Treatment of ocular disease

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 92 KB ASCII (Text) file named "233106-332748_Seq_Listing_ST25.txt," created on Oct. 12, 2012, at 3:07 pm.

FIELD

Methods for treating eye diseases or conditions characterized by vascular instability, vascular leakage, and neovacularization such as ocular edema, ocular neovascularization, diabetic macular edema, age-related macular degeneration, choroidal neovascularization, diabetic retinopathy, retinal vein occlusion (central or branch), ocular ischemia, ocular trauma, surgery induced edema, and uveitis.

BACKGROUND

The eye comprises several structurally and functionally distinct vascular beds, which supply ocular components critical to the maintenance of vision. These include the retinal and choroidal vasculatures, which supply the inner and outer portions of the retina, respectively, and the limbal vasculature located at the periphery of the cornea. Injuries and diseases that impair the normal structure or function of these vascular beds are among the leading causes of visual impairment and blindness. For example, diabetic retinopathy is the most common disease affecting the retinal vasculature, and is the leading cause of vision loss among the working age population in the United States. Vascularization of the cornea secondary to injury or disease is yet another category of ocular vascular disease that can lead to severe impairment of vision.

"Macular degeneration" is a general medical term that applies to any of several disease syndromes, which involve a gradual loss or impairment of eyesight due to cell and tissue degeneration of the yellow macular region in the center of the retina. Macular degeneration is often characterized as one of two types, non-exudative (dry form) or exudative (wet form). Although both types are bilateral and progressive, each type may reflect different pathological processes. The wet form of age-related macular degeneration (AMD) is the most common form of choroidal neovascularization and a leading cause of blindness in the elderly. AMD affects millions of Americans over the age of 60, and is the leading cause of new blindness among the elderly.

Choroidal neovascular membrane (CNVM) is a problem that is related to a wide variety of retinal diseases, but is most commonly linked to age-related macular degeneration. With CNVM, abnormal blood vessels stemming from the choroid (the blood vessel-rich tissue layer just beneath the retina) grow up through the retinal layers. These new vessels are very fragile and break easily, causing blood and fluid to pool within the layers of the retina.

Diabetes (diabetes mellitus) is a metabolic disease caused by the inability of the pancreas to produce insulin or to use the insulin that is produced. The most common types of diabetes are type 1 diabetes (often referred to as Juvenile Onset Diabetes Mellitus) and type 2 diabetes (often referred to as Adult Onset Diabetes Mellitus). Type 1 diabetes results from the body's failure to produce insulin due to loss of insulin producing cells, and presently requires the person to inject insulin. Type 2 diabetes generally results from insulin resistance, a condition in which cells fail to use insulin properly. Type 2 diabetes may have a component of insulin deficiency as well.

Diabetes is directly responsible for a large number of disease conditions, including conditions or diseases of the eye including diabetic retinopathy (DR) and diabetic macular edema (DME) which are leading causes of vision loss and blindness in most developed countries. The increasing number of individuals with diabetes worldwide suggests that DR and DME will continue to be major contributors to vision loss and associated functional impairment for years to come.

Diabetic retinopathy is a complication of diabetes that results from damage to the blood vessels of the light-sensitive tissue at the back of the eye (retina). At first, diabetic retinopathy may cause no symptoms or only mild vision problems. Eventually, however, diabetic retinopathy can result in blindness. Diabetic retinopathy can develop in anyone who has type 1 diabetes or type 2 diabetes.

At its earliest stage, non-proliferative retinopathy, microaneurysms occur in the retina's tiny blood vessels. As the disease progresses, more of these blood vessels become damaged or blocked and these areas of the retina send signals into the regional tissue to grow new blood vessels for nourishment. This stage is called proliferative retinopathy. The new blood vessels grow along the retina and along the surface of the clear, vitreous gel that fills the inside of the eye.

By themselves, these blood vessels do not cause symptoms or vision loss. However, they have thin, fragile walls and without timely treatment, these new blood vessels can leak blood (whole blood or a constituent thereof) which can result in severe vision loss and even blindness.

Also, fluid can leak into the center of the macula, the part of the eye where sharp, straight-ahead vision occurs. The fluid and the associated protein begin to deposit on or under the macula causing the patient's central vision to become distorted. This condition is called macular edema. It can occur at any stage of diabetic retinopathy, although it is more likely to occur as the disease progresses. About half of the people with proliferative retinopathy also have macular edema.

Uveitis is a condition in which the uvea becomes inflamed. The eye is shaped much like a tennis ball, hollow on the inside with three different layers of tissue surrounding a central cavity. The outermost is the sclera (white coat of the eye) and the innermost is the retina. The middle layer between the sclera and the retina is called the uvea. The uvea contains many of the blood vessels that nourish the eye. Complications of uveitis include glaucoma, cataracts or new blood vessel formation (neovascularization).

The currently available interventions for exudative (wet form) macular degeneration, diabetic retinopathy, diabetic macular edema, choroidal neovascular membrane, complications from uveitis or ocular trauma, include laser photocoagulation therapy, low dose radiation (teletherapy) and surgical removal of neovascular membranes (vitrectomy). Laser therapy has had limited success and selected choroidal neovascular membranes which initially respond to laser therapy have high disease recurrence rates. There is also a potential loss of vision resulting from laser therapy. Low dose radiation has been applied ineffectively to induce regression of choroidal neovascularization. Recently, vascular endothelial growth factor (VEGF) antagonists, ranibizumab and aflibercept, have been approved for use in age-related macular degeneration, diabetic macular edema and retinal vein occlusion (RVO).

(RVO) is the most common retinal vascular disease after diabetic retinopathy. Depending on the area of retinal venous drainage effectively occluded, it is broadly classified as either central retinal vein occlusion (CRVO), hemispheric retinal vein occlusion (HRVO), or branch retinal vein occlusion (BRVO). It has been observed that each of these has two subtypes. Presentation of RVO in general is with variable painless visual loss with any combination of fundal findings consisting of retinal vascular tortuosity, retinal hemorrhages (blot and flame shaped), cotton wool spots, optic disc swelling and macular edema. In a CRVO, retinal hemorrhages will be found in all four quadrants of the fundus, while these are restricted to either the superior or inferior fundal hemisphere in a HRVO. In a BRVO, hemorrhages are largely localized to the area drained by the occluded in the retinal vein.

There is therefore a long felt and substantial need for methods of treating diseases of the eye which are characterized by vascular instability, vascular leakage and neovascularization.

SUMMARY

Disclosed are agents that bind to the extracellular portion and inhibit human protein tyrosine phosphatase beta (HPTP.beta.). Also disclosed are methods for treating eye diseases or conditions characterized by vascular instability, vascular leakage, and neovacularization such as ocular edema, ocular neovascularization, diabetic macular edema, age-related macular degeneration, choroidal neovascularization, diabetic retinopathy, retinal vein occlusion (central or branch), ocular ischemia, ocular trauma, surgery induced edema, and uveitis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The monoclonal antibody R15E6 recognizes Endogenous HPTP.beta. on endothelial cells. (Panel A) Endothelial cell lysates are immunoprecipitated with a control antibody (Lane 1), with R15E6 (Lane 2) or with a mixture of anti-Tie2 and anti-VEGFR2 antibodies (Lane 3). Immunoprecipitates are resolved by SDS-PAGE, transferred to a PVDF membrane and probed by western blot with a mixture of R15E6, anti-Tie2 and anti-VEGFR2 antibodies. A single major high molecular weight band consistent with HPTP.beta. is seen with R15E6 (Lane 2) and not with the control antibody (Lane 1) or the mixture of anti-Tie2 and anti-VEGFR2 (Lane 3). (Panel B) Endothelial cells are subjected to FACS analysis with R15E6 (white peak) or a control with no primary antibody (black peak). The robust shift in fluorescence indicates that R15E6 binds to HPTP.beta. on the surface of intact endothelial cells.

FIG. 2. The monoclonal antibody R15E6 enhances Tie2 Receptor Activation in HUVECs. Tie2 activation is measured in human endothelial cells as described in Example 4. R15E6 dose dependently enhances both basal and Ang1-induced Tie2 activation.

FIG. 3. Is a graphical representation of the mean area of choroidal neovascularization in C57BL/6 mice 14 days post laser injury in eyes treated with intravitreal injection of 1 .mu.g or 2 .mu.g of an anti-VE-PTP extracellular domain antibody in one eye versus similar treatment of the fellow eye with control.

FIG. 4. Shows the mean area (mm.sup.2) of retinal neovascularization in C57BL/6 mice on day P17 after containment in a 75% oxygen atmosphere from P5 to P12 and intravitreal injection of an anti-VE-PTP extracellular domain antibody at P12 when the mice were returned to room air.

FIG. 5. Shows representative fluorescent micrographs of mouse retinas in the oxygen-induced retinopathy model after intravitreal injection of vehicle or 2 .mu.g of an anti-VE-PTP extracellular domain antibody.

FIG. 6. Shows the mean area (mm.sup.2) of retinal neovascularization in C57BL/6 mice on day P17 after containment in a 75% oxygen atmosphere from P5 to P12 followed by return to room air on P12 with subcutaneous administration of 1 mg/kg of an anti-VE-PTP extracellular domain antibody on days P12, 14 and 16.

FIG. 7. Shows the mean area (mm.sup.2) of retinal neovascularization in C57BL/6 mice on day P17 after containment in a 75% oxygen atmosphere from P5 to P12 followed by return to room air on P12 with subcutaneous administration of 2 mg/kg of an anti-VE-PTP extracellular domain antibody on days P12, 14 and 16.

DETAILED DESCRIPTION

General Definitions

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

The term "HPTP.beta.-ECD binding agent" and "specific binding agent" are used interchangeably herein and refer to a molecule that specifically binds to the extracellular portion of HPTP.beta., and variants and derivatives thereof, as defined herein, that inhibits the Tie2 dephosphorylase activity of HPTP.beta..

"Agent" as used herein refers to a "HPTP.beta. binding agent" unless otherwise noted.

"Specifically binds HPTP.beta.-ECD" refers to the ability of a specific binding agent of the present invention to recognize and bind to an epitope of the extracellular domain of HPTP.beta. with higher affinity than to other related and/or unrelated molecules. Specific binding agents preferentially bind to HPTP.beta. in a complex mixture of proteins and/or macromolecules. The specific binding agent is preferably selective for HPTP.beta.. "Selective" means that the agent has significantly greater activity toward HPTP.beta. compared with other related and/or unrelated molecules, not that it is completely inactive with regard to other molecules. For example, a selective agent may show 10-fold, 100-fold, or 1000-fold selectivity toward HPTP.beta. than to other related or unrelated molecules.

The term "anti-HPTP.beta.-ECD antibodies" refers to antibodies or antibody fragments that bind to the extracellular domain of HPTP.beta.. Anti-HPTP.beta.-ECD antibodies are a type of HPTP.beta.-ECD binding agent as defined herein.

The term "VE-PTP" refers to the mouse ortholog of HPTP.beta..

All percentages, ratios and proportions herein are by weight, unless otherwise specified. All temperatures are in degrees Celsius (.degree. C.) unless otherwise specified.

Ranges may be expressed herein as from one particular value to another particular value, the endpoints are included in the range. For example for the range from "1 mg to 50 mg" includes the specific values 1 mg and 50 mg. The antecedent "about" indicates that the values are approximate. For example for the range from "about 1 mg to about 50 mg" indicates that the values are approximate values. Additionally, when such a range is expressed, the range includes the range "from 1 mg to 50 mg." It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. For example the range "from 1 mg to 50 mg", includes the range "from 30 mg to 40 mg."

"Effective amount" means an amount of an active agent or combination of agents effective to ameliorate or prevent the symptoms, or prolong the survival of the patient being treated. An effective amount may vary according to factors known in the art, such as the disease state, age, sex and weight of the human or animal being treated. Although particular dosage regimes may be described in examples herein, a person skilled in the art would appreciate that the dosage regime may be altered to provide optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. In addition, the compositions of this disclosure can be administered as frequently as necessary to achieve a therapeutic amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

As used herein the term "inhibit" or "inhibiting" refers to a statistically significant and measurable reduction in activity, preferably a reduction of at least about 10% versus control, more preferably a reduction of about 50% or more, still more preferably a reduction of about 80% or more.

As used herein the term "increase" or "increasing" refers to a statistically significant and measurable increase in activity, preferably an increase of at least about 10% versus control, more preferably an increase of about 50% or more, still more preferably an increase of about 80% or more.

"HPTP beta" or "HPTP.beta." are used interchangeably herein and are abbreviations for human protein tyrosine phosphatase beta.

As used herein, "subject" means an individual. Thus, the "subject" can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. "Subject" can also include a mammal, such as a primate or a human. "Subject" and "patient" are used interchangeably herein. Preferably the subject is a human.

By "reduce" or other forms of the word, such as "reducing" or "reduction," is meant lowering of an event or characteristic (e.g., vascular leakage). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

The terms "treatment", "treating", "treat" and the like, refer to obtaining a desired pharmacologic and/or physiologic effect such as mitigating a disease or a disorder in a host and/or reducing, inhibiting, or eliminating a particular characteristic or event associated with a disorder (e.g., ocular edema). Thus, the term "treatment" includes, preventing a disorder from occurring in a host, particularly when the host is predisposed to acquiring the disease, but has not yet been diagnosed with the disease; inhibiting the disorder; and/or alleviating or reversing the disorder. Insofar as the methods of the present invention are directed to preventing disorders, it is understood that the term "prevent" does not require that the disease state be completely thwarted. Rather, as used herein, the term preventing refers to the ability of the skilled artisan to identify a population that is susceptible to disorders, such that administration of the compounds of the present invention may occur prior to onset of a disease. The term does not imply that the disease state is completely avoided.

Unless otherwise specified, diabetic retinopathy includes all stages of non-proliferative retinopathy and proliferative retinopathy.

Throughout the description and claims of this specification the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes one composition or mixtures of two or more such compositions.

Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

"Specifically binds HPTP.beta." refers to the ability of an agent of the present invention to recognize and bind to an epitope of the extracellular domain of HPTP.beta. with higher affinity than to the other related and/or unrelated molecules. The agent is preferably selective for HPTP.beta.. "Specific" means that the agent has significantly greater activity toward HPTP.beta. compared with other related and/or unrelated molecules, not that it is completely inactive with regard to other molecules. For example, a selective agent may show 10-fold, 100-fold, or 1000-fold selectivity toward HPTP.beta. than to other related or unrelated molecule.

The term "epitope" refers to any portion of any molecule capable of being recognized by and bound by a agent at one or more of the agent's antigen binding regions. Epitopes usually consist of distinct, recognizable surface groupings such as amino acids, sugars, lipids, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. Epitopes as used herein may be conformational or linear.

"Peptibody" is a molecule comprising an antibody Fc domain attached to at least one peptide. The production of peptibodies is generally described in WO2002/24782.

"Fragment" refers to a portion of an agent. A fragment may retain the desired biological activity of the agent and may be considered to be an agent itself. For example a truncated protein in which the amino terminus and/or carboxy terminus and/or an internal amino acid residue is deleted is a fragment of the protein and an Fab of an immunoglobulin molecule is a fragment of the immunoglobulin. Such fragments may also be connected to a another molecule by way of a direct connection (e.g. a peptide or disulfide bond) or by way of a linker.

