Compositions and methods for increasing bone mineralisation

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DESCRIPTION

Compositions and methods to increase the mineralization of the bone substance.

Technical field

The present invention relates generally to pharmaceutical products and methods, and, more specifically, to the manufacture of medicaments and compositions suitable for increasing the mineral content of bone. Such compositions and medicaments can be used to treat a wide variety of conditions, including, for example, osteopenia, osteoporosis, fractures, and other disorders in which bone mineral density is a hallmark of the disease.

Background of the invention

Two or three distinct phases of changes can occur in an individual's bone mass throughout life (see Riggs, West J. Med. 154: 63-77, 1991). The first phase occurs in both men and women, and continues until peak bone mass is achieved. This first phase is achieved through linear growth of endochondral growth plates, and radial growth due to a periosteal apposition rate. The second phase begins around age 30 for the trabecular bone (flat bones such as the vertebrae and pelvis) and around age 40 for the cortical bone (eg, long bones found in the extremities) and continues into adulthood. . This phase is characterized by slow bone loss, and occurs in both men and women. In women, a third phase of bone loss also occurs, most likely due to post-menopausal estrogen deficiencies. During this phase alone, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular compartment (see Riggs, supra).

Loss of mineral content in bone can be caused by a wide variety of conditions, and can lead to significant medical problems. For example, osteoporosis is a debilitating disease in humans characterized by marked decreases in skeletal bone mass and mineral density, structural deterioration of bone including degeneration of bone microarchitecture, and corresponding increases in bone fragility and susceptibility to fracture. in affected individuals. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone), a condition found in approximately 25 million people in the United States. Another 7-8 million patients in the United States have been diagnosed with clinical osteoporosis (defined as a bone mineral content greater than 2.5 standard deviations below that of mature young adult bone). Osteoporosis is one of the most costly diseases in the healthcare system, costing tens of billions of dollars annually in the United States. In addition to the costs related to health care, long-term residential care and lost work days are added to the financial and social costs of this disease. Worldwide, approximately 75 million people are at risk for osteoporosis.

The frequency of osteoporosis in the human population increases with age, and among Caucasians it is predominant in women (who comprise 80% of the pool of osteoporosis patients in the United States). The increased fragility and susceptibility to fracture of skeletal bone in the elderly is compounded by the increased risk of accidental falls in this population. More than 1.5 million osteoporosis-related bone fractures have been reported in the United States each year. Fractured hips, wrists, and vertebrae are among the most common injuries associated with osteoporosis. Hip fractures in particular are extremely uncomfortable and costly for the patient, and for women they are correlated with high mortality and morbidity rates.

Although osteoporosis has been defined as an increased risk of fracture due to decreased bone mass, none of the currently available treatments for bone disorders can substantially increase bone density in adults. There is a perception among all physicians that drugs are needed that can increase bone density in adults, particularly in the bones of the wrist, spine, and hip that are at risk for osteopenia and osteoporosis.

Current strategies for the prevention of osteoporosis may offer some benefit but cannot ensure resolution of the disease. These strategies include moderate physical activity (particularly weight-bearing activities) with the onset of advanced age, including adequate calcium in the diet, and avoiding the consumption of products containing alcohol or tobacco. For patients with clinical osteopenia or osteoporosis, all current therapeutic drugs and strategies are aimed at further reducing bone loss by inhibiting the process of bone absorption, a natural component of the bone remodeling process that occurs constitutively.

For example, estrogens are now being prescribed to slow bone loss. However, there is some controversy as to whether there is a long-term benefit for patients and whether there is any effect in all patients over 75 years of age. On the other hand, the use of estrogens is believed to increase the risk of breast or endometrial cancer.

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High doses of calcium in the diet, with or without vitamin D, have also been suggested for postmenopausal women. However, high doses of calcium often have unpleasant gastrointestinal side effects, and serum calcium levels must be continuously monitored (see Khosla and Riggs, Mayo Clin. Proc. 70: 978-982, 1995).

Other therapeutic agents that have been suggested include calcitonin, bisphosphonates, anabolic steroids, and boron fluoride. Such therapeutic agents, however, have undesirable side effects (eg, calcitonin and steroids can cause nausea and induce an immune reaction, bisphosphonates and sodium fluoride can inhibit fracture repair, even though bone density is moderately increased. ) that may prevent its use (see Khosla and Riggs, supra).

The entries AA393939 and AI11311 in the EMBL database have to do with Expressed Sequence Tags (EST) for which the function is not indicated.

No therapeutic strategy currently in practice involves a drug that stimulates or enhances the growth of new bone mass. The present invention provides compositions for the manufacture of drugs that can be used to increase bone mineralization, and thus can be used to treat a wide variety of conditions in which it is desired to increase bone mass. Additionally, the present invention provides other related advantages.

Compendium of the invention

The present invention provides a novel class or family of TGF-beta binding proteins, as well as assays to select compounds that increase bone mineral content and bone mineral density, compounds that increase bone mineral content and mineral density. of bone and the use of such compounds in the manufacture of medicaments for the treatment or prevention of a wide variety of conditions.

The invention provides an isolated nucleic acid selected from the group consisting of:

(a) an isolated nucleic acid molecule comprising SEQ ID NO. 1, 5, 9, 11, 13 or 15, or a complementary sequence thereof;

(b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecule of (a) under highly stringent conditions where hybridization is performed in 5 x SSPE, 5 x Denhardt's solution, and 0.5% SDS during night from 55 to 60 ° C; and (c) an isolated nucleic acid encoding a TGF-beta binding protein according to (a) and (b); where the nucleic acid is not the nucleic acid of any of the accession numbers AC003098, AA393939 and AI113131 of the EMBL database.

Within related aspects of the present invention, isolated nucleic acid molecules based on hybridization are provided to a portion of only one of the sequences identified above (eg, for (a) hybridization may be with a probe of at least 20 , 25, 50 or 100 nucleotides selected from nucleotides 156 to 539 or 555 to 687 of SEQ ID NO. 1). As should be readily apparent, the necessary stringent conditions to be used for hybridization can vary based on the size of the probe. For example, for a 25-mer probe highly stringent conditions could include 60 mM Tris pH 8.0, 2 mM EDTA, 5x Denhardt's solution, 6x SSC, 0.1% N-lauryl sarcosine (w / v ), 0.5% (w / v) NP-40 (nonidet P-40) overnight at 45 degrees Celsius, followed by two washes with 0.2x SSC / 0.1% SDS at 45-50 degrees . For a 100-mer probe under low stringent conditions, suitable conditions could include the following: 5x SSPE, 5x Denhardt, and 0.5% SDS overnight at 42-50 degrees, followed by two washes with 2x SSPE (or 2x SSC) / 0.1% SDS at 42-50 degrees.

Isolated nucleic acid molecules are provided that have homology to SEQ ID Nos. 1, 5, 7, 9, 11, 13, or 15, at a homology level of 50%, 60%, 75%, 80%, 90%, 95%, or 98% using a WiburLipman algorithm. Representative examples of such isolated nucleic acid molecules include, for example, nucleic acid molecules that encode a protein comprising Sequence ID Nos. 2, 6, 10, 12, 14, or 16, or have homology to these sequences at a 50%, 60%, 75%, 80%, 90%, 95%, or 98% level of homology using a Lipman-Pearson algorithm.

Isolated nucleic acid molecules are typically less than 100 kb in size, and in certain embodiments, less than 50 kb, 25 kb, 10 kb, or even 5 kb in size. Additionally, isolated nucleic acid molecules, in other embodiments, do not exist in a "library" of other unrelated nucleic acid molecules (eg, a BAC subclone as described in GenBank Accession No. AC003098 and No. EMB AQ171546). However, isolated nucleic acid molecules can be found in libraries of related molecules (eg, for genetic rearrangement, as described in US Patent Nos. 5,837,458, 5,830,721; and 5,811,238) . Finally, nucleic acid molecules isolated as described herein do not include

ES 2 272 093 T3 nucleic acid molecules encoding Dan, Cerberus, Gremlin, or SCGF (US Patent No. 5,780,263).

Also provided by the present invention are cloning vectors containing the nucleic acid molecules indicated above, and expression vectors comprising a promoter (eg, a regulatory sequence) operably linked to one of the nucleic acid molecules indicated above. Representative examples of suitable promoters include tissue-specific promoters, and virus-based promoters (eg, CMV-based promoters such as CMV 1-E, the SV40 early promoter, and MuLV LTRs). Expression vectors can also be based on, or derived from, viruses (eg, a "viral vector"). Representative examples of viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors, and retroviral vectors. Also provided are host cells that contain or comprise any of the vectors listed above (including, for example, host cells of human, monkey, dog, rat, or mouse origin).

In other aspects of the present invention, methods of producing TGFbeta-binding proteins are provided, comprising the step of culturing the aforementioned host cell containing the vector under conditions and for a time sufficient to produce the binding protein. TGF-beta. In additional embodiments, the protein produced by this method can be further purified (eg, by column chromatography, affinity purification, and the like). Thus, isolated proteins that are encoded by the nucleic acid molecules indicated above (eg, Sequences of ID Nos. 2, 4, 6, 8, 10, 12, 14 or 16) can be easily produced given the description of the subject request.

It should also be noted that the aforementioned proteins, or fragments thereof, can be produced as fusion proteins. For example, in one aspect, fusion proteins are provided that comprise a first polypeptide segment of a TGF-beta binding protein encoded by a nucleic acid molecule as described above, or a portion thereof of at least 10, 20, 30, 50, or 100 amino acids in length, and a second polypeptide segment comprising a non-TGF-beta binding protein. In certain embodiments, the second polypeptide can be a suitable tag for purification or recognition (eg, a polypeptide comprising multiple anionic amino acid residues - see US Patent No. 4,851,341), a marker (eg, the green fluorescent protein, or alkaline phosphatase), or a toxic molecule (eg, castor bean).

In another aspect of the present invention, antibodies are provided that are capable of specifically binding to the above-described class of TGF-beta binding proteins (eg, human BEER). In various embodiments, the antibody can be a polyclonal antibody, or a monoclonal antibody (eg, human or of mouse origin). In further embodiments, the antibody is a fragment of an antibody that retains the binding characteristics of a full-length antibody (eg, an F (ab ') fragment₂, F (ab)₂, Fab ', Fab, or Fv, or even a CDR). Also provided are hybridomas and other cells that are capable of producing or expressing the aforementioned antibodies.

In related aspects of the invention, methods are provided that detect a TGFbeta-binding protein, comprising the steps of incubating an antibody as described above under conditions and for a time sufficient to allow said antibody to bind to a protein. binding to TGF-beta, and detect the binding. In various embodiments the antibody can be attached to a solid support to facilitate washing or removal, and / or labeled (eg, with a label selected from the group consisting of enzymes, fluorescent proteins, and radioisotopes).

In other aspects of the present invention, isolated oligonucleotides that hybridize to a nucleic acid molecule are provided according to SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 0 18 or its complement, under very restrictive conditions. In additional embodiments, the oligonucleotide can be found in the sequence encoding SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, or 16. In certain embodiments, the oligonucleotide is at least 15, 20, 30, 50, or 100 nucleotides in length. In additional embodiments, the oligonucleotide is labeled with another molecule (eg, an enzyme, fluorescent molecule, or radioisotope). Also provided are primers that are capable of specifically amplifying all or a portion of the aforementioned nucleic acid molecules that encode TGF-beta binding proteins. As used herein, the term "specifically amplify" is to be understood to refer to primers that amplify the aforementioned TGF-beta binding proteins, and not other TGF-beta binding proteins such as Dan, Cerberus, Gremlin, or SCGF (US Patent No. 5,780,263).

In related aspects of the present invention, methods are provided for detecting a nucleic acid molecule encoding a TGF-beta binding protein, comprising the steps of incubating an oligonucleotide as described above under highly stringent conditions, and detecting the hybridization of said oligonucleotide. In certain embodiments, the oligonucleotide can be labeled and / or attached to a solid support.

In other aspects of the present invention, ribozymes are provided that are capable of cleaving RNA encoding one of the TGF-beta binding proteins (eg SEQ ID Nos. 2,6, 8,10,12,14, or 16) . Such ribozymes can be composed of DNA, RNA (including 2'-O-methylribonucleic acids), nucleic acid analogs (eg, nucleic acids having phosphorothioate linkages), or mixtures thereof. Also provided are nucleic acid molecules (eg, DNA or cDNA) that encode these ribozymes, and vectors that are capable of expressing or producing

ES 2 272 093 T3 ribozymes. Representative examples of vectors include plasmids, retrotransposons, cosmids, and virus-based vectors (eg, viral vectors generated at least in part from a retrovirus, adenovirus, or adeno-associated virus). Host cells (eg, human, dog, rat, or mouse cells) containing these vectors are also provided. In certain embodiments, the host cell can be stably transformed with the vector.

In additional aspects of the invention, methods are provided for producing ribozymes either synthetically, or by in vitro or in vivo transcription. In further embodiments, the ribozymes so produced can be further purified and / or formulated into pharmaceutical compositions (eg, the ribozyme or the nucleic acid molecule encoding the ribozyme together with a pharmaceutically acceptable carrier or diluent). Similarly, antisense oligonucleotides and selected antibodies or other molecules described herein can be formulated into pharmaceutical compositions.

In other aspects of the present invention, antisense oligonucleotides are provided comprising a nucleic acid molecule that hybridizes to a nucleic acid molecule according to SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, or 15, or the complement thereof, and wherein said oligonucleotide inhibits the expression of the TGFbeta-binding protein as described herein (eg, human BEER). In various embodiments, the oligonucleotide is 15, 20, 25, 30, 35, 40, or 50 nucleotides in length. Preferably, the oligonucleotide is less than 100, 75, or 60 nucleotides in length. As should be readily apparent, the oligonucleotide can consist of one or more nucleic acid analogs, ribonucleic acids, or deoxyribonucleic acids. Additionally, the oligonucleotide can be modified by one or more linkages, including for example, covalent linkages such as a phosphorothioate linkage, a phosphotriester linkage, a methylphosphonate linkage, a methylene (imino) linkage, a morpholino linkage, an amido linkage, a linkage polyamide, a short chain alkyl intersugar bond, a cycloalkyl intersugar bond, a short chain heteroatomic intersugar bond and a heterocyclic intersugar bond. A representative example of a chimeric oligonucleotide is provided in US Patent No. 5,989,912.

Yet another aspect of the present invention provides the use of a ribozyme as described above in the manufacture of a medicament for increasing bone mineralization in a warm-blooded animal where the medicament introduces an effective amount of ribozyme into the animal. In related aspects, such drugs introduce into a patient an effective amount of the nucleic acid molecule or vector described herein that is capable of producing the desired ribozyme, the drug being introduced under conditions that favor transcription of the nucleic acid molecule to produce ribozyme.

Transgenic, non-human animals are provided in other aspects of the invention. In one embodiment a transgenic animal is provided whose germ cells and somatic cells contain a nucleic acid molecule encoding a TGF-beta binding protein as described above that is operably linked to an effective promoter for expression of the gene, being introduced the gene into the animal, or ancestor of the animal, in an embryonic stage, provided that said animal is not human. In other embodiments, transgenic genetically modified animals are provided, comprising an animal whose germ cells and somatic cells comprise a disruption of at least one allele of an endogenous nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes a binding protein. to TGF-beta as described here, wherein the disruption prevents transcription of the messenger RNA from said allele compared to an animal without the disruption, provided that the animal is not a human. In various embodiments, the disruption is a deletion, substitution, or insertion in the nucleic acid. In other embodiments, the transgenic animal is a mouse, rat, sheep, pig, or dog.

In further aspects of the invention, kits are provided for the detection of TGF-beta binding protein expression, comprising a container comprising a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13 or 15; (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of (a); (c) a nucleic acid molecule that is a fragment of (a) or (b) of at least 15, 20, 30, 50, 75, or 100 nucleotides in length. Kits are also provided for the detection of a TGF-beta binding protein comprising a container comprising one of the antibodies to the TGF-beta binding protein described herein.

For example, in one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing the mineral content of bone, comprising the steps of (a) mixing one or more candidate molecules with the binding protein. TGF-beta encoded by the nucleic acid molecule according to claim 1 and a selected member of the TGF-beta family of proteins (eg, BMP 5 or 6), (b) determining whether the candidate molecule alters the signaling of the TGF-beta family member, alters or binds the TGF-beta binding protein to the TGF-beta family member. In certain embodiments, the molecule alters the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation. In this aspect of the present invention, the candidate molecule (s) may alter signaling or binding, for example, by decreasing (eg, inhibiting), or increasing (eg, enhancing) signaling or binding.

In yet another aspect, methods are provided for determining whether a selected molecule is capable of increasing the mineral content of bone, comprising the step of determining whether a selected molecule inhibits the binding of the TGF-beta binding protein to bone, or an analog of it. Among the representative examples

ES 2 272 093 T3 of bone or analogs thereof include hydroxyapatite and biopsied primary human bone samples.

In certain embodiments of the methods cited above, the selected molecule is contained in a mixture of molecules and the methods may further comprise the step of isolating one or more molecules that are functional in the assay. In still other embodiments, the TGF-beta family of proteins is bound to a solid support and the binding of the TGF-beta binding protein is measured or the TGF-beta binding proteins are bound to a solid support and are measures the binding of TGF-beta binding proteins.

Using methods such as those described above, a wide variety of molecules can be tested for their ability to increase bone mineral content by inhibiting the binding of TGF-beta binding protein to the TGF-beta family of proteins. . Representative examples of such molecules include proteins or peptides, organic molecules, and nucleic acid molecules.

In other related aspects the invention provides the use of a molecule identified from the analyzes cited herein in the manufacture of a drug that increases the mineral content of bone in a warm-blooded animal, where the drugs are administered to a warm-blooded animal. a therapeutically effective amount of a molecule identified from the assays cited herein. In another aspect, the invention provides the use of a molecule that inhibits the binding of TGF-beta binding protein to the TGF-beta super-family of proteins, including bone morphogenic proteins (BMPs), in the manufacture of a drug to increase the mineral content of bone in a warm-blooded animal. Medications provide a therapeutically effective amount of the molecule. Representative examples of suitable molecules include antisense molecules, ribozymes, ribozyme genes, and antibodies (eg, a humanized antibody) that specifically recognize and alter the activity of the TGF-beta binding protein.

In another aspect the present invention provides the use of cells that seek bone and that have had a vector that directs the expression of a molecule that inhibits the binding of the TGF-beta binding protein to the TGF-beta family of proteins. and bone morphogenic proteins (BMP) introduced into them, in the manufacture of a medicament for increasing the mineral content of bone in a warm-blooded animal. As used herein, it is to be understood that "cells seek bone" if they locate the bone matrix after peripheral administration. In one embodiment, the invention further comprises, prior to the introduction step, the isolation of bone marrow cells that seek bone. In a further embodiment, the bone seeking cells are selected from the group consisting of CD34 + cells and osteoblasts.

In other aspects of the present invention, molecules (preferably isolated) are provided that inhibit the binding of the TGF-beta binding protein to the TGF-beta super-family of proteins.

In additional embodiments, the molecules may be provided in the form of a composition, and may additionally comprise an inhibitor of bone resorption. Representative examples of such inhibitors include calcitonin, estrogen, a bisphosphonate, a growth factor having anti-resorption activity, and tamoxifen.

Representative examples of molecules that can be used in the aforementioned therapeutic contexts include, eg, ribozymes, ribozyme genes, antisense molecules, and / or antibodies (eg, humanized antibodies). Such molecules can be used, depending on their selection, to alter, antagonize, or agonize the signaling or binding of a member of the TGF-beta binding protein family as described herein.

With various embodiments of the invention, the molecules and drugs described above for treatment or prevention can be used in conditions such as osteoporosis, osteomalasia, periodontal disease, scurvy, Cushing's disease, bone fracture, and conditions due to limb immobilization. and steroid use.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and accompanying drawings. In addition, various references are shown herein that describe in more detail certain procedures or compositions (eg, plasmids, etc.)

Brief description of the drawings

Figure 1 is a schematic illustration comparing the amino acid sequence of Dan Humana; Human Gremlin; Cerberus Humana and Beer Humana. Arrows indicate the Cysteine ​​backbone.

Figure 2 summarizes the results obtained from the inspection of a variety of human tissues for the expression of a TGF-beta binding protein gene, specifically, the Human Beer gene. A semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) procedure was used to amplify a portion of the first strand cDNA gene synthesized from total RNA (described in more detail in Example 2A) .

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Figure 3 summarizes the results obtained from RNA in situ hybridization of mouse embryo sections, using a cRNA probe that is complementary to the mouse Beer transcript (described in more detail in Example 2B). Panel A is a 10.5 dpc embryo cross section. Panel B is a sagittal section of 12.5 dpc embryos and panels C and D are sagittal sections of 15.5 dpc embryos.

Figure 4 illustrates, by western blot analysis, the specificity of three different polyclonal antibodies for their respective antigens (described in more detail in Example 4). Figure 4A shows the specific reactivity of an anti-H antibody. Beer for the H. Beer antigen, but not H. Dan or H. Gremlin. Figure 4B shows the reactivity of an anti-H antibody. Gremlin for the H. Gremlin antigen, but not H. Beer or H. Dan. Figure 4C shows the reactivity of an anti-H antibody. Dan for H. Dan, but not for H. Beer or H. Gremlin.

Figure 5 illustrates, by western blot analysis, the selectivity of the TGF-beta binding protein, Beer, for BMP-5 and BMP-6, but not BMP-4 (described in more detail in Example 5).

Figure 6 demonstrates that the ionic interaction between TGF-beta binding protein, Beer, and BMP-5 has a dissociation constant in the range of 15-30 nM.

Detailed description of the invention

Definitions

Before explaining the invention in detail, it may help to understand the invention to show the definitions of certain terms and to list and define abbreviations that will be used later.

"Molecule" is to be understood to include proteins or peptides (eg, antibodies, recombinant binding pairs, peptides with a desired binding affinity), nucleic acids (eg, DNA, RNA, chimeric nucleic acid molecules, and nucleic acid analogs. such as PNA); and organic and inorganic compounds.

"TGF-beta" should be understood to include any known or novel member of the TGFbeta super-family, which also includes bone morphogenic proteins (BMPs).

It is to be understood that "TGF-beta receptor" refers to the specific receptor for a particular member of the TGF-beta super-family (including bone morphogenic proteins (BMPs)).

It is to be understood that "TGF-beta binding protein" refers to a protein with a specific binding affinity for a particular member or subset of members of the TGF-beta super-family (including bone morphogenic proteins (BMP )). Specific examples of TGF-beta binding proteins include the proteins encoded by Sequence ID Nos. 1, 5, 7, 9, 11, 13 and 15.

It should be understood that "the binding of the TGF-beta binding protein to the TGF-beta family of proteins and bone morphogenic proteins (BMPs)" refers to molecules that allow the activation of TGF-beta or morphogenic proteins. of bone (BMP), or allow the binding of members of the TGF-beta family including bone morphogenic proteins (BMP) to their respective receptors, separating or preventing the binding of TGFbeta with the TGF-beta binding protein. Such inhibition can be accomplished, for example, by molecules that inhibit the binding of the TGF-beta binding protein to specific members of the TGF-beta super-family.

"Vector" refers to an assembly that is capable of directing the expression of the desired protein. The vector must include transcriptional promoter elements that are operably linked to the gene (s) of interest. The vector can consist of deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (eg DNA-RNA chimeric). Optionally, the vector can include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the chosen host cell and the vector used, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences that confer inducibility of transcription, and markers can also be incorporated. selectable in the vectors described herein.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, a DNA molecule that encodes a TGF-binding protein that has been separated from the genomic DNA of a eukaryotic cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of the organism. The isolated nucleic acid molecule can be genomic DNA, cDNA, RNA, or consist of at least a part of nucleic acid analogs.

An "isolated polypeptide" is a polypeptide that is essentially free of contaminating cellular components, such as carbohydrates, lipids, or other proteinaceous impurities associated with the polypeptide in nature. In certain embodiments, a particular protein cleavage contains an isolated polypeptide if it appears nominally as a single band on the Coomassie Blue stained SDS-PAGE gel. "Isolated" when it refers

ES 2 272 093 T3 to organic molecules means that the compounds are more than 90 percent pure using methods that are well known in the art (eg, NMR, melting point).

"Sclerosteosis". Sclerosteosis is a term that was applied by Hansen (1967) (Hansen, HG, Sklerosteose. In: Opitz, H., Schmid, F., Handbuch der Kinderheilkunde. Berlin: Springer (pub.) 6 1967. pp. 351-355 ) to a disorder similar to van Buchem's generalized cortical hyperostosis but possibly differing in the radiological appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases. The jaw has an unusually square appearance in this condition.

"Humanized antibodies" are recombinant proteins in which the mouse complementarity determining regions of monoclonal antibodies have been transferred from mouse immunoglobulin heavy and light variable chains to a human variable domain.

As used herein, an "antibody fragment" is a portion of an antibody such as F (ab ')₂, F (ab)₂, Fab ', and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, a fragment of a monoclonal antibody to TGF-beta binding protein binds to an epitope on TGF-beta binding protein.

The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the light chain variable region, "Fv" fragments consisting of the heavy and light chain variable regions, recombinant single chain polypeptide molecules in which the light and heavy variable regions are connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.

A "detectable label" is a molecule or atom that can be conjugated to a radical of an antibody to produce a molecule useful for diagnoses. Examples of the labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, enzymes, and other radical labels.

As used herein, an "immunoconjugate product" is a molecule comprising an anti-TGF-beta binding protein antibody, or an antibody fragment, and a detectable label. An immunoconjugate product has more or less the same ability, or only slightly reduced ability to bind TGFbeta-binding protein after conjugation as before conjugation.

Abbreviations: TGF-beta - "Transforming Growth Factor-beta"; TGF-bBP - "Transforming Growth Factor-beta binding protein" (a representative TGF-bBP is named "H. Beer"); BMP - "bone morphogenic protein"; PCR - "polymerase chain reaction"; RT-PCR - PCR procedure in which RNA is first transcribed into DNA in the first step using reverse transcriptase (RT); CDNA - any DNA made by copying an RNA sequence in the form of DNA.

As noted above, the present invention provides a novel class of TGFbeta-binding proteins, as well as medicaments and compositions for increasing bone mineral content in warm-blooded animals. Briefly, the present inventions are based on the unexpected discovery that a mutation in the gene encoding a novel member of the TGF-beta-binding protein family produces a rare condition (sclerosteosis) characterized by mineral contents of bone that they are one to four times higher than in normal individuals. Thus, as discussed in more detail below, this discovery has led to the development of assays that can be used to select molecules that inhibit the binding of the TGF-beta binding protein to the TGF-beta family of proteins and bone morphogenic proteins (BMP), and of drugs that use such molecules to increase the mineral content of bone in warm-blooded animals (including, for example, humans).

Study of the disease known as sclerosteosis

Sclerosteosis is a term that was applied by Hansen (1967) (Hansen, HG, Sklerosteose. Opitz, H., Schmid, F., Handbuch der Kinderheilkunde. Berlin: Springer (pub.) 6 1967. pp. 351-355) to a disorder similar to van Buchem's generalized cortical hyperostosis but possibly differing in the radiological appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases.

Sclerosteosis is now known to be an autosomal semi-dominant disorder characterized by widely disseminated sclerotic lesions of the bone in the adult. The condition is progressive. Sclerosteosis also has an evolutionary aspect that is associated with syndactyly (two or more fingers are together). Sclerosteosis Syndrome is associated with large stature and many affected individuals reach a height of six feet or more. The mineral content of bone in homozygotes can be 1 to 6 times that of normal individuals and the mineral density of bone can be 1 to 4 times of normal values ​​(eg, of non-twin siblings).

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Sclerosteosis Syndrome occurs mainly in Afrikaaners of German descent in South Africa. Approximately 1/140 individuals of the Afrikaaner population are carriers of the mutated gene (heterozygous). The mutation shows 100% penetrance. There are anecdotal reports of increased spindle mineral density in heterozygotes with non-associated pathologies (syndactyly or overgrowth of the bony head).

At present it appears that there is no abnormality of the pituitary-hypothalamus axis in sclerosteosis. In particular, there appears to be no overproduction of growth hormone or cortisone. Furthermore, the levels of sex hormones are normal in affected individuals. However, markers of bone turnover (osteoblast-specific alkaline phosphatase, osteocalcin, procollagen type I propeptide C '(PICP), and total alkaline phosphatase, (see Comier, C., Curr. Opin. In Rheu. 7: 243, 1995) indicate that there is hyperosteoblastic activity associated with the disease but that there is normal to weakly decreased osteoclastic activity as measured by markers of bone resorption (pyridinoline, deoxypyridinoline, N-telopeptide, urinary hydroxyproline, acid-resistant acid phosphatases in plasma and galactosylhydroxylysine (see Coomier, supra)).

Sclerosteosis is characterized by the continuous deposition of bone throughout the entire skeleton during the lifetime of affected individuals. In homozygotes the continuous deposit of bone mineral leads to overgrowth of the bone in the areas of the skeleton in which there is an absence of mechanoreceptors (bony head, jaw, skull). In homozygous with sclerosteosis, overgrowth of the bony head bones leads to cranial compression and eventually death due to excessive hydrostatic pressure on the brainstem. In all other parts of the skeleton there is generalized and diffuse sclerosis. The cortical areas of long bones are greatly thickened resulting in a substantial increase in bone strength. The trabecular connections have an increased thickness which in turn increases the strength of the trabecular bone. Sclerotic bones normally appear opaque on x-rays.

As described in more detail in Example 1, the rare genetic mutation that is responsible for Sclerosteosis Syndrome has been localized to the region of human chromosome 17 that encodes a novel member of the TGF-beta-binding protein family ( a representative example of which is called "H. Beer"). As described in more detail below, based on this discovery, the mechanism of bone mineralization is more fully understood, allowing the development of assays for molecules that increase bone mineralization, and the use of such molecules in the manufacture of drugs to increase bone mineralization. the mineral content of bone, and in the treatment or prevention of a wide number of diseases.

TGF-beta super-family

The Transforming Growth Factor-beta (TGF-beta) super-family contains a variety of growth factors that share common sequence elements and structural units (at both secondary and tertiary levels). This family of proteins is known to exert a wide spectrum of biological responses on a wide variety of cell types. Many of them have important roles during embryonic development in pattern formation and tissue specification; in adults, they are involved, eg, in wound healing and bone repair and bone remodeling, and in modulation of the immune system. In addition to the three TGFbeta, the super-family includes Bone Morphogenic Proteins (BMPs), Activins, Inhibins, Growth and Differentiation Factors (GDF), and Glia-Derived Neurotrophic Factors. The primary classification is established by general sequence features that link a specific protein to a general sub-family. Further stratification within the sub-family is possible due to tighter sequence conservation among members of a smaller group. In certain cases, such as BMP-5, BMP-6, and BMP-7, this can be as high as 75 percent amino acid homology among members of the smaller group. This level of identity allows a single representative sequence to illustrate the key biochemical elements of the subgroup that separates it from the other members of the larger family.

TGF-beta signals by inducing the formation of hetero-oligomeric complexes of type I and type II receptors. The crystal structure of TGF-beta2 has been determined. The general fold of the TGF-beta2 monomer contains a compact, stable cysteine ​​knot-like structure formed by three disulfide bridges. The dimerization, stabilized by a disulfide bridge, is antiparallel.

Members of the TGF-beta family initiate their cellular action by binding to receptors with intrinsic serine / threonine kinase activity. This family of receptors consists of two subfamilies, called type I type II receptors. Each member of the TGF-beta family binds to a characteristic combination of type I and type II receptors, both of which are necessary for signaling. In the current model for TGFbeta activation, TGF-beta first binds to the type II receptor (TbR-II), which appears on the cell membrane in an oligomeric form with activated kinase. After that, the type I receptor (TbR-I), which cannot bind to the ligand in the absence of TbR-II, is recruited into the complex. TbR-II then phosphorylates TbR-I predominantly in a domain rich in glycine and serine residues (GS domain) in the juxtamembrane region, and thereby activates TbR-I.

So far, seven type I receptors and five type II receptors have been identified.

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Bone morphogenic proteins (BMPs) are key regulatory proteins in determining bone mineral density in humans.

A major advance in understanding bone formation was the identification of bone morphogenic proteins (BMPs), also known as osteogenic proteins (OP), that regulate cartilage and bone differentiation in vivo. BMP / OP induce endochondral bone differentiation through a cascade of events including cartilage formation, cartilage hypertrophy and calcification, vascular invasion, osteoblast differentiation, and bone formation. As described above, BMP / OP (BMP 2-14, and osteogenic proteins 1 and 2, OP-1 and OP-2) are members of the TGF-beta super-family. The surprising evolutionary conservation among members of the BMP / OP sub-family suggests that they are critical in the normal development and function of animals. Furthermore, the presence of multiple forms of BMP / OP raises an important question about the biological relevance of this apparent redundancy. In addition to chondrogenesis and post-fetal osteogenesis, BMP / OP play multiple roles in skeletogenesis (including the development of craniofacial and dental tissues) and in embryonic development and organogenesis of parenchymal organs, including the kidney. It is now known that nature has common (and rare) molecular mechanisms adapted to provide for the emergence of specialized tissues and organs. The BMP / OP super-family is an elegant example of natural parsimony in the programming of multiple specialized functions that display molecular isoforms with minor variation in amino acid units within highly conserved carboxy terminal regions.

