AU769970B2 - Novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION Name of Applicant: Address of Applicant: Actual Inventor(s): Boston Heart Foundation, Inc.

139 Main Street, Cambridge, Massachusetts 02142, United States of America LEES, Ann, M.

LEES, Robert, S.

LAW, Simon, W.

ARJONA, Aibal, A.

Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: "Novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis" The following statement is a full description of this invention, including the best method of performing it known to us: Q:\OPERUEH\Oct\2471113 Div 297.doc 26/10/01 -1A- NOVEL LOW DENSITY LIPOPROTEIN BINDING PROTEINS AND THEIR USE IN DIAGNOSING AND TREATING ATHEROSCLEROSIS Field of the Invention This invention related to novel polypeptides (LBPs) which bind to low density lipoprotein (LDL), polynucleotides which encode these polypeptides, and treatments, diagnoses and therapeutic agents for atherosclerosis.

Background of the Invention Atherosclerosis is the principle cause of heart attacks and strokes. It has been reported that about 50% of all deaths in the United States, Europe and Japan are due to atherosclerosis.

Atherosclerotic lesions in the arterial wall characterize atherosclerosis. Cholesteryl esters (CE) are present in these atherosclerotic lesions. Low density lipoprotein (LDL) has been shown to be the major carrier of plasma CE, and has been implicated as the agent by which CE enter the atherosclerotic lesions.

15 Scattered groups of lipid-filled macrophages, called foam cells, are the first visible signs of atherosclerosis and are described as type I lesions. These macrophages are reported to contain CE derived from LDL. The macrophages recongize oxidized LDL, by not native LDL, and become foam cells by phagocytosing oxidized LDL. Larger, more organized collections of foam cells, fatty streaks, represent type II lesions. These lesions further develop into complex lesions called plaques, 20 which can result in impeding the flow of blood in the artery.

It is widely believed that accumulation of LDL in the artery depends on the presence of functionally modified endothelial cells in the arterial wall. It has been reported in animal models of atherosclerosis that LDL, both native LDL and methylated LDL, accumulates focally and irreversibly only at the edges of regenerating endothelial islands in aortic lesions, where 25 functionally modified endothelial cells are present, but not in the centers of these islands where .i :endothelial regeneration is completed. Similarly, LDL accumulates in human atherosclerotic lesions. The mechanism by which the LDL accumulates focally and irreversibly in arterial lesions has not heretofore been understood.

Summary of the Invention It is an object of the invention to provide polypeptides which bind to LDL.

It is yet another object of the invention to provide a method for determining if an animal -2is at risk for atherosclerosis.

It is yet another object of the invention to provide a method for evaluating an agent for use in treating atherosclerosis.

It is yet another object of the invention to provide a method for treating atherosclerosis.

Still another object of the invention is to utilize an LBP (low density lipoprotein binding protein) gene and/or polypeptide, or fragments, analogs and variants thereof, to aid in the treatment, diagnosis and/or identification of therapeutic agents for atherosclerosis.

In one aspect, the invention features an isolated polynucleotide comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; or a polynucleotide capable of hybridizing to and which is at least about 95% identical to any of the above polynucleotides and wherein the encoded polypeptide is capable of binding to LDL; or a biologically active fragment of any of the above polynucleotides wherein the encoded polypeptide is capable of binding to LDL.

In certain embodiments, the polynucleotide comprises the nucleic acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

Another aspect of the invention is an isolated polypeptide comprising a polypeptide having the amino acid sequence as set forth in SEQ ID NO:1, SEQ IDMNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; or S a polypeptide which is at least about 95% identical to any of the above polypeptides and wherein the polypeptide is capable of binding to LDL; or a biologically active fragment-of any of the above polypeptides wherein the fragment is capable of binding to LDL.

Another aspectof the invention is a method for determining if an animal is at risk for atherosclerosis. An animal is provided. An aspect of LBP metabolism or structure is evaluated in the animal. An abnormality in the aspect of LBP metabolism or structure is diagnostic of •being at risk for atherosclerosis.

Another aspect of the invention is a method for evaluating an agent for use in treating atherosclerosis. A test cell, cell-free system or animal is provided. An agent is provided. The agent is administered to the test cell, cell-free system or animal in a therapeutically effective amount. The effect of the agent on an aspect of LBP metabolism or structure is evaluated. A change in the aspect of LBP metabolism or structure is indicative of the usefulness of the agent in treating atherosclerosis.

Another aspect of the invention is a method for evaluating an agent for the ability to alter the binding ofLBP polypeptide to a binding molecule, native LDL, modified LDL, e.g., methylated LDL or oxidized LDL, or an arterial extracellular matrix structural component. An agent is provided. An LBP polypeptide is provided. A binding molecule is provided. The agent, LBP polypeptide and binding molecule are combined. The formation of a complex comprising the LBP polypeptide and binding molecule is detected. An alteration in the formation of the complex in the presence of the agent as compared to in the absence of the agent is indicative of the agent altering the binding of the LBP polypeptide to the binding molecule.

Another aspect of the invention is a method for evaluating an agent for the ability to bind to an LBP polypeptide. An agent is provided. An LBP polypeptide is provided. The agent is contacted with the LBP polypeptide. The ability of the agent to bind to the LBP polypeptide is evaluated.

Another aspect of the invention is a method for evaluating an agent for the ability to.bind to a nucleic acid encoding an LBP regulatory sequence. An agent is provided. A nucleic acid encoding an LBP regulatory sequence is provided. The agent is contacted with the nucleic acid.

The ability of the agent to bind to the nucleic acid is evaluated.

Another aspect of the invention is a method for treating atherosclerosis in an animal. An animal in need of treatment for atherosclerosis is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the animal in a therapeutically effective amount such that treatment of the atherosclerosis occurs. In certain embodiments, the agent is an LBP polypeptide, LBP-1, LBP-2 or LBP-3, or a biologically -active fragment or analog thereof. In certain embodiments, the agent is a polypeptide of no more than about 100, 50, 30, 20, 10, 5, 4, 3 or 2 amino acid residues in length. In certain embodiments, the agent is a polypeptide having an amino acid sequence that includes at least about 20%, 60%, 80%, 90%, 95% or 98% acidic amino acid residues.

Another aspect of the invention is a method for treating an animal at risk for atherosclerosis. An animal at risk for atherosclerosis is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the animal in a therapeutically effective amount such that treatment of the animal occurs.

Another aspect of the invention is a method for treating a cell having an abnormality in structure or metabolism of LBP. A cell having an abnormality in structure or metabolism of LBP is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the cell in a therapeutically effective amount such that treatment of the cell occurs.

Another aspect of the invention is a pharmaceutical composition for treating atherosclerosis in an animal comprising a therapeutically effective amount of an agent, the agent being capable of altering an aspect of LBP metabolism or structure in the animal so as to result in treatment of the atherosclerosis, and a pharmaceutically acceptable carrier.

Another aspect of the invention is a vaccine composition for treating atherosclerosis in an animal comprising a therapeutically effective amount of an agent, the agent being capable of altering an aspect of LBP metabolism or structure in the animal so as to result in.treatment of the atherosclerosis, and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for diagnosing atherosclerotic lesions in an animal. An animal is provided. A labeled agent capable of binding to LBP, LBP-1, LBP-2 or LBP-3, present in atherosclerotic lesions is provided. The labeled agent is administered tb the 15 animal under conditions which allow the labeled agent to interact with the LBP so as to result in labeled LBP. The localization or quantification of the labeled LBP is determined by imaging so as to diagnose the presence of atherosclerotic lesions in the animal.

S Another aspect of the invention is a method for immunizing an animal against an LBP, LBP-1, LBP-2 or LBP-3, or fragment or analog thereof. An animal having LDL is 20 provided. The LBP or fragment or analog thereof is administered to the animal so as to stimulate antibody production by the animal to the LBP or fragment or analog thereof such that binding of the LBP to the LDL is altered, decreased or increased.

.Another aspect of the invention is a method of making a fragment or analog of LBP polypeptide, the fragment or analog having the ability to bind to native LDL and to modified LDL, methylated LDL, oxidized LDL; acetylated LDL, or cyclohexanedione-treated LDL.

An LBP polypeptide is provided. The sequence of the LBP polypeptide is altered. The altered S S LBP polypeptide is tested for the ability to bind to modified LDL and native LDL.

Yet another-aspect of the invention is a method for isolating a cDNA encoding an LBP.

A cDNA library is provided. The cDNA library is screened for a cDNA encoding a polypeptide which binds to native LDL and modified LDL, methylated LDL or oxidized LDL. The cDNA which encodes the polypeptide is isolated, the cDNA encoding an LBP.

The above and other features, objects and advantages of the present invention will be better understood by a reading of the following specification in conjunction with the drawings.

Brief Description of the Drawings Fig. I depicts the amino acid sequence of rabbit LBP-I (SEQ ID NO:1). Differences in amino acids between rabbit and human LBP-1 are depicted in bold type.

Fig. 2 depicts the amino acid sequence of rabbit LBP-2 (SEQ ID NO:2). Differences in amino acids-between rabbit and human LBP-2 are depicted in bold type.

Fig. 3 depicts the amino acid sequence of amino acids 86 to 317 of rabbit LBPr2 (SEQ ID NO:3).

Fig. 4 depicts the amino acid sequence of amino acids 66 to 317 of rabbit LBP-2 (SEQ ID NO:4).

Fig. 5 depicts the amino acid sequence of rabbit LBP-3 (SEQ ID NO:5). Differences in amino acids between rabbit and human LBP-3 are depicted in bold type.

Fig. 6 depicts the amino acid sequence of human LBP-I (SEQ ID NO:6). Differences in amino acids between rabbit and human LBP-1 are depicted in bold type.

Fig. 7 depicts the amino acid sequence of human LBP-2 (SEQ ID NO:7). Differences in amino acids between rabbit and human LBP-2 are depicted in bold type.

Fig. 8 depicts the amino acid sequence of human LBP-3 (SEQ ID NO:8). Differences in amino acids between rabbit and human LBP-3 are depicted in bold type.

20 Fig. 9 depicts the amino acid sequence of amino acids 14 to 33 of human or rabbit LBP-1, called BHF-I (SEQ ID NO:9).

Fig. 10 depicts the cDNA sequence encoding rabbit LBP-1 (SEQ ID NO:10) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human LBP-1 are depicted in bold type.

25 Fig. 11 depicts the cDNA sequence encoding rabbit LBP-2 (SEQ ID NO: 11) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human LBP-2 are depicted in bold type.

Fig. 12 depicts the cDNA sequence 256 to 1617 of rabbit LBP-2 (SEQ ID NO:12) and the corresponding amino acid sequence.

Fig. 13 depicts the cDNA sequence 196 to 1617 of rabbit LBP-2 (SEQ ID NO:13) and the corresponding amino acid sequence.

Fig. 14 depicts the cDNKsequence encoding rabbit LBP-3 (SEQ ID NO: 14) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human LBP-3 are depicted in bold type.

Fig. 15 depicts the cDNA sequence encoding human LBP-1 (SEQ ID NO:15) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human LBP-1 are depicted in bold type.

Fig. 16 depicts the cDNA sequence encoding human LBP-2 (SEQ ID NO:16) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human LBP-2 are depicted in bold type.

Fig. 17 depicts the cDNA sequence encoding human LBP-3 (SEQ ID NO:17) and the corresponding amino acid sequence. Differences in amino acids between rabbit and human to LBP-3 are depicted in bold type.

Fig. 18 depicts the cDNA sequence encoding BHF-1 (SEQ ID NO:18).

Fig. 19 corresponds to the amino acid sequence of rabbit LBP-1 (top sequence) in alignment with the amino acid sequence of human LBP-I (bottom sequence).

Fig. 20 corresponds to the amino acid sequence of rabbit LBP-2 (top sequence) in 15 alignment with the amino acid sequence of human LBP-2 (bottom sequence).

Fig. 21 corresponds to the amino acid sequence of rabbit LBP-3 (top sequence) in alignment with the amino acid sequence of human LBP-3 (bottonsequence).

Detailed Description In accordance with aspects of the present invention, there are provided novel mature human and rabbit polypeptides, LBP-1, LBP-2 and LBP-3, and biologically active analogs and fragments thereof, and there are provided isolated polynucleotides which encode such polypeptides. LBP is an abbreviation for low density lipoprotein (LDL) binding protein. The terms polynucleotide, nucleotide and oligonucleotide are used interchangeably herein, and the terms polypeptides,-proteins and peptides are used interchangeably herein.

This invention provides for an isolated polynucleotide comprising a polynucleotide :encoding the polypeptide having the amino acid sequence of rabbit LBP-1 as set forth in Fig. 1 (SEQ ID NO:1); rabbit LBP-2 as set forth in Fig. 2 (SEQ ID NO:2); 86 to 317 of rabbit LBP-2 as set forth in Fig 3 (SEQ ID NO:3); 66 to 317 of rabbit LBP-2 as set forth in Fig. 4 (SEQ ID NO:4); rabbit LBP-3 as set forth in Fig. 5 (SEQ ID NO:5); human LBP-1 as set forth in Fig. 6 (SEQ ID NO:6); human LBP-2 as set forth in Fig. 7 (SEQ ID NO:7); human LBP-3 as set forth in Fig. 8 (SEQ ID NO:8); 14 to 33 of human or rabbit LBP-1, called BHF-1, as set forth in Fig. 9 (SEQ ID NO:9); a polynucleotide capable of hybridizing to and which is at least about identical, more preferably at least about 90% identical, more preferably yet at least about identical, and most preferably at least about 98% identical to any of the above polynucleotides, and wherein the encoded polypeptide is capable of binding to LDL; or a biologically active fragment of any of the above polynucleotides wherein the-encoded polypeptide is capable of binding to LDL.

This invention also includes an isolated polynucleotide comprising a polynucleotide encoding the polypeptide having amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) of human LBP-2 as set forth in Fig. 7 (SEQ ID NO:7); amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) of rabbit or human LBP-I as set forth in Fig. 1 (SEQ ID NO:1) and Fig. 6 (SEQ ID NO:6); amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of rabbit LBP-2 as set forth in Fig. 2 (SEQ ID NO:2); amino acid residues 96-110 (SEQ ID N.:29) of rabbit LBP-3 as set forth in Fig. 5 (SEQ ID NO:5); amino acid residues 53-59 (SEQ ID NO:41) of human LBP-3 as set forth in Fig. 8 (SEQ ID NO:8); a polynucleotide capable of hybridizing to and which is at least about 80% identical, more S. preferably at least about 90% identical, more preferably yet at least about 95% identical, and most preferably at least about 98% identical to any of the above polynucleotides, and wherein the encoded polypeptide is capable of binding to LDL; or a biologically active fragment of any of the above polynucleotides wherein the encoded polypeptide is capable of binding to LDL.

By a polynucleotide encoding a polypeptide is meant a polynucleotide which includes only coding sequence for the polypeptide, as well as a polynucleotide which includes additional coding and/or non-coding sequences. Thus, the polynucleotides which encode for the mature polypeptides of Figs. 1-9 (SEQ ID NOS:1-9) may include only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequences 5' and/or 3' of the coding sequence for the mature polypeptide. The polynucleotides of the invention are also meant to include polynucleotides in which the coding sequence for the mature polypeptide is fused in the same reading frame to a polynucleotide sequence which aids in expression and/or secretion of a polypeptide from a host cell, a leader sequence. The polynucleotides-are also meant to include polynucleotides in which the coding sequence is fused in frame to a marker sequence which, allows for purification of the polypeptide.

The polynucleotides of the present invention may be in the form of RNA, DNA or PNA, cRNA, cDNA, genomic DNA, or synthetic DNA, RNA or PNA. The DNA may be doublestranded or single stranded, and if single stranded may be the coding strand or non-coding (antisense) strand.

In preferred embodiments, the polynucleotide comprises the nucleic acid of rabbit LBPas set forth in Fig. 10 (SEQ ID NO:10); rabbit LBP-2 as set forth in Fig. 11 (SEQ ID NO: 11); nucleotide 256 to 1617 of rabbit LBP-2 as set forth in Fig. 12 (SEQ ID NO:12); nucleotide 196 to 1617 of rabbit LBP-2 as set forth in Fig. 13 (SEQ ID NO:13); rabbit LBP-3 as set forth in Fig.

14 (SEQ ID NO:14); human LBP-1 as set forth in Fig. 15 (SEQ ID NO:15); human LBP-2 as set -forth in Fig. 16 (SEQ ID NO:16); humanLBP-3 as set forth in Fig. 17 (SEQ ID NO-17); or nucleotide 97 to 156 of rabbit LBP-1 or nucleotide 157 to 216 of human LBP-1, (BHF-1), as set forth in Fig. 18 (SEQ ID NO: 18).

In other preferred embodiments, the polynucleotide comprises the nucleic acid as set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:42.

The coding sequence which encodes the mature polypeptide may be identical to the coding sequences shown in Figs. 10-18 (SEQ ID NOS:10-18) or SEQ ID NOS:30-40 or 42, or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides as the-NA of Figs. 18 (SEQ ID NOS:I0-18) and SEQ ID NOS: 30-40 and 42.

This invention also includes recombinant vectors comprising the polynucleotides described above. The vector can be, a plasmid, a viral particle or a phage. In certain embodiments, the recombinant vector is an expression vector. The vectors may also include various marker genes which are useful in identifying cells containing such vectors.

This invention also includes a cell comprising such a recombinant vector. The recombinant vectors described hereinican be introduced into a host cell, by transformation, transfection or infection.

This invention also includes a method for producing an LBP comprising culturing such a cell under conditions that permit expression of the LBP.

This invention also includes an isolated polypeptide comprising a polypeptide having the amino acid sequence as set forth in Fig. 1 (SEQ ID NO:1); Fig.2 (SEQ ID NO:2); Fig. 3 (SEQ ID NO:3); Fig. 4 (SEQ ID NO:4); Fig. 5 (SEQ ID NO:5); Fig. 6 (SEQ ID NO:6); Fig. 7 (SEQ ID NO:7); Fig. 8 (SEQ ID NOTyor Fig. 9 (SEQ ID NO:9); or a polypeptide which is at least about identical, more preferably at least about 90% identical, more preferably yet at least about 95% identical, and most preferably at least about 98% identical to the above polypeptides, and wherein said polypeptide is capable of binding to LDL; or a biologically active fragment of any of the above polypeptides wherein the fragment is capable of binding to LDL. Differences in amino acids between the rabbit and human LBP-1, LBP-2 and LBP-3 genes are depicted in bold type in the figures. The differences in the amino acid sequences between rabbit and human LBPlo 1, LBP-2 and LBP-3 are also specifically shown in Figs. 19, 20 and 21, respectively.

This invention also includes an isolated polypeptide comprising a polypeptide having amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) as set forth in Fig. 7 (SEQ ID NO:7); amino acid residues 14-43 (SEQ ID NO'23) or 38-43 (SEQ ID NO:24) as set forth in Fig. 1 (SEQ ID NO:1) and Fig. 6 (SEQ.ID NO:6); amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) as set forth in Fig. 2 (SEQ ID NO:2); amino acid residues 96-110 (SEQ ID NO:29) as set forth in Fig. 5 (SEQ ID NO:5); and amino acid residues 53-59 (SEQ ID NO:41) as set forth in Fig. 8 (SEQ ID NO:8); or a polypeptide which is at least about 80% identical, more preferably at least about 90% identical, more preferably yet at least about 95% identical, and most preferably at least about 98% identical to the above polypeptides, and wherein said polypeptide is capable of binding to LDL; or a biologically active fragment of any of the above polypeptides wherein the fragment is capable of binding to LDL.

The polypeptides of the invention are meant to include, a naturally purified product, a chemically synthesized product, and a recombinantly derived product.

The polypeptides can be used, to bind to LDL, thereby inhibiting formation of atherosclerotic plaques. The polypeptides can also be used, in gene therapy, by expression of such polypeptides in vivo. The polypeptides can also be used in pharmaceutical or vaccine compositions. The polypeptides can also be used as immunogens to produce antibodies thereto, which in turn, can be used as antagonists to the LBP polypeptides.

Without being bound by any theory, it is believed that the LBPs provide the mechanism -by which atherosclerosis is promoted through LDL oxidation. The LBPs are believed to be required in order for focal, irreversible LDL binding to occur at the arterial wall, and that such binding is a critical early event in atherosclerosis because it allows the time necessary for LDL to be changed from its native state to a fully oxidized state. Since oxidized, but not native, LDL is a foreign protein, macrophages ingest it, first becoming the foam cells of type I lesions, and subsequently forming the fatty streaks of type II lesions. This invention also includes a method for determining if an animal is at risk for atherosclerosis. An animal is provided. An aspect of LBP metabolism or structure is evaluated in the animal. An abnormality in the aspect of LBP metabolism or structure is diagnostic of being at risk for atherosclerosis.

By atherosclerosis is meant a disease or condition which comprises several stages which blend imperceptibly into each other, including irreversible binding of LDL, LDL oxidation, macrophage recruitment, blockage ofthe artery and tissue death (infarction).

By animal is meant human as well as non-human animals. Non-humaiianimals include, mammals, birds, reptiles, amphibians, fish, insects and protozoa. Preferably, the non-himan animal is a mammal, a rabbit, a rodent, a mouse, rat or guinea pig, a primate, a monkey, or a pig. An animal also includes transgenic non-human animals. The term transgenic animal is meant to include an animal that has gained new genetic information from the S* introduction of foreign DNA, partly or entirely heterologous DNA, into the DNA of its cells; or introduction of a lesion, an in itr induced mutation, a deletion or other chromosomal rearrangement into the DNA of its cells; or introduction of homologous DNA into the DNA of its cells in such a way as to alter the genome of the cell into which the DNA is inserted, it is inserted at a location which differs from that of the natural gene or its insertion "results in a knockout or replacement of the homologous host gene or results in altered and/or regulatable expression and/or metabolism of the gene. The animal may include a transgene in all of its cells including germ line cells, or in only one or some of its cells. Transgenic animals of the invention can serve as a model for studying atherosclerosis or for evaluating agents to treat atherosclerosis.

SI, In certain embodiments, the determination for being at risk for atherosclerosis is done in a prenatal animal.

By LBP is meant a low density lipoprotein (LDL) binding protein which is capable of binding LDL and methylated LDL. By methylated LDL is meant that about 50% to about of the lysine residues of LDL have a methyl group chemically attached. Methylated LDL is not recognized by previously reported cell surface receptors. See. g Weisgraber et al., J. Biol.

Chem. 253:9053-9062 (1978). In certain embodiments, the LBP is also capable of binding oxidized LDL. In certain preferred embodiments, the binding of LDL to an LBP is irreversible.

In certain preferred embodiments, the LBP does not transport the LDL to any intracellular compartment. Examples of LBPs are LBP-1, LBP-2 and LBP-3 described herein.

By LBP metabolism is meant any aspect of the production, release, expression, function, action, interaction or regulation of LBP. The-metabolism of LBP includes modifications, e.g., covalent or non-covalent modifications, of LBP polypeptide. The metabolism of LBP includes modifications, covalent or non-covalent modifications, that LBP induces in other substances. The metabolism of LBP also includes changes in the distribution of LBP polypeptide, and changes LBP induces in the distribution of other substances.

Any aspect of LBP metabolism can be evaluated. The methods used are standard techniques known to those skilled in the art and can be found in standard references, e.g., Ausubel et al., ed., Current Protocols in Mol. Biology, New York: John Wiley Sons, 1990;- Kriegler, ed., Gene Transfer and Expression, Stockton Press, New York, NY, 1989; pDisplay gene expression system (Invitrogen, Carlsbad, CA); Preferred examples of LBP metabolism that o: can be evaluated include the binding activity of LBP polypeptide to a binding molecule, e.g., LDL; the transactivation activity of LBP polypeptide on a target gene; the level of LBP protein; the level of LBP mRNA; the level of LBP modifications, phosphorylation, glycosylation or acylation; or the effect of LBP expression on transfected mammalian cell binding of LDL.

By binding molecule is meant any molecule to which LBP can bind, a nucleic acid, a DNA regulatory region, a protein, LDL, a metabolite, a peptide mimetic, a nonpeptide mimetic, an antibody,-or any other type of ligand. In certain preferred-embodiments, the aspect of LBP metabolism that is evaluated is the ability of LBP to bind to native LDL and/or methylated LDL and/or oxidized LDL. Binding to LDL can be shown, by antibodies against LDL, affinity chromatography, affinity coelectrophoresis (ACE) assays, or ELISA assays. See Examples. In other embodiments, it is the ability of LBP to bind to an arterial extracellular matrix stuctural component that is evaluated. Examples of such components include proteoglycans, chondroitin sulfate proteoglycans and heparin sulfate proteoglycans; elastin; collagen; fibronectin; vitronectin; integrins; and related extracellular matrix molecules.

Binding to arterial extracellular matrix structural components can be shown by standard methods known to those skilled in the art, by ELISA assays. Primary antibodies to the LBP are then added, followed by an enzyme-conjugated secondary antibody to the primary antibody, which produces a stable color in the presence of an appropriate substrate, and color development on the plates is measured in a microtiter plate reader.

Transactivation of a target gene by LBP can be determined, in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, Pgalacto-sidase or luciferase, and co-transfected with an LBP expression vector. Such evaluations can be done in 3it or in vivo. Levels of LBP protein, mRNA or phosphorylation, can be measured, in a sample, a tissue sample, arterial wall, by standard methods known to those skilled in the art.

In certain embodiments, an aspect of LBP structure is evaluated, LBP gene structure or LBP protein structure. For example, primary, secondary or tertiary structures can be evaluated. For example, the DNA sequence of the gene is determined and/or the amino acid sequence of the protein is determined. Standard cloning and sequencing methods can be used as are known to those skilled in the art. In certain embodiments, the binding activity of an antisense nucleic acid with the cellular LBP mRNA and/or genomic DNA is determined using standard 15 methods known to those skilled in the art so as to detect the presence or absence of the target mRNA or DNA sequences to which the antisense nucleic acid would normally specifically bind.

The risk for atherosclerosis that is determined can be a reduced risk or an increased risk as compared to a normal animal. For example, an abnormality whichwould give a reduced risk is an inactive LBP polypeptide. An abnormality which would give an increased risk would be, an LBP polypeptide that has higher activity, LDL binding activity, than native LBP 0 polypeptide.

The invention also includes a method for evaluating an agent for use in treating atherosclerosis. A test cell, cell-free system or animal is provided. An agent is provided. The agent is administered to the test cell, cell-free system or animal in a therapeutically effective amount The effect of the agent on an aspect of LBP metabolism or structure is evaluated. A change in the aspect of LBP metabolism or structure is indicative of the usefulness of the agent in treating atherosclerosis.

