AU745324B2

AU745324B2 - Therapeutic methods comprising use of a neuregulin

Info

Publication number: AU745324B2
Application number: AU96042/98A
Authority: AU
Inventors: David I Gwynne; William Holt; Mark Marchionni; Robert N McBurney
Original assignee: Cenes Pharmaceuticals Inc
Current assignee: Cenes Pharmaceuticals Inc
Priority date: 1997-10-14
Filing date: 1998-10-08
Publication date: 2002-03-21
Anticipated expiration: 2018-10-08
Also published as: EP1028735A4; KR20010031103A; CA2306228A1; EP1028735A1; JP2001519400A; AU9604298A; WO1999018976A1

Description

WO 99/18976 PCT/US98/21349 -1- THERAPEUTIC METHODS COMPRISING USE OF A NEUREGULIN BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to methods for treatment of certain neurologicalrelated injuries and disorders comprising use of a neuregulin, or a fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or neuregulin fragment or derivative.

2. Background Nerve cell death (degeneration) can cause potentially devastating and irreversible effects for an individual and may occur e.g. as a result of stroke, heart attack or other brain or spinal chord ischemia or trauma. Additionally, neurodegenerative disorders involve nerve cell death (degeneration) such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Down's Syndrome and Korsakoffs disease.

Therapies have been investigated to treat nerve cell degeneration and related disorders, by limiting the extent of nerve cell death that may otherwise occur to an individual as well as promoting repair, remodeling and reprogramming after stroke or other neuronal injury. See, F. Seil, Curr Opin Neuro, 10:49-51 (1997); N. L.

Reddy et al., JMed Chem, 37:260-267 (1994); and WO 95/20950.

Certain growth factors have been reported to exhibit neuroprotective properties. In particular, nerve growth factor (NGF) has been evaluated in certain neuroprotective models. See, for example, G. Sinson et al., JNeurosurg, 86(3):511- 518 (1997); and G. Sinson et al., JNeurochem, 65(5):2209-2216 (1995). Osteogenic protein-1 (OP-1) has been evaluated in a rat model of cerebral hypoxia/ischemia for neuroprotective activity. G. Perides, Neurosci Lett, 1871):21-24 (1995). Glial cell line-derived neurotrophic factor (GDNF) was reported to exhibit trophic activity on certain populations of central neurons. Y. Wang et al., J Neurosci, 17(11):4341-4348 (1997). Small molecules also have been investigated as neuroprotective agents, such WO 99/18976 PCT/US98/21349 -2as MK-801. See B. Meldrum, Cereb Brain Metab Rev, 2:27-57 (1990); D. Choi, Cereb Brain Metab Rev, 2:27-57 (1990).

However, no effective pharmacotherapies are in regular clinical use for ischemia-induced brain injury or other such injuries and disorders. See, for example, Y. Wang et al., supra; G. Sinson et al., JNeurochem, JNeurochem, 65(5):2209 (1995).

It thus would be highly desirable to have new neuroprotective agents, particularly agents to limit the extent or otherwise treat nerve cell death (degeneration) that occur with stroke, heart attack or brain or spinal cord trauma, or to treat Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Down's Syndrome and Korsakoffs disease. It also would be desirable to have agents that promote repair, remodeling or reprogramming after stroke or other neuronal injury.

SUMMARY OF THE INVENTION The present invention provides methods for treatment and/or prophylaxis of certain neurological-related disorders, particularly treatment or prophylaxis of the effects of stroke, brain or spinal cord injury or ischemia, heart attack, optic nerve and retinal injury and ischemia and other acute-type conditions disclosed herein as well as chronic-type conditions, specifically epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Down's Syndrome, Korsakoffs disease, cerebral palsy and/or age-dependent dementia. Methods of the invention also include therapies for promoting repair, remodeling or reprogramming after stroke or other neuronal injury.

The methods of the invention comprise administration of an effective amount of neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or a neuregulin fragment or derivative gene therapy), to a patient suffering from or susceptible to such conditions.

Neuregulins are members of the epidermal growth factor (EGF) superfamily and include glial growth factor (GGF), acetylcholine receptor-inducing activity (ARIA), neu differentiation factor (NDF) and heregulins (HRF). See D. E. Wen et al., Cell, 69:559-572 (1992); W.E. Holmes et al., Science, 256:1205-1210 (1992); M.A.

Marchionni et al., Nature, 362:312-318 (1993); and D.L. Falls, Cell, 72:801-815 WO 99/18976 PCT/US98/21349 -3- (1993). A variety of neuregulins and fragments and derivatives thereof can be employed in the methods of the invention. For example, suitable agents have been disclosed in U.S. Patent 5,530,109 and PCT/US93/07491. Neuregulins also have been reported in U.S. Patent 5,367,060. Preferred neuregulins include regions shown in FIGS. 1-2 (SEQ ID NOS. 2 and also known as the E sequence. Preferred neuregulins or fragments or derivatives also include those that contain the C, C/D or C/D' sequences as shown in Figures 7, 8 and 9 respectively of the drawings, or those neuregulins or fragments or derivatives that have substantial homology to the peptide sequences shown in Figures 7, 8 or 9, e.g. at least about 70 percent homology, or at least about 80 percent homology, or more preferably at least about 90 or 95 percent homology to the peptide sequences shown in Figures 7, 8 or 9. Preferred nucleic acids and fragments and derivatives for use in the methods of the invention include those nucleic acids that include one or more nucleic acids sequences shown in Figures 7, 8 and 9 of the drawings, or those nucleic acids that that have substantial homology to the nucleic acid sequences shown in Figures 7, 8 or 9, e.g. at least about 70, 80, or 95 percent homology to the nucleic acid sequences shown in Figures 7, 8 or 9. A particularly preferred neuregulin is encoded by DNA obtainable from the clone pGGF2HBS11 (ATCC Deposit No. 75347). Also preferred are neuregulins encoded by DNA obtainable from GGF2BPP5, GGF2BPP2 and GGF2BPP4.

Typical patients that may be treated in accordance with the methods of the invention are persons suffering from brain or spinal cord trauma or ischemia, stroke, heart attack, hypoxia, hypoglycemia, post-surgical neurological deficits, decreased blood flow or nutrient supply to retinal tissue or optic nerve, retinal trauma or ischemia or optic nerve injury. Patients suffering from chronic-type conditions also may be treated in accordance with the invention, specifically subjects suffering from or susceptible to epilepsy, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Alzheimer's disease, Down's Syndrome, Korsakoffs disease, cerebral palsy and/or age-dependent dementia.

Also, as discussed above, a neuregulin or fragment or derivative thereof or nucleic acid encoding same, may be administered to promote repair, remodeling or reprogramming to a subject that has suffered stroke or other neuronal injury such as traumatic brain or spinal cord injury. In such cases, the therapeutic agent may be I-~IIIP-r~~--X- WO 99/18976 PCT/US98/21349 -4suitably administered to the subject over an extended period following the injury, e.g.

at least about 1, 2, 3, 4, 6, 8, 12 or 16 weeks following the injury.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) encoding a preferred neuregulin region (E segment of human GGF) and the amino acid sequence (SEQ ID NO:2) of that preferred region.

FIG. 2 shows a nucleotide sequence (SEQ ID NO:3) encoding a preferred neuregulin region (E segment of bovine GGF) and the amino acid sequence (SEQ ID NO:4) of that preferred region.

FIG. 3 shows nucleotide sequences (SEQ ID NOS:6-7) encoding further neuregulin regions (B segment of human and bovine GGF) and amino acid sequences (SEQ ID NOS:5 and 8) of those regions. Line 1 is the predicted amino acid sequence of bovine B segment, line 2 is a nucleotide sequence of bovine B segment, line 3 is a nucleotide sequence of human B segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human B segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 4 shows nucleotide sequences (SEQ ID NOS:I0-11) encoding further neuregulin regions (A segment of human and bovine GGF) and amino acid sequences (SEQ ID NOS:9 and 12) of those regions. Line 1 is the predicted amino acid sequence of bovine A segment, line 2 is a nucleotide sequence of bovine A segment, line 3 is a nucleotide sequence of human A segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human A segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:13) encoding a further neuregulin region segment of bovine GGF) and the predicted amino acid sequence (SEQ ID NO:14) of that region.

FIG. 6 shows nucleotide sequences (SEQ ID NOS:16-17) encoding further neuregulin regions (G segment of bovine and human GGF) and amino acid sequences (SEQ ID NOS:15 and 18) of that region. Line 1 is the predicted amino acid sequence of bovine G segment, line 2 is a nucleotide sequence of bovine G segment, line 3 is a WO 99/18976 PCT/US98/21349 nucleotide sequence of human G segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human G segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 7 shows nucleotide sequences (SEQ ID NOS:20-21) encoding further neuregulin regions (C segment of bovine and human GGF) and amino acid sequences (SEQ ID NOS:19 and 22) of those regions. Line 1 is the predicted amino acid sequence of bovine C segment, line 2 is a nucleotide sequence of bovine C segment, line 3 is a nucleotide sequence of human C segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human C segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 8 shows nucleotide sequences (SEQ ID NOS:24-25) encoding further neuregulin regions (C/D segment of human and bovine GGF) and amino acid sequences (SEQ ID NOS:23 and 26) of those regions. Line 1 is the predicted amino acid sequence of bovine C/D segment, line 2 is a nucleotide sequence of bovine C/D segment, line 3 is a nucleotide sequence of human C/D segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human C/D segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 9 shows nucleotide sequences (SEQ ID NOS:28-29) encoding a further neuregulin region segment of the human and bovine GGF) and the amino acid sequence (SEQ ID NO:27) of that region. Line 1 is the predicted amino acid sequence of the C/D' segment, line 2 is a nucleotide sequence of bovine C/D' segment and line 3 is a nucleotide sequence of human C/D' segment (nucleotide base matches are indicated with a vertical line).

FIG. 10 shows nucleotide sequences (SEQ ID NOS:31-32) encoding a further neuregulin region (D segment of the human and bovine GGF) and the amino acid sequence (SEQ ID NO:30) of that region. Line 1 is the predicted amino acid sequence of the D segment, line 2 is a nucleotide sequence of bovine D segment and line 3 is a nucleotide sequence of human D segment (nucleotide base matches are indicated with a vertical line).

WO 99/18976 PCT/US98/21349 -6- FIG. 11 shows nucleotide sequence (SEQ ID NO:34) encoding a further neuregulin region segment of bovine GGF) and the amino acid sequence (SEQ ID NO:33) of that region.

FIGS. 12A-12B show nucleotide sequences (SEQ ID NOS:36-37) encoding further neuregulin regions (H segment of human and bovine GGF) and amino acid sequences (SEQ ID NO:35 and 38) of that region. Line 1 is the predicted amino acid sequence of bovine H segment, line 2 is a nucleotide sequence of bovine H segment, line 3 is a nucleotide sequence of human H segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human H segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:40) encoding a further neuregulin region (K segment of bovine GGF) and the amino acid sequence (SEQ ID NO:39) of that region.

FIGS. 14A-14C show nucleotide sequences (SEQ ID NOS:42-43) encoding a further neuregulin region (L segment of bovine and human GGF) and amino acid sequences (SEQ ID NO:41 and 44) of that region. Line 1 is the predicted amino acid sequence of bovine L segment, line 2 is a nucleotide sequence of bovine L segment, line 3 is a nucleotide sequence of human L segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human L segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIG. 15 shows nucleotide sequences (SEQ ID NOS:46-47) encoding further neuregulin regions (F segment of bovine and human GGF) and amino acid sequences (SEQ ID NOS:45 and 48) of that region. Line 1 is the predicted amino acid sequence of bovine F segment, line 2 is a nucleotide sequence of bovine F segment, line 3 is a nucleotide sequence of human F segment (nucleotide base matches are indicated with a vertical line), and line 4 is the predicted amino acid sequence of human F segment shown where it differs from the bovine sequence set forth in line 1 of the figure.

FIGS. 16A-16C show the nucleotide sequence (SEQ ID NO:49) and deduced amino acid sequence (SEQ ID NO:50) of GGF2BPP4.

WO 99/18976 PCT/US98/21349 -7- FIGS. 17A-17B show the nucleotide sequence (SEQ ID NO:51) and deduced amino acid sequence (SEQ ID NO:52) of GGF2BPP2.

FIGS. 18A-18B show the nucleotide sequence (SEQ ID NO:53) and deduced amino acid sequence (SEQ ID NO:54) of DETAILED DESCRIPTION OF THE INVENTION As discussed above, preferred neuregulins for use in the therapeutic methods of the present invention include those disclosed in U.S. Patent 5,530,109 and PCT/US93/07491, incorporated herein by reference. Particularly preferred neuregulins comprise an amino acid sequence of the following formula:

WYBAZCX

wherein WYBAZCX is composed of amino acid sequences that include one or more sequences shown in FIGS. 1 through 15 (which includes SEQ ID NOS:2, 4, 5, 8, 9, 12, 14, 15, 18, 19, 22, 23, 26, 27, 30, 33, 35, 38, 39, 41, 44, 45 and 48), wherein W comprises the polypeptide segment F, or is absent; wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprise a polypeptide segment selected from the group consisting of C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/d D'H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL and C/D C/D' D' HKL, and preferably that either a) at least one ofF, Y, B, A, Z, C or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D'H, C/D C/D' D HL, C/D C/D' D' HKL, C/D'H, C/D C/D' H or C/D C/D' HL.

Particularly preferred neuregulins also include those polypeptides that include the segments FB polypeptides that include the segments FBA' the groups F, B and A' as defined herein including in the drawings); polypeptides that include the segments EBA the groups E, B and A as defined herein including in the drawings); polypeptides that include the segments EBA' the groups E, B and A' as defined herein including in the drawings); A the group A as defined herein WO 99/18976 PCT/US98/21349 -8including in the drawings); polypeptides that include the segments FEBA the groups F, E, B and A as defined herein including in the drawings); polypeptides that include the segments FBA' the groups F, B and A' as defined herein including in the drawings); and polypeptides that include the segments FEBA' the groups F, E, B and A' as defined herein including in the drawings).

