AU775722B2 - Effector proteins of rapamycin - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: WAJye /9 The Trustees of Columbia University in the City of New York and Ameriean Homo Produets Actual Inventor(s): Katherine Molnar-Kimber, Amedeo Failli, Thomas Joseph Caggiano, KOJI NAKANISHI, YANQIU CHEN Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: EFFECTOR PROTEINS OF RAPAMYCIN Our Ref: 654213 POF Code: 794/1481, 64292 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1sooeq EFFECTOR PROTEINS OF RAPAMYCIN This application is a divisional application of Australian Patent Application 17390/99, which in turn is a divisional of Australian Patent Application 13670/95, the entire contents of both of which are herein incorporated by reference.

This invention concerns effector proteins of Rapamycin. More particularly, this invention concerns novel Rapamycin-FKBP12 binding proteins of mammalian origin for identification, design and synthesis of immunomodulatory, anti-restenosis or anti-tumor agents.

BACKGROUND OF THE INVENTION Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus which was first characterized via its properties as an antifungal agent. It adversely affects the growth of fungi such as Candida albicans and Microsporum gypseum. Rapamycin, its preparation and its antibiotic activity were described in U.S. Patent No. 3,929,992, issued December 30, 1975 to Surendra Sehgal et al. In 1977 Martel, R. R. et al. reported on immunosuppressive properties of rapamycin against experimental allergic encephalitis and adjuvant arthritis in the Canadian Journal of Physiological Pharmacology, 55, 48-51 (1977). In 1989, Calne, R. Y. et al., in Lancet, 1989, no. 2, p. 227 and Morris, R. E. and Meiser, B. M. in Medicinal Science Research, 1989, No. 17, P. 609-10, separately reported on the effectiveness of rapamycin in inhibiting rejection in vivo in allograft transplantation. Numerous articles have followed describing the immunosuppressive and rejection inhibiting properties of rapamycin, and clinical investigation has begun for the use of rapamycin in inhibiting rejection in transplantation in man.

Rapamycin alone Patent 4,885,171) or in combination with picibanil Patent 4,401,653) has been shown to have antitumor activity. R. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that W d5kanUkMpeesNv 1739099.doc -3rapamycin is effective in the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB 3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules, also have been shown to be effective as immunosuppressive agents, therefore useful in preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978); and U.S. Patent 5,100,899].

Rapamycin has also been shown to be useful in preventing or treating systemic lupus erythematosus Patent 5,078,999], pulmonary inflammation Patent 5,080,899], insulin dependent diabetes mellitus [Fifth Int. Conf.

Inflamm. Res. Assoc. 121 (Abstract), (1990)], and smooth muscle cell proliferation and intimal thickening following vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 197 (1992)].

20 Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43 positions) have been shown to be useful as antifungal agents Patent 4,316,885) and used to make water soluble prodrugs of rapamycin Patent 4,650,803). Recently, the numbering convention for rapamycin has been changed; therefore according to Chemical Abstracts nomenclature, the esters 25 described above would be at the 31- and 42- positions. U.S. Patent 5,118,678 discloses carbamates of rapamycin that are useful as immunosuppressive, antiinflammatory, antifungal, and antitumor agents. U.S. Patent 5,100,883 discloses fluorinated esters of rapamycin. U.S. Patent 5,118,677 discloses amide esters of rapamycin. U.S. Patent 5,130,307 discloses aminoesters of 30 rapamycin. U.S. Patent 5,117,203 discloses sulfonates and sulfamates of rapamycin. U.S. Patent 5,194,447 discloses sulfonylcarbamates of rapamycin.

CWNWROIE Y1SPECMI35700<fV.DOC -4- U.S. Patent No. 5,100,899 (Calne) discloses methods of inhibiting transplant rejection in mammals using rapamycin and derivatives and prodrugs thereof. Other chemotherapeutic agents listed for use with rapamycin are azathioprine, corticosteroids, cyclosporin (and cyclosporin and FK-506, or any combination thereof.

Rapamycin produces immunosuppressive effects by blocking intracellular signal transduction. Rapamycin appears to interfere with a calcium independent signalling cascade in T cells and mast cells [Schreiber et al. (1992) Tetrahedron 48:2545-2558]. Rapamycin has been shown to bind to certain immunophilins which are members of the FK-506 binding proteins (FKBP) family. In particular, Rapamycin has been shown to bind to the binding proteins, FKBP12, FKBP13, FKBP25 [Galat A. et al., (1992) Biochemistry 31(8);2427-2437 and Ferrera A, et al., (1992) Gene 113(1):125-127; Armistead and Harding, Ann. Reports in Med. Chem. 28:207-215, 1993], and FKBP52 [WO 93/07269].

Rapamycin is able to inhibit mitogen-induced T cell and B cell proliferation as well as proliferation induced by several cytokines, including IL-2, 20 IL-3, IL-4 and IL-6 (reviewed by Sehgal et al., Med. Research Rev. 14: 1-22, 1994). It can also inhibit antibody production. Rapamycin has been shown to i: block the cytokine-induced activation of p70 S 6 kinase which appears to correlate with Rapamycin's ability to decrease protein synthesis accompanying cell cycle progression (Calvo et al., Proc. Natl. Acad. Sci. USA, 89:7571-7575, 25 1992; Chung et al., Cell 69:1227-1236, 1992; Kuo et al., Nature 358:70-73, 1992; Price et al., Science 257:973-977, 1992). It also inhibits the activation of cdk2/cyclin E complex (Flanagan et al., Ann. N.Y. Acad. Sci, in press; Flanagan et al. Mol. Cell Biol., in press; Flanagan et al., J.Cell Biochem. 17A:292, 1993).

Rapamycin's effects are not mediated by direct binding to p70 56 kinase and 30 cdk2/cyclin E, but by action of the Rapamycin-FKBP complex on upstream component(s) which regulate the activation status of the kinases.

C:1W INWORLJENNYMSPECNni It is generally accepted that the action of immunosuppressive drugs, such as Rapamycin, cyclosporine and FK506, is dependent upon the formation of a complex with their respective intracellular receptor proteins called immunophilins. While the binding of these immunosuppressants with their respective immunophilins inhibits the cis-trans peptidyl prolyl isomerase (PPIase) activity of immunophilins, PPIase inhibition is not sufficient to mediate the immunosuppressive activity (reviewed in Armistead and Harding, Annual Reports in Med. Chem, 28:207-215:1993). Two rapamycin analogs which are Diels Alder adducts, one with 4-phenyl-1,2,4-triazoline-3,5-dione, and the second with 4-methyl-1,2,4-triazoline-3,5-dione, bind to FKBP, inhibited its PPIase activity, yet they did not exhibit any detectable immunosuppressive activity. The phenyl-triazolinedione Diels Alder adduct at high molar excess has been shown to competitively inhibit rapamycin's effect on DNA synthesis in mitogen-stimulated murine thymocyte proliferation (Ocain et al., Biochem.

Biophys. Res. Commun. 192:1340, 1993). Recent evidence suggests that the binary immunophilin-drug complex such as cyclophilin-cyclosporin A and FKBP- FK506 gains a new function that enables it to block signal transduction by acting on specific target proteins. The molecular target of both cyclophilincyclosporin A and FKBP-FK506 complexes such as has been identified as the 20 Ca 2/calmodulin dependent serine/threonine phosphatase calcineurin Liu et al, Cell 66, 807, 1991; J. Liu et al, Biochemistry 31, 3896, 1992; W.M.

Flanagan, et al., Nature 352, 803, 1992; McCaffrey et al., J. Biol. Chem. 268, 3747, 1993; McCaffrey et al., Science 262:750, 1993).

25 Rapamycin's antifungal and immunosuppressive activities are mediated via a complex consisting of Rapamycin, a member of the FK506 binding protein (FKBP) family and at least one additional third protein, called the target of Rapamycin (TOR). The family of FKBPs is reviewed by Armistead and Harding (Annual Reports in Med. Chem, 28:207-215:1993). The relevant FKBP 30 molecule in Rapamycin's antifungal activity has been shown to be FKBP12 (Heitman et al., Science 253:905-909:1993). In mammalian cells, the relevant FKBPs are being investigated. Although two TOR proteins (TOR1 and TOR2) C WINWO DUENNYTSPECNKI 3870IV DOC 6 have been identified in yeast (Kunz et al., Cell 73:585-596:1993), the target of Rapamycin in human cells remains elusive. The carboxy terminus of yeast TOR2 has been shown to exhibit 20% identity with two proteins, the p110 subunit of phosphatidylinositol 3-kinase and VPS34, a yeast vacuolar sorting protein also shown to have PI 3K activity. However, J Blenis et al. (AAI meeting, May, 1993) have reported that Rapamycin-FKBP12 complex does not directly mediate its effects on PDGF stimulated cells via the p110, p85, PI 3K complex.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

DESCRIPTION OF THE INVENTION In one aspect this invention provides a protein of mammalian origin having a molecular weight of from about 50 kDa to 800 kDa which binds to a GST-FKBP-Rapamycin complex, or a fragment of such a protein.

In a further aspect this invention provides isolated, clones and expressed proteins which bind to a complex of GST-FKBP12-Rapamycin (GST denotes glutathione-s-transferase). These proteins are isolated from membrane preparations of Molt 4 T cell leukemia. The sizes of the four novel proteins are estimated by PAGE migration to be 125±12 kilodaltons (kDa), 148±14 kDa, 208±15 kDa and 210±20 kDa and will be referred to herein and in the claims that follow, as the 125 kDa, 148 kDa, 208 kDa, and 210 kDa proteins, 25 respectively. The four proteins may also be referred to herein as effector proteins.

The proteins of this invention can be used in screening assays, such as enzyme inhibitor assays and binding assays, to identify endogenous complexes 30 and ligands and novel exogenous compounds (like Rapamycin) which modulate their functions. They can also be used in assays to identify compounds with therapeutic benefit for W:\ciskaklspeces782S3-O.Ooc -7restenosis, immunomodulation and as antitumor agents. Cloning the proteins of this invention does not only allow the production of large quantities of the proteins, it also provides a basis for the development of related anti-sense therapeutics. The use of cDNA clones to generate anti-sense therapeutics with immunomodulatory activity (for use against transplantation rejection, graft versus host disease, autoimmune diseases such as lupus, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, type I diabetes, and diseases of inflammation such as psoriasis,dermatitis, eczema, seborrhea, inflammatory bowel disease, pulmonary inflammation, asthma, and eye uveitis), antirestenosis and anti-tumor activity is included within the scope of this invention.

The proteins of the present invention can be isolated from mammalian cells, such as cells of the T cell leukemia cell line, Molt 4 (ATCC 1582, American Type Cell Culture, 12301 Parklawn Drive, Rockville, MD, USA, 20852), the B cell lymphoma, BJAB, or normal human T cells. These mammalian cells can be lysed in a buffer containing protease inhibitors and reducing agent such as hypotonic buffer A (100 mM HEPES, pH 7.5, 20 mM KCI, 1 mM EDTA, 0.4 mM PMSF and 2 mM beta mercaptoethanol The cell nuclei and unbroken cells are cleared by centrifugation at a temperature which minimizes protein degradation. The membrane fraction of the cells can then be concentrated or pelleted by ultracentrifugation at 20 100,000 g. Detergent solubilization of the membrane pellet is carried out in a detergent containing buffer such as buffer B (50 mM Tris, pH 7.2, 100 mM NaCI, 20 mM KCI, 0.2 mM PMSF, 1 mM 2-ME, 2 mM CaCl 2 2 mM MgCI2, 5 p.g/ml aprotinin, leupeptin, pepstatin A and antipain), containing CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate; 12 mM) or Triton X 100 (polyethylene 25 glycol 4-isooctylphenyl ether). The solubilized membrane proteins can then be separated from the debris by 100,000g ultracentrifugation at a temperature which minimizes protein degradation. The supernatant containing solubilized membrane proteins is then preabsorbed with an affinity resin, such as glutathione resin, in the presence of protease inhibitors at a temperature which minimizes protein degradation..

30 After centrifugation to remove the resin from the supernatant, the supernatant is then incubated with complexed Rapamycin or Rapamycin analog to FKBP, such as GST- FKBPl2--Rapamycin at a temperature which minimizes protein degradation. The mixture of solubilized membrane proteins, incubated with complexed Rapamycin or Rapamycin analog to FKBP, such as GST-FKBPI2--Rapamycin, can then be -8incubated with the affinity resin to bind the complexes of rapamycin or rapamycin analog, FKBP fusion protein and binding proteins at a temperature which minimizes protein degradation. After most non-specific proteins are rinsed away using a detergent containing buffer, such as Buffer C (50 mM Tris, pH 7.2, 100 mM NaCI, 20 mM KCI, 0.2 mM PMSF, 1 mM 2-ME or 10 mM dithiothreitol, 0-5 mM CaCI2, 0-5 mM MgCl 2 5 gg/ml aprotinin, leupeptin, pepstatin A and antipain and 0.1% Triton X 100) (Polyethylene glycol 4-isooctyl phenyl ether), the proteins are eluted from the resin under denaturing conditions, such as a buffer containing sufficient detergent to dissociate it from resin Laemli buffer with or without glycerol or dye, as described by Laemli, Nature 227:680, 1970), or non-denaturing conditions such as a buffer containing an appropriate eluting compound for the affinity column, such as mM glutathione. The proteins can then be separated by size using SDS polyacrylamide gel electrophoresis (SDS-PAGE).

The present invention also includes the genomic DNA sequences for the abovementioned proteins, as well as the cDNA and anti-sense RNA and DNA sequences which correspond to the genes for the abovementioned proteins. The present invention further includes the proteins of other mammalian species which are homologous or equivalent at least in function to the abovementioned proteins, as well 20 as the DNA gene sequences for the homologous or equivalent proteins and the cDNA and anti-sense RNA and DNA sequences which correspond to the genes for the homologous or equivalent proteins.

@9.9 For the purposes of this disclosure and the claims that follow, equivalents of the proteins of this invention are considered to be proteins, protein fragments and/or truncated forms with substantially similar, but not identical, amino acid sequences to the proteins mentioned above, the equivalents exhibiting rapamycin-FKBP complex binding characteristics and function similar to the proteins mentioned above.

Therefore, in this specification and the claims below, references to the 125 kDa, 148 30 kDa, 208 kDa, and 210 kDa proteins of this invention are also to be understood to indicate and encompass homologous or equivalent proteins, as well as fragmented and/or truncated forms with substantially similar, but not identical, amino acid sequences of the 125 kDa, 148 kDa, 208 kDa, and 210 kDa proteins mentioned above.

These proteins or protein homologues or equivalents can be generated by similar isolation procedures from different cell types and/or by recombinant DNA methods and may be modified by techniques including site directed mutagenesis. For example, the genes of this invention can be engineered to express one or all of the proteins as a fusion protein with the fusion partner giving an advantage in isolation HIS oligomer, immunoglobulin Fc, glutathione S-transferase, FLAG etc).

Mutations or truncations which result in a soluble form can also be generated by site directed mutagenesis and would give advantages in isolation.

This invention further includes oligopeptide fragments, truncated forms and protein fragments that retain binding affinity yet have less than the active protein's amino acid sequences. This invention also includes monoclonal and polyclonal antibodies specific for the proteins and their uses. Such uses include methods for screening for novel agents for immunomodulation and/or anti-tumor activity and methods of measuring the parent compound and/or m'etabolites in biological samples obtained from individuals taking immunosuppressive drugs. The use of the cDNA clone to generate anti-sense therapeutics (Milligan et al, J. Med. Chem. 36:1923-1936, 1993) with immunomodulatory activity (transplantation rejection, graft versus host disease, autoimmune diseases such as lupus, myasthenia gravis, multiple sclerosis, 20 rheumatoid arthritis, type I diabetes, and diseases of inflammmation such as psoriasis, dermatitis.czema, seborrhea, inflammatory bowel disease, pulmonary inflammation, asthma. and eye uveitis), and anti-tumor activity is also included in the present invention.