In certain embodiments, the method employs two phases for evaluating an agent for use in treating atherosclerosis, an initial in yi phase and then an in vivo phase. The agentisadministered to the test cell or cell-free system in vito, and if a change in an aspect of LBP metabolism occurs, then the agent is further administered to a test animal in a therapeutically effective amount and evaluated in viYv for an effect of the agent on an aspect of LBP -13metabolism.

By cell is meant a cell or a group of cells, or a cell that is part of an animal. The cell can be a human or non-human cell. Cell is also meant to include a transgenic cell. The cell can be obtained, from a culture or from an animal. Animals are meant to include, natural animals and non-human transgenic animals. In certain embodiments, the transgenic cell or nonhuman transgenic animal has an LBP transgene, or fragment or analog thereof. In certain embodiments, the transgenic cell or non-human transgenic animal has a knockout for the LBP gene.

The test cell, cell-free system or animal can have a wild type pattern or a non-wild type pattern of LBP metabolism. A non-wild type pattern of LBP metabolism can result,.e.g., from under-expression, over-expression, no expression, or a temporal, site or distribution change.

Such a non-wild type pattern can result, from one or more mutations in the LBP gene, in a binding molecule gene, a regulatory gene, or in any other gene which directly or indirectly affects LBP metabolism. A mutation is meant to include, an alteration, in gross or fine structure, in a nucleic acid. Examples include single base pair alterations, missense or nonsense mutations, frameshifts, deletions, insertions and translocations. Mutations can be dominant or recessive. Mutations can be homozygous or heterozygous. Preferably, an aspect of LBP-1, LBP-2 or LBP-3 metabolism is evaluated.

An agent is meant to include, any substance, an anti-atherosclerosis drug. The agent of this invention preferably can change an aspect of LBP metabolism. Such change can be the result of any of a variety of events, including, preventingor reducing interaction between LBP and a binding molecule, LDL or an arterial extracellular matrix structural component; inactivating LBP and/or the binding molecule, by cleavage or other modification; altering the affinity of LBP and the binding molecule for each other, diluting out LBP and/or the binding molecule; preventing expression of LBP and/or the binding molecule; reducing synthesis of LBP and/or the binding molecule; synthesizing an abnormal LBP and/or binding molecule; synthesizing an alternatively spliced LBP and/or binding molecule; preventing or reducing proper conformational folding of LBP and/or the binding molecule; modulating the binding properties of LBP and/or the binding molecule; interfering with signals that are required to activate or deactivate LBP and/or the binding molecule; activating or deactivating LBP and/or the binding molecule in such a way as to prevent binding; or interfering with other receptors, ligands or other molecules which are required for the normal synthesis or functioning of LBP and/or the binding molecule. For example, the agent can block the binding site on LDL for LBPs expressed focally in the arterial wall extracellular matrix, or it could block the binding site on an LBP for LDL, or it could be bifunctional, it could block both binding sites.

Examples of agents include LBP polypeptide, LBP-1, LBP-2 or LBP-3, or a biologically active fragment or analog thereof; a nucleic acid encoding LBP polypeptide or a biologically active fragment or analog thereof; a nucleic acid encoding an LBP regulatory sequence or a biologically active fragment or analog thereof; a binding molecule for LBP polypeptide; a binding molecule for LBP nucleic acid, the LBP nucleic acid being, a nucleic acid comprising a regulatory region for LBP or a nucleic acid comprising a structural region for LBP or a biologically active fragment of LBP; an antisense nucleic acid; a mimetic of LBP or a binding molecule; an antibody for LBP or a binding molecule; a metabolite; or an inhibitory carbohydrate or glycoprotein. In certain embodiments, the agent is an antagonist, agonist or super agonist.

Knowledge of theexistence-of the sequence of the LBPs allows a search for natural or 15 artificial ligands to regulate LDL levels in the treatment of atherosclerosis. In certain embodiments, the agent is a natural ligand for LBP. In certain embodiments, the agent is an artificial ligand for LBP.

By analog is meant a compound that differs from naturally occurring LBP in amino acid sequence or in ways that do not involve sequence, or both. Analogs of the invention generally exhibit at least about 80% homology, preferably at least about 90% homology, more preferably yet at least about 95% homology, and most preferably at least about 98% homology, with substantially the entire sequence of a naturally occurring LBP sequence, preferably with a segment of about 100 amino acid residues, more preferably with a segment of about 50 amino acid residues, more preferably yet with a segment of about 30 amino acid residues, more 25 preferably yet with a segment of about 20 amino acid residues, more preferably yet with a *0 segment of about 10 amino acid residues, more preferably yet with a segment of about 5 amino acid residues, more preferably yet with a segment of about 4 amino acid residues, more preferably yet with a segment of about 3 amino acid residues, and most preferably with a segment of about 2 amino acid residues. Non-sequence modifications include, in vivo or in vio chemical derivatizations of LBP. Non-sequence modifications include, changes in phosphorylation, acetylation, methylation, carboxylation, or glycosylation. Methods for making such modifications are known to those skilled in the art. For example, phosphorylation can be modified by exposing LBP to phosphorylation-altering enzymes, kinases or phosphatases.

Preferred analogs include LBP or biologically active fragments thereof whose sequences differ frm the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish Li3P biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamnic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other examples of conservative. substitutions are shown in Table 1.

TableI CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid Code Replace with any of Alanine A D-Ala, Giy, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-bomo-Arg, Met, le, D- D-lle, Om, D-Orn, L-NMMA, L-NAME Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin, D=Gin Aspartic Acid D D-Asp, D-Asn, Asn, Giu, D-Glu, Gin, D-Gin Cysteine C D-Cys, S-Me-Cys, Met, D-Met, TVr, D-Thr Glutamine Q.D-Gin, Asn, D-Asn, Giu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gin 20 Glycine G Ala, D-Ala, Pro, D-Pro, P)-Ala Acp Histidine H D-His Isoleucine I D-le, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Vai, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, D-I1e, Omn, D-Orn 25 -Methionine M D-Met, S-Me-Cys, le, D-lle, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, TyrD-Thr, L-Dopa, His, D-His, Trp, D-Trp, Transor 5-phenyiproline, cis-3,4, or Proline P D-Pro, L-I-thioazoiidine-4-carboxylic acid, D-or L- 1 _______oxazolidine-4-carboxyiic acid Serine S D-Ser, Thr, D-Thr, alio-Thr, Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Th1r, Scr, D-Ser, allo-Thr, Met, Met(O), D-Met(O), Val, D-Val -Tryptophan W D-Trp, Phe, D-Pbe, Tyr, D-Tyr Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, le, D-Iie, Met, D-Met

S

S

S

S

*SS. 00.

0 -16- Amino acid sequence variants of a protein can be prepared by any of a variety of methods known to those skilled in the art. For example, random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein can be used, PCR mutagenesis (using, reduced Taq polymerase fidelity to introduce random mutations into a cloned fragment of DNA; Leung et al., BioTechnique 1:11-15 (1989)), or saturation mutagenesis (by, e.g, chemical treatment or irradiation of single-stranded DNA in vito, and synthesis of a complementary DNA strand; Mayers et al., Science 229:242 (1985)). Random mutagenesis can also be accomplished by, degenerate oligonucleotide generation (using, an automatic DNA synthesizer to chemically synthesize degenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A.G. Walton, Amsterdam: Elsevier, pp. 273-289 (1981)). Non-random or directed mutagenesis can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, by substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (ii) deleting the target residue, (iii) inserting residues of the same or a different class adjacent to the located site, or (iv) combinations of the above. For example, analogs can be made by in yit DNA sequence modifications of the sequences of Figs.

10-18 (SEQ ID NOS:-O-18). For example, in vit mutagenesis can be used to convert any of 20 these.DNA sequences into a sequence which encodes an analog in which one or more amino acid residues has undergone a replacement, a conservative replacement as described in Table 1.

Methods for identifying desirable mutations include, alanine scanning.mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman et al., DNA 2:183 (1983)); cassette mutagenesis (Wells et al., Gene 34:315 (1985)), combinatorial mutagenesis, and phage display libraries (Ladner et al., PCT International Appln.

No. W088/06630). The LBP analogs can be tested, for their ability to bind to LDL and/or 'to an arterial extracellular matrix component, as described herein.

Other analogs within the invention include, those with modifications which increase S: peptide stability. Such analogs may contain, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are, analogs that include residues other than naturally occurring L-amino acids, D-amino acids or non-naturally occurring or synthetic amino acids, P or y amino acids; and cyclic analogs.

Analogs are also meant to include peptides in which structural modifications have been introduced into the peptide backbone so as to make the peptide non-hydrolyzable. Such peptides are particularly useful for oral administration, as they are not digested. Peptide backbone modifications include, modifications of the amide nitrogen, the a-carbon, the amide carbonyl, or the amide bond, and modifications involving extensions, deletions or backbone crosslinks.For example, the backbone can be modified by substitution of a sulfoxide for the carbonyl, by reversing the peptide bond, or by substituting a methylene for the carbonyl group.

Such modifications can be made by standard procedures known to those skilled in the art. See, eg, Spatola, "Peptide Backbone Modifications: A Structure-Activity Analysis of Peptides o0 Containing Amide Bond Surrogates, Conformational Constraints, and Related Backbone- Replacements," in Chemistry and Biochemistry of Amino AEids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein Marcel Dekker, Inc., New York (1983).

An analog is also meant to include polypeptides in which one or more of the amino acid residues include a substituent group, or polypeptides which are fused with another compound, a compound to increase the half-life of the polypeptide, polyethylene glycol._ By fragment is meant some portion of the naturally occurring LBP polypeptide.

Preferably, the fragment is at least about 100 amino acid residues, more preferably at least about 50 amino acid residues, more preferably yet at least about 30 amino acid residues, more preferably yet at least about 20 amino acid residues, more preferably yet at least about 5 amino 20 acid residues, more preferably yet at least about 4 amino acid residues, more preferably yet at least about 3 amino acid residues, and most preferably at least about 2 amino acid residues in length. Fragments include, truncated secreted forms, proteolytic fragments, splicing fragments, other fragments, and chimeric constructs between at least a portion of the relevant gene, LBP-1, LBP-2 or LBP-3, and another molecule. Fragments of LBP can be generated by methods known to those skilled in the art. In certain embodiments, the fragment is biologically active. The ability of a candidate fragment to exhibit a biological activity of LBP can be assessed by methods known to those skilled in the art. For example, LBP fragments can be tested for their ability to bind to LDL and/or to.an arterial extracellular matrix structural component, as described herein. Also included are LBP fragments containing residues that are S" 30 not required for biological activity of the fragment or that result from alternative mRNA splicing or alternative protein processing events.

Fragments of a protein can be produced by any of a variety of methods known to those skilled in the art, recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated, by random shearing, restriction digestion or a combination of the above-discussed methods. For example, fragments of LBP can be made by expressing LBP DNA which has been manipulated in itro to encode the desired fragment, by-restriction digestion of any of the DNA lo sequences of Figs. 10-18 (SEQ ID NOS:10-18).

Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

An LBP or a biologically active fragment or analog thereof, or a binding molecule or a biologically active fragment or analog thereof, can, compete with its cognate molecule for the binding site on the complementary molecule, and thereby reduceor eliminate binding between LBP and the cellular binding molecule. LBP or a binding molecule can be obtained, from purification or secretion of naturally occurring LBP or binding molecule, from 20 recombinant LBP or binding molecule, or from synthesized LBP or binding molecule.

Therefore, methods for generating analogs and fragments andtesting them for activity are known to those skilled in the art.

An agent can also be a nucleic acid used as an antisense molecule. Antisense therapy is meant to include, administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize, bind, under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an LBP polypeptide, or mutant thereof, so as to inhibit expression of the encoded protein, by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

In certain embodiments, the antisense construct binds to a naturally-occurring sequence of an LBP gene which, is involved in expression of the gene. These sequences include, e.g., promoter, start codons, stop-codons, and RNA polymerase binding sites.

In other embodiments, the antisense construct binds to a nucleotide sequence which is not present in the wild type gene. For example, the antisense construct can bind to a region of an LBP gene which contains an insertion of an exogenous, non-wild type sequence. Alternatively, the antisense construct can bind to a region of an LBP gene which has undergone a deletion, thereby bringing two regions of the gene together which are not normally positioned together and which, together, create a non-wild type sequence. When administered in vivo to a subject, antisense constructs which bind to non-wild type sequences provide the advantage of inhibiting the expression of a mutant LBP gene, without inhibiting expression of any wild type LBP gene.

An antisense construct of the present invention can be delivered, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a iunique portion of the cellular mRNA which encodes an LBP polypeptide. An alternative is that .the antisense construct is an oligonucleotide which is generated ex viv and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA (duplexing) and/or genomic sequences (triplexing) of an LBP gene. Such oligonucleotides are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g.

exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate, phosphorodithioates and methylphosphonate analogs of DNA and peptide nucleic acids (PNA).

(See also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches 20 to constructing oligomers useful in antisense therapy have been reviewed. (Se. g, Van der Krol et aL, Biotechniques 6:958-976, (1988); Stein et al., Cancer Res. 48:2659-2668 (1988)).

By mimetic is meant a molecule which resembles in shape and/or charge distribution LBP or a binding molecule. The mimetic can be a peptide or a non-peptide. Mimetics can act as therapeutic agents because they can, competitively inhibit binding of LBP to a binding molecule. By employing, scanning mutagenesis, alanine scanning mutagenesis, linker Sscaiining mutagenesis or saturation mutagenesis, to map the amino acid residues of-a particular .LBP polypeptide involved in binding a binding molecule, peptide mimetics, diazepine or isoquinoline derivatives, can be generated which mimic those residues in binding to a binding molecule, and which therefore can inhibit binding of the LBP to a binding molecule and thereby interfere with the function of LBP. Non-hydrolyzable peptide analogs of suchresidues can be Sgenerated using, benzodiazepine (see, Freidinger et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); azepine (see, e,, Huffman et al., in Peptides: Chemistry and Biology, G.R. Marshalled., ESCOM Publisher: Leiden, Netherlands (1988)); substituted gamma lactam rings (ee, eg,, Garvey et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); keto-methylene pseudopeptides (ee, Ewenson et al., J. Med. Chem. 29:295 (1986); Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium)_Pierce Chemical Co. Rockland, IL (1985)); p-turn dipeptide cores (ee, Nagai et al., Tetrahedron Lett. 26:647 (1985); Sato et al., J. Chem. Soc. Perkin Trans. 1:1231 (1986)); or P-aminoalcohols (ee, Gordon et al., Biochem. Biophys. Res. Commun. 126:419 (1985); Dann et al., Biochem. Biophys. Res. Commun. 134:71 (1986)).

Antibodies are meant to include antibodies against any moiety that directly or indirectly affects LBP metabolism. The antibodies can be directed agaiit, LBP or a binding molecule, or a subunit or fragment thereof. For example, antibodies include anti-LBP-1, LBP-2 or LBP-3 antibodies; and anti-binding molecule antibodies. Antibody fragments are meant to include, Fab fragments, Fab' fragments,. F(ab') fragments, F(v) fragments, heavy chain' monomers, heavy chain dimers, heavy chain trimers, light chain monomers, light chain dimers, light chain trimers, dimers consisting of one heavy and one light-chain, and peptides that mimic the activity of the anti-LBP or anti-binding molecule antibodies. For example, Fab 2 fragments of the inhibitory antibody can be generated through, enzymatic cleavage. Both polyclonal and monoclonal antibodies can be used in this invention. Preferably, monoclonal antibodies are 20 used. Natural antibodies, recombinant antibodies or chimeric-antibodies, humanized antibodies, are included in this invention. Preferably, humanized antibodies are used when the subject is a human. Most preferably, the antibodies have a constant region derived from a human antibody and a variable region derived from an inhibitory mouse monoclonal antibody.

Production of polyclonal antibodies to LBP is described in Example 6. Monoclonal and humanized antibodies are generated by standard methods known to those skilled in the art.

Monoclonal antibodies can be-produced, by any technique which provides antibodies Sproduced by continuous cell lines cultures. Examples include the hybridoma technique (Kohler and Milstein, Nature 256:495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, A.R. Liss, Inc., pp. 77-96 (1985)). Preferably, humanized antibodies are raised through -conventional production and harvesting techniques (Berkower, Curr. Opin. Biotechnol. 7:622- 628 (1996); Ramharayan and Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)). In certain preferred embodiments, the antibodies are raised against the LBP, preferably the LDL-binding site, and the Fab fragments produced. These antibodies, or fragments derived therefrom, can be used, to block the LDL-binding sites on the LBP molecules.

Agents also include inhibitors of a molecule that are required for synthesis, post-translational modification, or functioning of LBP and/or a binding molecule, or activators of a molecule that inhibits the synthesis or functioning of LBP and/or the binding molecule. Agents include, cytokines, chemokines, growth factors, hormones, signaling components, kinases, phosphatases, homeobox proteins, transcription factors, editing factors, translation factors and post-translation factors or enzymes. Agents are also meant to include ionizing radiation, non-ionizing radiation, ultrasound and-toxic agents which caniC.g., at least partially inactivate or destroy LBP and/or the binding molecule.

An agent is also meant to include an agent which is not entirely LBP specific. For example, an agent may alter other genes or proteins related to arterial plaque formation. Such overlapping specificity may provide additional therapeutic advantage.

The invention also includes the agent so identified as being useful in treating atherosclerosis.

The invention also includes a method for evaluating an agent for the ability to alter the S.i. binding of LBP polypeptide to a binding molecule. An agent is provided. An LBP polypeptide 20 is provided. A binding molecule is provided. The agent, LBP polypeptide and binding molecule are combined. The formation of a complex comprising the LBP polypeptide and binding molecule is detected. An alteration in the formation of the complex in the presence of the agent as compared to in the absence of the agent is indicative of the agent altering the binding of the LBP polypeptide to the binding molecule.

In preferred embodiments, the LBP polypeptide is LBP-1, LBP-2 or LBP-3. -Examples of a binding molecule include native LDL, modified LDL, methylated LDL or oxidized LDL, and arterial extracellular matrix structural components.

Altering the binding includes, inhibiting or promoting the binding. The efficacy of the agent can be assessed, by generating dose response curves from data obtaiied using 30 various concentrations of the agent. Methods for determining formation of a complex are standard and are known to those skilled in the art, affinity coelectrophoresis (ACE) assays or ELISA assays as described herein.

-22- The invention also includes the agent so identified as being able to alter the binding of an LBP polypeptide to a binding molecule.

The invention also includes a method for evaluating an agent for the ability to bind to an LBP polypeptide. An agent is provided. An LBP polypeptide is provided. The agent is contacted with the LBP polypeptide. The ability of the agent to bind to the LBP polypeptide is evaluated. Preferably, the LBP polypeptide is LBP-1, LBP-2 or LBP-3. Binding can be determined, by measuring formation of a complex by standard methods known to those skilled in the art, affinity coelectrophoresis (ACE) assays or ELISA assays as described herein.

The invention also includes the agent so identified as being able to bind to LBP S-polypeptide.

The invention also includes a method for evaluating an agent for the ability to bind to a nucleic acid encoding an LBP regulatory sequence. An agent is provided. A nucleic acid encoding an LBP regulatory sequence is provided. The agent is contacted with the nucleic acid.

The ability of the agent to bind to the nucleic acid is evaluated. Preferably, the LBP regulatory sequence is an LBP-1, LBP-2 or LBP-3 regulatory sequence. Binding can be determined, e.g., by measuring formation of a complex by standard methods known to those skilled in the art, e.g., DNA mobility shift assays, DNase I footprint analysis (Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley Sons, New York, NY, (1989)).

The invention also includes the agent so identified as being able to bind to a nucleic acid encoding an LBP regulatory.sequence The invention also includes a method for treating atherosclerosis in an animal. An animal in need of treatment for atherosclerosis is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the animal in a therapeutically effective amount such that treatment of the atherosclerosis occurs.

In certain preferred embodiments, the agent is an LBP polypeptide, LBP-1, LBP-2 or LBP-3, or a biologically active fragment or analog thereof. The agent can be, the polypeptide as set forth in SEQD NOS: 1-9. Preferably, the agent is a polypeptide of no more than about 100 amino acid residues in length, more preferably of no more than about 50 amino acid residues, more preferably yet of no more than about 30 amino acid residues, more preferably yet of no more than about 20 amino acid residues, more preferably yet of no more than about amino acid residues, more preferably yet of no more than about 5 amino acid residues, more -23preferably yet of no more than about 4 amino acid residues, more preferably yet of no more than about 3 amino acid residues, and most preferably of no more than about 2 amino acid residues.

Preferably, the polypeptide includes at least about 20% acidic amino acid residues, more preferably yet at least about 40% acidic amino acid residues, more preferably yet at least about 60% acidic amino acid residues, more preferably yet at least about 80% acidic amino acid residues, more preferably yet at least about 90% acidic amino acid residues, more preferably yet at least about 95% acidic amino acid residues, and most preferably at least about 98% acidic amino acid residues. Acidic amino acid residues include aspartic acid and glutamic acid. An example of such an LBP polypeptide is BHF-1, which is a 20 amino acid length fragment of human or rabbit LBP-1 which contains amino acid residues-14 through 33. See Fig. 9 (SEQ ID NO:9). 45% of the amino acid residues of BHF-1 are acidic. The invention also includes biologically active fragments and analogs of BHF-1.

Other preferred acidic regions from the LBPs are amino acid residues 8 through 22 (SEQ ID NO:19), 8 through 33 (SEQ ID NO:20),23 through 33 (SEQ ID NO:21), and 208 through 217 (SEQ ID NO:22) of human LBP-2 as depicted in Fig. 7 (SEQ. ID NO:7); amino acid residues 14 through 43 (SEQ ID NO:23) and 38 through 43 (SEQ ID NO:24) of rabbit or human LBP-1 as depicted in Fig. 1 (SEQ ID NO:1) and Fig. 6 (SEQ ID NO:6); amino acid residues 105 through 120 (SEQ ID NO:25),-105 through 132 (SEQ ID NO:26), 121 through 132 (SEQ ID NO:27), and 211 through 220 (SEQ ID NO:28) of rabbit LBP-2 as depicted in Fig. 2 (SEQ ID .20 NO:2); amino acid residues 96 through 110 (SEQ ID NO:29) of rabbit LBP-3 as depicted in Fig.

5 (SEQ ID NO:5); and amino acid residues 53-59 (SEQ ID NO:41) of human LBP-3 as depicted in Fig. 8 (SEQ ID NO:8). The-invention is also, meant to include biologically active fragments and analogs of any of these polypeptides.

Other examples of agents include homopolymers and heteropolymers of any amino acid or amino acid analog. In certain preferred embodiments, the agent is a homopolymer of an acidic amino acid or analog thereof. In certain embodiments, the agent is a heteropolymer of one or more acidic amino acids and one or more other amino acids, or analogs thereof. For example, agents include poly(glu), poly(asp), poly(glu asp), poly(glu N),_oly(asp N) and poly(glu asp N).

By N is meant any amino acid, or analog thereof other than glu or asp. By poly(glu asp) is 30 meant all permutations of glu and asp for a given length peptide. A preferred peptide is poly(glu) of no more than about 10 amino acids in length, preferably about 7 amino acids in length.

-24- In certain preferred embodiments, the agent is an LBP nucleic acid or a biologically active fragment or analog thereof, a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or a biologically active fragment or analog thereof. The agent can be, a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOS:10-18. In other embodiments, the agent is an antisense molecule, one which can bind to an LBP gene sequence.

Treating is meant to include, preventing, treating, reducing the symptoms 9f, or curing the atherosclerosis. Administration of the agent can be accomplished by any method which allows the agent to reach the target cells. These methods include, injection, deposition, implantation, suppositories, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells bythe agent is obtained.

Injections can be, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal.

Implantation includes inserting implantable drug delivery systems, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, matrix erosion.and/or diffusion systems and non-polymeric systems, compressed, fused or partially fused pellets.

Suppositories include glycerin suppositories. Oral ingestion doses can be enterically coated.

Inhalation includes administering the agent with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed.

Administration of the agent can be alone or in combination with other therapeutic agents.

20 In certain embodiments, the agent can be combined with a suitable carrier, incorporated into a liposome; or incorporated into a polymer release system.

In certain embodiments of the invention, the administration can be designed so as to result in-sequential exposures to the agent over some time period, hours, days, weeks, months or years. This can be accomplished by repeated administrations of the agent by one of the methods described above, or alternatively, by a controlled release delivery system in which the agent is delivered to the animal over a prolonged period without repeated administrations.

By a controlled release delivery system is meant that total release of the agent does not occur immediately upon administration, but rather is delayed for some time. Release can occur in bursts or it can occur gradiually and continuously. Administration of such a system can be,rg., by long acting oral dosage forms, bolus injetions, transdermal patches or subcutaneous implants.

Examples of systems in which release occurs in bursts include, systems in which the agent is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to a specific stimulus, temperature, pH, light, magnetic field, or a degrading enzyme, and systems in which the agent is encapsulated by an ionically-coated microcapsule with a microcapsule core-degrading enzyme. Examples of systems in which release of the agent is gradual and continuous include, erosional systems in which the agent is contained in a form within a matrix, and diffusional systems in which the agent permeates at a controlled rate, through a polymer. Such sustained release systems can be, in the form of pellets or capsules.

The agent can be suspended in a liquid, in dissolved form or colloidal form. The liquid can be a solvent, partial solvent or non-solvent In many cases water or an.organic liquid can be used.

The agent can be administered prior to or subsequent to the appearance of atherosclerosis symptoms. In certain embodiments, the agent is administered to patients with familial histories of atherosclerosis, or who have phenotypes that may indicate a predisposition to atherosclerosis, or who have been diagnosed as having a genotype which predisposes the patient to atherosclerosis, or who have other risk factors, hypercholesterolemia, hypertension or smoking.