"Protein" is used herein interchangeably with peptide and polypeptide.

Peptides of the present invention include, but are not limited to amino acid sequences having from about 3 to about 75 amino acids, or from about 5 to about 50 amino acids, or from about 10 to about 25 amino acids. Peptides may be naturally occurring or artificial amino acid sequences.

A protein of the invention may be obtained by methods well known in the art, for example, using standard direct peptide synthesizing techniques such as via solid-phase synthesis. If the gene sequence is known or can be deduced then the protein may be produced by standard recombinant methods. The proteins may be isolated or purified in a variety of ways known to one skilled in the art. Standard purification methods include precipitation with salts, electrophoretic, chromatographic techniques and the like.

Agents may be covalently or non-covalently conjugated to a vehicle. The term "vehicle" refers to a molecule that prevents degradation and/or increase half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of the agent. Exemplary vehicles include, but are not limited, Fc domains of immunoglobulins and polymers, for example: polyethylene glycol (PEG), polylysine, dextran, a lipid, a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, or peptide that binds to a salvage receptor.

"Derivatives" include those binding agents that have been chemically modified in some manner distinct from insertion, deletion, or substitution variants. For example, wherein the binding agent is a protein, the carboxyl terminus may be capped with an amino group, such as NH.sub.2.

In some embodiments one or more molecules are linked together to form the agent. For example antibody fragments may be connected by a linker. In general, the chemical structure of the linker is not critical as it serves primarily as a space. In one embodiment, the linker is made of amino acids linked together by way of peptide bonds. In another embodiment, the linker is a non-peptide linker such as a non-sterically hindering C.sub.1-C.sub.6 alkyl group. In another embodiment, the linker is a PEG linker. It will further be appreciated that the linker can be inserted in a number of locations on the molecule.

Variants of an agent are included within the scope of the present invention. "Variant" or "Variants" as used herein means an agent having a protein or nucleotide sequence which is substantially similar to the protein or nucleotide sequence of the non-variant agent and which shares a similar activity of the non-variant agent. A protein or nucleotide sequence may be altered in various ways to yield a variant encompassed by the present invention, including substitutions, deletions, truncations, insertions and other modifications. Methods for such manipulations are well known in the art. See, for example, Current Protocols in Molecular Biology (and updates) Ausubel et al., Eds (1996), John Wiley and Sons, New York: Methods in Molecular Biology, Vol. 182, In vitro Mutagenesis Protocols, 2.sup.nd Edition, Barman Ed. (2002), Humana Press, and the references cited therein. For example, variants include peptides and polypeptides wherein amino acid residues are inserted into, deleted from and/or substituted into the known amino acid sequence for the binding agent. In one embodiment, the substitution of the amino acid is conservative in that it minimally alters the biochemical properties of the variant. In other embodiments, the variant may be an active fragment of a full-length protein, a chemically modified protein, a protein modified by addition of affinity or epitope tags, or fluorescent or other labeling moieties, whether accomplished by in vivo or in vitro enzymatic treatment of the protein, by chemical modification, or by the synthesis of the protein using modified amino acids.

Fusions proteins are also contemplated herein. Using known methods, one of skill in the art would be able to make fusion proteins of the proteins of the invention; that, while different from native form, may be useful. For example, the fusion partner may be a signal (or leader) polypeptide sequence that co-translationally or post-translationally directs transfer of the protein from its site of synthesis to another site (e.g., the yeast alpha-factor leader). Alternatively, it may be added to facilitate purification or identification of the protein of the invention (e.g., poly-His, Flag peptide, or fluorescent proteins).

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures are generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery and treatment of patients.

Sequence Listing

TABLE-US-00001 TABLE 1 SEQ ID NO: 1 Full length Human HPTP.beta. nucleotide sequence (X54131) SEQ ID NO: 2 Full length Human HPTP.beta. amino acid sequence (P23467) SEQ ID NO: 3 Extracellular Portion of Human HPTP.beta. with (His).sub.6Gly Tag SEQ ID NO: 4 Extracellular Portion of Human HPTP.beta. SEQ ID NO: 5 Full length mouse VE-PTP nucleotide sequence SEQ ID NO: 6 Full length mouse VE-PTP amino acid sequence SEQ ID NO: 7 Extracellular portion of mouse VE-PTP amino acid sequence

HPTP.beta.-ECD Binding Agents

Agents useful in the present invention include, but are not limited to, antibodies, proteins, darpins, peptides, aptamers, adnectins, peptibodies, or nucleic acids that bind specifically to the extracellular portion of HPTP.beta. and inhibit at least one phosphatase activity of HPTP.beta.. As used herein, "phosphatase activity" includes enzymatic activity and biologic activity where biological activity is measured by assessing Tie2 phosphorylation.

Agents useful in the present invention further include: antibodies, or antigen binding fragments thereof which bind to the extracellular portion of HPTP.beta. wherein the antibody or antigen-binding fragment inhibits at least one phosphatase activity of HPTP.beta.. These agents include monoclonal and polyclonal antibodies. An agent may be a fragment of an antibody, wherein the fragment comprises the heavy and light chain variable regions, or the fragment is an F(ab').sub.2, or the fragment is a dimer or trimer of an Fab, Fv, scFv, or a dia-, tria-, or tetrabody derived from the antibody.

For example, the agent may be, without limitation, an antibody or antibody fragment that binds the extracellular portion of HPTP.beta.; or in particular an antibody that binds an FN3 repeat of HPTP.beta., or more specifically an antibody that binds the first FN3 repeat of HPTP.beta..

Agents further include: the monoclonal antibody R15E6 which is described in U.S. Pat. No. 7,973,142, which is hereby incorporated in its entirety. (The mouse hybridoma, Balbc spleen cells (B cells) which may be used to produce the antibody are deposited with American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA on 4 May 2006, assigned ATCC No. PTA-7580) (Referred to herein as R15E6)), antibodies having the same or substantially the same biological characteristics of R15E6; antibody fragments of R15E6, wherein the fragment comprises the heavy and light chain variable regions; an F(ab')2 of R15E6; dimers or trimers of an Fab, Fv, scFv; and dia-, tria-, or tetrabodies derived from R15E6.

In particular, an agent suitable for use in the present invention is an antibody, antibody fragment, variant or derivatives thereof, either alone or in combination with other amino acid sequences, provided by known techniques. Such techniques include, but are not limited to enzymatic cleavage, chemical cleavage, peptide synthesis or recombinant techniques. The invention further embraces derivative agents, e.g. peptibodies.

Thus, one embodiment of an HPTP.beta.-ECD binding agent is an antibody, another embodiment is a protein, yet another embodiment is a peptide, and another embodiment is a darpin, another embodiment is an aptamer, another embodiment is a peptibody, still another embodiment is an adnectin, another embodiment is a nucleic acid. In some embodiments the HPTP.beta.-ECD binding agent is an monoclonal antibody, or is a polyclonal antibody. In particular embodiments, the HPTP.beta.-ECD binding agent is an antibody fragment that is capable of binding to HPTP.beta.-ECD. Preferably the HPTP.beta.-ECD binding agent is an antibody, or an antibody fragment, including but not limited to, an F(ab').sub.2, an Fab, a dimer of an Fab, an Fv, a dimer of an Fv, a scFv, a dimer of a scFv, a trimer of an Fab, a trimer of an Fv, a trimer of a scFv, minibodies, a diabody, a triabody, a tetrabody, a linear antibody, a protein, a peptide, an aptamer, a peptibody, an adnectin, or a nucleic acid, that binds to the extracellular portion of HPTP.beta.. In certain embodiments the HPTP.beta.-ECD binding agent is and F(ab').sub.2 of a monoclonal antibody. In some embodiments the HPTP.beta.-ECD binding agent comprises a plurality of HPTP.beta.-ECD binding sites, for example where the HPTP.beta.-ECD binding agent is an intact antibody or an F(ab').sub.2, or a dimer of an Fab, or a trimer of an Fab. For example, in some embodiments an HPTP.beta.-ECD binding agent is able to bind to two HPTP.beta. molecules simultaneously at the same or different epitope, thereby bringing the two HPTP.beta. molecules into close proximity with one and other. In other embodiments the HPTP.beta.-ECD binding agent is able to bind to three HPTP.beta. molecules simultaneously at the same or different epitope, thereby bringing the three HPTP.beta. molecules into close proximity with one and other. In another embodiment, the HPTP.beta.-ECD binding agent is the monoclonal antibody produced by hybridoma cell line ATCC No. PTA-7580. In yet another embodiment, the HPTP.beta.-ECD agent is an antigen binding fragment of the monoclonal antibody produced by hybridoma cell line ATCC No. PTA-7580. In still another embodiment, the HPTP.beta.-ECD binding agent is an antibody having the same or substantially the same biological characteristics the monoclonal antibody produced by hybridoma cell line ATCC No. PTA-7580 or an antigen binding fragment thereof.

Any of the embodiments of HPTP.beta.-ECD binding agents disclosed in the present application, may be covalently or non-covalently conjugated to a vehicle. The term "vehicle" refers to a molecule that affects a biological property of an agent. For example, a vehicle may prevent degradation, and/or increase half-life, absorption, reduce toxicity, reduce immunogenicity, or increase biological activity of the agent. Exemplary vehicles include, but are not limited to, Fc domains of immunoglobulins; polymers, for example: polyethylene glycol (PEG), polylysine, dextran; lipids; cholesterol groups (such as a steroid); carbohydrates, dendrimers, oligosaccharides, or peptides that binds to a salvage receptor. In some embodiments the vehicle is polyethylene glycol (PEG), in other embodiments the vehicle is polylysine, in yet other embodiments the vehicle is dextran, in still other embodiments the vehicle is a lipid

Water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337, which are incorporated herein in their entirety. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Particularly preferred are peptibodies covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the peptibodies, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving the therapeutic capacity for agents, e.g. peptibodies, and for humanized antibodies in particular, is described in U.S. Pat. No. 6,133,426. The invention also contemplates derivatizing the peptide and/or vehicle portion of the agents. Such derivatives may improve the solubility, absorption, biological half-life, and the like of the agents. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the agents and the like.

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies), single chain antibodies, e.g., antibodies from llama and camel, antibody fragments, e.g., variable regions and/or constant region fragments, so long as they exhibit a desired biological activity, e.g., antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

An "antigen binding fragment" as used herein is a fragment of an agent that binds to a portion of HPTP.beta. and inhibits the activity of HPTP.beta..

An "isolated antibody" is an antibody which has been identified, and/or separated, and/or recovered from its natural environment.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies may polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the four-chain unit is generally about 150 kilo Daltons (kDa). Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V.sub.H) followed by three constant domains (C.sub.H) for each of the alpha and gamma chains and four C.sub.H domains for mu and epsilon isotypes. Each L chain has at the N-terminus, a variable domain (V.sub.L) followed by a constant domain (C.sub.L) at its other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L is aligned with the first constant domain of the heavy chain (C.sub.H1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V.sub.H and V.sub.L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species may be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C.sub.H), immunoglobulins may be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. The gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in C.sub.H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

Members of the Camelidae family, e.g., llama, camel and dromedaries, contain a unique type of antibody, that are devoid of light chains, and further lack the C.sub.H1 domain (Muyldermans, S., Rev. Mol. Biotechnol., Vol. 74, pp. 277-302 (2001)). The variable region of these heavy chain antibodies are termed V.sub.HH or VHH, and constitute the smallest available intact antigen-binding fragment (15 kDa) derived from a functional immunoglobulin.

The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FR) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a .beta.-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V.sub.H; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop".

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations which include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope, i.e., a single antigenic determinant. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries, using the available techniques, e.g., Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 6851-6855 (1984)).

An "antibody fragment" comprises a portion of a multimeric antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, dimers and trimers of Fab conjugates, Fv, scFv, minibodies; dia-, tria- and tetrabodies; linear antibodies (See Hudson et al., Nature Med. Vol. 9, pp. 129-134 (2003)).

"Fv" is the minimum antibody fragment which contains a complete antigen binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, and are therefore included in the definition of Fv.

A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V.sub.H) and light chains (V.sub.L) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V.sub.H with the C-terminus of the V.sub.L, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two V.sub.H and two V.sub.L regions, yielding tandem scFvs. Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Consequently, diabody drugs could be dosed much lower than other therapeutic antibodies and are capable of highly specific targeting of tumors in vivo. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies are known and have been shown to exhibit an even higher affinity to their targets than diabodies.

The term "humanized antibody" or "human antibody" refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the V.sub.H and/or V.sub.L sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human V.sub.H and V.sub.L sequences to replace the corresponding nonhuman CDR sequences. Means for making chimeric, CDR-grafted and humanized antibodies are known to those of ordinary skill in the art (see, e.g., U.S. Pat. Nos. 4,816,567 and 5,225,539). One method for making human antibodies employs the use of transgenic animals, such as a transgenic mouse. These transgenic animals contain a substantial portion of the human antibody producing genome inserted into their own genome and the animal's own endogenous antibody production is rendered deficient in the production of antibodies. Methods for making such transgenic animals are known in the art. Such transgenic animals may be made using XenoMouse.RTM. technology or by using a "minilocus" approach. Methods for making XenoMice.RTM. are described in U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598 and 6,075,181. Methods for making transgenic animals using the "minilocus" approach are described in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, and WO 93/12227.

Humanization of a non-human antibody has become routine in recent years, and is now within the knowledge of one skilled in the art. Several companies provide services to make a humanized antibody, e.g., Xoma, Aries, Medarex, PDL and Cambridge Antibody Technologies. Humanization protocols are extensively described in technical literature, e.g., Kipriyanov and Le Gall, Molecular Biotechnol, Vol. 26, pp 39-60 (2004), Humana Press, Totowa, N.J.; Lo, Methods Mol. Biol., Vol. 248, pp 135-159 (2004), Humana Press, Totowa, N.J.; Wu et al., J. Mol. Biol. Vol. 294, pp. 151-162 (1999).

In certain embodiments, antibodies useful in the present invention may be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies may be used for transformation of a suitable mammalian host cell by known methods for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector), or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. The transformation procedure used may depend upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged lipids, and direct microinjection of the DNA into nuclei.

A nucleic acid molecule encoding the amino acid sequence of a heavy chain constant region, a heavy chain variable region, a light chain constant region, or a light chain variable region of an antibody, or a fragment thereof in a suitable combination if desired, is/are inserted into an appropriate expression vector using standard ligation techniques. The antibody heavy chain or light chain constant region may be appended to the C-terminus of the appropriate variable region and is ligated into an expression vector. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene may occur). For a review of expression vectors, see Methods Enzymol., Vol. 185, (Goeddel, ed.), 1990, Academic Press.

Identification of Specific Binding Agents

Suitable HPTP.beta.-ECD binding agents may be identified using a variety of techniques known in the art. For example, candidate agents can be screened for binding to HPTP.beta., and screened for activity. Generally, the candidate agents will first be screened for binding and those that show selective binding will then be screened to determine ability to inhibit the HPTP.beta.-mediated dephosphorylation of Tie2. In some cases however the candidate agents may be first screened in vivo for activity.