BMP antagonism

The sub-families of BMP and Activin are subject to significant post-translational regulation. There is an intricate extracellular control system, by means of which a high-affinity antagonist is synthesized and exported, and subsequently complexes selectively with BMPs or activins to disrupt their biological activity (WC Smith (1999) TIG 15 (1 ) 3-6). Some of these natural antagonists have been identified, and based on sequence divergence they appear to have evolved independently due to lack of conservation of the primary sequence. There has been no structural work to date on this class of proteins. Studies of these antagonists have highlighted a clear difference in interacting and neutralizing BMP-2 and BMP4. Furthermore, the inhibition mechanism appears to differ for the different antagonists (S. Iemura et al. (1998) Proc. Natl. Acad. Sci. USA 95 9337-9342).

Novel TGF-beta binding proteins

1. Antecedent re: TGF-beta binding proteins

As noted above, the present invention provides a novel class of TGFbeta-binding proteins possessing a nearly identical cysteine ​​(disulfide) scaffold when compared to Human DAN, Humana Gremlin, and Humana Cerberus, and SCGF (US Patent). United States No. 5,780,263) but has almost no homology at the nucleotide level (for background information, see generally Hsu, DR, Economides, AN, Wang, X., Eimon, PM, Harland, RM, “The Xenopus Dorsalizing Factor Gremlin Identifies a Novel Family of Secreted Proteins that Antagonize BMP Activities ", Molecular Cell 1: 673-683, 1998).

A representative example of the novel class of TGF-beta binding proteins is described in Sequence ID Nos. 1, 5, 9, 11, 13, and 15. Representative members of this class of binding proteins should be understood to include TGF-beta binding protein variants (eg, Sequence ID Nos. 5 and 7 ). As used herein, a "TGF-beta binding protein variant gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of Sequence ID Nos. 2, 10, 12, 14 or 16. Such variants include naturally occurring polymorphisms or allelic variants of TGF-beta binding protein genes, as well as synthetic genes that contain conservative amino acid substitutions for these amino acid sequences. Additional variant forms of a TGF-beta binding protein gene are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein. Variant TGF-beta binding protein genes can be identified by determining whether the genes hybridize to a nucleic acid molecule having the nucleotide sequence of Sequence ID Nos. 1, 5,7,9,11, 13, or 15 under stringent conditions. In addition, variant genes for the TGF-beta binding protein must encode a protein having a cysteine ​​backbone.

Alternatively, variant genes for the TGF-beta binding protein can be identified by sequence comparison. As used herein, two amino acid sequences have "100% sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximum correspondence. Similarly, two nucleotide sequences have "100% sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximum correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE Bioinformatics Computing Suite, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing two nucleotide or amino acid sequences by determining the optimal alignment are well known to those of skill in the art (see, for example, Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research ( ASM Press. Inc. 1997), Wu et al, (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins", in Methods in

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Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.), Guide to Human Genome Computing, 2^to Edition (Academic Press, Inc. 1998)).

A variant TGF-beta binding protein must have at least 50% amino acid sequence identity to Sequence ID Nos. 2,6,10,12,14, or 16 and preferably, an identity of more than 60%, 65%, 70%, 80%, 85%, 90%, or 95%. Alternatively, TGF-beta binding protein variants can be identified by having at least 70% nucleotide sequence identity to Sequence ID Nos. 1, 5, 9, 11, 13 or 15. On the other hand, the present invention contemplates variants of the TGF-beta binding protein gene that have an identity of more than 75%, 80%, 85%, 90%, or 95% with SEQ ID NO. 1. Regardless of the particular method used to identify a variant gene for a TGF-beta binding protein or a TGF-beta binding protein, a variant TGF-beta binding protein, or a polypeptide encoded by a protein gene Variant TGF-beta binding can be functionally characterized, for example, by its ability to bind to and / or inhibit the signaling of a selected member of the TGF-beta family of proteins, or by its ability to specifically bind an antibody to a TGF-beta binding protein.

Functional fragments of TGF-beta binding protein genes are included in the present invention. In the context of this invention, a "functional fragment" of a TGF-beta binding protein gene refers to a nucleic acid molecule that encodes a portion of a TGF-beta binding protein polypeptide that or it either possesses (1) the functional activity indicated above, or (2) specifically binds with an antibody to a TGF-beta binding protein. For example, a functional fragment of a TGF-beta binding protein gene described herein comprises a portion of the nucleotide sequence of SEQ ID Nos: 1, 5, 9, 11, 13, or 15.

2. Isolation of the TGF-beta binding protein gene

DNA molecules encoding a gene for a binding protein can be obtained by screening a human genomic or cDNA library using polynucleotide probes based, for example, on SEQ ID NO: 1.

For example, the first step in preparing a cDNA library is to isolate the RNA using methods well known to those of skill in the art. In general, RNA isolation techniques should provide a method of disrupting cells, a means of inhibiting RNAse-directed degradation of RNA, and a method of separating RNA from contaminating DNA, protein, and polysaccharides. For example, total RNA can be isolated by freezing the tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a phenol / chloroform solution to separate the proteins, and separating the RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)"]; Wu et al., Methods in Gene Biotechnology, pages 33-41] (CRC Press, Inc. 1997) ["Wu (1997)"]).

Alternatively, total RNA can be isolated by extracting the ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating the RNA from contaminants using differential centrifugation (see, for example, Ausubel (1995) on pages 4-1 to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly (A) + RNA must be isolated from the total RNA preparation. Poly (A) + RNA can be isolated from total RNA using the standard technique of oligo (dT) cellulose chromatography (see, for example, Ausubel (1995) on pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly (A) RNA⁺ using mechanisms well known to those of skill in the art (see, for example, Wu (1997) at pages 41-46). On the other hand, commercially available kits can be used to synthesize double-stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, Maryland), CLONTECH Laboratories, Inc. (Palo Alto, California), Promega Corporation (Madison, Wisconsin), and Stratagene Cloning Systems (La Jolla, California).

The basic approach to obtaining TGF-beta binding protein cDNA clones can be modified by constructing a subtracted cDNA library that is enriched for specific TGF-binding protein cDNA molecules. Mechanisms for constructing subtracted libraries are well known to those of skill in the art (see, for example, Sargent, "Isolation of Differentially Expressed Genes" in Meth. Enzymol. 152: 423, 1987, and Wu et al., (Eds.) "Construction and Screening of Substracted and Complete Expression cDNA Libraries", in Methods in Gene Biotechnology, pages 29-65 (CRC Press, Inc. 1997)).

Various cloning vectors are suitable for the construction of a cDNA library. For example, a cDNA library can be prepared in a bacteriophage-derived vector, such as a Jgl10 vector (see, for example, Huynh et al., "Construction and Screening cDNA in Jgl10 and Jgl11", in DNA Cloning: A Practical Approach Vol. I, Glover (ed.) Page 49 (IRL Press, 1985); Wu (1997) at pages 47-52).

Alternatively, double-stranded cDNA molecules can be inserted into a plasmid vector, such as a pBluescript vector (Stratagene Cloning Systems; La Jolla, California), LambdaGEM-4 (Promega Corp .; Madison, Wisconsin), or other commercially available vectors. Suitable cloning vectors can also be obtained from the American Type Culture Collection (Rockville, Maryland).

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In order to amplify the cloned cDNA molecules, the cDNA library is inserted into a prokaryotic host, using standard mechanisms. For example, a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained from Life Technologies, Inc. (Gaithersburg, Maryland).

A human genomic DNA library can be prepared by methods well known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing tissue with Sarkosyl detergent, digesting the lysate with proteinase K, clearing up insoluble remains of the lysate by centrifugation, precipitating the nucleic acid from the lysate using isopropanol, and purifying the resuspended DNA in a density gradient. cesium chloride.

DNA fragments that are suitable for the production of a genomic library can be obtained by randomly shearing genomic DNA or by partial digestion of genomic DNA with restriction endonucleases. Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, according to conventional mechanisms, such as the use of restriction enzyme digestion to provide appropriate ends, the use of alkaline phosphatase treatment to avoid the unwanted binding of DNA molecules, and ligation with appropriate ligase. Mechanisms for such manipulation are well known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307327).

Nucleic acid molecules encoding a TGF-beta-binding protein gene can also be obtained using polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences of the TGF-binding protein gene. -beta human, as described here. General methods for screening libraries with PCR are provided for example, by Yu et al., "Use of the Polymerase Chain Reaction to Screen Phage Libraries", in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.) Pages 211-215 (Humana Press, Inc. 1993). On the other hand, they describe mechanisms to use PCR to isolate related genes, for example, Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members", in Methods in Molecular Biology, Vol. 15: PCR Protocols : Current Methods and Applications, White (ed.), Pages 317-337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Rockville, Maryland).

A library containing cDNAs or genomic clones can be screened with one or more polynucleotide probes based on SEQ ID NO: 1, using standard methods (see, for example, Ausubel (1995) on pages 61 to 6-11).

Anti-TGF-beta binding protein antibodies, produced as described below, can also be used to isolate DNA sequences encoding TGF-beta binding protein genes from cDNA libraries. For example, antibodies can be used to screen lgt11 expression libraries, or antibodies can be used for immuno-screening after hybrid selection and translation (see, for example, Ausubel (1995) on pages 6-12 a 6-16; Margolis et al., "Screening λ expression libraries with antibody and protein probes", in DNA Cloning 2: Expression Systems, 2^to Edition, Glover et al., (Eds.) Pages 1-14 (Oxford University Press 1995)).

The sequence of a cDNA of a TGF-beta binding protein or of a genomic fragment of the TGF-beta binding protein can be determined using standard methods. On the other hand, the identification of genomic fragments that contain a promoter or regulatory element of the TGF-beta binding protein can be accomplished using well-established mechanisms, such as deletion analysis (see, generally, Ausubel (1995)).

Alternatively, a gene for a TGF-beta binding protein can be obtained by synthesizing DNA molecules using long mutually priming oligonucleotides and nucleotide sequences described here (see, for example, Ausubel (1995) on pages 8-8 to 8 -9). Established mechanisms using the polymerase chain reaction provide the ability to synthesize DNA molecules of at least two kilobases in length (Adang et al., Plant Molec. Biol. 21: 1131, 1993; Bambot et al., PCR Methods and Applications 2: 266, 1993; Dilton et al., "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes", in Methods in Molecular Biology, Vol. 15: PCR Protocols Current Methods and Applications, White (ed.), Pages 263-268, ( Humana Press, Inc., 1993); Holowachuk et al., PCR Methods Appl. 4: 299, 1995).

3. Production of TGF-beta binding protein genes

Nucleic acid molecules encoding variant TGF-beta binding protein genes can be obtained by screening various genomic or cDNA libraries with oligonucleotide probes having nucleotide sequences based on SEQ ID NOs: 1, 5, 9, 11, 13 , or 15, using the procedures described above. Variants of the TGF-beta binding protein gene can also be constructed synthetically. For example, a nucleic acid molecule can be devised that encodes a polypeptide that has a conservative amino acid change, compared to the amino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, or 16. That is, they can

ES 2 272 093 T3 obtain variants that contain one or more amino acid substitutions of SEQ ID NO: 2, 6, 8, 10, 12, 14 or 16, in which an alkyl amino acid is substituted by an alkyl amino acid in an amino acid sequence of the TGF-beta binding protein, an aromatic amino acid is substituted by an aromatic amino acid In an amino acid sequence of TGF-beta binding protein, a sulfur-containing amino acid is replaced by a sulfur-containing amino acid in an amino acid sequence of TGF-beta binding protein, a hydroxy-containing amino acid is substituted for a sulfur-containing amino acid in an amino acid sequence of the TGF-beta-binding protein, an acidic amino acid is substituted for an acidic amino acid in an amino acid sequence of the TGF-binding protein beta, an alkaline amino acid is substituted for an alkaline amino acid in an amino acid sequence of the TGF-beta binding protein, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in an amino acid sequence of the TGF-beta binding protein.

Among common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. In making such substitutions, it is important, where possible, to keep the cysteine ​​backbone outlined in Figure 1.

Conservative amino acid changes in the TGF-beta binding protein gene can be introduced by substituting nucleotides for the nucleotides cited in SEQ ID NO: 1. Such "conservative amino acid variants" can be obtained, for example, by mutagenesis. site directed, and the like (see Ausubel (1995) on pages 8-10 to 8-22, and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)). The functional capacity of such variants can be determined using a standard method, such as the assay described here. Alternatively, a variant TGF-beta binding protein polypeptide can be identified by the ability to specifically bind anti-TGF-beta binding protein antibodies.

Routine deletion analyzes of nucleic acid molecules can be performed to obtain "functional fragments" of a nucleic acid molecule that encodes a TGF-beta binding protein polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO: 1 can be digested with the Bal31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in an appropriate reading frame, and the expressed polypeptides are isolated and tested for their activity, or for the ability to bind anti-TGF-binding protein antibodies. beta. An alternative to exonuclease digestion is the use of oligonucleotide directed mutagenesis to introduce deletions or stop codons to specify the production of a desired fragment. Alternatively, specific fragments of a TGF-beta binding protein gene can be synthesized using the polymerase chain reaction.

Standard mechanisms for functional analysis of proteins are described, for example, by Treuter et al., Molec. Gen. Genet. 240: 113, 1993; Content et al., "Expression and preliminary deletion analysis of the 42 kea 2-5A synthesise induced by human interferon", in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), Pages 65-72 (Nijhoff 1987); Herschman, "The EGF Receptor", in Control of Animal Cell Proliferation, Vol. I, Boynton et al., (Eds.) Pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270-29270, 1995; Fukunaga et al., J. Biol. Chem. 270: 25291, 1995; Yamaguchi et al., Biochem. Pharmacol. 50: 1295, 1995; and Meisel et al., Plant Molec. Biol. 30: 1, 1996.

The present invention also contemplates functional fragments of a TGF-beta binding protein gene that have conservative amino acid changes.

A variant gene for the TGF-beta binding protein can be identified based on structure by determining the level of identity with the nucleotide and amino acid sequences of SEQ ID NO: 1, 5, 9, 11, 13, or 15 and 2, 6, 10, 12, 14, or 16, as discussed before. An alternative approach to identifying a variant gene based on structure is to determine whether a nucleic acid molecule encoding a variant TGF-beta binding protein gene can hybridize under stringent conditions to a nucleic acid molecule having the sequence of nucleotides of SEQ ID NOs: 1, 5, 9, 11, 13, or 15, or a portion thereof of a length of at least 15 or 20 nucleotides. As an illustration of stringent hybridization conditions, a nucleic acid molecule having a variant TGF-beta binding protein sequence can be joined to a nucleic acid molecule fragment having a sequence of SEQ ID NO: 1 in a buffer containing, for example, 5xSSPE (1xSSPE = 180 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA (pH 7.7), 5x Denhardt's solution (100xDenhardt = 2% bovine serum albumin (w / v ), Ficoll 2% (w / v), polyvinylpyrrolidone 2% (w / v) and SDS 0.5% incubated overnight at 55-60 ° C. Post-hybridization washes with high stringency are performed typically in 0.5xSSC (1xSSC = 150 mM sodium chloride, 15 mM sodium citrate) or in 0.5xSSPE at 55-60 ° C.

Regardless of the particular nucleotide sequence of a variant TGF-beta binding protein gene, the gene encodes a polypeptide that can be characterized by its functional activity, or by the ability to specifically bind to an anti-protein antibody of binding to TGF-beta. More specifically, variant TGF-beta binding protein genes encode polypeptides that exhibit at least 50%, and preferably more than 60, 70, 80, or 90% of the activity of the polypeptides encoded by the TGF gene. human TGF-beta binding protein described here.

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Four. Production of TGF-beta Binding Protein in Cultured Cells

To express a gene for a TGF-beta binding protein, a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene that is suitable for selection of cells that carry the expression vector.

Expression vectors that are suitable for the production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements that encode a bacterial origin of replication and an antibiotic resistance marker to provide for vector growth and selection of expression in a bacterial host; (2) eukaryotic DNA elements that control the initiation of transcription, such as a promoter, and (3) DNA elements that control the maturation of transcripts, such as a transcription / polyadenylation termination sequence.

The TGF-beta binding proteins of the present invention are preferably expressed in mammalian cells. Examples of mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587, human embryonic kidney cells ((293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK -21; ATCC CRL 8544), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40 transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the regulatory signals for transcription and translation can be derived from viral sources, such as adenovirus, bovine papillomavirus, simian virus, or the like, in which the regulatory signals are associated with a particular gene that has a high level of expression. Transcriptional and translational regulatory sequences can also be derived from mammalian genes, such as the actin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter from the mouse metallothionein I gene [Hamer et al., J. Molec. Appl. Genet. I: 273,1982], the Herpes virus TK promoter [McKnight, Cell 31: 355, 1982], the SV40 early promoter [Benoist et al., Nature 290: 304,1981], the Sarcoma virus promoter Rous [Gorman et al., Proc. Natl. Acad. Sci. USA 9: 6777, 1982], the cytomegalovirus promoter [Foecking et al., Gene 45, 101, 1980], and the mouse mammary tumor virus promoter (see, generally, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture ", in Protein Engineering Principles and Practice, Cleland et al (eds.), pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control the expression of the TGF-beta binding protein gene in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter ( Zhou et al., Mol. Cell. Biol. 10: 4529, 1990; Kaufman et al., Nucl. Acids Res. 19: 4485, 1991).

TGF-beta binding protein genes can also be expressed in bacterial, yeast, insect, or plant cells. Suitable promoters that can be used to express TGF-beta binding protein polypeptides in a prokaryotic host are well known to those of skill in the art and include promoters capable of recognizing T4, T3, Sp6, and T7 polymerases, promoters P_R and Pi from bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters from E. coli, the B. subtilis promoters, the Bacillus bacteriophage promoters, promoters from Streptomyces, the int promoter from bacteriophage lambda, the bla promoter from pBR322, and the CAT promoter from the chloramphenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol. 1: 277, 1987, Watson et al., Molecular Biology of the Gene, 4^to Ed. (Benjamin Cummins 1987), and by Ausubel et al., (1995).

Preferred prokaryotic hosts include E. coli and Bacillus subtilis. Suitable E. coli strains include BL21 (DE3), BL2 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4, DH5, DH51, DH51F ', DH51MCR, DH10B, DH10B / p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable Bacillus subtilis strains include BR151, YB886, M1119, M1120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (Ed.) (IRL Press 1985 )).

Methods for expressing proteins in prokaryotic hosts are well known to those of skill in the art (see, for example, Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.) page 15 (Oxford University Press 1995), Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc. 1995); and Georgiou, "Expression of Proteins in Bacteria", in Protein Engineering: Principles and Practice, Cleland et al., (eds.), page 101 (John Wiley & Sons, Inc. 1996).

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The baculovirus system provides an efficient means of introducing cloned TGF-beta binding protein genes into insect cells. Suitable expression vectors are based on the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as the Drosophila heat shock protein (hsp) promoter 70, the immediate early gene promoter (ie-1) and the delayed early 39K promoter from Autographa californica, the baculovirus p10 promoter, and the Drosophila metallothionein promoter. Suitable host and insect cells include cell lines derived from IPLB-Sf-21, a Spodoptera frugiperda pulp ovary cell line, such as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation, San Diego, CA), as well as Drosophila Schneider-2 cells. Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al., "Manipulation of Baculovirus Vectors", in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), Pages 147- 168 (The Humana Prees, Inc. 1991), by Patel et al., "The baculovirus expression system", in DNA Cloning 2: Expression Systems, 2^to Edition, Glover et al., (Eds.), Pages 205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols ( The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology", in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Promoters for yeast expression include promoters for GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include YIp-based vectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors, such as YEp13, and YCp vectors, such as YCp19. One skilled in the art will appreciate that there are a wide variety of vectors suitable for expression in yeast cells.

Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells. General methods for growing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).

An expression vector can be introduced into host cells using a variety of standard mechanisms including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like. Preferably, the transfected cells are selected and propagated to provide recombinant host cells comprising the expression vector stably integrated into the genome of the host cell. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991) . Methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are also provided by Ausubel (1995).

General methods for expressing and recovering foreign protein produced by a mammalian cellular system are provided, for example, by Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in "Protein Engineering: Principles and Practice, Cleland et al., (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard mechanisms for recovering protein produced by a bacterial system are provided, for example, by Grisshammer et al., "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2^to Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995). Established methods for the isolation of recombinant proteins from a baculovirus system are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc., 1995).

More generally, the TGF-beta binding protein can be isolated by standard mechanisms, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, HPLC, and the like. Additional variations in the isolation and purification of TGF-beta binding protein can be devised by those skilled in the art. For example, anti-TGF-beta binding protein antibodies, obtained as described below, can be used to isolate large amounts of protein by immunoaffinity purification.

5. Production of Antibodies to TGF-beta Binding Proteins

Antibodies to the TGF-beta binding protein can be obtained, for example, by using the product of an expression as the antigen. Particularly useful anti-TGF-beta binding protein antibodies "specifically bind" to the TGF-beta binding protein of SEQ ID Nos. 2, 6, 10, 12, 14, or 16, but not other TGF-beta binding proteins such as Dan, Cerberus, SCGF, or Gremlin. The antibodies of the present invention (including fragments and derivatives thereof) can be a polyclonal antibody or, especially, a monoclonal one. The antibody can belong to any class of immunoglobulin, and can be for example an IgG antibody, for example IgGi, IgG₂, IgG₃, IgG₄, IgE, IgM, or IgA. It can be of animal origin, for example mammalian, and can be for example a mouse, rat, human or other primate antibody. When desired the antibody can be an internalizing antibody.

Polyclonal antibodies to recombinant TGF-beta binding protein can be prepared using methods well known to those of skill in the art (see, for example, Green et al. "Production of Polyclonal Anti15

ES 2 272 093 T3 sera ", in Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press 1992); Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies", in DNA Cloning 2: Expression Systems, 2^to Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995)). Although polyclonal antibodies typically originate from animals such as rats, mice, rabbits, goats, or sheep, an anti-TGF-binding protein antibody of the present invention can also be derived from a sub-human primate antibody. General mechanisms for eliciting antibodies useful for diagnosis and therapy in baboons were found, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310, 1990.

The antibody must comprise at least one variable region domain. The variable region domain can be of any size or amino acid composition and will generally comprise at least one hypervariable amino acid sequence responsible for binding to antigen embedded in a framework sequence. In general terms the variable region domain (V) can be any suitable arrangement of heavy chain variable domains (V_H) and / or light (V_L) immunoglobulin. Thus, for example, the domain of the V region can be monomeric and be a V domain._H or V_L where they are capable of binding independently with acceptable affinity. Alternatively, the domain of the V region can be dimeric and contain VH-VH, VH-VL, or VLV dimers._L in which the strings V_H and V_L are non-covalently associated (hereinafter abbreviated as F_V). When desired, however, the chains may be covalently coupled either directly, for example via a disulfide bond between the two variable domains, or through a linker, for example a peptide linker, to form a single chain domain. (hereinafter abbreviated as scFV).

The variable region domain can be any naturally occurring variable domain or a designed version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA design mechanisms. Such engineered versions include those created for example from natural antibody variable regions by insertions, deletions or changes in the amino acid sequences of natural antibodies. Concrete examples of this type include those engineered variable region domains that contain at least one CDR and optionally one or more framework amino acids of an antibody and the remainder of the variable region domain of a second antibody.

The variable region domain may be covalently attached at a C-terminal amino acid to at least one other domain of the antibody or a fragment thereof. Thus, for example when a VH domain is present in the variable region domain it may be connected to a CH1 domain of the immunoglobulin or a fragment thereof. In a similar way a domain V_L can be connected to a C domain_K or a fragment of it. Thus for example the antibody can be a Fab fragment in which the antigen-binding domain contains associated VH and VL domains connected at their C-termini to a CH1 and CK domain respectively. The CH1 domain can be extended with additional amino acids, for example to provide a hinge region domain as found in a Fab 'fragment, or to provide additional domains, such as the CH2 and CH3 domains of the antibody.

Another form of antibody fragment is a peptide that encodes a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes that encode the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2 : 106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge Universisty Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).

Antibodies for use in the invention can be generally monoclonal (prepared by conventional immunization and cell fusion procedures) or in the case of fragments, derived therefrom using any suitable standard chemical mechanism eg, enzymatic reduction or cleavage and / or digestion, for example by pepsin treatment.

More specifically, monoclonal anti-TGF-beta binding protein antibodies can be generated using a variety of techniques. Rodent monoclonal antibodies to specific antigens can be obtained by methods known to those of skill in the art (see, for example, Kohler et al., Nature 256: 495, 1975; and Coligan et al. (eds.) Current Protocols in Immunology, 1: 2.5-1.2.7 (John Wiley & Sons 1991) ["Coligan"], Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al., (Eds.), Page 93 (Oxford University Press 1995)).

In summary, monoclonal antibodies can be obtained by injecting into mice a composition comprising a gene product of the TGF-beta binding protein, verifying the presence of antibody production by separating a serum sample, separating the spleen to obtain B -Lymphocytes, fusion of B-lymphocytes with myeloma cells to produce hybridomas, cloning of hybridomas, selection of positive clones that produce antibodies to the antigen, culturing the clones that produce the antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

ES 2 272 093 T3

Furthermore, an anti-TGF-beta binding protein antibody of the present invention can be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic priming. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain redirected disruptions of the endogenous heavy chain and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to produce human antibody screening hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368: 856, 1994; and Taylor et al., Int. Immun. 6: 579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established mechanisms. Such isolation mechanisms include Protein A-Sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and pages 2.9 .1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)", in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Human Press, Inc. 1992)).

For particular uses, it may be desirable to prepare anti-TGF-beta binding protein antibody fragments. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F (ab ')₂. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab 'monovalent fragments. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups that result from cleavage of the disulfide bonds. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and one Fc fragment directly. These methods are described, for example, by Goldenberg, US Patent No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89: 230,1960, Porter, Biochem. J. 73: 119,1959, Edelman et al., In Methods in Enzymology 1: 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10-2.10.4.

Other methods of antibody cleavage may also be used, such as heavy chain cleavage to form monovalent light chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques, as long as the fragments bind to the antigen that is recognized by the intact antibody.

Alternatively, the antibody may be a recombinant or genetically engineered antibody obtained through the use of recombinant DNA mechanisms involving manipulation and re-expression of DNA encoding the variable and / or constant regions of the antibody. Such DNA is known and / or readily available from DNA libraries including for example phage antibody libraries (see Chiswell, DJ and McCafferty, J. Tibtech. 10 80-84 (1992)) or can be synthesized as desired. Standard molecular biology and / or chemistry procedures can be used to sequence and manipulate DNA, for example, to introduce codons to create cysteine ​​residues, to modify, add or delete other amino acids or domains as desired.

Thereafter, one or more replicable expression vectors that contain the DNA and can be prepared and used to transform an appropriate cell line, eg a non-producing myeloma cell line, such as a mouse NSO line or a bacterial line, eg from E. coli, in which antibody production will take place. In order to obtain efficient transcription and translation, a DNA sequence from each vector must include appropriate regulatory sequences, namely a promoter and a leader sequence operably linked to the variable domain sequence. Concrete methods for producing antibodies in this manner are generally well known and used routinely. For example, they describe basic molecular biology procedures Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1989); DNA sequencing can be performed as described by Sanger et al. (PNAS 74, 5463, (1977)) and the Amersham International plc sequencing manual; and site-directed mutagenesis can be carried out according to the method of Kramer et al. (Nucl. Acids Res. 12, 9441, (1984)) and Anglian Biotechnology Ltd. manual. Additionally, there are numerous publications, detailing suitable techniques for the preparation of antibodies by manipulating DNA, creating expression vectors and transforming appropriate cells, for example as reviewed by Mountain A and Adair, JR in Biotechnology and Genetic Engineering Reviews. (ed. Tombs, MP, 10, Chapter 1, 1992, Intercept, Andover, UK) and in International Patent Specification No. WO 91/09967.

When desired, the antibody according to the invention may have one or more effector or reporter molecules attached to it and the invention extends to such modified proteins. The effector or reporter molecules may be attached to the antibody via any available amino acid side chain, amino terminal amino acid, or, where a localized carbohydrate functional group is present on the antibody, provided, of course, this does not adversely affect the properties. of binding and to the eventual utility of the molecule. Specific functional groups include, for example, any free amino, imino, thiol, hydroxyl, carboxyl, or aldehyde group. Anchoring of the antibody and the effector and / or reporter molecule (s) can be achieved via such groups and an appropriate functional group on the effector or reporter molecules. The connection can be direct or indirect, by means of group spacers or bridging formers.

ES 2 272 093 T3

Effector molecules include, for example, antineoplastic agents, toxins (such as pharmaceutically active toxins of bacterial or plant origin and fragments thereof eg ricin and fragments thereof), biologically active proteins, for example enzymes, nucleic acids and fragments thereof, eg DNA, RNA and fragments thereof, polymers of natural and synthetic origin eg polysaccharides and polyalkylene polymers such as polyethylene glycol and derivatives thereof, radionuclides, particularly radioiodide, and chelating metals. Suitable reporter groups include chelated metals, fluorescent compounds, or compounds that can be detected by NMR or ESR spectroscopy.

Specific antineoplastic agents include cytostatic and cytotoxic agents, for example, alkylating agents, such as nitrogen mustards (eg, chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracil mustard) and the derivatives thereof, triethylenephosphoramide, triethylene phosphoramide, triethylene phosphoramide , or cisplatin; antimetabolites, such as methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acid or fluoroocitric acid, antibiotics, such as bleomycins (eg bleomycin sulfate), doxorubicin, daunorubicin, mitomycin (eg actinomycin C) dactinomycin), plicamycin, calicamycin and derivatives thereof, or esperamycin and derivatives thereof, mitotic inhibitors, such as etoposide, vincristine or vinblastine and derivatives thereof, alkaloids, such as ellipticin; polyols such as taxicin-I or taxicin-II, hormones such as androgens (eg dromostanolone or testolactone), progestins (eg megestrol acetate or medroxyprogesterone acetate), estrogens (eg, dimethylstilbestrol diphosphate, polyestradiol phosphate or estramustine phosphate), or anti-estrogens (eg tamoxifen); anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea, hydrazines, such as procarbazine, or imidazoles, such as dacarbazine.

Particularly useful effector groups are calicamycin and derivatives thereof (see for example South African Patent Specifications Nos. 85/8794, 88 (8127 and 90/2839).

Chelated metals include chelates of di- or tri-positive metals having a coordination number from 2 to 8 inclusive. Specific examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd) and scandium (Sc). In general the metal is preferably a radionuclide. Specific radionuclides include

Tc^99m, Re¹⁸⁶, Co⁵⁸, Co⁶⁰, Cu⁶⁷, Au¹⁹⁵, Au¹⁹⁹, Au¹¹⁰, Pb²⁰³, Bi²⁰⁶, Bi²⁰⁷, In¹¹¹, Ga⁶⁷, Ga⁶⁸, Y⁸⁸, Y⁹⁰, Tb¹⁶⁰, Gd¹⁵³ and Sc⁴⁷.

The chelated metal can for example be one of the above types of chelated metal with any suitable polydentate chelating agent, for example acyclic or cyclic polyamines, polyethers, (eg crown ethers and derivatives thereof), polyamides, porphyrins, and carbocyclic derivatives.

In general, the type of chelating agent will depend on the metal used. A particularly useful group of chelating agents in the conjugates according to the invention, however, are the acyclic and cyclic polyamines, especially the polyaminocarboxylic acids, for example diethylene-triaminepentaacetic acid and derivatives thereof, and macrocyclic amines, eg derivatives cyclic tri-aza and tetra-aza (for example as described in International Patent Specification No. WO 92/22583); and polyamides, especially deferrioxyamine and derivatives thereof.

Thus for example when it is desired to use a thiol group on the antibody as an anchor point this can be achieved by means of a reaction with a thiol reactive group present on the effector or reporter molecule. Examples of such groups include an α-halocarboxylic acid or ester, eg, iodoacetamide, an imide, eg, maleimide, a vinyl sulfone, or a disulfide. These and other suitable joining procedures are generally and more specifically described in International Patent Specification Nos. WO 93/06231, WO 92/22583, WO 90/091195 and WO 89/01476.

Analysis to select molecules that increase bone density

As discussed above, the present invention provides methods for selecting and / or isolating compounds that are capable of increasing bone density. For example, in one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) mixing a selected molecule with TGF-beta binding protein and a selected member of the TGF-beta family of proteins, (b) determine whether the selected molecule stimulates signaling by the TGF-beta family of proteins, or it inhibits the binding of the TGF-beta binding protein to the TGF-beta family of proteins. In certain embodiments, the molecule enhances the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation.