The agent is-administered to the animal in a therapeutically effective amount. By therapeutically effective amount is meant that amount which is capable of at least partially 20 preventing or reversing atherosclerosis. A therapeutically effective amount can be determined on -an individual basis and will be based, at least in part, on consideration of the species of animal, the animal's size, the animal's age, the agent used, the type of delivery system used, the time of administration relative to the onset of atherosclerosis symptoms, and whether a single, multiple, or controlled release dose regimen is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

Preferably, the concentration of the agent is at a dose of about 0.1 to about 1000 mg/kg body weight/day, more preferably at about 0.1 to about 500 mg/kg/day, more preferably yet at about 0.1 to about 100 mg/kg/day, and most preferably at about 0.1 to about 5 mg/kg/day. The 30 specific concentration partially depends upon the particular agent used, as some are more effective than others. The dosage concentration of the agent that is actually administered is dependent at least in part upon the final concentration that is desired at the site of action, the -26method of administration, the efficacy of the particular agent, the longevity of the particular agent, and the timing of administration relative to the onset of the atherosclerosis symptoms.

Preferably, the dosage form is such that it does not substantially deleteriously affect the animal.

The dosage can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

In certain embodiments, various gene constructs can be used as part of a gene therapy protocol to deliver nucleic acids encoding an-agent, either an agonistic or antagonistic form of an LBP.polypeptide. For example, expression vectors can be used for in vivo transfection and expression of an LBP polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of, LBP polypeptide in a cell in which non-wild type LBP is expressed. Expression constructs of the LBP polypeptide, and mutants thereof, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the LBP gene to cells in vivo. Approaches include, insertion of the subject gene in viral vectof~ including, recombinant retroviruses, adenovirus, adenoassociated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids.

Viral vectors infect or transduce cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin T M (Life Technologies, Inc., Gaithersburg, MD) or derivatized antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection-of the gene construct or 20 Ca 3

(PO

4 2 precipitation carried out in yviv. The above-described methods are known to those skilled in the art and can be performed without undue experimentation. Since transduction of appropriate target cells represents the critical first step in gene therapy, choice ofthe particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, locally or systemically.- Administration can be directed to one or more cell types, and to one or more cells within a cell type, so as to be therapeutically effective, by methods that are known to those skilled in the art. In a preferred embodiment, the agent is administered to arterial wall cells of the animal. For example, a genetically engineered LBP gene is administered to arterial wall cells. In certain embodiments, administration is done in a prenatal animal or embryonic cell. It will be recognized that the particular gene construct provided for in in vivo transduction of LBP expression is also useful for in yitr transduction of cells, such as for use in the diagnostic assays described herein.

In certain embodiments, therapy of atherosclerosis is performed with antisense nucleotide analogs of the genes which code for the LBPs. Preferably, the antisense nucleotides have nonhydrolyzable "backbones," phosphorothioates, phosphorodithioates or methylphosphonates.

The nucleoside base sequence is complementary to the sequence of a portion of the gene coding for, LBP-1, 2 or 3. Such a sequence might be, ATTGGC if the gene sequence forthe LBP is TAACCG. One embodiment of such therapy would be incorporation of an antisense analog of a portion of one of the LBP genes in a slow-release medium, polyvinyl alcohol, which is administered, by subcutaneous injection, so as to release the antisense nucleotide analog over a period of weeks or months. In another embodiment, the antisense analog is incorporated into a polymeric matrix, polyvinyl alcohol, such that the gel can be applied locally to an injured arterial wall to inhibit LBP synthesis and prevent LDL accumulation, e.g., after angioplasty or atherectomy.

The invention also includes a method for treating an animal at risk for atherosclerosis. An animal at risk for atherosclerosis is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the animal in a therapeutically effective amount such that treatment of the animal occurs. Being at risk for atherosclerosis can result from, a family history of atherosclerosis, a genotype which-predisposes to atherosclerosis, or phenotypic symptoms which predispose to atherosclerosis, having hypercholesterolemia, hypertension or smoking.

20 The invention also includes a method for treating a cell having an abnormality in structure or metabolism of LBP. A cell having an abnormality in structure or metabolism of LBP is provided. An agent capable of altering an aspect of LBP structure or metabolism is provided. The agent is administered to the cell in a therapeutically effective amount such that treatment of the cell occurs.

In certain embodiments, the cell is obtained from a cell culture or tissue culture or an Sembryo fibroblast. The cell can be, part of an animal, a natural animal or a non-human transgenic animal. Preferably, the LBP is LBP-l, LBP-2 or LBP-3. The invention also includes a pharmaceutical composition for treating atherosclerosis in an animal comprising a therapeutically effective amount of an agent, the agent being capable of 30 altering an aspect of LBP metabolism or structure in the animal so as to result in treatment of the atherosclerosis, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, saline, liposomes and lipid emulsions.

-28- In certain preferred embodiments, the agent of the pharmaceutical composition is an LBP polypeptide, LBP-1, LBP-2 or LBP-3, or a biologically active fragment or analog thereof.

The agent can be, the polypeptide as set forth in SEQ ID NOS:1-9. Preferably, the agent is a polypeptide of no more than about 100 amino acid residues in length, more preferably of no more than about 50 amino acid residues, more preferably yet of no more than about 30 amino acid residues, more preferably yet of no more than about 20 amino acid residues, more preferably yet of no more than about 10 amino acid residues, more preferably yet of no more thanabout amino acid residues, more preferably yet of no more than about 4 amino acid residues, more preferably yet of no more than about 3 amino acid residues, and most preferably of no more than about 2 amino acid residues. Preferably, the polypeptide includes at least about 20%1acidic amino acid residues, more preferably yet at least about 40% acidic amino acid residues, more preferably yet at least about 60% acidic amino acid residues, more preferably yet at least about acidic amino acid residues, more preferably yet at least about 90% acidic amino acid residues, more preferably yet at least about 95% acidic amino acid residues, and most preferably at least about 98% acidic amino acid residues.

In certain preferred embodiments, the agent is an LBP nucleic acid, a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or a biologically active fragment or analog thereof. The agent can be, a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOS:10-18.

20 The invention also includes a vaccine composition for treating atherosclerosis in an animal comprising a therapeutically effective amount of an agent, the agent being capable of Saltering an aspect of LBP metabolism or structure in the animal so as to result in treatment of the atherosclerosis, and a pharmaceutically acceptable carrier.

The invention also includes a method for diagnosing atherosclerotic lesions in an animal.

25 An animal is provided. A labeled agent capable of binding to LBP present in atherosclerotic lesions is provided. The labeled agent is administered to the animal under conditions which allow the labeled agent to interact with the LBP so as to result in labeled LBP. The localization or quantification of the labeled LBP is determined by imaging so as to diagnose the presence of atherosclerotic lesions in the animal.

Preferably, the LBP is LBP-1, LBP-2 or LBP-3. The imaging can be performed by standard methods known to those skilled in the art, including, magnetic resonance imaging, gamma camera imaging, single photon emission computed tomographic (SPECT) imaging, or positron emission tomography (PET).

Preferably, agents that bind tightly to LBPs in atherosclerotic lesions are used for atherosclerotic imaging and diagnosis. The agent is radiolabeled with, ""Tc or another isotope suitable for clinical imaging by gamma camera, SPECT, PET scanning or other similar technology. Since LBPs occur in very early lesions, such imaging is more sensitive than angiographyor ultrasound for locating very early lesions which do not yet impinge on the arterial lumen to cause a visible bulge or disturbed flow. In addition to locating both early and more developed lesions, the imaging agents which bind to LBPs can also be used to follow the progress of atherosclerosis, as a means of evaluating the effectiveness of both dietary and pharmacological treatments.

Thus, a diagnostic embodiment of the invention is the ldaptation of, a peptide complementary to one of the LBPs, by radiolabeling it and using it as an injectable imaging agent for detection of occult atherosclerosis. The peptide is selected froni those known to bind to LBPs, RRRRRRR or KKLKLXX, or any other polycationic peptide which binds to the highly electronegative domains of the LBPs. For extracorporeal detection with a gamma scintillation (Anger) camera, technetium-binding ligands, COC, GGCGC, or GGCGCF, can be incorporated into the peptides at the N-terminus or C-terminus for 99mTc labeling. For- external imaging by magnetic resonance imaging (MRI), the gadolinium-binding chelator, diethylene triamine-penta-acetic-acid (DTPA), is covalently bound to the N- or C-terminus of the 20 peptides. In yet other embodiments, the LBP-binding peptides are covalently bound, to magnetic ion oxide particles by standard methods. known to those skilled in the art, e.g., conjugating the peptides with activated polystyrene resin beads containing magnetic ion oxide.

.The invention also includes a method for immunizing an animal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or analog thereof. An animal having.LDL is provided. An 25 LBP or fragment or analog thereof is provided. The LBP or fragment or analog thereof is administered to the animal so as to stimiulate antibody production by the animal to the LBP or fragment or analog thereof such that binding of the LBP to the LDL is altered, decreased or increased.

The invention also includes a method of making a fragment or analog of LBP 30 polypeptide, the fragment or analog having the ability to bind to modified LDL and native LDL.

An LBP polypeptideis provided.cThesequence of the LBP polypeptide is altered. The altered LBP polypeptide is tested for the ability to bind to modified LDL, methylated LDL, oxidized LDL, acetylated LDL, cyclohexanedione-treated LDL (CHD-LDL), and to native LDL.

The fragments or analogs can be generated and tested for their ability to bind to these modified LDLs and to native LDL, by methods known to those skilled in the art, as described herein. Preferably, they are tested for their ability to bind to methylated LDL and native LDL. The binding activity of the fragment or analog can be greater or less than the binding activity of the native LBP. Preferably, it is greater. In preferred embodiments, the LBP is LBP-1, LBP-2 or LBP-3.

The invention also includes a method for isolating a cDNA encoding an LBP. A cDNA library is provided. The cDNA library is screened for a cDNA encoding a polypeptide which o0 binds to native LDL and modified LDL, methylated LDL or oxidized LDL. The cDNA which encodes this polypeptide is isolated, the cDNA encoding an LBP.

The following non-limiting examples further illustrate the present invention.

EXAMPLES

Exrmple 1: Construction of a Rabbit cDNA Library This example illustrates the construction of a rabbit cDNA library using mRNA from balloon-deendothelialized healing rabbit abdominal aorta. Balloon-catheter deendothelialized rabbit aorta has been shown to be a valid model for atherosclerosis (Minick et al., Am. J. Pathol.

95:131-158 (1979).

20 The mRNA was obtained four weeks after ballooning to maximize focal LDL binding in the ballooned rabbit aorta. First strand cDNA synthesis was carried out in a 50 gl reaction mixture containing 4 gg mRNA; 2 pg oligo d(T) primer, methylation dNTP mix (10 mM each); 10 mMDTT; 800 units superscript II RT (Life Technologies, Gaithersburg, MD); 1 X first strand cDNA synthesis buffer (50 mM Tris-HC1, pH 8.3; 75 mM KCI; 5 mM MgC1), which was incubated for I hr at 37C. The reaction mixture was then adjusted to 250 il through the oaddition of 1 X second strand buffer (30 mM Tris-HCl, pH 7.5; 105 mM KCI; 5.2 mM MgCl); 0.1 mM DTT; methylation dNTP mix (10 mM each); 50 units E. Ecoi DNA polymerase 1,3 units RNase H; 15 units E. oli DNA ligase (all enzymes from Life Technologies), which was incubated for an additional 2.5 hr at 15 0 C. The resulting double-stranded cDNAs (dscDNA) 30 were then treated with 1.5 units T4 DNA polymerase (Novagen Inc., Madison, WI) for 20 min at I. 11 °C to make bluni-ended dscDNA. These were then concentrated by ethanol precipitation and EcoRl/Hind III linkers were attached to the ends by T4 DNA ligase (Novagen Inc.). The linkerligated cDNAs were treated with EcoRI and HindIII restriction enzymes to produce EcoRl and Hind III recognition sequences at their 5' and 3' ends, respectively. After the removal of linker DNA by gel exclusion chromatography, the dscDNAs were inserted into XEXlox phage arms (Novagen Inc.) in a unidirectional manner by T4 DNA ligase and packaged into phage particles according to the manufacturer's protocol (Novagen Inc.). A phage library of cDNAs containing 2 x 106 independent clones was established from 4 ug of mRNA.

Example 2: Identification of Rabbit cDNAs Encoding LDL Binding Proteins (LBPs This example illustrates a method of functionally screening a rabbit cDNA library so as to identify cDNAs encoding LBPs which bind to both native LDL and methyl LD-. Methyl LDL is not recognized by previously reported cell surface receptors. See. eg. Weisgraber et al., T.Biol. Chem. 253:9053-9062 (1978). A fresh overnight culture ofE. coi ERI647 cells (Novagen Inc.) was infected with the cDNA phage obtained from Example 1, and plated at a density of 2 x 104 plaque-forming units (pfu) in 150 nmm diameter plates containing 2 X YT agar. A total of 50 plates, equivalent to I x 10' phage, were plated and incubated at 37*C until the plaques reached 1 mm in diameter (5-6 hr). A dry nitrocellulose membrane, which had previously been saturated with 10 mM IPTG solution, was layered on top of each plate to induce the production of recombinant protein, as well as to immobilize the proteins on the membranes. The plates were incubated at 37*C for an additional 3-4 hr, and then overnight at 4"C.

20 The next day, the membranes were lifted from each plate and processed as follows.

.Several brief rinses in TBST solution (0mM Tris-HCl, pH 8.0; 150mM NaCil,.05% Tween 20); two 10-min rinses with 6M guanidine-HCl.in HBB (20mM HEPES, pH 7.5; 5mM MgCl 2 "1 mM DTT, and 5mM KC1); two 5-min rinses in 3M guanidine-HCl in HBB; a final brief rinse in TBSEN (TBS, ImM EDTA, 0.02% NaN 3

Y.

25 The membranes were then blocked for 30 min at room temperature in a solution of TBSEN with 5% non-fat dry milk, followed by 10 min in TBSEN with 1% non-fat-dry milk.

S Following blocking, the membranes were incubated with native human LDL (obtained as described in Example 11 or methylated human LDL (meLDL) (se Weisgraber et al., J. Biol.

Chem. 253:9053-9062 (1978)), at a concentration of 4 g/ml, in a solution containing 1 X 30 TBSEN, 1% non-fat dry milk, ImM PMSF, 0.5 X protease inhibitor solution (ImM e-amino caproic acid/lmM benzamidine). Incubation was for 4 hr at room temperature in a glass Petri dish with gentle stirring on a stirring table, followed by overnight at 4 0 C with no stirring.

-32- Specifically bound meLDL and native LDL were detected on the nitrocellulose membranes by antibodies against human LDL. Sheep anti-human LDL polyclonal antibodies (Boehringer Mannheim, Indianapolis, IN) were adsorbed with E. Wgij plys E cell extracts to abolish background. For adsorption, E. oli plys E cells were grown to log phase, spun down and resuspended in PBS containing 1 mM PMSF, 2 mM e-amino caproic acid, and I mM benzamidine.. The cell suspension then underwent 8 freeze-thaw cycles via immersion in liquid nitrogen and cold running tap water, respectively. The anti LDL antibodies/cell extract.solution were incubated with gentle stiiring for 1 hr at 4°C (1 ml of antibody solution/3 mg crude cell extract). Following incubation, the mixture was centrifuged (10,000 x g; 10 min; 4°C) and the supernatant was stored at 4°C in the presenceTrf 0.02% NaN 3 until use. The membranes were processed for im~fiunoscreening as follows: (i) three 5-min washes at room temperature in TBSEN containing 1% gelatin; (ii) 30 min incubation in PBS, pH 7.4 with 1% gelatin; (iii) two-hr room temperature incubation with gentle stirring in fresh PBS/gelatin solution containing adsorbed sheep anti-human LDL antibodies (Boehringer Manheim, Indianapolis, IN) (1:1000 dilution); (iv) three brief washes in TBS, pH 7.4; one-hr room temperature incubation with gentle stirring in PBS/gelatin solution containing donkey antisheep alkaline phosphatase-conjugated antibodies (Sigma, St. Louis, MO) (1:10,000 dilution); (vi) three brief washes with TBS, pH and (vii) development according to the manufacturer's instructions, using an alkaline phosphatase substrate development kit (Novagen 20 Inc.). Phage plaques which produced LBPs appeared as blue-colored "donuts" on the membranes.

The phage from Example 1 containing the LBP cDNAs were plaque-purified and converted into plasmid subclones by following a protocol called "Autosubcloning by Cremediated Plasmid Excision" provided by Novagen Inc. DNA sequences were obtained by the 25 dideoxynucleotide chain-termination method (Sanger et al., Proc. Natl. Acad. Sci., USA 74:5463-5467 (1977), and analyzed by an Applied Biosystems automated sequencer. The open reading frame (ORF) of each cDNA was determined from consensus sequences obtained from both the sense and antisense strands of the cDNAs. Sequencing confirmed that three previously unknown genes had been isolated. Since the genes were selected by functional screening for 30 LDL binding, the proteins coded by these genes were termed LDL binding proteins (LBPs), specifically, LBP-I, LBP-2 and LBP-3. The cDNA sequences for rabbit LBP-1, LBP-2 and -LBP-3 and the corresponding proteins are set forth in SEQ ID NOS:10-14.

Based on their respective cDNA coding sequences, the sizes of the recombinant proteins were determined to be 16.2 kDa for LBP-1, 40 kDa for LBP-2, and 62.7 kDa for LBP-3.

Examle 3: Northern Blot Analysis of Rabbit RNA Using LBP cDNA or cRNA This example illustrates the size and tissue distribution of LBP mRNAs. Total RNA was isolated from different rabbit tissues: adrenals, thoracic aorta, abdominal aorta, ballooned and reendothelialized abdominal aorta, heart, kidney, smooth muscle cells, lung and liver, by Trizol reagent (Life Technologies) and concentrated by ethanol precipitation. Gel electrophoresis of RNA was carried out in 1.2o agarose gel containing 1 X MOPS buffer (0.2M MOPS, pH 50mM sodium acetate; 5mM EDTA, pH 8.0) and 0.37M formaldehyde. Gels were loaded with pg total RNA from each tissue examined and electrophoresed at 100 volts for 2 hr in 1 X MOPS buffer. RNAs were blotted onto supported nitrocellulose membranes-(Schleicher Schuell, Keene, NH) and immobilized by baking at 80*C for 2 hr. Hybridization to radiolabeled LBP-1, LBP-2 and LBP-3cDNA or cRNA probes was.carried out by standard procedures known to those skilled in the art (see, g, Ausubel et al., Current Protocols in Molecular Biology; John Wiley Sons (1989)); signals were detected by autoradiography.

The results were as follows: the sizes of the mRNAs were about 1.3 kb for LBP-1, about 2.3-2.5 kb for LBP-2, and about 4.7 kb for LBP-3. LBP-1, LBP-2 and LBP-3 mRNA were found in all tissues tested, but the highest amount was in ballooned abdominal aorta.

Example 4: Isolation of Human LBP cDNAs •This example illustrates isolation of human LBP cDNAs. Human LBP cDNA clones were isolated from three cDNA libraries. A human fetal brain cDNA library was obtained from Stratagene, LaJolla, CA, a human liver and a human aorta cDNA library were obtained from Clontech, Palo Alto, CA, and screened with a radiolabeled cDNA probe derived from rabbit LBP-1, LBP-2 or LBP-3, according to the method described in Law et al., Gene Expression 4:77-84(1994). Several strongly hybridizing clones were identified and plaque-purified. Clones were confirmed to be human LBP-1, LBP-2 and LBP-3, by DNA sequencing using the -dideoxynucleotide chain-termination method and analysis by an Applied Biosystems automated sequencer. The cDNA sequences and the corresponding proteins for human LBP-1, LBP-2 and LBP-3 are set forth in SEQ ID NOS:15, 16 and 17, respectively. A comparison between the Scorresponding LBP-1, LBP-2 and LBP-3 protein sequences for rabbit and human are shown in Figs. 19, 20 and 21.

Exampki: Isolation of Recombinant LBP-1. LBP-2 and LBP-3 Rabbit Proteins from E. coli LBP cDNA was isolated from the original pEXlox plasmids obtained as described in Examples 1 and 2, and subcloned into the pPRoEX-HT vector (Life Technologies) for recombinant protein expression. Induction of the recombinant protein by IPTG addition to transformed E. coli DH 1B cultures resulted in the expression of recombinant protein containing a 6-histidine tag (N-terminal). This tagged protein was then purified from whole cell proteins by binding to Ni-NTA (nickel nitrilo-triacetic acid) as described in the protocol provided by the manufacturer (Qiagen, Inc., Santa Clara, CA). The preparation obtained after the chromatography step was approximately 90% pure; preparative SDS-PAGE was performed as the final purification step.

When required by the characterization lrocedure, iodination of LBPs was carried out using lodobeads (Pierce, Rockford, IL). The lodobeads were incubated with 500 pCi of Na'"I solution (17 Ci/mg) (New EngladN.uclear, Boston, MA) in a capped microfuge tube for 5 min iS at room temperature. The protein solution was added to the Iodobeads-Na' I microfuge tube and incubated for 15 min at room temperature. At the end of this incubation, aliquots were removed for the determination of total soluble and TCA-precipitable counts. The radiolabeled protein was then precipitated with cold acetone (2.5 vol; -20°C; 2.5 hr). Following this incubation, precipitated protein was collected by centrifugation (14,000 g; 1 hr, room temperature) and 20 resuspended in sample buffer (6 Mfurea/50 mM Tris, pH 8.0/2mM EDTA). Integrity of the protein preparation was assessed by SDS-PAGE.

The identities of the recombinant LBPs were confirmed using standard protein sequencing protocols known to those skilled in the art. (A Practical Guide for Protein and Peptide Purification for Microsequencing, Matsudaira, ed., Academic Press, Inc., 2d edition 25 (1993)). Analysis was performed using an Applied Biosystems Model 477A Protein Sequencer with on-line Model 120 PTH amino acid analyzer.

Example 6: Production of Antibodies to LBP-I. LBP-2 and LBP-3 This example illustrates the production of polyclonal antibodies to LBP- 1, LBP-2 and LBP-3. A mixture of purified recombinant LBP protein (0.5 ml; 200 g) andRIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, MT) was injected subcutaneously into male guinea pigs (Dunkin Hartley; Hazelton Research Products, Inc., Denver, PA) at 3-5 sites along the dorsal thoracic and abdominalregions of the guinea pig. Blood was collected by venipuncture on days 1 (pre-immune bleeding), 28, 49 and 70. Booster injections were administered on days 21 (100 pg; SC), 42 (50 g.gLSC), and 63 (25 jig; SC). The titer of the guinea pig antiserum was evaluated by serial dilution "dot blotting." Preimmune antiserum was evaluated at the same time. After the third booster of LBP protein, the titer against the recombinant protein reached a maximal level with a detectable colorimetric response on a dot blot assay of 156 pg.

Specificity of the polyclonal antibody for recombinant LBP-1, LBP-2 or LBP-3 was demonstrated using Western blot analysis. (Towbin et al., Proc. Natl. Acad; Sci. USA 76:4350 (1979)). The protein-antibody complex was visualized immunochemically with alkaline 0o phosphatase-conjugated goat anti-guinea pig IgG, followed by staining with nitro blue tetrazolium (BioRad Laboratories, Hercules, CA). Non-specific binding was blocked using 3% non-fat dry milk in Tris buffered saline (100 mM Tris; 0.9% NaCl, pH 7.4).

Example 7: Immunohistochemical Characterization This example illustrates the presence of LBPs in or on endothelial cells covering plaques, in or on adjacent smooth muscle cells, and in the extracellular matrix. In addition, co-localization of LDL and LBPs was demonostrated. These results were obtained by examining ballooned rabbit arterial lesions and human atherosclerotic plaques by immunohistochemical 20 methods.

Ballooned deendothelialized aorta was obtained from rabbits which had received a bolus injection of human LDL (3 mg; 24 hr prior to tissue collection. Human aortas containing atherosclerotic plaques were obtained from routine autopsy specimens. Tissues were fixed in 10% buffered formalin (.24 hr) and imbedded in paraffin using an automated tissue-imbedding machine. Tissue sections were cut (5-7 t) and mounted onto glass slides by incubating for 1 hr at 60"C. Sections were deparaffinized. After a final wash with deionizedH 2 0O, endogenous peroxidase activity was eliminated by incubating the sections with 1% HiO 2 /HO0 buffer for -min at room temperature. Sections were rinsed with phosphate buffered saline (PBS) for 5 min at room temperature and nonspecific binding was blocked with 5% normal goat serum or normal rabbit serum depending on the source of the secondary antibody (Sigma, St Louis, MO) S(1 hr, room temperature). Sections were then incubated with a 1:50 dilution (in 5% normal goat serum/PBS) of a guinea pig polyclonal antibody against the rabbit form of recombinant LBP-1, LBP-2 or LBP-3. Controls included preimmune serum as well as specific antisera to LBP-1, LBP-2, or LBP-3 in which the primary antibody was completely adsorbed and removed by incubation with recombinant LBP-1, LBP-2 or LBP-3 followed by centrifugation prior to incubation with the tissue sections. An affinity purified rabbit polyclonal antibody against human apolipoprotein B (Polysciences Inc.; Warrington, PA) was used at a dilution of 1:100 (in 5% normal rabbit serum/PBS). Sections were incubated for 2 hr at room temperature in a humidified chamber. At the end of incubation, sections were rinsed with PBS and incubated with a 1:200 dilution (in 5% normal goat serum/PBS) of goat anti-guinea pig biotinylated IgG conjugate (Vector Laboratories, Burlingame, CA) or a 1:250 dilution (in 5% normal rabbit serum/PBS) of rabbit anti-goat biotinylated IgG conjugate (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature in a humidified chamber. Sections were then rinsed with PBS and antigen-antibody signal amplifiedusing avidin/biotin HRPconjugate (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Sections were developed using DAB-substrate (4-6 min; room temperature) and counterstained with hematoxylin.

In the ballooned rabbit artery, immunohistochemistry with the anti-LBP-1, LBP-2 and LBP-3 antibodies showed that LBP-1, LBP-2 and LBP-3 were located in or on functionally modified endothelial cells at the edges of regenerating endothelial islands, the same location in which irreversible LDL binding has been demonstrated (Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). LBP-1, LBP-2 and LBP-3 were also found in or on intimal smooth muscle cells underneath the functionally modified endothelial cells, and to a lesser extent, in extracellular matrix. No LBP-1, LBP-2 or LBP-3 was detected in still deendothelialized areas, where LDL binding had been shown to be reversible (Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). Immunohistochemistry of ballooned rabbit aorta with anti-human apolipoprotein B antibodies showed the presence of LDL at the same locations as that found for LBP-1, LBP-2 and LBP-3.