Determination of Binding Activity

The selection of a suitable assay for use in identification of a specific binding agent depends on the nature of the candidate agent to be screened. One of skill in the art would be able to choose the appropriate assays for the particular candidate agent.

For example, where the candidates are antibodies or peptibodies, which comprises an Fc moeity, FACS analysis as described in Example 3B allows the candidate agent to be selected based on its ability to bind to cells, which express HPTP.beta.. The cell may endogenously express HPTP.beta. or may be genetically engineered to express HPTP.beta..

For other candidate agents such as aptamers, other techniques are known in the art. For example, aptamers which specifically bind to HPTP.beta. can be selected using a technique known as SELEX (systematic evolution of ligands by exponential enrichment) which selects specific aptamers through repeated rounds of in vitro selection.

Determination of Inhibitor Activity by Western Blot

As exemplified in Example 4, in one suitable assay HUVECs are cultured in serum free media in the presence or absence of various concentrations of candidate agent and lysates of the cells are prepared, immunoprecipitated with a Tie2 antibody, resolved by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Membrane-bound immunoprecipitated proteins are then serially western blotted with an antiphosphotyrosine antibody to quantify Tie2 phosphorylation followed by a Tie2 antibody to quantify total Tie2. Tie2 phosphorylation is expressed as the ratio of the anti-phosphotyrosine signal over the total Tie2 signal. Greater levels of the anti-phosphotyrosine signal indicate greater HPTP.beta. inhibition by the candidate agent.

Candidate agents that can be screened include, but are not limited to, libraries of known agents, including natural products, such as plant or animal extracts, biologically active molecules including proteins, peptides including but not limited to members of random peptide libraries and combinatorial chemistry derived molecular library made of D- or L-configuration amino acids, antibodies including, but not limited to, polyclonal, monoclonal, chimeric, human, single chain antibodies, Fab, F(ab).sub.2 and Fab expression library fragments and eptiope-binding fragments thereof.

As used herein "antibody fragments" include, but are not limited, to a F(ab').sub.2, a dimer or trimer of an Fab, Fv, scFv, or a dia-, tria-, or tetrabody derived from an antibody.

Methods

Disclosed are methods for the treatment of diseases or conditions of the eye, especially retinopathies, ocular edema and ocular neovascularization. Non-limiting examples of these diseases or conditions include diabetic macular edema, age-related macular degeneration (wet form), choroidal neovascularization, diabetic retinopathy, ocular ischemia, uveitis, retinal vein occlusion (central or branch), ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, uveitis, and the like. These diseases or conditions are characterized by changes in the ocular vasculature whether progressive or non-progressive, whether a result of an acute disease or condition, or a chronic disease or condition.

One aspect of the disclosed methods relates to diseases that are a direct or indirect result of diabetes, inter alia, diabetic macular edema and diabetic retinopathy. The ocular vasculature of the diabetic becomes unstable over time leading to conditions such as non-proliferative retinopathy, macular edema, and proliferative retinopathy. As fluid leaks into the center of the macula, the part of the eye where sharp, straight-ahead vision occurs, the buildup of fluid and the associated protein begin to deposit on or under the macula. This results in swelling that disturbs the subject's central vision. This condition is referred to as "macular edema." Another condition that may occur is non-proliferative retinopathy in which vascular changes, such as microaneurysms, may occur outside the macular region of the eye.

These conditions may or may not progress to diabetic proliferative retinopathy which is characterized by neovascularization. These new blood vessels are fragile and are susceptible to bleeding. The result is scaring of the retina, as well as occlusion or total blockage of the light pathway through the eye due to the over formation of new blood vessels. Typically, subjects having diabetic macular edema are suffering from the non-proliferative stage of diabetic retinopathy; however, it is not uncommon for subjects to only begin manifesting macular edema at the onset of the proliferative stage.

Diabetic retinopathy, if left untreated, can lead ultimately to blindness. Indeed, diabetic retinopathy is the leading cause of blindness in working-age populations.

Therefore, the disclosed methods relate to preventing, treating, controlling, abating, and/or otherwise minimizing ocular neovascularization in a subject having diabetes or a subject diagnosed with diabetes. In addition, subjects having or subjects diagnosed with diabetes can be alerted to or can be made aware of the risks of developing diabetes-related blindness, therefore the present methods can be used to prevent or delay the onset of non-proliferative retinopathy in subjects known to be at risk. Likewise, the present methods can be used for treating subjects having or being diagnosed with non-proliferative diabetic retinopathy to prevent progression of the condition.

The disclosed methods relate to preventing or controlling ocular neovascularization or treating a disease or condition that is related to the onset of ocular neovascularization by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof.

One aspect of this method relates to treating or preventing ocular neovascularization by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. One embodiment of this aspect relates to a method for treating ocular neovascularization comprising administering to a subject a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carrier or compatible excipient.

Thus, one embodiment of the present disclosure is a method of treating or preventing ocular neovascularization in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing ocular neovascularization in a subject, comprising administering an effective amount of a composition comprising an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carrier or compatible excipient. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of ocular neovascularization.

The disclosed methods also relate to preventing or controlling ocular edema or treating a disease or condition that is related to the onset of ocular edema by administering to a subject an HPTP.beta.-ECD binding agent.

One aspect of this method relates to treating or preventing ocular edema by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. One embodiment of this aspect relates to a method for treating ocular edema comprising administering to a subject a composition comprising: a. an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof; and b. one or more carriers or compatible excipients.

Thus, one embodiment of the present disclosure is a method of treating or preventing ocular edema in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing ocular edema in a subject, comprising administering an effective amount of a composition comprising HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. An embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of ocular edema.

Another disclosed method relates to preventing or controlling retinal edema or retinal neovascularization, or treating a disease or condition that is related to the onset of retinal edema or retinal neovascularization, by administering to a subject an HPTP.beta.-ECD binding agent. One aspect of this method relates to treating or preventing retinal edema or retinal neovascularization by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. One embodiment of this aspect relates to a method for treating retinal edema or retinal neovascularization comprising administering to a subject a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients.

Thus, one embodiment of the present disclosure is a method of treating or preventing retinal edema in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment is a method of treating or preventing retinal neovascularization comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. One embodiment of the present disclosure is a method of treating or preventing retinal edema in a subject, by administering a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Another embodiment is a method of treating or preventing retinal neovascularization by administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Another embodiment is the use of an HPTP.beta.-ECD binding agent in the treatment of retinal edema. A further embodiment is the use of an HPTP.beta.-ECD binding agent in the treatment of retinal neovascularization.

A further disclosed method relates to treating, preventing or controlling diabetic retinopathy, or treating a disease or condition that is related to the onset of diabetic retinopathy by administering to a subject an HPTP.beta.-ECD binding agent.

One aspect of this method relates to treating or preventing diabetic retinopathy by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. One embodiment of this aspect relates to a method for treating diabetic retinopathy comprising administering to a subject a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carrier or compatible excipient.

Thus, one embodiment of the present disclosure is a method of treating or preventing diabetic retinopathy in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing diabetic retinopathy in a subject, by administering a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of diabetic retinopathy.

A further disclosed method relates to a method for treating or preventing non-proliferative retinopathy comprising administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof.

Another embodiment of this aspect relates to a method for treating or preventing non-proliferative retinopathy comprising administering to a subject a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof; and one or more carrier or compatible excipient.

Thus, one embodiment of the present disclosure is a method of treating or preventing non-proliferative retinopathy in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing non-proliferative retinopathy in a subject, by administering a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of non-proliferative retinopathy.

Yet a further disclosed method relates to preventing or controlling diabetic macular edema, or treating a disease or condition that is related to the onset of diabetic macular edema by administering to a subject an HPTP.beta.-ECD binding agent.

One aspect of this method relates to treating or preventing diabetic macular edema by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. One embodiment of this aspect relates to a method for treating diabetic macular edema comprising administering to a subject a composition comprising: a) an effective amount of one or more of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof; and b) one or more carriers or compatible excipients.

Thus, one embodiment of the present disclosure is a method of treating or preventing diabetic macular edema in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing diabetic macular edema in a subject, by administering a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of diabetic macular edema.

Another embodiment of the present disclosure is a method for treating, or preventing age-related wet form macular degeneration edema in a subject, comprising administering an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure is a method of treating or preventing age-related wet form macular degeneration edema in a subject, by administering a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of age-related wet form macular degeneration edema.

A further embodiment is a method for treating, preventing or controlling choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, or uveitis, by administering to a subject an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof. Another embodiment is a method for treating, preventing or controlling choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, retinal angiomatous proliferation, macular telangiectasia, or uveitis, by administering to a subject a composition comprising an effective amount of an HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt thereof, and one or more carriers or compatible excipients. Yet another embodiment of the present disclosure is the use of an HPTP.beta.-ECD binding agent in the treatment of choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, retinal angiomatous proliferation, macular telangiectasia or uveitis.

Another embodiment is a composition for treating or preventing an ocular disorder, comprising an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carrier. Yet another embodiment is a composition for treating or preventing an ocular disorder, comprising an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carrier composition wherein the ocular disorder is ocular neovascularization, ocular edema, retinal neovascularization, diabetic retinopathy, diabetic macular edema, age-related macular degeneration, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, or uveitis.

In some embodiments, the HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof is used for treating an ocular disorder. In some embodiments, the HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof is used for treating an ocular disorder, wherein the ocular disorder is ocular neovascularization, ocular edema, retinal neovascularization, diabetic retinopathy, diabetic macular edema, age-related macular degeneration, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia or uveitis.

In still other embodiments, the HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof is used for the manufacture of a medicament for treating an ocular disorder. In some embodiments the ocular disorder is ocular neovascularization, ocular edema, retinal neovascularization, diabetic retinopathy, diabetic macular edema, age-related macular degeneration, choroidal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, ocular trauma, surgery induced edema, surgery induced neovascularization, cystoid macular edema, ocular ischemia, non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia or uveitis.

Dosing

Effective dosages and schedules for administering the HPTP.beta.-ECD binding agent may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of the agent that must be administered will vary depending on, for example, the subject which will receive the agent, the route of administration, the particular type of agent used and other drugs being administered to the subject. For example, guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical dose of the agent used alone might range from about 0.01 mg/kg to up to 500 mg/kg of body weight or more per day, or from about 0.01 mg/kg to about 50 mg/kg, or from 0.1 mg/kg to about 50 mg/kg, or from about 0.1 mg/kg to up to about 10 mg/kg, or from about 0.2 mg/kg to about 1 mg/kg, depending on the factors mentioned above.

One embodiment relates to a method for treating ocular edema and/or neovascularization comprising administering to a subject from about 0.01 mg/kg to about 50 mg/kg of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. Another iteration of this embodiment relates to administering to a subject from about 0.1 mg/kg to about 10 mg/kg by weight of the subject being treated, an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. A further iteration of this embodiment relates to a method for treating or preventing diseases or conditions related to ocular edema and/or neovascularization comprising administering to a subject from about 1 mg/kg to about 10 mg/kg by weight of the subject an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. Yet another iteration of this embodiment relates to a method for treating or preventing diseases or conditions related to ocular edema and/or neovascularization comprising administering to a subject from about 5 mg/kg to about 10 mg/kg by weight of the subject an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. In a further iteration of this embodiment relates to a method for treating or preventing diseases or conditions related to ocular edema and/or neovascularization comprising administering to a subject from about 1 mg/kg to about 5 mg/kg by weight of the subject an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof. In a yet further iteration of this embodiment relates to a method for treating or preventing diseases or conditions related to ocular edema and/or neovascularization comprising administering to a subject from about 3 mg/kg to about 7 mg/kg by weight of the subject an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof.

The dosing schedules for administration of an HPTP.beta.-ECD binding agent include, but are not limited to, once daily, three-times weekly, twice weekly, once weekly, three times, twice monthly, once monthly and once every other month.

Further disclosed are methods of treating or preventing one or more of the diseases or conditions described herein above related to ocular edema and/or neovascularization that are the result of administration of another pharmaceutically active agent. As such, this aspect relates to a method comprising administering to a subject a composition comprising: a) an effective amount of an HPTP.beta.-ECD binding agent or pharmaceutically acceptable salt thereof; b) one or more additional pharmaceutically active agents; and c) one or more carriers or compatible excipients.

The methods of the present invention may be combined with the standard of care, including, but not limited to, laser treatment.

Non-limiting examples of pharmaceutically active agents suitable for combination with an HPTP.beta.-ECD binding agent include anti-infectives, i.e., aminoglycosides, antiviral agents, antimicrobials, anticholinergics/antispasmotics, antidiabetic agents, antihypertensive agents, antineoplastics, cardiovascular agents, central nervous system agents, coagulation modifiers, hormones, immunologic agents, immunosuppressive agents, ophthalmic preparations and the like.

The disclosed method also relates to the administration of the disclosed agents and compositions. Administration can be systemic via subcutaneous or i.v. administration; or the HPTP-.beta. inhibitor will be administered directly to the eye, e.g., local. Local methods of administration include, for example, by eye drops, subconjunctival injections or implants, intravitreal injections or implants, sub-Tenon's injections or implants, incorporation in surgical irrigating solutions, etc.

The disclosed methods relate to administering an HPTP.beta.-ECD binding agent as part of a pharmaceutical composition. Compositions suitable for local administration are known to the art (see, for example, U.S. Pat. Publ. 2005/0059639). In various embodiments, compositions of the invention can comprise a liquid comprising an active agent in solution, in suspension, or both. As used herein, liquid compositions include gels. In one embodiment, the liquid composition is aqueous. Alternatively, the composition can take form of an ointment. In another embodiment, the composition is an in situ gellable aqueous composition. Such a composition can comprise a gelling agent in a concentration effective to promote gelling upon contact with the eye or lacrimal fluid in the exterior of the eye. Aqueous compositions of the invention have ophthalmically compatible pH and osmolality. The composition can comprise an ophthalmic depot formulation comprising an active agent for subconjunctival administration. The microparticles comprising active agent can be embedded in a biocompatible pharmaceutically acceptable polymer or a lipid encapsulating agent. The depot formulations may be adapted to release all or substantially all the active material over an extended period of time. The polymer or lipid matrix, if present, may be adapted to degrade sufficiently to be transported from the site of administration after release of all or substantially all the active agent. The depot formulation can be a liquid formulation, comprising a pharmaceutical acceptable polymer and a dissolved or dispersed active agent. Upon injection, the polymer forms a depot at the injections site, e.g., by gelifying or precipitating. The composition can comprise a solid article that can be inserted in a suitable location in the eye, such as between the eye and eyelid or in the conjuctival sac, where the article releases the active agent. Solid articles suitable for implantation in the eye in such fashion generally comprise polymers and can be bioerodible or non-bioerodible.

In one embodiment of the disclosed methods, a human subject with at least one visually impaired eye is treated with 2-4000 .mu.g of an HPTP.beta.-ECD binding agent via intravitreal injection. Improvement of clinical symptoms are monitored by one or more methods known to the art, for example, indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, electroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, optical coherence tomography and autorefaction. Subsequent doses can be administered weekly or monthly, e.g., with a frequency of 2-8 weeks or 1-12 months apart.