In other aspects of the invention, methods are provided for determining whether a selected molecule is capable of increasing the mineral content of bone, comprising the steps of (a) exposing a selected molecule to cells expressing the TGF-beta binding protein and (b) determine if the expression (or activity) of the TGF-beta binding protein of said exposed cells decreases, and determine from there if the compound is capable of increasing the mineral content of bone. In one embodiment, the cells are selected from the group consisting of spontaneously transformed or non-transformed normal human bone from bone biopsies and rat parietal bone osteoblasts. Such methods can be completed in a variety of assay formats including, for example, Countercurrent Immunoelectrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitations, Enzyme Linked Absorption Assays (ELISA), and sandwich assays (see US Patent Nos. 4,376,110 and 4,486,530, see also Antibodies: A Laboratory Manual, supra).

ES 2 272 093 T3

Representative elements of such analyzes are provided below in Examples 5 and 6. Briefly, a family member of the TGF-beta super-family or a TGF-beta binding protein first binds to a solid phase. , followed by the addition of a candidate molecule. The labeled TGF-beta super-family member or TGF-beta binding protein is then added to the assay, the solid phase washed, and the amount of TGF-beta super-family member bound. or labeling or TGF-beta binding protein of the solid support is determined. Molecules that are suitable for use in increasing bone mineral content as described herein are those molecules that decrease the binding of TGF-beta binding protein to a member or members of the TGF-beta super-family of in a statistically significant way. Obviously, the assays suitable for use in the present invention should not be limited to the embodiments described in Examples 2 and 3. In particular, numerous parameters can be altered, for example by binding TGF-beta to a solid phase, or by removing the solid phase completely.

In other aspects of the invention, methods are provided for determining whether a selected molecule is capable of increasing the mineral content of bone, comprising the steps of (a) exposing a selected molecule to cells expressing TGF-beta and (b) determine if the activity of TGF-beta from said exposed cells is altered, and determine from there if the compound is capable of increasing the mineral content of bone. Similar to the methods described above, a wide variety of methods can be used to assess expression changes of TGF-beta binding protein due to a selected test compound.

For example, in one aspect of the present invention methods are provided for determining whether a selected molecule is capable of increasing bone mineral content, comprising the steps of (a) mixing a selected molecule with TGF-beta binding protein and a selected member of the TGF-beta family of proteins, (b) determine whether the selected molecule up-regulates signaling from the TGFbeta family of proteins, or it inhibits the binding of the TGF-beta binding protein to the TGF-beta family of proteins. In certain embodiments, the molecule enhances the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation.

Similar to the methods described above, a wide variety of methods can be used to assess TGF-beta stimulation due to a selected test compound. One such representative method is provided below in Example 6 (see also Durham et al., Endo, 136: 1374-1380.

In still other aspects of the present invention, methods are provided for determining whether a selected molecule is capable of increasing the mineral content of bone, comprising the step of determining whether a selected molecule inhibits the binding of the binding protein to TGF-beta. to bone, or an analog thereof. As used herein, bone or analogs thereof should be understood to refer to hydroxyapatite or a surface composed of a powdered form of bone, ground bone, or intact bone. Similar to the methods described above, a wide variety of methods can be used to assess the inhibition of the localization of the TGF-beta binding protein in the bone matrix. One such representative method is provided below in Example 7.

It should be noted that while the methods cited herein may refer to the analysis of an individual test molecule, the present invention should not be limited to them. In particular, the selected molecule can be contained in a mixture of compounds. Thus, the cited methods may further comprise the step of isolating a molecule that inhibits the binding of the TGF-beta binding protein to a member of the TGF-beta family.

Candidate molecules

A wide variety of molecules can be tested for their ability to inhibit the binding of TGF-beta binding protein to a member of the TGF-beta family. Representative examples discussed in more detail below include organic molecules, proteins or peptides, and nucleic acid molecules. Although it should be apparent from the study below that the candidate molecules described herein can be used in the assays described herein, it should be readily apparent that such molecules can also be used in a variety of diagnostic and therapeutic settings.

1. Organic molecules

Numerous organic molecules can be tested for their ability to inhibit the binding of the TGF-beta binding protein to a member of the TGF-beta family.

For example, in one embodiment of the invention suitable organic molecules can be selected either from a chemical library, where chemical agents are analyzed individually, or from combinatorial chemical libraries in which multiple compounds are analyzed at once. , then deciphered to determine and isolate most of the active compounds.

Representative examples of such combinatorial chemical libraries include those described by Agrafiotis et al., "System and method of automatically generating chemical compounds with desired properties", US Patent No. 5,463,564; Armstrong, RW, "Synthesis of combinatorial arrays of organic compounds

ES 2 272 093 T3 through the use of multiple component combinatorial array syntheses ", WO 95/02566; Baldwin, JJ et al., "Sulfonamide derivatives and their use", WO 95/24186; Baldwin, JJ et al., "Combinatorial dihydrobenzopyran library", WO 95/30642; Brenner, S., "New kit for preparing combinatorial libraries", WO 95/16918; Chenera, B. et al., "Preparation of library of resin-bound aromatic carbocyclic compounds", WO 95/16712; Ellman, JA, "Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support," US Patent No. 5,288,514; Felder, E. et al., "Novel combinatorial compound libraries", WO 95/16209; Lerner, R. et al., "Encoded combinatorial chemical libraries", WO 93/20242; Pavia, MR et al., "A method for preparing and selecting pharmaceutically useful non-peptide compounds from a structurally diverse universal library", wO 95/04277; Summerton, JE and DD Weller, "Morpholino-subunit combinatorial library and method", US Patent No. 5,506,337; Holmes, C., "Methods for the Solid Phase Synthesis of Thiazolidinones, Metathiazonones, and Derivatives therof", WO 96/00148; Phillips, GB and GP Wei, "Solid-phase Synthesis of Benzimidazoles", Tet. Letters 37: 4887-90, 1996; Ruhland, B. et al., "Solid-supported Combinatorial Synthesis of Structurally Diverse ^ -Lactams", J. Amer. Chem. Soc. 111: 253-4, 1996; Look, GC et al., "The Indentification of Cyclooxygenase-I Inhibitors form 4-Thiazolidonone Combinatorial Libraries", Bioorg. and Med. Chem. Letters 6: 707-12, 1996.

2. Proteins and Peptides

Similarly, a wide range of proteins and peptides can be used as candidate molecules for inhibitors of binding of the binding protein to a member of the TGF-beta family.

to. Combinatorial Peptide Libraries

Peptide molecules that are putative inhibitors of TGF-beta binding protein binding to a member of the TGF-beta family can be obtained through combinatorial peptide library screening. Such libraries can be prepared by one of skill in the art (see eg, US Patent Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty Publication Nos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809, or purchased from commercially available sources (eg New England Biolabs Ph.D.® Phage Display Peptide Library Kit).

b. Antibodies

Antibodies that inhibit the binding of the TGF-beta binding protein to a member of the TGF-beta family can be readily prepared given the description provided herein. In the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (eg, Fab, and F (ab ')₂, variable regions F_v, or regions determining complementarity). As discussed above, antibodies are understood to be specific against the TGF-beta binding protein, or against a specific TGF-beta family member, if they bind with a K_to greater than or equal to 10⁷ M, preferably greater than or equal to 10⁸ M^-1, and do not bind to other TGF-beta binding proteins, or, they bind with a K_toless than or equal to 10⁶ M^-1. Furthermore, the antibodies of the present invention must block or inhibit the binding of the TGF-beta binding protein to a member of the TGF-beta binding family.

The affinity of a monoclonal antibody or binding standard; as well as inhibition of binding can be readily determined by one of ordinary skill in the art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

In summary, monoclonal antibodies can be easily generated by one of skill in the art from a variety of warm-blooded animals such as horses, cows, various birds, rabbits, mice, or rats. Typically, TGF-beta binding protein or a unique 13-20 amino acid peptide thereof (preferably conjugated to keyhole limpet hemocyanin by glutaraldehyde crosslinking) is used to immunize the animal via intraperitoneal, intramuscular injections. , infraocular, or subcutaneous, along with an adjuvant such as Freund's complete or incomplete adjuvant. After several booster immunizations, serum samples are collected and tested for reactivity with the protein or peptide. Particularly preferred polyclonal antisera will give a signal in one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity with the protein, larger amounts of antisera can easily be obtained either by weekly blood draws or by exsanguination of the animal.

Monoclonal antibodies can also be easily generated using conventional mechanisms (see US Patent Nos. RE 32,011,4,902,614,4,543,439, and 4,411,993, see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes , Plenum Press, Kenett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988.

Briefly, in one embodiment, an animal subject such as a rat or mouse is immunized with the TGF-beta binding protein or a portion thereof as described above. The proteins can be mixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resulting immune response. Between one and three weeks after the initial immunization the animal can be re-immunized with another booster immunization, and tested for reactivity with the protein using the assays described above. A

ES 2 272 093 T3 Once the animal has reached a plateau in its reactivity with the injected protein, it is sacrificed, and organs containing a large number of B cells such as the spleen and lymph nodes are harvested.

Cells obtained from the immunized animal can be immortalized by infection with a virus such as Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8 (4): 377-389, 1989). Alternatively, in a preferred embodiment, the harvested spleen and / or lymph node suspensions are fused with a suitable myeloma cell in order to create a monoclonal antibody secreting "hybridoma". Suitable myeloma lines include, for example, NS-1 (ATCC No. TIB 18), and P3X63-Ag 8,653 (ATCC No. CRL 1580).

Following fusion, cells are plated for tissue culture plates containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagle's Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as fetal bovine serum (FBS, ie, from Hyclone, Logan, Utah, or JRH Biosciences). Additionally, the medium must contain a reagent that selectively allows the growth of fused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After approximately seven days, the resulting fused cells or hybridomas can be screened for the presence of antibodies that are reactive against the TGFbeta-binding protein (depending on the antigen used), and that block or inhibit the binding of the TGF-beta binding protein to a member of the TGF-beta family.

A wide variety of assays can be used to determine the presence of antibodies that are reactive against the proteins of the present invention, including for example countercurrent immunoelectrophoresis, radioimmunoassays, radioimmunoprecipitations, enzyme-linked absorption assays (ELISA), spot blot analysis, Western blots, immunoprecipitation, inhibition or competitive assays, and sandwich analyzes (see US Patent Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). After numerous dilutions and clonal re-analyzes, a hybridoma that produces reactive antibodies against the desired protein can be isolated.

Other techniques can also be used to construct monoclonal antibodies (see William D. Huse et al., "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda", Science 246: 1275-1281, December 1989; see also L Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region Specific cDNA Library", Proc. Natl. Acad. Sci. USA 86: 5728-5732, August 1989; see also Michelle AltingMees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3: 1-9, January 1990). These references describe a commercial system available from Stratagene (La Jolla, California) that allows the production of antibodies via recombination mechanisms. Briefly, mRNA is isolated from a population of B cells, and used to create heavy and light chain immunoglobulin cDNA expression libraries in TImmunoZap (H) and ImmunoZap (L) vectors. These vectors can be individually screened or expressed simultaneously to form Fab fragments or antibodies (see Huse et al., Supra; see also Sastry et al., Supra). Positive plaques can be subsequently converted into a non-lytic plasmid that allows a high level of expression of the monoclonal antibody fragments from E. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, of antibodies can also be constructed using standard DNA recombination or enzymatic digestion mechanisms to incorporate the variable regions of a gene encoding a specifically binding antibody. In one embodiment, the genes encoding the variable region of a hybridoma that produces the monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers can be synthesized by one of ordinary skill in the art, or they can be purchased from commercially available sources. Stratagene (La Jolla) sells primers for mouse and human variable regions including, but not limited to, primers for V regions._{He has}, V_Hb, V_Hc, V_Hd, C_H1, V_L and C_L. These primers can be used to amplify the variable regions of the heavy or light chain, which can then be inserted into vectors such as ImmunoZAP® H or ImmunoZAP® L (Stratagene), respectively. These vectors can then be introduced into E. coli, yeast, or mammalian-based expression systems. Using these mechanisms, large quantities of a single chain protein can be produced containing a fusion of the V domains._H and V_L (see Bird et al., Science 242: 423-426, 1988). Furthermore, such techniques can be used to exchange a "mouse" antibody for a "human" antibody, without altering the binding specificity of the antibody.

Once suitable antibodies have been obtained, they can be isolated or purified by means of many mechanisms well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press , 1988). Suitable mechanisms include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these mechanisms.

c. Mutant TGF-beta binding proteins

As described herein and below in the Examples (eg, Examples 8 and 9), altered versions of the TGF-beta binding protein that compete with the ability of the native TGF-beta binding protein to block the activity

ES 2 272 093 T3 of a particular TGF-beta family member must lead to an increase in bone density. Thus, TGF-beta binding protein mutants that bind to the TGF-beta family member but do not inhibit the function of the TGF-beta family member would meet the criteria. Mutant versions must compete effectively with the endogenous inhibitory functions of the TGF-beta binding protein.

d. Protein production

Although various genes (or portions thereof) are provided herein, it should be understood that in the context of the present invention, reference to one or more of these genes includes those derived from genes that are substantially similar to genes (and , where appropriate, proteins (including peptides and polypeptides) that are encoded by genes and their derivatives). As used herein, a nucleotide sequence is believed to be "substantially similar" if: (a) the nucleotide sequence is derived from the coding region of the genes described above and includes, for example, portions of the sequence or allelic variations of the sequences discussed above, or alternatively, encodes a molecule that inhibits protein binding binding of TGF-beta to a member of the TGF-beta family, (b) the nucleotide sequence is capable of hybridization to the nucleotide sequences of the present invention under moderately stringent, highly stringent, or very stringent conditions (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) the DNA sequences are degenerate as a result of the genetic code of the DNA sequences defined in (a) or (b). Additionally, the nucleic acid molecule described herein includes both complementary and non-complementary sequences, as long as the sequences otherwise satisfy the criteria set forth herein. In the context of the present invention, highly stringent conditions represent standard hybridization conditions (eg, 5XSSPE, 0.5% SDS at 65 ° C, or equivalent).

The structure of the proteins encoded by the nucleic acid molecules described here can be predicted from the primary translation products using the hydrophobic character tracing function, for example, from P / C Gene or the Intelligenetics Suite (Intelligenetics, Mountain View, California), or according to the methods described by Kyte and Doolittle (J. Mol. Biol. 157: 105-132, 1982).

The proteins of the present invention can be prepared in the form of acidic or alkaline salts, or in neutral form. Furthermore, individual amino acid residues can be modified by oxidation or reduction. In addition, various substitutions, deletions, or additions can be made in the amino acid or nucleic acid sequences, the net effect of which is to preserve or enhance or further reduce the biological activity of the wild-type or mutant protein. On the other hand, due to the degeneracy of the genetic code, for example, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence.

Other derivatives of the proteins described herein include the conjugates of the proteins together with other proteins or polypeptides. This can be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins that can be added to facilitate protein purification or identification (see US Patent No. 4,851,341, see also, Hopp et al., Bio / Technology 6: 1204, 1988). Alternatively, fusion proteins such as Flag / TGF-beta binding protein can be constructed in order to aid in the identification, expression and analysis of the protein.

The proteins of the present invention can be constructed using a wide variety of mechanisms described herein. Additionally, mutations can be introduced at particular loci by synthesizing oligonucleotides that contain a mutant sequence, flanked by restriction sites that allow ligation to fragments that contain a natural sequence. After ligation, the resulting reconstructed sequence encodes a derivative having the desired insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segment-specific) mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the required substitution, deletion, or insertion. Exemplary methods of making the alterations shown above are described by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering; Principles and Methods, Plenum Press, 1981); and Sambrook et al., (supra). Derivatives by deletion or truncation of proteins (eg, a soluble extracellular portion) can also be constructed using convenient restriction endonuclease sites adjacent to the desired deletion. After restriction, the overhangs can be filled in, and the DNA religated. Exemplary alteration-making methods shown above are described by Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).

Mutations that are made in the nucleic acid molecules of the present invention preferably retain the reading frame of the coding sequences. Furthermore, mutations will preferably not create complementary regions that hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect mRNA translation. Although the site of the mutation can be predetermined, the nature of the mutation need not be predetermined per se. For example, in order to select optimal characteristics of mutants at a given site, random mutagenesis can be performed at the target codon and the expressed mutants screened for indicative biological activity. Alternatively, mutations can be introduced at specific loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow ligation to fragments of the natural sequence. After ligation, the

ES 2 272 093 T3 resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution, or deletion.

The nucleic acid molecules that encode the proteins of the present invention can also be constructed using mechanisms of PCR mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83: 340223406, 1986), by forcible nucleotide misincorporation (eg, Liao and Wise Gene 88: 107-111, 1990), or by using random mutagenized oligonucleotides (Horwitz et al., Genome 3: 112-117, 1989).

The present invention also provides for the manipulation and expression of the genes described above by culturing host cells that contain a vector capable of expressing the genes described above. Such vectors or vector constructs include cDNA-derived or synthetic nucleic acid molecules encoding the desired protein, which are operably linked to suitable transcriptional or translational regulatory elements. Suitable regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect, or plant genes. Selection of the appropriate regulatory elements is dependent on one of the host cells selected, and can be easily completed by one of ordinary skill in the art. Examples of regulatory elements include: a transcriptional promoter and enhancer or an RNA polymerase binding sequence, a transcriptional terminator, and a ribosome binding sequence, including a translation initiation signal.

Nucleic acid molecules encoding any of the proteins described above can be readily expressed by a wide variety of prokaryotic or eukaryotic host cells, including bacterial, mammalian, yeast or other fungal, viral, insect, or plant cells. Methods for transforming or transfecting such cells to express foreign DNA are well known in the art (see, eg, Itakura et al., US Patent No. 4,704,362; Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978; Murray et al., US Patent No. 4,801,542; Upshall et al., US Patent No. 4,935,349; Hagen et al., US Patent No. 4,784,950; Axel et al., US Patent No. 4,399,216; Goeddel et al., US Patent No. 4,766,075; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^toed., Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol. 104: 10671071, 1994; and Paszkowski et al., Biotech. 24: 387-392, 1992).

Suitable bacterial host cells for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium, and various species of the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill. normal in the art. Representative examples of bacterial host cells include DH5a (Stratagene, La Jolla, California).

Bacterial expression vectors preferably comprise a promoter that functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include ^ -lactamase (penicillinase) and the lactose promoter system (see Chang et al., Nature 275: 615, 1978), the T7 RNA polymerase promoter (Studier et al., Meth Enzymol. 185: 60-89, 1990) the lambda promoter (Elvin et al., Gene 87: 123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. In Enzymology 101 :: 155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolívar et al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101: 20-77, 1983 and Vieira and Messing, Gene 19: 259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, California).

Suitable yeast and fungal host cells for carrying out the present invention include, but are not limited to, Saccharomycespombe, Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces, and various species of the genus Aspergillus (McKnight et al., U.S. Patent No. 4,935,349). Suitable expression vectors for yeast and fungi include, but are not limited to, YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio / Technology 7: 169, 1989), YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978) , YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978) and derivatives thereof.

Preferred promoters for use in yeast include yeast glycolytic gene promoters (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-934, 1982) or alcohol dehydrogenase genes (Young et al., In Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., (Eds.), P. 355, Plenum New York, 1982; Ammerer, Meth. Enzymol, 101: 192-201, 1983). Useful examples of fungal promoters include those derived from the glycolytic genes of Aspergillus nidulans, such as the adh3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985). The expression units can also include a transcriptional terminator. An example of a suitable terminator is the adh3 terminator (McKnight et al., Ibid., 1985).

As with bacterial vectors, yeast vectors will generally include a selectable marker, which can be one of numerous genes that display a dominant phenotype for which a phenotypic analysis exists to allow selection of transformants. Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance, or allow a cell to utilize.

ES 2 272 093 T3 lize specific carbon sources, and include leu2 (Broach et al., Ibid.), Ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al., Ibid.) . Another suitable selectable marker is the cat gene, which confers chloramphenicol resistance to yeast cells.

Techniques for transforming fungi are well known in the literature, and have been described, for example, by Beggs (ibid.), Hinnen et al., (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978) , Yelton et al. (Proc. Natl. Acad. Sci. USA 81: 1740-1747, 1984), and Russell (Nature 301: 167-169, 1983). The host cell genotype may contain a genetic defect that is complemented by the selectable marker present in the expression vector. The choice of a particular host and selectable marker is within the level of one of ordinary skill in the art.

The protocols for yeast transformation are well known to those of ordinary skill in the art. For example, either the preparation of yeast spheroplasts with DNA (see Hinnen et al., PNAS USA 75: 1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153: 163, 1983). The transformation of fungi can also be carried out using polyethylene glycol as described by Cullen et al., (Bio / Technology 5: 369,1987).

Viral vectors include those that comprise a promoter that drives the expression of an isolated nucleic acid molecule encoding a desired protein as described above. A wide variety of promoters can be used in the context of the present invention, including, for example, promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviral promoters (Ohno et al., Science 265: 781-784, 1994) , the neomycin phosphotransferase promoter / enhancer, the late parvovirus promoter (Koering et al., Hum. Gene Therap. 5: 457-463, 1994), the Herpes TK promoter, the SV40 promoter, the metallothionein gene IIa enhancer / promoter, the cytomegalovirus immediate early promoter, and the cytomegalovirus immediate late promoter. In particularly preferred embodiments of the invention, the promoter is a tissue specific promoter (see, eg, WO 91/02805; EP 0,415,731; and WO 90/07936). Representative examples of suitable tissue-specific promoters include the neural-specific enolase promoter, the platelet-derived growth factor beta promoter, the bone morphogenic protein promoter, the human alpha1-chimerin promoter, the synapsin I promoter and the synapsin II promoter. In addition to the promoters indicated above, other virus-specific promoters (eg, retroviral promoters, (including those indicated above, as well as others such as HIV promoters), hepatitis-specific promoters, herpes (eg, EBV ), and bacterial, fungal, or parasitic (eg, malaria) in order to target a specific cell or tissue that is infected with a virus, bacteria, fungus, or parasite.

Suitable mammalian cells for carrying out the present invention include, but are not limited to COS, CHO, SaOS, osteosarcomas, KS483, MG-63, primary osteoblasts, and human or mammalian bone marrow stroma. Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Bone-specific promoters include bone sialo-protein and the osteocalcin promoter. Viral promoters include the cytomegalovirus immediate early promoter (Boshart et al., Cell 41: 521-530, 1985), the cytomegalovirus immediate late promoter, the SV40 promoter (Subramani et al., Mol. Cell. Biol 1: 854-864, 1981), MMTV LTR, RSV LTR, metallothionein-1, adenovirus E1a. Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., US Patent No. 4,579,821), a V promoter_K mouse (Bergman et al., Proc. Natl. Acad. Sci. USA 81: 70417045, 1983; Grant et al., Nucl. Acids Res. 15: 5496, 1987) and a V promoter_H mouse (Loh et al., Cell 33: 85-93, 1983). The choice of promoter will depend, at least in part, on the level of expression desired or the recipient cell line to be transfected.

Such expression vectors may also contain a group of RNA splice sites located downstream of the promoter and upstream of the DNA sequence encoding the peptide or protein of interest. Preferred RNA splice sites can be derived from adenovirus and / or immunoglobulin genes. Also contained in the expression vector is a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include SV40 early or late polyadenylation signals (Kaufman and Sharp, ibid.), Adenovirus 5 E1B region polyadenylation signal, and human growth hormone gene terminator (De Noto et al., Nucl. Acids Res. 9: 3719-3730, 1981). Expression vectors can include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer. Expression vectors can also include sequences encoding adenovirus Va RNAs. Suitable expression vectors can be obtained from commercial sources (eg, Stragene, La Jolla, California).

Vector constructs comprising cloned DNA sequences can be introduced into mammalian cells, for example, by calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725; Corsar and Pearson, Somatic Cell Genetics 7: 603, 1981 ; Graham and Vand der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), or DEAE-dextran-mediated transfection (Ausubel et al., (Eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987). To identify cells that have the cloned cDNA stably integrated, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The sea24

ES 2 272 093 T3 selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Massachusetts.

Mammalian cells containing a suitable vector are grown for a period of time, typically 1-2 days, to begin expressing the DNA sequence (s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable manner. For cells that have been transfected with an amplifiable, selectable marker, the drug concentration can be increased in steps to select for the increased sequence copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level. Cells meeting these criteria can then be cloned and scaled up for production.

Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods include calcium phosphate transfection, electroporation, lipofection, retroviral, adenoviral, and protoplast fusion-mediated transfection (see Sambrook et al., Supra). Naked vector constructs can also be absorbed by muscle cells or other suitable cells after injection into the muscle of a mammal (or other animal).

Numerous insect host cells known in the art may be useful in the present invention, in light of the subject specification. For example, the use of baculoviruses as vectors to express heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28: 215-224, 1990).

Numerous plant host cells known in the art may also be useful in the present invention in light of the subject specification. For example, the use of Agrobacterium rhizogen.es as a vector to express genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangadore) 11: 47-58, 1987).

In related aspects of the present invention, the proteins of the present invention can be expressed in a transgenic animal whose germ cells and somatic cells contain a gene encoding the desired protein and which are operably linked to an effective promoter for expression of the gene. Alternatively, transgenic animals lacking the desired gene (eg, knockout mice) can be prepared in a similar manner. Transgenic resemblances can be prepared in a variety of non-human animals, including mice, rats, rabbits, sheep, dogs, goats, and pigs (see Hammer et al., Nature 315: 680-683, 1985, Palmiter et al., Science 222 : 809-814, 1983, Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985, Palmiter and Brinster, Cell 41: 343345,1985, and US Patent Nos. 5,175. 383, 5,087,751,4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384). Briefly, an expression vector, including a nucleic acid molecule to be expressed in conjunction with suitably placed expression control sequences, is introduced into pronuclei of fertilized eggs, for example, by microinjection. Integration of the injected DNA is detected by DNA blot analysis from the tissue samples. It is preferred that the introduced DNA is incorporated into the germ line of the animal so that it passes into the progeny of the animal. Tissue-specific expression can be achieved through the use of a tissue-specific promoter, or through the use of an inducible promoter, such as the metallothionein promoter gene (Palmiter et al., 1983, ibid.), Which allow regulated expression of the transgene.

Proteins can be isolated, among other methods, by culturing suitable host systems and vectors to produce the recombinant translation products of the present invention. The supernatants of such cell lines, or protein inclusions or whole cells in which the protein is excreted into the supernatant, can then be prepared by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant can be first concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. After concentration, the concentrated product can be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support. Alternatively, anion or cation exchange resins can be used in order to purify the protein. As a further alternative, one or more reversed phase high performance liquid chromatography (RP-HPLC) steps can be employed to further purify the protein. Other methods of isolating the proteins of the present invention are well known to those of skill in the art.

A protein is believed to be "isolated" in the context of the present invention if no other (unwanted) protein is detected by SDS-PAGE analysis followed by Coomassie blue staining. In other embodiments, the desired protein can be isolated so that no other (unwanted) protein is detected according to SDS-PAGE analysis followed by silver staining.

3. Nucleic Acid Molecules

In other aspects of the invention, nucleic acid molecules are provided that are capable of inhibiting the binding of the TGF-beta binding protein to a member of the TGF-beta family. For example, in one embodiment, antisense oligonucleotide molecules are provided that specifically inhibit the expression of the sequences

ES 2 272 093 T3 nucleic acid binding protein TGF-beta (see generally, Hirashima et al., In Molecular Biology of RNA: New Perspectives (M. Inouye and BS Dudock, eds., 1987 Academic Press, San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of Gene Expression (JS Cohen, ed., 1989 MacMillan Press, London); Stein and Cheng, Science 261: 1004-1012, 1993; WO 95/10607; US Patent US Nos. 5,359,051; WO 92/06693; and EPA2-612844). Briefly, such molecules are constructed so that they are complementary to, and capable of forming Watson-Crick base pairs, with a transcribed TGF-beta binding protein mRNA sequence region. The resulting double-stranded acid interferes with the subsequent maturation of the mRNA, thereby preventing protein synthesis (see Example 10).

In other aspects of the invention, ribozymes are provided that are capable of inhibiting the binding of the TGF-beta binding protein to a member of the TGF-beta family. As used herein, "ribozymes" are intended to include RNA molecules that contain antisense sequences for specific recognition, and RNA cleavage enzymatic activity. The catalytic strand cleaves a specific site on a target RNA at a higher than stoichiometric concentration. A wide variety of ribozymes can be used in the context of the present invention, including, for example, the hammerhead ribozyme (eg, as described by Forster and Symons, Cell 48: 211-220, 1987; Haseloff and Gerlach, Nature 328 : 596-600, 1988; Walbot and Bruening, Nature 334: 196, 1988; Haseloff and Gerlach, Nature 334: 585, 1988); hairpin ribozyme (eg, as described by Haseloff et al., US Patent No. 5,254,678, issued October 19, 1993, and Hempel et al., European Patent Application No. 0,360,257, published March 26, 1990); and ribozymes based on Tetrahymena ribosomal RNA (see Cech et al., US Patent No. 4,987,071). The ribozymes of the present invention typically consist of RNA, but can also be composed of DNA, nucleic acid analogs (eg, phosphorothioates), or chimerics thereof (eg, DNA / RNA / RNA).

Four. Trademarks

The gene product or any of the candidate molecules described above and below can be labeled with a variety of compounds, including, for example, fluorescent molecules, toxins, and radionuclides. Representative examples of fluorescent molecules include fluorescein, Phycobili proteins, such as phycoerythrin, rhodamine, Texas red, and luciferase. Representative examples of the toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Phytolacca americana antiviral protein ("pokeweed"), tritin, Sigella toxin, and Pseudomonas exotoxin A. Representative examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Ph-105, Pd-109, In-111, I-123, I -125, I-131, Re-186, Re188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. Furthermore, the antibodies described above can also be labeled or conjugated to a pair of a ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein.

The methods for conjugating or labeling the molecules described herein with the representative labels shown above can be readily completed by one of ordinary skill in the art (see Trichothecene Antibody Conjugate, US Patent No. 4,744,981; Antibody Conjugate, US Patent No. US Patent No. 5,106,951; Fluorogenic Materials and Labeling Techniques, US Patent No. 4,018,884; Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, US Patent No. 4,897,255; and Metal Radionuclide Chelating Compounds for Improved Chelation Kinetics, US Patent No. 4,988,496; see also Inman, Methods In Enzymlogy, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jackoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; see also Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications", Anal. Biochem. 171: 1-32, 1988).

Pharmaceutical compositions

As noted above, the present invention also provides a variety of pharmaceutical compositions, comprising one of the molecules described above that inhibits the binding of TGF-beta binding protein to a member of the TGF-beta family together with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers can be non-toxic to recipients at the dosages and concentrations employed. Typically, the preparation of such compositions encompasses combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary suitable diluents.

Furthermore, the pharmaceutical compositions of the present invention can be prepared for administration by a variety of different routes. In addition, the pharmaceutical compositions of the present invention can be placed in containers, together with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as certain embodiments, relative amounts of excipient or diluent ingredients (eg, water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition.

ES 2 272 093 T3

Treatment methods

The present invention also provides for the manufacture of medicaments to increase the mineral content and mineral density of bone. In summary, numerous conditions result in the loss of mineral content of the bone, including, for example, disease, genetic predisposition, accidents that result in loss of use of a bone (eg due to a fracture), therapeutic agents that affect bone resorption, or that kill bone-forming cells and normal aging. By using the molecules described herein that inhibit the binding of the TGF-beta binding protein to a member of the TGF-beta family to make drugs such conditions can be treated or prevented. As used herein, it is to be understood that the mineral content of the bone has increased, if the mineral content of the bone has increased in a statistically significant manner (eg, greater than one half standard deviation), at a selected site.

The molecules described herein can be used in the manufacture of drugs to treat a wide variety of conditions that result from loss of mineral content from bone. Patients with such conditions can be identified through clinical diagnosis using well-known mechanisms (see, eg, Harrison's Principles of Internal Medicine, McGraw-Hill, Inc.). Representative examples of diseases that can be treated include dysplasias, in which there is abnormal growth or development of bone. Representative examples of such conditions include achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan disease, multiple hereditary exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis disease, sclerotic lesions, , pseudoarthrosis and pyogenic osteomyelitis.

Other conditions that can be treated or avoided using the drugs of the invention include a wide variety of causes of osteopenia (i.e., a condition that causes a greater than one standard deviation of mineral content or bone density below mineral content. skeletal peak in youth). Representative examples of such conditions include anemic states, conditions caused by steroids, conditions caused by heparin, bone marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic osteoporosis, osteopenia and congenital osteoporosis, alcoholism, disease chronic liver, senescence, post-menopausal status, oligomenorrhea, amenorrhea, pregnancy, diabetes melitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, transient regional osteoporosis, and osteomalacia.