25 In the human atherosclerotic plaques taken at routine autopsies, immunohistochemistry with the anti-LBP-l, anti-LBP-2 and anti-LBP-3 antibodies showed that LBP-1, LBP-2, and LBP-3 were also found in or on endothelial cells covering plaques and in or on adjacent smooth muscle cells. In the human tissue, there was greater evidence of LBP-1, LBP-2 and LBP-3 in extracellular matrix.

30 The results obtained with paraffin sections were identical to those of frozen sections.

-37- Examle 8: Affinity Coelectrophoresis (ACE) Assays of LBPs and LDL or HDL This example illustrates that binding occurs between LBP-1, LBP-2 or LBP-3 and LDL, and that this binding is specific, as illustrated by the fact that binding does not occur between LBP-1, LBP-2 or LBP-3 and HDL (high density lipoprotein).

Analysis of the affinity and specificity of recombinant rabbit LBP-1, LBP-2 or LBP-3 binding to LDL was carried out using the principle of affinity electrophoresis (Lee and Lander, Proc. Natl. Acad. Sci. USA 88:2768-2772 (1991)). Melted agarose 65"C) was prepared in hiM sodium MOPS, pH 7.0; 125 mM sodium acetate, 0.5% CHAPS. A teflon comb consisting of nine parallel bars (45 x 4 x 4 mm/3 mm spacing between bars) was placed onto GelBond film (FMC Bioproducts, Rockland, ME) fitted to a plexiglass casting tray with the long axis of the bars parallel to the long axis of the casting tray. A teflon strip (66 x 1 x 1 mm) was placed on edge with the long axis parallel to the short axis of the casting tray, at a distance of 4 mm from the edge of the teflon comb. Melted agarose (>65 C) was then poured to achieve a height of approximately 4 mm. Removal of the comb and strip resulted in a gel containing nine x 4 x 4 mm rectangular wells adjacent to a 66 x i mm slot. LDL or HDL samples were prepared in gel buffer (50mM sodium MOPS, pH 7.0, 125 mM sodium acetate) at twice the desired concentration. Samples were then mixed with an equal volume of melted agarose (in mM MOPS, pH 7.0; 125 mM sodium acetate; 50"C), pipetted into the appropriate rectangular wells and allowed to gel. The binding affinity and specificity of LBP-1 and LBP-3 was tested using several concentrations of LDL (540 to 14 nM) and HDL (2840-177 nM). A constant amount (0.003 nM 0.016 nM) of '"I-labeled LBP-1, LBP-2 or LBP-3 (suspended in 50 mM sodium MOPS, pH 7.0; 125 nM sodium acetate; 0.5% bromphenol blue; 6% (wt/vol) sucrose) was loaded into the slot Gels were electrophoresed at 70v/2hr/20C. At the end of the run, the gels were air dried and retardation profiles were visualized by exposure of X-ray films to the gels overnight at -70 0 C, with intensifying screens).

LDL retarded LBP-I, LBP-2 and LBP-3 migration through the gel in a concentrationdependent, saturable manner, indicating that LBP-1, LBP-2 and LBP-3 binding to LDL was highly specific. This conclusion is supported by the fact that HDL did not retard LBP-I, LBP-2 or LBP-3. A binding curve generated from the affinity coelectrophoresis assay indicated that LBP-1 binds to LDL with a Kd of 25.6 nM, that LBP-2 (rabbit clone 26) binds to LDL with a IK of 100 nM, and that LBP-3 (80 kDa fragment) binds to LDL with a I of 333 nM.

In addition to testing affinity and specificity of LBP-1, LBP-2 and LBP-3 binding to -38- LDL, the ability of"cold" non-radiolabeled) LBP-1, LBP-2.or LBP-3 to competitively inhibit radiolabeled LBP-1, LBP-2 or LBP-3 binding to LDL, respectively, was tested.

Competition studies were cared out using fixed concentrations of cold LDL and radiolabeled LBP-1 and increasing amounts of cold recombinant LBP-I (6-31 pM). The ACE assay samples and gel were prepared as described herein. Cold LBP-1 inhibited binding of radiolabeled LBP-1 to LDL in a concentration-dependent manner, cold LBP-2 inhibited binding of radiolabeled LBP-2 to LDL in a concentration-dependent manner, and cold LBP-3 inhibited binding of radiolabeled LBP-3 to LDL in a concentration-dependent manner.

Rabbit and human LBP-2 contain a long stretch of acidic amino acids at the amino terminal (rabbit LBP-2 amino acid residues 105 through 132 and human LBP-2 amino acid residues 8 through 33). The possibility that this segment of LBP-2 was the LDL binding domain was tested by subcloning two rabbit LBP-2 clones which differ from each other by the presence or absence of this acidic region (clone 26 and clone 45, respectively) into expression vectors, 6y standard methods known to those skilled in the art. ACE assays were then conducted in order to assess the affinity and specificity of the binding of these two clones to LDL. LDL retarded clone 26 derived radiolabeled LBP-2 migration through the gel in a concentration-dependent, saturable, manner while clone 45 derived radiolabeled LBP-2 migration was not retarded.

Competition studies using fixed concentrations of cold LDL and clone 26 derived radiolabeled LBP-2 and increasing concentrations of cold recombinant LBP-2/clone 26 and 20 LBP-2/clone 45 were carried out. Cold clone 26 derived LBP-2 inhibited binding of clone 26 .derived radiolabeled LBP-2 to LDL in a concentration-dependent manner. Clone 45 derived LBP-2, on the other hand, did not affect the binding of clone 26 derived radiolabeled LBP-2 to LDL. These results indicate that the long stretch of acidic amino acids contain a binding domain of LBP-2 to LDL.

Examle 9: Affinity Coelectrophoresis (ACE) Assays of LBP-1 or LBP-2 and LDL in the Presence of Inhibitors This example illustrates that binding between LBP-I or LBP-2 and LDL is inhibited by polyglutamic acid or BHF-1. The ability of a third compound to inhibit binding betweenrtwoproteins previously shown to interact was tested by a modification of the ACE assays described in Example 8. The third compound was added to the top or wells together with the radiolabeled protein. If the third compound inhibited binding, the radiolabeled protein would-run through the gel. If the third compound did not inhibit binding, migration of the radio-labeled protein was retarded by the protein cast into the gel.

Inhibition of LBP-1/LDL or LBP-2/LDL binding by polyglutamic acid (average MW about 7500, corresponding to about 7 monomers) was shown by casting a constant amount of LDL (148 nM) in all the rectangular lanes. A constant amount (1 pl) of '"I-labeled LBP-1 or LBP-2 (0.003 nM 0.016 nM) was loaded in the wells at the top of the gel, together with increasing concentrations ofpolyglutamic acid (obtained from Sigma) (0-0.4 nM). The gel was electrophoresed at 70 volts for 2 hr, dried and placed on X-ray film, with intensifying screens, overnight at -70 C before the film was developed to determine the retardation profile of LBP-1 and LBP-2. As the concentration of polyglutamic acid increased, retardation of radiolabeled LBP-1 and LBP-2 migration by LDL decreased in a concentration-dependent manner, which showed that polyglutamic acid inhibited binding between LBP-1, LBP-2 and LDL.

Inhibition of LBP-/LDL binding by BHF-1 was shown by casting a constant amount of LDL (148 nM) in all the rectangular lanes. A constant amount of '"I-labeled LBP-1 (0.003 nM 0.016 nM) was loaded in the wells at the top of the gel, together with increasing concentrations of BHF-1 (0-10 nM), obtained as described in Example 15. The gel was electrophoresed at volts for 2 hr, dried and placed on X-ray film, with intensifying screens,.overight at -70 0 C. The film was then developed to determine the retardation profile of '"I-LBP-l. As the concentration of BHF-1 increased, retardation of LBP-I by LDL decreased in a concentration-dependent 20 manner, which demonstrated that BHF-1 inhibited binding between LBP-1 and LDL.

Example 10: Affinity Coelectrophoresis (ACE) Assays for Identifying Fragments. Analogs and Mimetics of LBPs which Bind to LDL This example illustrates a method for identifying fragments, analogs or mimetics of LBPs which bind to LDL, and which thus can be used as inhibitors of LDL binding to LBP in the arterial walls, by occupying binding sites on LDL molecules, thereby rendering these sites unavailable for binding to LBP in the arterial wall.

Fragments of LBPs are generated by chemical cleavage or synthesized from the known amino acid sequences. Samples of these fragments are individually added (cold) to radiolabeled LBP as described in Example 8, to assess the inhibitory potency of the various fragments. By iterative application of this procedure on progressively smaller portions of fragments identified as inhibitory, the smallest active polypeptide fragment or fragments are identified. In a similar manner, analogs of the LBPs are tested to identify analogs which can act as inhibitors by binding to LDL. And, similarly, mimetics of LBP (molecules which resemble the conformation and/or charge distributions of the LDL-binding sites on LBP molecules) are tested in a similar fashion to identify molecules exhibiting affinities for the LDL-binding sites on LBP.

The affinities of the inhibitors so identified are at least as strong as the affinity of LDL itself for the LDL-binding sites on LBP. The inhibitors bind at least competitively, and some irreversibly and preferentially as well, to the LDL-binding sites, thereby rendering such sites unavailable for binding to humoral LDL.

Examle ll: ELISA Assays This example illustrates the use of an ELISA plate assay for the quantification of a test compound's capacity to inhibit the binding of LDL to a specific LBP.

The assay was carried out as follows: LDL was diluted in 50 mM NazHCO 3 pH 9.6/0.02% NaN 3 and added to the wells of a 96-well plate (ImmunoWare 96-Well Reacti-Biid EIA Polystyrene Plates; Pierce (Rockford, IL)) to achieve a final concentration ranging from 0.1 to 1 g/well. The plates were incubated for 6 hr at room temperature. At the end of the incubation period, the wells wereiwashed 3 times with Tris-buffered saline, pH 7.4 (TBS), and blocked overnight with 200 pl of 1% bovine serum albumin (BSA) in TBS/0.02% NaN 3 (Sigma; St. Louis MO) at room temperature. The wells were then incubated with 200 tl of LBP protein 20 (5-10 pg/well) in TBS and varying concentrations of the test compound. Plates were incubated for 1 hrat room temperature. The wells were then washed three times with TBS and blocked for *oo 2 hr with 200 tl of 1% BSA in TBS/0.02% NaN 3 at room temperature. At the end.of the *incubation period, the wells were washed 3 times with TBS and a 1:1000 dilution (in TBS/0.05% Tween 20) of the appropriate guinea pig anti-LBP protein polyclonal antibody was added to the 25 wells and incubated for 1 hr at room temperature. The wells were then washed 3 times with TBS/0.05% Tween 20; a 1:30,000 dilution of goat anti-guinea pig IgG alkaline phophatase conjugate (Sigma) was added to each well. Plates were incubated for 1 hr at room temperature.

The wells were washed 3 times with TBS/0.05% Tween 20 and a colorimetric reaction was carried out by adding 200 ml of p-nitrophenyl phosphate substrate (Sigma; St. Louis MO) to the 30 wells. The reaction was allowed to proceed for 30 min at room temperature and stopped with pl of 3N NaOH. The absorbance was determined at 405 nm using an ELISA plate reader. The test compound's effectiveness in blocking the binding of LDL to the recombinant protein was assessed by comparing the absorbance values of control and treated groups.

Alternatively, LBPs, rather than LDL, were bound to the plate. Recombinant LBP protein binding to LDL and the effect of varying concentration of the inhibitor on LBP-LDL binding was determined through the use of antibodies against LDL. This interaction was visualized through the use of a secondary antibody conjugated to a reporter enzyme alkaline phosphatase)..

ELISA plate assays were used to screen agents which can affect the binding of LBP proteins to LDL. For example, peptides derived from LBP-l and human LBP-3 protein sequences (BHF-1 and BHF-2, respectively) were synthesized and have been shown to reduce the binding of LDL to recombinant LBP-1 and LBP-2 in this format. These results were in agreement with those obtained with the ACE assays.

Example 12: Administration of Humanized Antibodies Against LBPs so as to Block LDL- Binding Sites on the LBPs This example illustrates administration to patients of humanized antibodies against LBP- 1, LBP-2 or LBP-3 so as to block LDL-binding sites on arterial LBP molecules. Mouse monoclonal antibodies are humanized by recombinant DNA techniques and produced by standard procedures known to those skilled in the art (Berkower, Curr. Opin. Biotechnol.

20 7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)) against SLBPs and/or.the LDL-binding sites on the LBPs. The corresponding Fab fragments are also produced, as described in Goding, Monoclonal Antibodies:Principles and Practice, Academic Press, New York, NY (1986). These antibodies are administered parenterally in sufficient quantity so as to block LDL-binding sites on the LBP molecules, 1-10 mg/kg daily. This prevents the irreversible arterial uptake of LDL that is required to facilitate oxidation ofthe LDL.

S.Exmple 13: Preparation of LDL This example illustrates the preparation of LDL. LDL was prepared from the plasma of normolipemic donors (Chang-et al., Arterioscler. Thromb. 12:1088-1098 (1992)). 100 ml of whole blood was placed into tubes containing 100 mM disodium EDTA. Plasma was separated from red blood cells By low-speesdcentrifugation (2,000 g; 30 min; Plasma density was adjusted to 1.025 gm/ml with a solution of KBr and centrifuged for 18-20 hr, 100,000 xg, 12 0

C.

-42- Very low density lipoproteins (VLDL) were removed from the tops of the centrifuge tubes with a Pasteur pipet The density of the infranate was raised to 1.050 gm/ml with KBr solution and centrifuged for 22-24 hr, 100,000 x g, 12°C. LDL was removed from the tops of the centrifuge tubes with a drawn out Pasteur pipet tip. Purity of the LDL preparation was checked by Ouchterlony double immunodiffusion against antibodies to human LDL, human HDL, human immunoglobulins, and human albumin. KBr was removed from the LDL solution by dialysis (1L, x 2, 16 hr) against 0.9% saline, pH 9.0, containing 1 mM EDTA and 10 pM butylated hydroxytoluene (BHT), the latter to prevent oxidation of LDL. Following dialysis, LDL protein was measured by the method of Lowry (Lowry et al., J. Biol. Chem. 193:265-275 (1951)), and io the LDL was stored at 4 0 C until use. LDL preparations were kept for no more than 4-6 weeks.

Example Prearation of HDL This example illustrates the preparation of HDL. HDL was prepared from plasma of normolipemic donors. 100 ml of whole blood was placed into tubes containing 100 mM disodium EDTA and plasma was collected by centrifugation (2000 g; 30 min; 4*C).

Apolipoprotein B containing lipropoteins present in plasma were then precipitated by the sequential addition of sodium heparin (5,000 units/ml) and MnCl, (1M) to achieve a final concentration of 200 unitsml and 0.46 M, respectively (Warnick and Albers, J. Lipid Res.

19:65-76 (1978)). Samples were then centrifuged (2000 g; I hr, The supernatant was 20 collected and density adjusted to 1.21 g/ml by the slow addition of solid KBr. HDL was separated by ultracentrifugation (100,000 g; >46 hr; 12°C). Purity of the HDL preparation was assessed via Ouchterlony double immunodiffusion test using antibodies against human HDL, Shumnan LDL, human immunoglobulins, and human albumin.- HDL samples were dialyzed against saline pH 9.0/1mM EDTA/10pM BHT (4L; 24 hr/4°C) and total protein was determined by the Lowry protein assay (Lowry et al., J. Biol. Chem. 193:265-275 (1951)). HDL was stored at 4°C until use. HDL preparations were kept for no longer than 2 weeks.

Example 15: Synthesis of BHF- This example illustrates the synthesis of BHF-1, a fragment of human or rabbit LBP-1 which contains amino acid residues 14 through 33. BHF-1 was synthesized using an Applied Biosystems Model 430A peptide synthesizer with standard T-Boc NMP chemistry cycles. The sequence of BHF-l is as follows: -43val-asp-val-asp-glu-tyr-asp-glu-asn-lys-phe-val-asp-gluglu-asp-gly-gly-asp-gly (SEQ ID NO:9) After synthesis, the peptide was cleaved with hydrofluoric acid/anisole (10/1 v/v) for 30 min at 10*C and then incubated for 30 min at 0*C. BHF-1 was then precipitated and washed three times with cold diethyl ether. Amino acid coupling was monitored with the ninhydrin test The BHF-1 peptide was purified to homogeneity by high performance liquid chromatography on a reverse phase Vydac C 4 column (2.24 X 25 cm) using a linear gradient separation (2-98% B in 60 min) with a flow rate of 9 ml/min. Buffer A consisted of 0.1% -iiifluoroacetic acid (TFA)/Milli Q water and Buffer B consisted of 0.085% acetonitrile. The gradient was run at room temperature and absorbance monitored at 210 and 277 nm.

Fast atom bombardment-mass spectrometry gave a protonated molecular ion peak at m/z= 2290.2, in good agreement with the calculated value. On amino acid analysis, experimental values for the relative abundance of each amino acid in the peptide were in good agreement with theoretical values. The lyophilized peptide was stored at Example 16: In Vitro Screening for Agents Which Inhibit Binding Between-LDL and LBPs This example illustrates in yitr screening for agents which inhibit binding between LDL and LBPs.

A candidate polypeptide for being an agent is chosen, LBP-1, LBP-2, LBP-3, BHF-1 or any other polypeptide. The shortest fragment of the polypeptide that inhibits LDL binding to 25 LBPs in itro is determined. Peptides are synthesized by standard techniques described herein.

Inhibition assays are performed using standard ELISA techniques for screening, and affinity coelectrophoresis (ACE) assays to confirm the ELISA results, as described herein. Short peptides ranging, from dimers to 20-mers are constructed across sequences of the candidate polypeptide whose chemical characteristics make them likely LDL binding sites, acidic regions. The ability of shorter and shorter lengths of the peptides to inhibit LDL binding in vito and to mammalian cells in culture is tested. For example, the effect of the peptide on inhibiting LDL binding in mammalian cells transfected to express an LBP gene is tested. Each of the peptides so identified as an inhibitor is tested with each ofLBP-1, LBP-2 and LBP-3, to -44determine whether a single inhibitor works against all three LBPs. Once the minimum active sequence is determined, the peptide backbone is modified so as to inhibit proteolysis, as discussed herein. For example, modification is accomplished by substitution of a sulfoxide for the carbonyl, by reversing the peptide bond, by substituting a Methylene for the carbonyl group, or other similar standard methodology. Se Spatola, A.F., "Peptide Backbone Modifications: A Structure-Activity Analysis of Peptides Containing Amide Bond Surrogates, Conformational Constraints, and Related Backbone Replacements," in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B.

Weinstein Marcel Dekker, Inc., New York (1983). The ability of these analogs to inhibit LDL binding to the LBPs in vitr is tested by ELISA and ACE assays in a similar manner asffor the natural peptides described above.

Example 17: In Vitro Screening With Cultured Mammalian Cells for Agents Which Inhibit Binding Between LDL and LPBs This example illustrates cell-based in 3itr screening of agents which have been shown by in vitr tests such as ACE assay and ELISA to be potential inhibitors of binding between LDL and LBPs.

Mammalian cells, such as 293 cells, which are commonly used for expression of 20 recombinant gene constructs, are-used to develop cell lines which express LBPs on the cell S. surface. This is done by subcloning LBP open reading frames (ORFs) into a mammalian expression plasmid vector, pDisplay (Invitrogen, Carlsbad, CA), which is designed to express the gene of interest on the cell surface. The use of mammalian cells to produce LBPs allows for their expression in a functionally active, native conformation. Therefore, stably transfected mammalian cell lines with surface expression of LBPs individually, or in combination, are particularly suitable for assaying and screening inhibitors that block LDL binding in cell culture, as well as to evaluate the cytotoxicity of these compounds.

A Specifically, LBP ORFs are amplified by PCR (Perkin Elmer, Foster City, CA) from cDNA templates using Taq polymerase (Perkin Elmer) and appropriate primers. The amplified LBP ORFs are purified by agarose gel electrophoresis and extracted from gel slices with the Bio- Rad DNA Purification kit (Bio-Rad, Hercules, CA). The purified DNAs are then cut with the restriction enzymes Bgl II and Sal-I (New England Biolabs, Beverly, MA) to generate cohesive ends, and purified again by agarose gel electrophoresis and DNA extraction as described above.

The LBP ORFs are then subcloned into the Bgl II/Sal I sites in the mammalian expression vector, pDisplay (Invitrogen) by ligation. Recombinant plasmids are established by transformation in E.li strains TOP10 (Invitrogen) or DH5 a (Life Technologies, Grand Island, NY). Recombinant pDisplay/LBP plasmid DNA is isolated from overnight E.co cultures with the Bio-Rad Plasmid Miniprep kit, cut with Bgl II/Sal I, and analyzed by agarose ge electrophoresis. LBP ORFs in successfully transformed clones are verified by automated dideoxy DNA sequencing. To transfect human kidney 293 cells, 1-2 ig of DNA is mixed with 6 pi lipofectamine reagent (Life Technologies) and incubated with the cells-as described in the Life Technologies protocol. LBP expression in transfected cells is confirmed by Western blot S0 analysis of cell extracts obtained 48 hr after transfection. To select for stably transfected 293 cells, the antibiotic G418 (Life Technologies) is added to the gfowth medium at a concentration of 800 pg/ml. Colonies resistant to G418 are tested for recombinant LBP expression by Western blot, and recombinant clones expressing LBPs are expanded, assayed for LDL binding and used to test compounds for their ability to inhibit LDL binding.

Exampe 18: In Vivo Screening for Agents Which Inhibit Binding Between LDL and LBPs This example illustrates in viv screening of agents which have been shown by in 3itn tests to be promising candidate inhibitors of binding between LDL and LBPs.

20 In vivo inhibitory activity is first tested in the healing balloon-catheter deendothelialized rabbit aorta model of arterial injury (Roberts et al., J. Lipid Res. 24:1160-1167 (1983); Chang et al., Arterioscler. Thomb. 12:1088-1098 (1992)). This model-was shown to be an excellent analog for human atherosclerotic lesions. Each candidate inhibitor is tested in five to ten ballooned rabbits, while an equal number of rabbits receive a control peptide, or placebo- Four weeks following aortic deendothelialization, when reendothelialization (healing) is partially complete, daily parenteral (intravenous or subcutaneous) or intragastric administration of the peptides and the analogs begins at an initial concentration of 10 mg/kg body weight, which is varied down, or up to 100 mg/kg depending on results. 30 min later, a bolus of intravenously injected '2sI (or labeled LDL is given to test the candidate inhibitor's ability in short term studies to inhibit LDL sequestration in healing arterial lesions. If 25 I-LDL is used, the animals are sacrificed 8-24 hr later, the aortas excised, washed and subjected to quantitative autoradiography of excised aortas, as previously described (Roberts et al., J. Lipid Res. 24:1160- 1167 (1983); Chang et al., Arterioscler. Thomb. 12:1088-1098 (1992)). If ""Tc-LDL is used, -46analysis is by external gamma camera imaging of the live anesthetized animal at 2-24 hr, as previously described (Lees and Lees, Syndromes of Atherosclerosis, in Fuster, ed., Futura Publishing Co., Armonk, NY, pp. 385-401 (1996)), followed by sacrifice, excision and imaging of the excised aorta. Immediately before the end of testing, the animals have standard toxicity tests, including CBC, liver enzymes, and urinalysis.

The compounds which are most effective and least toxic are then tested in short term studies of rabbits fed a 2% cholesterol diet (Schwenke and Carew, Arteriosclerosis 9:895-907 (1989)). Each candidate inhibitor is tested in five to ten rabbits, while an equal number of rabbits receive a control peptide, or placebo. Animals receive one or more doses per day of the candidate inhibitor, or placebo, for up to two weeks. Daily frequency of doses is determined by route of administration. If active drug or placebo are administered parenterally, they-are given 1- 3.times daily and the 2% cholesterol diet is continued. If drug or placebo are given orally, they are mixed with the 2% cholesterol diet Schwenke and Carew (Arteriosclerosis 9:895-907 (1989)) have shown that the LDL concentration in lesion-prone areas of the rabbit aorta is increased 22-fold above normal in rabbits fed a 2% cholesterol diet for 16 days, and that the increased LDL content precedes the histological evidence of early atherosclerosis. Therefore, analysis of the effect of the candidate inhibitors is tested two weeks after the start of cholesterol feeding by injecting '"I-LDL, allowing it to circulate for 8-24 hr, and then performing quantitative autoradiography on the excised aortas of both test and control animals. If 20 appropriate, quantitation of aortic cholesterol content is also carried out (Schwenke and Carew, Arteriosclerosis 9:895-907 (1989); Scwenke and Carew, Arteriosclerosis 9:908-918 (1989).

The above procedures identify the most'promising candidate inhibitors, as well as the best route and frequency of their administration. Inhibitors so identified are then tested in longterm studies of cholesterol-fed rabbits. These tests are carried out in the same way as the short- 25 term cholesterol feeding studies, except that inhibitor effectiveness is tested by injection of 125- LDL at longer intervals following the initiation of cholesterol feeding, and lesion-prone areas of the aorta are examined histologically for evidence of atherosclerosis. Testing times are at two, four, and six months. Major arteries areexamined grossly and histologically for evidence and extent of atherosclerosis. If necessary, other accepted animal models, such as atherosclerosissusceptible primates (Williams et al., Arterioscler. Thromib. Vase. Biol. 15:827-836 (1995) and/or Watanabe rabbits are tested with short- and long-term cholesterol feeding.

-47- Example 19: In Vivo Inhibition of Radiolabeled LDL Accumulation in the Ballooned Deendothelialized Rabbit Aorta via Induction of Active Immunity Against LBP Protein.

This example illustrates the effect that induction of immunity against LBP protein has on the accumulation of radiolabeled LDL in the ballooned deendothelialized rabbit aorta model of atherosclerosis.

Immunity was induced in male New Zealand White rabbits (Hazelton Research Products, Denver, PA) as follows: A mixture of purified human recombinant LBP-2 or BHF-1 peptide (1 io ml; 1 mg) and RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, MT) was injected subcutanously at 2-5 sites along the dorsal thoracic and abdominal regions of the rabbits. Blood was collected by venipuncture on days 1 (preimmune bleeding), 35, 63, and 91. Booster injections were administered on days 28 (500 pg; SC), 56 (250 pg; SC), and 84 (125 pg; SC).

The titer of the rabbits was evaluated by serial dilution using an ELISA plate format.

Preimmune serum was evaluated at the same time. After the third booster of LBP protein or peptide, the titer reached a maximal level with a detectable colorimetric response on an ELISA plate of 156 pg. Titer is defined as the maximum dilution of antibody which generates an absorbance reading of 0.5 above control in 30 min. Specificity of the polyclonal antibodies was demonstrated using Western blot analysis as described in Example 6.