The disclosed methods include administration of the disclosed agents in combination with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical formulation in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. In another aspect, many of the disclosed agents can be used prophylactically, i.e., as a preventive agent, either neat or with a pharmaceutically acceptable carrier. The ionic liquid compositions disclosed herein can be conveniently formulated into pharmaceutical compositions composed of neat ionic liquid or in association with a pharmaceutically acceptable carrier. See Remington's Pharmaceutical Sciences, 18th ed., Gennaro, A R. Ed., Mack Publishing, Easton Pa. (1990), which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the agents described herein and which is incorporated by reference herein. Such pharmaceutical carriers, most typically, would be standard carriers for administration of compositions to humans and non-humans, including solutions such as sterile water, saline and buffered solutions at physiological pH. Other agents can be administered according to standard procedures used by those skilled in the art. For example, pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics and the like.

Examples of pharmaceutically-acceptable carriers include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the disclosed agents, which matrices are in the form of shaped articles, e.g., films, liposomes, microparticles, or microcapsules. It will be apparent to those persons skilled in the art that certain carriers can be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Other agents can be administered according to standard procedures used by those skilled in the art.

Pharmaceutical formulations can include additional carriers, as well as thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the agents disclosed herein. Pharmaceutical formulations can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics and the like.

For the purposes of the present disclosure the term "excipient" and "carrier" are used interchangeably throughout the description of the present disclosure and said terms are defined herein as, "ingredients which are used in the practice of formulating a safe and effective pharmaceutical composition."

The formulator will understand that excipients are used primarily to serve in delivering a safe, stable and functional pharmaceutical, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may fill a role as simple and direct as being an inert filler, or an excipient as used herein may be part of a pH stabilizing system. The formulator can also take advantage of the fact the agents of the present invention have improved cellular potency, pharmacokinetic properties.

The disclosed agents can also be present in liquids, emulsions, or suspensions for delivery of active therapeutic agents. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active agent as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art, for example see Remington's Pharmaceutical Sciences, referenced above.

Kits

Also disclosed are kits comprising the agents and compositions to be delivered into a human, mammal, or cell. The kits can comprise one or more packaged unit doses of a composition comprising one or more agents to be delivered into a human, mammal, or cell. The unit dosage ampoules or multi-dose containers, in which the agents to be delivered are packaged prior to use, can comprise a hermetically sealed container enclosing unit dose of the composition, or multiples unit doses. The agents can be packaged as a sterile formulation, and the hermetically sealed container is designed to preserve sterility of the formulation until use.

EXAMPLES

Example 1

Production of HPTP.beta. Extracellular Domain Protein

Full length HPTP.beta. cDNA (SEQ ID NO:1) is cloned from a human placental library according to the manufacturer's (Origene) instructions. A cDNA encoding the entire soluble extracellular domain (ECD) of HPTP.beta. is cloned by PCR from the full length cDNA coding for amino acids 1-1621 with an added c-terminal His-His-His-His-His-His-Gly (6His-Gly) (SEQ ID NO:3). The resulting cDNA is cloned into mammalian expression vectors for transient (pShuttle-CMV) or stable (pcDNA3.1(-)) expression in HEK293 cells. To obtain purified HPTP.beta. ECD (.beta.ED), HEK293 cells transfected with a .beta.ECD expression vector are incubated in OptiMEM-serum free (Gibco) for 24 hours under normal growth conditions. The conditioned media is then recovered, centrifuged to remove debris, and 1 mL of washed Ni-NTA agarose (Qiagen) (500 .mu.L packed material) is added to each 10 .mu.L of cleared media and allowed to rock overnight at 4.degree. C. On the following day, the mixture is loaded into a column and washed with 20 bed volumes of 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8. The purified HPTP.beta. extracellular domain protein (SEQ ID NO:4) is then eluted with 200 .mu.L/elution in 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM Imidazole, pH 8. Fractions are analyzed for protein content using reducing-denaturing SDS-polyacrylimide gel electrophoresis and detected by silver stain (Invitrogen) and confirmed by mass spectrometry.

Example 2

Generation of Monoclonal Antibodies to HPTP.beta. Extracellular Domain

Purified HPTP.beta. extracellular domain protein is produced, for example by the procedure described in Example 1. For production of the HPTP.beta. extracellular domain immunogen, the purified HPTP.beta. extracellular domain-6-His protein is conjugated to porcine thyroglobulin (Sigma) using EDC coupling chemistry (Hockfield, S. et al., (1993) Cold Spring Habor Laboratory Press. Vol. 1 pp. 111-201, Immunocytochemistry). The resulting HPTP.beta. extracellular domain-thyroglobulin conjugate is dialyzed against PBS, pH 7.4. Adult Balb/c mice are then immunized subcutaneously with the conjugate (100-200 .mu.g) and complete Freund's adjuvant in a 1:1 mixture. After 2-3 weeks, the mice are injected intraperitoneally or subcutaneously with incomplete Freund's adjuvant and the conjugate in a 1:1 mixture. The injection is repeated at 4-6 weeks. Sera are collected from mice 7 days post-third-injection and assayed for immunoreactivity to HPTP.beta. extracellular domain antigen by ELISA and western blotting. Mice that display a good response to the antigen are boosted by a single intra-spleen injection with 50 .mu.l of purified HPTP.beta. extracellular domain protein mixed 1:1 with Alum hydroxide using a 31 gauge extra long needle (Goding, J. W., (1996) Monoclonal Antibodies: Principles and Practices. Third Edition, Academic Press Limited. p. 145). Briefly, mice are anesthetized with 2.5% avertin, and a 1 centimeter incision is created on the skin and left oblique body wall. The antigen mixture is administered by inserting the needle from the posterior portion to the anterior portion of the spleen in a longitudinal injection. The body wall is sutured and the skin is sealed with two small metal clips. Mice are monitored for safe recovery. Four days after surgery the mouse spleen is removed and single cell suspensions are made for fusion with mouse myeloma cells for the creation of hybridoma cell lines (Spitz, M., (1986) Methods In Enzymology, Vol. 121. Eds. John J, Lagone and Helen Van Vunakis. pp. 33-41 (Academic Press, New York, N.Y.)). Resulting hybridomas are cultured in Dulbeccos modified media (Gibco) supplemented with 15% fetal calf serum (Hyclone) and hypoxathine, aminopterin and thymidine.

Screening for positive hybridomas begins 8 days after the fusion and continues for 15 days. Hybridomas producing anti-HPTP.beta. extracellular domain antibodies are identified by ELISA on two sets of 96-well plates: one coated with the histidine tagged-HPTP.beta. extracellular domain and another one coated with a histidine-tagged bacterial MurA protein as a negative control. The secondary antibody is a donkey anti-mouse IgG labeled with horseradish peroxidase (HRP) (Jackson Immunoresearch). Immunoreactivity is monitored in wells using color development initiated by ABTS tablets dissolved in TBS buffer, pH 7.5. The individual HRP reaction mixtures are terminated by adding 100 microliters of 1% SDS and reading absorbance at 405 nm with a spectrophotometer. Hybridomas producing antibodies that interact with HPTP.beta. extracellular domain-6His, and not with the murA-6His protein are used for further analysis. Limiting dilutions (0.8 cells per well) are performed twice on positive clones in 96 well plates, with clonality defined as having greater than 99% of the wells with positive reactivity. Isotypes of antibodies are determined using the iso-strip technology (Roche). To obtain purified antibody for further evaluation, tissue culture supernatants are affinity purified using a protein A or protein G columns.

Six monoclonal antibodies immunoreactive to HPTP.beta.-ECD protein were isolated and given the following nomenclature, R15E6, R12A7, R3A2, R11C3, R15G2 and R5A8. Based on its reaction with the HPTP.beta.-ECD protein in ELISA and in western blots, R15E6 was selected for further study.

Example 3

The Monoclonal Antibody R15E6

The monoclonal antibody R15E6 was identified and characterized as described in Example 2 of the present application and in U.S. Pat. No. 7,973,142; the procedure and results are summarized below.

A. R15E6 Binds Endogenous HPTP.beta. as Demonstrated by Demonstrated by Immunoprecipitation.

Materials: Human umbilical vein endothelial cells (HUVECs), EGM media, and trypsin neutralizing solution from Cambrex; OPTIMEM I (Gibco), bovine serum albumin (BSA; Santa Cruz), phosphate buffered saline (PBS; Gibco), Growth Factors including Angiopoietin 1 (Ang1), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (R&D Systems), Tie2 monoclonal antibody (Duke University/P&GP), VEGF receptor 2 (VEGFR2) polyclonal antibody (Whitaker et. al), protein A/G agarose (Santa Cruz), Tris-Glycine pre-cast gel electrophoresis/transfer system (6-8%) (Invitrogen), PVDF membranes (Invitrogen), lysis buffer (20 mm Tris-HCl, 137 mm NaCl, 10% glycerol, 1% triton-X-100, 2 mM EDTA, 1 mM NaOH, 1 mM NaF, 1 mM PMSF, 1 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin).

Method: HUVECs were pre-treated for 30 min with antibody (in OPTIMEM) or OPTIMEM I alone. After removal of pre-treatment, cells were treated with Ang1 (100 ng/ml) for 6 minutes in PBS+0.2% BSA and lysed in lysis buffer. Lysates were run directly on a Tris-Glycine gel or immunoprecipitated with 2-5 .mu.g/ml Tie-2 antibody or 10 .mu.g/ml R15E6 antibody and protein A/G agarose. Immunoprecipitated samples were rinsed once with lysis buffer and boiled for 5 min in 1.times. times sample buffer. Samples were resolved on a Tris-Glycine gel, transferred to a PVDF membrane, and detected by western blot using the indicated antibodies (pTYR Ab (PY99, Santa Cruz), Tie-2, VEGFR2 and/or R15E6).

Results: By IP/western blotting, R15E6 recognizes a major, high molecular weight band consistent with the size of HPTP.beta. (FIG. 1, Panel A, Lane 2). The less intense, lower molecular weight bands likely represent less glycosylated precursor forms of HPTP.beta.. An immunoprecipitation (IP) with control, non-immune IgG shows no bands in the molecular weight range of HPTP.beta. (FIG. 1, Panel A, Lane 1), and a combined Tie2/VEGFR2 IP shows bands of the expected molecular weight (FIG. 1, Panel A, Lane 3). This result demonstrates that R15E6 recognizes and is specific for HPTP.beta..

B. R15E6 Binds Endogenous HPTP.beta. as Demonstrated by FACS Analysis

Materials: HUVECs, EGM media, and trypsin neutralizing solution from Cambrex; Secondary Alexfluor 488-tagged antibody from Molecular Probes; Hanks balanced salt solution (Gibco); FACSCAN flow cytometer and CellQuest software from Becton Dickenson.

Method: HUVECs are trypsinized, treated with trypsin neutralizing solution and rinsed with HBSS. R15E6 antibody (0.6 .mu.g) is added to 250,000 cells in 500 of HBSS and incubated on ice for 20 minutes. Cells were rinsed with 1 ml HBSS followed by adding 2 .mu.g of fluorescent-conjugated secondary antibody for 20 minutes on ice. Cells were rinsed and resuspended in 1 ml HBSS then analyzed on the FACSCAN flow cytometer with CellQuest software. Control cells were treated with fluorescent-conjugated secondary antibody only.

Results: By FACS analysis, intact HUVECs, R15E6 causes a robust shift (>90% of cells) in the fluorescence signal compared to the secondary antibody alone (FIG. 1, Panel B). This result indicates that R15E6 binds to endogenous HPTP.beta. presented on the surface of intact endothelial cells.

Example 4

R15E6 Enhances Tie2 Activation

R15E6 enhances Tie2 phosphorylation in the absence and presence of the angiopoietin 1 (Ang1), the Tie2 ligand.

Methods: HUVECs are cultured in serum free media as described above in the presence or absence of various concentrations of R15E6 and with or without added Ang1. Lysates are prepared, immunoprecipitated with a Tie2 antibody, resolved by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Membrane-bound immunoprecipitated proteins are then serially western blotted with an antiphosphotyrosine antibody to quantify Tie2 phosphorylation followed by a Tie2 antibody to quantify total Tie2. Tie2 phosphorylation is expressed as the ratio of the antiphosphotyrosine signal over the total Tie2 signal.

Results: R15E6 enhances Tie2 phosphorylation both in the absence and presence of Ang1 (FIG. 2). This result indicates that binding of R15E6 to HPTP.beta. on the surface of endothelial cells modulates its biological function resulting in enhanced activation of Tie2 in the absence or presence of ligand.

Example 5

Generation of Anti-VE-PTP Extracellular Domain Antibodies

A. Production of Mouse VE-PTP Extracellular Domain Protein (VE-PTP-ECD)

VE-PTP-ECD may be produced by any suitable method. Such methods are well known in the art. For example, VE-PTP-ECD can be produced using a method similar to Example 1 of the present disclosure where VE-PTP-ECD cDNA is used in place of cDNA encoding HPTP.beta.-ECD. SEQ ID NO: 5 provides a nucleotide sequence that encodes VE-PTP-ECD. SEQ ID NO: 7 provides the amino acid sequence of VE-PTP-ECD.

B. Generation of Antibodies to Ve-PTP ECD

Anti-VE-PTP antibodies are readily generated by methods that are well known in the art. For example, anti VE-PTP antibodies can be generated using the method of Example 2 of the present disclosure by substituting VE-PTP-ECD for the HPTP.beta. extracellular domain and immunizing rats with the resulting protein. The rat anti-mouse VE-PTP antibody used in the present studies was kindly provided by Dr. D. Vestweber (mAb 109). The antibody was generated as described in Baumer S. et al., Blood, 2006; 107: 4754-4762. Briefly, the antibody was generated by immunizing rats with a VE-PTP-Fc fusion protein. Immunization, hybridoma-fusion, and screening were conducted as described in Gotsch U., et al., J Cell Sci. 1997, Vol. 110, pp. 583-588 and Bosse R. and Vestweber D., Eur J. Immunol. 1994, Vol. 24, pp. 3019-3024.

The fusion protein was constructed such that the first 8 fibronectin type III-like repeats ending with the amino acid proline at position 732 of VE-PTP were fused in frame with the Fc part of human IgG1 (starting with amino acid proline at position 239). This construct cloned into pcDNA3 (Invitrogen) was stably transfected into CHO cells, and the fusion protein was purified by protein A Sepharose affinity purification.