Also preferred are nucleic acids that code for the above preferred polypeptides.

A "fragment" or "derivative" of a neuregulin refers to herein 1) a peptide in which one or more amino acid residues are with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) a peptide in which one or more of the amino acid residues includes a substituent group, or (iii) a peptide in which the mature protein is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Thus, a fragment or derivative for use in accordance with the methods of the invention includes a proprotein, which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide fragments and derivatives of the invention are of a sufficient length to uniquely identify a region of a neuregulin. Neuregulin fragments thus preferably comprise at least 8 amino acids, usually at least about 12 amino acids, more usually at least about 15 amino acids, still more typically at least about 30 amino acids, even more typically at least about 50 or 70 amino acids. Preferred fragments or derivatives for use in the methods of the invention include those that have at least about 70 percent homology (sequence identity) to any of the preferred sequences mentioned above, more preferably about 80 percent or more homology to any of the preferred sequences mentioned above, still more preferably about 85 to 90 percent or more homology to any of the preferred sequences mentioned above. Sequence identity or homology with respect to a neuregulin as referred to herein is the percentage of amino acid sequences of a neuregulin protein or fragment or derivative thereof that are identical with a specified sequence, after introducing any gaps necessary to achieve the maximum percent homology.

The neuregulin fragments and derivatives for use in the methods of the invention preferably exhibit good activity in standard neuroprotective assays such as WO 99/18976 PCT/US98/21349 -9the in vivo cerebral ischemia assay of Example 1, which follows. That assay includes the following steps: a) continuous intraventricular infusion of the protein fragment or derivative or vehicle alone to test rats for three days prior to inducing focal ischemic infarcts in right lateral cerebral cortex; and b) twenty-four hours after inducing ischemic infarcts, infarct volume in each test animal is determined by image analysis.

Preferably, a protein fragment or derivative of the invention provides at least about a reduction in infarct volume relative to vehicle-treated animals, more preferably about a 20% reduction in infarct volume, still more preferably about a 25% reduction in infarct volume relative to vehicle-treated animals in such an assay. References herein to in vivo cerebral ischemia assay are intended to refer to an assay of the above steps a) and which are more fully described in Example 1 which follows.

As discussed above, neuregulin nucleic acid fragments and derivatives are also provided for use in the methods.of the invention. Those fragments and derivatives typically are of a length sufficient to bind to a sequence of any of the nucleic acid sequences shown in Figures 1-15 of the drawings, including SEQ ID NOS:1, 3, 6, 7, 11, 13, 16, 17, 20, 21, 24, 25, 28, 29, 31, 32, 34, 36, 37, 40, 42 and 43 under the following moderately stringent conditions (referred to herein as "normal stringency" conditions): use of a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37 0

C.

Preferred neuregulin nucleic acid fragments and derivatives of the invention will bind to a sequence of any of the nucleic acid sequences shown in Figures 1-15 of the drawings, including SEQ ID NOS:1, 3, 6, 7, 10, 11, 13, 16, 17, 20, 21, 24, 25, 28, 29, 31, 32, 34, 36, 37, 40, 42 and 43 under the following highly stringent conditions (referred to herein as "high stringency" conditions): use of a hybridization buffer comprising 20% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a temperature of 42°C and remaining bound when subject to washing twice with that SSC buffer at 42 0

C.

The neuregulin nucleic acid fragments and derivatives preferably should comprise at least 20 base pairs, more preferably at least about 50 base pairs, and still more preferably a nucleic acid fragment or derivative of the invention comprises at least about 100, 200, 300 or 400 base pairs. In some preferred embodiments, the WO 99/18976 PCT/US98/21349 nucleic acid fragment or derivative is bound to some moiety which permits ready identification such as a radionucleotide, fluorescent or other chemical identifier.

Isolated neuregulin and peptide fragments or derivatives of the invention are preferably produced by recombinant methods, although suitable neuregulins also can be isolated from various sources. See the procedures disclosed U.S. Patent 5,530,109; U.S. Patent 5,367,060; and PCT/US93/07491, incorporated herein by reference. A wide variety of molecular and biochemical methods are available for generating and expressing neuregulin; see e.g. the procedures disclosed in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, N.Y. 1992) or other procedures that are otherwise known in the art. For example, neuregulin or fragments or derivatives thereof may be obtained by chemical synthesis, or more preferably by expression in bacteria such as E coli and eukaryotes such as yeast, baculovirus, or mammalian cell-based expression systems, etc., depending on the size, nature and quantity of neuregulin or fragment or derivative thereof. More particularly, a recombinant DNA molecule comprising a vector and a DNA segment encoding neuregulin, or a fragment or derivative thereof, can be constructed. Suitable vectors include e.g. baculovirus-derived vectors for expression in insect cells (see Pennock et al., Mol. Cell. Biol., 4:399-406 (1984)), T7-based expression vector for expression in bacteria (see Rosenberg et al., Gene, 56:125-135 (1987)) and the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J.

Biol. Chem., 263:3521-3527 (1988)). The DNA segment can be present in the vector operably linked to regulatory elements, a promoter polyhedron, T7 or metallothionein (Mt-I) promoters), or a leader sequence to provide for secretory expression of the polypeptide. The recombinant DNA molecule containing the DNA coding for a neuregulin or a fragment or derivative thereof can be introduced into appropriate host cells by known methods. Suitable host cells include e.g. prokaryotes such as E. coli, Bacillus subtilus, etc., and eukaryote such as animal cells and yeast strains, S. cerevisiae. Mammalian cells may be preferred such as J558, NSO, SP2-O or CHO. In general, conventional culturing conditions can be employed. See Sambrook, supra. Stable transformed or transfected cell lines can then be selected.

~==L~12~X WO 99/18976 PCT/US98/21349 -11 The expressed neuregulin or fragment or derivative thereof then can be isolated and purified by known methods. Typically the culture medium is centrifuged and the supernatant purified by affinity or immunoaffmity chromatography, e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinity protocol comprising use of monoclonal antibodies that bind neuregulins.

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

As discussed above, the present invention includes methods for treating and preventing certain neurological-related injuries and disorders, comprising the administration of an effective amount of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, to a subject including a mammal, particularly a human, in need of such treatment.

In particular, the invention provides methods for treatment and/or prophylaxis of nerve cell death (degeneration) resulting from hypoxia, hypoglycemia, brain or spinal cord ischemia, brain or spinal cord trauma, stroke, heart attack or drowning.

Typical candidates for treatment include e.g. heart attack, stroke and/or persons suffering from cardiac arrest neurological deficits, brain or spinal cord injury patients, patients undergoing major surgery such as heart surgery where brain ischemia is a potential complication and patients such as divers suffering from decompression sickness due to gas emboli in the blood stream. Candidates for treatment also will include those patients undergoing a surgical procedure involving extra-corporal circulation such as e.g. a bypass procedure.

The invention also provides methods for treatment which comprise administration of a neuregulin or fragment or derivative thereof, or nucleic acid WO 99/18976 PCT/US98/21349 -12encoding same, to a patient that is undergoing surgery or other procedure where brain or spinal cord ischemia is a potential risk. For example, carotid endarterectomy is a surgical procedure employed to correct atherosclerosis of the carotid arteries. Major risks associated with the procedure include intraoperative embolization and the danger of hypertension in the brain following increased cerebral blood flow, which may result in aneurysm or hemorrhage. Thus, an effective amount of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, could be administered pre-operatively or peri-operatively to reduce such risks associated with carotid endarterectomy, or other post-surgical neurological deficits.

The invention also is effective to promote and enhance recovery from acute nerve cell death and neurological conditions. Thus, for example, a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, could be administered to promote repair, remodeling or reprogramming to a patient that has suffered from stroke or other neuronal injury, suitably for an extended period as discussed above. A therapeutic agent of the invention also could be administered post-operatively to promote recovery from any neurological deficits that may have occurred to a patient that has undergone surgery.

The invention further includes methods for prophylaxis against neurological deficits resulting from e.g. coronary artery bypass graft surgery and aortic valve replacement surgery, or other procedure involving extra-corporal circulation. Those methods will comprise administering to a patient undergoing such surgical procedures an effective amount of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, typically either pre-operatively or peri-operatively.

The invention also provides methods for prophylaxis and treatment against neurological injury for patients undergoing myocardial infarction, a procedure that can result in ischemic insult to the patient. Such methods will comprise administering to a patient undergoing such surgical procedure an effective amount of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, typically either preoperatively or peri-operatively.

Also provided are methods for treating or preventing neuropathic pain such as may be experienced by cancer patients, persons having diabetes, amputees and other persons who may experience neuropathic pain. These methods for treatment comprise WO 99/18976 PCT/US98/21349 -13administration of an effective amount of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, to a patient in need of such treatment.

The invention also provides methods for treatment and prophylaxis against retinal ischemia or degeneration and resulting visual loss. For example, a neuregulin or fragment or derivative thereof, can be administered parenterally or by other procedure as described herein to a subject a suffering from or susceptible to ischemic insult that may adversely affect retinal function, significantly elevated intraocular pressures, diseases such as retinal artery or vein occlusion, diabetes or other ischemic ocular-related diseases. Post-ischemic administration also may limit retinal damage.

The invention also includes methods for treating and prophylaxis against decreased blood flow or nutrient supply to retinal tissue or optic nerve, or treatment or prophylaxis against retinal trauma or optic nerve injury. Subjects for treatment according to such therapeutic methods of the invention may be suffering or susceptible to retinal ischemia that is associated with atherosclerosis, venous capillary insufficiency, obstructive arterial or venous retinopathies, senile macular degeneration, cystoid macular edema or glaucoma, or the retinal ischemia may be associated with a tumor or injury to the mammal. Intravitreal injection also may be a preferred administration route to provide more direct treatment to the ischemic retina.

The invention further provides a method of treating Korsakoff's disease, a chronic alcoholism-induced condition, comprising administering to a subject including a mammal, particularly a human, an effective amount of a neuregulin or fragment or derivative thereof, in an amount effective to treat the disease.

Compounds of the invention are anticipated to have utility for the attenuation of cell loss, hemorrhages and/or amino acid changes associated with Korsakoffs disease.

The invention further includes methods for treating a person suffering from or susceptible to epilepsy, emesis, narcotic withdrawal symptoms and age-dependent dementia, comprising administering to a subject including a mammal, particularly a human, an effective amount of a neuregulin or fragment or derivative thereof, in an amount effective to treat the condition.

It will be appreciated that in some instances a neuregulin or a fragment or derivative thereof will be preferably administered to a subject rather than a neuregulin nucleic acid, particularly where a patient is suffering from or susceptible to an acute WO 99/18976 PCT/US98/21349 -14neurological injury that demands immediate therapy. For example, administration of a neuregulin polypeptide may be preferred to a patient suffering from stroke, heart attack, traumatic brain injury and the like where it is desired to deliver the active therapeutic as quickly as possible.

In the therapeutic methods of the invention, neuregulin peptides and nucleic acids may be suitably administered to a subject such as a mammal, particularly a human, by any of a number of routes including parenteral (including subcutaneous, intramuscular, intravenous and intradermal), oral, rectal, nasal, vaginal and optical (including buccal and sublingual) administration. A neuregulin protein or nucleic acid or fragment or derivative thereof may be administered to a subject alone or as part of a pharmaceutical composition, comprising the peptide or nucleic acid together with one or more acceptable carriers and optionally other therapeutic ingredients. The carriers should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Nucleic acids encoding a neuregulin or a neuregulin fragment or derivative can be administered to a patient by generally known gene therapy procedures. See, for example, WO 90/11092 and WO 93/00051. Thus, for instance, the nucleic acids may be introduced into target cells by any method which will result in the uptake and expression of the nucleic acid by the target cells. These methods can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, catheters, etc. Preferably, the administered nucleic acid codes for an appropriate secretory sequence to promote expression upon administration. Suitable vectors for administering a nucleic acid in accordance with the invention include chemical conjugates such as described in WO 93/04701, which has targeting moiety a ligand to a cellular surface receptor), and a nucleic acid binding moiety polylysine), viral vector a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety an antibody specific for a target cell) and a nucleic acid binding moiety a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox WO 99/18976 PCT/US98/21349 vectors, herpes virus vectors such as a herpes simplex I virus (HSV) vector [A.I.

Geller et al., J. Neurochem, 64:487 (1995); F. Lim et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); A.I. Geller et al., Proc Natl. Acad. Sci. U.S.A.:90 7603 (1993); A.I. Geller et al., Proc Natl. Acad. Sci USA, 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet., 3:219 (1993); Yang et al., J.

Virol., 69:2004 (1995)] and Adeno-associated Virus Vectors [Kaplitt, et al., Nat. Genet., 8:148 (1994)].

Pox viral vectors introduce the gene into the cell cytoplasm. Avipox virus vectors result in only a short-term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the specific condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include naked DNA, Ca 3 (PO4) 2 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofecton, cell microinjection, and viral vectors.

A vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell (Bobo et al., Proc. Natl. Acad.

Sci. USA, 91:2076-2080 (1994); Morrison et al., Am. J. Physiol., 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

Parenteral formulations for administration of a neuregulin or a fragment or derivative thereof may be in the form of liquid solutions or suspensions; for oral WO 99/18976 PCT/US98/21349 -16administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences". Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for a neuregulin or fragments or derivatives thereof include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

The concentration of a neuregulin or a fragment or derivative thereof, or nucleic acid encoding such polypeptides, administered to a particular subject will vary depending upon a number of issues, including the condition being treated, the mode and site of administration, the age, weight sex and general health of the subject, and other such factors that are recognized by those skilled in the art. Optimal administration rates for a given protocol of administration can be readily determined by those skilled in the art.

All documents mentioned herein are incorporated herein by reference in their entirety. The invention is further illustrated by the following non-limiting Examples.

Example 1 In vivo neuroprotection assay Neuregulins and neuregulin fragments and derivatives can be assessed for neuroprotective efficacy pursuant to the following assay.