In one aspect the present invention provides the use of a molecule that inhibits the binding of TGF-beta binding protein to a member of the TGF-beta family in the manufacture of a medicament to increase the mineral content or density of the bone of a warm-blooded animal providing the drug with an effective amount of the molecule. Examples of warm-blooded animals that can be treated include both vertebrates and mammals, including, for example, horses, cows, pigs, sheep, dogs, cats, rats and mice. Representative examples of therapeutic molecules include ribozymes, ribozyme genes, antisense oligonucleotides, and antibodies (eg, humanized antibodies).

In other aspects the present invention comprises introducing into bone-seeking cells a vector that directs the expression of a molecule that inhibits the TGF-beta binding protein to a member of the TGFbeta family, then such cells can be used in the manufacture of a drug to increase bone density in a warm-blooded animal. Briefly, bone-seeking cells can be obtained directly from the bone of patients (eg, cells obtained from bone marrow such as CD34 +, osteoblasts, osteocytes, and the like), from peripheral blood, or from cultures. .

A vector that directs the expression of a molecule that inhibits the binding of a TGF-beta binding protein to a member of the TGF-beta family is introduced into cells. Representative examples of suitable vectors include viral vectors such as herpes viral vectors (eg, US Patent No. 5,288,641), adenoviral vectors (eg, WO 94/26914, WO 93/9191; Kolls et al., PNAS 91 (1): 215-219, 1994; Kass-Eisler et al., PNAS 90 (24): 1993, 11498-502; Guzman et al., Circulation 88 (6): 2838-48, 1993; Guzman et al., Cir. Res. 73 (6): 1202-1207, 1993; Zabner et al., Cell 75 (2): 207-216, 1993; Li et al., Hum Gene Ther. 4 (4): 403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5 (10): 1287-1291, 1993; Vincent et al., Nat. Genet. 5 (2): 130-134, 1993; Jaffey col., Nat. Genet. 1 (5): 372-378, 1992; and Levrero et al., Gene 101 (2): 195-202, 1991), adeno-associated viral vectors (WO 95/13365; Flotte et al., PNAS 90 (22): 10613-10617, 1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap. 5: 457-463, 1994), poxvirus vectors (Panicali and Paoletti, PNAS 79: 4927-4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm. 193 (2): 653-660, 1993), and retroviruses (eg, EP 0,415,731; WO 90/07936; WO 91/0285; WO 94/03622; WO 93/25698; WO 93/25234; U.S. No. 5,219,740; WO 93/11239; WO 93/10218). In the same way, viral vectors can be constructed that contain a mixture of different elements (eg, promoters, envelope sequences and the like) from different viruses, or non-viral sources. In various embodiments, either the viral vector itself, or a viral particle containing the viral vector can be used in the methods and compositions described below.

In other embodiments of the invention, nucleic acid molecules that encode a molecule that inhibits the binding of a TGF-beta binding protein to a member of the TGF-beta family by themselves can be administered by a variety of techniques. , including, for example, administration to asialomucoid (ASOR) conjugated with poly-L-lysine DNA complexes (Cistano et al., PNAS 92122-92126, 1993), adenovirus-linked DNA

ES 2 272 093 dead T3 (Curiel et al., Hum. Gene Ther. 3 (2): 147-154, 1992), cytofectin-mediated introduction (DMRIE-DOPE, Vical, California), direct DNA injection (Acsadi and col., Nature 352: 815-818, 1991); DNA ligands (Wu et al., J. ofBiol. Chem. 264: 16985-16987, 1989); lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1989); liposomes (Pickering et al., Circ. 89 (1): 13-21, 1994; and Wang et al., PNAS 84: 7851-7855, 1987); microprojectile bombardment (Williams et al., PNAS 88: 2726-2730, 1991); and direct release of nucleic acids encoding the protein itself either alone (Vile and Hart, Cancer Res. 53: 3860-3864, 1993) or using PEG-nucleic acid complexes.

Representative examples of molecules that can be expressed by the vectors of the present invention include ribozymes and antisense molecules, each of which has been discussed in more detail above.

The determination of the increased bone mineral content can be solved directly through the use of x-rays (eg, Dual Energy X-ray Absorptometry or "DEXA"), or by inference through markers of bone turnover (specific alkaline phosphatase of osteoblasts, osteocalcin, type 1 procollagen, propeptide C '(PICP), and total alkaline phosphatase, see Comier, C., Curr. Opin. In Rheu. 7: 243, 1995), or bone resorption markers (pyridinoline, deoxypyridinoline, N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant acid phosphatases, and galactosylhydroxylysine; see Comier, supra). The amount of bone mass can be calculated from body weights, or using other methods (see Guiness-Hey, Metab. Bone Dis. And Rel. Res. 5: 177-181, 1984).

As will be apparent to one skilled in the art, the amount and frequency of administration will, of course, depend on factors such as the nature and severity of the indication being treated, the desired response, the patient's condition, and so on. Typically, the compositions can be administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration, and not by way of limitation.

Examples Example 1

Sclereostiosis maps for the long arm of human chromosome 17

Genetic mapping of the defect responsible for sclerosteosis in humans located the gene responsible for this disorder in the region of human chromosome 17 that encodes a novel member of the TGF-beta-binding protein family. In sclerosteosis, skeletal bone exhibits a substantial increase in mineral density relative to that of unaffected individuals. The head bone is also overgrowed. Patients with sclerosteosis are generally healthy although they may show varying degrees of syndactyly at birth and varying degrees of cranial compression and nerve compression in the skull.

Sclerosteosis-associated gene defect connection analysis was performed by applying the homozygous mapping method to DNA samples collected from 24 Afrikaaner families from South Africa in which the disease occurred. (Sheffield et al., 1994, Human Molecular Genetics 3: 1331-1335. "Identification of a Badet-Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to homozygosity mapping"). The Afrikaaner population of South Africa is generally homogeneous; the population descends from a small number of founders who colonized the area several centuries ago, and it has been isolated by geographic and social barriers since its founding. Sclerosteosis is rare worldwide outside the Afrikaaner community, suggesting that a mutation in the gene was present in the founder population and has increased in number with population growth. The use of homozygosity mapping is based on the assumption that DNA mapping markers adjacent to a recessive mutation are likely to be homozygous in affected individuals from consanguineous families and in isolated populations.

A group of 371 microsatellite markers (Research Genetics, Set 6) from autosomal chromosomes were selected to typify DNA pools from samples from patients with sclerosteosis. DNA samples for this analysis came from 29 sclerosteosis patients from 24 families, 59 members of unaffected families, and a group of unrelated control individuals from the same population. The pools consisted of 4-6 individuals, affected individuals, affected individuals from consanguineous families, unaffected parents and siblings, or unrelated controls. In pools of unrelated individuals and in most pools with affected individuals or family members, marker analyzes demonstrated numerous allele sizes for each marker. One marker, D17S1299, showed an indication of homozygosity: a band in some of the pools of affected individuals.

The 24 families with sclerosteosis were typified with a total of 19 markers in the D17S1299 region (at 17q12-q21). Affected individuals from each family were found to be homozygous in this region, and 25 of the 29 individuals were homozygous for a central haplotype; each had the same alleles between D17S1787 and D17S930. The other four individuals had one chromosome that matched this haplotype and a second that did not. In sum, the data convincingly suggested that this 3 megabase region contained the sclerosteosis mutation. Sequence analysis of most of the exons in this 3 megabase region identified a terminator mutation in the coding sequence for the novel TGF-binding protein (C> T mutation at position 117

ES 2 272 093 T3 of SEQ ID NO. 1 results in a stop codon). This mutation was shown to be unique to patients with sclerosteosis and carriers of Afrikaaner descendants. The identity of the gene was further confirmed by identifying a mutation in its intron (A> T mutation at position +3 of the intron) that results in inappropriate mRNA maturation in an unrelated, individual patient with diagnosed sclerosteosis.

Example 2

Tissue specificity of TGF-beta binding protein gene expression

A. Expression of the Human Beer Gene by RT-PCR

A first strand cDNA was prepared from the following total RNA samples using a commercially available kit ("Superscript Preamplification System for First Strand cDNA Synthesis", Life Technologies, Rockville, MD): human brain, human liver, human spleen, human thymus, human placenta, human skeletal muscle, human thyroid, human pituitary, human osteoblasts (NHOst from Clonetics Corp., San Diego, CA), human osteosarcoma cell line (Saos-2, ATCC # HTB-85), human bone, human bone marrow, human cartilage, Vervet monkey bone, Saccharomyces cerevisiae, and human peripheral blood monocytes. All RNA samples were purchased from a commercial source (Clontech, Palo Alto, CA), except for the following which were prepared by the company: human osteoblast, human osteosarcoma cell line, human bone, human cartilage, and Vervet monkey bone. These in-house prepared RNA samples were prepared using a commercially available kit ("TRI Reagent", Molecular Research Center, Inc., Cincinnati, OH).

PCR was performed on these samples, and additionally on a genomic sample as a control. The Beer effector oligonucleotide had the sequence 5'-CCGGAGCTGGAGAACAACAAG-3 '(SEQ ID NO: 19). The Beer antisense oligonucleotide primer had the sequence 5'-GCACTGGCCGGAGCACACC-3 '(SEQ ID NO: 20). Furthermore, PCR was performed using primers for the human beta-actin gene, as a control. The beta-actin-effector oligonucleotide primer had the sequence 5'-AGGCCAACCGCGAGAAGATGA CC-3 '(SEQ ID NO: 21). The antisense beta-actin oligonucleotide primer had the sequence 5'-GAAGT CCAGGGCGACGTAGCA3 '(SEQ ID NO: 22). PCR was performed using standard conditions in 25 µl reactions, with an annealing temperature of 61 degrees Celsius. 32 cycles of PCR were carried out with the Beer primers and 24 cycles with the beta-actin primers.

After amplification, 12 µl of each reaction was analyzed by agarose gel electrophoresis and ethidium bromide staining. See Figure 2A.

B. In-situ Hybridization of RNA from Mouse Embryonic Sections

The complete mouse Beer cDNA (Sequence ID No. 11) was cloned into the vector pCR2.1 (Invitrogen, Carlsbad, CA) in the antisense and effector directions using the manufacturer's protocol. S-labeled cRNA antisense and effector transcripts³⁵-alpha-GTP were synthesized using in vitro transcription reagents supplied by Ambion, Inc. (Austin, TX). In situ hybridization was performed according to the protocols of Lyons et al. (J. Cell Biol. 111: 2427-2436, 1990).

The mouse Beer cRNA probe detected a specific message expressed in the neural tube, blastemas, blood vessels, and ossification cartilage of developing mouse embryos. Panel A of Figure 3 shows expression in the apical ectodermal ridge (aer) of the blastema (l), blood vessels (bv), and neural tube (nt in its acronym in English). Panel B shows expression in the 4th ventricle of the brain (4). Panel C shows expression in the mandible (ma), cervical vertebrae (cv), occipital bone (oc), palate (pa) and blood vessels (bv). Panel D shows the expression in the ribs (r) and in the heart valve (va). Panel A is a 10.5 dpc embryo cross section. Panel B is a 12.5 dpc embryo sagittal section and panels C and D are 15.5 dpc embryo sagittal sections.

Ba = branchial arch, h = heart, te = telencephalon (forebrain), b = brain, f = frontal mass, g = intestine, j = mandible, li = liver, lu = lung, ot = otic vesicle, ao =, sc = spinal cord, skm = skeletal muscle, na = sinus, th = thymus, to = tongue, fl = forelimb, di = diaphragm.

Example 3

Expression and purification of recombinant beer protein A. Expression in COS-1 cells

The DNA sequence encoding the complete human Beer protein was amplified using the following oligonucleotide primers for PCR. The 5 'oligonucleotide primer had the sequence 5'-AAGCTTGGTACCATG

ES 2 272 093 T3

CAGCTCCCAC-3 '(SEQ ID NO: 23) and contained a site for the restriction enzyme HindIII (in bold) followed by 19 nucleotides of the Beer gene starting 6 base pairs before the presumptive amino terminal start codon (ATG). The 3 'oligonucleotide primer had the sequence 5'-AAGCTTCTACTTGTCATCGTCGTCCTTG TAGTCGTAGGCGTTCTCCAGCT-3' (SEQ ID NO: 24) and contained a site for the restriction enzyme HindIII (in bold) followed by a reverse complementary stop codon (CTA) Reverse complement of the FALG epitope (underlined, Sigma-Aldrich Co., St. Louis, MO) flanked by the reverse complement of nucleotides encoding the 5 carboxy terminal amino acids of Beer. The PCR product was cloned with TA ("Original TA Cloning Kit", Invitrogen, Carlsbad, CA) and individual clones were screened by DNA sequencing. A sequence verified clone was then digested by HindIII and purified on 1.5% agarose gel using commercially available reagents ("Qiaquick Gel Extraction Kit", Qiagen Inc., Valencia, CA). This fragment was then ligated to the phosphatase-treated, HindIII-digested plasmid pcDNA3.1, and plated on LB plates with 100 pg / ml ampicillin. Colonies carrying the desired recombinant in the appropriate orientation were identified by PCR-based screening, using a 5 'primer corresponding to the T7 promoter / primer site in pcDNA3.1 and a 3' primer with the sequence 5'-GCACTGGCCGGAGCACACC-3 '(SeC ID NO: 25) which corresponds to the inverse complement of the internal BEER sequence. The sequence of the cloned fragment was confirmed by DNA sequencing.

COS-1 cells (ATCC # CRL-1650) were used for transfection. 50 pg of the pcDNA-Beer-Flag expression plasmid were transfected using a commercially available kit following the protocols supplied by the manufacturer ("DEAE-Dextran Transfection Kit", Sigma Chemical Co., St. Louis, MO). The final medium after transfection was DMEM (Life Technologies, Rockville, MD) containing 0.1% Fetal Bovine Serum. After 4 days of culture, the medium was separated. The expression of recombinant BEER was analyzed by SDS-PAGE and Western Blot using anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St. Louis, MO). Purification of the recombinant BEER protein was performed using a M2 anti-FLAG affinity column ("Mammalian Transient Expression System", Sigma-Aldrich Co., St. Louis, MO). The profile of the column was analyzed via SDS-PAGE and Western Blot using monoclonal antibody M2 anti-FLAG.

B. Expression in SF9 insect cells

The sequence of the human Beer gene was amplified using PCR with standard conditions and the following primers:

Effector primer: 5'-GTCGTCGGATCCATGGGGTGGCAGGCGTTCAAGAATGAT-3 '(SEQ ID NO: 26)

Antisense primer: 5'-GTCGTCAAGCTTCTACTTGTCATCGTCCTTGTAGTCTAGGC

GTTCTCCAGCTCGGC-3 '(SEQ ID NO: 27)

The resulting cDNA contained Beer's coding region with two modifications. The N-terminal secretion signal had been removed and the FLAG (Sigma) epitope tag was fused in frame with the C-terminal end of the insert. The BamHI and HindIII cloning sites were added and the gene was subcloned into the pMelBac vector (Invitrogen) for transfer to a baculoviral vector using standard methods.

Recombinant baculoviruses expressing Beer protein were made using the Ba-N-blue transfection kit (Invitrogen) and purified according to the manufacturers instructions.

SF9 cells (Invitrogen) were maintained in TNM_FH medium (Invitrogen) containing 10% fetal calf serum. For protein expression, cultures of SF9 in flasks with an extender ("Spinner") at an MOI of greater than 10. Media and cell samples were taken daily for five days, and Beer's expression was monitored by Western blotting using anti-FLAG monoclonal antibody M2 (Sigma) or rabbit anti-Beer polyclonal antiserum.

After five days, the baculovirus-infected SF9 cells were collected by centrifugation and the cell-associated protein was extracted from the cell pellet using a high-salt extraction buffer (1.5 M NaCl, 50 mM Tris pH 7, 5). The extract (20 ml per 300 ml culture) was clarified by centrifugation, dialyzed three times against four liters of Tris-buffered saline (150 mM NaCl, 50 mM Tris pH 7.5), and rinsed again by centrifugation. This high salt content fraction was applied to Hitrap Heparin (Pharmacia: 5 ml bed volume), washed extensively with HEPES buffered saline (HEPES 25 mM 7.5, NaCl 150 mM) and bound proteins eluted with a gradient from 150 mM NaCl to 1200 mM NaCl. Beer's elution was observed at approximately 800 mM NaCl. The Beer-containing fractions were supplemented with 10% glycerol and 1 mM DTT and frozen at -80 ° C.

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Example 4

Polyclonal Antibody Preparation and Assay for Beer, Gremlin, and Dan A. Antigen Preparation

The DNA sequences of human Beer, human Gremlin, and human Dan were amplified using standard PCR methods with the following oligonucleotide primers:

Beer H

<td>Effector:</td><td>5'-GACTTGGATCCCAGGGGTGGCAGGCGTTC-3 '</td><td>(SEQ ID NO: 28)</td>

<td>Antisense:</td><td>5'-AGCATAAGCTTCTAGTAGGCGTTCTCCAG-3 '</td><td>(SEQ ID NO: 29)</td>

<td colspan="3">Gremlin H</td>

<td>Effector:</td><td>5'-GACTTGGATCCGAAGGGAAAAAGAAAGGG-3 '</td><td>(SEQ ID NO: 30)</td>

<td>Antisense:</td><td>5'-AGCATAAGCTTTTAATCCAAATCGATGGA-3 '</td><td>(SEQ ID NO: 31)</td>

<td colspan="3">DanH</td>

<td>Effector:</td><td>5'-ACTACGAGCTCGGCCCCACCACCCATCAACAAG-3 '</td><td>(SEQ ID NO: 32)</td>

<td>Antisense:</td><td>5'-ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTTCC-3 '</td><td>(SEQ ID NO: 33)</td>

In each case the listed primers amplified the entire coding region minus the secretion signal sequence. These include the restriction sites for subcloning in the bacterial expression vector pQE-30 (Qiagen Inc., Valencia) at the BamHI / HindIII sites for Beer and Gremlin, and at the SacI / HindIII sites for Dan. pQE30 contains a coding sequence for the 6x His tag at the 5 'end of the cloning region. The completed constructs were transformed into E. coli strain M-15 / pRep (Qiagen Inc.) and individual clones were verified by sequencing. Protein expression in M-15 / pRep and purification (binding of the 6xHis affinity tag to Ni-NTA coupled with Sepharose) were performed as described by the manufacturers (Qiagen, The QIAexpressionist).

Beer protein derived from E. coli was recovered in a significant amount using 6M guanidine solubilization and dialyzed at 2-4M to avoid precipitation during storage. The Gremlin and Dan proteins were recovered in a higher amount with solubilization in 6M guanidine and a post-purification guanidine concentration of 0.5M.

B. Production and testing of polyclonal antibodies

Polyclonal antibodies to each of the three antigens were produced in hosts such as rabbit and chicken using standard protocols (R&amp;R Antibody, Stanwood, WA; standard protocol for rabbit immunization and antiserum retrieval; Short Protocols in Molecular Biology, 2nd edition , 1992. 11.37-11.41 Contributors Helen M. Cooper and Yvonne Paterson; chicken serum was generated with Strategic Biosolutions Ramona, CA).

Rabbit antiserum and chicken egg IgY fraction were screened for activity via Western blot. Each of the three antigens was separated by PAGE and transferred to 0.45 pm nitrocellulose (Novex, San Diego, CA). The membrane was cut into strips each containing approximately 75 ng of antigen. The strips were blocked in 3% Blottig Grade Block (Bio-Rad Laboratories, Hercules, CA) and washed 3 times in 1X Tris buffered saline (TBS) / 0.02% Tween buffer. The primary antibody (pre-immunization blood draws, rabbit antiserum, or chicken egg IgY in dilutions ranging from 1: 100 to 1: 10,000 in blocking buffer) was incubated with the strips for one hour with gentle rocking. A second series of three 1X TBS / 0.02% TWEEN washes was followed by a one hour incubation with the secondary antibody (peroxidase-conjugated donkey anti-rabbit, Amersham Life Science, Piscataway, NJ; or anti-polli de peroxidase-conjugated donkey, Jackson ImmunoResearch, West Grove, PA). A final cycle of 3X washes of 1X TBS / 0.02% TWEEN was performed and the strips were developed with Lumi-Light Western Blotting Substrate (Roche Molecular Biochemicals, Mannheim, Germany).

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C. Antibody cross-reactivity assay

Following the protocol described in the previous section, Beer, Gremlin or Dan strips were incubated with dilutions (1: 5000 and 1: 10,000) of their respective rabbit antisera or chicken egg IgY as well as chicken egg antiserum or Igy. (1: 1000 and 1: 5000 dilutions) made for the remaining two antigens. Increased levels of non-matching antibodies were performed to detect low affinity binding to antibodies that can only be observed at high concentration. The protocol and duration of development are the same for all three binding events using the protocol described above. No cross-reactivity with antigen was observed for any of the antigens tested.

Example 5

Beer's interaction with proteins of the TGF-beta superfamily

The interaction of Beer with the proteins of different phylogenetic branches of the TGF-β super-family was studied using immunoprecipitation methods. TGF ^ -1, TGF ^ -2, TGF ^ -3, BMP-4, BMP-5, BMP-6, and GDNF were obtained from commercial sources (R &amp; D systems; Minneapolis, MN). A representative protocol is as follows. Partially purified Beer was dialyzed in HEPES buffered saline (25 mM 7.5 HEPES, 150 mM NaCl). Immunoprecipitations were performed in 300 µl of IP buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 1 mM EDTA, 1.4 mM ^ - mercaptoethanol, 0.5% triton X-100, and 10 glycerol %). 30 ng of recombinant human BMP-5 protein (R&amp;D systems) were applied to 15 µl of FLAG affinity matrix (Sigma, St. Louis MO)) in the presence and absence of 500 ng of Beer labeled with the FLAG epitope. The proteins were incubated for 4 hours @ 4 ° C and then the proteins associated with the affinity matrix were washed five times in IP buffer (1 ml per wash). Bound proteins were eluted from the affinity matrix in 60 microliters of 1X SDS PAGE sample buffer. Proteins were resolved by SDS PAGE and Beer associated with BMP-5 was detected by Western blotting using anti-BMP-5 antiserum (Research Diagnostics, Inc.) (see Figure 5).

Analysis of Binding to Ligand BEER

The FLAG-Beer protein (20 ng) is added to 100 µl of PBS / 0.2% BSA and adsorbed in each well of a 96-well microtiter plate previously coated with anti-FLAG monoclonal antibody (Sigma; St Louis MO ) and blocked with BSA in 10% PBS. This is done at room temperature for 60 minutes. This protein solution is separated and the wells are washed to remove unbound protein. BMP-5 is added to each well at concentrations ranging from 10 pM to 500 nM in PBS / 0.2% BSA and incubated for 2 hours at room temperature. The binding solution is removed and the plate is washed three times with 200 µl volumes of PBS / 0.2% BSA. BMP-5 levels are detected using anti-serum BMP-5 via ELISA (FM Ausubel et al. (1998) Current Protocols in Mol. Biol. Vol. 2 11.2.1-11.2.22). Specific binding is calculated by subtracting non-specific binding from total binding and analyzed by the LIGAND program (Munson and Podbard, Anal. Biochem., 107, pp. 220-239, (1980).

In a variation of this method, Beer is designed and expressed as a human Fc fusion protein. Similarly the BMP ligand is designed and expressed as a fusion with mouse Fc. These proteins are incubated together and the analysis is performed as described by Mellor et al. using time-resolved fluorescence detection (GW Mellor et al., J. of Biomol Screening, 3 (2) 91-99, 1998).

Example 6

Screening Assay for Inhibition of TGF-beta Binding Protein Binding to Members of the TGF-beta Family

The analysis described above is repeated with two exceptions. First, the BMP concentration is kept fixed at the previously determined Kd. Second, a library of antagonist candidates is added at a fixed concentration (20 pM in the case of the small organic molecule libraries and 1 pM in antibody studies). These candidate molecules (antagonists) for binding to the TGF-beta binding protein include organic compounds derived from commercial or internal libraries representing various chemical structures. These compounds are prepared as starting solutions in DMSO and are added to assay wells at &lt;1% of final volume under standard assay conditions. These are incubated for 2 hours at room temperature with BMP and Beer, the solution is separated and the bound BMP is quantified as described. Agents that inhibit 40% of BMP binding observed in the absence of compound or antibody are considered antagonists of this interaction. These are further evaluated as potential inhibitors based on titration studies to determine their inhibition constants and their influence on the binding affinity of the TGF-beta binding protein. Comparable specificity control assays can also be performed to establish the selectivity profile for the identified antagonist through studies using BMP ligand action-dependent assays (e.g., BMP / receptor competition study). BMP).

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Example 7

Inhibition of the localization of TGF-beta binding protein to the bone matrix

The evaluation of the inhibition of the localization in the bone matrix (hydroxyapatite) is carried out using modifications of the Nicolás method (Nicolás, V. Calcif Tissue Int. 57: 206, 1995). Briefly, the I-labeled TGF-beta binding protein¹²⁵ It is prepared as described by Nicolás (supra). Hydroxyapatite is added to each well of a 96-well microtiter plate equipped with a polypropylene filtration membrane (Polyfiltronic, Weymouth MA). TGF-beta binding protein is added to 0.2% albumin in PBS buffer. The wells containing the matrix are washed 3 times with this buffer. The adsorbed TGF-beta binding protein is eluted using 0.3M NaOH and quantitated.

Identification of the inhibitor is carried out by incubating the TGF-beta binding protein with test molecules and applying the mixture to the matrix as described above. The matrix is ​​washed 3 times with 0.2% albumin in PBS buffer. The adsorbed TGF-beta binding protein is eluted using 0.3M NaOh and quantitated. Agents that inhibit 40% of TGF-beta binding protein binding observed in the absence of compound or antibody are considered inhibitors of bone localization. These inhibitors are further characterized by dose-response studies to determine their inhibition constants and their influence on the binding affinity of the TGF-beta binding protein.

Example 8

Construction of TGF-beta binding protein mutants

A. Mutagenesis

A complete TGF-beta binding protein cDNA in pBluescript SK serves as a template for mutagenesis. Briefly, the appropriate primers (see study provided here) are used to generate the DNA fragment by polymerase chain reaction using Vent DNA polymerase (New England Biolabs, Beverly, MA). The polymerase chain reaction is run for 23 cycles in buffers provided by the manufacturer using an annealing temperature of 57 ° C. The product is then exposed to two restriction enzymes and after isolation using agarose gel electrophoresis, ligated back into pRBP4-503 from which the pairing sequence has been separated by enzymatic digestion. The integrity of the mutant is verified by DNA sequencing.

B. Expression in Mammalian Cells and Isolation of Mutant TGF-beta Binding Protein

The mutant TGF-beta binding protein cDNAs are transferred to the mammalian expression vector pcDNA3.1 described in Example 3. After verifying the sequence, the resulting constructs are transfected into COS-1 cells, and the secreted protein It is purified as described in Example 3.

Example 9

Animal Models-I

Generation of transgenic mice that overexpress the Beer gene

The -200 kilobase (kb) BAC clone 15G5, isolated from the CTIB mouse genomic DNA library (distributed by Research Genetics, Huntsville, AL) was used to determine the complete sequence of the mouse Beer gene and its 5 'and 3 border regions. '. A 41 kb SalI fragment, containing the entire gene body, plus -17 kb of the 5 'border sequence and -20 kb of the 3' border sequence was subcloned into the BamHI site of the SuperCos1 cosmid vector (Stratagene, La Jolla, CA) and propagated in E. coli strain DH101B. From this cosmid construct, a 35 kb MluI-AvlII restriction fragment (Sequence No. 6), including the complete mouse Beer gene, as well as the 17 kb and 14 kb 5 'and 3' border sequences, respectively, were then gel purified, using conventional means, and used for microinjection into mouse zygotes. (DNX Transgenics; US Patent No. 4,873,191). Founder animals in which the cloned DNA fragment had been randomly integrated into the genome were obtained at a frequency of 5-30% from the young born alive. The presence of the transgene was determined by performing Southern blot analysis of genomic DNA extracted from a small amount of mouse tissue, such as the tip of a tail. The DNA was extracted using the following protocol: the tissue was digested overnight at 55 ° C in lysis buffer containing 200 mM NaCl, 100 mM Tris pH 8.5, 5 mM EDTA, 0.2% SDS and 0, 5 mg / ml of Proteinase K. The next day, DNA was extracted once with phenol / chloroform (50:50), once with chloroform / isoamyl alcohol (24: 1) and precipitated with ethanol. After re-suspension in TE (10 mM Tris pH 7.5, 1.5 mM EDTA), 8-10 pg of each DNA sample were digested with a restriction endonuclease, such as EcoRI, subjected to gel electrophoresis and transferred to a charged nylon membrane, such as HybondN + (Amersham, Arlington Heights, IL). The resulting filter was then hybridized with a radioactively labeled fragment of DNA derived from the mouse Beer gene locus, and capable of recognizing both a

ES 2 272 093 T3 fragment of the endogenous gene locus as a fragment of a different size derived from the transgene. Founder animals were bred to normal non-transgenic mice to generate a sufficient number of transgenic and non-transgenic progeny in which to determine the effects of Beer gene overexpression. For these studies, animals of various ages (e.g. 1, day, 3 weeks, 6 weeks, 4 months) are subjected to numerous different tests designed to find out gross skeletal formation, bone mineral density, bone mineral content , the activity of osteoclasts and osteoblasts, the degree of endochondral ossification, the formation of cartilage, etc. The transcriptional activity of the transgene can be determined by extracting RNA from various tissues, and using RT-PCR analysis that takes advantage of individual nucleotide polymorphisms between the mouse strain from which the transgene is derived (129Sv / J) and the strain of mice used for DNA microinjection [(C57BL5 / J x SJL / J) F2].

Animal Models II

Disruption of the mouse Beer gene by homologous recombination

Homologous recombination in embryonic stem cells (ES) can be used to inactivate the endogenous mouse Beer gene and subsequently generate animals that carry the loss-of-function mutation. A reporter gene, such as the E. coli β-galactosidase gene, was designed in the redirect vector so that its expression was controlled by the endogenous Beer gene promoter and the translation initiation signal. In this way, the spatial and temporal patterns of the expression of the Beer gene can be determined in animals that carry a redirected allele.

The redirected vector was constructed by first cloning the neomycin resistant gene cassette (neo) driven by the drug-selectable phosphoglycerate kinase (PGK) promoter from pGT-N29 (New England Biolabs, Beverly, MA) into the cloning vector. pSP72 (Promega, Madson, WI). PCR was used to flank the PGKneo cassette with bacteriophage P1 1oxP sites, which are the recognition sites for P1 Cre recombinase (Hoess et al., PNAS USA, 79: 3398,1982). This allows for the subsequent removal of the neo resistance marker in redirected ES cells in ES cell derived animals (US Patent 4,959,317). The PCR primers were comprised of a sequence of 34 nucleotides (ntd) loxP, 15-25 ntd complementary to the 5 'and 3' ends of the PGKneo cassette, as well as sites for recognition by the restriction enzyme (BamHI in the effector primer and EcoRI in the antisense primer) for cloning into pSP72. The sequence of the effector primer was 5'-AATCTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCTG CAGGA TTCGAGGGGCCCCT-3 '(SEQ ID NO: 34); the sequence of the anti-sense primer was 5'-AATCTGAATTC CACCGGTGTTAATTAAATAACTTCGTATAATGTATGCTATACGAAGTTATAGATCTAGAGTCAGCTTCTGA-3 '(SEQ ID NO: 35).

The next step was to clone a 3.6 kb XhoI-HindIII fragment, containing the E. coli β-galactosidase gene and the SV40 polyadenylation signal from pSV / t (Clontech, Palo Alto, CA) into the plasmid pSP72-PGKneo. The "short arm" of the mouse Beer gene locus homology was generated by amplifying a fragment of the BAC 15G5 clone. The 3 'end of the fragment coincided with the Beer gene translation start site, and the anti-sense primer used in the PCR also included 30 ntd complementary to the 5' end of the β-galatosidase gene so that its region encoding could be fused with Beer's initiation site in frame. The approach chosen to introduce the "short arm" into the plasmid pSP72-egal-PGKneo was to linearize the plasmid at a site upstream of the β-gal gene and then co-transform this fragment with the "short arm" PCR product. and selecting the plasmids into which the PCR product was integrated by homologous recombination. The effector primer for "short arm" amplification included 30 ntd complementary to vector pSP72 to allow for this recombination event. The sequence of the effector primer was 5'-ATTTAGGTGACACTATAGAACTCGA GCAGCTGAAGCTTAACCACATGGTGGCTCACAACCAT-3 '(SEQ ID NO: 36) and the sequence of the anti-sense primer was 5'-AACGACGGCCAGTGAATCCGTA ATCATGCCGAGTGCA 3'.