Example 6

Intravitreal Injections of an Anti-VE-PTP ECD Antibody

Laser-induced Choroidal Neovascularization Model: The choroidal neovascularization model is considered to represent a model of neovascular age-related macular degeneration. Choroidal NV was generated as previously described. See To be T, et al., Am. J. Pathol. 1998, Vol. 153, pp. 1641-1646. Adult C57BL/6 mice had laser-induced rupture of Bruch's membrane in three locations in each eye and were then given 1 .mu.L intravitreal injections of 1 or 2 .mu.g of a VE-PTP-ECD antibody (IgG2a), in one eye and vehicle (5% dextrose) in the fellow eye. These treatments were repeated on day 7. Fourteen days after laser, the mice were perfused with fluorescein-labeled dextran (2.times.10.sup.6 average MW, Sigma, St. Louis, Mo.) and the extent of neovascularization was assessed in choroidal flat mounts by fluorescence microscopy. The area of CNV at each Bruch's membrane rupture site was measured by image analysis by an observer masked with respect to treatment group. The area of CNV is the average of the three rupture sites in one eye. As shown in FIG. 3, treatment with the VE-PTP-ECD antibody significantly reduced choroidal neovascularization at both 1 and 2 .mu.g doses versus treatment with vehicle control.

Example 7

Oxygen-Induced Ischemic Retinopathy

The oxygen-induced ischemic retinopathy model is considered to represent a model of proliferative diabetic retinopathy. Ischemic retinopathy was produced in C57BL/6 mice by a method described by Smith, L. E. H., et al. Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Vis. Sci. 35, 101-111 (1994).

C57BL/6 mice at postnatal day 7 (P7) and their mothers were placed in an airtight chamber and exposed to hyperoxia (75.+-.3% oxygen) for five days. Oxygen was continuously monitored with a PROOX model 110 oxygen controller (Reming Bioinstruments Co., Redfield, N.Y.). On P12, mice were returned to room air and under a dissecting microscope, a Harvard Pump Microinjection System and pulled glass pipettes were used to deliver a 1 .mu.l intravitreal injection of 1 or 2 .mu.g of a VE-PTP-ECD antibody was made in one eye and vehicle was injected in the fellow eye. At P17, the area of NV on the surface of the retina was measured at P17 as previously described. See Shen J, et al., Invest. Ophthalmol. Vis. Sci. 2007, Vol. 48, pp. 4335-4341. Briefly, mice were given an intraocular injection of 1 .mu.l containing 0.5 .mu.g rat anti-mouse PECAM antibody (Pharmingen, San Jose, Calif.). Twelve hours later, the mice were euthanized, the eyes fixed in 10% formalin. The retinas were dissected, incubated for 40 minutes in 1:500 goat anti-rat IgG conjugated with Alexa488 (Invitrogen, Carlsbad, Calif.), washed, and whole mounted. An observer masked with respect to treatment group examined the slides with a Nikon Fluorescence microscope and measured the area of NV per retina by computerized image analysis using ImagePro Plus software (Media Cybernetics, Silver Spring, Md.). FIG. 4 shows that treatment with the VE-PTP-ECD antibody significantly reduced retinal neovascularization at both 1 and 2 .mu.g doses versus treatment with vehicle control. FIG. 5 shows representative retinal whole mounts from a mouse treated with vehicle versus a mouse treated with 2 .mu.g of the VE-PTP-ECD antibody.

Example 8

Subcutaneous Injection of a VE-PTP-ECD Antibody

The oxygen-induced ischemic retinopathy model was conducted as described in Example 7 (containment in a 75% oxygen atmosphere from P5 to P12) for intravitreal dosing except that the VE-PTP-ECD antibody (1 mg/kg) was dosed subcutaneously at P12 when the mice were returned to room air and again on days P14 and P16 (three total doses). Neovascularization was assessed as described above on day (P17). FIG. 6 shows that subcutaneous dosing of the VE-PTP-ECD antibody reduces the area of retinal neovascularization.

Example 9

The experiment described in Example 8 was repeated at a subcutaneous dose of 2 mg/kg. (FIG. 7)

While a number of embodiments of this disclosure are described, it is apparent that the basic examples may be altered to provide other embodiments that utilize or encompass the HPTP.beta.-ECD binding agent, methods and processes of this invention. The embodiments and examples are for illustrative purposes and are not to be interpreted as limiting the disclosure, but rather, the appended claims define the scope of this invention.

SEQUENCE LISTINGS

1

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ctgtttacta actggaccca ggcacaagga 1740gacgtagaat tttaccaagt cttactgatc catgaaaatg tggtcattaa aaatgaaagc 1800atctccagtg agaccagcag atacagcttc cactctctca agtccggcag cctgtactcc 1860gtggtggtaa caacagtgag tggagggatc tcttcccgac aagtggttgt ggagggaaga 1920acagtccctt ccagtgtgag tggagtaacg gtgaacaatt ccggtcgtaa tgactacctc 1980agcgtttcct ggctcgtggc gcccggagat gtggataact atgaggtaac attgtctcat 2040gacggcaagg tggttcagtc ccttgtcatt gccaagtctg tcagagaatg ttccttcagc 2100tccctcaccc caggccgcct ctacaccgtg accataacta caaggagtgg caagtatgaa 2160aatcactcct tcagccaaga gcggacagtg cctgacaaag tccagggagt cagtgttagc 2220aactcagcca ggagtgacta tttaagggta tcctgggtgc atgccactgg agactttgat 2280cactatgaag tcaccattaa aaacaaaaac aacttcattc aaactaaaag cattcccaag 2340tcagaaaacg aatgtgtatt tgttcagcta gtccctggac ggttgtacag tgtcactgtt 2400actacaaaaa gtggacaata tgaagccaat gaacaaggga atgggagaac aattccagag 2460cctgttaagg atctaacatt gcgcaacagg agcactgagg acttgcatgt gacttggtca 2520ggagctaatg gggatgtcga ccaatatgag atccagctgc tcttcaatga catgaaagta 2580tttcctcctt ttcaccttgt aaataccgca accgagtatc gatttacttc cctaacacca 2640ggccgccaat acaaaattct tgtcttgacg attagcgggg atgtacagca gtcagccttc 2700attgagggct tcacagttcc tagtgctgtc aaaaatattc acatttctcc caatggagca 2760acagatagcc tgacggtgaa ctggactcct ggtgggggag acgttgattc ctacacggtg 2820tcggcattca ggcacagtca aaaggttgac tctcagacta ttcccaagca cgtctttgag 2880cacacgttcc acagactgga ggccggggag cagtaccaga tcatgattgc ctcagtcagc 2940gggtccctga agaatcagat aaatgtggtt gggcggacag ttccagcatc tgtccaagga 3000gtaattgcag acaatgcata cagcagttat tccttaatag taagttggca aaaagctgct 3060ggtgtggcag aaagatatga tatcctgctt ctaactgaaa atggaatcct tctgcgcaac 3120acatcagagc cagccaccac taagcaacac aaatttgaag atctaacacc aggcaagaaa 3180tacaagatac agatcctaac tgtcagtgga ggcctcttta gcaaggaagc ccagactgaa 3240ggccgaacag tcccagcagc tgtcaccgac ctgaggatca cagagaactc caccaggcac 3300ctgtccttcc gctggaccgc ctcagagggg gagctcagct ggtacaacat ctttttgtac 3360aacccagatg ggaatctcca ggagagagct caagttgacc cactagtcca gagcttctct 3420ttccagaact tgctacaagg cagaatgtac aagatggtga ttgtaactca cagtggggag 3480ctgtctaatg agtctttcat atttggtaga acagtcccag cctctgtgag tcatctcagg 3540gggtccaatc ggaacacgac agacagcctt tggttcaact ggagtccagc ctctggggac 3600tttgactttt atgagctgat tctctataat cccaatggca caaagaagga aaactggaaa 3660gacaaggacc tgacggagtg gcggtttcaa ggccttgttc ctggaaggaa gtacgtgctg 3720tgggtggtaa ctcacagtgg agatctcagc aataaagtca cagcggagag cagaacagct 3780ccaagtcctc ccagtcttat gtcatttgct gacattgcaa acacatcctt ggccatcacg 3840tggaaagggc ccccagactg gacagactac aacgactttg agctgcagtg gttgcccaga 3900gatgcactta ctgtcttcaa cccctacaac aacagaaaat cagaaggacg cattgtgtat 3960ggtcttcgtc cagggagatc ctatcaattc aacgtcaaga ctgtcagtgg tgattcctgg 4020aaaacttaca gcaaaccaat ttttggatct gtgaggacaa agcctgacaa gatacaaaac 4080ctgcattgcc ggcctcagaa ctccacggcc attgcctgtt cttggatccc tcctgattct 4140gactttgatg gttatagtat tgaatgccgg aaaatggaca cccaagaagt tgagttttcc 4200agaaagctgg agaaagaaaa atctctgctc aacatcatga tgctagtgcc ccataagagg 4260tacctggtgt ccatcaaagt gcagtcggcc ggcatgacca gcgaggtggt tgaagacagc 4320actatcacaa tgatagaccg cccccctcct ccacccccac acattcgtgt gaatgaaaag 4380gatgtgctaa ttagcaagtc ttccatcaac tttactgtca actgcagctg gttcagcgac 4440accaatggag ctgtgaaata cttcacagtg gtggtgagag aggctgatgg cagtgatgag 4500ctgaagccag aacagcagca ccctctccct tcctacctgg agtacaggca caatgcctcc 4560attcgggtgt atcagactaa ttattttgcc agcaaatgtg ccgaaaatcc taacagcaac 4620tccaagagtt ttaacattaa gcttggagca gagatggaga gcttaggtgg aaaacgcgat 4680cccactcagc aaaaattctg tgatggacca ctgaagccac acactgccta cagaatcagc 4740attcgagctt ttacacagct ctttgatgag gacctgaagg aattcacaaa gccactctat 4800tcagacacat ttttttcttt acccatcact actgaatcag agcccttgtt tggagctatt 4860gaaggtgtga gtgctggtct gtttttaatt ggcatgctag tggctgttgt tgccttattg 4920atctgcagac agaaagtgag ccatggtcga gaaagaccct ctgcccgtct gagcattcgt 4980agggatcgac cattatctgt ccacttaaac ctgggccaga aaggtaaccg gaaaacttct 5040tgtccaataa aaataaatca gtttgaaggg catttcatga agctacaggc tgactccaac 5100taccttctat ccaaggaata cgaggagtta aaagacgtgg gccgaaacca gtcatgtgac 5160attgcactct tgccggagaa tagagggaaa aatcgataca acaatatatt gccctatgat 5220gccacgcgag tgaagctctc caatgtagat gatgatcctt gctctgacta catcaatgcc 5280agctacatcc ctggcaacaa cttcagaaga gaatacattg tcactcaggg accgcttcct 5340ggcaccaagg atgacttctg gaaaatggtg tgggaacaaa acgttcacaa catcgtcatg 5400gtgacccagt gtgttgagaa gggccgagta aagtgtgacc attactggcc agcggaccag 5460gattccctct actatgggga cctcatcctg cagatgctct cagagtccgt cctgcctgag 5520tggaccatcc gggagtttaa gatatgcggt gaggaacagc ttgatgcaca cagactcatc 5580cgccactttc actatacggt gtggccagac catggagtcc cagaaaccac ccagtctctg 5640atccagtttg tgagaactgt cagggactac atcaacagaa gcccgggtgc tgggcccact 5700gtggtgcact gcagtgctgg tgtgggtagg actggaacct ttattgcatt ggaccgaatc 5760ctccagcagt tagactccaa agactctgtg gacatttatg gagcagtgca cgacctaaga 5820cttcacaggg ttcacatggt ccagactgag tgtcagtatg tctacctaca tcagtgtgta 5880agagatgtcc tcagagcaag aaagctacgg agtgaacaag aaaacccctt gtttccaatc 5940tatgaaaatg tgaatccaga gtatcacaga gatccagtct attcaaggca ttgagaatgt 6000acctgaagag ctcctggata aaaattattc actgtgtgat ttgtt 604521997PRTHomo sapiens 2Met Leu Ser His Gly Ala Gly Leu Ala Leu Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu Ala Glu Pro Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys Ala Ser Ser His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55 60 Ala Ala Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys 65 70 75 80 Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg Ile Ile 85 90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr Asp Pro Leu Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys Thr Thr Ser Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser Gly Lys Val Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn Asn Gln Lys Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr Ser Trp Asn Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser Lys Tyr Asn Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185 190 Phe Ser Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp 195 200 205 Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp Ser His 210 215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu Met Asp Lys Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp Lys His Ala Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly Tyr Leu Tyr Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu Gln Asn Tyr Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu Val Ser Asn Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser Leu Lys Val Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310 315 320 Asn Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val Leu 325 330 335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val Pro Gly Arg 340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly Glu Leu Ser Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro Asp Lys Val Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met Arg Ser Leu Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp Trp Glu Gln Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val Leu Leu Asn Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430 Met Asp Asp Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435 440 445 Ile Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly Arg 450 455 460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn Glu 465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro Val Ala Glu Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg Asp Leu Leu Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met Ala Thr Val Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser Ser Val Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555 560 His Val Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr 565 570 575 Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His Glu 580 585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu Thr Ser Arg Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu Tyr Ser Val Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser Val Ser Gly Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr Leu Ser Val Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn Tyr Glu Val Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680 685 Val Ile Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro 690 695 700 Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys Tyr Glu 705 710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro Asp Lys Val Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile Gln Thr Lys Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe Val Gln Leu Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800 Thr Thr Lys Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805 810 815 Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser Thr 820 825 830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp Val Asp Gln 835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr Arg Phe Thr Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile Leu Val Leu Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe Ile Glu Gly Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His Ile Ser Pro Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925 Thr Pro Gly Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930 935 940 His Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe Glu 945 950 955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr Gln Ile Met Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln Ile Asn Val Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly Val Ile Ala Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile Val Ser Trp Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr Asp Ile Leu Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg Asn Thr Ser Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045 1050 Asp Leu Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val 1055 1060 1065 Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly Arg Thr 1070 1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr Glu Asn Ser Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala Ser Glu Gly Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln Gly Arg Met Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165 1170 Ala Ser Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp 1175 1180 1185 Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe Asp Phe 1190 1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp Arg Phe Gln Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu Trp Val Val Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val Thr Ala Glu Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu Met Ser Phe Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285 1290 Glu Leu Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro 1295 1300 1305 Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu Arg 1310 1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr Val Ser Gly Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile Phe Gly Ser Val Arg Thr 1340 1345 1350 Lys Pro

Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn Ser 1355 1360 1365 Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370 1375 1380 Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Val Glu 1385 1390 1395 Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile Met 1400 1405 1410 Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val Gln 1415 1420 1425 Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro Pro Pro Pro Pro His Ile Arg Val Asn 1445 1450 1455 Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485 Thr Val Val Val Arg Glu Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490 1495 1500 Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His Asn 1505 1510 1515 Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys Cys 1520 1525 1530 Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser Phe Asn Ile Lys Leu 1535 1540 1545 Gly Ala Glu Met Glu Ser Leu Gly Gly Lys Cys Asp Pro Thr Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser Leu Pro 1595 1600 1605 Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Ala Ile Glu Gly Val 1610 1615 1620 Ser Ala Gly Leu Phe Leu Ile Gly Met Leu Val Ala Val Val Ala 1625 1630 1635 Leu Leu Ile Cys Arg Gln Lys Val Ser His Gly Arg Glu Arg Pro 1640 1645 1650 Ser Ala Arg Leu Ser Ile Arg Arg Asp Arg Pro Leu Ser Val His 1655 1660 1665 Leu Asn Leu Gly Gln Lys Gly Asn Arg Lys Thr Ser Cys Pro Ile 1670 1675 1680 Lys Ile Asn Gln Phe Glu Gly His Phe Met Lys Leu Gln Ala Asp 1685 1690 1695 Ser Asn Tyr Leu Leu Ser Lys Glu Tyr Glu Glu Leu Lys Asp Val 1700 1705 1710 Gly Arg Asn Gln Ser Cys Asp Ile Ala Leu Leu Pro Glu Asn Arg 1715 1720 1725 Gly Lys Asn Arg Tyr Asn Asn Ile Leu Pro Tyr Asp Ala Thr Arg 1730 1735 1740 Val Lys Leu Ser Asn Val Asp Asp Asp Pro Cys Ser Asp Tyr Ile 1745 1750 1755 Asn Ala Ser Tyr Ile Pro Gly Asn Asn Phe Arg Arg Glu Tyr Ile 1760 1765 1770 Val Thr Gln Gly Pro Leu Pro Gly Thr Lys Asp Asp Phe Trp Lys 1775 1780 1785 Met Val Trp Glu Gln Asn Val His Asn Ile Val Met Val Thr Gln 1790 1795 1800 Cys Val Glu Lys Gly Arg Val Lys Cys Asp His Tyr Trp Pro Ala 1805 1810 1815 Asp Gln Asp Ser Leu Tyr Tyr Gly Asp Leu Ile Leu Gln Met Leu 1820 1825 1830 Ser Glu Ser Val Leu Pro Glu Trp Thr Ile Arg Glu Phe Lys Ile 1835 1840 1845 Cys Gly Glu Glu Gln Leu Asp Ala His Arg Leu Ile Arg His Phe 1850 1855 1860 His Tyr Thr Val Trp Pro Asp His Gly Val Pro Glu Thr Thr Gln 1865 1870 1875 Ser Leu Ile Gln Phe Val Arg Thr Val Arg Asp Tyr Ile Asn Arg 1880 1885 1890 Ser Pro Gly Ala Gly Pro Thr Val Val His Cys Ser Ala Gly Val 1895 1900 1905 Gly Arg Thr Gly Thr Phe Ile Ala Leu Asp Arg Ile Leu Gln Gln 1910 1915 1920 Leu Asp Ser Lys Asp Ser Val Asp Ile Tyr Gly Ala Val His Asp 1925 1930 1935 Leu Arg Leu His Arg Val His Met Val Gln Thr Glu Cys Gln Tyr 1940 1945 1950 Val Tyr Leu His Gln Cys Val Arg Asp Val Leu Arg Ala Arg Lys 1955 1960 1965 Leu Arg Ser Glu Gln Glu Asn Pro Leu Phe Pro Ile Tyr Glu Asn 1970 1975 1980 Val Asn Pro Glu Tyr His Arg Asp Pro Val Tyr Ser Arg His 1985 1990 1995 31631PRTHomo sapiens 3Met Leu Ser His Gly Ala Gly Leu Ala Leu Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu Ala Glu Pro Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys Ala Ser Ser His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55 60 Ala Ala Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys 65 70 75 80 Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg Ile Ile 85 90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr Asp Pro Leu Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys Thr Thr Ser Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser Gly Lys Val Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn Asn Gln Lys Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr Ser Trp Asn Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser Lys Tyr Asn Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185 190 Phe Ser Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp 195 200 205 Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp Ser His 210 215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu Met Asp Lys Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp Lys His Ala Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly Tyr Leu Tyr Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu Gln Asn Tyr Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu Val Ser Asn Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser Leu Lys Val Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310 315 320 Asn Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val Leu 325 330 335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val Pro Gly Arg 340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly Glu Leu Ser Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro Asp Lys Val Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met Arg Ser Leu Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp Trp Glu Gln Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val Leu Leu Asn Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430 Met Asp Asp Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435 440 445 Ile Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly Arg 450 455 460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn Glu 465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro Val Ala Glu Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg Asp Leu Leu Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met Ala Thr Val Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser Ser Val Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555 560 His Val Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr 565 570 575 Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His Glu 580 585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu Thr Ser Arg Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu Tyr Ser Val Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser Val Ser Gly Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr Leu Ser Val Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn Tyr Glu Val Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680 685 Val Ile Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro 690 695 700 Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys Tyr Glu 705 710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro Asp Lys Val Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile Gln Thr Lys Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe Val Gln Leu Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800 Thr Thr Lys Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805 810 815 Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser Thr 820 825 830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp Val Asp Gln 835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr Arg Phe Thr Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile Leu Val Leu Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe Ile Glu Gly Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His Ile Ser Pro Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925 Thr Pro Gly Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930 935 940 His Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe Glu 945 950 955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr Gln Ile Met Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln Ile Asn Val Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly Val Ile Ala Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile Val Ser Trp Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr Asp Ile Leu Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg Asn Thr Ser Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045 1050 Asp Leu Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val 1055 1060 1065 Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly Arg Thr 1070 1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr Glu Asn Ser Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala Ser Glu Gly Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln Gly Arg Met Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165 1170 Ala Ser Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp 1175 1180 1185 Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe Asp Phe 1190 1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp Arg Phe Gln Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu Trp Val Val Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val Thr Ala Glu Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu Met Ser Phe Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285 1290 Glu Leu Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro 1295 1300 1305 Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu Arg 1310 1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr Val Ser Gly Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile Phe Gly Ser Val Arg Thr 1340 1345 1350 Lys Pro Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn Ser 1355 1360 1365 Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370 1375 1380 Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Val Glu 1385 1390 1395 Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile Met 1400 1405 1410 Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val Gln 1415 1420 1425 Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro Pro Pro Pro Pro His Ile Arg Val Asn 1445 1450 1455 Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485 Thr Val Val Val Arg Glu Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490 1495 1500 Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His Asn 1505 1510 1515 Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys Cys 1520 1525 1530 Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser Phe Asn Ile Lys Leu 1535 1540 1545 Gly Ala Glu Met Glu Ser Leu Gly Gly Lys Cys Asp Pro Thr Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe

Ser Leu Pro 1595 1600 1605 Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Ala Ile Glu Arg Gly 1610 1615 1620 Arg His His His His His His Gly 1625 1630 41621PRTHomo sapiens 4Met Leu Ser His Gly Ala Gly Leu Ala Leu Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu Ala Glu Pro Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys Ala Ser Ser His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55 60 Ala Ala Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys 65 70 75 80 Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg Ile Ile 85 90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr Asp Pro Leu Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys Thr Thr Ser Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser Gly Lys Val Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn Asn Gln Lys Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr Ser Trp Asn Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser Lys Tyr Asn Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185 190 Phe Ser Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp 195 200 205 Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp Ser His 210 215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu Met Asp Lys Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp Lys His Ala Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly Tyr Leu Tyr Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu Gln Asn Tyr Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu Val Ser Asn Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser Leu Lys Val Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310 315 320 Asn Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val Leu 325 330 335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val Pro Gly Arg 340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly Glu Leu Ser Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro Asp Lys Val Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met Arg Ser Leu Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp Trp Glu Gln Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val Leu Leu Asn Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430 Met Asp Asp Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435 440 445 Ile Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly Arg 450 455 460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn Glu 465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro Val Ala Glu Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg Asp Leu Leu Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met Ala Thr Val Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser Ser Val Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555 560 His Val Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr 565 570 575 Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His Glu 580 585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu Thr Ser Arg Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu Tyr Ser Val Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser Val Ser Gly Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr Leu Ser Val Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn Tyr Glu Val Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680 685 Val Ile Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro 690 695 700 Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys Tyr Glu 705 710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro Asp Lys Val Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile Gln Thr Lys Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe Val Gln Leu Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800 Thr Thr Lys Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805 810 815 Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser Thr 820 825 830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp Val Asp Gln 835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr Arg Phe Thr Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile Leu Val Leu Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe Ile Glu Gly Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His Ile Ser Pro Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925 Thr Pro Gly Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930 935 940 His Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe Glu 945 950 955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr Gln Ile Met Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln Ile Asn Val Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly Val Ile Ala Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile Val Ser Trp Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr Asp Ile Leu Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg Asn Thr Ser Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045 1050 Asp Leu Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val 1055 1060 1065 Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly Arg Thr 1070 1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr Glu Asn Ser Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala Ser Glu Gly Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln Gly Arg Met Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165 1170 Ala Ser Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp 1175 1180 1185 Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe Asp Phe 1190 1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp Arg Phe Gln Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu Trp Val Val Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val Thr Ala Glu Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu Met Ser Phe Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285 1290 Glu Leu Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro 1295 1300 1305 Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu Arg 1310 1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr Val Ser Gly Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile Phe Gly Ser Val Arg Thr 1340 1345 1350 Lys Pro Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn Ser 1355 1360 1365 Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370 1375 1380 Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Val Glu 1385 1390 1395 Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile Met 1400 1405 1410 Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val Gln 1415 1420 1425 Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro Pro Pro Pro Pro His Ile Arg Val Asn 1445 1450 1455 Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485 Thr Val Val Val Arg Glu Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490 1495 1500 Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His Asn 1505 1510 1515 Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys Cys 1520 1525 1530 Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser Phe Asn Ile Lys Leu 1535 1540 1545 Gly Ala Glu Met Glu Ser Leu Gly Gly Lys Cys Asp Pro Thr Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser Leu Pro 1595 1600 1605 Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Ala Ile Glu 1610 1615 1620 56199DNAMus musculus 5ggcacgaggg cggctgccac ggcccttgag catcgccagc cccgagggta gcgcgctgcg 60cccgcccgca gggcgcgctg agcgctcaac aagtggtacc cagaagtgcc ctggctggcc 120tcccagcgag atgctgaggc atggagccct aacggccttg tggataacac tgagcgtcgt 180gcagactgga gtggcagagc aagtgaaatg taacttcaca ctgttggagt ccagggtctc 240tagcttgtca gcgtctatcc agtggaggac tttcgcgtca ccctgtaact ttagcctcat 300ctacagcagt gatacctcgg ggcccatgtg gtgccatcct attcggatag acaactttac 360ctacggatgt aaccccaagg atttacaagc agggaccgtc tataacttca ggattgtttc 420tctggatgga gaagagagca ctctggtctt acagacagat ccgttgcctc ctgccaggtt 480tgaagtcaat cgggagaaaa cagcatcaac caccctgcag gtccggtgga ctccctcttc 540tggaaaagtc tcctggtatg aggtgcaatt atttgatcat aacaatcaaa agatacaaga 600agtccaagtt caagaaagta ccacctggag ccaatatact tttctgaacc tcactgaggg 660taacagttac aaagttgcca tcacagctgt ttcgggagaa aagcgctcct ttccggtgta 720tatcaatggc tctacagtac catctccagt gaaagatctt ggcatttccc ccaatcctaa 780ttctctccta atttcctggt ctcgtggttc tgggaatgtg gaacaataca ggctggtgct 840aatggataaa ggggccatcg ttcaagacac aaacgtggac aggcgtgata cttcttatgc 900ttttcacgag ctgacccctg gccacctcta caacctcact attgtcacca tggcctcggg 960actgcaaaac tccaggtgga aactggtgag gaccgctccc atggaagtct caaatctgaa 1020ggtgacaaat gacgggaggt tgacctctct aaatgtgaag tggcagaaac cccctgggga 1080tgtagattcc tacagcatta ccctgtctca ccaagggacc atcaaagaat ccaaaacatt 1140agcacctcct gttactgaaa ctcaatttaa ggacttagtc cctggacggc tttaccaagt 1200gaccatcagc tgcatctctg gtgagctctc tgctgagaag tcagcagcgg ggagaacagt 1260tccagaaaaa gtgaggaatc tggtttccta caacgagatt tggatgaagt cctttacagt 1320gaactggacg ccccctgctg gagattggga gcattatcgt atcgtgctct tcaatgaatc 1380cttggtcttg ctcaacacca cagtgggaaa ggaagaaacg cactatgcct tggatggctt 1440ggagctcata ccaggaagac agtatgagat agaagtcatt gttgagagcg gaaatctgcg 1500gaattccgag cgctgtcaag gcaggacagt acccctggct gtcctccagc ttcgcgtcaa 1560acacgctaac gaaacttcac tgggcatcac gtggcgggcc cctctaggcg aatgggagaa 1620atacatcatt tcgttgatgg acagagagct cttggtcatc cacaagtcac tctccaaaga 1680tgccaaagaa ttcactttta cagacctgat gcctggacgg aattacaagg ctactgtcac 1740tagcatgagt ggagatttaa aacagtcatc ttcaatcaaa ggaagaacag tgcctgccca 1800ggtgactgac ctgcacgtca acaaccaagg gatgaccagt agtctgttca ctaactggac 1860aaaggcactg ggagatgtag agttctacca agttttactg atccatgaaa atgtggttgt 1920caagaacgag agtgtttcca gtgataccag cagatacagc ttccgcgccc tgaaacccgg 1980cagcctctac tccgtggtgg tgaccacggt gagtggaggg atctcctccc ggcaggtggt 2040ggcggaagga agaacagtcc cgtccagcgt gagtggggtg acagtcaaca attctggccg 2100gaatgactac ctcagcgttt cctggctgcc ggcgcctgga gaagtggatc actacgtggt 2160gagcctctcc cacgagggca aggtggatca gttcctcatc atcgccaaat ctgtcagcga 2220gtgttccttc agctccctca ccccgggccg cctctacaac gtcactgtaa ccaccaagag 2280cggcaattat gcaagccact ccttcaccga ggaacggaca gtgccagaca aggtccaggg 2340aatcagtgtt agcaactctg ccagaagcga ctacttaaag gtgtcctggg tgcatgccac 2400tggagacttt gaccactatg aagtcaccat caaaaacaga gaaagcttca ttcaaaccaa 2460aaccatcccc aagtcagaaa atgagtgtga atttattgag ctggttcctg gacgcctgta 2520cagcgtcact gtcagtacaa agagtggaca atatgaagcc agtgaacagg ggacagggag 2580aacgatccca gagcctgtga aggatctcac ccttctcaac aggagtacgg aggatctcca 2640tgtgacttgg tcaagagcca atggggatgt tgatcagtac gaggtccagc tgctcttcaa 2700cgacatgaaa gtcttccctc atattcacct tgtgaacaca gcaactgagt ataagttcac 2760ggcgctcacg ccggggcgcc attacaaaat cctcgtcctg accatcagtg gcgatgtcca 2820gcagtcagcc ttcattgaag gcctcccagt tcccagcact gtcaaaaaca ttcacatttc 2880tgccaatgga gccacggata ggctgatggt aacctggagc cctggtggcg gggatgtgga 2940ctcctatgtg gtgtctgcat tcagacagga cgagaaggtt gactctcaga ccattcccaa 3000gcatgcctcg gagcacacgt tccacaggct ggaggccgga gccaagtaca ggatcgccat 3060tgtttctgtc agtgggtccc tgagaaacca gatagatgcg ctcggacaga cagtcccagc 3120gtctgtccag ggagtcgtcg cagccaatgc atacagcagt aattccttaa cagtaagttg 3180gcagaaagcc cttggtgtgg cagaaagata cgatatcctg cttctaaacg agaatgggct 3240tcttttgagc aacgtgtcag agccagctac ggcaagacag cacaaatttg aagatctaac 3300gccaggcaag aaatacaaga tgcagatcct gactgtcagc ggaggcctct tcagtaaaga 3360atctcaggct gaaggccgaa cagtcccagc agctgtcacc aatctgagga tcacagagaa 3420ctccagtaga tacctgtcct tcggctggac cgcctcggag ggtgaactca gctggtacaa 3480catcttcctc tacaacccag acaggactct tcaggagcga gctcaagttg acccgctagt 3540ccagagcttc tctttccaga acttgctaca aggcagaatg tacaagatgg tgattgtcac 3600tcacagtggg gagctgtcca atgagtcatt tatattcggc agaacagttc ctgctgccgt 3660gaaccatctc aaaggctccc atcggaacac gacagacagc ctgtggttca gctggagccc 3720agcctccggg gactttgact tctatgagct gattctttac aatcccaacg gcacgaagaa 3780ggagaactgg aaagaaaagg acgtgacaga gtggcgtttc caaggtcttg ttcctggaag 3840gaaatacacc ctgtatgtgg tgactcacag tggggacctc agcaataaag tcacagggga 3900gggcagaaca gccccaagtc ctccgagtct tttgtcattc gctgatgttg caaacacctc 3960cttggctatc acctggaagg gacccccaga ctggacagat tacaatgact