Mature male Long-Evans rats (Charles River, 250-350g) are allowed food and water ad libitum. Animals are anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic head holder (David Kopf Instruments, Tujunga, CA). The WO 99/18976 PCT/US98/21349 -17dorsal surface of the skull is exposed by midline incision, and a small burr hole (2 mm diameter) is drilled over the right lateral ventricle, 1.6 mm lateral and 0.9 mm posterior to bregma. A stainless steel cannula 0.020", O.D. 0.028", 2 cm long) is then inserted stereotaxically into the ventricle to a depth of 4.4 mm beneath the surface of the skull. The tubing is suitably bent at a 90° angle 1-1.6 cm from its tip and connected to polyethylene tubing 0.76 mm, O.D. 1.22 mm, 10 cm long) that is connected (by glue) to a mini-osmotic pump (Alzet 1007D, 100 l fill volume, pump rate 0.5 pl/hr; Alza Corp., Palo Alto, CA) implanted subcutaneously in the back. The cannula can be suitably fixed to the skull by orthodontic resin Culk Co., Milford, DE) bonded to two small machine screws stainless steel slotted) inserted in the skull. The pump, tubing, and cannula are primed before insertion with infusate solutions; a 3-0 nylon suture is inserted into the cannula during implantation to prevent obstruction by brain tissue. The wound is closed with 3-0 silk suture and cefazolin (10 mg, is administered. After surgery animals are suitably kept in individual cages and fed soft food.

Pumps are filled with vehicle alone (containing 127 mM NaC1, 2.6 mM KC1, 1.2 mM CaC1 2 0.9 mM MgCl 2 4.14 mM HEPES, 3 mM glycerin, 0.001% bovine serum albumin [BSA], and 0.01% fast green), or vehicle neuregulin or fragment or derivative thereof (100 tgm/ml). Heparin can be suitably used at relatively low doses, e.g. about 0.8 units/kg/day which is approximately 250-500 times less than a standard anticoagulant dose.

Three days after cannula implantation, animals are reanesthetized with 2% halothane and given atropine (0.15 mg/kg, Animals are then intubated and connected to a ventilator (SAR-830; CWE Inc., Ardmore, PA) delivering 1% halothane/70% nitrous oxide in oxygen. The right femoral artery and vein are cannulated for monitoring of mean arterial blood pressure (MABP; Gould RS3200 Blood Pressure Monitor, Gould Inc., Valley View, OH), and blood sampling.

Animals are then paralyzed with pancuronium bromide (0.5 mg/kg, Arterial blood gasses (Coring 178 Blood Gas Analyzer, Ciba Coming Diagnostic Corp., Medford, MA), blood glucose (Accu-Check Blood Glucose Analyzer, Boehringer Mannheim, Indianapolis, IN), and hematocrit are measured at least twice during surgery and the immediate post-operative period. The stroke volume and rate of the WO 99/18976 PCT/US98/21349 18ventilator are adjusted to maintain PaO 2 between 100-200 mm Hg and PaCO 2 between 30-40 mm Hg. Core body temperature may be monitored by rectal thermocouple (e.g.

Model 73ATA, Yellow Springs Instrument Co., Yellow Springs, OH) and maintained between 36-37 0 C with a homeothermic blanket control unit (Harvard Bioscience, South Natick, MA).

Focal ischemic infarcts are made in the right lateral cerebral cortex in the territory of the middle cerebral artery (MCA) by the method of Chen, et al., Stroke, 17:738-743 (1986). Both common carotid arteries are exposed by midline anterior cervical incision. The animal is placed in a lateral position and a 1 cm skin incision is then made at the midpoint between the right lateral canthus and the anterior pinna.

The temporal muscle is retracted, and a small (3 mm diameter) craniectomy is made at the junction of the zygoma and squamosal bone using a dental drill cooled with saline.

Using a dissecting microscope, the dura can be opened with fine forceps, and the right MCA can be ligated with two 10-0 monofilament nylon ties just above the rhinal fissure and transected between the ties. Both common carotid arteries then can be occluded by microaneurysm clips for 45 minutes. After removal of the clips, return of flow is visualized in the arteries. Anesthesia is maintained for 15 minutes, and animals are returned to individual cages and fed soft food after surgery.

Twenty four hours after cerebral infarction, animals are again weighed, and then sacrificed by rapid decapitation. Brains are removed, inspected visually for the anatomy of the middle cerebral artery as well as for signs of hemorrhage or infection, immersed in cold saline for 10 minutes, and sectioned into six standard coronal slices (each 2 mm thick) using a rodent brain matrix slicer (Systems, Warren, MI). Brains are also examined visually for the presence of dye (fast green) in the cerebral ventricles. Slices are placed in the vital dye 2,3,5-triphenyl tetrazolium chloride (TTC, Chemical Dynamics Co., NH) at 37 0 C in the dark for 30 minutes, followed by 10% formalin at room temperature overnight. The outline of right and left cerebral hemispheres as well as that of infarcted tissue, clearly visualizable by lack of TTC staining (Chen et al., Stroke, 17:738-743 (1986)), is outlined on the posterior surface of each slice using an image analyzer (MTI videocamera and Sony video monitor connected to a Bioquant IV Image Analysis System run on an EVEREX computer).

Infarct volume is calculated as the sum of infarcted area per slice multiplied by slice WO 99/18976 PCT/US98/21349 -19thickness. Both the surgeon and image analyzer operator are blinded to the treatment given each animal.

Volumes of infarcts among vehicle vs. neuregulin-treated animals can be compared by unpaired, two-tailed t-tests for each experiment, and by two-way analysis of variance (ANOVA; Exp. X Treatment) for combined data. A subsequent slice-by-slice analysis of infarct area among pooled neuregulin- vs. vehicle-treated animals is suitably done by repeated measures two-way ANOVA (Treatment X Slice).

Other anatomical and physiological measurements are compared among GDF-1- vs.

vehicle-treated animals by unpaired, two-tailed t-tests using the Bonferroni correction for multiple pairwise comparisons.

Example 2 In vivo behavioral assays For behavioral outcome studies, such as to assess recovery, repair and remodeling promoted by administration of a neuregulin or fragment or derivative thereof, or nucleic acid encoding same, a number of assays can be employed such as those described in G. Sinson et al., JNeurochem, 65(5):2209-2214 (1995); T.K.

McIntosh et al., Neuroscience, 28:233-244 (1989); and T.K. McIntosh et al., J Neurotrauma, 10:373-384 (1993).

Briefly, one suitable behavioral assay as described in G. Sinson et al., supra, entails that test animals (male Sprague-Dawley rats) receive preinjury training in a Morris Water Maze, a circular tank 1 m in diameter that is filled with 18°C water.

The water surface is made opaque with a covering of Styrofoam pieces. During training of the animals a submerged platform is present in the maze. Each test animal undergoes 20 training trials over a two day period during which they learn to locate the platform using external visual cues. Immediately following the last training trial, animals are anesthetized and subjected to a lateral (parasagittal) fluid-percussion (FP) brain injury. Briefly, a 5-mm craniectomy is performed over the left parietal cortex, midway between lamda and bregma. A hollow Leur-loc fitting is cemented to the craniectomy site. The injury is delivered after attaching the FP device. The injury should be of moderate severity (2.1-2.3 atm). After injury, the Leur-loc is removed, and the skin is sutured. Normothermia is maintained with warming pads until the animals being to ambulate.

WO 99/18976 PCT/US98/21349 At 72 hours, 1 week or 2 weeks after injury, animals are assessed for their ability to remember the learned task of locating the platform in the MWM. For this evaluation the platform is removed from the maze, and the animal's swimming pattern is suitably recorded with a computerized video system for 1 minute. The maze is separated in zones that are weighed according to the proximity to the platform's location. A memory score is generated by multiplying the weighted numbers by the time the animal spends in each zone and then adding the products.

Animals surviving for 1 or 2 weeks also can undergo evaluation of neurologic motor function. Briefly, one suitable assay provides that animals are scored from 0 (severely impaired) to 4 (normal) for each of the following: left and right forelimb during suspension by the tail; left and right hindlimb flexion when the forelimbs remain on a surface and the hindlimbs are lifted up and back by the tail; the ability to resist lateral pulsion to the left and right; and the ability to stand on an inclined plane in the left, right, and vertical positions. Scores are combined for each of the tests through The observer for the tests should be blinded to the animal's previous treatment.

The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

EDITORIAL NOTE FOR 96042/98 THE FOLLOWING SEQUENCE LISTING IS PART OF THE DESCRIPTION THE CLAIMS FOLLOW ON PAGE 21 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: Cambridge NeuroScience. Inc.

(ii) TITLE OF THE INVENTION: THERAPEUTIC METHODS COMPRISING USE OF A

NEUREGULIN

(iii) NUMBER OF SEQUENCES: 54 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Dike. Bronstein. Roberts Cushman. LLP STREET: 130 Water Street CITY: Boston STATE: MA COUNTRY: USA ZIP: 02109 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US98/21349 FILING DATE: 08-OCT-1998

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 60/062.109 S(B) FILING DATE: 14-OCT-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: Corless. Peter F REGISTRATION NUMBER: 33.860 REFERENCE/DOCKET NUMBER: 47440-PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-523-3400 TELEFAX: 617-523-6440

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 745 base pairs 0 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...744 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATG

Met 1 AGA TGG CGA CGC Arg Trp Arg Arg 5 GCC CCG CGC CGC TCC Ala Pro Arg Arg Ser 10 GGG CGT Gly Arg CCC GGC CCC CGG Pro Gly Pro Arg

S

GCC CAG CGC CCC Ala Gln Arg Pro 20 CTG CCA CTA CTG Leu Pro Leu Leu 35 GGC TCC GCC GCC CGC Gly Ser Ala Ala Arg 25 TCG TCG Ser Ser CCG CCG CTG CCG CTG Pro Pro Leu Pro Leu CTG CTG CTG Leu Leu Leu

GGG

Gly 40 ACC GCG GCC Thr Ala Ala CTG GCG Leu Ala CCG GGG GCG *Pro Gly Ala 144 GCG GCC Ala Ala 50 GGC AAC GAG GCG GCT Gly Asn Glu Ala Ala 55 CCC GCG GGG GCC Pro Ala Gly Ala CCG CCC AGC GTG GGA Pro Pro Ser Val Gly TCG GTG CAG GAG CTA Ser Val Gln Glu Leu 75 GTG CAC CCG CAG CGG Val His Pro GlnArg 90 GTG GTG ATC GAG GGA Val Val Ile Glu Gly CTC GAC AGG AAG GCG Leu Asp Arg Lys Ala 100 TCG GTG TGC TAC TCG Ser Val Cys Tyr Ser GCT CAG CGC GCC GCG Ala Gln Arg Ala Ala CGG CAG CAG GGG GCA Arg Gln Gln Gly Ala GCA GGG GCG TGG GGC Ala Gly Ala Trp Gly 110 GCG CTG GGG CCG CCC Ala Leu Gly Pro Pro 125 GTG CCC TCT TGG CCC Val Pro Ser Trp Pro 140 240 288 192 GCG GCG GCG Ala Ala Ala

GCG

Ala 105 GGC GAG Gly Glu 336 GGC GAT CGC Gly Asp Arg 115 GAG CCG CCA GCC GCG Glu Pro Pro Ala Ala 120 GGC CCA CGG Gly Pro Arg 384 GCC GAG Ala Glu 130 GAG CCG CTG CTC GCC Glu Pro Leu Leu Ala 135 GCC AAC GGG ACC Ala Asn Gly Thr 11- i 0

ACC

Thr 145 GCC CCG GTG CCC AGC Ala Pro Val Pro Ser 150 GCC GGC GAG CCC GGG Ala Gly Glu Pro Gly 155 GTG TGG GCG GTG AAA Val Trp Ala Val Lys 170 CTG GTG AAG GTG CAC Leu Val Lys Val His 165 AAG GAC TCG CTG CTC Lys Asp Ser Leu Leu 180 TTC CCC TCC TGC GGG Phe Pro Ser Cys Gly 195 GAG GAG GCG CCC TAT Glu Glu Ala Pro Tyr 160 GCC GGG GGC TTG AAG Ala Gly Gly Leu Lys 175 TGG GGC CAC CCC GCC Trp Gly His Pro Ala 190 AGG TAC ATC TTC TTC Arg Tyr Ile Phe Phe 205 480 528 576 624 ACC GTG CGC CTG GGG Thr Val Arg Leu Gly 185 AGG CTC AAG GAG GAC Arg Leu Lys Glu Asp 200 ccc c cc b c cc.

c 9 b c.

c c ccc.

C c c ccc.

b b b ATG GAG Met Glu 210 CCC GAC GCC AAC AGC Pro Asp Ala Asn Ser 215 ACC AGC CGC GCG CCG GCC GCC TTC CGA Thr Ser Arg Ala Pro Ala Ala Phe Arg 220 672 720 GCC Ala 225 TCT TTC CCC CCT CTG Ser Phe Pro Pro Leu 230 GAG ACG GGC CGG Glu Thr Gly Arg

AAC

Asn 235 CTC AAG AAG GAG GTC Leu Lys Lys Glu Val 240 AGC CGG GTG CTG TGC AAG CGG TGC G Ser Arg Val Leu Cys Lys Arg Cys 245 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 248 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Arg Trp Arg Arg Ala Pro Arg Arg Ser 1 5 10 Ala Gin Arg Pro Gly Ser Ala Ala Arg Ser 25.