The "long arm" of the Beer gene locus was generated by amplifying a 6.1 kb fragment of clone BAC 15G5 with primers that also introduced the sites for the rarecutting restriction enzymes SgrAI, FseI, AscI and PacI. Specifically, the sequence of the effector primer was 5'-ATTACCACCGGTGA CACCC GCTTCCTGACAG-3 '(SEQ ID NO: 38); the sequence of the effector primer was 5'-ATTACTTAATTAAA CATGGCGCGCCATATGGCC GGCCCCTAATTGCGGCGCATCGTTAATT-3 '(SEQ ID NO: 39). The resulting PCR product was cloned into the TA vector (Invitrogen, Carlsbad, CA) as an intermediate step.

The mouse Beer gene targeting construct also included a second selectable marker, the herpes simplex virus thymidine kinase 1 (HSVTK) gene under the control of the Rous sarcoma virus long terminal repeat (RSV LTR) element. The expression of this gene makes mammalian cells sensitive (and unviable) to ganciclovir, it is therefore a convenient way to select against cells resistant to neomycin in which the construct has been integrated through a non-homologous event (Patent of United States 5,464,764). The RSVLTR-HSVTK cassette was amplified from pSP1337 using the primers that allow subsequent cloning into the FseI and AscI sites of the "long arm" -ta vector plasmid. For this PCR, the sequence of the effector primer was 5'-ATTACGGCCGGCCGCAAAGG AATTCAAGA TCTGA-3 '(SEQ ID NO: 40); the antisense primer sequence was 5'-ATTACGGCGCGCCCCTCACAGGCCGCACCC AGCT-3 '(SEQ ID NO: 41).

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The final step in the construction of the redirected vector involved the cloning of the 8.8 kb SgrI-AscI fragment containing the "long arm" and the RSVLTR-HSVTK gene into the SgrAI and AscI sites of plasmid pSP72- "short arm" - egal-PGK neo. This redirected vector was linearized by digestion with either AscI or PacI prior to electroporation into ES cells.

Example 10

Antisense-mediated Beer inactivation

17 nucleotide antisense oligonucleotides are prepared in an overlapping format, such that the 5 'end of the first oligonucleotide overlaps the translation initiation AUG of the Beer transcript, and the 5' ends of successive oligonucleotides are produced in increments 5 nucleotides moving in a 5 'direction (up to 50 nucleotides further), relative to Beer's AUG. Corresponding control oligonucleotides are designed and prepared using compositions of equivalent bases but rearranged in sequence to inhibit any significant hybridization to the encoding mRNA. Release of the reagent for the cellular assay system is carried out through cationic lipid partitioning (PL Felgner, Proc. Natl. Acad. Sci. USA 84: 7413, 1987). 2 pg of antisense oligonucleotide are added to 100 µl of medium with the reduced serum (Opti-MEM 1 reduced serum medium; Life Technologies Gaithersburg MD) and this is mixed with Lipofectin reagent (6 µl) (Life Technologies, Gaithersburg MD) in the 100 µl of medium with reduced serum. These are mixed, allowed to complex for 30 minutes at room temperature, and the mixture is added to pre-seeded MC3T3E21 or KS483 cells. These cells are cultured and the mRNA is recovered. Beer's mRNA is monitored using RT-PCR in conjunction with Beer's specific primers. In addition, separate experimental wells are collected and protein levels are characterized by means of western blotting methods described in Example 4. Cells are harvested, resuspended in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl , 1 mM EDTA, 1% SDS) and soluble protein is collected. This material is applied to SDS PAGE in a denaturing gradient at 10 = 20). The separated proteins are transferred to nitrocellulose and western blotting is performed as before using the antibody reagents described. In parallel, the control oligonucleotides are added to identical cultures and the experimental conditions are repeated. The decrease in Beer protein or mRNA levels is considered significant if treatment with the antisense oligonucleotide results in a 50% change in any case compared to the control oligonucleotide with the same randomly oriented bases ("scrambled"). . This methodology allows the selective inactivation of the gene and the subsequent characterization of the phenotype of the mineralized nodules in the tissue culture model.

(Sequence goes to next page)

ES 2 272 093 T3

SEQUENCES

SEQ ID NO. 1: Human BEER cDNA (complete coding region plus 5 'and 3' UTR)

AGAGCCTGTGCTACTGGAA.GGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACT3GCCCTGTGTCTCGTCTGC:

CTGCTGGTACACACAGCCTTCCGT3TAGTGGA3GGCCAGGGGTGGCAGGCGTTCAAGAATGATGCCACGGA.-ATCATCCC:

CGAGCTCGGAGA.GTAC: CCCGAGCCTCGACCGGAGCTGGAGAA (: AACPAGACCATGAACCG'3GCGGAGAA.CGGAGGGCGGC

CTCCCCAGCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGAT

GGGCCGTGCC3GAGGGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCT3CCCAACGC

CA.TCGGGCGCGGCAAGTGGTGGCGACCTAGTGGGCCCG.ACTTCCGCTGGATCCCCGACC3CTACCGCGCGCAGCGCGTGC

AGCT: -: CTGTGTCCGGGTGGTGAGGCGCCGCGCGCGC-3C. “AGGTGCGCCTGGTGGCCTCGTGCAAGTGC.-AGC3CCTCACC

CGCTTCCACA.ACC.AGTCGGAGCTCAAGGACTTCGGGACCGA.GGCCGCTCGGCCGCA.GAAGGGCCGGAAGCC33GCCCCG

CGCCCGGAGCGCCAAAGCCAACCAGGCCGAGCTGGAGAACGCCTACTAaAGCCCGCCCGCGCCCCTCCCCAGCGGCGGGC

GCCCCGGCC'CTG.-ACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGC

A.ACCCAGGGCAGGGGGCTGAGACCTTCCAGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCGTCAGCCCGCCAGCTG

AGGGGTCCCACGGGGCAGGGGAGGGAATTGAGAGTCAC.AG.ACACTGAGCCACGCAGCCCCGCCTCTGGGGCCGCCTACCT

TTGCTGGTCCCAC'rTCAGAGGAGGCAGAAATGGAAGCATTTTCACCGCCCTGGGGTTTTAAGGGAGCGGTGTGGGAGTGG

CAAAGTCCAGGGACTGGTTAAG.AAAGTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAG.AAAGCCTGAGGCGTGC

CCAGAGCACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTAACCTTGAAC

TACACAATTCTCCTTCGGGACCTCPATTTCCACTTTGTAAAATGAGGGTGüAGGTGGGAATAGG.ATCTCGAGGAGACTAT

TGGCATATGATTCCAAGGACTCCAGTGCCTTTTGAATGGGCAGAGGTGAGAGAGAGAGAGAGAGAAAGAGAGAGAATGAATü

CA.CTTGCATTGATTCAGTGCCAAGGTCA.CTTCCAGAATTCAGAGTTGTGATGCTCTCTTCTGACAGCCAAAGATGAAAAA

CAAACAGAAA-A.WAAGTAAAGA.GTCTATTTATGGCTGACATATTTACGGCTGACAAACTCCTGGAAGAAGCTATGCTG

CTTCCCA5CCTGGCTTCCCCGGATGTTTGGCTACCTCCACCCCTCCATCTCAAAGAAATAACATCATCCATTGGGGTAGA aaaggagagggtccgagggtggtgggagggatagaaatcacatccgccccaacttcccaaagagcagcatccctcccccg

ACCCATAGCCATGTTTTAAAGTCACCTTCCGAAGAGAAGTGAAAGGTTCAAGGACACTGGCCTTGCAGGCCCGAGGGAGC

AGCCATCACAAACTCACAGACCAGCACATCCCTTTTGA.GACACCGCCTTCTGCCCACCACTCACGGACACATTTCTGCCT

AGAAAACAGCTTCTTACTGCTCTTACATGTGATGGCATATCTTACACTAAAAGAATATTATTGGGGGAAAAACTACAAGT

GCTGTACATATGCTGAGAAACTGCAGAGCATAATAGCTGCCACCCA.AAAATCTTTTTGAAAATCATTTCCAGACAACCTC

TTACTTTCTGTGTAGTTTTTAATTGTT.AAAAAAAAAAAGTTTTAAACAGAAGCAGATGACATATGAAAGCCTGCAGGACT

GGTCGTTTTTTTGGCAATTCTTCCACGTGGGACTTGTCCAC.AAGAATGAAAGT.AGTGGTTTTTAAAGAGTTAAGTTAC.AT

ATTTATTTTCTCACTTAAGTTATTTATGCAAAAGTTTTTCTTGTAGAGAATGACAATGTTAATATTGCTTTATGAATTAA

CAGTCTGTTCTTCCAGAGTCCAGAGACATTGTTAATAAAGACAATGAATCATGACCGAAAG

SEQ ID NO. 2: Human BEER protein (complete sequence) mqlflalclvcllvhtafrwegqgwq.afkndatexifel3eyfeffeelennktmnraenggrffhhffetkdvseysc RELHFTRYVTDGFCRSAKPVTELVCSGQCGFARLLPNAIGRGKWWRFSGFDFRCIPDRYRAQRVQLLCFGGEAFRARKVR LVASC KC KRLTRFHNQSELKDFGTEAARP QKGRKPRP RARSAKANQAELENAY

ES 2 272 093 T3

SEQ ID NO. 3: Human BEER protein containing Sclerosteosis terminator mutation

AGAGCCTGTGCTA.CTGGAA.GGTGGCGTGCCCTCCTCTGGCTGGTACCATGCAGCTCCCACTGGCCCTGTGTCTCGTCTGC

CTGCTGGTACACACA3CCTTCCGTG7AGTGGAGGGCTA.GGGGTGGCAGGCGTTCAA.GA.AT5ATGCCACGGAAA.TCA.TC £ ·. ·

CG.AGCTCGGAGAGTACCCCGAGCCTCCACCGGA.GCTGGA.GAACAA.CA_AGA.CC.ATGAA.CCGGGCGGAGAACGGA.GGGCGGC

CTCCCCACC.ACCCCTTTGAG.ACCAAA.GACGTGTCCGAGTACAÜCTGCCGCGAGCTGCACTTCA.CCCGCTACGTGACCGAT

GGGCCGTGCCGC.AGCGCC.AAGCCGGTCA.CCG.AGCTGGTGTGCTCCGGCCA.GTÜCGGCCCGGCGCGCCTGCTGCCC.AACGC gatcggccgcggc.aagtggtggcgaccta.gtgggcccgacttgccgcccca

A3CTGCTGTGTCCCGG7GGTGAGGCGCCGCGCGCGCGCAA.GGTGCGCCTGGTGGCCTCGT6CAA.GTGCAAGCGCCTCA.CC

CGCTTCCACAA.CCA.GTCGGA.GCTCAA.3GACTTCGGGACCGAGGCCGCTC6GCCGCAG.AAGGGCCGGAAGCCGCGGCCCCG cgcccggagcgccaaa.gccaaccaggccgagctggaga.acgcctactag.agcccgcccccggccg.agcccgcccccccgc

GCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTATATTTCATTGTAAATGCCTGC

AACCCAGGGCA6GGGGCTGAG.ACCTTCC.AGGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCA.GCCCGCCAGCT6

AGGGC-TCCCACGGGGCAGGGG.AGGG.-A.TTGAG.AGTCACAGACA.CTG.AGCCA.CGC.AGCCCCGCCTCTGGGGCCGCCTACCT

TTGCrGGTCCCA.C'¡'7CAGAGG.AGGCA.GAAATGGAA.GCA.TTTTC.ACCGCCCTGGGGTTTTAA.GGGA.GCGGTGTGGGAGTGG

GAAAGTCCAGGGACTGGTTAAGAAA.GTT3GATAAGA.TTCCCCCTTGCACCTCGCTGCCCA.TC.AGAAAGCCTGA.GGCGTGC

CCAGAGC.ACAAGACTGGGGGCAACTGTAGATGTGGTTTCTAGTCCTGGCTCTGCCACTAACTTGCTGTGTA.ACCTTG.A.AC

TA.CACAATTCTCCTTCGGGACCTCAA.TTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGAGACTAT

TGGCATA.TGA.TTCC.AAGGACTCCAGTGCCTTTTGAATGGGCAG.AGGTGAGA.GAGAGAGAGA.GAAAG.AGAGAGAA.TGAA.TG

CAGTTGCATTGA.TTCAGTGCCAAGGTCACTTCCAGAATTCAGA.GTTGTGA.TGCTCTCTTCTGACA.GCC.A.AA.GA.TGAAAAA

C.AAACACi.AAAAAAAAAA.GTAAAGAGTC7ATTTA, T6GCTGA.CATA.TTTA.CGGCTGA.CAAACTCCTGGAA.GAAGCTA.TGCTG

CTTCCC.AGCCTGGCTTCCCCGGA.TGTTTGGCTACCTCCACCCCTCCATCTCA.AAGAAATAACATCATCCATTGGGGTAGA aaaggagagggtccg.agggtggtggga.gggatagaa.atca.ca.tccgcccca? .Cttcccaaagagcagc.atccctcccccg .acccata.gccatgttttaaagtc.accttccgaagagaagtgaaaggttcaagg.acactggccttgcaggcccgagggagc agcc.atca.caaactc.aca.g.accagc.acatcccttttg.aga.caccgccttctgcccaccactcacggacaca.tttctgcct

AGAAAACA.GCTTCTTACTGCTCTTA.CA.TGTGATGGC.ATA.TCTTA.C.ACTAAAAGAATA.TT.ATTGGGGGAAAAACTACAAGT

GCTGTACA.TATGCTGAGAAACTGCA.GA.GCATAATAGCTGCCACCCAAAAA.TCTTTTTGA.AAATCA.TTTCCA.GACAACCTC

TTA.CTTTCTGTGTAGTTTTT.AATTGTTAAAAAAA.A¹AA.GTTTTAAACAG.AAGCACATGACATATGAAA.GCCTGCAGGAC7

GGTCGTTTTTTTGGCAA.TTCTTCCACGTGGGA.CTTGTCCACAA.GAATGAAAGTAGTGGTTTTT.AAAGAGTT.AAGTT.-.CAT

A.TTTATTTTCTCA.CTTAAGTTA.TTTA.TGCAAAAGTTTTTCTTGTAGAGAATGA.CAATGTTA.ATATTGCTTTA.TGAA.TTAA.

CA.GTCTGTTCTTCCAGA.GTCCA.GAGA.CATTGTTAATAAA.G.ACAA.TGAATC.ATG.ACCGAAA.G

SEQ ID NO. 4: Sclerosteosis Truncated Human BEER Protein

MQLFLALCLVCLLVHTAFRWEG *

ES 2 272 093 T3

SEQ ID NO. 5: Human BEER cDNA encoding Variant Protein (V10I)

A.GA.GCCTGTGCTA.CTGGAAGGTGGCGTGCCCTCCTCTGGCTGGTACC.ATGCAGCTCCC.ACTGGCCCTGTGTCTCATCTGC

CTGCTGGT.ACAC.AC.AGCCTTCCGTGTAGTGGAGGGCC.AGGGGTGGCAGGCGTTC.AAGAA.TG.ATGCC.ACGGAAATC.ATCCG

CGAGCTCGGAG.AGT.ACCCCGAGCCTCC.ACCGG.AGCTGGAGAACA.ACAAGA.CC.ATGAACCGGGCGGAGA.ACGGAGGGCGGC

CTCCCC.ACC.ACCCCTTTGA.GACCAAAG.ACGT6TCCG.AGT.AC.AGCTGCCGCGAGCTGCA.CTTC.RCCCGCT.ACGTGA.CCG.AT

GGGCCÜTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAACGC

CATCGGCCGCGGCAAGTGGTGGCGACCTAGTGGGCCCG.ACTTCCGCTGCATCCCCGACCGCTA.CCGCGCGCAGCGCGTGC

AGCTGCTGTGTCCCGGTGGTGAGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCA.CC

CGCTTCCA.CAACC.AGTCGGAGCTCAAGGACTTCGGGACCGAGGCCGCTCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCG

CGCCCGG.AGCGCCAAA.GCCAACC.AGGCCGAGCTGG.AGAACGCCT.ACTAGAGCCCGCCCGCGCCCCTCCCC.ACCGGCGGGC

GCCCCGGCCCTGAACCCGCGCCCCACATTTCTGTCCTC7SCGCGTGSTTTGATTGTTTATATTTCATTGTAAATGCCTGC

AACCCA.GGGCA.GGGGGCTGAGACCTTCCA.GGCCCTGAGGAATCCCGGGCGCCGGCAAGGCCCCCCTCAGCCCGCC.AGCTG

AGGGGTCCC.ACGGGGCA.GGGG.AGGGAATTGAGAGTC.ACAGACACTG.AGCCACGCA.GCCCCGCCTCTGGGGCCGCCTA.CCT ttgctggtcccacttcagaggaggcagaaatggaagcattttcaccgccctggggttttaagggagcggtgtgggagtgg gaaagtccagggactggttaagaaagttggataagattcccccttgcacctcgctgcccatcagaaagcctgaggcgtgc ccaga.gcacaagactgggggcaactgtagatgtggtttctagtcctggctctgccactaacttgctgtgtaaccttg.aac

t.ac.aca.attctccttcggg.acctc, aa.tttccactttgtaaaatgagggtgg.aggtgggaat.agga.tctcg.aggaga.cta.t tggcatatgattccaaggactccagtgccttttgaatgggcagaggtgagagagagagagaatgaatgagaga

CA.GTTGC.ATTG.ATTCAGTGCCAA.GGTC.ACTTCCAG.AA.TTC.AGAGTTGTGA.TGCTCTCTTCTG.AC.AGCC.AAAG.ATG.AAAAA

C.AAACAG.A.AAAA.AAAAAGTAAAGAGTCTATTTATGGCTGACAT.ATTTACGGCTGACAAACTCCTGG.AAG.AAGCTATGCTG cttcccagcctggcttccccggatgtttggctacctccacccctccatctcaaa.gaaat.aacatcatccattggggtag.a aaaggagagggtccgagggtggtgggagggat.agaaatcacatccgccccaacttcccaaagagcagcatccctcccccg acccatagcc.atgttttaaagtcaccttccgaagagaagtgaaaggttc.aaggacactggccttgcaggcccgagggagc

A.3CCATCA.CAAACTCACA.G.ACCAGCACATCCCTTTTGAX;, AC.ACCGC'TrTCTGCCCA.CCACTCACGGACAC.ATTTCTGCCT

a.gaaaacagcttcttactgctctta.catgtgatggcata.tcttacactaa.aa.gaatattattggggg.aaaaa.ctacaagt

GCTGT.ACATA.TGCTG.AG.AAACTGCAGA.GCATAATAGCTGCC.ACCCAAAA.ATCTTTTTGAAAA.TC.ATTTCCAGA.C.AA.CCTC tt.actttctgtgt.agtttttaattgtt.aaaa.aaaaaa.agtttt.acagacataca.gt.aaaaa.agtttt.

33TCGTTTTTTTGCC.AA.TTCTTCCA.CGTGGGA.CTTGTCC.ACA.AGAATGAAACTAGTGGTTTTTAAAG.AGTTAAGTT.ACA.T .ATTTATTTTCTC.ACTTA.AGTT.ATTT.ATGCAAAAGTTATACTCTAATGAAACTAGTGGTTTTTAAAG.AGTTAAGTT.ACA.T .ATTTATTTTCTC.ACTTA.AGTT.ATTT.ATGCAAAAGTTATACCTACTAATAGTAGTAGTAGTA

CA.GTCTGTTCTTCCA.GA.GTCCAGAG.ACATTGTTAATAAAGACA.ATGAATCATGA.CCGAA.AG

SEQ ID NO. 6: Human BEER Protein Variant (V10I)

LVASCKCKRLTR FHWQSELKDFGTEAARFQKGRKF Rt RARS.AKAIJQAE LENA. '. ·

ES 2 272 093 T3

SEQ ID NO. 7: Human Beer cDNA encoding the Variant Protein (P38R)

AG .-. GCCTGTGCTA.CTGGAAGGTGGCGTGCCCTCCTCTGGCTGGTA.CCA.TGCAGCTCCCA.CTGGCCCTGTGTCTCGTCTGC

CTGCTGGTACACA.CAGCCTTCCGTGTAGTGGAGGGCCAGGGGTGGCAGGCGTTCAAGAA.TGATGCCACGGAAA.TCA.TCCG

CGAGCTCGGA.GAGTA.CCCCGA.GCCTCCACCGGAGCTGGAGAA.CAACAA.GACCA.TGAACCGGGCGGAGAACGGAGGGCGGC

CTCCCCACCACCCCTTTGAGACCA.AAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTTCACCCGCTACGTGACCGAT

GGGCCGTGCCGCA.GCGCCAA.GCCGGTCACCGAGCTGGTGTGCTCCGGCCAGTGCGGCCCGGCGCGCCTGCTGCCCAA.CGC

CA.TCGGCCGCGGCAA.GTGGTGGCGACCTA.GTGGGCCCGACTTCCGCTGCATCCCCGACCGCTACCGCGCGCA.GCGCGTGC

A.GCTGCTGTGTCCCGGTGGTGA.GGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTCGTGCAA.GTGCAA.GCGCCTCACC

CGCTTCCACAA.CCAGTCGGAGCTCAA.GGACTTCGGGACCGA.6GCCGCTCGGCCGCAGAA.GGGCCGGAA.GCCGCGGCCCCG

CGCCCGGAGCGCCAAA.GCCAACCAGGCCGAGCTGGAGAACGCCTACTAGAGCCCGCCCGCGCCCCTCCCCACCGGCGGGC

GCCCCGGCCCTGAA.CCCGCGCCCCACATTTCTGTCCTCTGCGCGTGGTTTGATTGTTTA.TATTTCATTGTAAATGCCTGC aa.ccca.gggcagggggctgagaccttccaggccctgaogaa.tcccgggcgccggcaaggcccccctcagcccgccagctg aggggtcccacggggcaggggagggaattgagagtcacagacactgagccacgcagccccgcctctggggccgcctacct ttgctggtccca.cttcaga.ggaggcagaaatggaagcattttcaccgccctggggttttaagggagcggtgtggga.gtgg

GAAA.GTCCA.GGGACTGGTTAAGAAA.GTTGGATAAGATTCCCCCTTGCACCTCGCTGCCCATCAGAAAGCCTGAGGCGTGC ccaga.gcacaaga.ctgggggcaactgta.gatgtggtttctagtcctggctctgccatgatgaacttgact

TACAC.V.TTCTCCTTC5GGACCTCAATTTCCACTTTGTAAAATGAGGGTGGAGGTGGGAATAGGATCTCGAGGA.GACTAT

TGGCATA.TGATTCCAAGGA.CTCCAGTGCCTTTTGAATGGGCAGA.GGTGAGAGAGAGAGAGAGAGAAAGAGAGAGAGAATGAATG

C.AGTTGCA.TTGA.TTCA.GTGCCAA.GG ~ CA.CTTCCAGAA.TTCA.GA.GTTGTGA.TGCTCTCTTCTSA.CA.GCCAAAGA.TGAAAAA caaa.c.agaaaaaaaaaagtagaaa.gact.gatt.cattta.t. .ctcctggaa.gaagctatgctg cttccca.gcctggcttccccgga.tc-ttggctacctcca.cccctcca.tctcaaa.gaa®.taaca.tcatccattggggta.ga.

bb.bgGA.GAGGGTCCGA.GGGTGGTG3GAGGGATAGAAA.TCA.CA.TCCGCCCCAA.CTTCCCAAAGAGCAGCA.TCCCTCCCCCG

ACCCA.TA.GCCA.TGTTTT.AAAGTCA.CCTTCCG.A.AGA.GAA.GTGAAAGGTTCAA.GGA.CA.CTGGCCTTGCA.ÜGCCCGA.GGGA.GC

A, GCCATCAC.AAACTCA. TCTTTTTüAAAATCA.TTTCCA.GA.CA.ACCTC tta.ctttctgtgt.agtttttaa.tt37taaaaaaaaaaa.gttttaaa.ca.gaa.gcaca.tga.ca.ta.tgaaa.gcctgcagga.ct ggtcccagtttgatt. agttaa.gtta.ca.t

A.TTTA.TTTTCTCACTTAA.GTTATT7A.TGCAAAAGTTTTTC7TGTA.GAGAA.TGA.CAA.TGTTAA.TATTGCTTTATGAA.TTAA.

cagtctgttcttccagagtccagagacattgttaataaagacaatgaatcatgaccgaaag

SEQ ID NO. 8: Human BEER Protein Variant (P38R)

MQLFLA.LCLVCLLVHTA.FRWEGQGWQA.FKNDA.TEIIREbGEypEFFFELENNKTMMRA.ENGGRPFHHFFETKPVGEySC

RELHFTRYVTDGFCRSAKFVTELVC'GGQCGFARLuFHAIGRGKWWRFSGFDFRCIFDRyRAQRVQLLCFGGEAFRA.RKVR

LVA, ECKCKRLTRFHNQSELKDFGTEAA.RPQKGRKFRPRARSA.KAHOA.ELEHA.y

ES 2 272 093 T3

SEQ ID NO. 9: Vervet BEER cDNA (complete coding region)

ATGCAGCTCCCACTGGCCCTGTGTCTTGTCTGCCTGCTGGTACACGCAGCCTTCCG'rGTAGTGGAGGGCCAGGGGTGGCA ggccttcaagaatgatgccacggaaatcatccccgagctcggagagtaccccgacagcactccacc

AGACCATGAACCGGGCGGAGAATGGAGGGCGGCCTCCCCACCACCCCTTTGAGACCAAAGACGTGTCCGAGTACAGCTGC

CGAGAGCTGCACTTCACCCGCTACGTGACCGAtGGGCCGTGCCGCAGCGCCAAGCCAGTCACCGAGTTGGTGTGCTCCGG

CCAGTGCGGCCCGGCACGCCTGCTGCCCAACGCCATCGGCCGCGGCAAGTGGTGGCGCCCGAGTGGGCCCGACTTCCGCT

GCATCCCCGACCGCTACCGCGCGCAGCGTGTGCAGCTGCTGTGTCCCGGTGSTGCCGCGCCGCGCGCGCGCAAGGTGCGC

CTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGTCCCGAGGCCGC

TCGGCCGCAGAAGGGCCGGAAGCCGCGGCCCCGCGCCCGGGGGCCAAAGCCAATCAGGCCGAGCTGGAGAACGCCTACT

AG

SEQ ID NO. 10: Vervet BEER protein (complete coding sequence)

ΜΟΕΡΙΑ, ΕΟΕνθΕΙ.νΗΑΑΡΚννεθ0ΟΗ0ΑΓΚΝ0ΑΤΕΙΙΕΕΰ · 3ΕϊΡΕΡΡΕΕΕΕΜΝΚΤΜΝΡΑΕΙΊ6ΟΡΡΕΗΗΡΡΕΤΚΙ) ν3Εϊεθ

RELHFTRYVTDGECRSAKFVTELVG8GQCGFARLLPNAIGKGKWWRr33FPFRCIFDRYRAQRVQLLCFGGAAFRARKVR

LVASCKCMLTRFHNQSELKDFGEEAAREQKGRKPRFRARGAKANQAELENAY

SEQ ID NO. 11: Mouse BEER cDNA (complete coding region)

ATGCAGCCCTCACTAGCCCCGTGGCTCATCTGCCTACTTGTGCACGC77-CCTTCTG7GCTGTGGAGGGCCAGGGGTG-3CA.

TGAACCGGGCGGAGAATGGAGGCAGACCTCCCCACCATCCC7ATGACGCCAAAGGTGTGTCCGAG7ACAGCTGCCGCGAG

CTGCACTACACCCGCTTCCTGACAGACGGCCCATGCCGCAGCGCCA.AGCGGGTGACCGAG7TG'3TGTGCTCCGGCCAGTG

CG3CCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCG7GAAGTGGTGGCGCCCGAACGGACCGGATTTCCGGTGCA7CC cggatcgctaccgcgcgcagcgggtgcagct6CTgtgccccg5gggcgggcgtgtgccccg5gggcgggcgcg

GCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACGAG7CGGAGCTCAAGGACTTCGGGCCGGAGACC3CGCGGCC

GCAGAAGGGTCGCAAGCCGCGGCCCGGCGCCCGGGGAGCCAAAGCCAACCA &amp; GCGGAGCTGGAGAACGCCTACTAGAG

SEQ ID NO. 12: Mouse BEER protein (complete sequence)

MQFSLAFCLICLLVHAAFCAVEGQGWQAFRNDATEVIFGLGEYFEPFFENHQTMNRAENGGRFFHHFYDAKDVSEYSCRE

LHYTRFLTDGECRSAKFVTELVCSGQCGFARLLENAIGRVKWWRFMGEDFRCIFDRYRAQRVQLLCEGGAAFRSRKVRLV

ASCKCKRLTRFHHQSELKDFGFETARFQKGRKPRFGARGAKAHQAELEIIAY

ES 2 272 093 T3

SEQ ID NO. 13: Rat BEER cDNA (complete coding region plus 5 'UTR)

GAGGACCGAGTGCCCTTCCTCCTTCTGGCACCATGCAGCTCTCACTAGCCCCTTGCCTTGCCTGCCTGCTTGTACATGCA

GCCTTCGTTGCTGTGGAGAGCCAGGGGTGGCAAGCCTTG4AGAATGATGCCACAGAAATCATCCCGGGACTCAGAGAGTA

CCCAGAGCCTCCTCAGGAACTAGA.GAACAACCAGACCATGAACCGGGCCGAGAACGGAGGCAGACCCCCCCACCATCCTT

ATGACACCAAAGACGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCGTGACCGACGGCCCGTGCCGCAGT

GCCAAGCCGGTCACCGAGTTGGTGTGCTCGGGCCAGTGCGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGTGAA

GTGGTGGCGCCCGAACGGACCCGACTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCG

GCGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAG

7CGGAGCTCAAGGACTTCGGACC? 5AGACGGCGCGGCCGCAG.AAGGGTCGCAAGCCGCGGCCCCGCGCCCGGGv .-. 3 - A.