The proteins of this invention can also be made by recombinant DNA techniques familiar to those skilled in the art. That is, the gene of the protein in S, question can be cloned by obtaining a partial amino acid sequence by digestion of the protein with a protease, such as Lysine C, and isolating the resulting protein fragments by microbore HPLC, followed by fragment sequencing (Matsudaira in A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press (San Diego, CA, 1989)). The determined sequence can then be used to make oligonucleotide probes which can be used to screen a human cDNA library directly or generate probes by polymerase chain reaction. The library can be generated from human T cells or the cell lines, Molt 4, Jurkat, or other etc. to obtain clones. These clones can be used to identify additional clones containing additional sequences until the protein's full gene, i.e. complete open reading frame, is cloned.

It is known in the art that some proteins can be encoded by an open reading frame which is longer than initially predicted by the size of the protein. These proteins may represent cleavage products of the precursor protein translated from the complete open reading frame (eg. IL-1 beta) or proteins translated using a downstream start codon (eg. HepatitisB surface antigen). In view of this knowledge, it is understood that the term cDNA as used herein and in the claims below refers to cDNA for the gene's complete open reading frame or any portions thereof which may code for a protein of this invention or the protein's fragments, together or separate, or truncated forms, as previously discussed.

In a complementary strategy, the gene(s) for the proteins of this invention may be identified by interactive yeast cloning techniques using FKBP12:RAPA as a trap for cloning. These strategies can also be combined to quicken the identification of the clones.

The relevant cDNA clone encoding the gene for any of the four proteins can 20 also be expressed in E. coli, yeast, or baculovirus infected cells or mammalian cells using state of the art expression vectors. Isolation can be performed as above or the cDNA can be made as a fusion protein with the fusion partner giving an advantage in isolation HIS oligomer, immunoglobulin Fc, glutathione S-transferase, etc).

Mutations which result in a soluble form can also be generated by site directed mutagenesis and would give advantages in isolation.

The uses of such cDNA clones include production of recombinant proteins.

Further, such recombinant proteins, or the corresponding natural proteins isolated from mammalian cells, or fragments thereof (including peptide oligomers) are useful in generation of antibodies to these proteins. Briefly, monoclonal or polyclonal antibodies are induced by immunization with recombinant proteins, or the corresponding natural proteins isolated from mammalian cells, or fragments thereof (including peptide oligomers conjugated to a carrier protein keyhole limpet hemocyanin or bovine serum albumin)) of animals using state of the art techniques. The antibodies can be -11used in the purification process of the natural proteins isolated from mammalian cells or recombinant proteins from E. coli, yeast, or baculovirus infected cells or mammalian cells, or cell products.

The uses of such cDNA clones include production of recombinant proteins.

Further, such recombinant proteins, or the corresponding natural proteins isolated from mammalian cells, are useful in methods of screening for novel agents such as synthetic compounds, natural products, exogenous or endogenous substrates for immunomodulation and/or antitumor activity. The natural products which may be screened may include, but are not limited to, cell lysates, cell supernatants, plant extracts and the natural broths of fungi or bacteria. As an example of a competitive binding assay, one of these proteins attached to a matrix (either covalently or noncovalently) can be incubated with a buffer containing the compounds, natural products, cell lysates or cell supernatants and a labeled rapamycin:FKBP complex. The ability of the compound, natural products, exogenous or endogenous substrates to competitively inhibit the binding of the complex or specific antibody can be assessed.

Examples of methods for labeling the complex include radiolabeling, fluorescent or chemiluminescent tags, fusion proteins with FKBP such as luciferase, and conjugation to enzymes such as horse radish peroxidase, alkaline phosphatase, acetylcholine 20 esterase (ACHE), etc. As an example of an enzymatic assay, the proteins are incubated in the presence or absence of novel agents such as synthetic compounds, natural products, exogenous or endogenous substrates with substrate and the enzymatic activity of the protein can be assessed. Methods of measuring the parent compound and/or metabolites in biological samples obtained from individuals taking immunosuppressive drugs can also be assessed using these proteins.

This invention includes a method for identifying substances which may be useful as immunomodulatory agents or anti-tumor agents, the method utilizing the following steps: a) combining the substance to be tested with one of the four mammalian proteins (125 kDa, 148kDa, 208 kDa or 210 kDa) of this invention, with the protein being bound to a solid support: -12b) maintaining the substance to be tested and the protein bound to the solid support of step under conditions appropriate for binding of the substance to be tested with the protein, and c) determining whether binding of the substance to be tested occurred in step This invention also includes a method for identifying substances which may be useful as immunomodulatory or anti-tumor agents which involves the following steps: a) combining a substance to be tested with one of the mammalian proteins of this invention, the protein being bound to a solid support: b) maintaining the substance to be tested and the protein bound to the solid support of step under conditions appropriate for binding of the substance to be tested with the protein, and c) determining whether the presence of the substance to be tested modulated the activity of the mammalian protein.

This invention further includes a method for detecting, in a biological sample, rapamycin, rapamycin analogs or rapamycin metabolites which, when complexed with a FKBP, bind to one of the four listed proteins of this invention, the method comprising the steps of: a) combining the biological sample with a FKBP to form a first mixture containing, if rapamycin, rapamycin analogs or rapamycin metabolites are present in the biological sample, a rapamycin:FKBP complexes, rapamycin ~analog:FKBP complexes, or rapamycin metabolite:FKBP complexes; b) creating a second mixture by adding the first mixture to one of the proteins of this invention, the protein bound to a solid support; -13c) maintaining the second mixture of step under conditions appropriate for binding the rapamycin:FKBP complexes, rapamycin analog:FKBP complexes, or'rapamycin metabolite:FKBP complexes, if present, to the protein of this invention; and d) determining whether binding of the rapamycin:FKBP complexes, rapamycin analog:FKBP complexes, or rapamycin metabolite:FKBP complexes and the protein occurred in step Also included in this invention is the use of the cDNA clones to generate antisense therapeutics. This can be accomplished by using state of the art techniques, such as those described in Milligan et al, J. Med. Chem. 36:14:1924-1936. For the purposes of this disclosure and the claims that follow, antisense RNA and DNA are understood to include those RNA and DNA strands derived from a cDNA clone which encodes for one of the four proteins (125 kDa, 148 kDa, 208 kDa or 210 kDa) of the present invention which have a native backbone or those which utilize a modified backbone. Such modifications of the RNA and DNA backbones are described in Milligan et al, J. Med. Chem. 36:14:1924-1936. The antisense compounds created by the state of the art techniques recently described (Milligan et al, J. Med. Chem.

20 36:14:1924-1936) can be useful in modulating the immune response and thus useful in the treatment or inhibition of transplantation rejection such as kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small bowel, and skin allografts, and heart valve xenografts; in the treatment or inhibition of autoimmune diseases such as luptis, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis; and S 25 diseases of inflammation such as psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, and eye uveitis. The antisense molecules of this invention can have antitumor, antifungal activities, and antiproliferative activities. The compounds of this invention therefore can be also useful in treating solid tumors, adult "T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases such as restenosis and atherosclerosis. Thus, the present invention also comprises methods for treating the abovementioned maladies and conditions in mammals, preferably in humans. The method comprises administering to a mammal in need thereof an effective amount of the relevant antisense therapeutic agent of this invention.

-14- When administered for the treatment or inhibition of the above disease states, the antisense molecules of this invention can be administered to a mammal orally, parenterally, intranasally, intrabronchially, transdermally, topically, intravaginally, or rectally.

It is contemplated that when the antisense molecules of this invention are used as an immunosuppressive or antiinflammatory agent, they can be administered in conjunction with one or more other immunoregulatory agents. Such other immunoregulatory agents include, but are not limited to azathioprine, corticosteroids, such as prednisone and methylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506, OKT-3, and ATG. By combining the complexes of this invention with such other drugs or agents for inducing immunosuppression or treating inflammatory conditions, the lesser amounts of each of the agents are required to achieve the desired effect. The basis for such combination therapy was established by Stepkowski whose results showed that the use of a combination of rapamycin and cyclosporin A at subtherapeutic doses significantly prolonged heart allograft survival time. [Transplantation Proc. 23: 507 (1991)].

Treatment with these antisense compounds will generally be initiated with small 20 dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached. Precise dosages will be determined by the administering physician based on experience with the individual subject treated. In general, the antisense compounds of this invention are most desirably administered at a concentration that will afford effective results without 25 causing any harmful or deleterious side effects.

In light of the therapeutic value of the abovementioned antisense compounds, *this invention also includes pharmaceutical compositions containing the antisense RNA and antisense DNA compounds derived from cDNA clones which encode for the 125 kDa, 148 kDa, 208 kDa and 210 kDa proteins of this invention.

This invention also comprises the following process for isolating the proteins of this invention, as well as the proteins isolated therefrom: A process for isolating proteins from mammalian cells, the process comprising the steps of: 1. The mammalian cells of interest are grown and harvested. As mentioned previously, the cells may be of T cell origin T cell lymphomas, leukemias, normal human T cells), B cell origin EBV transformed B cells, normal human B cells), mast cells, or other cell sources sensitive to rapamycin. The cells may be processed shortly after harvesting or may be stored frozen, such as in pellets, prior to processing. The cells which are kept frozen may be stored in a dry ice and ethanol bath, stored frozen at -70-80' C until use. This step of growing and harvesting the cells of interest may be seen as the first step of this process or as merely preparatory for the present process.

2. Cells are lysed in a buffer containing a buffering agent (e.g.HEPES, Tris, pH low salt (e.g.10 -50 mM NaCI or KCI), chelating agent 1-2 mM EDTA), protease inhibitors (e.g.0.4 mM PMSF) and a reducing agent 2 mM 2-ME or 1-20 mM Dithiothreitol) at a temperature which minimizes protein degradation 4 It should be understood that the mammalian cells may be treated in any manner capable of producing cell lysis, including sonic lysis and 20 douncing.

3. Unbroken cells and cell nuclei are precleared from lysates by centrifugation at a temperature which minimizes protein degradation 4 Centrifugation at, for example, 1600g for 10 minutes has been found sufficient to preclear the unbroken cells and cell nuclei from the lysates. This step, while not mandatory, provides a clearer preparation for the steps that follow.

4. The membrane fraction in the precleared lysate is then concentrated, such as by ultracentrifugation. An example of this concentration would be ultracentrifugation at 100,000 g for 1-1.5 hours.

The membrane proteins transmembrane, integral and membrane associated proteins) are then solubilized. This may be accomplished by incubating the pellet of Step 4 in a buffer containing a detergent which solubilizes the -16proteins without detrimentally denaturing them, a buffering agent 20-50 mM Tris or HEPES, pH salt 100 200 mM NaCI 20 mM KC1), reducing agent 1-2 mM 2-ME or 1 20 mM dithiothreitol), protease inhibitors 0.2 mM PMSF, 5 g.g/ml aprotinin, leupeptin, pepstatin A and antipain), divalent cations 0- 5 mM CaCI 2 0-5 mM MgCl 2 at a temperature which minimizes protein degradation 4 C) Examples of detergents useful in this step are CHAPSO cholamidopropyl)dimethylammonio]- 1-propane sulfonate) or Triton XI00 (polyethylene glycol 4-isooctylphenyl ether). After this step, the mixture contains solubilized membrane proteins and non-solubilized cellular debris.

6. The solubilized membrane proteins are separated from the nonsolubilized cellular debris, such as by ultracentrifugation (eg 100,000g for 1-1.5 hours) at a temperature which minimizes protein degradation 4 7. The supernatant containing solubilized membrane proteins is incubated with an affinity resin in a buffer containing a buffering agent (e.g.20-50 mM Tris or HEPES, pH salt 100 200 mM NaCI 20 mM KCI), reducing agent 1-2 mM 2-ME or 10 20 mM dithiothreitol), protease inhibitors 0.2 mM PMSF, 5 p.g/ml aprotinin, leupeptin, pepstatin A and antipain), divalent cations 0-5 mM CaC12, 0-5 mM MgCl 2 at a temperature and time which allows the absorption of the proteins which bind to affinity resin directly, and minimizes protein degradation 4 8. The resin is then removed from the supernatant by centrifugation at a temperature which minimizes protein degradation 4 9. The supematant is then incubated with Rapamycin or Rapamycin analog (IC50 in LAF 500nM) complexed to fusion protein of FKBP12 +protein which enhances the isolation of the desired effector protein and through which the fusion protein binds to an affinity resin or affinity column, such as GST-FKBP12, Histidine oligomer -FKBPI2, biotin-FKBP12, etc., in a buffer containing a buffering agent 20-50 mM Tris or HEPES, pH salt 100 200 mM NaCI mM KC1), reducing agent 1-2 mM 2-ME or 1 20 mM dithiothreitol), protease inhibitors 0.2 mM PMSF, 5 .g/ml aprotinin, leupeptin, pepstatin A and antipain), -17divalent cations 0-5 mM CaCI 2 0-5 mM MgCI 2 at a temperature and for a time which allows binding of the effector proteins to the fusion FKBP protein:Rapamycin or analog complexes and minimizes protein degradation 4 *C and 1-2 hours).

10. The mixture of Step 9 containing the effector proteins and fusion FKBP protein:Rapamycin complexes is incubated with an affinity resin at a temperature and for a time which allows binding of the complexes of the effector proteins and fusion FKBP protein:Rapamycin or analog to the affinity resin and minimizes protein degradation 4 'C and 0.5-2 hours).

11. Most non-specific proteins are rinsed away from the resin using a buffer which dissociates binding of non-specific proteins but not the complex between the desired proteins and RAPA-FKBP, such as a buffer containing a buffering agent (e.g.20-50 mM Tris or HEPES, pH salts 100 1000 mM NaCI, KCI), reducing agent 1-2 mM 2-ME or 10 20 mM dithiothreitol), protease inhibitors 0.2 mM PMSF, 5 gg/ml aprotinin, leupeptin, pepstatin A and antipain), divalent cations 0-5 mM CaC12, 0-5 mM MgCl 2 and detergent which dissociates binding o. of non-specific proteins but not the complex between the four proteins and RAPAfusion FKBP protein such as Triton X100 (Polyethylene glycol 4-isooctyl phenyl 20 ether).

12. The effector proteins and the fusion FKBP protein:Rapamycin complexes are eluted from the resin using an appropriate buffer, such as a buffer containing sufficient detergent to dissociate it from resin Laemli buffer with or 25 without glycerol or dye, Laemli, Nature 227:680, 1970), or an appropriate eluting i compound for the affinity column, such as glutathione, histidine.

13. The effector proteins can then be separated by size. This may be accomplished in any manner which separates the proteins by size, including, but not limited to, polyacrylamide gel electrophoresis and size exclusion column chromatography.

It might also be useful to compare the proteins isolated by a control procedure, that is a procedure which substitutes buffer for the rapamycin or rapamycin analog with -18an IC50 in LAF 500 nM in step 8, can be used to more easily distinguish proteins which bind to the rapamycin:FKBP complex.

The proteins of this invention can also be made by recombinant DNA techniques familiar to those skilled in the art. That is, the gene of the protein in question can be cloned by obtaining a partial amino acid sequence by digestion of the protein with an appropriate endopeptidase, such as Lysine C, and isolating the resulting protein fragments by microbore HPLC, followed by fragment sequencing (Matsudaira in A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, San Diego, CA 1989). The determined sequence can then be used to make oligonucleotide probes which can be used to screen a human cDNA library, such as those for human T cells, Molt 4, Jurkat, etc, to obtain clones.(Sambrook, Fritsch, and Maniatas, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, 1989) These clones can be used to identify additional clones containing additional sequences until the protein's full gene is cloned (Sambrook, Fritsch, and Maniatas, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, 1989). In a complementary strategy, the gene(s) may be identified by interactive yeast cloning techniques using FKBPl2:RAPA as a trap for cloning (Chien et al., Proc. Natl. Acad. Sci. 88: 9578-9582, 1991). These strategies 20 can also be combined to quicken the identification of the clones.