Leu Pro Leu Leu Leu Leu Leu Gly Thr Ala Gly Arg Pro Gly Pro Arg Ser Pro Pro Ala Leu Ala Leu Pro Leu Pro Gly Ala i. i ~-C 40 Ala Ala Gly Asn Glu Ala Ala Pro Ala Gly Ala Ser Val Cys Tyr Ser 55 Ser Pro Pro Ser Val Gly Ser Val Gin Glu Leu Ala Gin Arg Ala Ala 70 75 Val Val Ile Glu Gly Lys Val His Pro Gin Arg Arg Gin Gin Gly Ala 90 Leu Asp Arg Lys Ala Ala Ala Ala Ala Gly Glu Ala Gly Ala Trp Gly 100 105 110 Gly Asp Arg Glu Pro Pro Ala Ala Gly Pro Arg Ala Leu Gly Pro Pro 115 120 125 Ala Glu Glu Pro Leu Leu Ala Ala Asn Gly Thr Val Pro Ser Trp Pro 130 135 140 Thr Ala Pro Val Pro Ser Ala Gly Glu Pro Gly Glu Glu Ala Pro Tyr 145 150 155 160 Leu Val Lys Val His Gin Val Trp Ala Val Lys Ala Gly Gly Leu Lys 165 170 175 Lys Asp Ser Leu Leu Thr Val Arg Leu Gly Thr Trp Gly His Pro Ala e 180 185 190 Phe Pro Ser Cys Gly Arg Leu Lys Glu Asp Ser Arg Tyr Ile Phe Phe 195 200 205 Met Glu Pro Asp Ala Asn Ser Thr Ser Arg Ala Pro Ala Ala Phe Arg P: P210 215 220 Ala Ser Phe Pro Pro Leu Glu Thr Gly Arg Asn Leu Lys Lys Glu Val 225 230 235 240 Ser Arg Val Leu Cys Lys Arg Cys 245 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 252 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 3...251 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CC CAT CAA GTG TGG GCG GCG AAA GCC GGG GGC TTG AAG AAG GAC TCG 47 His Gin Val Trp Ala Ala Lys Ala Gly Gly Leu Lys Lys Asp Ser 1 5 10 CTG CTC ACC GTG CGC Leu Leu. Thr Val Arg TGC GGG CGC CTC AAG Cys Gly Arg Leu Lys CTG GGC GCC TGG GGC Leu Gly Ala Trp Gly 25

CAC

His CCC GCC TTC CCC TCC Pro Ala Phe Pro Ser TTC TTC ATG GAG CCC Phe Phe Met Glu Pro GAG GAC AGC AGG Glu Asp Ser Arg 40 TAC ATC Tyr Ile GAG GCC AAC Glu Ala Asn AGC AGC GGC Ser Ser Gly GGG CCC Gly Pro 55 GGC CGC CTT CCG AGC CTC CTT CCC Gly Arg Leu Pro Ser Leu Leu Pro CCC TCT Pro Ser CGA GAC GGG CCG Arg Asp Gly Pro

GAA

Glu 70 CCT CAA GAA GGA GGT CAG CCG GGT GCT Pro Gin Glu Gly Gly Gin Pro Gly Ala a..

a a a a *aa.

a

GTG

Val CAA CGG TGC G 252 Gin Arg Cys INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 83 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: His 1 Leu Gly Gin Val Trp Ala Ala Lys Ala Gly Gly 10 Leu Lys Lys Asp Ser Leu Thr Val Arg Arg Leu Lys Leu Gly Ala Glu Asp Ser Trp Gly His Pro 25 Arg Tyr Ile Phe Gly Arg Leu Pro Ala Phe Phe Met Pro Ser Cys Glu Pro Glu Leu Pro Pro Ala Asn Ser Ser Arg Asp Gin Arg Cys Ser Gly Gly Pro 55 Pro Ser Gin Gly Pro Glu 70 Gin Glu Gly Gly Pro Gly Ala INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 59 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser 1 5 10 Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr 25 Lys Phe Lys Trp Phe Lys Asn Gly Ser Glu Leu Ser Arg 40 Pro Gin Asn Ile Lys Ile Gin Lys Arg Pro Gly 50 Val Ala Gly Ser Ser Leu Lys Asn Lys S Sr

C

5

S

INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 178 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...177 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCT TGC CTC CCC GCT TGA AAG AGA TGA AGA GTC AGG Leu Pro Pro Arg Leu Lys Glu His Lys Ser Gin Glu 1 5 10 GTT CCA AAC TAG TGC TTC GGT GCG AGA CCA GTT CTG Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu 25 AGT CTG TGG CAG Ser Val Ala Gly AAT ACT CCT CTC Tyr Ser Ser Leu TCA AGT TCA AGT GGT TCA AGA ATG GGA GTG AAT TAA GCC GAA AGA ACA 144 Lys Phe Lys Trp Phe Lys Asn Gly Ser Glu Leu 40 Ser Arg Lys Asn Lys AAC CAC AAA ACA TCA AGA TAC AGA AAA GGC CGG G Pro Gly Asn Ile Lys Ile Gin Lys Arg Pro Gly INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 178 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...177 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 178 a.

a a a a a .a a a a

CCT

Leu 1 TGC CTC CCC GAT Pro Pro Gin Leu 5 TGA AAG AGA TGA Lys Glu His Lys AAA GCC AGG AAT CGG CTG CAG Ser Gin Glu Ser Ala Ala Gly 10 CCA GTT CTG AAT ACT CCT CTC Ser Ser Glu Tyr Ser Ser Leu GTT CCA AAC TAG TCC TTC GGT GTG Ser Lys Leu Val Leu Arg Cys Glu TCA GAT TCA AGT GGT TCA AGA ATG Arg Phe Lys Trp Phe Lys Asn Gly 40 GGA ATG AAT TGA ATC GAA AAA ACA Asn Glu Leu Asn Arg Lys Asn Lys AAC CAC Lys Gly AAA ATA TCA AGA TAC AAA AAA AGC CAG G Asn Ile Lys Ile Gin Lys Arg Pro Gly INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 59 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear i (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Pro Pro Gin Leu Lys Glu Met Lys Ser Gin Glu Ser Ala Ala Gly 1 5 10 Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu 25 Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys 40 Pro Gin Asn Ile Lys Ile Gln LysLys Lys Pro Gly INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: f Lys Ser Glu Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu 1 5 10 Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala 20 25 Asn Ile Thr Ile Val Glu Ser Asn Ala 35 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 122 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY' Coding Sequence LOCATION: 2...122 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID G AAG TCA GAA CTT CGC ATT AGC AAA GCG TCA CTG GCT GAT TCT GGA 46 Lys Ser Glu Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly 1 5 10 GAA TAT ATG TGC AAA GTG ATC AGC AAA CTA GGA AAT GAC AGT GCC TCT 94 Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser GCC AAC ATC ACC ATT GTG GAG TCA AAC G 122 Ala Asn Ile Thr Ile Val Glu Ser Asn Ala INFORMATION FOR SEQ ID NO:11: *0 SEQUENCE CHARACTERISTICS: LENGTH: 122 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 2...122 OTHER INFORMATION: *i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: G AAG TCA GAA CTT CGC ATT AAC AAA GCA TCA CTG GCT GAT TCT GGA 46 Lys Ser Glu Leu Arg Asn Ser Lys Ala Ser Leu Ala Asp Ser Gly 1 5 10 GAG TAT ATG TGC AAA GTG ATC AGC AAA TTA GGA AAT GAC AGT GCC TCT 94 Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser 25 GCC AAT ATC ACC ATC GTG GAA TCA AAC G 122 Ala Asn Ile Thr Ile Val Glu Ser Asn Ala INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lys Ser Glu Leu Arg Asn Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu 1 5 10 Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala 25 Asn Ile Thr Ile Val Glu Ser Asn Ala INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear *o.

(ix) FEATURE: :o NAME/KEY: Coding Sequence LOCATION: 84...272 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCTAAAACTA CAGAGACTGT ATTTTCATGA TCATCATAGT TCTGTGAAAT ATACTTAAAC CGCTTTGGTC CTGATCTTGT AGG AAG TCA GAA CTT CGC ATT AGC AAA GCG TCA 113 Lys Ser Glu Leu Arg Ile Ser Lys Ala Ser 1 5 CTG GCT GAT TCT GGA GAA TAT ATG TGC AAA GTG ATC AGC AAA CTA GGA 161 Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly 20 AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTG GAG TCA AAC GGT AAG 209 Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Gly Lys AGA TGC CTA CTG CGT GCT ATT TCT CAG TCT CTA AGA GGA GTG ATC AAG Arg Cys Leu Leu Arg Ala Ile Ser Gin Ser Leu Arg Gly Val lie Lys 50 GTA TGT GGT CAC ACT TGAATCACGC AGGTGTGTGA AATCTCATTG TGAACAAATA A Val Cys Gly His Thr AAATCATGAA AGGAAAACTC CACTCCATAA GGTGAAATAG TATGTTTGAA ATATCTTATG GGTCCTCCTG TAAAGCTCTT ACCTGAAATA TATATAGATT ATTT 373 417 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 63 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

S

Ser Glu Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu 25 Asn Ile Thr Ile Val Glu Ser Asn Gly 40 Gly Asn Asp Ser Ala Ser Ala Leu Arg Ala Lys Arg Cys Leu Gly His Thr Ile Ser Gin Ser Leu Arg Gly Val Ile Lys Val Cys 55 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal 0 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: Glu Ile Thr Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr 1 5 10 Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Thr 25 Ser Ser Ser INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Val Ser Asn Thr (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...100 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

AG

Glu 1 ATC ACC ACT GGC ATG CCA GCC TCA ACT GAG Ile Thr Thr Gly Met Pro Ala Ser Thr Glu 5 ACA GCG TAT GTG TCT Thr Ala Tyr Val Ser TCA GAG TCT CCC ATT AGA ATA TCA GTA TCA ACA Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 25 TCT TCA T Ser Ser Ser INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

GAA

Glu GGA ACA AAT ACT Gly Thr Asn Thr (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...100 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AG ATC ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA GCA TAT GTG TCT 47 Glu Ile Thr Ile Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser 1 5 10 TCA GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA GAA GGA GCA AAT ACT Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr 25 TCT TCA T 102 Ser Ser Ser INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 35 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: SGlu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser 1 5 10 Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr 25 Ser Ser Ser INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 0 (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser Thr Ser Thr Ala Gly Thr Ser Leu Val Lys Cys Ala Glu Glu Cys Phe Met Val Lys Lys Glu Lys Thr Phe Cys Val Asn Gly Gly 25 Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 128 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: .r NAME/KEY: Coding Sequence LOCATION: 3...128 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CC ACA TCC ACA TCT ACA GCT GGG ACA AGC CAT CTT GTC Thr Ser Thr Ser Thr Ala Gly Thr Ser His Leu Val 1 5 10 AAG TGT GCA Lys Cys Ala GAG AAG GAG AAA ACT TTC TGT GTG AAT Glu Lys Glu Lys Thr Phe Cys Val Asn AAA GAC CTT TCA AAT CCC TCA AGA TAC Lys Asp Leu Ser Asn Pro Ser Arg Tyr GGA GGC Gly Gly 25 TTG TGC Leu Cys GAG TGC TTC ATG GTG Glu Cys Phe Met Val INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 128 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 3...128 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CT ACA TCT ACA TCC ACC ACT GGG ACA AGC CAT Thr Ser Thr Ser Thr Thr Gly Thr Ser His 1 5 10 CTT GTA AAA TGT GCG Leu Val Lys Cys Ala GAG TGC TTC ATG GTG Glu Cys Phe Met Val GAG AAG GAG AAA ACT TTC TGT GTG AAT Glu Lys Glu Lys Thr Phe Cys Val Asn AAA GAC CTT TCA AAC CCC TCG AGA TAC Lys Asp Leu Ser Asn Pro Ser Arg Tyr GGA GGG Gly Gly 25 a..

TTG TGC Leu Cys 128 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val Lys Thr 1 5 10 Lys Glu Lys Thr Phe Cys Val Asn Gly Gly 25 Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Cys Ala Glu Met Val Lys Glu Cys Phe INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids I i. TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Lys Cys Gin Pro Gly Phe Thr Gly Ala Arg 1 5 10 Met Lys Val Gin Thr Gin Glu Cys Thr Glu Asn Val Pro r INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...69 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

AAG

Lys 1 TGC CAA CCT GGA TTC ACT GGA GCG AGA Cys Gin Pro Gly Phe Thr Gly Ala Arg 5 10 TGT ACT GAG AAT Cys Thr Glu Asn GTG CCC Val Pro ATG AAA GTC CAA ACC CAA GAA Met Lys Val Gin Thr Gin Glu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear r-i L (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...69 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID TGC CAA CCT GGA Cys Gin Pro Gly TTC ACT GGA GCA Phe Thr Gly Ala

AGA

Arg 10

TGT

Cys ACT GAG AAT Thr Glu Asn GTG CCC Val Pro

S

*SSS

ATG AAA GTC CAA AAC CAA GAA Met Lys Val Gin Asn Gin Glu INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Cys Gin Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Lys Val Gin Asn Gin Glu INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gin Asn Tyr Val Met 1 5 10 Ala Ser Phe Tyr INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...60 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AAG TGC CCA AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATG 48 Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gin Asn Tyr Val Met 1 5 10 :..GCC AGC TTC TAC Ala Ser Phe Tyr INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...60 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ;^liiij~ i AAG TGC CCA AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATG 48 Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gin Asn Tyr Val Met 1 5 10 GCC AGC TTC TAC Ala Ser Phe Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu 1 5 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...33 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AGT ACG TCC ACT CCC TTT CTG TCT CTG CCT GAA TAG 36 Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu 1 5 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...33 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AGT ACG TCC ACT CCC TTT CTG TCT CTG CCT GAA TAG 36 Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu 1 5 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