20 On day 93, the abdominal aorta of immunized and control rabbits was deendothelialized S using a Fogarty number 4 embolectomy catheter (Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). Four weeks after ballooning, rabbits received a bolus injection of 125I labeled LDL (1 ml; Blood samples were collected atTfrintervals for 8 hr, and 24 hr post injection. Blood samples were centrifuged for 30 min at 2000 rpm (40*C) and total activity present in the serum was determined using.a Gamma counter. Total TCA precipitable counts were determinined by addition of TCA to the serum to a final concentration of 10% followed by incubation for 10 min at 4°C. Serum samples were then centrifuged (2000 rpm; 30 min; and total activity present in the supernate was determined. TCA precipitable counts were calculated by substration: total soluble counts minus counts present in the supernate after TCA precipitation. Blood samples for the determination of antibody titers were collected priorto the injection of the radiolabeled LDL.

S After 24 hr, the rabbits were injected intravenously with 5% Evan's blue dye which was allowed to circulate for 15 min. Areas of the aorta in which the endothelial covering is absent stain blue while those areas covered by endothelium remain unstained. At the end of the incubation period, the rabbits were euthanized and the abdominal and thoracic aorta were dissected out, rinsed, and fixed overnight in 10% TCA at room temperature. The aortas were then rinsed exhaustively with physiological saline, weighed, counted, blotted dry and placed onto X-ray film in order to visualize the pattern of radiolabeled LDL accumulation in the deendothelialized rabbit abdominal aorta.

Immunization of rabbits against recombinant human LBP-2 or BHF-1 peptide altered the pattern of radiolabeled LDL accumulation in the ballooned deendothelialized abdominal aorta.

When corrected for dosage,,and percent reendothelialization, immunized-ballooned rabbits had lower accumulation of radiolabeled LDL compared to nonimmune-ballooned rabbits. These results indicate that active immunization against LBP provides an effective means by which-the accumulation of LDL in the injured arterial wall can be modified.

Example 20: Screening Agents in Humans Wicdh Inhibit Binding Between LDL and LBPs" Human studies are carried out according to standard FDA protocols for testing of new drugs for safety (Phase efficacy (Phase II), and efficacy compared to other treatments (Phase III). Subjects, who are enrolled into studies after giving informed consent, are between the ages of 18 and 70. Women who are pregnant, or likely to become pregnant, or subjects with diseases 20 other than primary atherosclerosis, such as cancer, liver disease, or diabetes, are excluded.

Subjects selected for study in FDA Phase II and Phase III trials have atherosclerotic disease previously documented by standard techniques, such as ultrasound and/or angiography, or are known to be at high risk of atherosclerosis by virtue of having at least one first degree relative with documented atherosclerosis. Subjects themselves have normal or abnormal plasma-lipids.

Initial testing includes 20-50 subjects on active drug and 20-50 subjects, matched for age, sex, and atherosclerotic status, on placebo. The number of subjects is pre-determined by the number needed for statistical significance. Endpoints for inhibitor efficacy includes ultrasound measurements of carotid artery thickness in high risk subjects, as well as in subjects with known carotid or coronary disease; atherosclerotic events; atherosclerotic deaths; and all-cause deaths in all subjects. Non-invasive analysis (carotid artery thickness by ultrasound) as per Stadler (Med.

and Biol. 22:25-34 (1996)) are carried out at 6- to 12-month intervals for 3 years.

Atherosclerotic events and deaths, as well as all-cause deaths are tabulated at 3 years.

Oral dosage of drug in FDA Phase I trials ranges from 0.01 to 10 gm/day, and is -49determined by results of animal studies, extrapolated on a per kg basis. Based on data obtained from Phase I studies, the dose range and frequency are narrowed by Phase II and I trials. If parental administration of drug is determined by animal studies to be the only effective method, parental administration in human subjects is tested by injection, as well as by the transdermal and nasal insufflation routes. Testing of parental drug follows the same outline as that for oral administration.

The optimal treatment schedule and dosage for humans is thus established.

Example 21: Treating an Individual Having Atherosclerosis with BHF- 1 This example illustrates a method for treating an individual having atherosclerosis with an LBP fragment, BHF-1, so as to decrease the levels of arterially bound LDL in the individual.

BHF-1I is obtained as described therein. The BHF-1 is administered to the mammal intravenously as a bolus or as an injection at a concentration of 0.5-1.0 mg/kg body weight. Such administrations are repeated indefinitely in order to prevent the development or progression of symptomatic atherosclerosis, just as is done currently with cholesterol-lowering drugs. Stable subjects are "examined twice yearly to evauate the extent of any athereosclerotic disea y physical exam and "non-invasive studies, such as carotid artery thickness, ultrasound, and/or gamma camera imaging of "":the major arteries, to determine if atherosclerosis lesions are present, and, if previously present, have regressed or progresses. Such a regimen results in treatment of the atherosclerosis; Those skilled in the art will be able to ascertain using no more than routine oo*o*experimentation, many equivalents of the specific embodiments of the invention described herein.

These and all other equivalents are intended to be encompassed by the following claims.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

~Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

EDITORIAL NOTE APPLICATION NUMBER 83641/01 The following Sequence Listing pages 50 to 87 are part of the description. The claims pages follow on pages 88 to 94.

SEOUENCE LISTING GENERAL INFORMATION: APPLICANT: Lees, Ann M.

Lees, Robert S.

Law, Simon W.

Arjona, Anibal A.

(ii) TITLE OF INVENTION: NOVEL LOW DENSITY LIPOPROTEIN

BINDING

PROTEINS AND THEIR USE IN DIAGNOSING AND TREATING

ATHEROSCLEROSIS

(iii) NUMBER OF SEQUENCES: 42 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Banner Witcoff Ltd.

STREET: One Financial Center CITY: Boston STATE: MA COUNTRY: USA (F)ZIP: 02111 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 and WordPerfect 6.1 CURRENT APPLICATION DATA: APPLICATION NUMBER: Not available S FILING DATE: November 26,1997 CLASSIFICATION: Not available (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Greer, Helen REGISTRATION NUMBER: 36,816 REFERENCE/DOCKET NUMBER: 3983/59819 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-345-9100 TELEFAX: 617-345-9111 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 151 amino acids TYPE: amino acid -Si1- TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Ser Lys Asn Thi. Val Ser Ser Ala Arg Phe Arg Lys Val Asp Val 1 510 Asp (flu Tyr Asp Giu Asn Lys Phe Val Asp Glu Glu Asp Gly Gly Asp 25 Gly Gin Ala Gly Pro Asp Glu Gly Glu Val Asp Ser Cys Leu Arg Gin 40 Gly Asn Met Thr Ala Ala Leu Gin Ala Ala Leu Lys Asn Pro Pro Ile 55 Asn Thr Arg Ser Gin Ala Val Lys Asp Arg Ala Gly Ser Ile Val Leu 70 75 Lys Val..Leu Ile Ser Phe Lys Ala Gly Asp Ile Glu Lys Ala Val Gin.

90 Ser Leu Asp Arg Asn Gly Val Asp Leu Leu Met Lys Tyr Ile Tyr Lyi 100 105 110 Gly Phe Glu Ser Pro Ser Asp Asn Ser Ser Ala Val Leu Leu Gin Trp, -115 120 125 His Glu Lys Ala Leu Ala Ala Gly Gly Val Giy Ser Ile Val Arg Vai 130- 13514 Leu Thr Ala Arg Lys Thr Val .*145 150 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 317 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 0*Asp Cys Arg Ser Ser Ser Asn Asn Arg Xaa Pro Lys Gly Giy Ala Ala 1 10 Arg Ala Gly Gly Pro Ala Arg Pro Val Ser Leu Arg Glu Val Val Arg -25 Tyr Leu Giy Gly Ser Ser Gly Ala Gly Giy Arg Leu Tbhr Arg Giy Arg 40 -52- Val Gin Gly Leu Leu Giu Glu Glu Ala Ala Ala Arg Gly Arg Leu Giu Ar!4 Thr Arg Ala Gly Giu -Asp Asp Pro Glu 130 Gly Glu 145 Leu Cys Gly Ser *Gly Gly Pr6 Leu 210 Phe-- Gly 225 Trp _Thr Glu -Gin Leu Leu Gly Pro 290 Gly His 305 Arg Pro Glu Glu Ser Arg Gly Gly Ser 195 Pro Cys Val Ala Met 275 Ala Phe Leu Gly Pro Ala Arg Val -100 Asp Asp Asp Arg Gly Pro Pro His Thr Arg 180 Ala Ser Pro Gly Pro Ala Met Asp 245 'Dhr Ala 260 Gin Arg Leu Lys Ala Leu Ala 70 Ala Ser Ala Leu Giu Lys Asp Asp Asp 120 Pro Ala Gly 135 Gin Thr Ala 150 Pro Gly Gin Gin Vai Phe Ser Thr Thr 200 Lys Pro Ala 215 Gly Arg Lys 230 Val Val Giu Phe Gin Giu, Thr Asp Val 280 le'Tjr Giu __295 Leu Arg Glu 105 Val Al a Lys Glu Ser 1,85 Gly Leu Glu Tyr7 Gin 265 Leu His Pro Arg Gly AspArg 75 Ala AlaArg Asn Lys 90 Giu Giu Glu Giu Giu 110 Val Ser Gi u Gly Ser 125 Gin His His.Gin Leu 140 Glu Arg Ala Lys Giu 155 Giu Gly Arg Gly Pro 170 Met Ala Ala Leu Ser 190 Pro Asp Ser Pro Ser 205 Pro Gly Ala Asp Gly 220 Lys- Pro Ala Asp Pro 235 Phe Thr Glu Ala Gly 250.

Giu Ile Asp Gly Lys 270 Thr Gly Leu Ser Ile 295 His Ile Lys-Val Leu 300 Pro Arg Glu Asn Trp Ala 175 Lys Pro Thr Val Phe 255 Ser Arg Gin Giy Ala Giu Val Gly .Ser 160 Ala Glu Val pro Glu 240 Pro Leu Leu Gin .a *a

S

Giu Asp Asp Asp Pro Giu Giy Phe.Leu Gly 310 315 INFORMAT1ON FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 232 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly Giu Glu Arg Va-1 Leu Asp Pro Gin Pro Gin Ser Lys Gly 145 Val Phe Thr Ile Glu Lys'Giu Asp Asp Val Ala Gly Ala Thr Ala Lys Gly Gin Glu Val Phe Ser 100 Thr Thr Gly 115 Pro Ala Leu 130- Arg Lys Glu Val Glu Tyr Gin Giu Gin 180 Asp Val Leu 195 Tyr Glu His 210 Glu Val Gin Giu Giu- Met Pro Pro Lys Phe 165 Glu Thr His Glu Ser His Arg 70 Gly Ala

ASP

Gly Pro 150 Thr Ile Gly Ile Glu Glu- His 55 Ala Arg Ala Ser Ala 135 Ala Glu Asp Leu Lys 215 Glu Giy 40 Gin Lys Gly Leu Pro 120 Asp Asp Ala Gly Ser 200 Val Giy Giu 25 Ser Leu Glu Pro Ser 105 Ser Gly Pro Gly Lys 185 Ile Giu Giu Asn Trp Ala 90 Lys Pro Thr Val Phe 170 Ser Arg Glu Val Gly Ser 75 Ala Glu Vai Pro Glu 155 Pro Leu Leu

ASP

Pro Gly Leu Gly_ Gly Pro Phe 140 Trp Giu ILeu G)ly Gly 220 Asp Giu Giu Cys Ser Giy Leu 125 Gly Thr Gin Leu Pro 205 Giu Ser Arg Gly Gly Ser 110 Pro.

Cys Vai Ala Met 190 Ala Asp Asp Gly Pro Thr Ala Pro Pro Met Thr 175 Gin Leu

ASP

Arg Pro His Arq Ser Glk Ala Asp 160 Ala Arg Lys is Leu Gin Gin His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu 225 230 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 252 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTON: SEQ ID, NO*4 Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly Arg 1 5 1015 Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly .25 Giu Glu Arg Val Leu Glu Lys Giu Glu Glu Glu Giu Glu GlUT G-u Asp 40 Asp Giu Asp Asp Asp Asp Asp Val Val Ser Glu Gly Ser Glu Val Pro s0 55. Glu Ser Asp Arg Pro Ala Gly Ala Gin His His Gin Leu Asn Gly Gly 70 75 Glu Arg Giy Pro Gin Thx Ala Lys Glu Arg Ala Lys Glu Trp Ser.Leu 90 Cys Gly Pro His Pro Gly Gin Glu Glt Gly Arg Gly Pro Ala Ala Gly 100 105' 110 Ser -Gly Thr Arg Gin Val Phe Ser Met Ala Ala Leu Ser Lys Glu Gly *115 120 125 Gly Ser Ala Ser Ser Th~r Thr Gly Pro Asp Ser -Pro Ser Pro Val Pro 130 135 140 Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe -145 150 *.155 160 Gly Cys Pro Ala Gly Arg Lys. JGlu Lys Pro Ala Asp Pro Val Giu Trp 1 170 175 Thr Val Met Asp Val Val Glu Tyr Pile Thir-.Glu Ala Gly Phe Pro Glu *180 185 190 Gin Ala Thr Ala Pile Gin Giu Gin Glu Ile Asp Gly- Lys Ser Leu Leu 195 200 205 Leu Met Gin. Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly 210 215 220 Pro Ala Leu Lys Ile Tyr Giu His His Ile Lys Val Leu Gin Gin Gly 225 230 235 240 His Phe Giu Asp Asp Asp Pro Giu Gly Phe Leu Gly 245 250 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 557 amino acids TYPE: amino acid TOPOLOGY: linear "xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Asn Gin Asp Lys Lys Asn Gly Ala-Ala Lys Gin Pro Asn Pro S Arg Gin Gly 65 Ile Asp Arg Val Ile 145 Glu Ser Pico Ala Ala Leu Giy Ala Asn 130 Arg Lys Ser Pro Gly Arg Pro Giy Leu. Cys Ser Thr Vai Gin 100 Tyr Val 115 Gly Giu Thr Ser Lys Lys Gly Pro Arg Asp Tyr 85 -dly Ala Lys Asp Ala 165 Gin- Ala Pro Val 70 Cys Glu Arg Glu' Giu Lys Pro Pro Glu 55 Ser Val Pro Asn, Thr 135 Val Giy Giu Ala 40 Gly Giu Asp Pro Gly 120 Ser Gly Leu Ala 25 Arg Ala Glu Glu 105 Giu Lys Asp_ Gly Gly Glu Gin Leu Asn 90 Pro Pro Ala Arg Lys 170 Ala Mla Mla Ser 75 Gin Glu Giu Glu Asp 155 Giu Glu Giu Lys Arg Gly Asp Pro Pro 140 His Ile Gly Gly Thr Gin Mla Ala Gly 125 Gly Arg Thr Ala Gin Ala Ser Ala Gin Leu Glu Pro Giy Glu Lys 110 Thr Pro Thr Giu Arg Pro Leu Leu 175 Gly Ser Pro Asp Giu Ser Val Giu Gin- 160 Met Gin Thr Leu 180 Thr Leu Ser Thr Glu Giu Lys Leu Mla Ala Leu 190 -56- Cys Lys Lys Tyr Ala Gilu Leu Leu Giu Giu His Arg Asn Ser Gin Lys Gin Asp 225 Leu Giu Val Glu Leu 305 Giu Val Ser Gin 385 Leu Lys Arg Glu Met 210 His Giu Giu Thr Gin 290 Ala His Asp Arg Gin 370 Gin Ser met Ser Lys 450 195 Lys Leu Ser Giy Ser 275 His Glu Ile.

Ala Hi.s 355 Arg Leu Lys Thr Arg 435 Thr Leu Arg Leu Val 260 His Asn Arg Asp Lys 340 Gin Met Ala Ser Lys 420 Tr p Leu Leu Gin Giy Giu 230 Cys Arg- 245* Gin Arg Phe Gin Giu Arg Leu Lys 310 Lys Val 325 Leu Gin Arg' Giu Cys Giu Leu Tyr 390 Ser Giu 405 Ly's Ile Glu Ser Arg Asp Lys 215 His Giu Ala Met Asn 295 Lys Phe Gin Lys Leu 375 Thr Val Lys Ser Lys 455 200 Lys Ser Leu Arg Thr 280 Ser Leu Lys Ala Asp 360 Met Giu Phe Lys Asn 440 Glu 205 Gin Ser Lys Ala Gin Arg 250 Giu Giu 265 Leu Asn Lys Leu Ile Giu His Lys 330 Gin Giu 345 Phe Leu Lys Gin Lys Phe Thr Thr 410 Leu Giu 425 Lys Ala Leu Glu 4

C

C. eC

C

C

'C C C,.

*4 C C C C

C.

Gin Leu'Vai 220 Ile Leu Ala 235 His Asn Arg Giu Giu Lys Asp Ile Gin 285 Arg Gin Giu 300 Gin Tyr Giu 315 Asp Leu Gin Met Leu Lys Leu Lys Giu 365 Gin Giu Thr 380 Glu Glu Phe 395 Phe.-Lys Gin Lys Giu Thr Leu Leu Giu 445 Gly Leu Gin 460 Gin Arg Ser Arg 270 Leu Asn Leu Gin Giu 350 Ar&a His Gin Giu Thr 430 Met Val Giu Lys Ser Lys 240 Leu Lys 255 Lys Gill Gin Met Met Giu Arg Giu 320 Gin Leu 335 Al A 'Giu Val Giu Leu Lys Asn Thr 400 Met Glu 415 Met Tyr Ala Glu Lys Ile Gin Arg Leu Giu Lys-Leu Cys Arg Ala Leu Gln Thr Giu Arg Asn Asp 465 475 480 -57- Leu Asn Lys Arg Val Gin Asp Leu Ser 485 Sex Asp Ser Gly Pro Glu Arg Arg Pro 500 505 Glu Gin Gly Val Giu Gly Pro Gly Ala 515 520 Ala.Thr Asp Ala Ser Cys Cys.Ala Gly 530 535 Gly Gin Thr Gly Pro Gin Glu Pro Thr 545 550 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 151 amnino acids TYPE: amino acid TOPOLOGY: l inear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ser Lys Asn Thr Val Ser Ser Ala Ala 490 -Giu Gin Ala Thr Arg 10 Asp Val Ala.

Arg Asp 90 Leu Ser Gly Pro Val Pro Ala 555 Phe Giu Asp Leu Ala 75 Ile Met Ala Gly Ala Pro Ser 540 Thr Arg Glu Ser Lys Gly Glu Lys Met Gin Gly Pro Val 495 Thr Thr Ser Lys 510 Asn Ser Pro Arg 525 Thr Glu Ala Ser Ala S 1 Asp Gly Gly Asn Lys Ser Gly Giu Gin Asn 50 Thr Val Leu Phe Tyr Ala 35 Met Lys Leu Asp Glu 115 Asp 20 Gly Thr Ser Ile Lys 100 Ser

S

Giu Pro Ala Gin Ser 85.

Asn Pro Asn Asp Ala Ala 70 Phe Gly Ser Lys Glu Leu 55 Val Lys Val Asp Plie Gly 40 Gin

LYS

Ala Asp Asn 120 Val 25 Giu Ala Asp Asn Leu 105 Ser Lys Asp Cys Asn Ser Lys Tyr Leu 125 Val Asp Val is Gly Gly Asp Leu Arg Gin Pro Pro Ile Ile Val-.Leu Ala Val Gin Ile Tyr Lys 110 Leu..Gln Trp His Glu Lys Ala Leu Ala Ala Gly Gly Val Gly Ser Ile Val Arg Val 130 135 140 -58- Leu Thr Ala Arg Lys Thr Val 14 5 150 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERJSTICS: LENGTH: 217 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Glu Glu Azrg Val Leu Glu Lys Giu Giu 1 Glu

ASP

Pro His 65 Arg Ser Gly Pro Asp 145 A14 Arg Asp Arg Gin Gin Gin Val Lys Gly 130 Val Phe Thr Glu Pro Ser Gly Val Ala Pro 113s Arg Val Gin Asp Giu Ala Ala Gin Phe Thr 100 Ala Lys Glu Glu Val 180 5

MP

Gly

LYS

Asp Ser Gly 'Leu Giu Tyr Gin 165 Leu Asp Val Ala Gin Glu Arg 55 Glu Gly 70 Met Ala Pro Asp Pro Gly Lys Pro.

135 Phe Thr 150 Giu Ile Thr Gly Ser His 40 .Val Arg Ala Ser Ala 120 Ser Giu Asp Leu Lys 200 Giu 25 His Lys Gly Met Pro 105 Asp Asp Ala Gly Ser 185 Giu Gly Gin G1u Pro Asn 90 Ser Gly Pro Gly Lys 170 Ile Glu Ser Leu Trp Ala 75 Lys Pro Thr Val Phe 155 Ser Arg Asp Glu Asn Thr Pro Giu Val Pro Giu 140 Pro Leu Leu Asp Asp Giu Val Pro Giu Gly Giu Arq Pro Cys Gly Gly Ser Gly Gly Gly Thr Pro Leu Pro 110 Phe Gly Cys 125 Trp Thr Val Giu Gin Ala Leu Leu Met 175 Gly Pro Ala 190 Asp Ser Gly Pro Thr Ala Pro Pro Met Thr 160 Gfi Leu Lys Ile Tyr 195 Giu His His Ile Val Leu Gin Gin Gly His Phe Giu 205 -59- Asp Asp Asp Pro Asp Gly Phe Leu Gly 210 215 INFORMATION FOR-SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 530 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Lys Ser Ser Pro Gly Gin Pro Giu Ala Gly Pro Giu Gly Ala Gin Giu 1 5 10 Arg Pro Ser Gin Ala Ala Pro Ala Val Glu Ala Glu Gly.Pro Gly Ser 25 Ser Gin Ala Pro Arg Lys Pro Glu Gly Ala Gin Ala Arg Thr Ala Gin _40 Ser Gly Ala Leu Arg Asp Val Ser Glu Giu Leu Ser Arg Gin Leu Glu 55 Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gin Gly Gly Pro Gly 9...65 70 75 Giu Asp Gly Ala Gin Gly Glu Pro Ala Glu Pro Glu Asp Ala Glu Lys 90 *Ser Arg Thr Tyr Val Ala Arg Asn Gly Giu Pro Glu Pro Thr Pro Val 9...100 105 .110- Val Tyr Gly Gli Lys Giu Pro Ser Lys Gly Asp PLro Asn Thr Glu Glu 115 120 125 Ile Arg..Gln Ser Asp Glu Val Gly Asp Arg Asp His Arg Arg Pro Gin 130 135 140 -Glu Lys Lys Lys Ala Lys Gly Leu Gly Lys Giu Ile Thr Leu Leu Met *145 150 15- 160 Gln Thr Leu Ann Thr Leu Ser Thr Pro. Glu Giu Lys Leu Ala Ala Leu 165 1170 175 Cys Lys Lys Tyr Ala -Giu Leu Leu Giu Glu His Arg Ann Ser Gin Lys 180 185 190 Gin Met Lys Leu Leu Gin Lys Lys Gin- Ser Gin Leu-Val Gin Glu Lys 195 200 205 Asp Leu 225 Giu Val Giu Leu Giu 305 Val Giu Ser Gin Leu 385 Lys Arg Glu Gin -Leu 465 His 21 0 Giu Giu Thr Gin Ala 290 His Asp Arg.

Gin Gin 370 Ser Met.

Ser

LYS

Arg 450 Asn Leu Ser Gly Ser His 275 Glu Ile Ala His Arg 355 Leu Lys Thr Arg Thr 435 Leu Lys Arg Leu Val His* 260 Asn Arg Asp Lys Gin 340 Met Ala Ser Lys Trp 420.

Val Glu Arg Giy Giu-His Set Lys Ala Val Leu Ala Arg Ser Lys Cys Arg 230 Gin Arg 245 Phe Gin Gilu Arg Leu Lys Lys Val 310 Le;GF-ln 325 Arg Giu Cys Giu Lou Tyr Sor Giu 390 Lys Ile .405 Giu Ser Arg Asp Lys Leu Val Gin 470 215 Giu 6 0 8*0S6 **go :000fS 0@@0 0*0* 5 0 00 556.

go Ala Val Asn- Lys 295 Phe Gin Lys Leu Thr 375 Vai Lys Ser Lys *Cys 455 Asp Leu Gin Arg Arg Glu Giu 250 Thr Leu Asn 265 Ser Lys Lou .280 Leu Ile Giu Lys-His Lys Ala Gin Giu '330 Asp Phe Lou 345 Met Lys Gin 360 Giu Lys Pho Phe Thr Thr Lys Lou Giu 410 Asn Lys Ala 425 Giu Leu Giu 440 Arg Ala Lou Leu Ser Ala 220 fi s Asn.

235 Giu Glu Asp Ile Arg Gin Gin Tyr 300 Asp Lou 315 Met Leu Lou Lys Gin Giu Giu Giu 380 Phe Lys 395: Lys Giu Lou Lou Gly Lou Gin Thr 460 Gly Gly 475 Arg Lys Gin Giu 285 Giu Gin Lys Giu Thr 365 Phe Gin Thr Giu Gin 445 Giu Gin Sor Lou Lys 240 Arg Lys Glu 255 Lou Gin Met 270 Aen Met Giu Lou Arg Giu GIP-Gln Lou 320 Giu Ala Giu 335 Ala Vai Giu 350 His Lou LYS Gin Asn Thx Giu Met Giu 400 Thr Met Tyr 415 Met Ala Giu 430 vAi'Lye -le Arg_ Asfl Asp Giy Ser Lou 480 Ala Gin Ala 495 Thr Asp Sot Gly Pro Giu Arg Arg Pro Giu Gly Pro Giy 485 490 -61- Pro Ser Ser Pro Arg Val Thr Glu Ala Pro Cys Tyr Pro Gly Ala Pro 500 505 510 Ser Thr Glu Ala Ser Gly Gin Thr Gly Pro Gin Glu Pro Thr S9er Ala 515 520 525 Arg Ala 530 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH-. 20 amino acids- TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Val Asp Val Asp Glu Tyr Asp Glu Asn Lys Pkie Val-Asp Glu Glu Asp I1 10 Gly Gly Asp Gly INFORMATION FOR SEQ ID NO: 10: SEQUENCE CHARACTERISTICS: LENGTH: 1404 basepairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAGCCTCGCA GCGGTCGGGG CGCCCGCG GAGGCTCGAG GGCGcGGGC GGCGGCG ATG TCG AAG AAC ACG Met Ser Lys Asn Thr 1 5 GAT GAG TAC GAC GAG AspGiu Tyr Asp Glu .20 GTG TCG TCG GCG CGG TTC CGG AAG GTG Val Ser Ser Ala Arg.Phe.Arg Lys Val GAC GTG Asp Val AAc AAG TTC GTG GAC Asn Lys Phe Val Asp 25 GAG GAA GAC GGC GGC GAC Glu Glu Asp Gly Gly Asp GGC CAG GCG GGG Gly Gin Ala Gly 35.