The relevant cDNA clone can also be expressed in E.coli, yeast, or baculovirus infected cells or mammalian cells using state of the art expression vectors. Isolation can be performed as above or the cDNA can be made as a fusion protein with the fusion 25 partner giving an advantage in isolation HIS oligomer, immunoglobulin Fc, glutathione S-transferase, etc). Mutations which result in a soluble form can also be generated by site directed mutagenesis and would give advantages in isolation.

Homologs in the mouse, rat, monkey, dog and other mammalian species can be obtained using similar procedures. In addition, upon isolation of the human clone of the proteins, the clone can be used to screen for homologs in other mammalian species.

These homologs can also be used to develop binding assays and to set up high through put screening assays for compounds, endogenous ligands, exogenous ligands with immunomodulatory activity.

-19- Compounds, endogenous ligands and exogenous ligands having such immunomodulatory activity would can be useful in modulating the immune response and thus useful in the treatment or inhibition of transplantation rejection such as kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small bowel, and skin allografts, and heart valve xenografts; in the treatment or inhibition of autoimmune diseases such as lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis; and diseases of inflammation such as psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, and eye uveitis.

The compounds, endogenous ligands and exogenous ligands mentioned above can also have antitumor, antifungal activities, and antiproliferative activities. The compounds of this invention therefore can be also useful in treating solid tumors, adult T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases such as restenosis and atherosclerosis.

*0 EXAMPLE 1 The proteins of the present invention were isolated utilizing a fusion protein of 20 glutathione S-transferase--FK506 binding proteinl2 (GST-FKBP). GST-FKBP is produced by a recombinant E. coli containing the plasmid, pGEX-FKBP. The cells were grown, induced with IPTG and the fusion protein was isolated using standard technology described in D.B. Smith and K.S. Johnson, Gene 67, 31, 1988 and K.L.

Guan and J.E. Dixon, Anal. Biochem. 192, 262, 1991. The solution containing glutathione and GST-FKBP12 was exchanged 5x using a Centricon 10 filtration unit (Amicon) to remove the glutathione and exchange the buffer.

Molt 4 cells (Ix 10 9 were grown in standard media (RPMI 1640 containing 100 U/ml pennicillin, 100 ug/ml L-glutamine, 10% FCS). The cells were harvested and rinsed 3x with PBS (50mM phosphate buffer, pH 7.0, 150 mM NaCI), flash frozen in dry-ice ethanol bath and stored at -80°C. On ice, the cells were thawed and lysed using a dounce homogenizer with B pestle in 5 ml of buffer A (10 mM Hepes, pH 7.5, mM KCI, 1 mM EDTA, 0.4 mM PMSF and 2 mM 2-ME). After the debris was cleared by centrifugation at 1600g for 10 min. and the membrane fraction was concentrated by 100,000g centrifugation (1 hour), the 100,000 g pellet was incubated in 3 ml buffer B (50 mM Tris, pH 7.2, 100 mM NaCI, 20 mM KCI, 0.2 mM PMSF, 1 mM 2-ME, 2 mM CaCI2, 2 mM MgCl2, 5 pg/ml aprotinin, leupeptin, pepstatin A and antipain), containing 12 mM CHAPSO for two hours at 4°C. The solubilized membrane proteins were separated from the debris by a 100,000 g centrifugation. After preabsorption of the supernatant for 3-18 hours with 0.4 ml glutathione sepharose resin swollen in buffer B, the supernatant was incubated with complexed Rapamycin-GST-FKBP12 (preformed by incubation of 660 ug GST- FKBP 60 ug RAPA in buffer B for 1-2 hours, 40C) for two hours at 4°C. The supernatant was then incubated for 2 hours at 4°C with 100 ul glutathione resin (1:1 Buffer Nonspecific proteins were rinsed 5x with buffer C (buffer B 0.1% Triton x 100) and the proteins eluted from the resin in Laemli buffer by incubation at 950C for 3 minutes and microcentrifugation. The proteins were separated by size using a 7% SDS-PAGE followed by silver stain. Four bands corresponding to proteins of molecular weights of 210kDa, 208 kDa, 148 kDa, and 125 kDa were present in higher concentrations in the sample containing RAPA GST-FKBP12 vs GST- FKBP alone.

The mitogen-stimulated thymocyte proliferation assay called the LAF 20 (Lymphocyte Activation Factor assay, also known as the comotigen-induced lymphocyte proliferation procedure), can be inhibited by rapamycin or analogs such as demethoxyrapamycin and indicates relative activity of rapamycin analogs in immunosuppression. The same proteins were isolated using GST-FKBP complexed with the immunosuppressive analog, demethoxyrapamycin (Table 1).

The Diels Alder adducts bound to FKBP12 and inhibited PPlase activity of FKBP12 but did not exhibit detectable immunosuppressive activity and thus do not bind to the target of rapamycin. The use of these two compounds complexed with GST- FKBP12 in the analogous isolation procedure (ie. replacing rapamycin:GST- FKBP12) yielded background levels of the 210kDa proteins (no rapamycin)(Table FK506, is an immunosuppressive compound which binds to FKBP and mediates at least some of its effects through the binding of the FK506-FKBP complex with calcineurin. FK506 when complexed with GST-FKBP in an analogous procedure yielded only background levels of the 210 kDa protein (Table 1).

C:'WWORD'oUENNYlSPEC 1370IV.DOC -21- TABLE 1 Comparison of Binding of Rapamvcin Analo2--FKBP12 complexes to 210 kDa Protein Compound 210 kDa LAF PPase(Ki) RAPA 6 nM 0.12nM demethoxyrapamycin 58nM 4.4 nM Diels Alder adduct (phenyl) >1000nM 12 nM Diels Alder adduct (methyl) >1000nM 12 nM FK506 3nM* 0.4 nM none (FKBP) mechanism of action is different) It is known that rapamycin must bind to a member of the FKBP family in order to mediate its effects. To verify that the proteins of this invention bind to the complex RAPA-GST-FKBP and not individually to rapamycin or FKBP12, a modified isolation 20 procedure was employed. The modification consists of using a rapamycin-42biotin glycinate ester in place of rapamycin (both exhibit equivalent immunosuppressive activity in the LAF assay), no exogenous FKBP and a strepatavidin-conjugated resin in place of glutathione-resin. Only background levels of the 210 kDa protein was isolated using this modified isolation procedure.

*The 210 kDa protein was isolated using the GST--FKBP12--rapamycin complex from BJAB cells (B cell lymphoma) and normal human T lymphocytes *'purified by Ficoll-Hypaque and T cell columns.

The results of the partial amino acid composition analysis are set forth in Table 2, below. It should be noted that the percentage of the basic amino acids was not determined.

-22- TABLE 2 Peak Number Component Retention Peak Response Time Area Factor Peak Concentration ame Height No

I

2 3 4 5 6 2)0 7 8 9 Asp/Asn Thr Ser GluIGln Prp, Gly Ala Val Met Die Leu nLeu 9.38 11.09 12.06 12.47076 0.02344 0.05 142 0.30 13.05 2.92898 0.00000 0.00985 0.068 13.78 6.43968 0.00000 0.01995 0.15 a a 15.68 16.87 18.24 22.35 22.90 23.73 2 6.0 6 28.81 29.39 32.28 34.10 35.09 36.27 25.47273 0.00000 2 1.50384 0.00000 0.05285 0.59 0.14 0.04645 -0.44 16.69160 0.00000 0.03113 0.36 4.83196 3.00560 5.73202 20.48232 0.00000 0.00605 0.11 0.2326 0.02331 0.00782 0.0 1372 0.0699 0. 1383 0.4453 0.02174 0.04286 -23- TABLE 2 (Cont'd) Peak Component Retention Peak Response Peak Concentration Number Name Time Area Factor Heisht 11 Tyr 38.33 1.44792 0.02618 0.00226 0.0379 12 Phe 40.05 1.25017 0.02703 0.00187 0.0338 13 His 47.79 1.50905 0.02553 0.00580 0.0385 14 51.80 12.66136 0.00000 0.01960 0.0000 15 Lys 53.34 9.90767 0.02283 0.02274 0.2262 Totals 146.53645 0.33436 Not Determined 144.29 EXAMPLE 2 The 210 kDa (210±20 kDa) protein of this invention was isolated from 4 x 20 1011 Molt 4 cells using the affinity matrix protocol as described previously. Bound proteins were eluted from the affinity matrix with lx Laemli buffer without glycerol and dye (0.0625 M Tris-HCI, ph6.8, 2% SDS, 0.37M b-mercaptoethanol) and were concentrated 3 consecutive times by centrifugation using centricon 100 (Amicon, Beverly, MA) at 4 "C the first two times and at 18 °C the third time. The concentrated sample was eluted from the centricon 100 filter by incubating 2 hours at room temperature with an equal volume of 2 x laemli buffer without glycerol and dye the first 2 x and 2 x laemli buffer the third time. The proteins in the sample were separated by PAGE on a 1.5mm thick 7% polyacrylamide gel The proteins were transferred to polyvinylidine difluoride, PVDF, (Biorad, Hercules, CA) in 10 x Tris/glycine buffer 30 (Biorad) containing 0.037% SDS at 50 mAmps at 4 °C overnight. The proteins on the PVDF were stained with amido black (Biorad) in 10% ethanol, 2% acetic acid and the appropriate band was excised, rinsed with PBS and water and stored frozen.

-24- Sequencing The protein (approx. 3 ug) on the PVDF membrane was digested in situ with trypsin using a modification described by J. Fernandez et al, (Anal.Biochem. 201: 255- 64, 1992 Briefly, the PVDF was cut into 1 mm 2 pieces, prewet, and the protein digested in a 100mM Tris-HCI, pH buffer containing 10% acetonitrile, and 1% reduced triton (CalBiochem) with 0.2ug trypsin at 37 "C for 6 hours followed by addition of 0.2 ug trypsin and incubation overnight. The fragments were eluted from the membrane by sonication and the buffer containing the fragments were separated by microfuge centifugation. The membranes were backextracted 2x 50 ul buffer was added to membranes, sonicated, and centrifuged in a microfuge and solution pooled with the original buffer containing the eluted fragments.) The sample (140-145 ul) was separated by narrow bore high performance liquid chromatography using a Vydac C18 2.1mm x 150 mm reverse phase column on a Hewlett Packard HPLC 1090 with a 40 diode array detector as described previously by W.Lane et al, (J.Protein Chem., 10(2): 151-60, 1991). Multiple fractions were collected and measured for absorption at multiple wavelengths (210, 277 and 292 nm). Optimal fractions were chosen for sequencing based on resolution, symmetry, and ultraviolet absorption and spectra (210 nm, 277 nm and 292 An aliquot of the optimal fractions was analyzed for S"20 homogeneity and length of fragment by matrix assisted laser desorption time of flight mass spectrometry, MALDE-TOF-MS, on a Finnigan lasermat. Selected optimal fractions were sequenced by automated Edman degradation on an Applied Biosystems 477A protein sequencer using microcartridge and manufacturer's recommended chemistry cycle.

Sequence comparison Comparison was performed using the Intelligenetics suite (Intelligenetics, CA).

Sequences Utilizing the methods mentioned above, it was determined that the 210 kDa (210±20 kDa) protein of this invention contains peptide fragments, four of which have amino acid sequences as shown below: a) ILLNIEHR; b) LIRPYMEPILK; c) DXMEAQE; and d) QLDHPLPTVHPQVTYAYM(K) Those skilled in the art will recognize the one-letter symbols for the amino acids in question (the definitions for which can also be seen at page 21 of the text Biochemistry, Third Edition, W.H. Freeman and Company, 1988 by Lubert Stryer).

Those so skilled will also understand that the X in sequence c) indicates an as yet unidentified amino acid and the parentheses in sequence d) indicates that the amino acid in the position in question is possibly lysine.

As mentioned previously, the present invention includes fragmented or truncated forms of the proteins mentioned herein. This includes proteins which have as part or all of their amino acid sequence one or more of the four sequences listed as a)above. For the purposes of the claims, below, the proteins referred to as including 20 one or more of the "internal amino acid sequences" are understood to be any protein which contains one of the sequences listed above, whether the protein is comprised wholly of one or more of the sequences or whether one or more of the sequences mentioned above form any portion of the protein. This is understood to include all locations on the protein's amino acid sequence including, but not limited to, those 25 sections of the protein which initiate and terminate the protein's amino acid chain.

These partial amino acid sequences were compared with sequences in the Genbank database. There was identity with the sequence, accession number L34075 (Brown et al., Nature 369, 756-758 (1994)). The cDNA of the SEP gene was cloned 30 as follows: Two micrograms of Molt 4 cDNA (Clontech, Palo Alto,CA) in 1 x PCR buffer (10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1 mM MgCI, 200 jM dATP, 200 M dTTP, 200 jiM dCTP, 200 gM dGTP; Perkin Elmer, with 1 unit Taq polymerase (Perkin Elmer),was amplified by Polymerase chain reaction (PCR) at 94 C for 30 sec., 66 C for 4 min for 30 cycles, 72 C for 10 min by three separate reactions containing one of the following pairs of oligomers: -26- CGATCGGTCGACTGCAGCACTGGGGATTGTGCTCTC and GCGGCCGCAGCFITCTTCATGCATGACAACAGCCCAGGC; or GCGGCCGCAAGCTTCAAGTATGCAAGCCTGTGCGGCAAGA and CGATCGGTCGACACCTTCTGCATCAGAGTCAAGTGGTCA; or GCGGCCGCAAGCCCTCAGCTCACATCCTTAGAGCTGCA and

CGATCGGTCGACTTATTACCAGAAAGGGCACCAGCCAATATA

The oligonucleotides were synthesized and isolated by methods previously described and known in the art (Chemical and Enzymatic Synthesis of Gene Fragments, ed. by H.G.Gassin and Anne Lang, Verlag Chemie, FLA, 1982). The resulting PCR products named SEP3, SEP4, and SEPS, respectively, were incubated at 15 C overnight in buffer containing T4 DNA ligase (1 unit) and 50 ng pcII which was modified to efficiently ligate PCR products (TA cloning kit, Invitrogen, San Diego, CA) to yield PCR-pcII ligated products. The PCR-pcII products were transformed into competent E. coli INValphaF cells obtained commercially from Invitrogen. Miniprep DNA was prepared using the Quiagen miniprep kits (Quiagen, Chatsworth, CA) and the clones containing the appropriate sized PCR product were identified by restriction enzyme digestion with commercially available HindIII or Sal I, electrophoresis, and comparison to standards. Sep2 and Sepl cDNA was made using the TimeSaver cDNA synthesis Kit (Pharmacia, Piscataway, NJ) with the first strand synthesis reaction containing oligodT (0.13 jgg) and 250 pmoles of CGATCGGTCGACCAGATGAGCACATCATAGCGCTGATGAor

CGATCGGTCGACAAATTCAAAGCTGCCAAGCGTTCGGAG,

respectively. Sep2 and SepI second strand synthesis was performed using the TimeSaver cDNA synthesis kit with the addition of 250 pmoles of SGCGGCCGCAAGCTTGGCTCGAGCAATGGGGCCAGGCA or 30 GCGGCCGCAAGCTTAAGATGCTTGGAACCGCACC 30 GCGGCCGCAAGCTTAAGATGCTGGAACCGCACCTGCCG, respectively. The Sep2 and Sep1 cDNA was then amplified by PCR using -27- CGATCGGTCGACCAGATGAGCACATCATAGCGCTGATGA and GCGGCCGCAAGCTTTGGCTCGAGCAATGGGGCCAGGCA or GCGGCCGCAAGCTTAAGATGCTTGGAACCGCACCTGCCG and

CGATCGGTCGACAAATTCAAAGCTGCCAAGCGTTCGGAG,

respectively as described above. The Sep2 PCR products were cloned into pcII using the TA cloning kit (Invitrogen). The Sep 1 PCR products were digested with Hind m and Sal I, separated from the pcII vector by agarose electrophoresis. The Sepl (Hindml-SalI) fragment was isolated using the Sephaglas bandprep kit from Pharmacia and cloned into the HindIII and Sal I sites of pUC19 as described (Sambrook et al., Molecular Cloning Cold Spring Harbor, 1989). Ligation of the isolated Sep2(HindEII, AspI) and Sep3(AspI, Sail) fragments or Sep4(HindIII, AccIII/MroI) and Sal I) fragments into pUC18(HindIII, Sall) vector and transformation of competent E. coli INValphaF cells (Invitrogen) was performed by techniques known to those skilled in the art (Sambrook et al., Molecular Cloning Cold Spring Harbor, 1989) to obtain pUC18-Sep 23 and pUC18-Sep45 which contain o. nucleotides 1468- 5326 and 4964 7653, respectively, of the full length clone shown in the attached Sequence No. 1. Ligation of the pUC19-Sepl (EcoRV, Sail), Sep23 (EcoRV, BstEII) and Sep45 (BstEII, Sail) fragments and transformation of competent 20 E. coli INValphaF cells (Invitrogen) can be performed by techniques known to those skilled in the art (as described by Sambrook et al., Molecular Cloning Cold Spring Harbor, 1989) to obtain the full length clone. The nucleic acid sequence coding for this protein is shown in Sequence No. 1.