S

Lys His Leu Gly Ile Glu Phe Met Glu 1 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...27 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AAG CAT CTT GGG ATT GAA TTT ATG GAG 27 Lys His Leu Gly Ile Glu Phe Met Glu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 190 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein FRAGMENT TYPE: internal a (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Ala Glu Glu Leu Tyr Gin Lys Arg Val Leu Thr Ile Thr Gly Ile 1 5 10 Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Val Tyr Cys 20 25 Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu Arg Gin Ser 35 40 Leu Arg Ser Glu Arg Asn Thr Met Met Asn Val Ala Asn Gly Pro His 50 55 His Pro Asn Pro Pro Pro Glu Asn Val Gin Leu Val Asn Gin Tyr Val 65 70 75 Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu 90 Ser Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr 100 105 110 Thr Val Thr Gin Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu 115 120 125 Ser Ile Ile Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val Glu 130 135 140 Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu Asn 145 150 155 160 Gly Leu Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg His Ala Arg 165 170 175 Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg 180 185 190 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 569 base pairs TYPE: nucleic acid STRANDEDRESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1 569 OTHER INFORMATION: (xi) SEUENCE DESCRIPTION: SEQ ID NO:36: r r r r r r tr rr r r c r r r r AAA GCG GAG GAG Lys Ala Glu Glu 1 TGC ATC GCG CTG Cys Ile Ala Leu TAC CAG AAG AGA GTG Tyr Gln Lys Arg Val 10 CTC ACC Leu Thr ATT ACC GGC AT Ile Thr Gly Ile GTG GTC TAC TGC Val Val Tyr Cys CTC GTG GTT GGC ATC Leu Val Val Gly Ile 25 ATG TGT GTG Met Cys Val AAA ACC AAG Lys Thr Lys 35 AAA CAA CGG AAA Lys Gln Arg Lys AAG CTT CAT GAC CGG CTT CGG CAG AGC Lys Leu His Asp Arg Leu Arg Gln Ser 40 ATG ATG AAC GTA GCC AAC GGG CCC CAC Met Met Asn Val Ala Asn Gly Pro His CTT CGG Leu Arg 50 TCT GAA AGA AAC ACC Ser Glu Arg Asn Thr 55 192

CAC

His CCC AAT CCG CCC CCC Pro Asn Pro Pro Pro 70 GAG AAC GTG CAG CTG Glu Asn Val Gln Leu 75 AGC GAG CAT A-FT GTT Ser Glu His Ile Val GTG AAT CAA TAC GTA Val Asn Gln Tyr Val GAG AGA GAG GCG GAG Glu Arg Glu Ala Glu 240 TCT AAA AAT GTC Ser Lys Asn Val AGC TCT TTT TCC Ser Ser Phe Ser 100 ACT GTC ACT CAG Thr Val Thr Gln 115 288 ACC AGT CAC TAC Thr Ser His Tyr ACT TCG ACA GCT CAT CAT TCC ACT Thr Ser Thr Ala His His Ser Thr 105 110 AGC TGG AGC AAT GGA CAC ACT GAA Ser Trp Ser Asn Gly His Thr Glu 125 336 ACT CCC AGT Thr Pro Ser

CAC

His 120 384 0 AGC ATC Ser Ile 130 ATT TCG GAA AGC CAC Ile Ser Glu Ser His 135 TCT GTC ATC GTG ATG TCA TCC GTA GAA Ser Val Ile Val Met Ser Ser Val Glu 140 432

AAC

Asn 145 AGT AGG CAC AGC AGC Ser Arg His Ser Ser 150 CCG ACT GGG GGC CCG Pro Thr Gly Gly Pro 155

AGA

Arg GGA CGT CTC AAT Gly Arg Leu Asn 160 480 GGC TTG GGA GGC CCT CGT GAA TGT AAC AGC Gly Leu Gly Gly Pro Arg Glu Cys Asn Ser 165 170 TTC CTC AGG CAT GCC AGA Phe Leu Arg His Ala Arg 175 528 GAA ACC CCT GAC TCC Glu Thr Pro Asp Ser 180 TAC CGA GAC TCT Tyr Arg Asp Ser CCT CAT AGT GAA AG Pro His Ser Glu Arg 190 569 06e

S.

0

S

OS

0*See *0

S..

0O S 0O S *0 *5

S

0 5505

S

5 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 569 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...569 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: AAA GCG GAG GAG CTG Lys Ala Glu Glu Leu 1 5 TGC ATC GCC CTC CTT Cys Ile Ala Leu Leu TAC CAG AAG AGA GTG Tyr Gin Lys Arg Val 10 GTG GTC GGC ATC ATG Val Val Gly Ile Met 25 CTG ACC ATA ACC GGC ATC Leu Thr Ile Thr Gly Ile TGT GTG GTG GCC TAC TGC Cys Ala Val Val Tyr Cys AAA ACC AAG AAA Lys Thr Lys Lys CTT CGG TCT GAA Leu Arg Ser Glu CAG CGG AAA AAG CTG CAT GAC CGT CTT CGG CAG AGC Gin Ser Gin Arg Lys Lys Leu His Asp Arg Leu Arg CGA AAC AAT ATG ATG AAC ATT Arg Asn Asn Met Met Asn Ile GCC AAT GGG CCT CAC Ala Asn Gly Pro His CCT AAC CCA CCC CCC Pro Asn Pro Pro Pro GAG AAT GTC CAG CTG Glu Asn Val Gin Leu 75 GTG AAT CAA TAC GTA Val Asn Gin Tyr Val GAG AGA GAA GCA GAG Glu Arg Glu Ala Glu 240 TCT AAA AAC GTC ATC Ser Lys Asn Val Ile TCC AGT GAG Ser Ser Glu CAT ATT GTT His Ile Val 90 288 ACA TCC TTT TCC ACC AGT CAC TAT ACT Thr Ser Phe Ser Thr Ser His Tyr Thr 100 105 ACT GTC ACC CAG ACT CCT AGC CAC AGC Thr Val Thr Gin Thr Pro Ser His Ser 115 120 TCC ACA GCC CAT CAC TCC ACT Ser Thr Ala His His Ser Thr 110 TGG AGC AAC GGA CAC ACT GAA Trp Ser Asn Gly His Thr Glu 125 336 384 AGC ATC Ser Ile 130 CTT TCC GAA AGC CAC Leu Ser Glu Ser His 135 TCT GTA ATC GTG ATG TCA TCC GTA GAA Ser Val Ile Val Met Ser Ser Val Glu 140 AGT AGG CAC AGC AGC Ser Arg His Ser Ser 150 CCA ACT GGG GGC Pro Thr Gly Gly GAA TGT AAC AGC Glu Cys Asn Ser 170 CCA AGA GGA CGT CTT AAT Pro Arg Gly Arg Leu Asn 155 160 TTC CTC AGG CAT GCC AGA Phe Leu Arg His Ala Arg 175 432 480 528 569 GGC ACA GGA GGC CCT Gly Thr Gly Gly Pro 165 GAA ACC CCT GAT TCC Glu Thr Pro Asp Ser 180 TAC CGA GAC TCT Tyr Arg Asp Ser 185 CCT CAT AGT Pro His Ser GAA AG Glu Arg 190 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 190 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ii Lys Ala Glu Glu Leu Tyr Gin Lys Arg Val Leu Thr Ile Thr Gly Ile 1 5 10 Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Ala Val Val Tyr Cys 25 Lys Thr Lys Lys Gin Arg Lys Lys Leu His Asp Arg Leu Arg Gin Ser 40 Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn Gly Pro His 55 His Pro Asn Pro Pro Pro Glu Asn Val Gin Leu Val Asn Gin Tyr Val 70 75 Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu 90 Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr 100 105 110 Thr Val Thr Gin Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu 115 120 125 Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val Glu 130 135 140 S. Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu Asn 145 150 155 160 Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg His Ala Arg 165 170 175 Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg 180 185 190 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 47 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear :i (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala His Arg Ser Lys 1 5 10 Cys Met Gin Ile Gin Leu Ser Ala Thr His Leu Arg Ala Ser Ser Ile 25 Pro His Trp Ala Ser Phe Ser Lys Thr Pro Trp Pro Leu Gly Arg 40 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 141 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 2...141 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID AAC CTT ATA GCT GAG CTA AGG AGA AAC AAG GCC CAC AGA TCC Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala His Arg Ser A CAT His 1 a AAA TGC ATG CAG ATC CAG CTT TCC GCA ACT CAT CTT AGA GCT TCT TCC Lys Cys Met Gin Ile Gin Leu Ser Ala Thr His Leu Arg Ala Ser Ser 25 ATT CCC CAT TGG GCT TCA TTC TCT AAG ACC CCT TGG CCT TTA GGA AG Ile Pro His Trp Ala Ser Phe Ser Lys Thr Pro Trp Pro Leu Gly Arg 40 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 217 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser 5 10 Tyr 1 Pro His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met 25 Val Ser Ser Thr Thr Val Ser Met Pro Ser Met Ala Val 40 Val Asp Phe Ser Pro Pro 30 Ser Pro Phe I Val Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg 55 Glu Lys Tyr Asp His His Ala Gin Gin Phe Asn Ser Phe His Cys Asn 70 75 Pro Ala His Glu Ser Asn Ser Leu Pro Pro Ser Pro Leu Arg Ile Val 90 Glu Asp Glu Glu Tyr Glu Thr Thr Gin Glu Tyr Glu Pro Ala Gin Glu 100 105 110 Pro Val Lys Lys Leu Thr Asn Ser Ser Arg Arg Ala Lys Arg Thr Lys 115 120 125 Pro Asn Gly His Ile Ala His Arg Leu Glu Met Asp Asn Asn Thr Gly 130 135 140 Ala Asp Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly 145 150 155 160 Glu Asp Thr Pro Phe Leu Ala Ile Gin Asn Pro Leu Ala Ala Ser Leu 165 170 175 Glu Ala Ala Pro Ala Phe Arg Leu Val Asp Ser Arg Thr Asn Pro Thr 180 185 190 Gly Gly Phe Ser Pro Gin Glu Glu Leu Gln Ala Arg Leu Ser Gly Val 195 200 205 Ile Ala Asn Gin Asp Pro Ile Ala Val 210 215 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 730 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 2...652 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: G TAT GTA TCA GCA ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA GAT 46 Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp 1 5 10 TTC CAC ACG CCA AGC TCC CCC AAG TCA CCC CCT TCG GAA ATG TCC CCG 94 Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro 25 CCC GTG TCC AGC ACG ACG GTC TCC ATG Pro Val Ser Ser Thr Thr Val Ser Met 40 TTC GTG GAA GAG GAG AGA CCC CTG CTC Phe Val Glu Glu Glu Arg Pro Leu Leu 55 CCC TCC ATG GCG GTC AGT CCC Pro Ser Met Ala Val Ser Pro CTT GTG ACG CCA CCA CGG CTG Leu Val Thr Pro Pro Arg Leu CGG GAG Arg Glu AAG TAT GAC CAC CAC Lys Tyr Asp His His 70 GCC CAG CAA TTC AAC TCG TTC CAC TGC Ala Gin Gin Phe Asn Ser Phe His Cys CCC GCG CAT GAG AGC Pro Ala His Glu Ser AAC AGC CTG CCC CCC Asn Ser Leu Pro Pro 90 u r r r r r r GTG GAG GAT GAG GAA Val Glu Asp Glu Glu 100 GAG CCG GTT AAG AAA Glu Pro Val Lys Lys 115 AAG CCC AAT GGT CAC Lys Pro Asn Gly His 130 TAT GAA Tyr Glu ACG ACC CAG Thr Thr G1n 105 AGC CCC TTG AGG ATA Ser Pro Leu Arg Ile TAC GAA CCA GCT CAA Tyr Glu Pro Ala Gin 110 CGG GCC AAA AGAACC Arg Ala Lys Arg Thr 125 CTC ACC AAC AGC AGC Leu Thr Asn Ser Ser 120 ATT GCC CAC Ile Ala His 135 GGC GCT Gly Ala 145 GAC AGC AGT AAC TCA GAG Asp Ser Ser Asn Ser Glu 150 AGG TTG GAA ATG GAC AAC AAC ACA Arg Leu Glu Met Asp Asn Asn Thr 140 AGC GAA ACA GAG GAT GAA AGA GTA Ser Glu Thr Glu Asp Glu Arg Val 155

GGA

Gly 160 GAA GAT ACG CCT TTC Glu Asp Thr Pro Phe 165 CTG GCC ATA CAG AAC Leu Ala Ile Gin Asn 170 TTC CGC CTG GTC GAC Phe Arg Leu Val Asp 185 CCC CTG GCA GCC AGT Pro Leu Ala Ala Ser 175 AGC AGG ACT AAC CCA Ser Arg Thr Asn Pro 190 CTC GAG GCG GCC CCT Leu Glu Ala Ala Pro 180 ACA GGC GGC TTC TCT Thr Gly Gly Phe Ser 195 CCG CAG GAA GAA Pro Gin Glu Glu 200 TTG CAG GCC AGG CTC TCC GGT Leu Gin Ala Arg Leu Ser Gly 205 GTC TAA AAC CGA AAT ACA CCC Val 622 GTA ATC GCT Val Ile Ala 210 AAC CAA GAC CCT ATC GCT Asn Gin Asp Pro Ile Ala 215 670 ATA GAT TCA CCT GTA AAA CTT TAT UT ATA TAA TAA AGT TAA ATT AAA CAA ATT CCA CCT INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 730 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: a

