMATION OF SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 373 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: Wing Lys Leu Val Cys Tyr Phe Thr Asn Trp Wing Gln Tyr Arg Gln Gly 1 5 10 15 Glu Wing Arg Phe Leu Pro Lys Asp Leu Aep Pro Ser Leu Cye Thr His 20 25 30 Leu He Tyr Wing Phe Wing Gly Met Thr Asn His Gln Leu Ser Thr Thr 35 40 45 Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys 50 55 60 Met Asn Pro Lys Leu Lys Thr Leu Leu Wing He Gly Gly Trp Asn Phe 65 70 75 80 Gly Thr Gln Lys Phe Thr Asp Met Val Wing Thr Wing Asn Asn Arg Gln 85 90 95 Thr Phe Val Asn Ser Wing He Arg Phe Leu Arg Lys Tyr Ser Phe Asp 100 105 not Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Wing 115 120 125 Val Aep Lys Glu Arg Phe Thr Thu Leu Val Gln Aep Leu Ala Asn Ala 130 135 140 Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser 145 150 155 160 Wing Wing Val Pro Wing Gly Gln Thr Tyr Val Asp Wing Gly Tyr Glu Val 165 170 175 Asp Lys He Wing Gln Aen Leu Asp Phe Val Asn Leu Met Wing Tyr Asp 180 185 190 Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr 195 200 205 Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp .Ala 210 215 220 Wing Val Gln Gln Trp Leu Gln Lys Gly Thr Pro Wing Ser Lys Leu He 225 230 235 240 Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Wing Being Being 245 250 255 Aup Thr Arg Val Gly Wing Pro Wing Thr Gly Se Gly Thr Pro ßly P «a 260 265 270 Phe Thr Lys Glu Gly Gly Mßt Leu Wing Tyr Tyr Glu Val Cys Ser Txp 275 230 285 Lys Gly Wing Thr Lys Gln Arg lie Gln Asp Gln Lyß Val Pro Tyr He 290 295 300 Phe Arg Asp Asn Glp Trp Val Gly Phe Aßp Asp Val Glu Ser Phe Ly »3G5 310 315 220 Thr Lys Val Se Tyr Leu Lye Gln Lys Gly Leu Gly Gly Ala Met Val 325 330 335 Trp Ala Leu Aep Leu Aap Asp Phé Wing Gly Phe Ser Cyß Asn Gla Gly 340 345 350 Arg Tyr Pro Leu He Gl_? Thr Leu Arg Gln Glu Leu Ser lßu Pro Tyr 355 360 365 Leu Ser Se Gl Thr 370 (2) INFORMATION OF SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) .TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: TGATACGGTA CCGCCCCATG GCTGACTA (2) INFORMATION OF SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs áfc (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear 5 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: desc / = "primer" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17; GCAAGTTTGG CGCGAAA.TCG

Claims (35)

1. A purified, isolated polynucleotide encoding the amino acid sequence of human chitinase of SEQ ID NO:

2. The polynucleotide of claim 1 which is a DNA.

3. The DNA of claim 2 contains the protein encoding the nucleotides of SEQ ID NO: 1.

4. The purified, isolated polynucleotide encoding amino acids 1 to 445 of SEQ ID NO: 2.

5. The polynucleotide of the claim 4 which is a DNA.

6. The DNA of claim 5 comprises nucleotides 65 to 1402 of SEQ ID NO: 1.

7. A purified, isolated polynucleotide encoding the amino acid sequence of human chitinase of SEQ ID NO: 4.

8. The polynucleotide of claim 7 which is a DNA.

9. The DNA of claim 8 comprises the protein encoding the nucleotides of SEQ ID NO: 3.

10. A purified, isolated polynucleotide encoding amino acids 1 to 445 of SEQ ID NO: 4. •

11. The polynucleotide of claim 10 which is a DNA. The DNA of claim 11 comprising nucleotides 90 to 1427 of SEQ ID NO: 3. 13. A purified, isolated polynucleotide encoding human chitinase selected from the group consisting of: (a) a double-stranded DNA that comprises the protein encoding the portions of the sequence set forth in SEQ ID NO: 3; (b) a DNA that hybridizes under severe conditions to a non-coding strand of the DNA of (a); and (c) a DNA which, except for the redundancy of the genetic code, hybridizes under severe conditions to a non-coding strand of the DNA sequence of (a) or (b). 14. The polynucleotide of claim 13 which is a DNA. "fifteen. A vector comprising the DNA of claim 2, 3, 5, 6, 8, 9, 11, 12 or 14. 16. The vector of claim 15 which is an expression vector, wherein the DNA binds in a manner operant to a DNA sequence control expression. 17. A host cell stably formed or transfected with the DNA of claim 2, 3, 5, 6, 8, 9, 11, 12 or 14, in a form that allows expression in the host cell of human chitinase. 18. A method for producing human chitinase which consists of: culturing the host cell of claim 17 in a nutrient medium and isolating human chitinase from the host cell or the nutrient medium. 19. A purified, isolated polypeptide produced by the method of claim 18. 20. A purified, isolated polypeptide comprising the amino acid sequence of human chitinase of SEQ ID NO: 2. 21. A purified, isolated polypeptide comprising the amino acid sequence of human chitinase SEQ ID NO: 4. 22. A purified, isolated polypeptide comprising amino acids 1 to 445 of human chitinase of SEQ ID NO: 2. 23. A purified, isolated polypeptide comprising amino acids 1 to 445 of human chitinase of SEQ ID NO: 4. 24. A fragment of the human chitinase polypeptide that lacks 1 to about 72 c-terminal amino acid residues of mature human chitinase. 25. The fragment of the human chitinase polypeptide of SEQ ID NO: 14. 26. A purified, isolated polynucleotide comprising the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14. 27. The polynucleotide of claim 26 which is a DNA. 28. The human chitinase polypeptide analog of SEQ ID NO: 15. 29. A purified, isolated polynucleotide encoding the amino acid sequence of SEQ ID NO: 15. 30. A hybridoma cell line that produces a monoclonal antibody which is specifically reactive with the polypeptide of claims 19, 20, 21, 22, 23 or 28. 31. The monoclonal antibody produced by the hybridoma 10 of claim 30. 32. A pharmaceutical composition containing a polypeptide of any of the claims 19 to 25 or claim 28. 33. A method of treating a fungal infection 15 which consists in administering to a subject a therapeutically effective amount of a human chitinase.The method of claim 33 further comprises administering to an individual a therapeutically effective amount of an antifungal agent other than chitinase. 35. A method of reducing the amount of animitic agent other than chitinase necessary to exert an antifungal activity in an individual consists in administering to the individual an amount of an effective human chitinase to improve the antifungal activity of the animal. 25 antifungal agent different from chitinase.