25 A fusion protein, called glutathione S transferase-sirolimus effector protein, GST-SEP, was engineered by subcloning the Sep4 and Sep5 fragments into the plasmid, pGEX-KG (Guan, K. and Dixon, J.E. (1991) Anal. Biochem. 192, 262-267) as follows. Briefly, Sep4 was digested with commercially available HindlII restriction enzyme, the restriction site was filled in with the Klenow fragment of DNA polymerase (Gibco), and the DNA was extracted with phenol-chloroform and ethanol precipitated using techniques known by those skilled in the art (Sambrook et al., Molecular Cloning Cold Spring Harbor, 1989). The SEP4 (HindIII-Klenow) was further digested with MroI restriction enzyme, separated from the pcII vector by agarose electrophoresis and isolated as the fragment SEP4-HindlII-Klenow-MroI. Sep5 fragment was prepared by -28digestion with SalI and Mrol, separated from the pclI vector by agarose electrophoresis and isolated as the fragment SEP5-SalI-MroI. pGEX-KG (Guan, K. and Dixon, J.E.

(1991) Anal. Biochem. 192, 262-267) was digested with Nco I, filled in with the Klenow fragment of DNA polymerase and the DNA was extracted with phenolchloroform and ethanol precipitated, using techniques of those skilled in the art (Sambrook et al., Molecular Cloning Cold Spring Harbor, 1989). pGEX-KG (Ncol, Klenow) was further digested with Sal I, separated from the undigested vector by agarose electrophoresis and isolated as the vector pGEX-KG-Ncol-Klenow-SalI, using techniques of those skilled in the art. Ligation of the vector, pGEX-KG-NcoI-Klenow- Sall and Sep 4 (HindIII, Mrol) and Sep5 (Mrol, Sail) fragments and transformation into E. coli strain INValphaF cells (Invitrogen) using techniques of those skilled in. the art yielded the plasmid, pGEX-Sep45. The DNA and protein sequence of this fusion protein is shown in Sequence No. 2.

Flag sequences can be added at the amino terminal end, within SEP or at the carboxy terminus of SEP, SEP4,5 or other fragments using an oligonucleotide which includes the coding sequence for Asp Tyr Lys Asp Asp Asp Asp Lys. The fusion protein can be isolated by affinity chromatography with anti-flag specific antibodies using the commercially available kits from IBI, New Haven, Conn.

Transformed host cells containing the pUC19-Sepl (EcoRV, Sall), Sep23 (EcoRV, BstEII) and Sep45 (BstEII, Sal) fragments as shown in Sequence No. 1 were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA, on 2 March 1995 25 and were assigned the following accession numbers: oo a -29- Sample (all in INV alpha ATCC Accession Number pucl8-Sep 23 69753 pucl8-Sep 45 69754 pGEX-Sep 45 69755 puc-19-Sep 1 69756 EXAMPLE 3 The 210 kDa protein of this invention was also isolated by the techniques described in Example 1 utilizing the following rapamycin analogs: a) 42-Deoxy-42-[1 -(1,1-dimethylethoxy)-2-oxoethoxy] rapamycin (which is described in U.S. Patent. No. 5,233,036); b) 42-[O-[(1,1-Dimethylethyl)dimethylsilyl]] rapamycin (described in U.S. Patent. No. 5,120,842); c) Rapamycin 42-ester with N-[1,1-dimethylethoxy)carbonyl]-Nmethylglycine (described in U.S. Patent. No. 5,130,307); d) Rapamycin 42-ester with 5-(1,1-dimethylethoxy)-2-[[(1,1acid ethyl acetate solvate three 20 quarter hydrate (see U.S. Patent. No. 5,130,307); e) Rapamycin 42-ester with 1dimethylethoxy)carbonyl]glycylglycine hydrate (see U.S. Patent. No.

5,130,307); and f) Rapamycin 42-ester with N2, N6-bis[(1,1- 25 dimethylethoxy)carbonyl]-L-lysine (see U.S. Patent. No. 5,130,307).

C:1WINWORDUENNYMSPECNeu35f70Drv O0C SEQUENCE LISTING GENERAL INFORMATION: INVENTORS: Molnar-Kimber, Katherine L.

Failli,Amedeo F.

Caggiano,Thomas J.

Nakanishi, Koji Chen, Yanqiu (ii) TITLE OF INVENTION: Effector Rapamycin (iii) NUMBER OF SEQUENCES: 2 Proteins of COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3.50 inch, 1.4 Mb storage COMPUTER: Apple Macintosh OPERATING SYSTEM: Macintosh 7.1 SOFTWARE: Microsoft Word (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/312,023 FILING DATE: 26-SEPTEMBER-1994 APPLICATION NO: US 08/207,975 FILING DATE: 08-MARCH-1994 -31- INFORMATION FOR SEQ. ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: amino acids TYPE: nucleic acid STRANDEDNESS: sense orientation of the double-stranded DNA strand TOPOLOGY: linear (ii) MOLECULE TYPE: sense orientation of doublestranded cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (vi) ORIGINAL SOURCE: ORGANISM: Molt 4 human T-cell leukemia cells STRAIN: ATCC Strain CRL 1582 (xi) SEQUENCE DESCRIPTION: AAG ATG CTT GGA ACC GGA CCT GCC Met Leu Gly Thr Gly Pro Ala SEQ. ID NO: 1 GCC GCC ACC ACC 27 Ala Ala Thr Thr 10 GCT GCC ACC ACA TCT AGC 54 Ala Ala Thr Thr Ser Ser AAG AGC CGG AAT GAG GAA 108 Lys Ser Arg Asn Glu Glu AAT GTG AGC GTC CTG CAG CAG TTT GCC AGT GGC CTA 81 Asn Val Ser Val Leu Gln 20 ACC AGG GCC AA 35 Thr Arg Ala Lys

GAG

Glu

TTT

Phe

ATG

MET

55

GAA

Glu AGT CAA Ser Gln TTG GTT Leu Val GCC GCC Ala Ala GAG GAG Glu Glu TCC AGC Ser Ser Gln Phe Ala 25 AAG GAG CTC 135 Lys Glu Leu TCT ACT CGC 189 Ser Thr Arg 60 TCA GAT GCC 243 Ser Asp Ala Ser Gly Leu CAG CAC TAT Gln His Tyr 45 TTC TAT GAC Phe Tyr Asp 65 AAT GAG AGG Asn Glu Arg 30 GTC ACC Val Thr CAA CTG Gln Leu

ATG

MET

GAA CTC CGA 162 Glu Leu Arg

AAA

Lys

ACC

Thr

GGT

Gly

CGA

Arg AAC CAT CAC ATT 216 Asn His His Ile GGC ATC TTG GCC 270 Gly Ile Leu Ala ATT GGC AGA TTT 324 Ile Gly Arg Phe 105 ATA GCT AGC CTC ATA GGA GTG GAA GGT GGG AAT GCC 297 'Ile Ala Ser Leu Ile Gly Val Glu Gly Gly Asn Ala 95 100 -32- GCC AAC TAT CTT CGG AAC CTC CTC CCC TCC AAT GAC CCA OTT GTC ATG GA-A ATG Ala Asn Tyr Leu 110* Arg GCA TCC Ala Ser TAC GTG Tyr Val 145 GAG GGC Glu Gly AAG GCC ATT Lys Ala Ile 130 GAA TTT GAG Olu*Phe Glu Asn Leu Leu 115 GGC COT CTT Gly Arg Leu OTO AAG CGA Val Lys Arg 150 OCA OCT OTC Ala Ala Val Pro Ser Asn Asp Pro 120 000 GAC Gly Asp Val Val MET 0 u 378

MET

125

GAO

432 Glu 0CC ATG 405 Ala MET 135

OCA

Ala ACT TTT Thr Phe 140

ACC

Thr

OCT

Ala 0CC 459 Ala

CTG

513 CTG GAA TG Leu Giu Trp 155 OTT CTC COT Val Leu Arg

CTO

Leu

GOT

Gly COG AGA Arg Arg 165

CAT

His GAG CTO Olu Leu 175 OCT GAC COC AAT 486 Ala Asp Arg Asn 160 0CC ATC AGC GTC 540 Ala Ile Ser Val *0 CCT ACC Pro Thr 180 25 T

G

Val Trp 30 TOT CTG Cys Leu

TTC

Phe TTC TTC Phe Phe CCC AA-A Pro Lys

CAG

Gin 185 Leu 170 CA OTO CAA 567 Gin Val Gin CCC TTC TTT Pro Phe Phe 190

GAC

Asp AAC ATT TTT OTO 0CC 594 Asn Ile Phe Val Ala 195 0CC 0CC CTT COT 0CC 648 Ala Ala Leu Arg Ala

GAC

Asp 200 CAG 0CC ATC COT GAG 621 Gin Ala Ile Arg Giu 205 ACC CAG COT GAG CCG 675 Thr Gin Arg Giu Pro OGA GCT OTA Gly Ala Val 210 AAG GAG ATG Lys Oiu MET ATT CTC ACA Ile Leu Thr 220 CAC ACA TTT His Thr Phe CAG AAG Gin Lys 230 CCT CAG Pro Gin 215

TGG

702 Trp TAC AGO Tyr Arg 235 GAA GAA Giu Giu 240 GCA GAG 729 Ala Giu

GAG

Glu 45

GAG

Oiu 270

GAA

AAO GOC ATO Lys Gly MET 255 CTO OTC CGA Leu Val Arg ATC ACA CAG

AAT

Asn 225 AAG OGA TTT Lys Oly Phe 245 ATC CAT GGA Ile His Gly

OAT

Asp

GAO

0hz

COO

Arg ATC AOC Ile Ser 275 CAG CAG OAT GAT CGO 783 Asp Asp Arg 260 AOC ATO GAG 837 Ser MET Giu CTG GTA CAC OGA GAO Oly Giu 280 GAC AAG ACC TTG 0CC AAA -756 Thr Leu -A16- Ly s 250 TTG ATC CTT AAC 810 Leu Ile Leu Asn 0CC TTO Ala Leu 265

COT

Arg

CTG

Leu AGA GAA GAA ATO OAA 864 Arg Oiu Giu MET Giu 285 AAA OAT CTC ATG GGC 918 Lys Asp Leu MET Oly 305 Oiu Ile Thr 290 Gin Gin Gin TAC TOC Tyr Cys 300 Leu Val 295 His Asp Lys -33- TTC GGA ACA AA.A CCT CGT CAC ATT ACC CCC TTC ACC AGT TTC CAG GCT GTA CAG Phe Gly Thr Lys Pro 310 TCA AAT Ser Asn Arg His Ile Thr Pro Phe 315 Thr Ser Phe Gin 320 Ala Val Gin CCC CAG CAG Pro Gin Gin 325 GCC TTG Ala Leu 330 GTG GGG 999 Val Gly

CTG

Leu CTG GGG Leu Gly 335

TAC

Tyr

AGC

Ser TCT CAC CAAk GGC 1026 Ser His Gin Gly 340 CTG GTG GAG AGC 1080 Leu Val Giu Ser

CTC

Leu ATG GGA TTT GGG MET Gly Phe Gly 345 TGT TGC AGA GAC Cys Cys Arg Asp

CGG

Arg 360

ACC

Thr

TTG

Leu 365 TCC CCC AGT CCA 1053 Ser Pro Ser Pro 350 ATG GAG GAG AA 1107 MET Glu Glu Lys

GCT

Ala

TTT

AAG TCC ACC Lys Ser Thr 355 GAT CAG GTG Aso Gin Val CTG AAPA TGC Leu Lys Cys 380 TTG CCC CGC Leu Pro Arg AGG AAT Arg Asn TTG GCT Leu Ala 400 AGC AAG AAC TCG 1161 Ser Lys Asn Ser 385 GCA TTC CGA CCT 1215 Ala Phe Arg Pro TGC CAG Cys Gin 375 TGG GTG 1134 Trp Val

CTG

Leu

TCT

Ser 405 ATC CA-A ATG ACA Ile Gin MET Thr 390 GCC TTC ACA GAT Ala Phe Thr Asp

ATC

Ile CTT A.AT TTG 1188 Leu Asn Leu 395 CAG TAT CTC 1242 Gin Tyr Leu 370

ACC

CAA GAT Gin Asp 415

ACC

Thr

ATG

MET

AAC CAT Asn His GCC CTG Ala Leu

GCC

Ala 420 CTA AGC 1269 Leu Ser TGT GTC AAG Cys Val Lys 425 TCT GTG GCT Ser Val Ala A.AG GAG Lys Glu GTG AGG Val Arg 410 AAG GAA CGT ACA 1296 Lys Giu Arg Thr 430 TCT GAG TTT. AAG f350 Ser Giu Ph'-Lys

GCG

Ala

GTC

Val 450 GCC TTC CAA Ala Phe Gin 435 TAT TTG CCT Tyr Leu Pro TTC GCC CAT A Phe Ala His L 470 ATC AGC ATG C Ile Ser MET L

AG

y s CGC GTG Arg Val 455 AGG CAG Arg Gin GGG CTA CTT 1323 Gly Leu Leu 44,0 CTG GAC ATC 1377 Leu Asp Ilie AAG GCA ATG 1431 Lys Ala MET 475 ATC CGA Ile Arg 460 CAG GTG Gin Val GCG GCC Aia Ala GAC GCC Asp Ala 480 CTG CCC CCA Leu Pro Pro 465 ACA GTC TTC Thr Val Phe AAG GAC 1404 Lys Asp ACT TGC 1450' Thr Cys 485 A.AG GAG 445 TG GCT CGA GCA ATG GGG CCA GGC ATC CAG CAG GAT ATC 1485 eu Ala Arg Ala MET Gly Pro Gly Ile Gin Gin Asp Ile Lys Giu 490 49';cn -34- CTG CTG GAG CCC ATG CTG GCA GTG GGA CTA AGC CCT GCC CTC ACT GCA GTG CTC 1539 1566 Leu Leu 505 Glu Pro MET Leu CGT CAG Arg Gin Ala 510 Val Gly Leu Ser Pro Ala Leu Thr Ala Val Leu

TAC

Tyr

CTG

Leu 540 GAC CTG AGC Asp Leu Ser 525 AAA ATG CTG Lys MET Leu ATT CCA CAG 1593 Ile Pro Gin 530 GTC CTT ATG 1647 Val Leu MET CTA AAG AAG GAC ATT Leu Lys Lys Asp Ile 535 CAC AAA CCC CTT CGC His Lys Pro Leu Arg 520 CAA GAT GGG CTA 1620 Gin Asp Gly Leu TCC CTG Ser Leu 545 CCC AAG GGC Pro Lys Gly 560 GCC AGC GAT Ala Ser Asp

CTG

Leu

GCC

Ala CAT CAG CTG GCC 1701 His Gin Leu Ala 565 AGC ATC ACT CTT 1755 Ser Ile Thr Leu