GTATGTGTCA

CTCCCCCAAA

GCCTTCCATG

ACCAAGGCTG

CAACCCCGCG

GGAGTATGAA

TAGCCGGCGG

CAGCAACACA

TGAAGATACG

TGCCTTCCGC

AATCCAGGCC

GCCATGACCA

TCGCCCCCTT

GCGGTCAGCC

CGGGAGAAGA

CATGACAGTA

ACGACCCAAG

GCCAAAAGAA

AGCTCCCAGA

CCTTTCCTGG

CTGGCTGACA

AGGCTGTCTA

CCCCGGCTCG

CGGAAATGTC

CCTTCATGGA

AGTTTGACCA

ACAGCCTCCC

AGTACGAGCC

CCAAGCCCAA

GCAGTAACTC

GCATACAGAA

GCAGGACTAA

GTGTAATTGC

TATGTCACCT

TCCACCCGTG

AGAAGAGAGA

TCACCCTCAG

TGCTAGCCCC

AGCCCAAGAG

TGGCCACATT

AGAGAGTGAA

CCCCCTGGCA

CCCAGCAGGC

TAACCAAGAC

GTAGATTTCC

TCCAGCATGA

CCTCIACTTC

CAGTTCAGCT

TTGAGGATAG

CCTGTTAAGA

GCTAACAGAT

ACAGAAGATG

GCCAGTCTTG

CGCTTCTCGA

CCTATTGCTG

TAATAAAGTA

ACACGCCAAG

CGGTGTCCAT

TCGTGACACC

CCTTCCACCA

TGGAGGATGA

AACTCGCCAA

TGGAAGTGGA

AAAGAGTAGG

AGGCAACACC

CACAGGAAGA

TATAAAACCT

TTCCACCTTA

120 180 240 300 360 420 480 540 600 660 720 730 AAATAAACACA TAGATTCACC TGTAAAACTT TATTTATA

AATTAAACAA

INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 218 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Tyr 1 His Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe Thr Pro Ser Sen Pro Lys Ser Pro Pro Sen Glu Met 25 Val Ser Ser Met Thr Val Ser Met Pro Sen Met Ala Val 40 Ser Pro Pro Ser Pro Phe 9 Asn Glu Glu Glu Arg Glu Asn Val Glu Lys Ser 145 Gly Leu Ala Val Lys Pro Glu Pro Pro 130 Ser Glu Glu Gly Ile 210 Lys Ala Asp Val 115 Asn G1n Asp Ala Arg 195 Ala Phe His Glu 100 Lys Gly Ser Thr Thr 180 Phe Asn Asp Asp Glu Lys His Ser Pro 165 Pro Ser G1n Pro His 70 Ser Tyr Leu lle Asn 150 Phe Ala Thr Asp Leu Leu His Pro Asn Ser Glu Thr Ala Asn 120 Ala Asn 135 Ser Glu Leu Gly Phe Arg G1n Glu 200 Pro lle 215 Leu Gin Leu Thr 105 Ser Arg Ser Ile Leu 185 Glu Ala Val Gin Pro 90 Gin Arg Leu Glu G1n 170 Ala Ile Val Thr Phe 75 Ala Glu Arg Glu Thr 155 Asn Asp Gln Pro Ser Pro Glu Lys 125 Asp Asp Leu Arg Arg 205 Arg Phe Leu Pro 110 Arg Ser Glu Ala Thr 190 Leu Leu Arg His His Arg Ile Ala G1n Thr Asn Thr Arg Val 160 Ala Ser 175 Asn Pro Ser Ser

S

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Glu Arg Arg Glu Gly Lys Gly Lys Gly Lys Gly Gly Lys Lys 1 5 10 Asp Arg Gly Ser Gly Lys Lys Pro Val Pro Ala Ala Gly Gly Pro Ser 25 Pro Ala INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS:

S

LENGTH: 559 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 460...559 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: AGTTTCCCCC CCCAACTTGT CGGAACTCTG GGCTCGCGCG CAGGGCAGGA

GGCGGCTGCC

TGCGAGCGCG

CCAGCGGCGC

AGTCCCAGGT

GCTCCCCCCC

AAACTTTTCC

CGGGAGCCGT

CAGGCGATGC

CCGGACCGAG

GCCAGCAGGA

GGCCCGGACC

ACGCCGCGCG

CGAAGCCGAT

CCGCGCAGAG

GAGCGCGGGC

GCAGCGACAG

GCCACCCCGC

GCACGTTGCG

CGCCTCGGCC

CCCAGCCCTC

CGTGCACTTC

CGGACGGTAA

GAGCGGACCG

GAGNCGTGCG

TCCCCGCGCT

CGGTCGCTGG

GGACCCAAAC

TCGCCTCTCC

CGGCGGGAAC

ACCGGGACGG

CCCCGCCGGC

CCCGCCTCCA

TTGTCGCGCG

GCGGAGCGGC

CTCCTCGGGC

CGAGGACTCC

AGCGCCCGCC

GACAGGAGAC

CTCCGGGGAC

TCGCCTTCGC

CGC AGA Arg Arg 120 180 240 300 360 420 474 522

S.

b

S

TCGGGCGAG ATG TCG GAG Met Ser Glu GAA GGC AAA GGC AAG GGG AAG GGC GGC AAG AAG Glu Gly Lys Gly Lys Gly Lys Gly Gly Lys Lys 15 GAC CGA GGC TCC GGG Asp Arg Gly Ser Gly AAG AAG CCC GTG CCC GCG GCT GGC GGC CCG AGC CCA G Lys Lys Pro Val Pro Ala Ala Gly Gly Pro Ser Pro Ala INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 231 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 132...231 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

CGCGAGCGCC

CGATCCGAGC

TCTCCGGCGA

TCAGCGCGGC CGCTCGCTCT CCTTGGACCA AACTCGCCTG G ATG TCC GAG CGC AAA Met Ser Glu Arg Lys 1 5 CCCCCTCGAG GGACAAACTT TTCCCAAACC CGCCGAGAGC CGTCCGCGTA GAGCGCTCCG GAA GGC AGA GGC AAA GGG AAG GGC Glu Gly Arg Gly Lys Gly Lys Gly 120 170 AAG AAG Lys Lys AAG GAG CGA GGC TCC GGC Lys Glu Arg Gly Ser Gly 20 AAG AAG CCG Lys Lys Pro GAG TCC GCG GCG GGC Glu Ser Ala Ala Gly 218 AGC CAG AGC CCA G Ser Gin Ser Pro INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

S

Si *55 S S

S

S.

S

Met 1 Glu Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys 5 10 Arg Gly Ser Gly Lys Lys Pro Glu Ser Ala 25 Lys Lys Pro Ser Ala Gly Gly Pro Ala INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 1764 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 2...1681 OTHER INFORMATION: -ci-- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: G AAG TCA GAA CTT CGC ATT AGC AAA GCG TCA CTG GCT GAT TCT GGA GAA 49 Lys Ser Glu Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu 1 5 10 TAT ATG TGC AAA GTG ATC AGC AAA CTA GGA AAT GAC AGT GCC TCT GCC 97 Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala 25 AAC ATC ACC ATT GTG GAG TCA AAC GCC ACA TCC ACA TCT ACA GCT GGG 145 Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr Ser Thr Ala Gly 40 ACA AGC CAT CTT GTC AAG TGT GCA GAG AAG GAG AAA ACT TTC TGT GTG 193 Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val 55 AAT GGA GGC GAC TGC TTC ATG GTG AAA GAC CTT TCA AAT CCC TCA AGA 241 70 75 TAC TTG TGC AAG TGC CAA CCT GGA TTC ACT GGA GCG AGA TGT ACT GAG 289 Tyr Leu Cys Lys Cys Gin Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu 90 :.AAT GTG CCC ATG AAA GTC CAA ACC CAA GAA AAA GCG GAG GAG CTC TAC 337 Asn Val Pro Met Lys Val Gln Thr Gin Glu Lys Ala Glu Glu Leu Tyr 100 105 110 CAG AAG AGA GTG CTC ACC ATT ACC GGC ATT TGC ATC GCG CTG CTC GTG 385 Gin Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val 115 120 125 GTT GGC ATC ATG TGT GTG GTG GTC TAC TGC AAA ACC AAG AAA CAA CGG 433 Val Gly Ile Met Cys Val Val Val Tyr Cys Lys Thr Lys Lys Gin Arg 130 135 140 AAA AAG CTT CAT GAC CGG CTT CGG CAG AGC CTT CGG TCT GAA AGA -AAC 481 Lys Lys Leu His Asp Arg Leu Arg Gin Ser Leu Arg Ser Glu Arg Asn 145 150 155 160 ACC ATG ATG AAC GTA GCC AAC GGG CCC CAC CAC CCC AAT CCG CCC CCC 529 Thr Met Met Asn Val Ala Asn Gly Pro His His Pro Asn Pro Pro Pro 165 170 175 GAG AAC GTG CAG CTG GTG AAT CAA TAC GTA TCT AAA AAT GTC ATC TCT 577 ixir; ,ifi~L Glu Asn Val AGC GAG CAT Ser Glu His 195 Gin Leu Val Asn Gin 180 ATT GTT GAG AGA GAG Ile Val Glu Arg Glu 200 Val Ser Lys Asn Val Ile Ser 190 GCG GAG AGC TCT TTT TCC ACC AGT Ala Glu Ser Ser Phe Ser Thr Ser 205 625 CAC TAC His Tyr 210 ACT TCG ACA GCT CAT CAT Thr Ser Thr Ala His His 215 TCC ACT Ser Thr

AGT

Ser 225 CAC AGC TGG AGC AAT His Ser Trp Ser Asn 230 GGA CAC ACT GAA Gly His Thr Glu TCA TCC GTA GAA Ser Ser Val Glu 250 ACT GTC ACT CAG ACT CCC Thr Val Thr Gin Thr Pro 220 AGC ATC ATT TCG GAA AGC Ser Ile Ile Ser Glu Ser 235 240 AAC AGT AGG CAC AGC AGC Asn Ser Arg His Ser Ser 255 CAC TCT GTC ATC GTG His Ser Val Ile Val 245 CCG ACT GGG GGC CCG AGA GGA CGT CTC Pro Thr Gly Gly Pro Arg Gly Arg Leu 260 265 GAA TGT AAC AGC TTC CTC AGG CAT GCC Glu Cys Asn Ser Phe Leu Arg His Ala 275 280 AAT GGC TTG GGA GGC CCT CGT Asn Gly Leu Gly Gly Pro Arg 270 AGA GAA ACC CCT GAC TCC TAC Arg Glu Thr Pro Asp Ser Tyr 285 CGA GAC Arg Asp 290 TCT CCT CAT AGT GAA Ser Pro His Ser Glu 295 AGA CAT AAC CTT ATA GCT GAG CTA AGG Arg His Asn Leu Ile Ala Glu Leu Arg 300 913 AGA AAC AAG GCC CAC Arg Asn Lys Ala His 305 ACT CAT CTT AGA GCT Thr His Leu Arg Ala 325 AGA TCC AAA TGC ATG Arg Ser Lys Cys Met 310 TCT TCC ATT CCC CAT Ser Ser Ile Pro His 330 CAG ATC CAG CTT TCC GCA G1n ile Gin Leu Ser Ala 315 320 TGG GCT TCA TTC TCT AAG Trp Ala Ser Phe Ser Lys 335 1009 ACC CCT TGG CCT TTA GGA AGG TAT GTA Thr Pro Trp Pro Leu Gly Arg Tyr Val 340 345 CGT ATG TCA CCT GTA GAT TTC CAC ACG Arg Met Ser Pro Val Asp Phe His Thr 355 360 TCA GCA ATG ACC ACC CCG GCT Ser Ala Met Thr Thr Pro Ala 350 CCA AGC TCC CCC AAG TCA CCC Pro Ser Ser Pro Lys Ser Pro 365 1057 1105 CCT TCG GAA ATG TCC CCG CCC GTG TCC AGC ACG ACG GTC TCC ATG CCC 1153 Pro Ser 370 Glu Met Ser Pro Pro 375 Val Ser Ser Thr Thr 380 Val Ser Met Pro ATG GCG GTC AGT CCC Met Ala Val Ser Pro 390 TTC GTG GAA GAG GAG Phe Val Glu Glu Glu 395 CGG GAG AAG TAT GAC Arg Glu Lys Tyr Asp 410 GTG ACG CCA CCA CGG Val Thr Pro Pro Arg 405 TTC AAC TCG TTC CAC Phe Asn Ser Phe His 420 CCC AGC CCC TTG AGG Pro Ser Pro Leu Arg 435 AGA CCC CTG CTC CTT Arg Pro Leu Leu Leu 400 CAC CAC GCC CAG CAA His His Ala Gin Gin 415 AGC AAC AGC CTG CCC Ser Asn Ser Leu Pro 430 1201 1249 1297 1345 TGC AAC CCC GCG Cys Asn Pro Ala 425 ATA GTG GAG GAT Ile Val Glu Asp 440 CAT GAG His Glu GAG GAA TAT GAA ACG ACC CAG Glu Glu Tyr Glu Thr Thr Gin 445

S..

S* Sr

S..

S.