CCG GAC GAG Pro Asp Glu GGC_ GA G GTG Gly--Glu Val 40 GAC 'TCG _TGC CTG Asp Ser Cys-lieu CGG CAA Arg Gin 201 249 GGG AAC ATG ACA GCC GCC CTG CAG GCG GCG CTG AAG AAC CCT CCC ATC Gly Ann Met Thr Ala Ala Leu Gin Ala Ala Leu Lys Ann Pro Pro Ile so 55 AAC ACC Asn Thr AAG GTG Lys Val AG AGC CAG GCG GTG AAG GAC Arg Ser Gin Ala Val Lys Asp 70 CTC ATC TiC-C TTRC-AG GCC GGC Leu Ile Ser Phe Lys Ala -Gly COG GCA GGC AGC ATC GTG CTG Arg Ala Gly Ser-Ile Val Leu 75 GAC ATA GAA AAG GCC GTG CAG Asp Ile Glu Lys Ala Val Gin CTC ATG AAG TAC ATC TAC AAG Leu Met Lys Tyr le Tyr Lys 110 AGC GCC GTO CTC CTG CAG TGG Ser Ala Val Leu Leu GJln-Trp TCC CTG GAC Ser Leu Asp GGC TTC GAG Gly Phe Glu 115 CAC GAG AAG His Glu -Lys 130 CTO ACT.GCA

AGG

Arg 100

AGC

Ser

GCG

Ala AAC GGC GTG GAC CTG Asn Gly Val Asp Leu 105 CCC TCC GAC*AAC AGC Pro Ser Asp Asn Ser 120 CTO OCT OCA-OGA GGA Leu Ala Ala Gly Gly GTO GGC TCC Val Gly Ser =T COT GTC Val Arg Val; AGO AAA ACC GTG TAGCCTGGCA OGAACGGGTO CCTGCCGGG Leu Thr Ala Arg Lys Thr Val 145 150 0

AGCGGGAGCT

CGTCTGTTCC

CCTTCGTGAG

TGTAAGACCC

TTTTGTCGTG

GGAAATGATC

CTCTTCCCTT

GGCCGCCGTC

GCCGCGTCCC

GCCCCAfTGC

GACAGACGCC

GAGCACACTG

TCCTGGGAGA

CTGCCTTCAG TGTCCTTGAG GAGGATCTG GTCAGAATTI' TTrCATTTAAA AAAAAAAAAA ATTTAAGCAG AGTGAGTTTC CTOCTCAAGG GAAGCCTCCC GCTCCCAGGA GACAGCATGC CCCGGAGTC TTCAGGGCGA CATTCTTGGT TCAGCTCAGT uTTCTAGAA CACGGCCGGC CAGGCGAGCG CGAGTCGCCG

CAACGGTAGC

GAGGCCAGTT

AGACTGGCAG

TGGAACCPIGT

TGCAGAGCAG

GCGGTCACGC

CAGGGACCTC

GTTTTCAAAA

GCTTGACGGT

GGCAGGAATT

GCCGGTACAA AGACCAAAAC GCCCAGATGC CAGCTTCGCT TTTTCCCTTT CCCGTGTCTG AATCGGAGTG GCGCAGAGGG TCCTGTI!CAT

CGCCGCTGCC

TCAGGATTAC

ACGCGCCGTG

GTGTCGCCGG

TCCTAACTCA

CTATTATGCA

GGTGCCCCCC

.CAGAGCCCCT

GGCACTTCCT

AGAAGACTGG

GGAACGTGCT

TGTCACAGCT

CCACACACTC

CTGTGGGCG

ATAAGGTTTC

CGTCCGGCTG

CTGGQTTG

TGCTGGTCA

AAACTGGACC

(ccccaCCCec- GGGCAGGAGC

GTGCCTCCCA

ATCCGATCCA

ACCGTGTA

CCAdAACAAA

GTGCTGTTT

489 540 600 660- 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 TGATACCTGC TTTTTGTT GTTTTGTAAA AATGATGCAC TTGAGAAAAT AAAACGTCAG TGTTGACAAA AAAAAA AAAA 1404- INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGT4: 1617 base pairs.

TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GAC TGC CGC AGC AGC AGC AAC AAC CGC TAG CCG AAG GGT GGC GCG GCG Asp Cys Arg Ser Ser Ser Asn Awn Arg Xaa Pro Lys Gly Gly Ala Ala 10 CGG GCC GGC GGC CCG GCG CGG CCC GTG AGC CTG CGG GAA Arg Ala Gly Gly Pro Ala Arg Pro Val Ser Leu Arg Glu GTC GTG CGC Val Val Arg TAC CTC Tyr Leu GGG GGT AGC Gly Gly Ser AGC GGC GCT GGC GGC CGC CTG ACC CGC GGC CGC Ser Gly Ala Gly Gly Arg Lgeu Thr Arg Gly Arg 40 GTG CAG Val Gin so GGT CTG CTG GAA GAG GAG GCG GCG GCG CGG GOC CGC CTG GAG Gly- Leu Leu Glu Glif Glu Ala Ala Ala Arg Gly Arg Leu Glu CGC ACC CGT CTC GGA GCG CTT GCG CTG Arg Thr Arg Leu Gly Ala Leu Ala Leu 70 CGG GCG CCA CCG GCC GCC AGC GCC CGC Arg Ala.Pro, Pro Ala Ala Ser Ala Arg as CCC CGC Pro -Arg 75 GCG OCO Ala Ala 90 GOG GAC AGG CCC Gly Asp Arg Pro

GGA.

Gly CGG AAC AAG Arg Asn Lys AGA OCT Arg Ala GOC GAG GAG CGA GTG CTI' Gly Glu Glu GAC GAC GAG Asp Asp Glu 115 CCC GAG AGC Pro Glu Ser 130 Arg Va1;:-Leu 100.

GAA AAG GAG Glu Lys Glu 105 GAG GAG-GAG 'GAG Glu Glu Glu Glu GAG GAG GAA Glu Glu Glu 110 GAC GAC GAC GAC GAC GTC Asp Asp Asp Asp Asp Val 120 GAT CGT CCC GCG GOT GCG Asp Arg Pro Ala Gly Ala OTG TCC GAG GGC TCG GAG GTG Val Ser G11i Gly S er Olu Val 125 CAG CAT CAC CAG CTG AAT GGC Gin His-His Gin Leu Asn Gly 140

GGC

Gly 145 GAG COC GGC CCG Glu Arg Gly Pro CAG ACC Gin Thr 150 GCC AAG GAG ;CGG GCC AAG GAG TGG TCG Ala Lys Giu Arg Ala Lys Glu Trp Ser 155 160 CTG TGT GGC CCC Leu Cys Gly Pro CCT GOC CA%3 GAG GAA 000 Pro Gly Gin Glu.Giu Gly 170 CGG GG CCG GCC GCG Arg Gly Pro Ala Al~a 175 GGC AGT GGC Gly Ser Gly

ACC

Thr 180 CGC CAG GTG Arg Gin Val TCT TCG ACC Ser Ser Thr TTC TCC ATG Phe Ser Met 185 GCG GCC TTO Ala Ala Leu AGT AAG GAG 576 Ser Lye Glu 190 GGG GGA TCA GCC Gly Gly Ser Ala 195* GGG CCI. GAC TCC Gly Pro Asp Ser TCC CCG OTG Ser Pro Val 624 -64- CCA GCC CTC CCA GGA CCT TTG CCC CCC GGG AAG Pro Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly 210

GGC

Gly GCC GAT GGG ACC CCC Ala Asp Gly Thr Pro 220 GCA GAC CCC GTG GAG Ala Asp Pro Val Glu

TTT

Phe 225

TGG

Trp, TGC CCT GCC Cys Pro Ala ACA GTC ATG Thr Val Met GAG CAA GCC- ACG Glia Gin Ala Thr 260 CTG CTC ATG GAG Leu Leu Met Gin 275

GAC

Asp 245

OCT

Ala cGc Arg GGG CGC AAA GAG AAG Gly Arg Lys Glu Lys 230 GTC GTG GAG TAC TTC Val Val. Glu Tyr Phe 250 TTC GAG GAG GAG GAG Phe Gin Glu Gin Glu 265 ACC GAT GTC CTC ACC Thr Asp---Val Leu Thr GAG GCG GGC Glu Ala Gly 240 TTc CCT Phe Pro 255 ATC GAC GGC AAG TCC CTG Ile Asp Gly Lys Ser Leu 270 GGC CTG TCC ATC CGC CTG Gly Leu -Ler Ile Arg Leu 285 280 AAA ATC TAT GAG Lys Ile Tyr Glu GGG CGA Gly Pro 290 OCT, CAC Gly His 305

GCG

Ala

TTG

Leu 295 GAC CdG Asp Pro CAC CAT ATC AAG GTG CTG GAG CAG His His Ile Lys Val. Leu Gin Gin 300 GAA GGC TTC'CTG GGA .TGAGCACAGA GlTiGly Phe Leu Gly 315 TTC GAG GAC GAT Phe Glu Asp Asp 310 GCCGCCGCGC CCCTTGTCCC CACCCCCACC CCGCCTGGAC a a, CCCAAG4GTGT

GATTCTGGTA

CTCTGGACCT

TGCTCATTT

AGAAAAAAAG

CGGCCCAAcc

CGTTTTTTGG

GCCCTGTCGG

CccTcTcTcc

CCGAAGGGTT

CCGAGAGGCC

GGGGGCGGGG

GCCCCCAGCA

GCCCCCAGAT

GAAAAAAAAA-

AGGTGTTTAT

TGCGTGGC.CT

CGQGAGGAGC

CGTTGGTrTCT

GTI-TTTT

AGGAGCTGGA

CCTTGCTGTG

CAccccTccc

CAGCCCCCTC

AATTGGG.GAG

TCCTATATAT

TTCTTCCCTC

TGGGAATGGG

GTTGTCGCTC

TTTTAAATAA

CTGGGAGGC

CTCATTGCTA

GGAGCCTGGA

CCATTCCTGC

GAGGGGTGCG

cccccccAcc

TGTCGCCTGG

CCTCCCTCCT -GTCCCAGGAC GGGGCTGGGA AGGTGCCCCA ATATATATAT' GTrTTGTI'CT CCACCACCAC TCATGGCCCC AGGAGGGTGG GACCTTGGGT CAGCTGGCTG TATTGCTTv17T AATTTTAAAA AAAGGAAAAA

CTCCATGTCA

GACCTACCCT

CCGTGTGTGT

GACTCTGGCC

ATTTTTTAAA

AGATCCTCCT

GCCTGTTTT

AGCCCTGCTC

CTGTCTCCCA

TAATATrGCA

AAAAAA-

1021 1081 1201 1261 1321 1381.

1441 1501 1561 1617 INFORMAT1ON FOR SEQ ID NO-12: SEQUENCE CHARACTERISTICS: LENGTH: 1362 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCC AGC GCC.CGC GCG GCG CGG AAC AAG AGA GCT GGC Ala Ser CTT GAA Leu Glu GAC GAC Asp Asp CCC GCG Pro Ala s0 CAG ACC Gin Thr

CCT..-GGC

Pro Gly CAG GTG Gin Val TCG ACC Ser Thr AAG CCA Lys Pro 130 GGG CGC Gly Arg 145 GTC GTG Val Val.

rrc CAG Phe Gin Arg

GAG-

Giu

GTC

Val

GCG

Ala

AAG

Lys

GAG

Glu

TCC

Ser 100

GGG

Gly

CTC

Leu

GAG

Giu

TAC

Tyr

CAG

Gin 180 Ala

GAG

Glu

GTG

Val

CAG

Gin

GAG

Glu

GAA

Glu

ATG

Met

CCT

Pro

CCA

Pro

AAG

Lys

TTC

Phe 165

GAG

Giu Ala Arg Asn Lys GAG GAG GAG GAG Glu Glu Giu Giu 25 TCC GAG GGC TCG Ser .Glu Gly Ser CAT CAC CAG CTG His His Gin Leu .55 CGG GCC AAG GAG Arg Ala Lye Glu 70 GOG CGG GGG CCG Gly Arg Gly Pro GCG GCC TTG AGT Ala Ala.Leu Ser 105 GAC TCC CCG TCC Asp Ser -Pro 3cr 120 GGA GCC GAT GGG Gly Ala Asp Gly 135 CCG GCA GAC CCC Pro Ala Asp Pro 150 ACC GAG GCG GGC Thr Glu Ala Gly ATC GAC GGC AAG Ile Asp Gly -Lys 185 Arg

GAG

Giu

GAG

Giu

AAT

Asn

TGG

Trp Gcc Ala 90

AAG

Lys

CCG

Pro

ACC

Thr

GTG

Val

TTC.

Phe 170

TCC

Ser Gly

GAC

Asp cc Pro

GGC

Gly

CTG

Leu

GGC

Gly

GGG

Gly

CCT

Pro Phe 140

TGG

Trp,

GAG

Glu

=TG

Leu GAG GAG CGA GTG Glu Glu Arg Val GAC GAG GAC GAC Asp Glu Asp Asp GAG AGC GAT CGT Giu Ser Asp Arg GAG dGC GGC CCG Glu Arg.Gly Pro TGT*GGC CCC CAC Cys Gly Pro His so AGT GGC ACC CGC Ser Gly Thr Arg GGA TCA GCC TCT Gly Serl a Ser 110 TTG CCC CCC GGG Leu Pro Pro Gly 125 GGC TGC CCT GCC Gly Cys Pro Ala ACA GTC ATG GAC Thr Val Met Asp 160 CAA. GCC ACG GCT Gin Ala Thr Ala 175 CTC ATG.CAG CGC Leu Met Gin Arg 190 ACC GAT GTC CTC ACC GGC CTG TCC ATC CGC CTG GGG CCA GCG TTG AAA Thr Asp Vai 195 Leii Thr Gly Leu Ser Ile 200 Arg Leu Gly Pro 205 Ala Leu Lys -66- ATC TAT GAG CAC CAT ATC AAG GTG CTG CAG CAG GGT CAC TTC GAG GAC Ile Tyr Giu His His Ile Lys Val Leu Gin Gin Gly His Phe Giu Asp 210 215 220 GAT GAC CCG GAA GGC TTC CTG GGA TGAGCACAGA GCCGCCGCGC CCCTTGTCCC Asp Asp Pro Glu Gly Phe Leu Gly CACCCCCACC CCGCCTGGAC CCATTCCTGC CTCCATGTCA

AGGAGCTGGA

CCTTGCTGTG

CACCCCTCCC

CAGCCCCCTC

AATTGGGGAG

TCCTATATAT

TTCTTCCCTC

TGGGAATGGG

GTTGTCGCTC

CTGGGCAGGC

CTCATTGCTA

GGAGCCTGGA

CCTCCCTCCT

GGGGCTGGGA

ATATATATAT

CCACC6ACCAC

AGGAGGGTG

CAGCTGGCTG

GAGGGGTGCG GACCTACCCT CCCCCCCACC CCGTGTGTGT TGTCGCCTGG GACTCTGGCC GTCCCAGGAC ATTTTTTAAA AGGTGCCCCA AGATCCTCCT GTTTTGTTCT GCCTGTTITT TCATGGCCCC AGCCCTGCTC GACCTTGGGT CTGTCTCCCA TATTGCT7"T TAATATTGCA

CCCAAGGTGT

GATTCTGGTA

CTCTGCACCT

TGCTCATTTT

AGAAAAAAAG

CGGCCCAACC

CGTTTTTTGG

GCCCTGTCGG

CCCTcTcTcc

CCGAAGGGTT

CCCAGAGGCC 786 GGGGGCGWG 846 GCCCCCAGCA 906' GCCCCCAGAT 966 GAAAAAAAAA 1026 AGGTGTTTAT 1086.

TGCGTGGCCT 1146 CGGGAGCAGC 1206 CGTTGGTTCT 1266 GTTTTTTTT 1326 1362 TTTTAAATAA AATTTTAAAA AAAGGAAAAA AAAAAA *44* INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 1422 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ACC COT CTC GGA GrCG'CTT GCG CTG CCC CGC GGG GAC AGG CCC GGA CGG Thr Arg Leu Gly Ala Len Ala Leu PZr6 Arg Gly Asp Arg Pro Gly Arg 1 5 10 1 GCGd CCA CCG GCC GCC AGC GCC CGC GCG GCG CGG AAC-AAG AGA GCT GGC Al1a Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly 25 GAG GAG*CGA GTG CTT GAA AAG GAG GAG GAG GAG GAG GAG GAG GAA GAC Giu Giu Arg Vai Leu Giu Lys Gin Giu Gin Giu Gin Giu Giu Glu Asp 40 GAC GAG GAC GAC GAC GAC GAC GTC GTG TCC GAG GGC TCG-.GAG GTG CCC Asp Glu Asp Asp Asp Asp Asp Val Vai Ser Gin Gly Ser Glu Val Pro 55 -67- GAG AGC GAT CGT Giu Ser Asp Arg GAG CGC GGC CCG Giu Arg Gly Pro CCC GCG GGT GCG CAG CAT CAC CAG CTG AAT GGC Pro Ala Gly Ala Gin His His Gin Leu Ag Gly

GGC

Gly 240 CAG ACC GCC AAG GAG CGG GCC AAG GAG TGG TCG CTG Thr Ala. Lys Glu Arg Ala Lys Glu Trp Ser Leu GGC CCC CAC CCT Gly Pro His Pro 100 GG6C CAG Gly Gin GAG GAA Glu Giu 105 GGG CGG GG Gly Arg Gly CCG GCC GCG GGC Pro Ala Ala Gly 110 AGT AAG GAG GGG Ser Lys Giu Gly 125 AGT GGC ACC Ser Gly Thr 115 CGC GAG GTG TTC TCC ATG G.CG GCC TTG Arg Gin Val Phe Ser Met Ala Ala Leu 120 384 GGA TCA Gly Scr 130 GCC TCT TCG ACC Ala Ser Ser Thr

ACC

Thx 135 GGG CCT GAC Gly Pro Asp TCC CCG TCC CCG.GTG CCT Ser Pro Ser Pro Val Pro 140

TTG

Leu 145 CCC'CCC GOG AAG CGA GCC CTC CCA GGA Pro Pro Gly Lys Pro Ala Leu Pro Gly 150 GCC GAT Ala Asp 155 GCA GAC Ala Asp GGG ACC CCC Gly thr Pro TTT 48S0 Phe 160 GGC TGC CCT GCC Gly Cys Pro Ala GGG CGC Gly-Arg 165 AAA GAG AAG Lys Glu Lys CCC GTG GAG TGG Pro Val Giu Trp 175 GGC-TTC CCT GAG Gly Phe Pro Giu 190 ACA GTC ATG Thr Val Met CAA GCC ACG Gin Ala Thr 195 GTC GTG GAG TAC Val Val Giu Tyr ACC GAG GCG Thr Glu Ala 576 624

S

GCT'TTC GAG GAG AlaPhe Gin Glu GAG ATC GAC GGC Giu Ile Asp Gly TCC CTG CTG- Ser Leu Leu CTC ATO Leu Met 210 CAG CGC ACC GAT Gin Arg Thr Asp CTC ACC GGC CTG Leu Thr Gly Leu

TCC

Ser 220 ATC CGC CTG GGG Ile Arg Leu Gly

CCA

Pro 225 GCG TTG'AAA ATC Ala Lou :Lys Ile GAG CAC CAT ATC Glu His His Ilie GTG CTG GAG GAG Val Leu Gin Gin GGT Gly 240 CAC TTC GAG GAC His Phe Gu Asp

GAT

Asp 24S GAC CCG GAA GGC Asp Pro Giu Gly TTC CTG GGA Phe Leu Gly 250

TGAGCACAGA

GCCGCCGCGC CCCTTGTCCC CACCCCCACC CCGCCTGGAC CCATTCCTGC CTCCATGTCA 826 CCCAAGGTGT CCCAGAGGCC AGGAGCTGGA CTGGGCAGrGC GAGGGGTGCG GACCTACCCT 886 GATTCTGGrA GGGGGCGGGG CCTTGCTGTG CTGATTGCTA CCCCCCCACC CCGTGTGTGT 946 CTCTGGACCT GCCCCCAGCA GACCCCTCCC GGAGCCTGGA TGTCGCCTGG GACTCTGGCC 1006 TGCTCATTT GCCCCCAGAT CAGCCCCCTC CCTCCCTCCT GTCCGAGGAC ATTTTTTAAA 1066 -68- AGAAAAAAAG GAAAAAAAAA AATTGGGGAG GGGGCTGGGA CGGCCCAACC AGGTGTrTAT TCCTATATAT ATATATATAT CGTTTTTTGG TGCGTGGCCT TTCTTCCCTC CCACCAC CAC GCCCTGTCGG CGGGAGCAGC TGGGAATGGG AGGAGGGTGG CCCTCTCTCC CGTTGGTTCT GTTGTCGCTC CAGCTGGCTG CCGAAGGGTT GTTTTTTTN-ri TTTTAAATAA AATTTTAAAA INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 4722 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTGTGAAATA GCBACTGTGT-.TTCTCAAGGA TCCAATCCCA

AGGTGCCCCA

GTrTTGTTCT rcTCAGCCCC

GACCTTGGGT

TATTGCTITTT

kAAGGAAAAA

AGATCCTCCT

GCCTGTTTTT

AGCCCTGCTC

CTGTCTCCCA

TAATATTGCA

AAAAA

1126 1186 1246 1306 1366 1422 RCCTAAGGTG GCAGCGCACP.

ATG AAG AAT CAA GAC AAA AAG ABC-GGG GCT GCC AAA CAG CCC AAC-CCC Met Lys Asn AAA A$3C AGC Lys Ser Ser Gin Asp Lys CCG OA C AG Pro Gly Gin Lys Asn Gly Ala Ala Lys Gin Pro Asn Pro 10 CCG GAB GCB GGA GCG GAG GGB GCC CAG 000 Pro Glu Ala Gly Ala Glu Gly Ala Gin Gly 25 108 156 204 CGG CCC GGC COG Arg Pro Gly Arg CAG GCT CCC G00 Gin Ala Pro Glv CCG GCC CCC GCC CGA GAB GCC GAA GOT GCC AGC AGC Pro- Ala Pro Ala Arg Glu Ala Glu Gly Ala Ser Ser OCT CAG CCT Ala Gin Pro AGO CCG Arg Pro GGG OCT CAB GCC Gly Ala Gin Ala GGG GCO Gly Ala ATA CTC le Leu

AAA

Lys CTC TOT-GAT GTC TCT GAG GAG CTG Lou Cys Asp Val Ser Giu Glu Leii

AOC

Sei 7!

CA(I

CGC CAG TTG Arg Gin Leu G00 GCC CCG Gly Ala Pro GAA GAC Giu Asp so8 GOT GAG Gly Glu 252 300 348.

0* a a AGT ACA TAC Ser Thr Tyr 85 TOT GTO GAC ABC AAC Cys Val Asp Asn BAn GAT GGG GTC Asp Gly Val CGC GCC TAT Arg Ala Tyr 115 CAGb GOT Gin Gly 100 GAG CCC CC?' Gliu Pro Pro CCT GAB GAT GCA Pro Giu Asp Ala GAG BAG TCT Glu Lys Ser 110 ACC CCA OTA Thr Pro Val GTG GCA AGO BAT Val Ala Arg Asn 000 Gly 120 CCG GAG CCG Pro Glu Pro GTC AAT GGC Val Asn- Gly 130 GAG AAG GAG ACC TCC AAG GCA GAG Giu Lys Giu Thr Ser Lys Ala Giu' 135 CCG GGC ACG GAA GAG Pro Gly Thr Giu Giu 140

ATC

Ile 145 CGG ACG AGC GAT GAG GTC GGA GAC CGA Arg Thr Ser Asp Glu Val Gly Asp Azg 150 GAC CAC CGG AGG CCA GAG Asp His Arg Arg Pro Gin 155 160 540 GAA AAG AAG AAG Giu Lys Lys Lys AAG GGT CTG GGA .AAG GAG ATC Lys Gly Leu Giy Lys Giu Ile ACG CTG CTG ATG Thr Leu Leu Met GAG ACA CTG Gin Thr Leu TGC AAG AAG Cys Lys Lys 195

AAC

Aen 180 ACG CTG AGC ACC Thr Leu Ser Thr CGA GAG Pro Glu 185 GAG AAG CTQ GCG GCT CTG Giu Lys Leu Ala-Ala Leu TAT GCG GAA CTG CTC GAG GAG CAC CGG Tyr Ala Giu Leu Leu Giu Giu His Arg

AAC

Asn 205 TCG GAG AAG Ser Gin Lys 588 636 684 73.2 780 GAG ATG Gin Met 210 AAG CTG CTG GAG Lys Leu Leu Gin AAG GAG AGC GAG Lys Gin Ser Gin-

CTG

Leu 220 GTG CAD GAG AAS Val Gin Giu Lys

GAC

Asp 225 GAC CTG CGT GGC His Leu Arg Gly CAC AGC AAG GCC His Ser Lys Ala C TO GCC CGC AGC Leu Ala Arg Scr CTC GAG AGC CTG Leu Glu Ser Leu CGG GAG CTG Arg Giu Leu GAG -CGG Gin Arg 250 CAC AAC CGC TCG His Asia Affg. 5cr CTC AAG Leu Lys 255 GAA GAA GOT Giu Giu Gly GTG ACG TCA Val Thr Ser 275 GAG GAG GAC Giu Gin His 290 CTG GCC GAG Lau Ala Giu 305 GAG GAC ATC Glu His Ile GTG GAC GCC Val Asp Ala GAG CGA GCC COP.