TCT

Ser 550 CCT GGC Pro Gly CTC CGA Leu Arg

CTC

Leu 570

ACG

Thr CAC CCA His Pro 555 ACC CTC Thr Leu GGC ATG 1674 Gly MET CCT GAG 1728 Pro Glu 575 TTT GAA 1782 Phe Glu GTG GGC Val Gly 580

GCC

Ala TTT G.AA GGC Phe Glu Gly 595

CAC

His TCT CTG ACC Ser Leu Thr 600 AAG GAG ATC Lys Glu Ile CAA TTT 1809 Gin Phe 585 GTT CGC CAC Val Arg His 605 ACG CTT GGC AGC Thr Leu Gly Ser 590 TGT GCG GAT CAT Cys Ala Asp His

AAC

Asn AGT GAG CAC Ser Glu His 615 CTC ACA Leu Thr 630 GCA GTG Ala Val

CCC

Pro

CAA

Gin TCC ATC CAC Ser Ile His 635

CTC

Leu CGC ATG 1863 Arg MET 620 ATC AGT 1917 Ile Ser GAG GCT Glu Ala GCC CGC ACC TGC Ala Arg Thr Cys 625 GCT CAT GTG GTT Ala His Val Val GGC CAT Gly His 640

TCC

Ser

AGC

Ser 645 TTC CTG 1836 Phe Leu 610 CGC CTG 1890 Arg Leu CAG ACC 1944 Gln-Thr GTG GTG Val Val 650 GAT CCT GAC Asp Pro Asp GAT GCA CAC Asp Ala His 685 GCA GAT GTG CTT 1971 Ala Asp Val Leu 655

AGC

Ser AAA CTG CTC Lys Leu Leu 660 TTG GCG TCC Leu Ala Ser GTA GTT Val Val CTG GAC Leu Asp 680 TTT GTG CCT GAC Pro Asp 670 CTG GCC ATT CGC TAC TGT GTC 2025 Ile Arg Tyr Cys Val 675 CAG GCG GAG AAC TTG 2079 GGG ATA ACA 1998 Gly Ile Thr 665 GAG CGC TTT 2052 Glu Arg Phe GCT CTG AAT 2106 Ala Leu Asn 700 CAG GCC TTG Leu Ala Gin Ala Glu Asn Leu Gin Ala 690 695 Leu Phe Val GAC CAG GTG TTT GAG ATC CGG GAG CTG GCC ATC TGC ACT GTG GGC CGA CTC AGT Asp Gin Val AGC ATG A.AC Ser MET Asn 720 TTG ACA GAG Leu Thr Giu Phe 705 2133 Giu Ile Arg Giu Leu 710 CCT GCC TTT GTC Pro Ala Phe Vai 725 TTG GAG CAC AGT Leu Giu His Ser ATG CCT 2187 MET Pro GGG ATT 2241 Gly Ile Ala Ile TTC CTG Phe Leu 730 Cys Thr CGC AAG Arg Lys Val1 715 2160 Gly Arg Leu Ser ATG CTC MET Leu GAG CAG Giu Gin

ATC

Ile 735 CAG ATT 2214 Gin Ile

GGA

Gly ATG CTG MET Leu CCT ATT Pro le 775 740 GGG CAC CTG Gly His Leu 760 CTG A.AG GCA Leu Lys Ala 745 GTC TCC AAT GCC 2295 Val Ser Asn Ala AGA ATC AAAk Arg Ile Lys 750 CGA CTC ATC Arg Leu Ile AGT GCC GGC 2268 Ser Ala Arg 755 TAC ATG GAG 2322 Tyr MET Giu

CCC

Pro 765 CGC CCC Arg Pro 770 TTA ATT Leu Ile 780 TTG AAA 2349 Leu Lys

C

C.

C

CCA

Oro GGT GTG ATC Gly Val Ile 795

AAT

Asn

AAT

Asn GTC CTG GCA 2403 Val Leu Aia 800 TGG GTT GAT 2457 Trp Val Asp CTG AAA Leu Lys ACA ATA Thr Ile GAA CTT Giu Leu 820

GAT

Asp 785

CCA

Pro

GAC

Asp

TTG

Leu 805 CCT GAT GCA AAC 2376 Pro Asp Pro Asn 790 GCA GAG GTT AGT 2430 Ala Gin Val Ser GGA GA.A Gly Giu TTT ATT Phe Ile GGC CTG Gly Leu 810 CTC GAG Leu Gin

GAA

Glu

GAT

Aso ATG AGG MET Arg TCC TCT Ser Ser

A.AA

Ly s 815 ATC ATC ATG Ile Ile MET 825 CTG TGG ACC Leu Trp Thr GAG TTG Gin Leu 830 GTG GCC AGC Val Ala Ser 850 GAG GTG CTA Giu Val Leu TTG TTG GCC AAA 2511 Leu Leu Ala Lys 835 ACT GGC TAT GTA 2565 Thr Giy Tyr Vai

AGG

Arg

GTA

Val 855

AAG

Ly s GAG GTG GCT Gin Val Ala 840 GAG CCC TAG Glu Pro Tyr GAG ATG 2484 Asp MET CTG GGA -2538 .Le& Gly 845 CGT ACT 2592 Pro Thr AGG AAG Arg Lys 860

TAG

Ty r

TTG

Leu

AGA

Arg

CTT

Leu 865

GAG

Glu

CTG

Leu

AAT

Asn 870

TTA

Leu TTT CTG 2619 The Leu ACT GAG Thr Giu 875

GAG

Gin

AAC

Asn GAG GGT ACA GGC 2646 Gin Gly Thr Arg 880 GCT TAG AAG GAG 2700 Pro Tyr Lys His GCC ATC Ala Ile 885 CGT GTG Arg Val GGG CTT TTA GGG GCT TTG GAT 2673 Giy Leu Leu Giy Ala Leu Asp 890 895 -36- AAA GTG AAC ATT OGC ATO ATA GAC CAG TCC COO GAT GCC 2727 Ly s 900 Val Asn le Gly MET Ile Asp Gin 905 AAG TCA AGT CAG OAT TCC 2781 Lys Ser Ser Gin Asp Ser Ser Arg Asp Ala 910 TCT GAC TAT AGC TCT OCT OTC AOC CTO 2754 Ser Aia Val Ser Leu 915 TCA GAA Ser Oiu OTC AAC Val Asn

TCC

Ser 920 ACT AOT Thr Ser ATO OGA AAC TTO MET Oly Asn Leu 940 ATO COO ATC TTC MET Arg Ile Phe 925 CCT CTG OAT 2835 Pro Leu Asp Ser Asp Tyr GAO TTC TAC Giu Phe Tyr 945 TCA CTC TCT Ser Leu Ser Ser 930 CCA OCT OTO Pro Ala Val 950 CAT CAT CAC His His His OAA ATO CTO 2808 Oiu MET Leu 935 TCC ATO OTO 2862 Ser MET Val ACC ATO OTT 2916 Thr MET Val 970 OTO CAG TTC 2970 Vai Gin Phe 0CC CTG Ala Leu 955

CGA

Arg 960 GAC CAG 2889 Asp Gin

OTC

Val1 CAG 0CC ATC Gin Ala Ile 975 CCC CAG GTC Pro Gin Val ACC TTC Thr Phe ATO CCC MET Pro 995 965 ATC TTC A.AG TCC 2943 le Phe Lys Ser 980 ACO TTC CTT AAT 2997 Thr Phe Leu Asn

CTO

Leu

GTC

Val1 OGA CTC AAA, TOT Gly Leu Lys Cys 985 a 4.

CTG

Leu 990 ATT CGA Ile Arg 1000 ATC COO G AA TTT Ile Arg Giu Phe 1010 CAC ATC AGA CCT His Ilie Arg Pro

TTO

Leu TTC CAG CAG CTO 3051 Phe Gin Gin Leu 1015 ATO OAT GAA ATA 3105 MET Asp Glu Ile

OGA

Gly ATO TTG OTO MET Leu Val 1020 ACC CTC ATG Thr Leu MET GTC TOT OAT GOG 0CC 3024 Val Cys Asp Gly Ala 1005 TCC TTT OTO AAO AGC 3078 Ser Phe Val Lys Ser 1025 AGA GAA TTC TOO GTC 3132 Arg Giu Phe .Trp- Val

TAT

Tyr

GTC

Val ATO AAC ACC TCA MET Asn Thr Ser 1045 1030 ATT CAG Ile Gin AGC ACO ATC 3159 Ser Thr Ile 1050 AAO CTC TAC 3213 1035 ATT CTT CTC ATT Ile Leu Leu Ile 1055 CTG CCC CAG CTG Leu Pro Gin Leu 1040 GAG CAA ATT Glu Gin Ile GTO OTA 3186 Val Val 1060 ATO CTG 3240 MET Leu OCT CTT 000 GOT OAA TTT Ala Leu G1 y GOIy Olu Phe 1065 COT GTC TTC ATO CAT GAC Lys Leu Tyr 1070 AAC AOC CCA GOC COC ATT OTC ATC CCA Ile Pro 1075 TCT ATC

CAC

His AAG TTA CTG 3294 Arq Val Phe 1080 3267 MET His Asp Asn Ser Pro Gly Arg 1085 1090 Ile Val Ser Ile Lys Leu Leu 1095 -37- GCT GCA *ATC CAG Ala Ala Ile Gin 1100 CCT CCT ATT GTT Pro Pro Ile Val GCA GCG CTA GAG Ala Ala Leu Glu 1135 GCC TCC CGG ATC Ala Ser Ara Ile CTG TTT GGC GCC AAC 3321 Leu Phe Gly Ala Asn 1105 AAG TTG TTT GAT GCC 3375 Lys Leu Phe Asp Ala 1120 ACT GTG GAC CGC CTG 3429 Thr Val Asp Arg Leu 1140 ATT CAC CCT ATT GTT 3483 Ile His Pro ile Val CTG GAT Leu Asp CCT GAA Pro Giu 1125 GAC TAC Asp Tyr illc GCT CCA Ala Pro

CTG

Leu CAT TTA His Leu CTG CTG 3348 Leu Leu 111.5 CGA AA2G 3402 Arg Lys CTG CCA TCT Leu Pro Ser 1130 ACG GAG TCC CTG Thr Glu Ser Leu 1145 CGA ACA CTG GAC Arg Thr Leu Asp

GAT

Asp

CAG

Gin 1165 TTC ACT GAC TAT 3456 Phe Thr Asp Tyr 1150 AGC CCA GA.A CTG 3510 Ser Pro Giu Leu

CGC

Arg 1170 25 115~ TCC ACA GCC Ser Thr Ala ATG GAC ACG MET Asp Thr 1175 116 CTG TCT 3537 Leu Ser 0 TCA CTT GTT Ser Leu Val 1180

TTT

Phe

GTG

Val2 1200 CAG CTG GGG Gin Leu Gly 1185 CGA CAC CGA Arg His Arg TAC CAA ATT TTC Tyr Gin Ile Phe 1190 CAT CAG CGC TAT His Gin Arg Tyr

ATT

Ile

GAT

Asp CCA ATG GTG AAT 3591 Pro MET Val Asn 1195 GTG CTC ATC TGC 3645 Val Leu le Cys

AA

Ly s

AGA

Arg GTT CTG Val Leu ATT GTC 7ie Val AAG AAG 3564 Lys Lys ATC AAT 3618 Ile Asn 1205 CTT GCT 3672 Leu Ala AAG GGA TAC ACA Lays Gly Tyr Thr 1220 GAT GAA GAG Asp Glu Glu 1225 GGG GAT GCA Giy Asp Ala 121( GAG CAT Glu Asp TTG GCT Leu Ala 1245 0 CCT TTG Pro Leu 123 AGT GGA Ser Gly 0 ATT TAC 3699 Ile Tyr 121

CAG

Gin 45 GTC AGC Val Ser 1260 GAT GAC

ACC

Thr

ATC

Ile 5 AAC CTC CAA Asn Leu Gin 1265 GAA TGG CTG CCA GTG GAA 3753 Pro Val Glu 1250 AAG GCC TGG 3807 Lys Ala Trp AGA CGG CTG 3861 Arg Arg Leu 1285 CAT CGG His Arg 1235 ACA GGA Thr Gly GGC GCT Gly Ala 1270 AGC CTG C ALTG CTT M1ET Leu CCC ATG Pro MET AGG AGT GGC CAA 3726 Arg Ser G1j Gin 1240 AAG AAA CTG CAC 3780 Lys Lys Leu His 1255 ;CC AGG AGG kla Arg Arg AG CTG CTG GTC TCC AAA 3834 Val Ser Lys 1275 AAG GAC TCA TGG CTG Asp Asp Trp 1280 Leu Glu Trp Leu Ser Leu Glu Leu Leu Lys Asp Ser 1290 1295 -38- TCA TCG Ser Ser CCC TCC CTG CGG Pro Ser Leu Arg 1300

TCC

Ser TGC TGG GGC CTG 3915 Gys Trp Ala Leu 1305 GCC AGG GAT Ala Arg Asp 1315 CTC TTC AAT GGT GCA TTT GTG TCC Leu Phe Asn 3969 Ala Ala Phe Val Ser 1320 GCA CAG Ala Gin TGC TGG Cys Trp 1325 TTG GCG Leu Ala

TCT

Ser

CTC

Leu GAA CTG AAT GAA 3996 Glu Leu Asn Glu 1330 ACC TGA CAA GAG 4050 Thr Ser Gin Asp GCG TAC AAC Ala Tyr Asn 1310 CCG ATG 3942 Pro MET

GAT

Asp CAA CAG GAT GAG Gin Gin Asp Glu 1335 GCT GAA GTC ACA Ala Glu Val Thr CTC ATC AGA AGC ATG GAG 4023 Leu Ile Arg Ser Ile 1340 GAG ACC CTC TTA AAC 4077 Gin Thr Leu Leu Asn Glu 1345

ATC

Ile 1350 TTG GCT Leu Ala 1360

GAA

Glu GAC AA2G Asp Lys AGA GCT Arg Ala

GGC

Gly 137( 1351 CCC GTG GCA Pro Leu Pro CTG AGA GAT 4131 Leu Arg Asp 1375 GCA TAT GCC 4185 Ala Tyr Ala

GAG

Asp

AAA

Lys AAT GGC ATT Asn Gly Ile 1380 GCA CTA CAC Ala Leu His TTC ATG GAA CAC AGT 4104 Phe MET Glu His Ser 1365 GTT CTG CTG GGT GAG 4158 Val Leu Leu Gly Glu 1395 TAC A.A GAA CTG GAG 4212 Tyr Lys Glu Leu Glu 1400 TTC GAG Phe Gin 1405 GCC AAG TGG Ala Lys Gys 139 AAA GGC CCC Lys Gly Pro

GGA

Arg 0 139 k-G Lys

TTT

Phe 1440 CTA GAG GAG CCG Leu Gin Gin Pro 1425 GGA GAG GTG GAG Gly Glu Leu Giu ACC CCT GCC ATT GTA 4239 Thr Pro Ala Ile Leu 1410 GAG GGA GGG GCC GGA 4293 Glu Ala Ala Ala Gly 1430 ATC CAG GGT ACC TGG 4347 Ile Gin Ala Thr Trp 5

GAA

Glu

GTG

Va1

TCT

Ser 141

TTA

Leu GTC ATG AGG Leu Ile Ser ATT AAT AAT 4266 Ile Asn Asn 1420 ATG AAA CAC 4320 MET Ly-His GA-A TAT GCG Glu Tyr Ala 1435 AAA CTG CAC Lys Leu His GAT GCC CTT GTG GCC Asp Ala Leu Val Ala 1460 CTG ATG GTG GGC CGC Leu MET Leu Gly Arg 1480 TAT GAG Tyr Aso TAT GAG Tyr Glu 1450 GAG AGG GAC GAG TGG GAG 4374 Glu Trp Glu 1455 GAC CCA GAGC AAG AXAA ATG 4401 Lys Lys MET 1465 AAC A-AG 4428 Asp Thr Asn Lys Asp Asp Pro Glu 1470 1475 ATG CGC TGC CTC GAG GCG TTG GGG GAA TGG GGT CAA CTC 4455 4482 MET Arg Cys Leu Glu Ala Leu 1485 Gly Glu Trp 149C Gly Gin Leu -39- CAC CAG CAG TGC TGT His Gin Gin Cys Cys 1495 ATG GCC CGG ATG GCT MET Ala Arg MET Ala GAA AAG TGG ACC CTG 4509 Giu Lys Trp Thr Leu 1500 GCT GCA GCT GCA TGG 4563 Ala Ala Ala Ala Tro 1520 ATG ATC CCT CGG GAC 4617 MET Ile Pro Arg Asp GTT AAT GAT Val Asn Asp 1505 GAG ACC Giu Thr CAA GCC AAG 4536 Gin Ala Lys 1510 GAC AGC ATG 4590 Asp Ser MET GGT TTA GGT GAG TOO Gly Leu Gly Gin Trp 1525