S

S S

S

*SSS

*5S5

S

GAG TAC Glu Tyr 450 GAA CCA GCT CAA Glu Pro Ala Gin GAG CCG GTT AAG AAA CTC ACC AAC AGC AGC Glu Pro Val Lys Lys Leu Thr Asn Ser Ser 455 460 AAG CCC AAT GGT CAC ATT GCC CAC AGG TTG Lys Pro Asn Gly His Ile Ala His Arg Leu 475 480 1393 1441

CGG

Arg 465 CGG GCC AAA AGA ACC Arg Ala Lys Arg Thr 470 GAA ATG GAC AAC AAC Glu Met Asp Asn Asn 485 ACA GGC GCT GAC AGC Thr Gly Ala Asp Ser 490 GTA GGA GAA GAT ACG Val Gly Glu Asp Thr 505 AGT AAC TCA GAG AGC GAA Ser Asn Ser Glu Ser Glu 495 CCT TTC CTG GCC ATA CAG Pro Phe Leu Ala Ile Gin 510 1489 1537 ACA GAG GAT Thr Glu Asp AAC CCC Asn Pro GAC AGC Asp Ser 530 CTG GCA GCC AGT CTC Leu Ala Ala Ser Leu 515 AGG ACT AAC CCA ACA Arg Thr Asn Pro Thr 535

GAG

Glu 520 GCG GCC CCT GCC TTC CGC CTG GTC Ala Ala Pro Ala Phe Arg Leu Val 525 1585 1633 GGC GGC TTC TCT CCG CAG GAA GAA TTG Gly Gly Phe Ser Pro Gin Glu Glu Leu 540

CAG

Gin 545 GCC AGG CTC TCC GGT Ala Arg Leu Ser Gly 550 GTA ATC GCT AAC CAA Val Ile Ala Asn Gln 555 GAC CCT ATC GCT GTC T Asp Pro Ile Ala Val 560 1682 AAAACCGAAA TACACCCATA GATTCACCTG TAAAACTTA TTTTATATAA TAAAGTATTC 1742

I

CACCTTAAAT TAAACAAAAA AA 1764 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 560 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Ser Glu Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu 1 5 10 Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala 20 25 Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr Ser Thr Ala Gly 35 40 SThr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val 50 55 Asn Gly Gly Asp Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 70 75 Tyr Leu Cys Lys Cys Gin Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu 85 90 Asn Val Pro Met Lys Val Gin Thr Gin Glu Lys Ala Glu Glu Leu Tyr 100 105 110 Gin Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val 115 120 125 Val Gly Ile Met Cys Val Val Val Tyr Cys Lys Thr Lys Lys Gin Arg S130 135 140 Lys Lys Leu His Asp Arg Leu Arg Gin Ser Leu Arg Ser Glu Arg Asn 145 150 155 160 Thr Met Met Asn Val Ala Asn Gly Pro His His Pro Asn Pro Pro Pro 165 170 175 Glu Asn Val Gin Leu Val Asn Gin Tyr Val Ser Lys Asn Val Ile Ser 180 185 190 Ser Glu His Ile Val Glu Arg Glu Ala Glu Ser Ser Phe Ser Thr Ser 195 200 205 His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gin Thr Pro 210 215 220 Ser His Ser Trp Ser Asn Gly His Thr Glu Ser Ile Ile Ser Glu Ser 225 230 235 240 His Ser Val Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser 245 250 255 '4 a..

a pp..

a a a 4 a Pro Glu Arg Arg 305 Thr Thr Arg Pro Ser 385 Val Phe Pro Glu Arg 465 Glu Thr Asn Asp Gln 545 Thr Cys Asp 290 Asn His Pro Met Ser 370 Met Thr Asn Ser Tyr 450 Arg Met Glu Pro Ser 530 Ala Gly Asn 275 Ser Lys Leu Trp Ser 355 Glu Ala Pro Ser Pro 435 Glu Ala Asp Asp Leu 515 Arg Arg Gly 260 Ser Pro Ala Arg Pro 340 Pro Met Val Pro Phe 420 Leu Pro Lys Asn Glu 500 Ala Thr Leu Pro Phe His His Ala 325 Leu Val Ser Ser Arg 405 His Arg Ala Arg Asn 485 Arg Ala Asn Ser Arg Leu Ser Arg 310 Ser Gly Asp Pro Pro 390 Leu Cys Ile Gin Thr 470 Thr Val Ser Pro Gly 550 Arg Glu 295 Ser Ser Arg Phe Pro 375 Phe Arg Asn Val Glu 455 Lys Gly Gly Leu Thr 535 Val His 280 Arg Lys Ile Tyr His 360 Val Val Glu Pro Glu 440 Pro Pro Ala Glu Glu 520 Gly Ile 265 Ala His Cys Pro Val 345 Thr Ser Glu Lys Ala 425 Asp Val Asn Asp Asp 505 Ala Gly Ala 270 Arg Asn Met His 330 Ser Pro Ser Glu Tyr 410 His Glu Lys Gly Ser 490 Thr Ala Phe Asn Glu Leu Gin 315 Trp Ala Ser Thr Glu 395 Asp Glu Glu Lys His 475 Ser Pro Pro Ser Gin 555 Thr Ile 300 Ile Ala Met Ser Thr 380 Arg His Ser Tyr Leu 460 Ile Asn Phe Ala Pro 540 Asp Pro 285 Ala Gin Ser Thr Pro 365 Val Pro His Asn Glu 445 Thr Ala Ser Leu Phe 525 Gin Pro Asp Glu Leu Phe Thr 350 Lys Ser Leu Ala Ser 430 Thr Asn His Glu Ala 510 Arg Glu Ile Ser Leu Ser Ser 335 Pro Ser Met Leu Gin 415 Leu Thr Ser Arg Ser 495 Ile Leu Glu Ala Tyr Arg Ala 320 Lys Ala Pro Pro Leu 400 Gin Pro Gin Ser Leu 480 Glu G1n Val Leu Val 560 Gly Arg Leu Asn Gly Leu Gly Gly Pro Arg INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 1140 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...840 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CAT CAA His Gin 1 GTG TGG GCG Val Trp Ala 5 GCG AAA GCC GGG GGC Ala Lys Ala Gly Gly 10 TTG AAG AAG GAC TCG CTG Leu Lys Lys Asp Ser Leu CTC ACC GTG CGC CTG GGC GCC TGG GGC Leu Thr Val Arg Leu Gly Ala Trp Gly 25 GGG CGC CTC AAG GAG GAC AGC AGG TAC Gly Arg Leu Lys Glu Asp Ser Arg Tyr 40 CAC CCC GCC TTC CCC TCC TGC His Pro Ala Phe Pro Ser Cys ATC TTC TTC ATG GAG CCC GAG Ile Phe Phe Met Glu Pro Glu 48 96 144 192

S

p t

S

*SSS

S

GCC AAC Ala Asn AGC AGC GGC GGG CCC Ser Ser Gly Gly Pro 55 GGC CGC CTT CCG Gly Arg Leu Pro AGC CTC CTT CCC CCC Ser Leu Leu Pro Pro CAG CCG GGT GCT GTG Gin Pro Gly Ala Val CGA GAC GGG CCG GAA CCT Arg Asp Gly Pro Glu Pro 70 CAA GAA GGA G1n Glu Gly CAA CGG TGC GCC Gin Arg Cys Ala TCT GTG GCA GGT Ser Val Ala Gly 100 TAC TCC TCT CTC Tyr Ser Ser Leu 115

TTG

Leu 85 CCT CCC CGC TTG AAA Pro Pro Arg Leu Lys 90 GAG ATG AAG AGT CAG GAG Glu Met Lys Ser Gin Glu 240 288 336 384 TCC AAA CTA GTG CTT Ser Lys Leu Val Leu 105 AAG TTC AAG TGG TTC Lys Phe Lys Trp Phe 120 CGG TGC GAG ACC AGT TCT GAA Arg Cys Glu Thr Ser Ser Glu 110 AAG AAT GGG AGT GAA TTA AGC Lys Asn Gly Ser Glu Leu Ser 125 CGA AAG Arg Lys 130 AAC AAA CCA GAA AAC Asn Lys Pro Glu Asn 135 ATC AAG ATA CAG AAA AGG CCG GGG AAG Ile Lys Ile Gin Lys Arg Pro Gly Lys 140 432

TCA

Ser 145 GAA CTT CGC ATT AGC Glu Leu Arg Ile Ser 150 AAA GCG TCA CTG GCT Lys Ala Ser Leu Ala 155 GAT TCT GGA GAA TAT Asp Ser Gly Glu Tyr 160 480 0 ATG TGC AAA GTG ATC AGC Met Cys Lys Val Ile Sen 165 AAA CIA GGA AAT GAC Lys Leu Gly Asn Asp 170 AGI GCC TCT GCC AAC Sen Ala Sen Ala Asn 175 ATC ACC AUT GTG Ile Thr Ile Va] 180 AGC CAT CTT GTC Sen His Leu Va] 195 GAG TCA AAC GCC Glu Sen Asn Ala ACA ICC ACA TCT ACA GCT GGG ACA Thn Sen Thr Sen Thn Ala Gly Thr 185 190 AAG GAG AAA ACT TTC TGT GIG AAT Lys Glu Lys Thr Phe Cys Va] Asn 205 528 576 624 AAG TGT GCA Lys Cys Ala

GAG

Glu 200 GGA GGC GAG Gly Gly Glu 210 TGC TTC ATG GTG Cys Phe Met Va] 215 AAA GAC CTT TCA AAT Lys Asp Leu Sen Asn CCC TCA AGA TAC Pro Sen Ang Tyr

TTG

Leu 225 TGC AAG TGC CAA CCT Cys Lys Cys Gin Pro 230 GGA TTC ACT GGA GCG Gly Phe Thn Gly Ala 235 AGA TGI ACT GAG AAT Arg Cys Thn Glu Asn 240

S.'

*5

*SSOP

S.

*5* 5' 5

S

*5O@

S

9 5

S

GTG CCC ATG Val Pro Met AAA GTC CAA ACC CAA GAA AAG Lys Val Gin Thr Gin Glu Lys 245 250 IGC CCA AAT GAG Cys Pro Asn Glu TTT ACT Phe Thn 255 GGT GAT CGC TGC CAA AAC TAC GTA ATG GCC Gly Asp Ang Cys Gin Asn Tyr Va] Met Ala 260 265 AGC TTC TAC AGT ACG TCC Sen Phe Tyn Sen Thr Sen 270 768 816 870 ACT CCC TTT Thr Pro Phe 275 CTG TCT CIG CCT GAA TAGCGCATCT CAGTCGGTGC CGCUCTTG Leu Sen Leu Pro Glu 280

TTGCCGCATC

GACTGCCTCI

CGIGACTAGT

ATCTTGAATT

GCCTTGAAAA

TCCCCICAGA TTCCNCCTAG GCCTGTCGCA TGAGAACATT GGGCTCIGAG CTACTCGTAG ACTGTGATAC GACATGATAG GTCAAAAAAA AAAAAAAAAA

AGCIAGATGC

AACACAAGCG

GTGCGTAAGG

TCCCTCTCAC

GTTTTACCAG

ATTGTATGAC

CTCCAGTGTT

CCAGTGCAAT

GTCTAACATT

TTCCTCTGTC

ICTGAAATG

GACAATAAAG

930 990 1050 1110 1140 INFORMATION FOR SEQ ID NO:52 SEQUENCE CHARACTERISTICS: LENGTH: 280 amino acids TYPE: amino acid SIRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein i FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: His Gin Val

S

See...

06

S

0@S 0*@@O Oe e Oe 000 OeS

S.

0 S. S 0500

@SSS

S

r

S

C

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S

SO..

1 Leu Gly Ala Ser Gin Ser Tyr Arg Ser 145 Met Ile Ser Gly Leu 225 Val Gly Thr Thr Arg Asn Arg Arg Val Ser Lys 130 Glu Cys Thr His Gly 210 Cys Pro Asp Pro Val Leu Ser Asp Cys Ala Ser 115 Asn Leu Lys Ile Leu 195 Glu Lys Met Arg Phe 275 Trp Ala Ala 5 Arg Leu Gly Lys Glu Asp Ser Gly Gly Gly Pro Glu 70 Ala Leu Pro Gly Ser Lys 100 Leu Lys Phe Lys Pro Glu Arg Ile Ser 150 Val Ile Ser 165 Val Glu Ser 180 Val Lys Cys Cys Phe Met Cys Gln Pro 230 Lys Val G1n 245 Cys Gin Asn 260 Leu Ser Leu Lys Ala Ser Pro 55 Pro Pro Leu Lys Asn 135 Lys Lys Asn Ala Val 215 Gly Thr Tyr Pro Ala Trp Arg 40 Gly G1n Arg Val Trp 120 Ile Ala Leu Ala Glu 200 Lys Phe Gln Val Glu 280 Gly Gly 25 Tyr Arg Glu Leu Leu 105 Phe Lys Ser Gly Thr 185 Lys Asp Thr Glu Met 265 Gly 10 His Ile Leu Gly Lys 90 Arg Lys Ile Leu Asn 170 Ser Glu Leu Gly Lys 250 Ala Leu Pro Phe Pro Gly 75 Glu Cys Asn Gin Ala 155 Asp Thr Lys Ser Ala 235 Cys Ser Lys Ala Phe Ser Gin Met Glu Gly Lys 140 Asp Ser Ser Thr Asn 220 Arg Pro Phe Lys Phe Met Leu Pro Lys Thr Ser 125 Arg Ser Ala Thr Phe 205 Pro Cys Asn Tyr Asp Pro Glu Leu Gly Ser Ser 110 Glu Pro Gly Ser Ala 190 Cys Ser Thr Glu Ser 270 Ser Ser Pro -Pro Ala Gin Ser Leu Gly Glu Ala 175 Gly Val Arg Glu Phe 255 Thr Leu Cys Glu Pro Val Glu Glu Ser Lys Tyr 160 Asn Thr Asn Tyr Asn 240 Thr Ser INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 1652 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 459.. .1181 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

AGTTTCCCCC

GGCGGCTGCC

TGCGAGCGCG

CCAGCGGCGC

GTCCCAGGTG

CTCCCCCCCA

AACTTTTCCC

CCCAACTTGT

CAGGCGATGC

CCGGACCGAG

GCCAGCAGGA

GCCCGGACCG

CGCCGCGCGC

GAAGCCGATC

CGGAACTCTG

GAGCGCGGGC

GCAGCGACAG

GCCACCCCGC

CACGTTGCGT

GCCTCGGCCC

CCAGCCCTCG

GGCTCGCGCG

CGGACGGTAA

GAGCGGACCG

GAGCGTGCGA

CCCCGCGCTC

GGICGCTGGC

GACCCAAACT

CAGGGCAGGA

TCGCCTCICC

CGGCGGGAAC

CCGGGACGGA

CCCGCCGGCG

CCGCCICCAC

TGTCGCGCGT

GCGGAGCGGC

CTCCTCGGGC

CGAGGACTCC

GCGCCCGCCA

ACAGGAGACG

TCCGGGGACA

CGCCITCGCC

120 180 240 300 360 420 GGGAGCCGTC CGCGCAGAGC GTGCACTTCT CGGGCGAG ATG TCG GAG CGC AGA Met Sen Giu Ang Arg GAA GGC AAA GGC AAG GGG AAG GGC GGC Glu Gly Lys Gly Lys Gly Lys Gly Gly AAG AAG Lys Lys GAC CGA GGC Asp Ang Gly TCC GGG Sen Gly AAG AAG CCC GTG Lys Lys Pro Val CGC TTG AAA GAG Arg Leu Lys Glu CCC GCG GCT GGC GGC CCG AGC CCA GCC TTG CCT CCC Pro Ala Ala Gly Gly Pro Sen Pro Ala Leu Pro Pro 30 521 569 617