AGCCAACGAGGCGGAOCTGGAGAACGCCTACTAG

SEQ ID NO. 14: Rat BEER protein (complete sequence)

PQLSLAFCLACLLVHAAFVAVESQGWQAFk'HPATEI IFGLREYEEF FQELENMQTMIIRAEHGGRF FHHFYDTKD.'íEY /. ' REUlYTRFVTüGFCRSAKFVTELVCSGQCGEARLLFtlAIGRVKFWRFNGFDFRCIEORYRAQRVQLLCEGGAAEñSRKVA LVASCKCKR LTR F HMQSE LKDFGF ETAR PQKGRKTIQ RFAELGAKHAY

SEQ ID NO. 15: Bovine BEER cDNA (partial coding region)

<img file="ES2272093T3_D0001.tif" />

AT.SGCA.OAGAAATC.ATCCCCGAGCTGGGCGAGTACCCCGAGCCTCTGCCAGAGCTGAACAACAAGACCATjAAC ¿vv'iAACGGAGGGAGACCTCCCCACCACCCCTTTGAGACCAAAGACGCCTCCGAGTACAGCTGCCGCAGGAGG

CÍGCTACÜTÜACCGATGGGCCGTGCCGCAGCGCCAAGCCGGTCACCGAGCTGGTGTGCTCGGGCCAGTGCGGCC

GCCTGCTGCCCAA.CGCCATCGGCCGCGGCAAGTGCTGGCGCCCAAGCGGGCCCGACTTCCGCTGCATCCCCGAC

CGCGCCÍCAGCGGGTGCAGCTGTTGTGTCCTGGCGGCGCGGCGCCGCGCGCGCGCAAGGTGCGCCTGGTGGCCTC

3T ~ A \ ". TVAAGCC.CCTCACTCGCTTCCACAACCAGTCCGAGCTCAAGGACTTCGGGCCCGAGGCCGCGCG3CCGCAAA

3G - .; CCGGA. '".- TGCGGCCCCGCGCCCGGGGCACCAAAGCCAGCCGGGCCGA

SEQ ID NO. 16: Bovine BEER protein (partial sequence - missing signal sequence and last 6 residues)

ÍIDATF.I i FELGEYEEELFELNi'lKTMNRAENGGRPPHHFFETKOASEYSCRELHFTRYVTDGFCRSAKPVTELVCSGQCGF ARI.l.FMAIGRGKWWRF'SGFDFRCI FDRHNYR.AKRVCFRKLCRGAVRLAKRVCFRKLCRGARLQAQRVCFRKCVGARLFKASDAREFARKLCRGAVRLAQRVQLKCVGA

GRKLR F RASGTKASKA

ES 2 272 093 T3

SEQ ID NO. 17: MliI-AvlII Restriction Fragment used to make mouse Beer transgene

<img file="ES2272093T3_D0002.tif" />

GA.CCGA.GCTGGA.CCAGCGCA.TTCGTGACACCGTCTCCTTCGAACTTATTCGCAATGuA.GTGTCATTC.ATCAAGGACNGCC

TGA.TCGCAAATGGTGCTA.TCCACGCAGCGGCAATCGAAAACCCTCAGCCG3TGACCAA.TA, TCTACAA.CA.TCAGCCTTGGT

a.tcctgcgtgatgagccagcgcagaacaaggtaa.ccgtcagtgccga.taagttcaaa.gttaaacctggtgttgataccaa cattgaaacgttgatcgaaaacgcgctgaaaaacgctgctgaatgtgcggcgctggatgtca.caaagcaaa.tggcagcag ac.aag.aaagcga.tgga.tgaactggcttcctatgtccgcacggccatca.tg.atgg.aatgtttccccggtggtgtt.atctgg cagcagtgccgtcgatagtatgcaattga.taa.tta.tta.tcatttgcgggtcctttccggcgatccgccttgttacggggc

GGCGACCTCGCGGGTTTTCGCTATTTA.TGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCA.TAACTTAATGT

TTTTA.TTTAAAATACCCTCTGAAAAGAAAGGAAACGACA.GGTGCTGAA.AGCGA.GCTTTTTGGCCTCTGTCGTTTCCTTTC

TCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCAACAAAAAGCAGA.GCT7A.TCGATGATAAGCGGTCAAACATGA.GAAT

TCGC5GCC5CATAATACGACTCA.CTA.TA.GGGA.TCGACGCCTA.CTCCCC5CGCA.TGAAGCGGA.GGA.GCTGGA.CTCCGCA.TG

CCCAGAGACGCCCCCCAACCCCCAAA.GTGCCTGACCTCA.GCCTCTACC .-. 5CTCTGGCTTGGGCTTGGGCGGG5TCAAGGC

TACCACGTTCTCTTAACAGGTGGCTGGGCTGTCTCTTGGCCGCGCGTCATGTGACA.GCTGCCTAGTTCTGCAGTaA.GGTC

ACCGTGGAA.TGTCTGCCTTCGTTGCCA.TGGCAACGGGA.TGA.CGTTA.CAATCTGGGTGTGGAGCTTTTCCTGTCCGTGTCA

GG.AAATCCAAATA.CCCTAAAATACCCTAGAAGAGGAAGTAGCTGA.GCCAAGGCTTTCCTGGCTTCTCCAGATAAAGTTTG acttagatggaaaaaaacaaaatgataaagacccgagccatctgaaaattcctcctaa.ttgcaccactaggaaatgtgta tattattgagctcgtatgtgttctta.ttttaaaaagaaaactttagtcatgttattaat.aagaatttctcagcagtggga

GAGAACCAA.TATTAACACCAAGA.TAAAAGTTGGCATGA7CCACATTGCAG5AAGATCCA.CGTTGGGTTTTCATGAATGTG

AAGACCCCATTTA7TAAAGTCCTAAGCTCTGTTTTTGCACACTAGGAAGCGATGGCCGGGA.TGGCT3AGGGGCTGTAA.GG

ATCTTTCAATGTCTTACATGTGTGTTTCCTGTCCTGCACCTAGGACCTGCTGCCTAGCCTGCAGCAGAGCCAGAGGGGTT

TCACATGATTAGTCTCAGACACTTGGGGGCAGGTTGCATGTACTGCATCGCTTATTTCCATACGGAGCACCTACTATGTG

TCAAACACCATATGGTGTTCACTCTTCAGAACGGTGGTGGTCA.TCA.TGGTGCA.TTTGCTGACGGTTGGATTGGTGGTAGA.

GAGCTGAGATATATGGACGCA.CTCTTCAGCATTCTGTCAACGTGGCTGT5CA.TTCTTGCTCCTGAGCAAGTGGCTAAA.CA

GACTCACAGGGTCAGCCTCCA.GCTCA.GTCGCTGCATA.GTCTTAGGGAACCTCTCCCAGTCCTCCCTA.CCTCAACTATCCA

A.GAAvCCAGG3GGCTTGGCGGTCTCAG5A.GCCTGCTTGCTGGGGGACA.GGTTGTTGAGTTTTATCTGCAGTAGGTTGCCT

AGSCATA.GTGTCAGGACTGATGGCTGCCTTGGAG.AACA.CATCCTTTGCCCTCTATGCAAATCTGACCTTGACATGGGGGC

GCTGCTCAGCTGGGAGGATCAACTGCATACCTAAAGCCAAGCCTAAAGCTTCTTCGTCCACCTGAAACTCCTGGACCAAG

GGGCTTCCGGCACATCCTCTCAGGCCAGTGAGGGAGTCTGTGTGAGCTGCACTTTCCAATCTCAGGGCGTGAGAGGCAGA

GGGAGGTGGGGGCAGAGCCTTGCAGCTCTTTCCTCCCATCTGGACAGCGCTCTGGCTCAGCAGCCCATATGAGCACAGGC

ACA.TCCCCACCCCACCCCCACCTTTCCTGTCCTGCAGAATTTAGGCTCTGTTCACGGGGGGGGGGGGGGGGGGCAGTCC

TATCCTCTCTTAGGT.AGACAGGACTCTGCA.G3AGA.CACTCCTTTGTAAGATACTGCA.CTTTAAA.TTTGGA.TGTTGTGAGG

GGAAAGCGAAGGGCCTCTTTGA.CCATTCAGTCAAGGTACCTTCTAACTCCCA.TCGTATTGGGGGGCTACTCTAGTGCTAG

ACATTGCAGAGAGCCTCAGAACTGTAGTTACCAGTGTGGTA.GGA.TTGATCCTTCA.GGGAGCCTGACATGTGACAGTTCCA.

TTCTTCACCCAGTCA.CCGAACATTTA.TTCAGTACCTA.CCCCGTAACA.GGCACCGTAGCAGGTACTGAGGGA.CGGACCACT

CAAAGAACTGACAGACCGAAGCCTTGGAA.TA.TAAACA.CCAAAGCATCAGGCTCTGCCAACAGAACACTCTTTAACACTCA

GGCCCTTTAACACTCAGGACCCCCA.CCCCCACCCCAAGCAGTTGGCA.CTGCTA.TCCACA.TrTTA.CAGAGAGGAAAAACTA ggcacaggacgat.ataagtggcttgcttaagcttgtctgcatccaggtAXatggcagggctggatcattgacccaga

ES 2 272 093 T3

ACTCTA.GGGTCTATTTT7CTTTTTTCTCGTTGTTCGAATCTGGGTCTTACTGGGTAAACTC.AGGCTAGCCTCACACTCAT

ATCCTTCTCCCATGGCTTACGAGTGCTAGGATTCCAGGTGTGTGCTACCATGTCT5ACTCCCTGTAGCTTGTCTATACCA

TCCTCACAACATAGGAATTGTGATAGCAGCACACACACCCGAAGGAGCTGGGGAAATCCCACAGA.GGGCTCCGCAGGATG

ACAGGCGAATGCCTACACAGAAGGTGGGGAAÜGGAAGCAGAGGGAACAGCATGGGCGTGGGACCACAAGTCTATTTGGGG

AA3CT3CCGGTAACCGTATATGGCTGGGGTGAGGGGAGAGGTCATGAGATGAGGCAGGAAGAGCCACAGCAGGCAGCGGG

TACGGGCTCCTTATTGCCAAGA.GGCTCGGATCTTCCTCCTCTTCCTCCrrCCGGGGCTGCCTGTTCATTTTCCACCACTG

CCTCCCATCZC.AG'GTCTGTGGCTCAGGACATCACCCAGCTGCAGAAACTGGÍV / ATCACCCACGTCCTG.AA.TGCTGf.-CGAGG

SCAGOTCCTTCATGCACGTCAACACCAGTGCTAGCTTCTACGAGGATTtrrüüCATCACCTACTTGGGCATGAAGGCCAAT

GA.TACGCAGGAGTTCAACCTCAGTGCTTACTTTGAAAGGGCCA.CAGATTTC.ATTG.ACCAGGCGCTGGCCCATAAAAATGG

7.ii. ;;. 5.ii.BCGTACATTCCGGCACCCATGGAGCGTA.AGCCCTCTGGGACCTGCTTCCTCC.®A.AGAGGCCCCCACTTGAA^AND.^toTO

GGTTCC.AGAAAG.ATCCCAA.AATATGCCACC.^toACTAGGGATTAAGTGTCCTACATGTGAGCCGATGGGGGCCACTGCATAT

AGTCTGTGCCATAGACATGACAATGGATAAT.AAT.ATTTCAGACAGAGAGCAGGAGTTAGGTAGCTGTGCTCCTTTCCCTT

TAATTG.AGTGTGCCCATTTTTTTATTCATGTATGTGTATACATGTGTGTGC.ACACATGCCATAGGTTG.ATACTGAACACC

STCTTC.AATCGTTCCCCACCCCACCTTATTTTTTGAGG &amp; AGGGTCTCTTCCCTGATCCTGGGGCTCATTGGTTTATCTAG

GCTCZrrüGCCAGTGAGCTCTGGAGTTCTGCTTTTCTCTACCTCCCTAGCCGTGGGACTGCAGGGGC.ATGTGCTGGGCCAG

3CTTTTATGTCGCGTTGGGGATCTGAACTTAGC? RCCCTAGGCCTGAGCACCGTAAA5ACTCTGCCACATCCCCAGCCTGT

TTGAGCAAGTGAACCATTCCCCAGAATTCCCCCAGTGGGGCTTTCCTACCCTTTTATTGGCTAGGCATTCATGAGTGwTC

ACCTCGCCAGAGG.AATGAGTGGCCACGACTGGCTCAGGGTCAGCAGCCTAGAGATACTGGGTTA^to.3TCTTCCTGCCGCTC ·

GCTCCCTGCAGCCGCAGACAGAAAGTAGGACTGAATGAGAGCTGGCTAGTGGTCAGACAGGACAGAAGGCTGAGAGGGTC acagggcagatgtcagcagagcagacaggttctccctctgtgggggaggggtggcccactgcaggtgtaattggccttct ttgtgctcc.atagaggcttcctgggt.acacagcagcttccctgtcctggtgattcccaaagagaa.ctccctaccactgga cttacag.aagttctattgactggtgtaacggttcaacagctttggctcttggtggacggtgcatactgctgtatcagctc aagagctcattcacg.aatg.aacacacacacacacacacacacacacacacacaca.agctaattttgatatgccttaacta

GCTCAGTGACTGGGCATTTCTGAACATCCCTGAAGTTAGCACACATT'rCCCTCTGGTGTTCCTGGCTT.AACACCTTCTAA

ATCTATATTTTATCTTTGCTGCCCTGTTACCTTCTGAGAAGCCCCTAGGGCCACTTCCCTTCGCACCTACATTGCTGGAT

GGTTTCTGTCCTGCAGCTCTTAAATCTGATCCCTCTGCCTCTGAGCCATGGGAACAGCCCAATAACTGAGTTAGACATAA

AAACGTCrCTAGCCAA^toACTTCAGCTAAATTTAGACAATAAATCTTACTGGTTGTGGAATCCTTAAGATTCTTCATGACC tccttcacatggcacgagtatgaagctttattacaattgtttattgatcaaactaactcataaaaagccagttgtctttc

ACCTGCTCAAGGAAGGAACAAAATTCATCCTTAACTGA.TCTGTGCACCTTGCACAATCC.ATACGAATATCTTAAGAGTAC taagattttggttgtgaoagtcacatgt-tacagaa.tgtacagctttgacaaggtgcatccttgggatctgccgactattgactgcatgacttgggatttgccgactagtgcatgcatgactggatttgccgactagtgcatgcatgactggatttgccgactagtccag

AGCGGGTCACTTCAGCATCCCGATGACGAATCCCGTCAAAGCTGTACATTCTGTAACAGACTGGGAAAGCTGCAGACTTT

AAGGCCAGGGCCCTATGGTCCCTCTTAATCCCTGTCACACCCAACCCGAGCCCTTCTCCTCCAGCCGTTCTGTGCTTCTC

ACTCTGGATAGATGGAGAACACGGCCTTGCTAGTTAAAGGAGTGAGGCTTCACCCTTCTCACATGGCAGTGGTTGGTCAT

CCTCATTCAGGGAACTCTGGGGCATTCTGCCTTTACTTCCTCTTFTTGGAeTACAGGGAATATATGCTGACTTGTTTTGA

CCTTGTGTATGGGGAGACTGGATCTTTGGTCTGGAATGTTTCCTGCTAGTTTTTCCCCATCCTTTGGCAAACCCTATCTA

ES 2 272 093 T3

TATCTTACGACTAGGCATAGTGGCCCTCGTTCTGGAGCCT-íCCTTCAGGCTGGTTCTCGGGGACCATGTCCCTGGTTTCT

CCCCAGCA.TATG3TGTTCACAGTGTTCACTGCGGGTGGT7GCTGAACAAAGCGGGATTGCATCCCAGAGCTCCGGTGCC

TTGTGGGTACACTGCTAAGATAAAATGGATACTGGCCTCTCTCTGACCACTTGCAGAGCTCTGGTGCCTTGTGGGTACAC

TGCTAAGATAAAATGGATACTGGCCTCTCTCTATCCAC7TGCAGGACTCTAGGGAACAGGAATCCATTACTGAGAAAACC

AGG &lt;K5CTAGGAGCAGGGAGGTAGCTGGGC.AGCTGAA3TGCTTGGCGACTAACCAATGAATACCAGAüTTTGOATCTCTAG

AATAGTCTTAAAATCTGGGTGGGCAGAGTGGCCTGCCTGTAATCCCAGAACTCGÜGAGGCGGAGACACKíGAATCATCAGA

GCAAACTGGCTAACCAGAATAGCAAAACACTGAGCTCTGGGCTCTGTGAGAGATCCTGCCTTAACATATAAGAGAGAGAA

TAAAACATTG.AA-3AAGACAGTA3ATGCCAATTTTAAGCCCCCACATGCACATGGACAAGTCTCCGTTTGA.AC:ACACATAT

GCACTCATGTGAACCAGGCATGCACACTCGGGCTTATCACACACATAATTTGAAAGAGAGAGTGAGAGACWAGAGTSCAC attagagttcacaggaaagtgtgagtgagcacacccatgcacacagacatgtgtgccagggagtaggaaaggagcctggg

TTTGTGTAT.A “.G .-. GGGAGCCATC .-. TGTGTTTCTAAÜG .-. G ~, GCGTGTG.ⁿAGGAGGC3TTGTGTGGGCTG3GACTGGAGCA7

GGTTGT.AACTGAGCATGCTCCCTGTGGGAAACAGGAGGGTGGCCACCCTGCAGAGGGTCCCACrTGTCCAGCGGGATCAGT

AAAAGCCCCTGCTGAGAACTTTAGGTAA.TAGCCAGAÍw3AGAAAC &lt;iTAGGAAAGTG &lt;»CGGACTCCCATCTCTGATGTAG

GAGGATCTGGGCAAGT.AGAGGTGCGTTT &amp; AGGTAGAAAGAGGGOTGCAGAGGAGATGCTCTTAATTCTvGGTCAGCAGTT

TCT7TCCAAATAATGCCTGTGA3GAGGTGTAGGTGGTGGPCATTCACTCAC7CAGCAGAGGGATGATGATGCCCGGTGGA

TGCTGG? AATGGCCGAGCATC.AACCCTGGCTCTGG.AAGAACTCCATCTTTCAG.iAGGAGAGTGGATCTG7GTATGGCCAG

CGGGGTCACAGGTGCTTGGGGCCCCTGGGGGACTCCTAGCACTGGGTGATGTTrATCuAGTG-JTCTTGTGTGCCACKíCAC

TGGCCTGGGGCTTTGTTTCTGTCTCTGTTTTGTTTCGTTTTTTGAGACAGACTCTTGCTATGTATOCGTGTCAATCTTGG

A.ATCTCACTGCA.TAGCCCAGGCTGCGGA.GAGAGGGGAGGGCAATAGGCCTTGTAAGCAAGCCACACTTCAG.AGACTAGAC

TCCACCCTGCGAATGATGACAGGTCAGAGCTGAGTTCCGGAAGATTTTTTTCCAGCTGCCAGGTGGAGTGTGGAGTGGC

AGCTAGCGGCAAGGGTAGAGGGCGAGCTCCCTGTGCAGGAGAAATGCAAGCAAGAGATGGCAAGCCAGTGAGTTAAGCAT tctgtgtggggagcaggtggatgaagagagaggctgggctttcgcctctggggggggggtgaggtgag

GAGGAGGGCAGCTCCCTGCAGTGTGATGAGATTTTTCCTGACAGTGACCTTTGGCCTCTCCCTCCCCCACTTCCCTTCTT

TCCTTTCTTCCCACCATTGCTTTCCTTG7CCTTGACAAATTCTGAGTTTCCACTTCACTGGTGATGCAGACG3AAACAGA

AGCCGTGTGTGTGTGTG7GTG7GTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGTATGTGTGTGTGTGTGTTTGTGT

GTATGTGTGTCAGTGCGAATGGCTCATAGTCTGCAGGAAGGTGGGCAGGAAGGAATAAGCTGTAGGCTGAGGCAGTGTGG gatgcagggagagaggagaggagggataccagagaaggaaattaagggagctacaagagggcattgttgggtgt

TGTGTGTGTTGTTTATATTTGTATTGGAAATA.CATTCTTTTAAA.AAATACTTATCCA.TTTATTTATTTTTATGTGCACGT

GTGTGTGCCTGCATGAGTTCATGTGTGCGACGTGTGTGCGGGAACCCTTGGAGGCCACAAGGGGGCATCTGATCCCCTGG

AACTGGAGTTGGAGGAGGTTGTGAGTCCCCTGApATGTTTGCTGGG.AA.CTGAACCCCGGTCCTATGCAAGAGCAGGAAGT

GCAGTTATCTGCTGAGCCATCTCTCCAGTCCTGAAATCCATTCTCTTAAAATACACGTGGCAGAGACATGATGGGATTTA

CGTATGGATTTAATGTGGCGGTCATTAAGTTCCGGCACAGGCAAGCACCTGT.AAAGCCATCACCACAACCGCAACAGTGA

ATGTGACCATCACCCCCATGT'TCTTCATGTCCCCTGTCCCCTCCATCCTCCATTCTCAAGC.ACCTCTTGCTCTGCCTCTG

TCGCTGGAGAACAGTGT6CATCTGCACACTCTTATGTCAGTGAAGTCACACAGCCTGCACCCCTTCCTGGTCTGAGTATT

TGGGTTCTGACTCTGCTATCA.CACAC7ACTGTACTGCATTCTCTCGCTCTCTTTTT7TAAACATATTTTTATTTGTTTGT gtgtatgcacatgtgccacatgtgtacagatactatggaggccagaagaggcgatggccgtccctggacagctggaggag

ES 2 272 093 T3

GGGAGCGTGTGAGC7GCG7GGTG7GGGTGC7GGGA.ACCA.-AC77GA. *. 7GTAA.AGG¹AGCACTT7TAACTGCTüAGG '-' AG · .:

TCTGAGTACCCTTCTTCAT7TCTCCGCC7GGGTTCCATTvTA.7GGACACATG7A.GCT.AGAATATCTTGCTTA7CTAA77A

TGTACATTGTTTrGTGCTAAGAGAGAGTAATGCTCTATA.GCCTGAGCTGGCCTCA .-. CCTTGCCATCCTCCTGCCTCAGGC

TCCTCCTCCTGAGTGCTAGGATG.ACAGGCGAGTGGTAA.CTTAC.ATGGTTTCATGTTTTGTTQAAGACTGAAGGATAACAT

TGA7AGAGAGAAGG7CTGG3TG^to.CAAAGTüTGCAGTTGAC7G.AATGGGACAACCCGT3.ATCAAGAAAGAAAACTG-.GGG ·? CTGGAGAGATGGCA.CTGACTGCTCTTCCAGA.GGTCCGS.RGTTCAATTCCCAGC.AACCACCAT.ATGGTAGGGCA.

TAACGAGATCTGACGCCCTCTTCTGGTGTüTCTGAAGAC.AGCTAGAGTGTA.CTCACATAAAATAAAT.AAATCTTTAAAAG

ACAGACACACACACAATTACCACCCCAGAAAwCCCACTCCATGTTCCCTCCCACGTGTCTGCCTACAGTACTCGCAGGaccact-sttcaggcttctaagaacctggtttacttgggcctcttttctgctctgtggagccacacatttgtgtgccat

ACACGTTCT7TCTAGTAA.GT7GCATATTACTC75CGTTT7TACATGTATTTATTTA7TGTAGTTGTGTGTGCGTG7GGGC

CCA7GCATGGCACAGTGTGTGGGGATGTCAGAG7AT7G7GAACAGGGGACAG77C7T7TCT7CAATCATGTGGGTTCCAG

AGG7TGAAC7CAGGTCA.TCATGTGTGGCA.GCA.AATGCG777A.CCCA.CTGAGA.CA72TGCATATTC7TTTT7TTTCCCC7G

AGG7GGGGGCTTGTTCCA7.AGCCCAAACTGGCTTTGCAC77GCA.GT?CAAAG7GAC7CCCTGTCTCCA';C7CTTAGAG7A

77G5AA7TAGGA7G7G7AC7ACCACACCTGACTGGA7CAT7AAT7G77TGATGGGGGCGGGG.^sAGGGCACATGCTGCA «

7GAAGGGATGAC7GGACTGGACATGAGCGTGGAAGCCAGAGAACAGC7TCAGTC7AATGCTC7CCCAA.C7GAGCTAT77C

GGT7TGCCAGAGAACAACTTACAGAAAGTTCTCAGTGí: CATGTGGA.TTCGGGGTTGGAGTTCAACTCATCAGCTTGACAT

TGGCTCCTCTACCCAC7GAGCCTTC7CACTACTCTCTACCTAGA7CA ?? AATTCTTTTTTA.ⁿTO.^to.AGACTTAT7AGGGGGC

7GGAGAGATGGCTCAGCCGTTAAGAGCACCGAATGCCC7TGCAGAGGTCC7GAGTTCA.ATTCCGAGCA7GCCA77GC7GG

ÜCAGTAGGGGGCGCAGGTG7TCAACGTGAG7AGCTGTTGCCAGTTTTCCGCGGTGGAGAACC7CTTGACACCCTGCTG?

CCTGG7CATTCTGGG7GGGTGCATGGTÜA7ATGCT7GTTG7ATGGAASACT77GACTGTTACAGTGAAG7TGGSC77CCA

CAGTTACCACGTCTCCCCTGTTTCTTGCAGGCCGGG7GC7TG7CCAT7GCCGCGAGGGC7ACAGCCGCTCCCCAACGC7A

GTTATCGCCTACCTCATGATGCGGCAG.AAGATGGACGTCAAGTCT3CTCTGAGTACTGTGAGGCAGAA7CGTGAGATCGG

CCCCA^toCG^to7GGCTTCCTGGCCCA^to.CTCTGCG^to.GCTCRATGACAGACTAGCG^toAGGAGGGGAAGGTGAAACTCTAGGGTG

CCCACAGCCTCT77TCCAÜAGGTCT3ACTGGGAGGGCCC7GGCAGCCATGT7TAGG.AAACACAGTATACCCAC7CCCTGC

ACCACCAGACACGTGCCCACATCTGTCCCACTCTGGTCCTCGGGGGCCACTCCACCCTTAGGGAGCACATGAAGAAGC7C

CCT.RAGAAGT7C7GCTCC7TAGCCATCCT7TCCTGTAATTTATGTCTCTCCCTGAGGTGAGGTTCAGGTT7A7GTCCC7G

TCTGTGGCATAGA7ACATCTCAGTGACCCAGGGTGGGAGGGCTATCAGG3TGCATGGCCCGGGACACGGGCAC7CTTGAT

GACCCCTCCCCC.ACCTGGG7TC77CCTGTGTGG7CCAGAACCACGAGCC7GGTAAAGGAACTA7GCAAACACAGGCCCTG

ACCTCCCCATG7CTGT7CCTGGTCCTCACAGCCCGACACGCCCTGCTGAGGCAGACGAATGACATTAAGTTCTGAAGCAG

AGTGGAGATA.GA7TAGTGACTAGA77TCCAAAAAGAAGGAAAAAAAAGGCTGCATTTTAAAATTATTTCCTTAGAATTAA

AGATACTAGATAGGGGCCCTTGGG7AAGCAAATCCATTTTTCCCAGAGGCTAfCTTGATTCTTTGGAATG7TTAAAGTGT

GCCTTGCCA.GAGAGCT7ACÜATCTATA7C7GCTGCTTCAGAGCCTTCCC7GAGGATGGCTCTGTTCCTTTGC7TGTTAGA

AGAGCGATGCCTTGGGCAGGGTTTCCCCCTTTTCAGAATACAGGGTGTAAAGTCCAGCCTATTACAAACAAACAAACAAA

CAAACAAACAAAGGACCTCCATTTGGAGAATTGCAAGGATTTTATCCTGAATTATAGTGTTGGTGAGTTCAAGTCATCAC

GCCAAG7GCTTGCCA7CC7SG77GC7ATTCTAAGAA7A^to.TTAGGAGGA3GAACC7AGCCAATTGCAGCTCATGTCCG7GG

GTGTGTGCACGGGTGCATA7GTTGG.AAGGGGTGCCTGTCCCCTTGGGGACAGAAGGAAA^to.TGAAAGGCCCCTCTGCTCAC

ES 2 272 093 T3

CCTGGCCAT7TACGGGAGGCTCTGCTGGTTCCACGGTGTC? GTGCAGGATCCTGAAA.CTGACTCGCTGGACAGA^BACGAG acttggcggcaccatgagaatggagagagaga.gagcaaagaaagaaaca.gcctttaaaagaactttct.aagggtggtttt tgaacctcgctggaccttgtatgtgtgcacatttgccagagattgaacataatcctcttgggacttcacgttctcattat ttgtatgtctccggggtcacgcagagccgtcagccaccaccccagcacccggcacataggcgtctcataaaagcccattt tatgagaaccagagctgtttgagtaccccgtgtatagagagagttgttgtcgtggggcacccggatcccagcagcctggt

TGCCTGCCTGTA5GATGTCTTACAC4GAGTTTGCAGAGAAACCTTCCTTGGAGGG.AV.GAAATATCAGGGATTTTTGTTGA

ATATTTCAAATTCAaCTTTAAGTGTAAGACTCAGCAGTGTTCATGGTTAAGGTAAGGAACATGCCTTTTCCAGAGCTGCT

GCAAGAGGCAGGAGAAGCAGACCTGTCTTAGGATGTCACTCCCAGGGTAAAG.ACCTCTGATCACAGCAGGAGCAGAGCTG

TGCAGCCTGGATGGTCATTGTCCCCTATTCTGTGTGACCACAGCAACCCTGGTCACATAGGGCTGGTCATCCTTTTTTTT

TTTTTTTTTTTTTTTTTTGGCCCAGAATGAAGTGACCATAGCCAAGTTGTGTACCTCAGTCTTTAGTTTCCAAGCGGCT

CTCTTGCTCAATACAATGTGCATTTCAAAATAACACTG7AGAGT7GACAGAACTGGTTCATGTGTTATGAGAGAGGAAAA

GAGAGGAAAGAACAAAACAA.AACAAAACACCACAAACCAAAAACATCTGGGCTAGCC.AGGCATGATTGCAA.TGTCTACAG

GCCCAGTTCATGAGAGGCAGAGACAGGAAGACCGCCGAAAGGTCAAGGATAGCATGGTCTACGTATCGAGACTCCAGCCA

GGGCTACGGTCCCAA &amp; ATCCTAGGTTTTGGATTTTGGGCTTTGGTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTG

TCCTGGAACTCGCrCTGTAGACCAGGCTGGCCTCAAACTTAGAGATCTGCCTGACTCTÜCCTTTGAGGGCTGGGACGAAT

GCCACCAC7GCCCAACTAAGATTCCATTAAAAAAAAAAAAAGTTCAA5ATAATTAAGAGTTGCCAGCTCGTT.AAAGCT.AA

GTAGAAGCAGTCTCAGGCCTGCTGCTTGAGGCTGTTCTTGGCTTGGACCTGAAATCTGCCCCCAACAGTGTCCAAGTGCA

CATGACTTTGAGCCATCTCCAGAGAAGGAAGTGAAAATTGTGGCTCCCCAGTCGATTGGGACACAGTCTCTCTTTGTCTA

GGTAACACAT3GTGACACATAGCATTGAACTCTCCACTCTGAGGGTGGGTTTCCCTCCCCCTGCCTCT7CTGGGTTGGTC

ACCCCATA.ÜGACAGCCACAGGACAGTCACTAGCACCTACTGGAAACCTCTTTGTGGGAACATGAAG.AAAGAGCCTTTGGG

AGATTCCTGGCTTTCCATTAGGGCTGAAAGTAC.AACGGTTCTTGGTTGGCTTTGCCTCGTGTTTATAAAACTAGCTACTA

TTCTTCAGGTAAAATACCGATGTTGTGGAAAAGCCAACCCCGTGGCTGCCCGTGAGTAGGGGTGGGGTTGGGAATCCTG

GATAGTGTTCTATCCATGGA »AGTGGTGGAATAGGAATTAAGGGTGTTCCCCCCCCCCCCAA.CCTCTTCCTCAGACCCAG ccactttctatgacttataaacatccaggtaaaaattacaaacataaaaatggtttctctggctctcaatttgtctattaatggtttctctggctgactttgtctattgtcattgtcactggtgactgattgattgattgttgttg

a.actcttggcccatactaaggctcttctgggctccagcactcttaagttattttaagaattctcacttggcctttagcac acccgccacccccaagtgggtgtggataatgccatggccagcaggggücactgttgaggcgggtgcctttccaccttaag ttgcttatag7atttaagatgctaaatgttttaatcaagagaa.gca.ctgatcttataatacgaggat. ° agagattttctc acaggaaattgtctttttcataa.ttcttttacaggctttgtcctgatcgtagcatagagagaatagctggatatttaact tgtattccattttcctctgccagcgtta.ggttaactccgtaaaaagtgattcactggaccgaagaggctcagagggcagg ggatggtggggtgaggcagagcactgtcacctgccaggcatgggaggtcctgccatccgggaggaaaaggaaagtttagc ctctagtctaccaccagtgttaacgcactctaaagttgtaaccaaaataaatgtcttacattacaaagacgtctgttttg tgtttccttttgtgtgtttgggctttttatgtgtgctttataactgctgtggtggtgctgttgttagttttgaggtagga tctcaggctggccttgaacttctgatcgcctgcccctgcccctgcccctgcccctgtccctgcctccaagtgctaggact

AAAAGCACATGCCACCACACCAGTACAGCATTTTTCTAACATTTAAÁAATAATCACCTAGGGGCTGGAGAGAGGGTTCCA

GCTAAGAGTGCACACTGCTCTTGGGTAGGACCTGAGTTTAGTTCCCAGAACCTATACTGGGTGGCTCCAGGTCCAGAGGA

ES 2 272 093 T3

TCCAGGACCTCTGGCCTCCATGG37ATCTC &lt;TC7TAG7ACATACCCACATACAGATACACAr; ATAAA^toATA^toAAT'3AAGC

CTTTAA^toAACCTCCTAAAACCTAGC-'CTTGGA3GTAC3ACTCTGGA^toAGCTGGCA7ACTGTGTAAGTCCATCTCATGGTG

TTCTGGCTAACGTA^to.GACTTAC .-. 3AGACAG^toAAAGA .-. C7CAGGC &lt;TGTGCTGGGGGTTGGGATGGAGG^toAGAGGGATGAGT

<td>AGGSGGAGCACGGGG. &amp; ACTTGGvC.</td><td>i.3TGAA^toATTCT773CAGGACACTAG.AGGAGGATAAATACCA5TCATTGCAfCCAC</td>

<td>TACTGGAC.WTCCAGGGAATTA? ·:</td><td>3CTGGGTGAAAA3 .-. 3AAG5CCCCAG5TATTGGCTGCATTGGCTGCATTTGCGTAAC</td>

<td>ATTTTTTTAAATTGAAAAGAAAAA.</td><td>3ATGTAAATCAA337TAGA7GAGT3GTTGCTGTGAGCTGAGAGCTGGGGTGAGTGA</td>

<td>GACATGTwAW.CTCCATCAAAA</td><td>-.3CGACAG.AAAG.^to.-. 33GGCTGTGGTGACAGCTACCTOTAATCTCCACCTCCGGGAG</td>

GTGATCAAGGTTAGCCCTCA'3CT .-. 3CCTGTGüTGCA7G .-. 3ACC (: TGTT7CAA^toTO^to.CTTT.^toAT^toAAGA^BAT^toATG.AAA^toAA aACATCA.GGGCAGATCCTTGGGS7CA.AAGGCGGACA3G33A3TCTCGTG-3TAA.GGTCGTGTAGAAGC &amp; 3ATGCATGAGCA