GAA

Glu 1530 1515 GAA TAC ACC TGT Giu Tyr Thr Cys ACC CAT Thr His 1540 GAT GGG GCA TTT TAT AGA Asp Gly 1535 GCT GTG CTG GCA Ala Val Leu Ala 1550 AAG GCC AGG GAC- Lys Ala Arg Asp

CTG

Leu

CTG

Leu CAT CAG GAC CTC 4671 His Gin Asp Leu 1555 CTG GAT GCT GAA 4725 Leu Asp Ala Giu

TTC

Phe

TTA

Leu 157~ 4644 Ala Phe Tyr Arg 1545 CAG TGC ATT GAC 4698 Gin Cys le Asp TCC TTG GCA CAA Ser Leu Ala Gln 1560 ACT GCA ATG GCA Thr Ala MET Ala

GGA

Gly 1565 GAG AGT TAC 4752 Giu Ser Tyr AGT CGG Ser Arg 1585 GCA TAT Ala Tyr 1570 GGG GCC ATG Gly Ala MET 1590 OTT TCT 4779 Val Ser TGC CAC ATG Cys His MET 159~ CTG TCC Leu Ser 1580 GAG CTG GAG GAG 4806 Glu Leu Giu Giu 1600 CGC CAG ATC TGG 4860 Arg Gin Ile Trp

OTT

Val ATC CAG TAC Ile Gin Tyr 1605 AAA CTT Lys Leu GTC CCC GAG CGA2 4833 Val Pro Giu Arg 1610 TGC CAG CGT ATC 4887 Cys Gin Arg ile

CGA

Arg

OTA

Val GAG ATC Giu Ile GAG GAC Glu Asp

ATC

Ile 1615

C

TGG GAG Trp Giu 1620 40 ATG GTG ME Val TAT GCA Tyr Ala

AGA

Arg

CGG

Arg CTG CAG GGC Leu Gin Gly 1625 TCC CTT GTG Ser Leu Val

GTC

Val 1640 AGC CTG Ser Leu AGC CCT 4941 Ser Pro 1645 ACT GGC 4995 Ser Gly 1630 CAT GAA His Giu AGO CTG Arg Leu 1665 GAC ATG Asp MET 1650 GCT CTT Ala Leu TOO CAG Trp Gin AGA ACC Arg Thr GCT CAT Ala His 1670

AAA.

Ly s 1635 TGG CTC AAG 4968 Trp Leu Lys 1655 AAA ACT TTA Lys Thr Leu PTC CTT 4914 Ii& Leu TGC GGC AAG Cys Gly Lys 1660 GTG TTG CTC CTG GGA GTT GAT CCG TCT CGG CAA CTT GAC CAT CCT CTG CCA ACA 5049 5076 Val Leu Leu Leu Gly Val Asp Pro Ser Arg Gin Leu Asp His Pro Leu Pro Thr 1675 1630 1685 1690 GTT CAC CCT CAG GTG ACC TAT Val His Pro Gin Val Thr Tyr 1695 AAG ATC GAT GCC TTC CAG CAC Lys Ile Asp Ala Phe Gin His 1710 1715 GCC CAG CAT CCC ATC GCT ACT Ala Gin His Ala Ile Ala Thr 1730 CTC ATG GCC CGA TGC TTC CTG Leu MET Ala Arg Cys Phe Leu 1750 ATC AAT GAG ACC ACA ATC CCC le Asn Clu Ser Thr Ile Pro GCC TAC ATC AAA AAC ATG TGG AAXG ACT CCC CC 5103 5130 Ala Tyr MET Lys Asn MET Trp Lys Ser Ala Arg 1700 1705 ATG CAC CAT TTT GTC CAG ACC ATC CAG CAA CAG 5157 MET Gin His Phe Val Gin Thr GAG GAC 5211 Ciu Asp 1735 AAA CTT 5265 Lys Leu AAA CTG 5319 1720 CAG CAG CAT AAC CAG Gin Gin His Lys Gin 1740 CGA GAG TGG CAG CTG Gly Giu Trp Gin Leu 1755 CTG CAC TAC TAC AC Leu Cmn Tyr Tyr Ser 1775 5184 MET Gin Gin Gin 1725 GAA CTC CAC AAC 5238 Giu Leu His Lys 1745 AAT CTA CAG GC 5292 Asn Leu Gin Gly 1760 CCC CC Ala Ala 1765 25 CAC GAC CC His Asp Arg 30 GCT GTG CTA Ala Val Leu 1800 CGT CAT GCC Arg His Ala AGC TGG TAC Ser Trp Tyr 1785 1770 AAG CCC TGG 5373 Lys Ala Trp 179( ACA GAG 5346 Thr Giu 1780 TTC GAA 5400 Phe Glu CAC TAC AAA CAT His Tyr Lys His 1805 AGC GGG CCC AAC Ser Giy Ala Asn CAG AAC 5427 Gin Asn ATC ACC 5481 Ile Thr 1825 ACC ACC 5535 Ser Thr CAT CC TGG His Ala Tro 0 CAA CCC CC Gin Ala Arg 1810 AAC CCC ACC Asn Ala Thr GCA GTG ATG Ala Val MET 1795

AAC

Asn

CAT

Asp

ACT

GAG AAG AAG AAA CTG 5454 Clu Lys Lys Lys Leu 1815 CCC CCC ACC ACG CC 5508 Ala Ala Thr *Thi- Ala 1835 ACT GAG ACC GAG CC 5562 Ser Ciu Ser Ciu Ala 1850 1820 CCC ACT CCC ACC Ala Thr Ala Thr GAG AGC ACC GAG Clu Ser Thr Glu 1855 CAT CTC TCC AAA ACC ACT Thr Thr 1840

GCC

Ala GAG GC Ciu Gly 1845 1830 AGC AAC Ser Asn AAC AGC CCC ACC CCA TCG CCC 5589 Asn Ser Pro Thr Pro Ser Pro 1860 ACC CTC CTG ATC TAC ACG GTG 5643 CTC CAG AAC Leu Gin Lys 1865 CCT CCC GTC AAG CTC ACT GAG 5616 Lys Val Thr Giu 1870 CAG CCC TTC TTC 5670 Asp Leu Ser Lys Thr Leu Leu MET Tyr Thr Val Pro Ala Val Gin Gly Phe Phe 1875 1880 1885 -41- CGT TC Arg Ser 1890 ACC TTA Thr Leu ATG TGG TTG TGA GGA Ile Set Leu Set Arg 1* 1895 TGG TTT GAT TAT GGT Trp Phe Asp Tyr Gly GGG AAG 5697 Gly Asn GAG TGG 5751 His Trp, AAC GTC GAG Asn Leu Gin 1900 GAT ACA GTG AGA GTT CTC 5724 Asp Thr Leu Arg Val Leu 1905 A.T GAG GCC TTA GTG GAG 5778 Asn Giu Ala Leu Val Glu

GGG

G ly

GTG

Val1 1910 AAkA GCC ATG GAG Lys Ala Ile Gin 1930 ATT GAT ACG CCC Ile Asp Thr Pro 1915 ATT GAT ACC 5805 Ile Asp Thr

GGA

Pro

TGG

Trp 1935

GTG

Val1 GAT GTC Asp Val CTA GAG Leu Gin 1920 GTT ATA GCT Val Ile Pro 1940 1925 CAG GTC ATT 5832 Gin Leu Ile GGA AGA Ala Arg 194~ ACA GAG Thr Asp

AGA

Arg CCC TTG 5859 Pro Leu ATT GGT CGG Ile Gly Arg 1965 195C TAG GAG Tyr His GGA GGT Gly Arg 195~ GTG ATG Leu Ile GTG ATT Leu Ile

S.

TGT AAG Ser Lys 1980 GGG GAG GGG 5913 Pro Gin Ala 1970 GGG GAG AAT 5967 Arg His Asn TAG GGA Tyr Pro 1975 AAG AAG Asn Lys GAG GAG GTT GTG 5886 His Gin Leu Leu 1960 GTG AGA GTG GGT 5940 Leu Thr Val Ala ATT GTG AAG AAG 5994 Ile Leu Lys Asn

TGT

Ser AGG AGG AGA GGG Thr Thr Thr Ala 1985 GGA GGG Ala Ala 1990 ATG TGT GAG GAG MET Gys Giu His 2000 GTG ATtCGGA GTG Leu Ilie Arg Val GGA TGT GGT TTG AGG AAC Ser Asn GGG ATG Ala Ile 2020 TAG TTT AGG GTG GTG 6021 Thr Leu Val 2005 GTG TGG GAT 6075 Leu Trp, His GGG GAA AGG

GAG

Gin 1995

GAG

Gin GAG ATG Giu MET 2025 AAG GTG GGG ATG ATG Ala MET MET 2010 TGG GAT GAA Trp His Glu AAA GGG ATG

GTG

Val1

GGG

Gly AGG GAG GAG 6048 Ser Glu Giu 2015 GTG GAA GAG '6102

S.

2030 TTT GAG Phe Giu 6129 Ala Set Arg Leu Tyr Phe Gly Giu Arg Asn Vai Lys Gly MET GTG GTG 6156 Val1 Leu 2050 2035 2040 2045 GAG GGG TTG GAT GGT ATG ATG GAA CGG GGG GGG GAG AGT GTG AAG GAA AGA TGG 6183 6210 Giu Pro Leu His Ala MET MET Giu Arg Gly Pro Gin Thr Leu Lys Giu Thr Ser 2055 2060 2065 TTT AAT GAG GGG TAT GGT GGA GAT TTA ATG GAG GGG GAA GAG TGG TGG AGG AAG 6237 6264 Phe Asn Gin Ala Tyr Gly Arq Asp Leu MET Giu Ala Gin Glu Trp Gys Arg Lys 2070 2075 2080 2085 -42- TAC ATG A.AA TCA GGG AAT GTC AAG GAC CTC ACC CAA GCC TGG GAC CTC TAT TAT Tyr MET CAT GTG His Val CA.A TAT Gin Tyr 2125 Lys Ser 2090 6291 Gly Asn Val Lys Asp Leu Thr Gin 2095 Ala Trp 2100 Aso Leu TTC CGA CGA ATC Phe Arg Arg Ile 2110 GTT TCC CCA AAA Val Ser Pro Lys TCA AAG CAG 6345 Ser Lys Gin CTG CCT CAG Leu Pro Gin 2115

CTC

Leu ACA TCC TTA Thr Ser Leu 2120 GA-A TTG GCT Giu Leu Ala 6318 Ty r Tyr 2105 GAG CTG 6372 Giu Leu GTG CCA 6426 Val Pro 2140 CCG TCT 6480 Pro Ser

GGA

G ly ACA TAT GAC CCC AAC Thr Tyr Asp Pro Asn 2145 CAA GTC ATC ACA TCC Gin Val le Thr Ser CTT CTG ATG TGC 6399 Leu Leu MET Cys 2130 CAG CCA ATC ATT 6453 Gin Pro Ile Ile 2150 AAG CAG AGG CCC 6507 Lys Gin Arg Pro CGG GAC CTT Arg Asp Leu 2135

TTG

Leu 2 16C0

CGC

Arg

CGG

Arg 2170 ATT CAG TCC ATA Ile Gin Ser Ile 2155

GCA

Ala AAA TTG Lys Leu 2165 'S S

S

*5 S S 9*

S

S

S

S

S*

AAC GGA CAT GAG Asn Gly His Glu 2180 GAG CGT GTG ATG Glu Arg Vai MET

TTT

Phe

CAG

Gin GTT TTC CTT CTA 6561 Val Phe Leu Leu 2185 CTC TTC GGC CTG 6615 Leu Phe Gly Leu

AAA

Ly s

GGC

Gly CAT GAA His Giu 2190 ACC CTT Thr Leu ACA CTT ATO GGC AGC 6534 Thr Leu MET GlY Ser 2175 GAT CTG CGC CAG GAT 6588 Asp Leu Arg Gin Asp 2195 CTG GCC AAT GAC CCA 6642 Leu Ala Asn Asp ?ro 2200 ACA TCT CTT Thr Ser Leu 2215 ACC AAC TCG Thr Asn Ser

CGG

Arg

GGC

AAA AAC CTC AGC ATC 6669 Lys Asn Leu Ser Ile 2220 CTC ATT GGC TGG GTT 6723 Leu Ile Gly Trp Val 2240 AGG GAG AA2G AAG AAG 6777 Arg Giu Lys Lys Lys GTT AAC Val Asn 2205 CAG AGA Gin Arg CCC CAC Pro His

TAC

Ty r 2225

TGT

Cy s GCT GTC Aia Val GAC ACA Asp Thr 2245 AAC ATC Asn Ile CTG ATG 2210 ATC CCT TTA TCG 6696 Ile Pro Le'- Ser 2230 CTG CAC GCC CTC 6750 Leu His Ala Leu GAG CAT CGC ATC 6804 Giu His Ar Ile 2265 CAG AAG GTG GAG 6858 ATC CGG Ile Arg 2250 ATG TTG 2235 GAC TAC Asp Tyr 2255 GCT CCG GAC TAT GAC 6831 ATC CTT CTC Ile Leu Leu 2260 CAC TTG ACT CGG ATG MET Leu Arg MET Ala Pro Asp 2270 Tyr Asp 2275 TrAspHis Leu Thr Leu MET Gin Lys Val Glu 22752280 2285 -43- GTG TTT GAG CAT CCC GTC AAT AAT ACA GCT GGG GAC GAC CTC CCC A-kG CTG CTG Val Phe Glu His Ala Val.

1 2290 6885 Asn Asn Thr Ala Gly Asp Asp Leu 2295 TGC CTG AAA Tro Leu Lys 2305

AGC

Ser CCC ACC TCC Pro Ser Ser 2310 GAG GTG TCG TTT

CAC

Glu Val Trp CGT TCT Arg Ser CAC CCA His Pro 2340 TTA GCG GTC Leu Ala Val 2325

ATG

MET

TCA ATG GTT GGG 6993 Ser MET Val Cly 2330 CTG CAC CGT CTG 7047 Leu Asp Arg Leu Phe Asp 231~ TAT ATT Tyr Ile ACT GC Ser Cly 2350 5 CC A AGA Arg Arg 6912 Ala Lvs Leu Leu 2300 ACC AAT TAT ACC 6966 Thr As' Tyr Thr 2320 CTC CCA CAT ACA 7020 Leu Cly Asp Arg TTA GC Leu Cly 2335

TCC

Ser

AAC

As n *CTC ATC Leu MET 2345 TTT CAC Phe Clu

AAC

Lys 6* ge 0O S 6@ 0@S@ 0* 0 0* 0 S *0 *50* 0 0*00*0

S

0 *0SS

S

5* S S

*S

0 0*0S TTT CCC CAC TC Phe Cly Asp Cys 2360 CCA TTT AGA CTA Pro Phe Arg Leu CC-C A.AC TAC AGA Cly Asn Tyr Arg 2395 CAC ACT CTC ATC Asp Ser Val MET

CTT

Va 1 CCT ATC 7101 Ala MET 2365 TTC ACC 7155 Leu Thr

ACC

Thr

CCA

Arg CAC AAC Clu Lys 2370 ATC GAG MET Clu ATC CTC CAC ATT CAC 7074 Ile Leu His Ile Asp 2355 TTT CCA GAG AAC ATT 7128 Phe Pro Clu Lys Ile 2375 TT ACA CCC CTC CAT 7182 Val. Thr Clvy Leu Asp ACA AGA Thr Arg 2380

ATC

M ET AAT CCT Asn Ala 2385 2390 35 ATC ACA TCC CAC ACA 7209 le Thr Cys His Thr 2400 CCC CTC CTC CAA GCC 7263 Ala Val Leu Clu Ala

GTG

Val1

TTT

ATC GAG CTC MET Glu Val.