ttgagctgca 4020gtggttccct ggagatgcac ttaccatctt caacccctac agcagcagaa agtcagaagg 4080acgcattgtg tacgggcttc acccagggag gtcctatcaa ttcagtgtca agactgtgag 4140cggggactcc tggaaaacct acagcaaacc aatttctggg tctgtgagga caaagccaga 4200caagatacaa aacctgcatt gccgccccca gaactccacg gccattgcct gctcttggat 4260acctcctgac tccgactttg atggctacag cattgagtgc cgaaaaatgg atacccaaga 4320aatcgagttt tccagaaagc tggagaaaga aaaatcactg ctcaacatca tgatgttagt 4380acctcataag aggtacctgg tgtccatcaa ggtgcagtcg gccggcatga ccagtgaggt 4440ggttgaagat agcaccatca ccatgataga ccgcccgcct caaccgcctc cacacatccg 4500tgtgaatgaa aaggatgtgc taatcagcaa atcttccatc aactttactg tcaactgcag 4560ctggttcagc gacaccaacg gagcggttaa atactttgct gtggtggtga gagaggccga 4620cagcatggat gagttgaagc cagaacagca gcaccctctc ccttcctacc tggagtacag 4680acacaacgcc tccatccgag tctaccagac caattatttt gccagcaaat gtgctgaaag 4740tcccgacagc agttctaaaa gtttcaacat taagcttgga gcagagatgg acagcctcgg 4800tggcaaatgt gatcccagtc agcagaaatt ctgtgatgga ccgctgaagc cacacaccgc 4860ctacagaatc agcatccggg cttttacaca gctatttgac gaggacttga aagagttcac 4920caaacctctc tactcggata cgttcttctc tatgcccatc accacagagt cagagccctt 4980gtttggagtt attgaaggtg tgagtgctgg cctgtttcta attggcatgc tggtggccct 5040tgttgccttc ttcatctgca gacagaaagc tagccacagc agggaaaggc catctgcccg 5100gctcagcatt cgtagggacc ggcctttgtc tgtccatctg aatctgggcc agaaaggcaa 5160ccggaaaact tcttgcccca taaagatcaa tcagtttgaa gggcatttca tgaagctgca 5220ggcagactcc aactaccttc tatccaagga atatgaggac ttaaaagacg tgggtagaag 5280ccagtcatgt gacattgccc tcttgcctga gaatcgaggg aaaaatcgat acaacaacat 5340attgccttat gatgcctcaa gagtgaagct ctcgaatgtc gatgacgacc cttgctctga 5400ctacatcaac gccagctaca tccccggtaa caacttcaga cgagaataca tcgccactca 5460gggaccgctt ccaggcacca aggatgactt ctggaagatg gcgtgggagc agaacgttca 5520caacatcgtc atggtgaccc agtgtgttga aaagggccga gtgaagtgtg accattactg 5580gccagcagac caggaccccc tctactacgg tgatctcatc ctacagatgg tctcggagtc 5640cgtgctcccc gagtggacca tcagggagtt taagatatgc agtgaagaac agttggatgc 5700acacagactc atccgtcact ttcactacac ggtgtggcca gaccatgggg tcccagagac 5760cacccagtcc ctgatccaat ttgtgaggac agtcagggac tacatcaaca gaagccccgg 5820ggctgggccc accgtagtgc actgcagcgc tggtgtgggc agaacaggga cgttcgttgc 5880cctggaccgg atcctccagc agttggactc taaggactcc gtggacattt atggggcagt 5940gcatgaccta agactccaca gggttcacat ggtccagacc gagtgtcaat atgtgtatct 6000gcatcagtgt gtaagagacg tcctcagagc aaagaaactg cggaacgagc aagagaaccc 6060cctgtttccg atttatgaga atgtgaatcc agagtatcac agagatgcaa tctactcgag 6120acattaagaa ttcacctgaa gatcccctgg ataaaagcgt ttcactgtgt gactttaaaa 6180aaaaaaaaaa aaaaaaaaa 619961998PRTMus musculus 6Met Leu Arg His Gly Ala Leu Thr Ala Leu Trp Ile Thr Leu Ser Val 1 5 10 15 Val Gln Thr Gly Val Ala Glu Gln Val Lys Cys Asn Phe Thr Leu Leu 20 25 30 Glu Ser Arg Val Ser Ser Leu Ser Ala Ser Ile Gln Trp Arg Thr Phe 35 40 45 Ala Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Ser Gly 50 55 60 Pro Met Trp Cys His Pro Ile Arg Ile Asp Asn Phe Thr Tyr Gly Cys 65 70 75 80 Asn Pro Lys Asp Leu Gln Ala Gly Thr Val Tyr Asn Phe Arg Ile Val 85 90 95 Ser Leu Asp Gly Glu Glu Ser Thr Leu Val Leu Gln Thr Asp Pro Leu 100 105 110 Pro Pro Ala Arg Phe Glu Val Asn Arg Glu Lys Thr Ala Ser Thr Thr 115 120 125 Leu Gln Val Arg Trp Thr Pro Ser Ser Gly Lys Val Ser Trp Tyr Glu 130 135 140 Val Gln Leu Phe Asp His Asn Asn Gln Lys Ile Gln Glu Val Gln Val 145 150 155 160 Gln Glu Ser Thr Thr Trp Ser Gln Tyr Thr Phe Leu Asn Leu Thr Glu 165 170 175 Gly Asn Ser Tyr Lys Val Ala Ile Thr Ala Val Ser Gly Glu Lys Arg 180 185 190 Ser Phe Pro Val Tyr Ile Asn Gly Ser Thr Val Pro Ser Pro Val Lys 195 200 205 Asp Leu Gly Ile Ser Pro Asn Pro Asn Ser Leu Leu Ile Ser Trp Ser 210 215 220 Arg Gly Ser Gly Asn Val Glu Gln Tyr Arg Leu Val Leu Met Asp Lys 225 230 235 240 Gly Ala Ile Val Gln Asp Thr Asn Val Asp Arg Arg Asp Thr Ser Tyr 245 250 255 Ala Phe His Glu Leu Thr Pro Gly His Leu Tyr Asn Leu Thr Ile Val 260 265 270 Thr Met Ala Ser Gly Leu Gln Asn Ser Arg Trp Lys Leu Val Arg Thr 275 280 285 Ala Pro Met Glu Val Ser Asn Leu Lys Val Thr Asn Asp Gly Arg Leu 290 295 300 Thr Ser Leu Asn Val Lys Trp Gln Lys Pro Pro Gly Asp Val Asp Ser 305 310 315 320 Tyr Ser Ile Thr Leu Ser His Gln Gly Thr Ile Lys Glu Ser Lys Thr 325 330 335 Leu Ala Pro Pro Val Thr Glu Thr Gln Phe Lys Asp Leu Val Pro Gly 340 345 350 Arg Leu Tyr Gln Val Thr Ile Ser Cys Ile Ser Gly Glu Leu Ser Ala 355 360 365 Glu Lys Ser Ala Ala Gly Arg Thr Val Pro Glu Lys Val Arg Asn Leu 370 375 380 Val Ser Tyr Asn Glu Ile Trp Met Lys Ser Phe Thr Val Asn Trp Thr 385 390 395 400 Pro Pro Ala Gly Asp Trp Glu His Tyr Arg Ile Val Leu Phe Asn Glu 405 410 415 Ser Leu Val Leu Leu Asn Thr Thr Val Gly Lys Glu Glu Thr His Tyr 420 425 430 Ala Leu Asp Gly Leu Glu Leu Ile Pro Gly Arg Gln Tyr Glu Ile Glu 435 440 445 Val Ile Val Glu Ser Gly Asn Leu Arg Asn Ser Glu Arg Cys Gln Gly 450 455 460 Arg Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn 465 470 475 480 Glu Thr Ser Leu Gly Ile Thr Trp Arg Ala Pro Leu Gly Glu Trp Glu 485 490 495 Lys Tyr Ile Ile Ser Leu Met Asp Arg Glu Leu Leu Val Ile His Lys 500 505 510 Ser Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp Leu Met Pro 515 520 525 Gly Arg Asn Tyr Lys Ala Thr Val Thr Ser Met Ser Gly Asp Leu Lys 530 535 540 Gln Ser Ser Ser Ile Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp 545 550 555 560 Leu His Val Asn Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp 565 570 575 Thr Lys Ala Leu Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His 580 585 590 Glu Asn Val Val Val Lys Asn Glu Ser Val Ser Ser Asp Thr Ser Arg 595 600 605 Tyr Ser Phe Arg Ala Leu Lys Pro Gly Ser Leu Tyr Ser Val Val Val 610 615 620 Thr Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Ala Glu Gly 625 630 635 640 Arg Thr Val Pro Ser Ser Val Ser Gly Val Thr Val Asn Asn Ser Gly 645 650 655 Arg Asn Asp Tyr Leu Ser Val Ser Trp Leu Pro Ala Pro Gly Glu Val 660 665 670 Asp His Tyr Val Val Ser Leu Ser His Glu Gly Lys Val Asp Gln Phe 675 680 685 Leu Ile Ile Ala Lys Ser Val Ser Glu Cys Ser Phe Ser Ser Leu Thr 690 695 700 Pro Gly Arg Leu Tyr Asn Val Thr Val Thr Thr Lys Ser Gly Asn Tyr 705 710 715 720 Ala Ser His Ser Phe Thr Glu Glu Arg Thr Val Pro Asp Lys Val Gln 725 730 735 Gly Ile Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Lys Val Ser 740 745 750 Trp Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr Ile Lys 755 760 765 Asn Arg Glu Ser Phe Ile Gln Thr Lys Thr Ile Pro Lys Ser Glu Asn 770 775 780 Glu Cys Glu Phe Ile Glu Leu Val Pro Gly Arg Leu Tyr Ser Val Thr 785 790 795 800 Val Ser Thr Lys Ser Gly Gln Tyr Glu Ala Ser Glu Gln Gly Thr Gly 805 810 815 Arg Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Leu Asn Arg Ser 820 825 830 Thr Glu Asp Leu His Val Thr Trp Ser Arg Ala Asn Gly Asp Val Asp 835 840 845 Gln Tyr Glu Val Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro His 850 855 860 Ile His Leu Val Asn Thr Ala Thr Glu Tyr Lys Phe Thr Ala Leu Thr 865 870 875 880 Pro Gly Arg His Tyr Lys Ile Leu Val Leu Thr Ile Ser Gly Asp Val 885 890 895 Gln Gln Ser Ala Phe Ile Glu Gly Leu Pro Val Pro Ser Thr Val Lys 900 905 910 Asn Ile His Ile Ser Ala Asn Gly Ala Thr Asp Arg Leu Met Val Thr 915 920 925 Trp Ser Pro Gly Gly Gly Asp Val Asp Ser Tyr Val Val Ser Ala Phe 930 935 940 Arg Gln Asp Glu Lys Val Asp Ser Gln Thr Ile Pro Lys His Ala Ser 945 950 955 960 Glu His Thr Phe His Arg Leu Glu Ala Gly Ala Lys Tyr Arg Ile Ala 965 970 975 Ile Val Ser Val Ser Gly Ser Leu Arg Asn Gln Ile Asp Ala Leu Gly 980 985 990 Gln Thr Val Pro Ala Ser Val Gln Gly Val Val Ala Ala Asn Ala Tyr 995 1000 1005 Ser Ser Asn Ser Leu Thr Val Ser Trp Gln Lys Ala Leu Gly Val 1010 1015 1020 Ala Glu Arg Tyr Asp Ile Leu Leu Leu Asn Glu Asn Gly Leu Leu 1025 1030 1035 Leu Ser Asn Val Ser Glu Pro Ala Thr Ala Arg Gln His Lys Phe 1040 1045 1050 Glu Asp Leu Thr Pro Gly Lys Lys Tyr Lys Met Gln Ile Leu Thr 1055 1060 1065 Val Ser Gly Gly Leu Phe Ser Lys Glu Ser Gln Ala Glu Gly Arg 1070 1075 1080 Thr Val Pro Ala Ala Val Thr Asn Leu Arg Ile Thr Glu Asn Ser 1085 1090 1095 Ser Arg Tyr Leu Ser Phe Gly Trp Thr Ala Ser Glu Gly Glu Leu 1100 1105 1110 Ser Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Arg Thr Leu Gln 1115 1120 1125 Glu Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe Ser Phe Gln 1130 1135 1140 Asn Leu Leu Gln Gly Arg Met Tyr Lys Met Val Ile Val Thr His 1145 1150 1155 Ser Gly Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val 1160 1165 1170 Pro Ala Ala Val Asn His Leu Lys Gly Ser His Arg Asn Thr Thr 1175 1180 1185 Asp Ser Leu Trp Phe Ser Trp Ser Pro Ala Ser Gly Asp Phe Asp 1190 1195 1200 Phe Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu 1205 1210 1215 Asn Trp Lys Glu Lys Asp Val Thr Glu Trp Arg Phe Gln Gly Leu 1220 1225 1230 Val Pro Gly Arg Lys Tyr Thr Leu Tyr Val Val Thr His Ser Gly 1235 1240 1245 Asp Leu Ser Asn Lys Val Thr Gly Glu Gly Arg Thr Ala Pro Ser 1250 1255 1260 Pro Pro Ser Leu Leu Ser Phe Ala Asp Val Ala Asn Thr Ser Leu 1265 1270 1275 Ala Ile Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp 1280 1285 1290 Phe Glu Leu Gln Trp Phe Pro Gly Asp Ala Leu Thr Ile Phe Asn 1295 1300 1305 Pro Tyr Ser Ser Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu 1310 1315 1320 His Pro Gly Arg Ser Tyr Gln Phe Ser Val Lys Thr Val Ser Gly 1325 1330 1335 Asp Ser Trp Lys Thr Tyr Ser Lys Pro Ile Ser Gly Ser Val Arg 1340 1345 1350 Thr Lys Pro Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn 1355 1360 1365 Ser Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe 1370 1375 1380 Asp Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Ile 1385 1390 1395 Glu Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile 1400 1405 1410 Met Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val 1415 1420 1425 Gln Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile 1430 1435 1440 Thr Met Ile Asp Arg Pro Pro Gln Pro Pro Pro His Ile Arg Val 1445 1450 1455 Asn Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr 1460 1465 1470 Val Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr 1475 1480 1485 Phe Ala Val Val Val Arg Glu Ala Asp Ser Met Asp Glu Leu Lys 1490 1495 1500 Pro Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His 1505 1510 1515 Asn Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys 1520 1525 1530 Cys Ala Glu Ser Pro Asp Ser Ser Ser Lys Ser Phe Asn Ile Lys 1535 1540 1545 Leu Gly Ala Glu Met Asp Ser Leu Gly Gly Lys Cys Asp Pro Ser 1550 1555 1560 Gln Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr 1565 1570 1575 Arg Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu 1580 1585 1590 Lys Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser Met 1595 1600 1605 Pro Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Val Ile Glu Gly 1610 1615 1620 Val Ser Ala Gly Leu Phe Leu Ile Gly Met Leu Val Ala Leu Val 1625 1630 1635 Ala Phe Phe Ile Cys Arg Gln Lys Ala Ser His Ser Arg Glu Arg 1640 1645 1650 Pro Ser Ala Arg Leu Ser Ile Arg Arg Asp Arg Pro Leu Ser Val 1655 1660 1665 His Leu Asn Leu Gly Gln Lys Gly Asn Arg Lys Thr Ser Cys Pro 1670 1675 1680 Ile Lys Ile Asn Gln Phe Glu Gly His Phe Met Lys Leu Gln Ala 1685 1690 1695 Asp Ser Asn Tyr Leu Leu Ser Lys Glu Tyr Glu Asp Leu Lys Asp 1700 1705 1710 Val Gly Arg Ser Gln Ser Cys Asp Ile Ala Leu Leu Pro Glu Asn 1715 1720 1725 Arg Gly Lys Asn Arg Tyr Asn Asn Ile Leu Pro Tyr Asp Ala Ser 1730 1735 1740 Arg Val Lys Leu Ser Asn Val Asp Asp Asp Pro Cys Ser Asp Tyr 1745 1750 1755 Ile Asn Ala Ser Tyr Ile Pro Gly Asn Asn Phe Arg Arg Glu Tyr 1760 1765 1770 Ile Ala Thr Gln Gly Pro Leu Pro Gly Thr Lys Asp Asp Phe Trp 1775 1780 1785 Lys Met Ala Trp Glu Gln Asn Val His Asn Ile Val Met Val Thr 1790 1795 1800 Gln Cys Val Glu Lys Gly Arg Val Lys Cys Asp His Tyr Trp Pro 1805 1810 1815 Ala Asp Gln Asp Pro Leu Tyr Tyr Gly Asp Leu Ile Leu Gln Met 1820 1825 1830 Val Ser Glu Ser Val Leu Pro Glu Trp Thr Ile Arg Glu Phe Lys 1835 1840 1845 Ile Cys Ser Glu Glu Gln Leu Asp Ala His Arg Leu Ile Arg His 1850 1855 1860 Phe His Tyr Thr Val Trp Pro Asp His Gly Val Pro Glu Thr Thr 1865 1870 1875 Gln Ser Leu Ile Gln Phe Val Arg Thr Val Arg Asp Tyr Ile Asn 1880 1885 1890 Arg Ser Pro Gly Ala Gly Pro Thr Val Val His Cys Ser Ala Gly 1895 1900 1905 Val Gly Arg Thr Gly Thr Phe Val Ala Leu Asp Arg