<td>CGTGCCGCAGGCATCATGAGA.G .-. 3</td><td>CCCTAGGTAAGTAAG3ATGGATGTGAGTCTGTCGGCGTC5GCGCACTGCACGTCCT</td>

<td>• 3GCTGTGGTGCTGGACTGGCATC7</td><td>7? 3GTGA3CTGT3G .-. 3GGGAAATGGGTAGGGAGATCATAA^toATCCCTCCGAATTAT</td>

<td>TTCAAGAACTGTCTATTACAAT7.-.</td><td>7CTC.^toTO^toA.?ATT.-AA.AAAAAAGAAG.AATTAAAAAAC.^toAA^toAACCTATCCAGGTG7G</td>

<td>GTGGTGTGCACCTATAGCCACGG3</td><td>C.AC7TGG.AAAGC7 3 3.AGC.^toAGAG3A7GGCGAGTTTG.^toAGGTATCTGGGGCTGTACA</td>

<td>GCAAGACCGTCGTCCCCAAACCA-,</td><td>A.GCAAACAGCAAACGCATTATGTCACACAAGAGTGTTTATAGTGAGCGGCCTCGCT</td>

<td>GAGAGCAT GGGvTGGGGGT GGGG3</td><td>7GGGGGACAüAAA7 .-. TCTA^toACTGCAGTCAATAGGG^toTCCACTGAGACCCTGGGGC</td>

<td>TTGACTGCAGCTTAACCTTGGGAA</td><td>A.7GAT.AAGGGTTTT3TGTTGAGTA^toAAGC.ATCGATTACTGACTTAACCTC.^toAATGA</td>

AGAAAAAGAAAAAAAGAAAACAA.CAAAAGGCAAACC.-AGGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGC

TCTTCCGA^to.GGTCCAGAGTTCAAA? CCCA.GCAACCA.C .-.? 3GT3GCTCACAACCATCTGTAACGAGATATGATGCCCTCTT

<td>CTGGTGTGTC7GAAGACAGCTAC.¹</td><td>.GTGTACTTACATA7AATAAATA^toATCTTA^toAAAAAAAPAAAAAAAAAAAAGCCAAA</td>

<td>CCGAGC.AAACCAGGCCCCCAAACí</td><td>.G.-AGGCAGGCACG .-. CGGC.AGGCACCACGAGCCATCCTGTGAAAAGGCAGGGCTACC</td>

<td>CATGGGCCGAGGAGGGTCCAGAGí</td><td>• .g.ataggctggtaagctcagtttctctgtataccctttttcttgttgacact.acttc</td>

<td>AATTACAGA.TAAAATAACAAATAJ</td><td>A.CAAAATCTAGAGCCTGGCCACTCTCTGCTCGCTTGATTTTTCCTGTT.ACGTCCAO</td>

<td>CAGGTGGCGGAAGTGTTCCAAGG. ·</td><td>-.CAGATCGC.ATCATT.-AGGTGGCCAGCAT.AATCTCCCATCAGCAGGTGGTGCTGTG.A</td>

G.AACCATTATGGTGCTCACAGAATCCCGGGCCCAGGAGCTGCCCTCTCCCAAGTCTGGAGCAATAGGAAAGCTTTCTGGC

CCAGAC.AGGGTTAACAGTCCAGATTCCAGAGCAGGGGAAAAGGAGACTGGAGGTCAC.AGA.CAAAAGGGCCAGCTTCTAAC

AACTTCACAGCTCTGGTAGGAGAGATA.GATCACCCCC.AACAATSGCCACAGCTGGTTTTGTCTGCCCCGAAGGAAACTGA

CTTAGGAAGCAGGTATCAGAGTCCCCTTCCTGAGGGGACTTCTGTCTGCCTTGTAAAGCTGTCAGAGCAGCTGCATTGAT

GTGTGGGTGACAGAAGATGAAAAGGAGGACCCAGGCAGATCGCCACAGATGGACCGGCCACTTACAAGTCGAGGCAGGTG

GCAGAGCCTTGCAGAAGCTCTGCAGGTGGACGACACTGATTCATTACCCAGTTAGCATACCACAGCGGGCTAGGCGGACC

ACAGCCTCCTTCCCAGTCTTCCTCCAGGGCTGGGGAGTCCTCCAACCTTCTGTCTCAGTGCAGCTTCCGCCAGCCCCTCC

TCCTTTTGCACCTGAGGTGTGAACCCTCCCTCCTCTCCT7CTCCCTGTGGCATGGCCCTCCTGCTACTGGAGGCTGAGCA

TTGGATTTCTTTGTGCTTAGATAGACCTGAGATGGCTTTCTGATTTATATATATATATCCATCCCTTGGATCTTACATCT

AGGACCCAGAGCTGTTTGTGATACCATAAGAGGCTGGGGAGATGATATGGTAAGAGTGCTTGCTGTACAAGCATGAAGAC

ATGAGTTCGAATCCCCAGCAACCATGTGGAAAAATAACC7TCTAACCTCAGAGTTGAGGGGAAAGGCAGGTGGATTCTGG

GGGCTTACTGGCCAGCTAGCCAGCCTAACCTAAATGTCTCAGTCAGAGATCCTGTCTC'AGGGAATA^to.CTTGGGAGAATGA

CTGAGAAAGACACCTCCTCAGGTCTCCCATGCACCCACACAGACACACGGGGGGGGGGTAATGTAATAAGCTAAGAAATA

ES 2 272 093 T3

ATGAGGGAAA7GATTTTTTGCT.AAGAAATGAAATTC73TGT7GüCCGC.AAGAAGCCTGGCC.A3GGAAGG.AACTGGCTTTG

GCACACCAGCC7ATAAGTCACCATGAGTTCCCTGGC7AAGAATCACATGTAATGGAGCCCAGG7CCCTCTTGCCTGGTGG

TTGCCTCTCCCACTGGTTTTGAAGAGAAATTCAAGAGA3ATCTCCTTGGTCAGAATTGTAGG73CTGAGCAATGTGGAGC

TGGGGTCAATGSGATTCCTTTAAAGGCATCCTTCCCAGGGCTGGGTCATACTTCAATAGTAGGGTGCTTGCACAGCAAGC

GTGAGACCCTAGGTTAGAGTCCCCAGAATCTGCCCCCAACCCCCC.W.AAGGCATCCTTCT5CCTCTGGGTGGGTGGGGG

GAGCAAACACCTTTAACTAAGACCATTAGCTGGCAGSGGTAACAAAT-SACCTTGGCTAGAGGAATTTGGTCAAGCTGGAT

TCCGCCTTCT3TAGAAGCCCCACTTGTTTCCTTTGTTAAGCTGGCCCACAGTTTGTTTTGA5.AAT5CCTGAGGGGCCCA «

GGAGCGAGACA.ATTAAAA'GCCAAGCTCATTTTGATATTTGAAÍACCACAGCCTGACTGCCCT 3CCCGTGGGAGGTACTGG

GAGACCTG3CTGTGTCCCTCKXTCACCAACGCCCGCGCCCCCAACACACACTCCTCGGGTCAGCTGGGAGGTGCCAGCAG

CAATTTÍWAAGTTTACTGAGCTTGAGAAGTCTTGGGAGGGCTGACGCT.AAGCACACCCCTTCTGCACCCCCCCCCACCCC acccccgtgaggaggagggtgaggaaacatgggaccagccctgctccagcccgtccttattgaggctggcatgagg

3GGí'T7T.AAAAA3GCAACCGTATCTAGGCTGGACACTGGAGCCTGTGC7ACCG .-. G? '3CCC7CCTCCACCTGGCAGCATGC. • GCC · ::'!

7TC-.GG.AAT &lt;r \ 70CCAC.AGAGGTGATCCCAGGGCT7GGA.G.AGTACCCCGAGCCTCCTCCTGAGAACA^to.CCAGACCAT6A4

CCG'r.; Í:&gt; AAAG.AA7GGAGGCA3ACCTCCCCACCATCCCTATGACGCCAAAGGTACGGGATGAAGAAGCACATTAGTGGGGG

3-3 33-3 ATCCTGiGAGGTGACTGGGGTGGTTTTAGCATCTTCTTCAGAGGTTTGTGTGGGTGGCTAGCCTCTGCTACATCA

3í¡X-AG + iACA'.A71l'GCCTGGAAGAATACTAGCACAGCATTAGAACCTGGAGGGCÁGCATTGGGGGGCTGGTAGAGAGC

O. • G.AAGGC .--. Í33G7GGAGGCTGAGGTCAGCCGAAGCTGGCATTAACACG3GCAT-3GGCTTG7ATGATGGTCCAGAGAATG

7GCTC'.TA-v .- .; A7GAGGACACAGGTCAGATCTAGC7GCTGACCAGTGGGGAAG7GATATGG73AGGCTGGATGCCAGATG

CCATCCAT3-.:77 ÜTACTATATCCCACATGACCACCACATGAGGTAAAGAAGGCCCCAGCTTGAAGATGGAGAAACCGAGA •&gt;:^:CTC-CT'3A3ATAAAGTCACCTGGGAGT.AAGAAGA3CTGAG.ACTGGAAGCTGGT7TGATCCAGATaC.AAGGCAACCCTAG

ATT ':'. 3C-TT7-3.3-37GGG.AACCTGA.AGCCAGGAGG.AATCCCTTTAGTTCCCCCTTGCCC.AGGGTCTGCTCAATG.AGCCCAG.A • 330 T •• '• OT.AAAAGAACAGGGTTTGTAGGCCGACGCA.

AGG “ACTTGCCCTGAGCTTGCACCCTGACCCCAGCTTTGCCTCATTCCTGAGGACAGCAGAAACTGTGGAGGC

AG .-. OC&gt; '.'. GC.A &lt;: AG.Aü.AGATGCCTGGGGTGGGGGTGGGGGTATCACGCACGGAACTAGCAGCAATGAATGGGGTGGGGTGG

CACCTGGAGGGACACTCCAGAGAAATGACCTTGCTGGTCACCATTTGTGTGGGAGGAGAGCTCATTTTCCAGCTTGCCAC

CArATGCTGTCGCTCCTGTCTCCTAGCCAGTAAGGGATGTGGAGGAAAGGGCCACCCCAAAGGAGCATGCAATGCAGTCA

CGTTT'n'GCAGAGG.AAGTGCTTG.ACCTAAGGGCACTATTCTTGGAAAGCCCCAAAACT.AGTCCTTCCCTGGGCAAACAGG

CCTCC ~ Cr.ACATACCACCTCTGCAGGGGTGAGTAAATTAAGCCAGCC.ACAGAAGGGTGGCA.AGGCCTACACCTCCCCCCT

G7TGTGCCCC0CCCCCCCCCGTGA.AGGTGCATCCTGGCCTCTGCCCCTCTGGCTTTGGTACTGGGATTTTTTTTTTCCTT

T7ATGTC.ATA7T3ATCCTG.ACACCATGGAACTTTTGGAGGTAGACAGGACCCACAC.ATGGATT.AGTTAAAAGCCTCCCAT

CCATCTAACCTCATGGTAGG.AGATAGAGCATGTCCAAG.AGAGGAGGGCAGGC.ATCAGACCTAGAAGATATGGCTGGGCAT

CCAACCCAATCTCCTTCCCCGGAG.AACAGACTCT.AAGTCAGATCCAGCCACCCTTGAGTAACCAGCTCAAGGTACACAGA

ACAAGAGAGTCTGGTATACAGCAGGTGCTAAACAAATGCTTGTGGTAGCAAAAGCTA.TAGGTTTTGGGTCAGAACTCCGA

CCCAAGTCGCGAGTG.AAGAGCG.AAAGGCCCTCTACTCGCCACCGCCCCGCCCCCACCTGGGGTCCTATAACAGATC'ACTT

TCACCCTTGCGGGAGCCAGAGAGCCCTGGCATCCTAGGTAGCCCCCCCCGCCCCCCCCCCGCAAGCAGCCCAGCCCTGCC

ES 2 272 093 T3

77TGGGGC.^toAGT7CT777CTCAGC? 7GG^toCCTGTGAT.^toATGAGGGGGTTGüACGCGCCGCCTTTGGTCGC7TTC.AAG7CT

AA7G.^toA7TCTTATCCCTACCACC7GCCCTTCTACCCCGC7CCTCCACAGCA6CTGTCCTGATTTAT7.ACCTTCAAT7.^tAC

C7CCAC7CCTTTCTCCATCTCCTGG3ATACCGCCCCTGTCCCAGTGGCTGG7AAAGGAGCT7AGGA.AGGACCAGAGCCAG

GTG7GGCTAGAG3CT, ACCAGGGAGGGCTGGGGATGAGGAGCTA^toACTGGAAGAGTGTTTGGTTAGTAGGCACA.^toAGCCT7

GGGTGGGATCCCTAGTACCGGAGAAGTGGAGATGGGCGCTGAGAAGTTCAAGACCATCCATCCTTAACTACACAGCCAGT

TTGAGGCCAGCCTGGGCTA.CA.TAAAAACCCAATCTGA.AAAGCTGCCA.ATTCTGATTCn'GTGCCACGTAGTGCCCGATGTA

A7ñGTGGATGAAG7GTTGAATCCTGGGíiGAACCTA7TTTAC^to.GATGTGGGiÍ^toAAAGCAACTTT.AAGTACCCT «CGCAGA

GATCAC^toAA5AAAG7.-AGTGACA-3A3CTCCA3TGTTTCATCCCTGi &lt;GTTCCAAGGACAGGGAC4AG.AGA .-. GC (: AG.3G7GCW

ATGTCACTGCTCCCCGGTGCCTXCTTCCTATAATCCATACAGATTCGAAAGCGCA &amp; SGCAGGTTTGGAAAAAGAGAG.AAG • 337GGAAGGAGCAGACC^toGTfTGG ~ CTAGGCTGCAG7CGC7CACGC^to.TCCCTCTCTCCGCAGATGTGTCCGAGTACAGCT

G7CGCGAGC7GCACTACACCCGC7TCCTGACAGACGGCCCATGCCGCAGCGCCAAGCCGGTCACCGAGT7GGTGTGC7CC

GGCCAG7GCGGCCG ~ GCGCGGC7GCTGCCC.^toACGCCA.7CGGGCGCG7GAAGTGGTGGCGCCCG^toACGGACCGGATT7CC ·?

~ 7GC.^to.7CCCGGA7CGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGGGGCGCGGCGCC5CGCTCGCGC.AAGGTGC.

G7C7GGTGGCCTCGTGCAA3TGCAA.3CGCCTCACCCGC7TCCA.CAACCAGTCGGAGCTCAAGGA.CTTC5GGGCGG.AGA.CC

GCGCGGCCGCAGAA.GGGTCGCAAGCCGCGGCCCGGCGCCCGGGüAGCCAAAGCCAACCAGGCGGAGCTGGAGAACGCCTA

CT.AGAGCGAGCCCGCGCCTATGCAGCCCCCCCGCGCGATCCGATTCGTTTTCAGTGT.AAA.GCCTGCAGCCCAGGCCAÜGGGT

GCC.AAACTTTCCA.GACCGTGTGGA.GTTCCCAGCCCA.5TAGAGA.CCGCAGGTCCTTCTGCCCGCTGCGGG3GATGGGGAGG

GGGTGGGGTTCCCGCGGGCCAGGA.GAGGAAGCTTGA.GTCCCAGACTCTGCCTAGCCCCGGGTGGGATGGGGGTCTTTCTA.

CCCTCGCCGGACCTATA.CAGGAGAA.GGCAGTGTTTCCACCTTAAAGGGAAGGGAGTGTGGAACGAAAGACCTGGGACTGG

TTATGGACÜTACA.GTAAGATCTACTCCTTCCACCCAAATGTAAAGCCTGCGTGGGCT.AGATAGGGTTTCTGACCCTGACC

TSGCCACTGAGTGTGATGTTGGGCTACGTGGTTCfCTTTTGGTACGGTCTTCTTTGTAAAA.TAGGGACCGGAACTCTGCT

G.AGATTCCAAGGATTGGGGTACCCCGTGTAGACTGGTGAGAGAGAGGAGAACAGGGGA.GGGGTTAGGGGAGAGATTGTGG tgggcaaccgcctagaagaagctgtttgttggctcccagcctcgccgcctcacaggtttggcttcccc

TCAAATCTGCCTTCAAATCCATATCTGGGA.TAGGGAAQGCCAGGGTCCGAGAGATGGTGGAAGGGCCA.GAAATCA.CACTC

CTGGCCCCCCGAAGAGCAGTGTCCCGCCCCCAACTGCCTTGTCA.TA.TTGTAAAGGGATTTTCTACACAACAGTTTAA.GGT

CGTTGGAGCAAACTGGGCTTGCCAGTCACCTCCCATCCTTGTCCCTTGCCAGGACACCACCTCCTGCCTGCCACCCACGG

ACACATTTCTGTCTAGAAACAGAGCGTCGTCGTGCTGTCCTCTGAGACAGCATATCTTACATTAAAAAGAAT.AATACGGG

GGGGGGGGGCGGAGGGCGCAAGTGTTATACATATGCTGA.GAAGCTGTCAGGCGCCACAGCACCACCCACAATCTTTTTGT

AAATCATTTCCACACACCTCTTACTTTCTGTGTAGATTTTAATTGTTAAAAGGGGAGGAGAGAGAGCGTTTGTAACAGAA

GCACATGGAGGGGGGGGTAüGGGGGTTGGGGCTGGTGAGTTTGGCGAACTTTCCATGTGAGACTCATCCACAAAGACTGA

AAGCCGCGTTTTTTTTTTTAAGAGTTCAGTGACATATTTATTTTCTCATTTAAGTTATTTATGCCAACA.TTTTTTTCTTG

TAGAGA^toAGGCAGTGTTAATATCGCTTTGTGAAGCACAAGTGTGTGTGGTTTTTTGTTTTTTGTTTTTTCCCCGACCAGA

GGCA.TTGTTAAT.AAA.GA.CAATGAA.TCTCGAGCA.GGA.GGCTGTGGTCTTGTTTTGTCAACCACACACAATGTCTCGCCA.CT

GTCATCTCACTCCCTTCCCTTGGTCACAAGACCCAAACCTTGACAACACCTCCGACTGCTCTCTGGTAGCCCTTGTGGCA

ATACGTGTTTCCTTTGAAAAGTCACATTCATCCTTTCCTTTGCAAACCTGGCTCTCATTCCCCAGCTGGGTCATCGTCAT

ACCCTCACCCCAGCCTCCCTTTA.GCTGACCACTCTCCACACTGTCTTCCAAAAGTGCACGTTTCACCGAGCCAGTTCCCT

ES 2 272 093 T3

GG7CCAG ~ .TCATCCCATTGCTCCTCC7TGTCCAGACCCT7C7CCCACAAAGA7GT7CATC7CCC.ACTCCATCAAGCCCC

AGTGGCCCTGCGGCTATCCCTGTC7CTTCAGTTAGCTG^toATCTACTTGC7G^B.CACCACATGAATTCCTTCCCCTGTCTTA

AGGTTCATGGAACTCTTGCCTGCCCCTGAACCTTCCAGGACTGTCCCAGCGTCTGATíiTGTCCTCTCTCTTGT.AAAGCCC

CACCCCACTATTTGATTCCC.^aB.TTCTAGATCTTCCCTTGT7CATTCCTTCACGGGATAGTGTCTCATCTGGCC.AA.GTCCT

GCTTGATATTGGGATAAATGCAAAGCCAAGTACAATTGAGGACCACiTTCATCATTGGGCCAAGCTTTTTCAAAATGTGAA ttttacacctatagaagtgtaaaagccttccaaagcagaggcaatgcctggctcttccttcacatgcaggt

TATGGGTCTGGTGGGGTAGTACATTCATAAACGCAACACTAGGGGTGTGAAAGCAAGATGATTGGGAGTTCGAGGCaAAT

CTTGGCTATGAGGCCCTGTCTCAACCTCTCCTGCCTCCCTCGAGGGTTTTGTTTTGTTTTGTTTTTTTGATTTGA'ACTG

CA.AC'ACTTTAAATCCAGTCAAGTGCATCTTTGCGTGAGGGGAACTCTATCCCTAATATAAGCTTCCATCTTGATTTGTGT

A.TGTGCA.CA.CTGGGGGTTGAACCTGGGCCTTTGTACCTGCCGGGCAAGCTCTCTACTGCTCTAAACCCA.GCCCTCACTGG

CTTTCTGTTTCAACTCCCAATGAATTCCCCTAAATG.AATTATCAATATCATGTCTTTGAAAAATACCATTGAGTGCTGCT

GGTGTCCCTGTGGTTCCAGATTCCAGG.AAGGACTTTTCAGGG.AATCCAGGCATCCTCAAGAATGTCTTAGAGCAGGAGGC catggaga.ccttggccagccccacaaggcagtgtggtgcaga.gggtgaggatggaggcaggcttgacaattga

CAGCíGTACTCAGGATTAAAAAGCTTCCCCCAAAACAATTCCA.AGATCAGTTCCTGGTACTTGCACCTGTTCAGCTATGCA

GAGC'CCAGTGGGCATAGGTGA^to.GA.CACCGGTTGTACT5TCATGTACTAACTGTGCTTCAGAGCCGGCAGAGA, C'AA^to.TAAT

GTTATGGTGACCCCAGGGGACAGTGATTCCAGAAGGAACACAGAAGAGAGTGCTGCTAGAGGCTGCCTGAAGGAGAAGGG gtcccacactctctaagcaaagactccactcacataaagacacaggctgaccaccagatcagagctggccgtggatgcattagggaggcatcaggcatgtggatgcattagggaggcatcatgcatgcatgaggcatgcatgaggcatgcatgcatgaggcatgcatgaggcatcat⁰.

acccaaacacacctaggacttgctttctgggacagacagatgcaggagagactaggttgggctgtgatcccattaccaca

TO^B.GA5GüA^BTO^BTO^B.G^BTO^BAAACA^toAC^BARrAAACAAAA^BTO^BAACAAAACA^toAACAAAA&gt; AAAACCCAAGGTCCAAATTGTA

GGTC.AGGT7AGAG7TTATTT^BTGGAAAGTTA7A7TC7ACCTCCATGGGGTCTACAAGGCTGGCGCCCATCAGAAAGAACA

AACAACAGGCTCATCTGGGAGGGG7GGTACTCTATGGCAGGGAGCACGTGTGC7TGGGG7ACAGCCAGACACGGGGCTTG

TAT7AR7CACAGC.CCT7GTATT.^B.ATAGGCTGAGAGTC.AA5CAGACAGAGAGACAGAAGGAAACACACACACACACACACA

CACACACACACACACACACACATGCACACACCACTCACTTCTCAC7CGAAGAGCCCC7ACTTACA.7TC7A^B.GAACAAACC

ATTCCTCC7C.ⁿ.T.ⁿ.^BAGGAGAC.AAAG7TGCAG.^to.AA.CCCAA.^toAGA.GCCACAGGGTCCCCACTCTCTTTGA.^toATGAC7TGGAC

TTG7TGCAGGGAAGACAGAGGGG7C7GCAGAGGCTTCCTGGGTGACCCAGAGCCACAGACAC7GA.^BATCTGGTGCTGAGA

CCTGTATA^BACCCTCTTCCACAGGTTCCCTGAAAGGAGCCCACATTCCCCAACCCTGTCTCCTGACCACTGAGGATGAGA

GCACT7GGGCCT7CCCCA.TTCTTGGA.GTGCACCC7GGTTTCCCCATCTGAGGGCACATGAGGTCTCAGGTCTTGGGAA^B.G

TTCCACAACTATTGA^BAGTGTTCTTGTTTTGT7TGTGATTTAATTTAGGTGTATGAGTGCTTT7GCTTGAATATATGCCT

G7G7AGCATTTACAAGCCTGG7GCC7GAGGAGA7CAGAAGATGGCATCAGATACCCTGGAACTGGACTTGCAGACAGTTA

TCAXICCACTGTGTGGGTGCTAGGAACAGAACCTGGATCCTCCGGAAGAGCAGACAGCCAGCGCTCTTAGCCACTAAGCCA

7CACTGAGG7TC77TCTGTGGCTAA^toGAGACAGG.^to.GACAAAGGAGAGTTTC.7TTTAGTCAATAGGACCATGAATGTTCCT

CGTAACGTGAGACTAGGGCAGGGTGA.TCCCCCAGTGACACCGATGGCCCTGTGTAGTTATTAGCAGCTCTAGTCTTATTC

C7TAATAAGTCCCAGTTTGGGGCAGGAGATATGTATTCCCTGC7TTGAAGTGGCTGAGGTCCAGTTATCTACTTCCAAGT

ACTTGTTTCTCTTTCTGGAGTTGGGGAAGCTCCCTGCCTGCCTGTAAATG75TCCATTCTTCAACCTTAGACAAGATCAC

TTTCCCTGAGGAGTCAGGCCAGTCCAAAGCCCTTCAATTTAGCTTTCATAAGGAACACCCCTTTTGTTGGGTGGAGGTAG

ES 2 272 093 T3

CACTTGCCTTGAATCCC^toGC^toTT^toAGAAGGCAGAGACAGTCGGATC7C7G7G^toG7TCACAGCCAGCC7GGTCTACGGAGT

GAGTTCCAAGA.CAGCCAGGCCTA.CACAGAGAAACCCTGTCTCGAAAAAAACAAAAACAAAAGAAATAAAGAAAAAGAAAA

CAAAAACGA.ACAAACAGAAAAACAAGCCAGAGTGTT73TCCCGGTATTTTATTAATCATATTTTTGTCCCT7TGCCATTT

TAGACTAAAAGACTCGGGAA.AGCAGGTCTCTCTCTGTTTCTCATCCGGACACACCCAGAACCAGATGTATGGAAGATGGG

agcaaggagtatgtggtgcatagcaaacgaggaagtttgcacagaacaacactgtgtgtactgatgtgcaggtatgggca TAATGTGCTGCAGTTGCACATCTGGGGCTGGGTGGATTGGTTAGATGGCA7GGGCTGGGTGTGGTTACGA7GA7rüCAGG catgcaagcagaagccaagggacagccttagggtagtgtttccacaga.cccctccccccttttaacatgggcatctctca

TTGG'JCTGGAGOTTGCCAACTGGGCTGGGCTGGCTAGCTTGTA.GGTCCCAGGGATCTGCATA.TCTCTGCCTCCXXAGTGC

TGGGATTACAGTCATATATGAGCACACCTGGCTTTTTTATGTGGGTTCTGGGCTTTGAACCCAGATCTGAGTGCTTGCAA

GGCAATCGGTTGAATGACTGCTTCATCTCCCCAGACCCTGGGATTCTA7777GTATTAAAG7ATTTCTATT.-AATCAATG

AGCCCCTGCCCC7GCACTCAGCAGTTCTTAGGCCTGCTGAGAGTCAAGTGGGGAGTÜAGAGCAAGCCTCGAGACCCCATC

AGCGAAGCAG^toGGACAAAG? AATGA^toAACTTGGGAT7CG^toGGCTCGGGA7ATGGAGATACAGA.^toA.GGGTC^-AG00.^toAGGAA

ATGAACCAGATGAATAGAGGCAGGAAGGGTAGGGCCCTGCATACATGGAACCTGGTGTACATGTTATCTGCA.TGGGGTTT

GCATTGC.^toATGGCTCTTCAGGAGGTTCACCACACTÜGG^toAACAGAAGCCA.^aaAAG^toAGAGTAGGTGGTGTT33AGTCAGA

TACTGTCAGTCATGCCTüAAGA^toATGGAAGCAATTAACGATGCGCCGCAAT? AGGATATTAGCTCCCTGAAG .-. AAGGCAA

GAAGCTGGGCTGTGGGCACTGAAGGGAGCTTTGAATGATGTCACATTCTCTGTATGCCTAGCAGGGCAGTATTGGAGACT

ÜA.GACTTGACTTGTGTGTCCATATGATTCCTCCTTTTCCTACAGTCATCTGGGGCTCCTGAGCTTCGTCCTTGTCCAAGA

ACCTCGAGCTGGCAGTGGGCA.GCTGCAGTGATAGATGTCTGCAAGAAAGATCTGAAAAGAGGGAGGAAGATGAAGGACCC

AGAGGACCACCGACCTCTGCTGCCTGACAAAGCTGCAGGACCAGTCTCTCCTACAGATGGGAGACAGAGGCGAGAGATGA

ATGG7CAGGGGAGSAGTCAGAGAA^toGGAGAGGGTGAGGCAGAGACCAAAGGAS3GAAACACTTGTGCTCTACA-3CTACTG

ACTGAGTACCAGCTGCGTGGCAGACAGCCAATGCC.AAGGCTCGGCTGATCATGGCACCTCGTGGGACTCCTAGCCCAGTG

CTGGCAGAGGGGAGTGCTGAATGGTGCA.TGGTTTGGATATGATCTGAATGTGGTCCAGCCCTAGTTTCCTTCCAGTTGCT

GGGATAAAGCACCCTGACCAAAGCTACTTTTTTGTTTGTTTGTTTTGGTTTGGTTTTGTTTGGTTTTTCGAGjCAGGGTT

TCTCTGTATCACCCTAGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCCCCTGCCTCTGC

CTCCTAAGTGCTGGAATTAAAGGCCTGCGCCACCACTGCCGGCCCAAAGCTACTTTAAGAGA.GAG.AGAGGAA7GTATAAG

TATTATAATTCCAGGTTATAGTTCATTGCTGTAGAATTGGAGTCTTCATATTCCAGGTAATCTCCCACAGACATGCGACA

AAACAACCTGTTCTACGAAATCTCTCATGGACTCCCTTCCCCAGTAATTCTAAACTGTGTCAAATCTACAAGAAATAGTG

AC.^to.GTCACAGTCTCTAACGTTTTGGGCATGAGTCTGAAGTCTC.^to.T7GCTAAGTACTGGGAAGATGAAAACTTTA.CCTAGT

GTCAGCATTTGGAGCAGAGCCTTTGGGATTTGAGATGGTCTTTTGCAGAGCTCCTAATGGCTACATGGAGAGAGGGGGCC

TGGGA.GAGACCCATACACCTTTTGCTGCCTTATGTCACCTGACCTGCTCCTTGGGAAGCTCTAGCAAGAAGGCCTTCCCT

GGATCACCCACCACCTTGCACCTCCAGAACTCAGAGCCAAATT.AAACTTTCTTGTTACTGTCGTCAAAGCACAGTCGGTC

TGGGTTGTA.TCACTGTCAATGGGAAACAGACTTGCCTGGATGGAT.AA.CTTGTA.CATTGCATAATGTCTAGAAATGAAAAG

TCCTATAGAGAAA.^toAG.AAAATTAGCTGGCACACAGATAGAGGCCCTGGAGGAGGCTGGCTTTGTCCTCCCCGAGGAGGTG

GCGAGTAAGG7GTAAATGTTCATGGATGTAAATGGGCCCATATATGAGGGTCTGGGGTAAC.AAGAAGGCCTGTGAATATA

AAGCACTGAAGGTATGTCTAGTCTGGAGAAGGTCACTACAGAGAGTTCTCCAACTCAGTGCCCATACACACACACACA

CACACACACACACACACACACACACACACACCACAAAGAAAAAAAGGAAGAAAAATCTGAGAGCAAGTACAGTA.CTTAAA

ES 2 272 093 T3

ATTGTGTGA.TT3TGTGTGTGACTCT5.ATGTCACATGCTCATCTTGCCCTATGAGTTGAAAACCA «ATGGCCCCT5A.GAG3

CA.TA.ACAA.CCACA.CTGTTGGCTGTGTGCTC.ACGTTTTTCTTAAAGCGTCTGTCTGGTTTGCTGCTA.GCA.TCA.GGCA.3A.CT

TGCAGCAGAC7ACATATGCTCAGCCCTGA.AGTCCTTCTAGGGTGCATGTCTCTTCAGAATTTCAGAAAGTCATCTG7S5C

TCCAGGAX: C3CCTGCACTCTCCCTCTGCCGCGAGGCTGCA.GA.CTCTAGGCTGGGGTGGA.AGCA.ACGCTTA.CCTCTGGGA.C

AAGT.ATA.ACA.?GTT'3GCTTTTCTTTCCCTCTGTGGCTCCAA.CCTGGA.C.ATAAA.ATAG.ATGCAA.GCTGTGT.AA.TAA_ATA.TT

TCCTCCCGTOCA.CTTAGTTCTCAACAA.TAACTACTCTGAGAGCA.CTTA.TTAATA.GGTGGCTTA6ACA.T.AAGCTTT33CTC

A.TTCCCCCA.CTA.GCTCTTA.CTTCTTTAACTCTTTCAA.ACCA.TTCTGTGTCTTCC.ACA.TGGTTA.GTTA.CCTCTCCTTCCA.T

CCTGGTTCGCTTCTTCCTTCG.AGTCGCCCTCA.GTGTCTCTA.GGTGA.TGCTTGTA.AGAT.ATTCTTTCTA.CAA.AGCTGA.GA.G

TGGTGGCACTC7GCGAGTTCA.AA.GCCA.GCCTGATCTA.CACAGCAAGCTCCAGGA.TATCCAGGGCA.ATGTTGGGA.AA.ACCT

TTCTCAAACA.-_ AAAGA.GGGGTTCAGTTGTCA.GGA.GG.AGA.CCCATGGGTT.AAGAA.GTCT.AG.ACG.AGCCATGGTGATGCATA.