2405

CTG

Leu

CCA

Arg

GTC

Val.

40

AGC

Arg 243( 45 241 CTC ATC CAC Leu MET Asp 5 TCC TAC TCT CCT Ser Tyr Ser Ala 2450 CCA CCC CAT AAC ACA AAT ACC Thr Asn Thr 2435 CCC CAC TCA (My CII' Ser AAA ACG CCC 242( .kAA GC 7317 Lys Cly CTC CA.A 7371 Val. Glu 2455 ACC ACA 7425 0h AAC AAC Asn Lys 2440 ATT TTC ile Leu CTC CCA TAT CAC CCC TTC Tyr Asp Pro Leu 2425 CCA TCC CCA ACC Arg Ser Arg Thr CAC CCT CTC CA.A Asp Cly Va. Clu 2460 CA.A TCT ATT CAT *GAG CAC -LAG 7236 Clu His Lys 2410 CTC AAC TCC 7290 Leu Asri-Trp ACC ACC CAT 7344 Arg Thr Asp 2445 CTT CCA GAG 7398 Leu Gly Clu 2465 TCT TTC ATT Pro Ala His Lays Lys Thr Cly Thr Thr Va].

2470 2475 Pro Clu Ser Ile His Ser Phe Ile 2480 -44- GGA GAC GGT TTG GTG AAA CCA GAG GCC CTA AAT AAG AAA GCT ATC CAG 7470 Gly Asp Gly 2485 Leu Val Lys Pro Glu Ala 2490 GAT AAG CTC ACT GGT 7533 Asp Lys Leu Thr Gly Leu Asn Lys Lys 2495 CGG GAC TTC TCT Arg Asp Phe Ser

AAC

Asn AGG GTT CGA Arg Val Arg 2505 Ala Ile Gin CAT GAT GAC His Asp Asp 2515 ACA TCC CAT Thr Ser His ATT ATT 7506 Ile Ile 2500 ACT TTG 7560 Thr Leu

GAT

Asp 252C

CTC

Leu GTT CCA ACG CAA GTT GAG Val Pro Thr Gin Val Glu 2525 TGC CAG TGC TAT ATT GGC Cys Gin Cys Tyr Ile Gly 251 CTG CTC 7587 Leu Leu TGG TAC 7641 Trp Tyr 2545 0 ATC AAA CAA GCG Ile Lys Gin Ala 2530 CCT TTC TGG TAA Pro Phe Trp GAA AAC 7614 Glu Asn 2535 2540 *o INFORMATION FOR SEQ. ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1140 amino acids TYPE: nucleic acid 25 STRANDEDNESS: sense orientation of the doublestranded DNA strand TOPOLOGY: linear (ii) MOLECULE TYPE: sense orientation of double-stranded cDNA to mRNA DESCRIPTION: Sequence No. 2 illustrates a portion of the GST-SEP fusion protein beginning at the linker sequence between the GST and portions of the protein.

(iii) HYPOTHETICAL: no (iv) ANTISENSE: no (vi) ORIGINAL SOURCE: ORGANISM: Molt 4 human T-cell leukemia cells 40 STRAIN: ATCC Strain CRL 1582 (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 2 ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA CCC ACT CGA 27 54 435 MET Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg 1 5 10 CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG TAT GAG CGC GAT 81 108 Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp 25 30 OAA GGT GAT AAA TGG CGA AAC AAA AAG TTT GAkA TTG GGT TTG GAG TTT CCC AA2T Glu Giy Asp Lys Trp Arg Asn Lys

CTT

Leu

CGT

Arg

GAG

Glu CGT TAT TAT Pro Tyr Tyr TAT ATA GGT Tyr Ile Ala ATT TCA ATG Ile Ser MET 135 Lvs Phe 45 Giu Leu Gly Leu Glu 162 Phe Pro Asn ATT GAT Ile Asp 60 GOT OAT Gly Asp GTT AAA TTA ACA GAG 189 Val Lys Leu Thr Gin 65 ATG TTO GOT GGT TGT 243 MET Leu Gly Gly Cys

TCT

Ser

CCA

Pro ATO 0CC ATC ATA 116 MET Ala Ile le

GAG

Asp

CTT

Leu AAO GAG AAC Lys His Asn 80 GAA OGA GO Oiu Oly Ala

A.AA

Lys OTT TTG 297 Val Leu 100 ACT GTC 351 Thr Leu GAT ATT Asp Ile AAA OTT Lys Val 1220 85 AGA TAG GOT Arg Tyr Giy 105 OAT TTT GTT Aso Phe Leu GAG GT GA 270 Oiu Arg Ala OTT TG AGA 324 Val Ser Arg AGG ALAO GTA 378 Ser Lys Leu 12-5 TAT TTA AAT 432 Tyr Leu Asn ATT OCA TAT le Ala Tyr 110

AGT

Se r AAGAG TTT Lys Asp Phe 115

GAIN

Giu 25

CGT

Dro

GOT

Gly 145 35

TTA

Leu GAA ATO CG AAA Giu MET Leu Lys 130 OAT GAT OTA AGG Aso His Val Thr

ATO

MET

GAT

His 150 TTCGOAA OAT GT 405 Phe Olu Asp Arg 135 GGT GAG TTG ATO 459 Pro Aso Phe MET

TTA

Le u TOT CAT Cys His TAT GAG Tyr Asp

AA

Ly s 140

ACA

Thr

TTG

Leu GGT GTT OAT OTT OTT 486 Ala Leu Asp Val Val 160 TAG ATO Tyr MET GAG CCA Asp Pro AAA COT Lys Arg ATA OCA le Ala 200 GCA AAA Pro Lys 165 ATT OAA OCT Ile 01u Ala 185 TOG GCT TTO Trp Pro Leu ATO TG MET Cys ATC CCA Ile Pro GAG GOC Gin Oly 205 OTT CG Val Pro

CTO

Leu 170 OAT OCO 513 Asp Ala CAA ATT OAT 567 Gin Ile Asp 190 TOG CAA 0CC 621 Trp Gin Ala GT GOT OGA 675 155 TTC CGA Phe Pro AAO TAG Lys Tyr AG TTT Thr Phe 210 TGC CCO

AAA

Ly s 175 TTA OTT Leu Val TTG AAkA TCG Leu Lys Ser 195 GOT GOT GG Gly Gly Oiy OGA ATT TCG TOT TTT AAA 540 Cys.Phe- Lys 180 AOC AAG TAT 594 Ser Lys Tyr OAC CAT GCT 648 Asp His Pro 215 GOT GOT GOT 702

TGGOAT

Ser Aso 220

CG

Leu Arg Oly 225 Oly Ser Pro Oly Ile 230 Ser Oly Oiy Oly -46- GGT GGT GGA ATT CTA GAC GAC TCC ATG AGC TTC AAG TAT GCA AGC CTG TGC GGC Gly Gly 235 Gly Ile Leu Asp 240 CTG GCT Leu Ala 729 AAG AGT GGC AGG Lys Ser Gly Arg 255 Asp Ser CTT GCT Leu Ala 260 MET Ser CAT AAA 783 His Lys Phe 245 Lys Tyr

ACT

Thr TTA GTG Leu Val 265 ACA GTT Thr Val Ala Ser TTG CTC Leu Leu CAC CCT His Pro 285 ATC GAT Ile Asp Leu 250 756 Cys Gly GAT CCG Asp Pro TAT GCC Tyr Ala 290 TCT CGG CAA CTT GAC Ser Arg Gin Leu Asp 275 TAC ATG AAA AAC ATG Tyr MET Lys Asn MET CAT CCT CTG CCA 837 His Pro Leu Pro 280 TGG AAG AGT GCC 891 Trp Lys Ser Ala CTG GGA GTT 810 Leu Gly Val 270 CAG GTG ACC 864 Gin Val Thr GCC TTC CAG 918 Ala Phe Gin 305 GCC ATC GCT 972 Ala Ile Ala

CGC

Arg 300

AAG

Lys

CAC

His 4.

ACT

Thr 325 ATG CAG CAT MET Gin His 310 GAG GAC CAG Glu Asp Gin TTT GTC Phe Val CAG CAT Gin His 330 295 CAG ACC ATG 945 Gin Thr MET 315 AAG CAG GAA 999 Lys Gin Glu CAG CAA Gin Gin CTG CAC Leu His 335 CAG GCC CAG Gin Ala Gin 320 AAG CTC ATG Lys Leu MET

CAT

His GCC CGA Ala Arg 340 TGC TTC 1026 Cys Phe CTG AAA CTT Leu Lys Leu 345 CCC AAA GTG Pro Lys Val

GGA

Gly

GAG

Glu TGG CAG CTG AAT 1053 Trp Gin Leu Asn 350 TAC TAC AGC GCC 1107 Tyr Tyr Ser Ala

CTA

Leu

GCC

Ala 370 CAG GGC ATC AAT Gin Gly Ile Asn 355 ACA GAG CAC GAC Thr Glu His Asp

GAG

Glu AGC ACA ATC 1080 Ser Thr Ile 360 AGC TGG TAC 1134 Ser Trp-Tyr CTG CAG Leu Gin 365

CGC

Arg AAG GCC Lys Ala 390 CAT CAG His Gin AAC ATC Asn Ile

TGG

Trp

CAT

His AAC CAA Asn Gin 400 ACC AAC GCG TGG GCA Ala Trp Ala 385 GCC CGC GAT Ala Arg Asp GCC ACC ACT GTG ATG 1161 Val MET GAG AAG 1215 Glu Lys 405 GCC GCC 1269 AAC TTC GAA Asn Phe Glu 390 AAG AAA CTG Lys Lys Leu ACC ACG GCC GCT GTG Ala Val CGT CAT Arg His 410 GCC ACT 375 CTA CAC TAC AAA 1188 Leu His Tyr Lys 395 GCC AGC GGG GCC 1242 Ala Ser Gly Ala GCC ACC ACC ACT 1296 Thr Asn Ala Thr Thr Ala Ala Thr Thr Ala Ala Thr Ala Thr Thr Thr 420 425 430 -47- GCC AGC ACC GAG GGC AGC AAC ACT GAG AGC GAG GCG GAG AGC ACC GAG AAC AGC 1323 1350 Ala Ser Thr Glu Gly Ser Asn Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser 435-- 440 450 CCC ACC Pro Thr GCA TCG CGG Pro Ser Pro 455 CTG GAG AAG AAG 1377 Leu Gin Lys Lys GTC ACT GAG GAT CTG TCC AAA ACC CTC 1404 Val1 460 Thr Giu CTG ATG TAG Leu MET Tyr 470

AG

Thr GTG GGT CC Val Pro Ala 475 CTC GAG CAT Leu Gin Asp Asp Leu GGT TGG Arg Ser Ser 465 Lys Thr Leu

GGA

Arg GGG AAGC AAC Gly Asn Asn 490 GAG TGG GGA His Trp Pro GTG GAG 1431 Val Gin ACA GTG 1485 Thr Leu 495 GAG CC 1539 Clu Ala GGG TTG Gly Phe AGA GTT Arg Val TTA GTG Leu Val 515

TTG

Phe 480 ATG TGG TTG TGA 1458 Ile Ser Leu Ser 485 TGG TTT GAT TAT 1512 Trp Phe Asp Tyr

GGT

G ly 505 GAT GTG Asp Val 510

A.AT

As n GTG ACC TTA Leu Thr Leu 500 GAG GGG GTG Giu Gl1Y Val AAA GGG Lys Ala 520 ATG GAG 1566 Ile Gin ATT GAT ACC TGG le Asp Thr Trp 525 AGA CCC TTG GTG Arg Pro Leu Val

CTA

Leu GAG GTT ATA GGT 1593 Gin Val Ile Pro 530 GGT GTG ATT GAG 1647 Arg Leu le His

GAG

Gin GTG ATT Leu Ile GTT GTG Leu Leu

GGA

Ala 535

AGA

Arg'

ATT

Ile GAT AGG CCC 1620 Aso Thr Pro 540 GGT GGG TAG 1674 Gly Arg Tyr

GGA

G ly 545

GAG

Gin 550 GAG CCC His Pro 560 GAG GCC Gin Ala ACA GAG Thr Asp TGT AAG Ser Lys

ATT

Ile 555 GTG ATG TAG Leu Ile Tyr 565 GGA CCC AAC Aia Ala Asn GGA GTG 1701 Pro Leu AAG ATT 1755 Lvs Ile ACA CTG GGT Thr Val Ala 570 GTG AAG AAG Leu Lys Asn TGT ACC AGG ACA '1728 Ser Thr .Thl Thr 575 GAG GAG AGG AAC 1782 Glu His Ser Asn

GGG

Ala GGG GAG AAT Arg His Asn 580 GTG GTG GAG Leu Val Gin ATG TGT MET Gys 590

ACC

Thr 595

GTG

Leu GAG GGG Gin Ala 600 585 ATG ATG GTG 1809 ME T MET Val AGG GAG Ser Giu 605 GTG GAA Leu Giu

GAG

C iu

GTG

Leu ATG GGA GTG Ile Arg Val.

610 TGT GGT TTG Ser Arg Leu GGG ATG 1836 Ala Ile TAG TTT 1890 Tyr Phe 630 TCG CAT Trp His 615 GAG ATG TGG CAT GAA GGG 1863 Giu MET Trp His Clu Cly 620 GAG GGA Giu Ala 625 -48- GGG GAA AGG A.C GTG AA GGC ATG TTT GAG GTG CTG GAG CCC TTG CAT 1917 GCT ATG 1944 Ala MET Gly Giu Arg Asn Val Lys Gly MET Phe Glu Val Leu Glu Pro Leu His ATG GAA CGG MET Giu Arg 650 CGA GAT TTA Arg Aso Leu

GGC

Gly

ATG

MET

CCC CAG ACT CTG AAG 1971 Pro Gin Thr Leu Lys 655 GAG GCC C AA GAG TGG 2025 Giu Ala Gin Glu Trp GAA ACA TCC Glu Thr TGC AGG rCy Ar~- Ser 660

AAG

Ly s

TAT

Tyr TTT A.AT Phe Asn TAC ATG Tyr MET 680 CAT GTG His Val CAG GCC TAT GGT 1998 Gin Ala Tyr Gly 665 TC-A GGG AAT 2052 Lys Ser Gly Asn 670

GTC

Val1 685

AAG

Ly s

GAC

Aso CTC ACC CAA GCC Leu Thr Gin Ala 690 CTG CCT CA'G CTC Leu Pro Gin Leu 675 TGG GAC 2079 Trp Asp ACA TCC 2133 ~1 CTC TAT Leu Tvr 695 TTC CGA Phe Arg 700 CGA ATC 2106 Arg Ile TCA AAG CAG Ser Lys Gin 705 CTT CTG ATG Leu Leu MET

TTA

Leu TGC CGG GAC Cys Arg Asp 725 Thr Ser 710 CTT GAA TTG 2187 Leu Giu Leu GAG CTG C .A TAT Giu Leu Gin Tyr 715 GTG CCA GGA ACA Val Pro Gly Thr

GTT

Val1 TCC C CA AAA.