Ile Leu Gln 1910 1915 1920 Gln Leu Asp Ser Lys Asp Ser Val Asp Ile Tyr Gly Ala Val His 1925 1930 1935 Asp Leu Arg Leu His Arg Val His Met Val Gln Thr Glu Cys Gln 1940 1945 1950 Tyr Val Tyr Leu His Gln Cys Val Arg Asp Val Leu Arg Ala Lys 1955 1960 1965 Lys Leu Arg Asn Glu Gln Glu Asn Pro Leu Phe Pro Ile Tyr Glu 1970 1975 1980 Asn Val Asn Pro Glu Tyr His Arg Asp Ala Ile Tyr Ser Arg His 1985 1990 1995 71619PRTMus musculus 7Met Leu Arg His Gly Ala Leu Thr Ala Leu Trp Ile Thr Leu Ser Val 1 5 10 15 Val Gln Thr Gly Val Ala Glu Gln Val Lys Cys Asn Phe Thr Leu Leu 20 25 30 Glu Ser Arg Val Ser Ser Leu Ser Ala Ser Ile Gln Trp Arg Thr Phe 35 40 45 Ala Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Ser Gly 50 55 60 Pro Met Trp Cys His Pro Ile Arg Ile Asp Asn Phe Thr Tyr Gly Cys 65 70 75 80 Asn Pro Lys Asp Leu Gln Ala Gly Thr Val Tyr Asn Phe Arg Ile Val 85 90 95 Ser Leu Asp Gly Glu Glu Ser Thr Leu Val Leu Gln Thr Asp Pro Leu 100 105 110 Pro Pro Ala Arg Phe Glu Val Asn Arg Glu Lys Thr Ala Ser Thr Thr 115 120 125 Leu Gln Val Arg Trp Thr Pro Ser Ser Gly Lys Val Ser Trp Tyr Glu 130 135 140 Val Gln Leu Phe Asp His Asn Asn Gln Lys Ile Gln Glu Val Gln Val 145 150 155 160 Gln Glu Ser Thr Thr Trp Ser Gln Tyr Thr Phe Leu Asn Leu Thr Glu 165 170 175 Gly Asn Ser Tyr Lys Val Ala Ile Thr Ala Val Ser Gly Glu Lys Arg 180 185 190 Ser Phe Pro Val Tyr Ile Asn Gly Ser Thr Val Pro Ser Pro Val Lys 195 200 205 Asp Leu Gly Ile Ser Pro Asn Pro Asn Ser Leu Leu Ile Ser Trp Ser 210 215 220 Arg Gly Ser Gly Asn Val Glu Gln Tyr Arg Leu Val Leu Met Asp Lys 225 230 235 240 Gly Ala Ile Val Gln Asp Thr Asn Val Asp Arg Arg Asp Thr Ser Tyr 245 250 255 Ala Phe His Glu Leu Thr Pro Gly His Leu Tyr Asn Leu Thr Ile Val 260 265 270 Thr Met Ala Ser Gly Leu Gln Asn Ser Arg Trp Lys Leu Val Arg Thr 275 280 285 Ala Pro Met Glu Val Ser Asn Leu Lys Val Thr Asn Asp Gly Arg Leu 290 295 300 Thr Ser Leu Asn Val Lys Trp Gln Lys Pro Pro Gly Asp Val Asp Ser 305 310 315 320 Tyr Ser Ile Thr Leu Ser His Gln Gly Thr Ile Lys Glu Ser Lys Thr 325 330 335 Leu Ala Pro Pro Val Thr Glu Thr Gln Phe Lys Asp Leu Val Pro Gly 340 345 350 Arg Leu Tyr Gln Val Thr Ile Ser Cys Ile Ser Gly Glu Leu Ser Ala 355 360 365 Glu Lys Ser Ala Ala Gly Arg Thr Val Pro Glu Lys Val Arg Asn Leu 370 375 380 Val Ser Tyr Asn Glu Ile Trp Met Lys Ser Phe Thr Val Asn Trp Thr 385 390 395 400 Pro Pro Ala Gly Asp Trp Glu His Tyr Arg Ile Val Leu Phe Asn Glu 405 410 415 Ser Leu Val Leu Leu Asn Thr Thr Val Gly Lys Glu Glu Thr His Tyr 420 425 430 Ala Leu Asp Gly Leu Glu Leu Ile Pro Gly Arg Gln Tyr Glu Ile Glu 435 440 445 Val Ile Val Glu Ser Gly Asn Leu Arg Asn Ser Glu Arg Cys Gln Gly 450 455 460 Arg Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn 465 470 475 480 Glu Thr Ser Leu Gly Ile Thr Trp Arg Ala Pro Leu Gly Glu Trp Glu 485 490 495 Lys Tyr Ile Ile Ser Leu Met Asp Arg Glu Leu Leu Val Ile His Lys 500 505 510 Ser Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp Leu Met Pro 515 520 525 Gly Arg Asn Tyr Lys Ala Thr Val Thr Ser Met Ser Gly Asp Leu Lys 530 535 540 Gln Ser Ser Ser Ile Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp 545 550 555 560 Leu His Val Asn Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp 565 570 575 Thr Lys Ala Leu Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His 580 585 590 Glu Asn Val Val Val Lys Asn Glu Ser Val Ser Ser Asp Thr Ser Arg 595 600 605 Tyr Ser Phe Arg Ala Leu Lys Pro Gly Ser Leu Tyr Ser Val Val Val 610 615 620 Thr Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Ala Glu Gly 625 630 635 640 Arg Thr Val Pro Ser Ser Val Ser Gly Val Thr Val Asn Asn Ser Gly 645 650 655 Arg Asn Asp Tyr Leu Ser Val Ser Trp Leu Pro Ala Pro Gly Glu Val 660 665 670 Asp His Tyr Val Val Ser Leu Ser His Glu Gly Lys Val Asp Gln Phe 675 680 685 Leu Ile Ile Ala Lys Ser Val Ser Glu Cys Ser Phe Ser Ser Leu Thr 690 695 700 Pro Gly Arg Leu Tyr Asn Val Thr Val Thr Thr Lys Ser Gly Asn Tyr 705 710 715 720 Ala Ser His Ser Phe Thr Glu Glu Arg Thr Val Pro Asp Lys Val Gln 725 730 735 Gly Ile Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Lys Val Ser 740 745 750 Trp Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr Ile Lys 755 760 765 Asn Arg Glu Ser Phe Ile Gln Thr Lys Thr Ile Pro Lys Ser Glu Asn 770 775 780 Glu Cys Glu Phe Ile Glu Leu Val Pro Gly Arg Leu Tyr Ser Val Thr 785 790 795 800 Val Ser Thr Lys Ser Gly Gln Tyr Glu Ala Ser Glu Gln Gly Thr Gly 805 810 815 Arg Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Leu Asn Arg Ser 820 825 830 Thr Glu Asp Leu His Val Thr Trp Ser Arg Ala Asn Gly Asp Val Asp 835 840 845 Gln Tyr Glu Val Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro His 850 855 860 Ile His Leu Val Asn Thr Ala Thr Glu Tyr Lys Phe Thr Ala Leu Thr 865 870 875 880 Pro Gly Arg His Tyr Lys Ile Leu Val Leu Thr Ile Ser Gly Asp Val 885 890 895 Gln Gln Ser Ala Phe Ile Glu Gly Leu Pro Val Pro Ser Thr Val Lys 900 905 910 Asn Ile His Ile Ser Ala Asn Gly Ala Thr Asp Arg Leu Met Val Thr 915 920 925 Trp Ser Pro Gly Gly Gly Asp Val Asp Ser Tyr Val Val Ser Ala Phe 930 935 940 Arg Gln Asp Glu Lys Val Asp Ser Gln Thr Ile Pro Lys His Ala Ser 945 950 955 960 Glu His Thr Phe His Arg Leu Glu Ala Gly Ala Lys Tyr Arg Ile Ala 965 970 975 Ile Val Ser Val Ser Gly Ser Leu Arg Asn Gln Ile Asp Ala Leu Gly 980 985 990 Gln Thr Val Pro Ala Ser Val Gln Gly Val Val Ala Ala Asn Ala Tyr 995 1000 1005 Ser Ser Asn Ser Leu Thr Val Ser Trp Gln Lys Ala Leu Gly Val 1010 1015 1020 Ala Glu Arg Tyr Asp Ile Leu Leu Leu Asn Glu Asn Gly Leu Leu 1025 1030 1035 Leu Ser Asn Val Ser Glu Pro Ala Thr Ala Arg Gln His Lys Phe 1040 1045 1050 Glu Asp Leu Thr Pro Gly Lys Lys Tyr Lys Met Gln Ile Leu Thr 1055 1060 1065 Val Ser Gly Gly Leu Phe Ser Lys Glu Ser Gln Ala Glu Gly Arg 1070 1075 1080 Thr Val Pro Ala Ala Val Thr Asn Leu Arg Ile Thr Glu Asn Ser 1085 1090 1095 Ser Arg Tyr Leu Ser Phe Gly Trp Thr Ala Ser Glu Gly Glu Leu 1100 1105 1110 Ser Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Arg Thr Leu Gln 1115 1120 1125 Glu Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe Ser Phe Gln 1130 1135 1140 Asn Leu Leu Gln Gly Arg Met Tyr Lys Met Val Ile Val Thr His 1145 1150 1155 Ser Gly Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val 1160 1165 1170 Pro Ala Ala Val Asn His Leu Lys Gly Ser His Arg Asn Thr Thr 1175 1180 1185 Asp Ser Leu Trp Phe Ser Trp Ser Pro Ala Ser Gly Asp Phe Asp 1190 1195 1200 Phe Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu 1205 1210 1215 Asn Trp Lys Glu Lys Asp Val Thr Glu Trp Arg Phe Gln Gly Leu 1220 1225 1230 Val Pro Gly Arg Lys Tyr Thr Leu Tyr Val Val Thr His Ser Gly 1235 1240 1245 Asp Leu Ser Asn Lys Val Thr Gly Glu Gly Arg Thr Ala Pro Ser 1250 1255 1260 Pro Pro Ser Leu Leu Ser Phe Ala Asp Val Ala Asn Thr Ser Leu 1265 1270 1275 Ala Ile Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp 1280 1285 1290 Phe Glu Leu Gln Trp Phe Pro Gly Asp Ala Leu Thr Ile Phe Asn 1295 1300 1305 Pro Tyr Ser Ser Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu 1310 1315 1320 His Pro Gly Arg Ser Tyr Gln Phe Ser Val Lys Thr Val Ser Gly 1325 1330 1335 Asp Ser Trp Lys Thr Tyr Ser Lys Pro Ile Ser Gly Ser Val Arg 1340 1345 1350 Thr Lys Pro Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn 1355 1360 1365 Ser Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe 1370 1375 1380 Asp Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Ile 1385 1390 1395 Glu Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile 1400 1405 1410 Met Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val 1415 1420 1425 Gln Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile 1430 1435 1440 Thr Met Ile Asp Arg Pro Pro Gln Pro Pro Pro His Ile Arg Val 1445 1450 1455 Asn Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr 1460 1465 1470 Val Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr 1475 1480 1485 Phe Ala Val Val Val Arg Glu Ala Asp Ser Met Asp Glu Leu Lys 1490 1495 1500 Pro Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His 1505 1510 1515 Asn Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys 1520 1525 1530 Cys Ala Glu Ser Pro Asp Ser Ser Ser Lys Ser Phe Asn Ile Lys 1535 1540 1545 Leu Gly Ala Glu Met Asp Ser Leu Gly Gly Lys Cys Asp Pro Ser 1550 1555 1560 Gln Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr 1565 1570 1575 Arg Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu 1580 1585 1590 Lys Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser Met 1595 1600 1605 Pro Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly 1610 1615