CCTTTC.ATCCA-AGCA.CTT.AGGAGGCAAA.G.AAAGGTGAA.ACTCTTTGACTTTGAGGCC.AGCT.AGGTT.ACATA.GTGA.TA.CCC

TGCTT.AGTG73T3TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTA.ATTT.AAAAGTCTA.AAAATGCA.TTCTTTTA.AAAA

TA.TGTAT.AA3T.ATTTGCCTGCAC.A7ATGTATGTATGTA.TGT.ATACC.ATGTGTGTGTCTGGTGCTGAAGG.ACTAGGCAT.AG

A.CTCCCTAGAA.CTA.GA.GTCA.T.AGACAGTTGTGACA.CTCCCC.AACCCCCCA.CCA.TGTGGGTGCTTG.AAGCT.AAACTCC7GT

CCTTTGT.AAAGCA.GCA.GGTGTCTA.TGA.ACCCTG.AACCATCTCTCCA.GTCTCCAGA.TGTGCA.TTCTC.AA.AG.AGGAGTCCTT

CATATTTCCCT.AAACTGAACA.TCCTTRTCAGTGAGC4TCCTCGA.GTCACCPAAGOTACTGCA.AACCCTCTTAGGG.AACAT

TCACTA.TTCA.CTTCTA.CTTGGCTCATGAA-ACTT.AAGT.ACACACACACAAACAC.ACA.CA.CA.CACACA.G.AGTC, ATGCACTCA.

CAAAAGCA.TGCATGTA.CACCATTCTT.ATTA.GA.CTA.TGCTTTGCT.AAAAGA.CTTTCCTAGA.TACTTT.AAAAC.ATCA.C7TCT

GCCTTTTGGTGGGCA.GGTTCCAAG.ATTGGTACTGGCGTACTGGA.AACTG.AACA.AGGTAGAGA.TCTAG.AAATCACAGCAGG tca.g.aagggccagcctgtac.aaga.ga.ga.gttccacaccttccaggacctcaggctg

CCCAAGAAA.CTA.GTGCGTTTCCTGTA.TGCATGCCTCTCAGA.GATTCCAT.AAGA.TCTGCCTTCTGCCAT.AACATCTCCTGC

A.TCCAG.ACAA.GCCT.AGGGGA.AGTTG.AGA.GGCTGCCTGAGTCTCTCCC.ACAGGCCCCTTCT7GCCTGGCAGTA.TTTT7TTA.

TCTGGA.GG.AGA.GGAATC.AGGGTGGGA.ATGA.TCA.AATAOAATTA.TCAAGG.AAAAAGTAA.AAAACAT.ATA.TATATA.TA.T.ATT

A.ACTGA.TCTA.GGGA.GCTGGCTCAGCA.GTT.AAGA.GTTCTGGCTGCCCTTGCTTCA.GATCTTGCTTTG.ATTCCCAGC.ACCCA

CA.TGATGGCTTTCAA.CTGT.ATCTCTGCTTCCAGGGG.ATCCAACAGCCTCTTCTGACCTCC.ATAGACAAGACCT.AGTCCTC tgc.aa.gagcacc.aaatgctcttatctgttg.atcc.atctctctagcctc.atggaccaactc.atctctctagcctc.atggaccaactc.atctctctagcctc.atggaccaagatc.atctctctagcctc.atggaccaactc.atctctctagcctc.atggaccaactc.atctctttg

CCCATTTTACG.AAGA.TGTCACTGCCCAGTCATTTGCCATGAGTGGATATTTCGATTCTTTCT.ATGTTCTCACCCTTGCAA

TTTA.TAAGAAAGAT.ATCTGCATTTGTCTCCTG.AGAG.AACAAAGGGTGGA.GGGCTACTGAGATGGCTCT.AGGGGTA.AAG3T

GCTTGCCA.CAAAATCTGA, CAA.CTTA, AGTTTGGTCTTGG.AATCCACATGGTGGAGAGAGAG.AAGA.GA.TTCCCGTAAGTTGT

CCTCAA.ACTTCCCACAC.ATGTGCTGTGGCTTA.TGTGT.AACCCCAA.TAAGTAA.AGA.TAGTTTTAAACACTA.CATAA.GGTAG

GGTTTCTTCA.TGACCCCAAGGAATGA.TGCCCCTGATAGAGCTTA.TGCTG.AAACCCCA.TCTCCATTGTGCCA.TCTGGAA.AG .AGA.CAATTGCA.

TGGCGGCGTAATGACCTCCA.CCA.TGA.TGTT.ATCC.AGC.ATGAAGGTCCTCA.CC.AGAAGTC.AT.ACAAA.TCTTCTTAGGCTTC

CAGAGTCGTGAGC.AAAAAA.AGCACACCTCT.AAATA.AATTA.ACTAGCCTCAGGT.AGTT.AACCACCGAAAATGAACCA.AGGC

A.GTTCTAATA.C.AAAACCACTTCCCTTCCCTGTTC.AAACCACA.GTGCCCTATTATCTAAAAGA.T.AAACTTC.AAGCCA.AGCT tttaggttgccagta.ttta.tgtaacaacaaggcccgttgacacacatctgtaactcct.cagggagc

ES 2 272 093 T3

GACAGGTGGAGCCCTGGAGTTTGAATTCCAGGTTCTGTGAGAAACTCTGTTTGAAAAGACAATATGCíTGAGTGACCCGG ·? A.GGA.TATCTGATATTGACTTCTG3CCA.ACACACA.GCCA.TCTCTGCACA7C7GTA.GTTGCAAGCCTTTTGCACTAAG77TG GCCAGAGTCAGAGTTTGCA.A5TGT? 7GTGGACTGA.ATGCACGTGTTGCTTGACGACA.^or.GTCACCCTCCTTCTCAA. ·? CTA.GCAGCACTGGCTTCGGCCAGC7GCTCA.TTCAAGCCT CTTTGCAGA.G7CATCACGGGGATGGGGGAGCAGG3CCCC7C CCTAGAAA.CCAAGCCTGTGGTTG? TTCAAGCCT CTTTGCAGA.G7CATCACGGGGATGGGGGAGCAGG3CCCC7C CCTAGAAA.CCAAGCCTGTGGTTG? TTCAAGCCT.

CA.GTCGGGTGTTGCG0TGTTA.GGT7ATTTCTGTGTC7'3C.AGA.AAA.CA.GTGCAA.CCTGGACAAAAGAAAT.AAA.T5A.TA.TCA.

TTTTTCATTCA.GGCAACTAGA.TTCCGTGGTACAAAA.GGCTCCCTGGüüAA.CGAGGCCGGGA.C.'AGCGCGGCTCCTGA.GTCG

CTATTTCCGTCTGTCAA.CTTCTCTAA.TCTCTTGATTTCCTCCCTCTGT&gt; T73TTTCCTTCCTCTTGCTGGGGCCCA.GT55A. GTCTGTGT.ACTCACAGGGAGGAGGGTGGC.W3CCCTGSTCCTCTACGGGCT5GGGGAAGGGGGGAAGCTGTCGGCCC.AG TGACTTTTTCCCCTTTCTCTTTTTCTTAGAAACC.AGTCTC.A.ATTTAA.G.AT.AA.TGAGTCTCCTCATTCACGTGT3CTC.ACT .A7TCA.TAGGGACTTATCCACCCCCGCCCTGTCAATC73GCTAAGTAAG.ACAA.3TCAAA.TTTAAAAGGGAACGTTTTTCT.A AAAA.TGTGGCTGGACC5TGT5CCGGCA.CGAAACCA.-333.ATGGCGGTCT. "A. G77ACA.TGCTCTCTGCCAGCCCCGGTGCC7

TTTCCTTTC5GAAA.GGA.GA.CCCG3A.3GTAAAA.CGAA.-3TTGCCAA.CTTTT3.ATGATGGTGTGCGCCGGGTGACTCTTTAAA.

ATGTCA.TCCA.TACCTGGG.A7.AGGG. \ AGGCTCTTC.AGGG.AGTCATCTAGCCC7CCCTTCAGG.AAAAGA.TTCCACTTCCG-3T

TTAGTTAGCTTCC.ACCTGGTCCCTTA.TCCGCTGTCTCTGCCCA.CTAGTCCTC.ATCCATCCGGTTTCCGCCCTCATCCACC

TTGCCCTTTTAGTTCCTA.G.AAAGC.AGCACCGT.AG7C77GGCA.GGTGGGCC.ATT'3GTCACTCCGCTA.CCACTGTT.ACC.ATG

GCCA.CCAA.GGTGTCATTTAA.A7A.TG.AGCTCACTG.AGTCCTGCGGGATGGCTTGGTTGGTAA.TA.TGCTTGCTGCAAAA.TCG

TGAGAA, CTGGAGTTCAA.TTCCCAGC.ACATGGA.TGTATTTCCAGCACCTGG-A.GGC.AGGGAGCA.GAGATCTTAAA.GCTCCT

GGCCAGACAGCCCAGCC7AA7TAGTAA7CAGTGAGAGACCCTGTCTCAAGAA.ACAAGATGGAACATCAAAGGTCA.ACCT

TTGTCTCCA.C.ACA.C.ACAAA.TACA.CA.CA.TGCACATACATCA.CACACAGGCAA.ACACATGCA.CACACCTGAACA.CCCTCC.A

CAAATACATACATAAAAAAATAAAT.ACA.TACACA.CA.7.ACA.TACATACACCAA.CATTCCCTCTCCTTA.GTCTCCTGGCTA.C gctcttgtca.cccccactaaggcttcaacttcttcta.tttcttca.tcttgactcctctgta.ctttgcatgccttttccag caaaggcttttctttaaa.tctccgtca.ttcataaactccctctaaatttcttcccctgcccttttctttctctctaggga cataaa.gaca.caca.ctacaaagtcaccgtgggaccagtttattcacccacccacccctgcttctgttcatccggccagct aagta.gtcc.aa.cctctctggtgctgta.ccctggaccctggcttcaccac. agctcctcca.tgctacccagccctgcaaacc ttcagcctagcctctggttctccaaccagcacaggcccagtctggcttcta.tgtcctagaaatctccttcattctctcca tttccctcctgaatctacca.ccttctttctcccttctcctgacctctaatgtcttggtcaaacgattacaa.ggaagccaa.

tga »atta.gca.gtttggggta.cctca.ga.gtcagcaggggagctgggatgaa.ttcacatttccaggcctttgctttgctcc ccggattctgacaggcagttccgaa.gctgagtccagg.aagctgaatttaaaatcacactccagctgggttctgaggca.gc cctaccaca.tca.gctcgccctgactgagctgtgtctsggtggcagtggtgctggtggtgctggtggtgctggtggtggtg gtggtggtggtggtggtggtggtggtggtgtgtgtgtgtgttttctgcttttacaaaa.cttttctaattcttatacaaag gacaaatctgcctcatataggcagaaagatgacttatgcctatataaga.tataaagatgactttatgccacttattagca atagttactgtcaaaagtaa.ttcta.tttatacacccttatacatggtattgcttttgttggagactctaaaatccagatt atgtatttaaaaaaaaattccccagtccttaaaaggtgaagaatggaccc.agatagaaggtcacggcacaaggatatgccagtgactgactgactgactgactgactgagtcctgcatgactgactgactgactgagtccttaaaaggtgaagaatggaccc.

a.ctggca.gccctgatg.aggta.ccagaga.ttgctaagtggaggaata.caggatc.agaccca.tggaggggcttaaagcgtga

ES 2 272 093 T3

C ~ GTAGCA3CCCTCCGCTGA3GGGCTCCAGGTGGGCGCCCAAGGTGCTGCAGT6GGAGCCACATGAGAGGTGATGTCTTG

GAGTCACCTCGGGTACCATTGTTTAGGGAGGTGGGGATTTGTGGTGTGGAGACAGGCAGCCTCAAGGATGCTTTTCAACA

ATGGTTGATGA.GTTGGAACTAAAACAGGGGCCATCACACTGGCTCCCATAGCTCTGGGCTTGCCAGCTTCCACATCTGCC

CCCCACCCCCTGTCTGGCACCAGCTCAAGCTCTGTGATTCTACACATCCAAAAGAGG.AAGA.3TAGCCTACTGGC; CATGCC

ACCTCTTCTGGACCATCAGGTGAGAGTGTGGCAAGCCCTAGGCTCCTGTCGAG6ATGGAGGGCTGCCAGATAGGATGCTC agctatctcctgagctgg. »Actattttaggaataaggattatgcccgcccggggttggccagcagccagcagcct

TTGCGTAAAAGCAAGTGCTGTTGATTTATCTAAAAACAGAGCCGTGGACCCACCCACAGGACAAGTATGTATGCATCTGT ttcatgtatctgaaaagcgacacaaccatttttttcacatcatggcatcttcctaacccccattcttttttgttttgttgt

TT-3AGACAGGGTTTCTCTGTGTAGTCCTGGCTGTCCTGGAACTCACTTTGTAGACCAG-3CTGGCCTCGAACTCAGAAATC

CTGGGATTAAAGGTGTGTGCCACCACGCCCGGCCCTAACCCCCATTCTTAATGGTGATCCAGTGGTTGAAATTTCGGGCC acacacatgtccattagggattagctgctgtcttctgagctacctggtacaatctttatcccctggctggcctgggctcctc

ATCCCTGACTCGGGCCCGATCAAGTCCAGTTCCTGGGCCCGATCAAGTCCAGTTCCTGGGCCCGAACAAGTCCAGTCCCT

AGCTCGATTAGCTCATCCTGGCTCCCTGGCCTGTTCTTACTTACACTCTTCCCCTTGCTCTGGACTTGTTGCTTTCTTTA

CTCAAGTTGTCTGCCACAGTCCCTAAGCCACCTCTGTAAGACAACT.AAGATAATACTTCCCTCAAGCACGGAAAGTCCTG

AGTCACCACACCCTCTGGAGGTGTGTGGACACATGTTCATGCGTGTGGTTGCGCTTACGTACGTGTGC

SEQ ID NO. 18: Human BEER Genomic Sequence (This gene has two exons, at positions 161-427 7 3186-5219)

<td>tagaggagaa</td><td>gtctttgggg</td><td>agggtttgct</td><td>ctgagcacac</td><td>ccctttccct</td><td>ccctccgggg</td><td> 60</td>

<td>ctgagggaaa</td><td>catgggacca</td><td>gccctgcccc</td><td>agcctgtcct</td><td>cattggctgg</td><td>catgaagcag</td><td> 120</td>

<td>agaggggctt</td><td>sooooaggcg</td><td>accgtgtctc</td><td>ggccggagac</td><td>cagagcctgt</td><td>gctactggaa</td><td> 180</td>

<td>ggtggcgcgc</td><td>cctcctctgg</td><td>ctggtaccat</td><td>gcagctccca</td><td>ctggccctgt</td><td>gtctcgtctg</td><td> 240</td>

<td>cccgctggta</td><td>cacacagcct</td><td>tccgtgtagt</td><td>ggagggccag</td><td>gggcggcagg</td><td>cgttcaagaa</td><td> 300</td>

<td colspan="2">tgatgccacg * gaaatcatcc</td><td>ccgagctcgg</td><td>agagtacccc</td><td>gagcctccac</td><td>cggagctgga</td><td> 360</td>

<td>gaacaacaag</td><td>accatgaacc</td><td>gggcggagaa</td><td>cggagggcgg</td><td>cctccccacc</td><td>acccctttga</td><td> 420</td>

<td>gaccaaaggt</td><td>atggggtgga</td><td>ggagagaatt</td><td>cttagtaaaa</td><td>gatcctgggg</td><td>aggttttaga</td><td> 480</td>

ES 2272093 T3 aacttctcct cgggaggcct ggaagactgg ggtagaccca gtgaagattg ctggcctctg 540 ccagcactgg tcgaggaaca gtcctgcccg gaggtggggg aagaatggct cgctggtgca 600 gccttcaaat tcaggcgcag aggcacgagg caacagacgc tggtgagagc ccagggcagg 660 gaggacgccg gggtggtgag ggtatggcat cagggcacca gaacaggctc aggggctcag 720 aaaagaaaag gcctcaaaga atctcctcct gggaatatag gagccacgcc cagctgctgg 780 caccaccggg aagggaacaa ggtaagggag cctcccatcc acagaacagc acctgtgggg 840 caccggacac tctatgccgg tggcggctgt ccccaccaca cagacccaca tcatggaacc 900 cccaggaggc gaacccccag ctcgaagggg aagaaacagg ttccaggcac tcagtaactc 960 ggtagtgaga agagctgagg cgtgaacctg gtttgatcca actgcaagac agccccggtg 1020 tgtggggggg tgtgggggac agatctccac aaagcagtgg ggaggaaggc cagagaggca 1080 cccccgcagc gcgcatcgcc cacggcctgc ccagggagct ggcacttgaa ggaatgggag 1140 cccccggcac agctttagcc cctgacatgg gtgcagctga gtccaggccc tggaggggag 1200 agcagcatcc tctgtgcagg agtagggaca tctgtcctca gcagccaccc cagtcccaac 1260 cccgccccat tccaggggag ggagaaggaa gaggaaccct gggttcctgg tcaggcctgc 1320 ccaggtgaca acagagaagc gtgtgcatct ggctctataa ttggcaggaa tcctgaggcc 1380 atqqqgacqt ctgaaat-gar 'cttcagact acgagcttcc ci.yi.uci.ctg gccatcatcc 1440 aggcggcaga gaagtccact gcccaggctc ctggacccca gccctccccg cctcacaacc 1500 tgctgggact atggggtgct aaaaagggca accgcatggg aggccagcca ggaccctccg 1560

ES 2272093 T3 tcttcaaaat ggaggacaag ggcgcctccc cccacagctc cccttctagg caaggtcagc 1620 tgggctccag cgactgcctg aagggctgta aggaacccaa acacaaaatg tccaccttgc 1680 cgagaggcca tggactccca cagcccctga ggaagccaca tgctcaaaac aaagtcatga 1740 tctgcagagg aagtgcctgg cctaggggcg ctattctcga aaagccgcaa aatgccccct 1800 tccctgggca aatgcccccc tgaccacaca cacattccag ccctgcagag gtgaggatgc 1860 aaaccagccc acagaccaga aagcagcccc agacgatggc agtggccaca tctcccctgc 1920 tgtgcttgct cttcagagtg ggggtggggg gtggccttct ctgtcccctc tctggtttgg 1980 tcttaagact atttttcatt ctttcttgtc acattggaac tatccccatg aaacctttgg 2040 gggtggactg gtactcacac gacgaccagc tatttaaaaa gctcccaccc atctaagtcc 2100 accataggag acatggtcaa ggtgtgtgca ggggatcagg ccaggcctcg gagcccaatc 2160 tctgcctgcc cagggagtat caccatgagg cgcccattca gataacacag aacaagaaat 2220 gtgcccagca gagagccagg tcaatgtttg tggcagctga acctgtaggt tttgggtcag 2280 agctcagggc ccctatggta ggaaagtaac gacagtaaaa agcagccctc agctccatcc 2340 cccagcccag cctcccatgg atgctcgaac gcagagcctc cactcttgcc ggagccaaaa 2400 ccccagggaa ggtgctggga gtggagtccg gagatgcagc ccagcctttt gggcaagttc 2460 ttttctctgg ctgggcctca gtattctcat tgataatgag ggggttggac acactgcctt 2520 tgattccttt caagtctaac gaattcctgt cctgatcacc tccccttcag tccctcgcct 2580 ccacagcagc tgccctgatt tattaccttc aattaacctc tactcctttc tccatcccct 2640

ES 2272093 T3 gtccacccct cccaagtggc tggaaaagga atttgggaga agccagagcc aggcagaagg 2700 tgtgctgagt acttaccctg cccaggccag ggaccctgcg gcacaagtgt ggcttaaatc 2760 ataagaagac cccagaagag aaatgataat aataatacat aacagccgac gctttcagct 2820 atatgtgcca aatggtattt tctgcattgc gtgtgtaatg gactaactcg caacgcctgg 2880 ggcggcccat tttgcagaca ggaagaagag agaggttaag gaacttqccc aaqatqacac 2940 ctgcagtgag cgatggagcc ctggtgtttg aaccccagca gtcatttggc tccgagggga 3000 cagggcgcgc aggagagccc tccaccagct ctagagcatc tgggaccttc ctgcaataga 3060 tgttcagggg caaaagcctc tggagacagg cttggcaaaa gcagggctgg ggtggagaga 3120 gacgggccgg tccagggcag gggtggccag gcgggcggcc accctcacgc gcgcctctct 3180 ccacagacgt gtccgagtac agctgccgcg agctgcactt cacccgctac gtgaccgatg 3240 ggccgcgccg cagcgccaag ccggtcaccg agctggtgtg ctccggccag tgcggcccgg 3300 cgcgcctgct gcccaacgcc atcggccgcg gcaagtggtg gcgacctagt gggcccgact 3360 tccgctgcat ccccgaccgc taccgcgcgc agcgcgtgca gctgctgtgt cccggtggtg 3420 aggcgccgcg cgcgcgcaag gtgcgcctgg tggcctcgtg caagtgcaag cgcctcaccc 3480 gcttccacaa ccagtcggag ctcaaggact tcgggaccga ggccgctcgg ccgcagaagg 3540 gccggaagcc gcggccccyc ycccyyaycy ccaaagccaa ccaggccgag ctggagaacg 3600 cctactagag cccgcccgcg cccctcccca ccggcgggcg ccccggccct gaacccgcgc 3660 cccacatttc tgtcctctgc gcgtggtttg attgtttata aatgcctgca tttcattgta 3720

ES 2272093 T3 acccagggca gggggccgag accttccagg ccctgaggaa ccccgggcgc cggcaaggcc 3780 cccctcagcc cgccagctga ggggtcccac ggggcagggg agggaatcga gagtcacaga 3840 cactgagcca cgcagccccg cctctggggc cgcctacctt tgctggtccc acCCcagagg 3900 aggcagaaat ggaagcattt tcaccgccct ggggttttaa gggagcggtg cgggagtggg 3960 gactggtcaa aaagtccagg gaaagttgga caagattccc ccttgcacct cgccgcccat 4020 cagaaagcct gaggcgtgcc cagagcacaa gactgggggc aactgcagat gtggtttcta 4080 gtcctggctc tgccactaac ttgctgtgta accttgaact acacaattct ccttcgggac 4140 ctcaatttcc actttgtaaa atgagggtgg aggtgggaat aggatctcga ggagactatc 4200 ggcacatgat tccaaggact ccagtgcctt ttgaatgggc gagagagaga agaggtgaga 4260 gaaagagaga gaatgaatgc agttgcattg atccagtgcc aaggtcactt ccagaattca 4320 gagttgtgat gctctcttct gacagccaaa gatgaaaaac aaacagaaaa aaaaaagtaa 4380 agagtctact tacggctgac atatttacgg ccgacaaact cctggaagaa gctatgctgc 4440 ttcccagcct ggcttccccg gatgtttggc tacctccacc cccccatctc aaagaaataa 4500 catcatccat tggggtagaa aaggagaggg tccgagggtg gtgggaggga tagaaatcac 4560 acttcccaaa atccgcccca cctcccccga gagcagcatc cccatagcca tgttttaaag 4620 tcaccttccg aagagaagtg aaaggttcaa ggacactggc ettgcaggcc cgagggagca 4680 gccatcacaa actcacagac cagcacatcc cttttgagac accgccttct gcccaccact 4740 tttccgccta cacggacaúa gaaaacagct tctcactgct cttacatgtg acggcatatc 4800

ES 2272093 T3 ttacactaaa agaatactat tgggggaaaa actacaagtg ctgtacatat gctgagaaac 4860 tgcagagcat aatagctgcc acccaaaaac atcatttcca ctttttgaaa gacaacctct 4920 gtagttttta tactctctgt attgttaaaa aaaaaaagtt gcacatgaca etaaacagaa 4980 tatgaaagcc tgcaggactg gtcgtttttt tggcaattct tccacgtggg acttgtccac 5040 aagaatgaaa gtagtggttt ttaaagagtt aagttacata tttattttct cacttaagtt 5100 atttatgcaa aagtttttct tgtagagaat gacaatgtta atattgcttt atgaactaac 5160 agtctgttct tccagagtcc agagacattg ttaataaaga caatgaatca tgaccgaaag 5220 gatgcggtct cattttgtca accacacacg acgtcatttc tgecaaagtt gacacccttc 5280 cagagcccca ccttggtcac accttggaca cacctttgac tgctccctgg tggcccctgt 5340 ggcaattacg tcttcctttg aaaagtcatg tttatccctt cctttccaaa cccagaccgc 5400 atttcttcac ccagggcatg gtaataacct cagccttgta tccttttagc agcctcccct 5460 ccatgctggc ttccaaaatg ctgttctcat tgtatcactc aagccttcca ccctgctcaa 5520 tagctccccc ttgcccagga tcaagtgcag tttccctatc tgacatggga ggccttcCct 5580 gcttgactcc cacctcccac tccaccaagc ttcctactga ccccaaatgg tcatgcagat 5640 ccctgcttcc ttagtttgcc atccacactt agcaccccca ataactaatc ctctttcttt 5700 ttacttgtca aggattcacs tctcttccce Laacccccca gagatgttcc aatctcccat 5760 gatccctctc tcctctgagg ttccagcccc ttttgtctac accactactt tggttcctaa 5820 ttctgttctc catttgacag tcattcatgg aggaccagcc tggccaagtc ctgcttagta 5880

ES 2272093 T3 ctggcataga caacacaaag ccaagtacaa ttcaggacca gctcacagga aacttcatct 5940 tcttcgaagt gtggatttga tgcctcctgg gtagaaatgt aggatcttca aaagtgggcc 6000 acttctctca agcctcctgc aagtctcgcc tccccaaggt gtcttaatag tgctggatgc 6060 tagctgagtt agcatcttca gatgaagagt aaccctaaag ttactcttca gttgccctaa 6120 ggtgggatgg ccaactggaa agctttaaac taagtccagc ctaccttggg ggaacccacc 6180 cccacaaaga aagctgaggt ccctcctgat gacttgtcag tttaactacc aataacccac 6240 ttgaattaat catcatcatc aagtctttga taggtgtgag tgggtatcag tggccggtcc 6300 cttcctgggg ctccagcccc cgaggaggcc tcagtgagcc cctgcagaaa atccatgcat 6360 tcagggccca catgagcgtc gcaggtagga gaatatgaga aacagagaca tcttccatcc 6420 gtgcggtcca ccgagaggca gtgggtgggg acacgggctc tgggtcaggt ttgtgttgtt 6480 ttttgagaca tgtttgtttg gagtctcgct ctattgccca ggctggagtg cagtgtcaca 6540 atctcggctt actgcaactt ctgccttccc ggattcaagt gatcctcctg cctcagcctc 6600 cagagtagct gggattacag gtgcgtgcca ccacgcctgg ctaatttttg tatttttgat 6660 agagacgggg tttcaccatg ttggccaggc tagtctcgaa ctcttgacct caagtgatct 6720 gcccgcctcg gcctcccaaa gtgctgggat tacaggcgtg agccaccaca cccagcccca 6780 ggttggtgtt tgaatctgag gagactgaag caccaagggg ttaaatgttt tgcccacagc 6840 catacttggg ctcagttcct tgccctaccc ctcacttgag ctgcttagaa cctggtgggc 6900 acatgggcaa taaccaggtc acactgtttt gtaccaagtg ttatgggaat ccaagatagg 6960

ES 2272093 T3 agtaatttgc tctgtggagg ggatgaggga tagtggttag ggaaagcttc acaaagtggg 7020 tgttgcttag agattttcca ggtggagaag ggggcttcta ggcagaaggc atagcccaag 7080 caaagactgc aagtgcatgg ctgctcatgg gtagaagaga atccaccatt cctcaacatg 7140 taccgagtcc ttgccacgtg caaggcaaca tgggggtacc aggaattcca agcaatgtcc 7200 aaacctaggg tctgctCtct gggacctgaa gatacaggat ggatcagccc aggctgcaat 7260 cgagggggaa cccattacca aaaaacctga aggctaaatt gtaggtcggg tcagaggtta 7320 tttatggaaa gttatactct acctacacgg ggtctataag cctggcgcca accagaaaag 7380 gaacaaacaa cagacctagc tgggaggggc agcatcttgt tgtagggggc ggggcacatg 7440 ttctgggggt acagccagac tcagggcttg catcaatagt gacagacaga ctgagagtaa 7500 tctctctctc tctctctctc gggatagaag gaaataggtc cctctctctc actctctctc 7560 tctctcacac acacacacag acacacacac acgctctgta ggggtctact tatgctccaa 7620 gtacaaatca ggccacattt acacaaggag gtaaaggaaa agaacgttgg aggagccaca 7680 ggaccccaaa attccctgtt ttccttgaat caggcaggac ttacgcagct gggagggtgg 7740 gaagccacct agagcctgca gcgagtaagc caagttcaga gtcacagaca ccaaaagctg 7800 gtgccatgtc ccacacccgc ccacctccca cctgctcctt gacacagccc tgtgccccac 7860 aacccggctc ccagatcatc yaLi_ái_ay &lt;-L cLyyyycccg caccgtcctt cctgccacat 7920 ccccacccca ttcttggaac ctgccctctg tcttctccct tgtccaaggg caggcaaggg 7980 ctcagctatt gggcagcttt gaccaacagc tgaggcccct tttgtggctg gagatgcagg 8040

ES 2272093 T3 aggcagggga atattcctct tagtcaatgc gaccatgtgc ctggtttgcc cagggtggtc 8100 tcgtttacac ctgtaggcca agcgtaatta ttaacagctc ccacttctac tctaaaaaat 8160 gacccaatc.t gggcagtaaa ttatatggtg cccatgctat taagagctgc aacttgctgg B220 gcgtggtggc tcacacctgt aatcccagta ctttgggacg tcaaggcggg tggatcacct 8280 gttagagact gaggtcacga gcatggcaaa ggcctggcca accccatctt tactaaaaat 8340

<td>acaaaaatta</td><td>gcaaggcatg</td><td>gtggcatgca</td><td>cctgtaatcc</td><td>caggtactcg</td><td>ggaggctgag</td><td> 8400</td>

<td>acaggagaat</td><td>ggcttgaacc</td><td>caggaggcag</td><td>aggttgcagt</td><td>gagccaagac</td><td>tgtgccactg</td><td> 8460</td>

<td>ccctccagcc</td><td>ctggcaacag</td><td>agcaagactt</td><td>catctcaaaa</td><td>gaaaaaggat</td><td>actgtcaatc</td><td> 8520</td>

<td>accgcaggaa</td><td>gaacccaggt</td><td>aatgaatgag</td><td>gagaagagag</td><td>gggctgagtc</td><td>accatagtgg</td><td> 8580</td>

cagcaccgac tcctgcagga aaggcgagac actgggtcat gggtactgaa gggtgccctg 8640

<td>aatgacgttc</td><td>tgcttcagag</td><td>accgaacctg</td><td>agccctgaaa</td><td>gtgcatgcct</td><td>gttcatgggt</td><td> 8700</td>

<td>gagagactaa</td><td>attcatcatt</td><td>ccttggcagg</td><td>tactgaatcc</td><td>tttcttacgg</td><td>ctgccctcca</td><td> 8760</td>

<td>atgcccaatt</td><td>tccctacaat</td><td>tgtctggggt</td><td>gcctaagctt</td><td>ctgcccacca</td><td>agagggccag</td><td> 8820</td>

<td>agctggcagc</td><td>gagcagccgc</td><td>aggtaggaga</td><td>gacaggtacc</td><td>cataagggag</td><td>gtgggaaaga</td><td> 8880</td>

<td>gagatggaag</td><td>gagaggggtg</td><td>cagagcacac</td><td>acctcccctg</td><td>cctgacaact</td><td>tcctgagggc</td><td> 8940</td>

<td>tggtcatgcc</td><td>agcagattta</td><td>aggcggaggc</td><td>aggggagatg</td><td>gggcgggaga</td><td>ggaagtgaaa</td><td> 9000</td>

<td>aaggagaggg</td><td>tggggatgga</td><td>gaggaagaga</td><td>gggtgatcat</td><td>tcattcattc</td><td>cattgctacc</td><td> 9060</td>

<td>gactggatgc</td><td>cagctgtgag</td><td>ccaggcacca</td><td>ccctagctct</td><td>gggcatgtgg</td><td>ttgtaatctt</td><td> 9120</td>

gg agcctcat ggagctcaca gggagtgctg gcaaggagat ggataatgga cggataacaa 9180 ataaacattt agtacaatgt ccgggaatgg aaagttctcg aaagaaaaat aaagctggtg 9240 agcatataga cagccctgaa ggcgatt1gagg cgcaggcagg 93