Gin Arg Ala -Arg GAG GAG GAG AAG Glu Glu G~lu Lys CGC AAG GXG Arg Lys Glti 270 CTG GAG .ATG=_ Leu.Gin Met CAC TTC GAG ATG His Phe'Gln' Met Thr 280 CTC AAC GAC ATT Leu Asn Asp Ile AAC GAG Asn Giu COG CTC Arg Leu CGC AAC TCC AAG CTG CGC GAG Arg Aqn Scr Lys Leu Arg Gin 295 300 GAG AAC ATO GAG Giu Asn Met Glu 876 924 972 1020 1068 1116 AAG AAG LYS LYS 310 CTG ATT GAG Leu Ile Glu TAC GAG CTG Tyr Giu Leu CGA GAA Arg Glu 320 CAG CTG Gin Leu 335 GAC AAA GTC TTC AAA GAC AAG Asp Lys Vai Phe Lys -His Lys 325 330 AAG CTC GAG GAG GCC GAG GAG Lys Leu Gin Gin Ala Gln Giu 340- 345 OAT -CTG CAD GAG Asp Leu Gin Gin ATG CTG AAG GAG GCA 0A9 Met.Leu Lys Giu Al *a Giu 350 CTC CTG Leu Leu GAG CGG CAC GAG CGG GAG AAG GAC TTT Giu Arg His Gin Arg Giu Lys Asp Phe AAG GAG GCC GTG GAG Lys Glu Al-_AVal Giu

TCC

Ser

CAG

Gin 385

CT?

Leu

AAG

Lys

CGT

Arg

GAG

Giu

GAG

Gin 46S

CTG

Leu z jCC Ser

GA:G

Giu 0CC Ala

GGC

355 GAG AGO ATG TGC Gin Arg Met Cys 370 GAG CT? GCC CTA Gin Leu Ala Leu TCC AAA AGC AGC Ser Lys Ser Ser 405 ATO ACA AAG AAG Met Thr Lys Lys 420 TCC CGG TOG GAG Ser Arg Trp Giu 435 AAA ACA CTC CG Lys Thr Leu Arg 450 COG CTG GAG AAG Arg Leu Giu Lye AAC AAG AGO GTG Aen Lye Arg-Vai 485 GAC AGC GOT CC? Asp Ser Gly Pro.

500 CAG GOT GTC GAG Gin Gly Val Giu ACA GAC GCT TCC Thr Asp Ala Ser 530 CAG ACA GGG CCC 360 GAG CTG ATG Giu Leu Met 375 TAC ACA GAG Tyr Thr Oiu 390 GAG OTG TTC Giu Vai Phe ATC AAG AAG Ile Lys Lys AGC AGC AAC Ser Ser Asn 440 GAC AAA GAG -Asp Lye Giu 455 CTG TGC CG Lou Cys Arg 470 CAG GAG CTG Gin Asp Lou GAG COG AGO Giu Arg Arg GGC CCC 000 Giy Pro Gly 520 TGC TGC GCA Cys.Cys Ala 535 CAG GAG CCC 365 AAG CAA GAG GAG--ACC GAC CT0 AAG Lys Gin Gin Oiu Thx His Lou Lye 380 AAG TTT GAG GAG TTC GAG AAC AC? Lye Phe Giu Giu Phe Gin Aen Thr 395 400 ACC AC-A TTC AAA GAG GAA ATG GAA Thr Thr Phe Lye Gin Giu Met Giu 410 415 CT0 GAG AAA GAG ACC ACC ATG TAC Lou Giu Lys i Thr Thr Met Tyr 425 430 AAG 0CC CG CT? GAG ATG GC? GAG Lye Ala Lau Lou Giu Met Ala Giu 445 CTG GAA GGC CTG GAG GTG AAA ATC Lou Glu Gfy Lou-Gin Val Lye Ile 460 GCA CTG GAG ACA GAG eGC AAT GAC Ala Lou Gin Thr Giu Arg Asn Asp 475 480 AG? 0CC GOT GOC CAG GOC CCC GTC Ser Ala Gly Gly Gin Gly Pro Val 490 495 CCA GAG CCC GCC ACC ACC TCC AAG Pro Oiu Pro Ala Thr Thr Ser Lye 505 510 GCT CAA GTA CCC AAC TC? CGA AGO Ala Gli-Val Pro Asn Ser Pro Axg 525 GOT OCA CCC AGC ACA GAG GGA TCA Gly Ala Pro Ser Thr Giu Ala Ser 540 ACC-ACT GCC ACT 0CC TAGAGAGCTT Thr Thr Ala Thr Ala 555.

CAGCCCAGCC AGGCCGGCC CAGCCCAGGC -AGGATGTTC? GACCTGGCTG GCACCTGACC TTACATGCAT ATGGCACAGA TGCAAGGCCr 1164 1212 1260 1!308 1356 140A 1452 1500 1548 1596 1644 1692 1741 1801 1861 1921 Gly Gin Thr Giy Pro Gin Giu, Pro 545 550 GOTOCTOGOG TOTGCCAGGA AGGGAGCAGC TCCGATGCA AGCAGTCCGG TGCGAGGCC CCTGCAGTC TTGGATTTTG TGGGTCAGTI -71- CACACATrTG TGTCTCTAAG TGTACTGTGG AAGCCAGCGG CTCCCTIGTG AGCTGAAGTC -CGCAGTGACC TGCAGGACGA GTTCCTTGPLG CTCCCGGCCC TTCAGAGCTC CTGACAGGTG TGGGTAGCTG GGCTCTCCTA GCCTCCCCCA GGQQATGCCC AGGCGTGGCC TTGGCAGGCA CCTCGAGCAG GGGCAGGAGT GTTTCTGGTG TGGGCGCTGC TTCCCCCGCC CTCTGGTGAT AGCCTTACCA TTCACAGGTG CCTCTCCAGC GATTCCTTCT GAAGGTGTTC GTTTCQTTTr GACCCTCTGA GGAAGAGGGG TGCTGTAGCA TGCCCTCCCC CCCCTCGCCP 4

TGGCTCCTCG

GAGATCAGGT TGTCTGTTCA GAQAGAAGAG GCGGGCCTCA ATCCCGGGAG GCCGCCCGGT TGCTGGTCCC CAGGACCAGC TCCTCGTTTG

GCTTGCATCG

TTACGGAGGA

CTTTCCCTGC

ATACACACCC

GAGGCGCCAT

CTGGGACTA

GGATGATGCG

GCCAGGACCA,

CCGCACAGTG

GTTTTGTTGC

GTGGTCCCTG

GCCGCCTTGG

GCGTCTGATG

TCCCGTCACT

CTGTATGGGT

TCCCTGGTTC

.TTCTGiCCCAT

AGAGCTGCAG

CTCTGCTGGC

CTGCTGGC6T GCTG1CGTAGG

CTGCTATTCC

CAGGGTTCAT

TGAATGGCAC

CCCCGCCACA

GAGTGTTGCC

TCCTGCTCAG

CGCCAAGCCC

CTGCAcCCTT

GGGGTGACGA

GACGGCGTCT

CTGCTTTGAG

AGCCTI'GACC

TGCTTCTCTT

GTGATTGGGC

CTCGCTGGTC

GGCCAGTGAT

AGTGACAAGA

ACGTGACGOT

CGTGCCTGGC

TGCTGAGGTT

GCTTTGCCGC

GTTGTCCCCG

CAGTTTCTGC

TCGTCCCTGG

GCGGGAGCTG

AGCTCTGCAC

ACTGGCCAGT

CGAGGGACAG

GTGCTTGGGG

TTCATCTTCC

GAGACCCGCA

GCCXI-rGCTG

GTGCAAGCAG

CGAGATTGTG

GGAGGCTTTC

ATGCAAGGTG

GAAAGCCAAG

TGGACAGATG

GCACTGCCAT

GCTGAGACCC

GCACTTCTCT

GACTTGGAGA

TG7CTCTCCTG

AGGAGCCCCG

GCTTCTCAGT

TCATCCAAAG

TTGTATTGAG

TCCAGTGTCC

TCTGTTTGGT

CAGCTTGCCT

TGCAGTGCGT

TTCCTGCCCC

TACCTCTQTG

CTGCTGTCTG

GGTGGCTCCC

CTGTGCAGAG

CGCCACGTCT

TTTGCTTCTT

ACAGTCCACG

AGTAC;GCCCA

CTGICTAATCC

TGAMTCCACG

TCTCTTCTGA

CCCTTCCCTC

TGGCTAGGCG

AGGCCTACGG

1981 2041 2101 2161' 222 1 2281 2341 2401 2461 2521 2581 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481; 3541 3601 3661 00* CCACTCCACC TAACTGCAAT CCCAAGAAGT AGCCTTCTTTi-.

ACAG3GTGAGG CCTGAGACTC GGGGCGGCCT CTGTGTGCTG CATTTGAdTA CTGGCTCAGG CCAGCTGG-GC TCAGGGAGAG GCAGTATI'TC TTGQAAGCTG CTCAGCCTTT AACTTGGATC AGCTACGTAT GGCTGAGGCC CTGGCATATC TrETAGTTCAC TGCCACCCTG GGCTGAATCC ATGCCTTCAC TGGGAATGGC AGCCCCTGTG CCAGACTGAG CCAGGGTGCA GGAP.GGAGGC

CCTTGGGGTT

GGGATTTGr

AGCGGCTGAC

CACACCGCTG

AGGGAGGGCT

CCCCAGACTG

TTTTGTTGTC

CCTCACATAA

AGCTGGCAGG

CTCAATGC

CACCCCCTGT

CTCTGCCGCC

GTACAAGAAC

AGGTAGCTGC

S. S

S.

-72- TGGCCTCCAT CCTGGCTTGC CTCACTTCAG CTACCTCGCA

TGGATTCCAG

CAGCTCAGGA

GGCATCTAGG

CTGTCTCTCC

GCCCACATI'G

GCCAGTGCCT

CTGTGTTGGT

GAGCCCAGAG

GCTTCGTGTA

TCAGCGGTGT

TTAGCTCAGG

CTAACGCCTG

TAGAGCTGCT

GGTGGGGGGA TGGGAAGCTG ATGTGGGCCT CAGGTAGGGG CCAGTCTAGA TGGCGACCCC AGCTGCTCGC TTGCTCTGCT GCCTGGAACC TGGACCAGAG TCCTCACCCG GTGGCTTCCA T1'CTTACGTT ~GG'~ CCCTTGGCGC CGCCAGCAGT TGACTCAGCT -TCGTGTGTGA CAGCCGCACT TTGATTTGTT CAACTTTTCT GTAAGGAGAA CTTCCCCCAC TCCCPACCAG CCAGCTCTCC CATCTTGCAC.

CPLGGGGGCAG

AGAGCCTTTG

TTCTCTTCCT

TTCCAAAGTC

GCAAGGGAGG

TGCCTGTGAC

CCTCCAGGGG

AACTTGTGTC

GTCCAGCGGA

TGTTTTCCAT

TGTTAACTTT

GTGGCCACCG

CGCACAAAGG

CCTTATGGCC

,GGTTTTTGTT

GACTTGATTT

TAGCCCAGGG

GTGGCTCTCA

CTCCACTCCA

CTCCATTGAC

AGCCCAGGTA

TGGCCTATCC

CTCAGATTTA

ACCACAGCAG

CGGACCTTGT

GTGCGT.CACG

GAGGTTTG

CTGTAAP.GAT

AGA=CCACC

TGGCCGCCCC

TGGCCATCTA

TTTGTTTTTG

TGATGTATT

A

GTGGGGCTAT

CTAGGCTTCC

CCCATTTGCA

CAAATCGTAC

CCCAGGTGC...

TTGAGTGATA

GGACCAAGAG

GTGAGGCTCG

CCAGCTGAGC

TGACCTAGAC

GAdfTGGGC- GCTTATT1'AA

AGGAGGCCAA

AGGGACAGCC

GATTGTTTAP.

TTTrTGTTT

TAAGCCACAT

3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 438r 4441 4501 4561 4621 4681 4722 AGGCACCTGC CTGGGGGA.GG GGCTTCTCTr

AGTTGTGCTG

TGTCTACTTT

ACAGCTTTTT TTGGTTTTM TGGTATTCAC AACAGCCAGG a a a a.

S.

a a a.

TAAATAAPLGA GTCTG.TTGCC TTAAAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID NO: SE QUENCE CHARACTERISTICS: LENGTH: 1928 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:.linear (xi) SEQUENCE DESCRIPTON: SEQ ID NO: GACGCCTCAG AGCGGAACAG GGAAGTGAAT CAGGCGCCGG GTAGTGGGTT GCTGGGCTGG GCTTGCTGAG GTAGAGGCAG CGCCAAGAAG AGGCCTTTGC CGCTGGTCGG GALTTGGG -ATG TCG AAG AAC ACA GTG TCG TCG GCC CGC TTC CGG AAG GTG GAC GTG Met Ser Lys Asn Thr Val Ser Ser Ala Arg Phe Arg Lye Val Asp Val 1 5 -10 117 165 GAT GAA TAT Asp Glu-Tyr GGC CAG GCC Gly Gin Ala GGA AAC ATG Gly Asn Met GAC GAG Asp Glu AAC AAG TTC GTG Asn Lys Phe Val 25

GAC

Asp GGG CCC GAC GAG Gly Pro Asp Glu GGC GAG GTG Gly- Giu Val 40 ACA GCT GCC CTA CAG GCA GCT Thr Ala Ala Leu Gin Ala Ala AAC ACC Asn- Thr AAG GTG Lys Val AAG AGT CAG LyqSer Gin.

GCA GTG Ala Val .70 AAG GAC CGG Lys Asp Arg GAA GAA GAT GGG GGC GAC Glu Giu Asp Ply Gly Asp GAC TCC TGC CTG CGG CAAk Asp Ser Cys Lou Arg Gin CTG AAG AAC CCC CCT ATC.

Lou Lys Asn Pro Pro Ile GCA GGC AGC ATT GTC TTG Ala Gly Ser Ile Val Leu 75 ATA GAA AAG GCA GTT CAA Ile Glu Lys. Ala Vai Gin ATG AAG TAT ATr TAT AAA Met Lys Tyr Ile TyrLys 110 GCT ATG--7A CTG--CAA TG Ala Met Leu Lou Gin Trp CTC ATC TCT TTT AAA GCT AAT Lou Ile Ser Phe Lys-Ala Asn 213 261 309 357 405 4t3 501 549 .600 TCT CTG GAC Ser Leu Asp GGA TXT GAG Giy Phe Giu CAT GAA AAG His.Giu Lys AAT GGT GTrG GAT Asn 04y Vai Asp CTC CTA Lou Leu 105 AGC AGT Ser Ser CCG TCT GAC Pro Ser Asp Ak Asn 12 0

GGA

Giy GCA CTT GCT GCT Ala Lou Ala Ala 13 GGA OTA GG0 TCC. ATT GTT COT GTC.

Giy Vai Gly Ser Ile Vai Arg Val 130 TTG ACT Leu Thr 145 GCA. AGA AAA ACT GTG Ala Arg Lys Thr Vai 150 TAGTCTOGCA GGAAGTGGAT TATCTGCCTC

GGGAGTGOGGA*

TAGCATCTGT

ACATATCAGT

GAAGTTTC

AATTTACAT1!

TTTGGTTTTG

T1'AACATTGC

TAAGTGGTG

TCTGTACTGT

AT=ATTGT

ATTGCTGGTA CAAAGACCAA AACAACCAAA-TGCCACCGCT GCCCTGTGGG TTCTCTCAGC TTTGCCTTCT TGCTTTTTCA TATCTGTAAA GAAAAAAA~- TGTCCCTTNA ATGAAAATTG GGATAATATA:'GAAOAAALTTG TGTTAAAATA ATCCTTTCAA AACCATTTCA GTGATGTTTA TACCAATCTG TATATAGTAT CAAGITTAA TTGTGCAACT TTTAACCCTG TTGGCTGGTT TTTGGT1'CTG TATTAT AACTAPATACT GAAAAATTG GTCAGAATrI' GAGGCCAGTT TTGCTAGTCA GGAAATGA7A TTrATAAAAA ATATGAGAGA CTGQCAGCTA AAAACTGGAC CATATTTCCC TTATTTAATA AGCAAAATAT .GTTITGGAA GTGAATACCA CTGCTAAGTT ATAGCTTTGT TTTTGCTTGC CTCCTCATTA GGGTTTAAGT ATGCTACTTI' CTCTCAGCAT C CAATAATCA TGGCCCCTCA GGTCACGCAG GGTTCAGAGC AAGAAGTCTT GCTTTATAcA AATGTATCCA 660 720 780 840 900 .960 1020 1080 1140 1200 1260 -74-.

TAAAATATCA

TTTGGAAAAA

AATTTAAACT

CCTTAGACAT

TGTCTTCAGT

ATACACATTC

AATTGTTTAC

AAGTrATAAA

TGCTTAGTAT

AGAAATTCTA

TTTGTAAAAA

GAGCTTGTTG GGCATGAACA TCAAACTTTT GTTCCACTAA ACTGCAAATC AGAAAGAATG ATGCAGAA AGAAAGAAAA CTGGGCAGCC TCTGAATGAA ATGCTACTrT CTTAGAAAT TATGAGGTAT ACAPLCTAGTA TTTAAGATAC CATI'TAATAT GTTCTTCAGG GTAGTTGGGA TCTCAAAAGA TTrGGTI'CAG TGTGTTTTAG CTCAGTGTTT TCTAAAAAAA GAAA.CTGCCA TTTGTTGGAC AAACCAAATC AGTTCTCAAA AAATQACCGG TATCGAGTAG CTCTAAAACA AACCACCTGA CCAAGAGGGA TTACATTGGA TGCCAGTTTT GTAATCACTG ACTTATGTGC TAAACTCTIT GCTGTI'TTG ATACCTGCTT TTNGITTCAT TGATAAAACT TCAGAAAATA AAATGTCAGT GTTGAATAAT

TATGGCTCTG

ACTATGGTGT

ATAATAGCTG

GCCCCGTAAA

ATCCAAACAA

CACAGCAAAA

TGCTTATAAAL

AGTGAGCTTG

AAACTGGTGC

Ti L.GTTTTGT

TAAAAAAAAA

1320 1380 1440' 1500 1560 1620 1660 1740 1800 1860 1920 1925

AAAAA

INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 1207 base pairs (B)TIYPE: nucleic acid STRAND)EDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GAA GAG CGA GTA. CTT GAG AMA GAA GAG GAA GAA Giu Giu Arg Val Leu Giu Lys Glu Giu Gl~u Glu 1 5 -00000 &00 0 as0 GAT GAT GAT Asp--kap Asp

GAT

Asp CAA GAA GAA G"T GAT GT TCA GAG GGC TCT CAA GTG CCC GAG AGT Glu Asp Glu Glu Asp Asp Val Ser Giu Gly Ser Giu Val Pro Glu Ser 25 GAC CGT CCT GCA GGT GCC CAG CAC CAC CAG CTT AAC GGC GAG CGG GGA Asp Arg Pro Ala Gly Ala Gin His His Gin Leu Asn Gly Glu Arg Gly 40 CCT CAG AGT GCC AAG GAG AAGG GTC .AAGU GAG TGG ACC CCC TGC GGA CCG Pro Gin Ser Ala Lys Giu Arg Val Lys Glu Trp, Thr Pro Cys Gly Pro 55 CAC CAG GGC CAG GAT GAA GGG CGG GGq-CCA GCC CCG GGC AGC GGC ACC His Gin Gly Gin Asp Glu Gly Arg Gly Pro Ala Pro Gly-Ser Gly Thr 70 75 96 144 192 240 ATG AAC AAG CGC CAG GTG TTC TCC ATG GCA GCC GAA GGG GGA ACA GCT Arg Gin Val Phe Ser Met Ala Ala met Asn Lys Glu Gly Gly Thr Ala TCT GTT GCC Ser Val Ala GGC AAA CCA Gly Lys Pro 115 CCC GGG CGC Pro Gly Arg

ACC

Thy 100

GCC

Ala

AAA

Lys GGG CCA GAC Gly Pro Asrp CTA CCT GGG Leu Pro Gly GAG AAG CCA Glu Lys Pro 135 TAT TTr ACT Tyr Phe Thr TCC CCG TCC CCC Ser Pro Ser Pro 105 GCC GAC GGG ACC Ala Asp'Oiy Thr 120 TCT GAT CCC GTC Ser Asp Pro Val GAG GCT GGA TTC Giu Ala Gly Phe GTG CCT Val Pro CCC TTT Pro Phe' 125 GAG TGG Giu Trp 140 CM GAG Pro Glu TTG CCC CCA Lou Pro Pro 110 GGC TGT CCT Gly Cys Pro ACC GTG ATG Thr Val Met 336 -384 432 480 528 130

GTC

Val

GAT

Asp 145

GCT

Ala GTC GAA Val Giu CAG GCG Gin Ala

ACA

Thr 160 TTC CAA GAG CAG Phe Gin GluGin 165 ACA GAT GTG CTC" Thr Asp Val Leu ATT.GAT GGC Ile Asp Gly

AAA

Lys 170

ATC

Xle TTG CTG CTC Lou Leu Leu ATG GAG Met Gin 175

CGC

Arg ACC GGC CTG Thr Gly Leu CGC CTC GGG Arg Leu Gly 180

GAG

Glu CCA GCC CTG Pro Ala Leu 190 CAC TTT GAG His Phe Giu AAA ATC TAC Lys Ile Tyr 195 GAT GAT GAC Asp Asp Asp CAC CAC His His ATC AAG GTG Ile-Lye Val .200 TTC TrA GGC Phe Lem Giy 215 CTT CAG CPSA GGC Lou.Gin Gin Giy 205 CCC GAT -GGC Pro Asp Gly TGAGCGCCCA GCCTCACCCC .1 0 0 0.

:0..0 TGCCCCAGCC. CATTCCGGCC CCCATCTCAC CCAAGATCCC GGGGCACCC TCAGCCCTCA TAACAGATTC CAAGGAGAGG TTGCCCCT1'G TGTCTGTCTC ACAGACATCT GCTCCTCAGC TTGCTCCTTA AACCCCAGG;T GGCTGACCCT CCCCACCCA.G AAAAAATGAA ATGTGGGGGG CTTCTGATCT CCCCAAGATC TTCCTGTAT AAATGTF~GG ATCTGCCTGT TrATTTI'GGT CCTACCACCC ATGCCCCCCT TCTCAGTCTG CCCCTGGCCT TGGGTTGGGG TTCCTCGGGC CITTCTC TC CTCCCTCTTT CCAGCTGGCT GTATTGCTTT TTAATATTGC ACCGAAGGTT INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: CCAGAGTCCA GzGAGCTGGAC GGALCCCTCTT GTCCTTATCT ACGTCGGTGT GGGGAGGGGA TCCAGGACAT T1'AGGAAAA CTCT 'CCGTT GAGCCAGATG G =GGTCTT TCCTCCCTCC CCAGCCCCTA GGGGACTAGC TCTITCTGTT GATrGTCGCT TT1'TAAALTAA AATTrTA 731 791 851 911 971 1031 1091 1151 1208 -76- LENGTH: 4697 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CA AAA AGC AGC CCA GOP. CAA. CCG GAA GCA GGA CCC GAG GOP. GCC CAG Lys Ser Ser Pro Gly Gin Pro Glu Ala Gly Pro Giu Gly Ala Gin GAG CGG CCC AGC CAG GCG GCT CCT GlU Arg Prar Ser-Gin.Ala Ala Pro AGC AGC CAG GCT CCT CGG AP.GCCG Ser Ser Gin Ala Pro Arg Lys Pro GCA GTA GAP. GCA GAP. GGT Ala Val Giu Ala Giu Gly 25 CCC GGC Pro Gly GAG GGG OCT Glu Giy Ala CAA GCC AGA ACG GCT Gin Ala Arg Thr Ala CTG AGC CGC CAA. CTG Leu Ser Arg Gin Leu CAG TCT G00 GCC CTT Gin Ser Gly. Ma Leu so GAP. GAC ATA CTG AGC Giu Asp Ile Leu Ser -GGC GAG GAT 000 GCA Ply Giu Asp Gly Ala 80 CGT GAT GTC TCT GAG GAG Kfq Asp Vai Ser Giu Giu 191 239 ACA TAC TGT Thx Tyr Cys 70 CAG GOT GAG Gin Ply Giu 85

GTG-

Val GAC AP.T AAC CAG*GG Asp Ain Asn Gin Gly 7S GGC CCC Gly Pro CCG GCT GAP. CCC GAP. OAT GCA Pro Ala Giu Pro Giu Asp Ala AAG TCC -COO ACC TAT GTG GCA AGO AP.T Lys Ser Arg Thx Tyr Vai Ala Arg Asn

GG

Gly 105 GAO CCT GAP. CCA Giu Pro Glu Pro ACT CCA thr Pro 110 ACA GAP.

Thr Giu GTA GTC TAT GOP.

Vai Val Tyr. Gly.

GAG P.TC CGG GAG Glu Ile Arg Gin 1-30 GAG AAG1GAP. CCC TCC AP.G Giu Lys Olu Pro Ser Lys 1 GGG' OAT -CCP.

Gly Asp Pro AGT GAC GAG GTC Ser Asp Glu Vai 135 GOP. GAC CGA GAC Gly Asp Arg Asp COP. AGO CCA Arg .Arg Pro GAG GAG AAG AAA AAA GCC AAG 01ln Glu Lys Lys Lys Ala Lys 145. 150 GOT TTG GGG Gly Lou Giy AAG GAG Lys Giu 155 ATC ACG TTG CTG Ile Thr Lou Lou

ATG

Met 160 CAG ACA TTG Gin Thr Lou AAT ACT Asn Thr 165 CTG AGT ACC CCA GAG GAG Leu Ser Thr Pro Giu Glu 170 AAG CTG GCT Lys- Lou -Ala 0* CTG- TGC AAG AP.G TA GCT GAP. CMG =T GAG GAG CAC CGGAAT TGA CAG Lou Cys Lys Lys Tyr Ala Giu Lou Lou Giu Giu His Arg Asn Ser. Gin 180 185 190 AAG CAG ATG Lys Gin Met AAG GAC CAC Lys Asp His 210

AAG

Lys 195 CTC CTA CAG AAA Leu Leu Gin Lys CAG AGC CAG.CTG Gin Ser Gin Lou GTG CAA GAG Val Gin Glu 205..