2160 Ser Pro Lys 720 GAC CCC AAC 2214 Asp Pro Asn

GCT

Ala CAG CCA ATC Gin Pro Ile 740 AAG CAG AGG Lys Gin Arg

ATT

Ile CGC ATT CAG Arg Ile Gin 745 CGG AAA TTG Arg Lys Leu

TAT

Tyr 735 TCC ATA 2241 Ser Ile ACA CTT 2295 Thr Leu 765 730 GCA CCG TCT Ala Pro Ser 750 ATG GGC AGC M ET Gly Ser TTG CAA Leu Gin AAC GGA Asn Gly 770 GTC ATC ACA TCC 2268 Vai Ile Thr Ser 755 CAT GAG TTT GTT 2322 His Glu Ph6- Val

CCC

Pro

TTC

Phe 775 760 CTT CTA AA Leu Leu Lys GGC CAT Gly His 780 AAC ACC Asn Thr GPA GAT CTG 2349 Glu Asp Leu CTT CTG GCC 2403 Leu Leu Ala CGC CAG GAT GAG Arg Gin Asp Glu 785 AAT GAC CCA ACA Asn Asp Pro Thr 805 CGT GTG ATG Arg Val MET 790 TCT CTT CGG Ser Leu Arg TTC GGC CTG GTT Phe Gly Leu Val '795 CTC AGC ATC CAG CAG CTC 2376 Gin Leu AAA AAC 2430 Lys Asn 810 CTC ATT 2484 Leu Ile AGA TAC GCT GTC ATC CCT TTA TCG 2457

ACC

Leu Ser Ile Gin Arg Tyr Ala Val Ile Pro Leu Ser Thr 815 820 AAC TCG GGC Asn Ser Gly 825 -49- GGC TGG GTT CCC CAC TGT GAC ACA CTG CAC GCC CTC ATC CGG GAC TAC AGO GAG 2511 Thr Leu Gly Trp 830 AAG A.AG Lys Lys Val Pro His Cys Asp 835 His Ala CAT COC His Arg Leu 840 Ile Arg 2 53 8 Asp Tyr Arg Giu 843 COG ATG OCT CCO 2592 Arg MET Ala Pro AAO ATC CTT Lys Ile Leu 850 GAC CAC TTG Asp His Leu

CTC

Leu

ACT

Thr AAC ATC GAG 2565 Asn Ile Oiu 855 CTO ATO CAG 2619 Leu MET Gin ATC ATO Ile MET GAG OTO Glu Val

TTG

Leu 860

GAC

Asp 865

TAT

Tyr AAT AAT ACA OCT Asn Asn Thr Ala 885 TCC GAO OTO TG Ser Oiu Val Trp 000 Oly

TTT

Phe 905 870 GAC GAC CTG 0CC 2673 Asp Asp Leu Aia 890 GAC CGA AGA ACC 2727 Asp Arg Arg Thr AAO OTO Lys Val 875 AAG CTO Lys Leu TTT GAG Phe Oiu CTG A.AA Leu Lys

CAT

His 880 0CC OTC 2646 Ala Val CTG TOO Leu Trp 895 AGC CCC AOC 2700 Ser Pro Ser 900 OCO OTC ATO 2754 Ala Val MET A.AT TAT Asn Tyr 910

ACC

Thr COT TCT TTA Arg Ser Leu 915 TCA ATO Ser MET 920 OTT 000 Vai Oly TAT ATT Tyr Ile AGT 000 Ser Oly

TTA

Leu 925 GOC CTG 2781 Gly Leu OGA OAT AGA CAC Oly Asp Arg His 930 CAC ATT GAC TTT His le Asp Phe

CCA

Pro 000 Gly TCC AAC CTO ATO 2808 Ser Asn Leu MET 935 GAC TOC TTT GAG 2862 Asp Cys Phe Glu

CTO

Leu OAC COT Asp Arg OCT ATO Ala MET

CTO

Leu 940 AAO ATC CTG 2835 Lys Ile Leu 945 A-AO TTT CCA 2889 Lys Phe Pro

OTT

Val 955 ACC COA Thr Arg AAT OCT Asn Ala

GAO

Olu 960 GAG AAG Oiu L.ys 965 ATT CCA Ile Pro ATO TTO ACC MET Leu Thr 975 TOC CAC ACA Cys His Thr CTG OAA Leu Oiu 1010

GCC

Al a OTO ATO Val1 MET 995 TTT OTC Phe ValI ATO GAG OTT ACA 2943 MET Glu Val Thr 980 GAG OTO CTO COA 2997 Olu Val Leu Arg TAT GAC CCC TTG 3051

OGC

Gly

GAO

Glu CTG OAT GOC Leu Asp Oly 985 CAC AAO GAC His Lys Asp 950 TTT AGA CTA Phe Arg Leu 970 AAC TAC AGA Asn Tyr Arg AOT OTC ATO Ser Val MET 1005 CTO ATO GAC Leu MET Asp ACA AGA 2916 Thr- Arg ATC ACA 2970 Ile Thr 990 0CC OTO 3024 Ala Val ACA A.AT 3078 Thr Asn 1025 1000 CTO AAC TOG AGO Tyr Asp 1015 Pro Leu Leu Asn Trp Arg 1020 ACC A-A GGC AAC AAG CGA TCC CGA ACG AGG ACG GAT TCC TAC TCT GCT GGC CAG Thr Lys Gly Asn Lys 030 TCA GTC GAA ATT TTG Ser Val Glu Ile Leu 3105 Arg Ser Arg Thr Arg 1035 GAC GGT GTG GAA CTT 3159 Asp Gly Val Glu Leu Thr Asp Ser Ty r S er 1040 3132 Ala Gly Gil.

GGA GAG Gly Glu 1055

CCA

Pro 1045 GGG ACC ACA GTG Giy Thr Thr Val 1065 CCA GAG GCC CTA Pro Glu Ala Leu 1050

CCA

Pro

AAT

Asn

C

*CC.

CTC ACT Leu Thr hioO GAG CTG Glu Leu GGC TGG 1085 GGT CGG GAC Gly Arg Aso GAA- TCT ATT CAT 3213 Glu Ser Ile His 1070 AAG AAA GCT ATC 3267 Lys Lys Ala Ile TTC TCT CAT GAT 3321 Phe Ser His Asp 1105 CAA GCG ACA TCC

CAG

Gin

TCT

Ser 1090 GAC ACT TTG GAT Asp Thr Leu Asp 1110 CAT GA.A A-zC CTC 1095 GTT CCA ACG Val Pro Thr TGC CAG TGC TTC ATT GGA Phe Ile Gly 1075 ATT ATT AAC Ile Ile Asn CAAz GTT 3348 Gin Val 1115 TAT A TT 3402 GCC CAT AAG A.AA ACG 3186 Ala His Lys Lys Thr 1060 GAC GGT TTG GTG AA 3240 Asp Gly Leu Val Lys 1080 AGG GTT CGA GAT AAG 3294 Arg Val Arg Asp Lys CTC ATC A-k Leu Ilie Lys Gin Ala 1120 TAC CCT TTC TGG TAA 3375 Thr Ser His Giu Asn Leu Cys Gin Cys Tyr le 1125 1130 Gly Trp Tyr Pro Phe Tro 1135 1140

C

Claims (27)

1. A protein of human origin having a molecular weight as determined by SDS PAGE of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa, or a fragment of such a protein, which binds to an FKBP-Rapamycin complex.

2. A protein which exhibits FKBP-Rapamycin complex binding characteristics comprising at least part of the amino acid sequence of a protein of human origin having a molecular weight as determined by SDS PAGE of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa, which binds to an FKBP-Rapamycin complex.

3. The protein of claims 1 or 2, wherein the protein of human origin has a 15 molecular weight as determined by SDS PAGE of about 210 kDa. i

4. A DNA which encodes the protein of human origin or the fragment thereof according to claim 1, or the protein exhibiting FKBP-Rapamycin i complex binding characteristics according to claim 2.

5. A DNA as claimed in claim 4, wherein the DNA is a cDNA.

6. The DNA of claims 4 or 5, wherein the protein of human origin has a molecular weight as determined by SDS PAGE of about 210 kDa. 2 7. Antisense RNA derived from a cDNA clone as defined in claim

8. Antisense DNA derived from a cDNA clone as defined in claim

9. Antisense RNA derived from a cDNA according to claim Antisense DNA derived from a cDNA according to claim

11. A monoclonal or polyclonal antibody specific for the proteins of claim 1. Y:MaryNKI NO DELETE MR\78263-01doc 52

12. Use of a monoclonal or polyclonal antibody according to claim 11 for screening for novel agents for immunomodulation and/or anti-tumor activity.

13. Use of a monoclonal or polyclonal antibody according to claim 11 for measuring the parent compound and/or metabolites in biological samples obtained from individuals taking immunosuppressive drugs.

14. A process for isolating a protein having a molecular weight as determined by SDS PAGE of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa, which binds to an FKBP-Rapamycin complex from mammalian cells, the process comprising: a) lysing the mammalian cells in the presence of a buffering agent, a chelating agent, a protease inhibitor, and a reducing agent and preferably low salt at a temperature which minimizes protein degradation, 15 the lysing creating unbroken cells, cell nuclei, and lysates, the lysates i; including cellular membrane fractions and cellular debris; i b) preclearing unbroken cells and cell nuclei from the lysates at a temperature which minimizes protein degradation to create a precleared lysate; c) concentrating the cellular membrane fractions of the mammalian cells from the precleared lysate, the membrane fractions containing S- membrane proteins; d) solubilizing the membrane proteins in a buffer containing a detergent which solubilizes the proteins, without detrimentally denaturing the 25 proteins, at a temperature which minimizes protein degradation, resulting in solubilized proteins and mammalian cellular debris; e) separating the solubilized proteins from the mammalian cellular debris; f) incubating a solution containing the solubilized proteins in a buffer, the buffer containing a buffering agent, a reducing agent, one or more protease inhibitor(s), divalent cations and preferably a salt, with an affinity matrix to absorb to the affinity matrix those proteins which have a binding affinity to the affinity matrix at a temperature which allows binding to the affinity matrix and minimizes protein degradation; Y:VMaylNKI NO DELETE MR\78263-01 dOC 53 g) separating the affinity matrix from the solution of step at a temperature which minimizes protein degradation, yielding a solution containing solubilized proteins which do not bind to the affinity matrix in step h) incubating the solution with Rapamycin or a Rapamycin analog in LAF 500nM) complexed to a fusion protein of FKBP12+protein, the fusion protein enhancing the isolation of the desired about 125 kDa protein, about 148 kDa, about 208 kDa, or about 210 kDa protein yielding a mixture containing the desired proteins having a molecular weight of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa bound to fusion FKBP protein:Rapamycin complexes or fusion FKBP protein:Rapamycin analog complexes; i) incubating the mixture containing the desired proteins having a molecular weight of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa bound to fusion FKBP protein:Rapamycin complexes or 15 fusion FKBP protein:Rapamycin analog complexes with an affinity matrix which binds to the fusion protein at a temperature and for a time which allows the binding to the affinity matrix and minimizes protein degradation, bound to fusion FKBP protein:Rapamycin complexes or fusion FKBP protein:Rapamycin analog complexes; j) rinsing the affinity matrix containing the bound complexes with a buffer which dissociates binding of proteins other than the desired about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa proteins; k) eluting the about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa protein:fusion FKBP protein:Rapamycin complexes or the about 125 *l 25 kDa, about 148 kDa, about 208 kDa, or about 210 kDa protein:FKBP protein:Rapamycin analog complexes from the affinity matrix with a buffer; I) separating the proteins eluted in step by size.

15. A method for identifying an immunomodulatory or anti-fungal or anti- proliferative or anti-tumor agent, comprising the steps of: a) combining a substance to be tested with a protein as claimed in claim 1 which binds to an FKBP-Rapamycin complex, the protein preferably being bound to a solid support; Y:\MarNKI NO DELETE MR\7823-01 doc 54 b) maintaining the substance to be tested and the protein (preferably bound to the solid support) of step under conditions appropriate for binding of the substance to be tested with the protein; and c) determining whether binding of the substance to be tested occurred in step

16. A method for identifying an immunomodulatory or anti-fungal or anti- proliferative or anti-tumor agent, comprising the steps of: a) combining a substance to be tested with a protein as claimed in claim 1 which binds to an FKBP-Rapamycin complex, the protein preferably being bound to a solid support; b) maintaining the substance to be tested and the protein (preferably bound to a solid support) of step under conditions appropriate for binding of the substance to be tested with the protein; and 15 c) determining whether the presence of the substance to be tested modulated the activity of the protein which binds to an FKBP-Rapamycin complex.

17. A method for detecting, in a biological sample, Rapamycin, Rapamycin analogs or Rapamycin metabolites which, when complexed with a FKBP, bind to a mammalian protein having a molecular weight as determined by SDS PAGE of about 125 kDa, about 148 kDa, about 208 kDa, or about 210 kDa which binds to an FKBP-Rapamycin complex, the method comprising the steps of: S. 25 a) combining the biological sample with a FKBP to form a first mixture containing, if Rapamycin, Rapamycin analogs or Rapamycin metabolites are present in the biological sample, Rapamycin:FKBP complexes, Rapamycin analog:FKBP complexes, or Rapamycin metabolite:FKBP complexes; b) creating a second mixture by adding the first mixture to a protein as claimed in claim 1 which binds to a GST-FKBP-Rapamycin complex, the protein preferably being bound to a solid support; c) maintaining the second mixture of step under conditions appropriate for binding the Rapamycin:FKBP complexes, Rapamycin analog:FKBP Y:W2,wiNKI NO DELETE MRW78263-0.doc complexes, or Rapamycin metabolite:FKBP complexes, if present, to the protein which binds to a GST-FKBP-Rapamycin complex; and d) determining whether binding of the Rapamycin:FKBP complexes, Rapamycin analog:FKBP complexes, or Rapamycin metabolite:FKBP complexes and the protein occurred in step

18. Antisense RNA derived from a cDNA clone as defined in claim 5 for use for the treatment of a medical condition selected from the group consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases.

19. Use of an immunomodulatory amount of antisense RNA derived from a cDNA clone as defined in claim 5 for the preparation of a medicament.for the 15 treatment of a medical condition selected from the group consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases. C C

20. Antisense DNA derived from a cDNA clone as defined in claim 5 for use for the treatment of a medical condition selected from the group consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases. C

21. Use of an immunomodulatory amount of antisense DNA derived from a cDNA clone as defined in claim 5 for the preparation of a medicament for the treatment of a medical condition selected from the group consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases.

22. The antisense RNA or DNA according to claims 18 or 20 for the use specified therein, or the use of claims 19 or 21, wherein the transplantation Y:wVasNKI NO DELETE MRt782e3-Iodo= 56 rejection is against kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small bowel, a skin allograft, or a heart valve xenograft.

23. The antisense RNA or DNA according to claims 18 or 20 for the use specified therein, or the use of claims 19 or 21, wherein the autoimmune disease is selected from the group consisting of lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis.

24. The antisense RNA or DNA according to claims 18 or 20 for the use specified therein, or the use of claims 19 or 21, wherein the disease of inflammation is selected from the group consisting of psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, and eye uveitis. The antisense RNA or DNA according to claims 18 or 20 for the use 15 specified therein, or the use of claims 19 or 21, wherein the hyperproliferative S: vascular disease is restenosis or atherosclerosis.

26. A method of treating a medical condition selected from the group consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases, the method comprising administering a therapeutically effective amount of antisense RNA derived from cDNA clone according to claim S 4 25 27. A method of treating a medical condition selected from the group *****consisting of transplantation rejections, autoimmune diseases, diseases of inflammation, solid tumors, T-cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases, the method comprising administering a therapeutically effective amount of antisense DNA derived from cDNA clone according to claim

28. A method according to claim 26 or 27 when the transplant rejection is against kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small bowel, a skin allograft, or a heart valve xenograft. Y:\WryWKI NO DELETE MR%782&03-.doc 57

29. A method according to claim 26 or 27 wherein the autoimmune disease is selected from the group consisting of lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis.

30. A method according to claim 26 or 27 wherein the disease of inflammation is selected from the group consisting of psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, and eye uveitis.

31. A method according to claim 26 or 27 wherein the hyperproliferative vascular disease is restenosis or atherosclerosis. DATED: 7 June 2004 PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK and WYETH Y:WasyNKI NO DELETE MR7826301dOC