ATG

Met AAG ATG CAG Lys Sen Gin 45 GAG TCT GTG GCA GGT TCC AAA CTA Glu Sen Val Ala Gly Sen Lys Leu GTG CTT Va] Leu CGG TGC GAG ACC AGT Arg Cys Glu Thr Sen 60 TCT GAA TAC ICC TCT CIC AAG TTC AAG Sen Glu Tyr Sen Sen Leu Lys Phe Lys TTC AAG AAT GGG AGT Phe Lys Asn Gly Sen GAA TTA AGC CGA AAG Glu Leu Sen Ang Lys 80 AAC AAA CCA CAA AAC Asn Lys Pro Gin Asn ATC AAG ATA CAG AAA AGG CCG GGG AAG TCA GAA CIT CGC ATT AGC Ile Lys Ile Gin Lys Ang Pro Gly Lys Sen Glu Leu Ang Ile Sen 95 -100 GCG TCA CTG GCT Ala Sen Leu Ala GAT TCT GGA GAA Asp Sen Giy Glu TAT ATG IGC AAA Tyr Met Cys Lys GTG ATC AGC AAA Val Ile Sen Lys 809 0 CTA GGA AAT Leu Gly Asn 120 GAC AGT GCC TCT GCC Asp Ser Ala Sen Ala 125 AAC ATC ACC ATT GTG GAG TCA AAC Asn Ile Thr Ile Val Glu Ser Asn 130 857 GAG ATC Glu Ile 135 ACC ACT GGC ATG CCA Thr Thr Gly Met Pro 140 GCC TCA ACT GAG Ala Sen Thr Glu ACA GCG TAT GTG TCT Thn Ala Tyr Va] Sen 145 905

TCA

Sen 150 GAG TCT CCC ATT AGA Glu Sen Pro Ile Arg 155 ATA ICA GTA TCA ACA Ile Sen Val Sen Thn 160 TCT ACA GCT GGG ACA Sen Thn Ala Giy Thn 175 GAA GGA ACA AAT Glu Giy Thn Asn

ACT

Thn 165 953 TCT TCA ICC ACA TCC Sen Sen Sen Thn Sen 170 AGC CAT CTT GTC AAG Sen His Leu Val Lys 180 1001 TGT GCA GAG AAG GAG AAA ACT TTC TGT Cys Ala Giu Lys Giu Lys Thn Phe Cys 185 190 ATG GTG AAA GAC CTT TCA AAT CCC TCA Met Vai Lys Asp Leu Sen Asn Pro Sen 200 205 GTG AAT GGA GGC GAG TGC TTC Val Asn Gly Gly Giu Cys Phe 195 AGA TAC TTG TGC AAG TGC CCA Arg Tyr Leu Cys Lys Cys Pro 210 1049 1097 AAT GAG Asn Glu 215 TTT ACT GGT GAT Phe Thn Gly Asp

CGC

Arg 220 TGC CAA AAC TAC Cys Gin Asn Tyr GTA ATG GCC AGC TTC Val Met Ala Sen Phe 225 GAA TAGGCGCATG Glu 1145

TAC

Tyn 230 AGI ACG TCC ACT CCC Sen Thn Sen Thn Pro 235 TTT CTG TCT CTG CCT Phe Leu Sen Leu Pro 240 1191

CTCAGTCGGT

GCGTTTTACC

CGATTGTATG

GGCTCCAGTG

ACCCAGTGCA

CGUCCACGG

TTAAGTTGTA

TCUTCTGAC

GCCGCTTTCT

AGGTCTAACA

ACTTCCTCTG

TTTCTGAAAT

ATGACAATAA

GACAGTCCCT

ACCAGTACAC

AAATAAACAG

TGTTGCCGCA

UGACTGCCT

TCCGTGACTA

TGATCTGAA

AGGCCTTGAA

CTTCTTTATA

ACTTGAAATG

AATAAAAAAA

TCTCCCCTCA

CTGCCTGICG

GTGGGCTCTG

TTACTGTGAT

AAGTCTCACT

AAATGACCCT

ATGGTAAGTT

AAAAAAAAAA

GATTCAACCT

CATGAGAACA

AGCTACTCGT

ACGACATGAT

TTTATTGAGA

ATCCTTGAAA

CGCTTCGGTT

AGAGCTAGAT

TTAACACAAG

AGGTGCGTAA

AGTCCCTCTC

AAATAAAAAT

AGGAGGTGTG

CAGAATGTGT

1251 1311 1371 1431 1491 1551 1611 1652 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 241 amino acids L TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: Met Ser Glu Arg Arg Glu Gly Lys Gly Lys Gly Lys Gly Gly Lys Lys 1 5 10 Asp Arg Gly Ser Gly Lys Lys Pro Val Pro Ala Ala Gly Gly Pro Ser 25 Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gin Glu Ser Val 40 Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser 55 Ser Leu Lys Phe Lys Trp Phe Lys Asn Gly Ser Glu Leu Ser Arg Lys 65 70 75 Asn Lys Pro Gln Asn Ile Lys Ile Gin Lys Arg Pro Gly Lys Ser Glu 90 Leu Arg Ile Ser Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys S.1 100 105 110 .ii: Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr S 115 120 125 Ile Val Glu Ser Asn Glu Ile Thr Thr Gly Met Pro Ala Ser Thr Glu 130 135 140 Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160 S Glu Gly Thr Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Ala Gly Thr 165 170 175 Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn 180 185 190 Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 195 200 205 Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gin Asn Tyr 210 215 220 Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro 225 230 235 240 Glu

Claims

1. A method of treating a mammal suffering from or susceptible to ischemia or heart attack, comprising administering to the mammal a therapeutically effective amount of a neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or a fragment or derivative of a neuregulin.

2. A method of treating a mammal suffering from or susceptible to effects of hypoxia or hypoglycemia, comprising administering to the mammal a therapeutically effective amount of a neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or a fragment or derivative of a neuregulin.

3. The method of Claim 1, wherein the neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or a fragment or derivative of a neuregulin is administered after the subject has suffered ischemia 15 or heart attack.

4. The method of Claim 3, wherein the neuregulin, or fragment or derivative of a neuregulin, or a nucleic acid encoding a neuregulin or a fragment or derivative of a neuregulin is administered to the subject for at least about two 2 weeks after the subject has suffered ischemia or heart attack.

5. A method of any one of Claims 1 to 4, wherein a neuregulin or a fragment or derivative thereof is administered to the mammal.

6. A method of Claim 5, wherein the neuregulin or fragment or derivative thereof comprises an amino acid sequence of the following formula: WYBAZCX 25 wherein WYBAZCX is composed of amino acid sequences that include one or more sequences shown in FIGS. 1 through 15 (which includes SEQ ID NOS:2, 4, 8, 9, 12, 14, 15, 18, 19, 22, 23, 26, 27, 30, 33, 35, 38, 39, 41, 44, 45 and 48), wherein W comprises the polypeptide segment F, or is absent; wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprise a polypeptide segment selected from the group consisting of C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D'H, C/D D' HL,C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL and C/D C/D' D' HKL, and preferably that either 10/01/02,mcl 1229.claims,21 e I I -22- a) at least one of F, Y, B, A, Z, C or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D HL, C/D C/D' D' HKL, C/D' H, C/D C/D' H or C/D C/D' HL.

7. The method of Claim 5, wherein the neuregulin or fragment or derivative thereof a) has at least one of F, Y, B, A, Z, C or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL; C/D C/D' D, C/D D H, C/D D' HL, C/D D' HKL, C/D C/D' D' C/D C/D' D HL, C/D C/D' D' HKL, C/D' H, C/D C/D' H or C/D C/D' HL.

8. The method of Claim 5, wherein the neuregulin or fragment or derivative thereof comprises FBA polypeptide segments, FEBA polypeptides 15 segments, EBA polypeptide segments, EBA' polypeptide segments or FEBA' polypeptide segments.

9. A method of Claim 5, wherein the neuregulin is encoded by a nucleic acid that comprises one of SEQ ID NOS:49, 51 and 53.

10. A method of Claim 5, wherein the neuregulin or fragment or derivative thereof is encoded by a nucleic acid that comprises a sequence that has at least about 70% sequence identity to one of SEQ ID NOS:49, 51 and 53.

11. A method of Claim 5, wherein the neuregulin or fragment or derivative thereof is encoded by a sequence that hybridizes to one of SEQ ID NOS:49, 51 or 53 under normal stringency conditions. 25 12. A method of Claim 5, wherein the neuregulin or fragment or derivative thereof is encoded by a sequence that hybridizes to one of SEQ ID NOS:49, 51 or 53 under high stringency conditions.

13. A method of Claim 5, wherein the neuregulin or fragment or derivative has at least about 70% sequence identity to SEQ ID NOS:50, 52 or 54.

14. A method of Claim 5, wherein the neuregulin or fragment or derivative thereof is encoded by a nucleic acid that comprises a sequence that has at least about 70% sequence identity to one of SEQ ID NO:20 (Figure SEQ ID NO:21 (Figure SEQ ID NO:24 (Figure SEQ ID NO:25 (Figure SEQ ID NO:28 (Figure or SEQ ID NO:29 (Figure 9). 10/01/02,mc11229.claims,22 i 1-- -23- A method of Claim 5, wherein the neuregulin or fragment or derivative thereof is encoded by a sequence that hybridizes to one of SEQ ID (Figure SEQ ID NO:21 (Figure SEQ ID NO:24 (Figure SEQ ID (Figure SEQ ID NO:28 (Figure or SEQ ID NO:29 (Figure 9) under normal stringency conditions.

16. A method of Claim 5, wherein the neuregulin or fragment or derivative comprises a sequence that has at least about 70% sequence identity to any of the peptide sequences shown in Figures 7, 8 or 9 of the drawings.

17. A method of Claim 5, wherein the neuregulin or fragment or derivative comprises a sequence that has at least about 80 percent homology to any of the peptide sequences shown in Figures 7, 8 or 9.

18. A method of Claim 5, wherein the neuregulin or fragment or derivative comprises a sequence that has at least about 90 percent homology to any of the peptide sequences shown in Figures 7, 8 or 9. 15 19. A method of Claim 5, wherein the neuregulin or fragment or Sderivative comprises a sequence that has at least about 95 percent homology to any of the peptide sequences shown in Figures 7, 8 or 9.

20. A method of Claim 5, wherein the neuregulin or fragment or derivative comprises a sequence that is shown in Figures 7, 8 or 9.

21. A method of any one of Claims 1 to 4, wherein a nucleic acid encoding a neuregulin or a fragment or derivative thereof is administered to the mammal.

22. A method of Claim 21, wherein the nucleic acid is SEQ ID NO:49, 51 or 53, or the complement thereof. 25 23. A method of Claim 21, wherein the nucleic acid or fragment or derivative thereof encodes a neuregulin or neuregulin fragment or derivative that comprises an amino acid sequence of the following formula: WYBAZCX wherein WYBAZCX is composed of amino acid sequences that include one or more sequences shown in FIGS. 1 through 15 (which includes SEQ ID NOS:2, 4, 8, 9, 12, 14, 15, 18, 19, 22, 23, 26, 27, 30, 33, 35, 38, 39, 41, 44, 45 and 48), wherein W comprises the polypeptide segment F, or is absent, wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the olypeptide segment G or is absent; and wherein X comprise a polypeptide 10/01/02,mcl 1229.claims,23 >2.Z -24- segment selected from the group consisting of C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL and C/D C/D' D' HKL.

24. The method of Claim 23, wherein the neuregulin or neuregulin fragment or derivative a) has at least one of F, Y, B, A, Z, C or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D H, C/D D' HL, C/D D' HKL, C/D C/D' D' H, C/D C/D' D HL, C/D C/D' D' HKL, C/D' H, C/D C/D' H or C/D C/D' HL. The method of Claim 23, wherein the neuregulin or neuregulin fragment or derivative comprises FBA polypeptide segments, FEBA polypeptides segments, EBA polypeptide segments, EBA' polypeptides segments or FEBA' polypeptide segments. 15 26. A method of Claim 21, wherein the nucleic acid comprises a sequence that hybridizes to SEQ ID NO:20 (Figure SEQ ID NO:21 (Figure 7); SEQ ID NO:24 (Figure SEQ ID NO:25 (Figure SEQ ID NO:28 (Figure or SEQ ID NO:29 (Figure 9) under normal stringency conditions. 2. 27. A method of Claim 21, wherein the nucleic acid comprises a sequence that hybridizes to SEQ ID NO:20 (Figure SEQ ID NO:21 (Figure 7); SEQ ID NO:24 (Figure SEQ ID NO:25 (Figure SEQ ID NO:28 (Figure or SEQ ID NO:29 (Figure 9) under high stringency conditions.

28. A method of Claim 21, wherein the nucleic acid comprises a sequence that has at least about 70 percent homology to any of the nucleic acid 25 sequences shown in Figures 7, 8 or 9.

29. A method of Claim 21, wherein the nucleic acid comprises a sequence that has at least about 80 percent homology to any of the nucleic acid sequences shown in Figures 7, 8 or 9. A method of Claim 21, wherein the nucleic acid comprises a sequence that has at least about 90 percent homology to any of the nucleic acid sequences shown in Figures 7, 8 or 9.

31. A method of Claim 21, wherein the nucleic acid comprises a sequence that has at least about 95 percent homology to any of the nucleic acid equences shown in Figures 7, 8 or 9. 10/01/02,mc11229.claims,24 i N- l

32. A method of Claim 21, wherein the nucleic acid comprises a sequence shown in Figures 7, 8 or 9.

33. A method of any one of Claims 1 to 32 wherein the administered neuregulin fragment or derivative, or the administered nucleic acid encodes a neuregulin fragment or derivative exhibits at least about a 10% reduction in infarct volume in an in vivo cerebral ischemia assay.

34. A method of any one of Claims 1 to 33 wherein the mammal is human. DATED this 10 th day of January, 2002 CeNes Pharmaceuticals, Inc. By their Patent Attorneys: CALLINAN LAWRIE 41 U b 4jc) e *oo *o S S S 10/01/02,mcl 1229.claims,25