GCC CGC AGC Ala Arg Ser CTG CGC GGT GAG Leu Arg Gly Giu AGC AAG GCC GTC Ser Lys Ala Val AAG CTT Lys Leu 225 GAG AGC CTA TGC Giu Ser Leu Cys GAG CTG CAG CGG Giu Lou Gin Arg CAC AAC CGC TCC- CTC His Asn Arg Ser Lou 235 GAG GAG AAG CGC AAG Giu Giu Lys Arg Lys 255 71-9

AAG

Ly's 240 GAA GAA GGT GTG Giu Glu Gly Val CGG GCC Arg Al a CGG GAG GAG Arg Giu Giu 250 7 67 GAG GTG ACC TCG Glu Val Thr Ser

CAC

His 260 TTC CAG GT ACA =T AAT GAC An~ CG Phe Gin Val Thr Lou Asri Asp Ile Gin 265 CTG CAG Lou Gin 270 ATG GAA CAG Met Giu Gin GAG CTG GCT Glu-Leu Ala 290 GAG GAG CAT Giu Giu-His 305 AAT GAG CGC AAC Asn Giu Arg Asn AAG CTG CGC Lys Leu Arg CAA GAG AAC ATG Gin Giu Asn Met 285 GAG AGG...CTC-AAG Giu Arg Leu Lys

AAG

Lys 2 95 CTG ATT GAG CAG TAT Leu le Giu Gin Tyr 300 GAG CTG CGC Giu Leu Arg ATC GAC AAA Ile Asp Lys TTC AAA CAC AAG Phe Lys His Lys CTA CAA GAG CAG Lou GinGin Gin 959

CTG

Leu 320 GTG GAT GCC AAG Val Asp Ala Lys

CTC

Lou 325 CAG CAG GCC CAG Gin Gin Ala Gin ATO CTA AAG GAG Met Lou Lys Giu

GCA

Ala 335 1007 GAA GAG CGG CAC GAG CGG GAG Giu Giu Arg His Gin Arg Gi u 340 GAG TCC CAG AGG ATO TGT GAG Giu Ser Gin.Arg Met Cys Giu 355 AAG GAT =T CTC MT AAA GAG GMA GTA Lys Asp Ph. Lou Lou Lys Giu Ala Vai 345 350 1055 CTG ATG AAG Lou Met Lys 360 CAG CAA GAG Gin Gin Giu ACC CAC CTG Thr His Lou 365 1103 AAG CAA CAG CTT GCC CTA TAC ACA Lys Gin Gin. Lou Ala Lou Tyr Thr 370 375 GAG AAG TTr GAG Giu Lys Phe Giu GAG TTC CAG -AAC Giu -Phe Gin An 390 AAG GAG rOAG ATO Lys Gin'Giu Met 1199 ACA CT Thr Lou 385 GAA AAG Giu Lye 400 TCC AAA AGC.

Ser Lye Ser ATG ACT AAG Met Thr.;Lys AGC GAG Ser Giu 39.0 GTA TTC ACC ACA Val Phe Thr Thr a.

AAG ATC AAG AAG CTG GAG LYe le'Lye Lys Lou Giu 405 410 AAA GAA ACC ACC ATG 1247 Lys Glu Thr Thr Met TAC CGG TCC Tyr.Arg Ser GAG GAG AAA Giu Giu Lys ATC CAA CGG le Gin Arg 450 GAC CTG AAC Asp Leu Asn CGG TGG GAG Arg Trp, Giu 420 ACA GTC CGG Thr Val Arg 435 CTG GAG AAG Leu Giu Lye AAG AGG GTA Lys Arg Vai AGC AGC AAC Ser Ser Asn -78- AAG GCC Lys Aia 425 CTG GAG Leu Glu GAT AAA Asp Lye CTG TGC Leu Cys 455 GAG GAC Gin Asp 470 GAG AGG Giu Arg

GAA

Giu CTG CTT GAG Leu Leu Giu GGC CTG GAG Giy Leu Gin 445 GAG ACA GAG Gin Thr Glu ATG GOT Met Ala 430 GTA AAA Val Lye CGC AAT Arg Asn 440 CGG GCA Arg IAia 12

OTG

Leu 460

CTC

Leu 480

GCA

Ala ACT GAC AGT GGC CCT Thr Asp Ser Gly Pro 485 CCC AGO TCC CCC AGG Pro Ser Sgi..Pzo Arg 500 CTO AGT GOT Leu Ser Ala AGG CCA GAG Arg Pro Glu 490 GAA GCG COT Giu Ala Pro

GGT

Gly 475 Gly

TGC

Cys GGC GAG .GGC TCC Gly Gin Gly Ser OCT GGG GOT CAA Pro Gly Ala Gin 495 TAOCGCA GGA GCA Tyr Pro Gly Ala 510 GAG CCC ACC TOO Glu Pro Thr Ser 1343 1391 1439 1487 1535 1 583 GTC AGA Val Thr S05

GGG

Gly CCG'AGC ACA GAA GCTA GGC CAG AC." Pro Ser Thr Giu Ala Ser Gly Gin Thr COT CAA Pro Gin GOC AGG GCC Ala Arg Aia TAGAGAGCCT GGTGTTGGGT GATGCTGGGA AGGGAGCGGC AGCCCAGCCA 1642 G;GCCTGGCCC ATAAAAGGCT GACeTGGCTG GCATCTGGCA

GGGCATTTTG.CAAGGCCTTG

TGGGGATGGG GGTGTGTACA GAGGGGCTG;T CATCTG;TAGC TCTGTCTGAT TGWCC AccAAGG;TCT TG.ACTCATT CTAGCTGCTT GGAGGCTCCT CCTGGCAGGG CGCTCAGAGC TTGCGTGGGA TGATGTGCTG AGTGCCAGGA CCAGGCCAA6T AGCCTGCACA AATGATTGAC CCCATGCTGA GCAGCCCATT GCTGAAGCCA CTTGCAATTT TGGATTTGT GGGTCAGTTr CAAATGCATT TATACCTGTA AGTGTACADT GATGAAGTCA GTGGCTI'GTC TGTGAGCTGA TGCCATCACA GTGAGTTGGC.AGAAGTGACT GACCTCCC TGCCCTTTCA GAGCTCAAAA TGTCTI'GTGA GCAGGGCTTG CTI!GGTCAGC TTGATTCTCT AGACCTGGAA AAGGTGTOCC TGGGATTTCC TGCCTGGAAC AAGGGACCTG GTGAGGAGCC CCT TGGGCAT CGCTTCCCCT GATGCTTCTC AGTAGCCTTA TCATTCACAG AAGAGATCAC CCAAAGGATT ATTTCTGAAG G7GATGTTCTT

TACGTACATA

GGGCTTGCAT

AGAGTCTTGA

TGAGCATTTC

CAAGTAATAC

TCAGGCCCTC

TAGGCAGAGC

GAGAATGTTT

GCCCTTTGGT

GTGCCTCTCT

GTGTT1TTTT 1702 1762 1822 1882 1942 2002 2062 2122 2182 2242 2302 2362 2422 CTTTATTTCT TTTTOTTTTT TITTTTTTOCT TTTTCTTITT TTrTTGCACA TGAOAGTGTT -79- TGTATTGAGG ACCTTCCAAG TCCAGTGTCC CACCTCCTTC TTCCTGTTTG GTGAQATCAG TTGCCACAAG CTTACCTGTG CTGTCTCCAT GCAGCAGTTG TTTCTGCTTC-tGCCCCCAC TCCTGGCACC TCCCTGCCTG TGTTGTCTAA CAGGTAAA.

ATGGCTGCAG ACAG-11G-TET TGTACTGTGT AGAGCCATTC TTAGCCAGTG T1TQTGCCAA ATAGGATGGT TAGGGCTTTG GTCCCTTCAT CTTCCATACC AGTTGGTTCA CAAAGAATGT ATGAA6TGGCA CACCATT1'CT GAGTTGCCTG CTATTCTCTG AGTGTTGAGA TCTCTGCCAT GACCTTTTTC TTCCCCTTTG CCAGTAGGCC TTTTCCTTAC AACTCACAGA GTT.rGCTAG CCCCTTGAAG AGAGGGGAAA GCTACCTCTT AATGCCTGTG AGGCC-ACAGT GALGCAGGTGG CAAGGTGCAG CCTTTGCTCA TdAGGTTCCC TACCTTTTTC CTGCTTCTGC TTTCCAAAGT ATGAGTGAAG GCCAGTGTCT AGGACCCAAG ATCTGTGTIG AGGTGAAGCT CAAGAGCGCA

GAAAAGGGAT

ACCACCCCAC

GTTGTTTGTG

GGTTTCAGTC

CTGGGTCCCA

CCCCACATGT

TCCTI'GGGGA

ATGAAATTGA

CTGTGTCCTG

TATATGCTGA

GCAGTTTTCG

CTTCTTGCTG

TAGCCCACTC

TAAGTACTGA

CCTTCTCCTG

GTGTATCCTT

CTCTGGCTGA

AATCCCTCTT

CCCAGCCCTT

GCGAGAGAGG

GOCCTACAGT

GAGGGACTGG

CCCCPLGCTGG

CAGCTCCPLTC

TICTCTTCAT

AGCCCAGGTC

TCCTCAhCCTG

GTTTCTTAGA

TGGCTCTGCT

GCTGTACCAG

TTGGCTCCTT

GTAAAAGAAA

CTGAGAGGCC

TGTCCAGCTG

GCAATCCTCA

TTCTACTTCT

ACTATTGACT

TCTACCCCC

ATGfl'CTGCT

GGGACAACAG

TTTTTC.TTTG

TTTACCCT

ATCATGTGTG

CCCCAGGGCAL

CACATCTAGG

GATACTGCTG

GTCTGGAGTA

GTGCCAGGCT

TGCAGGGAAG

GGCCCACATA

AGCTGCTGGA

GTTTCCCAGG

CATGTCTAGA

TGACCAAATC

CTGGGCCAGA

GGTGGTCCCA

TTGCTAGCTT

AATAGTAAAT

TGGTGCCTGG GTGCCTGGCC TGCCATCTTG ATGCTGAGGT

GGAAAGGGCT

ACCACCAGTT

CCTCTTTGGC

AGATTTGTCC

TCCTGTGTGG

GGGCCCCAGA

ACTCCAGTAC

GTTGCAAACT

AATGACTCAG

AACTAGTCAT

TACCTTAAAT

TCTGATGGCT

CCCATCAGCA

TTCATGGGTT

TGATTCTATT

GGCCCATAGC

A&TCCATAAA

ATAACTACTA

TGCCAGGGTA

ACCAAGATGG

TGTfCCCAG

CTCTCAGATA

ACTGAQACCA GAGATGGCAA GGTACCACTG ATCTGCATCA 2482 ,.2S42 2602 2662 2722 2782 2842 2902 2962 3022 3082 3143 3202 3262 3322 .3382 3442 3502 3562 3622 3682 3742 3802 3862 3922 3982 4042 4102 4162 a a.

a a

TGCCCTCAAG

TCCTGTGAAG

GTCCTTGCCT

AAGCTGGTAC

AGGCAGAGGT

ATTGCCTGAC

TCCCAGTGTG

TCAGGAATGT

CCTTCAGGCC

AACCAATCAC

TGCAGGGGAG

CACTTGTGAC

TTCCTCCAGG

TGTTTTCAGG

CAGCTGTGTG

TGTTTCCCAT

TCTTCT1'GCT

AAGAGCTGCC

ATGCACCTTC

TCACACTTCAk

GTGGTG;TAGG

GGGCCCCAGG

AGTCTGCAGP.

TACAGCTGCT

GTGCCTATCC

CCTCAGTTTT

GGACCACAGC

GCCTTGTCCA

GCTGAGAGCT TCATGTCCAC CAGATTCTGA GAGGTGTCAG TGTTGTrI'GT TTTCCATGAG GTTATCGGAC CATGGGCTGA AGACTGTTAT TTCTGTAAAG ATGGTTATTT AACCCTCCTC GAGGGCTGAC CCGGAGGCCA GTGG.AGCTGC CTGGTGTCCA CTGAGCTGAT TCTCCAGCTG CTGCCCCAGC CTTTCCGCCT ACCCCAGGGA CAGCCAGGCA CCTGCTCCTC TTGCCCTTCC CTGTCCCTGT AACTGCTTTC CTTATGGCCC AACCCGGCCA CACTGGCAGC TTTTTTGTCT CCTTTGGGTA TTCACAACAG TATTTTAAAC CACATTAAAT AAAGAGTCTG TTGCCTTAAA CAGCACTTI-r

GCTCAGGCAC

CACCCCATCA

CGGGGGAGGG

TGCACAGCAC

TGGGGGAAAG

CTCAGACTTG

CCAGGQACTT

AAAAAAAAAA

rTTTTATT

TTTCTGTAGG

CGGTGGCCCT

CCAAGGCCTG

AGAGGTGGTC

GAGCTGCCTT

TTTGAAGCTG

GATTTTGATG

AAAAA

4222 4282 4342 4402 4462 4522 4582 4642 4697 a a INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: fingle TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GTG GAC GTG GAT GAG TAC GAC GAG AAC AAG TTC GMG GAC GAG GAA GAC Val Asp Val. Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp 1 5 10 1 GGC GGC GAC GGC Gly Gly Asp Gly INFORMATION FOR SEQ ID, NO: 19- SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp Glu Glu Asp Asp 15 10 48 -81- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp Glu Glu Asp Asp Val 1 5 10 Ser Glu Gly Ser Glu Val Pro Glu Ser Asp 2) INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Val Ser Glu Gly Ser Glu Val Pro Glu Ser Aap 1 5 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids, TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Glu Asp Asp Asp Pro Asp Gly Phe Leu Gly 1 5 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: -82- Val Asp Val Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp 10 Gly Gly Asp Gly Gin Ala Gly Pro Asp Glu Gly Glu Val Asp 25 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:- Asp Glu Gly Glu Val Asp 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS;-.

LENGTH:. 16-amino acids TYPE: amino acid TOPOLOGY: ineau- (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Glu Glu Glu Glu Glu Glu G2lu Asp Asp Glu Asp Asp Asp-.Asp Asp 1 5 10 1 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Gtlu Asp Asp Asp Asp Asp 10 Val Val Ser Glu Gly Ser Glu Val Pro-Glu Ser Asp *20 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:27: Val Val Ser Glu Gly Ser Glu Val Pro Glu Ser Asp 1 5 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: .10 amino acids- TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:28: Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala 1 5 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids.

TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Glu Asp Gly Val Gin Gly G lu Pro Pro Glu Pro -Glu Asp Ala Glu 1. S 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS:- LENGTH: 45 bases* TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GAA GAG OAA GAA GAT GAT GAT GAA GAT GAA GAT GAA GAA GAT GAT *Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp 91U Glu Asp Asp 2. 510 is INFORMATION FOR SEQ ID, NO:3 1: SEQUENCE CHARACTERISTICS: LENGTH: 78 bases TYPE: nucleic acid 4(C) STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 1: GAA GAG GAA GAA GAT GAT GAT GAk GAT GMA GAT GAA GAA GAT GAT GTG Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp Glu Glu Asp Asp Val 1 5 10 1 TCA GAG GGC TCT OAA GTG CCC GAG AGT GAC 78 Ser Glu Gly Ser Glu Val Pro Glu Ser Asp INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 33 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: OTG TCA GAG GGC TCT GAA GTG CCC GAG AGT GAC 33 Val. Ser Glu Gly Ser Glu Val. Pro Glu Ser Asp 1 5 INFORMATION FOR SEQ ID-NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 30 bases TP:nucleic ai STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GA93-OAT. GAT GAC CCC OAT GOC TrC TTA GGC *a.Glux Asp Asp Asp Pro Asp Gly Phe Leu Gly A- 5 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: -nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GTG GAC GTrG GAT GAA TAT GAC GAG AAC AAG IITC GTG GAC GAA GAA GAT 48 Val Asp Val Asp Glii-Tyr-Asp Glu Asn Lys Phe Val. Asp Glu Glu Asp 1 510 GGG GGC GAC GGC CAG GCC GGGCCC GAC GAG GGC GAG GTG GAC Gly Gly Asp Gly Gin Ala Gly Pro Asp Glu Gly Glu Val Asp 25 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GAC GAG GGC GAG..GTG GAC 1s Asp Glu Gly Giu Val Asp 1 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 48 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCEDESCRIPTION: SEQ ID NO:36: *GAG GAG GAG GAG GAG GAG GAG GAA GAC GAC GAG GAC GAC GAC GAC GAC 48 Glu Glu Giu Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Asp Asp 1 5 10 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 84 bases TYPE: nucleic acid STRANDEDNE9SS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQi1D NO:37: GAG GAG GAG GAG GAG GAG GAG GAA GAC GAC GAG GAC GAC GAC GAC GAC 48 Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Glxi Asp Asp Asp Asp ASP- ~10 1 GTC GTG TCC GAG GGC TCG GAG GTG CCC GAG AGC GAT 84 Val Val Ser Glu Gly Ser Glu Val Pro Giu Ser Asp INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 36 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION:SEQ ID N0:38.

GTC GTG TCC GAG GGC TCG GAG GTG CCC GAG AGC GAT 36 Val Val Ser Giu Gly Ser Glu Val Pro Gilu Ser Asp 15 INFORMATI1ON FOR SEQ ID NO:39: SEQUENCE CHA1RACiTERISTICS: LENGTH: 30 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CCC CCC GGG AAG CCA GCC CTC dCCA GGA GCC Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala INFORMATION FOR SEQ ID (i)-SEQUENCE CHARACTERISTICS: LENGTH: 45 bases_ TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTON: SEQ ID GAG GAT GGG GWC CAD GGT GAD CCC CCT GAA CCT GAA GAT GCP. GAG Giu Asp Giy Val Gin Gly Gin Pro Pro, Giu Pro Giu Asp Ala Giu 1 5 10 1 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids -87- TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Arg Asp Val Ser Glu Glu Leu 1 INFORMATION FOR SEQ- ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 21 bases TYPE: nucleic acid STRANDEDNESS: single- TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CGT GAT GTC TCT--AG GAG CTG .21 Arg Asp Val Ser Glu Glu Leu

Claims (39)

1. An antibody or fragment thereof that specifically binds to a polypeptide consisting of the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

2. The antibody or fragment thereof of claim 1, comprising an antibody fragment selected from the group consisting of an Fab fragment, an Fab' fragment, and an F(ab') 2 fragment.

3. The antibody or fragment thereof of claim 1, comprising an antibody fragment selected from the group consisting of an F(v) fragment, a heavy chain monomer, a heavy chain dimer, a heavy chain trimer, a light chain monomer, a light chain dimer, a light chain trimer, and a dimer consisting of one heavy and one light chain.

4. The antibody or fragment thereof of claim 1, comprising an antibody that is a monoclonal antibody. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof is a humanized antibody or fragment thereof. o 6. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof is a chimeric antibody or fragment thereof.

7. The antibody or fragment thereof of claim 6, wherein the chimeric antibody or fragment thereof contains a constant region derived from a human antibody and a variable region derived from a mouse antibody.

8. The antibody or fragment thereof of claim 1, wherein the antibody or S 30 fragment thereof is a human antibody or antibody or fragment thereof. *o*o P:\OPERJEHRes Ctm\20033D0 /4 7 13 cIn, d-O01II 2/03 -89-

9. The antibody or fragment thereof of any one of claims 1-8, wherein the antibody or fragment thereof blocks binding of LDL to the polypeptide. The antibody or fragment thereof of any one of claims 1-9, wherein the antibody or fragment thereof comprises a label.

11. The antibody or fragment thereof of claim 10, wherein the label is a radiolabel.

12. The antibody or fragment thereof of any one of claims 1-9, wherein the antibody or fragment thereof comprises a technetium-binding ligand.

13. The antibody or fragment thereof of any one of claims 1-9, wherein the antibody or fragment thereof comprises a gadolinium-binding chelator.

14. The antibody or fragment thereof of claim 13, wherein the gadolinium- binding chelator is diethylene triamine penta-acetic acid (DTPA). A preparation of polyclonal antibodies that that specifically bind to a .o..6 0 0 20 polypeptide consisting of the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. t th 16. The preparation of claim 15, wherein the antibodies block binding of LDL o to the polypeptide.

17. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1-14 and a pharmaceutically acceptable carrier.

18. A cell line that produces the antibody of claim 17.

19. The cell line of claim 18, wherein the cell line is a hybridoma. P \OPER\EH\Rc CsIU\2003\DccU47 III cms de-01/1 2/03 A method for identifying a candidate agent that modulates LBP-1, LBP-2, or LBP-3 metabolism or structure, the method comprising: contacting in vitro a candidate agent and an LBP-1, LBP-2, or LBP-3 polypeptide; and evaluating the effect of the candidate agent on LBP-1, LBP-2, or LBP-3 metabolism or structure.

21. The method of claim 20, wherein the method comprises: contacting in vitro LBP-1, LBP-2, or LBP-3 polypeptide, an LBP-1, LBP-2, or LBP-3 binding molecule, and a candidate agent; and measuring the formation of a complex containing the LBP-1, LBP-2, or LBP-3 polypeptide and the LBP-1, LBP-2, or LBP-3 binding molecule, wherein a reduction in the formation of the complex in the presence of the candidate agent as compared with in the absence of the candidate agent indicates that the candidate agent inhibits the binding of LBP-1, LBP-2, or LBP-3 to the LBP-1, LBP-2, or LBP-3 2 binding molecule.

22. The method of claim 20, wherein the method comprises: measuring the binding of the candidate agent to the LBP-1, LBP-2, or LBP-3 20 polypeptide, to thereby identify a candidate agent that binds to an LBP-1, LBP-2, or LBP-3 Spolypeptide.

23. The method of any one of claims 20-22, wherein the LBP-1, LBP-2, or LBP-3 polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence that binds to LDL and is at least 80% identical to the sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, .or SEQ ID NO:8; an amino acid sequence that binds to LDL and comprises the sequence of SEQ ID .i 30 NO:9, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:29; and P \OPEREHcs Cms\203\De\247 1113 clmsdm.0112/03 -91 an amino acid sequence that binds to LDL and is identical to a fragment of at least amino acid residues of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

24. The method of claim 23, wherein the LBP-1, LBP-2, or LBP-3 polypeptide comprises an amino acid sequence that binds to LDL and is at least 80% identical to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

25. The method of claim 24, wherein the LBP-1, LBP-2, or LBP-3 polypeptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

26. The method of any one of claims 20-25, wherein the candidate agent is an LBP-1, LBP-2, or LBP-3 fragment, analog or mimetic.

27. The method of any one of claims 20-25, wherein the candidate agent is a nucleic acid, antibody, metabolite, carbohydrate, glycoprotein, peptide, or non-peptide mimetic. *oooo :28. The method of claim 21, wherein the LBP-1, LBP-2, or LBP-3 binding molecule is LDL. S29. The method of claim 28, wherein the LDL is native LDL.

30. The method of claim 28, wherein the LDL is modified LDL.

31. The method of claim 30, wherein the modified LDL is methylated LDL or •oxidized LDL. *ooo* P \OPER\VEHlRc Clm-\20030 Q471113 drdoc- -1/12/03 92

32. The method of claim 21, wherein the LBP-1, LBP-2, or LBP-3 binding molecule is an extracellular matrix component.

33. The method of claim 32, wherein the extracellular matrix component is a proteoglycan.

34. The method of claim 20, wherein the method comprises evaluating the effect of the candidate agent on the primary, secondary, or tertiary structure of LBP-1, LBP-2, or LBP-3. The method of claim 20, wherein the method comprises evaluating the effect of the candidate agent on conformational folding of LBP-1, LBP-2, or LBP-3.

36. The method of claim 20, wherein the method comprises evaluating the effect of the candidate agent on LBP-1, LBP-2, or LBP-3 expression.

37. The method of claim 20, wherein the method comprises evaluating the effect of the candidate agent on the release of LBP-1, LBP-2, or LBP-3.

38. The method of claim 20, wherein the method comprises evaluating the effect of the candidate agent on the distribution of LBP-1, LBP-2, or LBP-3.

39. An antisense molecule that hybridizes under cellular conditions to the nucleic acid of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 in a manner that inhibits expression of the LBP-1, LBP-2, or LBP-3 polypeptide encoded by the nucleic acid.

40. A method of treating atherosclerosis in a subject, the method comprising administering to the subject an amount of the antibody or fragment thereof or preparation 30 of polyclonal antibodies of any one of claims 1-16 effective for treating atherosclerosis. P \OPERUEHlRes Ct,\2003\Dec\2471 103 cl--dO Oll1)2/03 -93

41. A method of treating atherosclerosis in a subject, the method comprising administering to the subject an amount of the pharmaceutical composition of claim 17 effective for treating atherosclerosis.

42. A method of diagnosing atherosclerosis in a subject, the method comprising administering to the subject an amount of the antibody or fragment thereof or preparation of polyclonal antibodies of any one of claims 1-16 effective for diagnosing atherosclerosis.

43. The method of claim 42, wherein the diagnosing comprises: administering to the subject the antibody or fragment thereof under conditions that allow the antibody or fragment thereof to interact with LBP-1, LBP-2, or LBP-3 present in an atherosclerotic lesion so as to result in labeled LBP-1, LBP-2, or LBP-3; and determining the localization or quantification of the labeled LBP-1, LBP-2, or LBP-3 by imaging so as to detect the presence of an atherosclerotic lesion in the subject.

44. The method of claim 43, wherein the imaging is selected from the group consisting of magnetic resonance imaging, gamma camera imaging, single photon emission computed tomographic imaging, and positron emission tomography. 20 45. A method of treating atherosclerosis in a subject, the method comprising administering to the subject an amount of an LBP-1, LBP-2, or LBP-3 polypeptide effective for treating atherosclerosis. s

46. A method of immunizing a subject, the method comprising administering to the subject an amount of an LBP-1, LBP-2, or LBP-3 polypeptide effective for immunizing the subject against the LBP-1, LBP-2, or LBP-3 polypeptide. *00

47. A method of diagnosing atherosclerosis in a subject, the method comprising administering to the subject an LBP-1, LBP-2, or LBP-3 polypeptide effective for 30 diagnosing atherosclerosis. P lOPER\JEH\Rce Clmrsk2003\Dcc247 113 cis dm.OlI 2/03 -94-

48. A method of treating atherosclerosis in a subject, the method comprising administering to the subject an amount of the antisense molecule of claim 39 effective for treating atherosclerosis.

49. An antibody according to any one of claims 1 to 14, a preparation according to any one of claims 15 to 16, a pharmaceutical composition according to claim 17, a cell line according to any one of claims 18 to 19, a method according to any one of claims to 38 or 40 to 48 or an antisense molecule according to claim 39 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 1st day of December, 2003 BOSTON HEART FOUNDATION, INC. by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) e* 4 S S