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  T-cell death-inducing epitopesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 12 To 15 Peptide Repeating Units In Known Peptide ChainThe Patent Description & Claims data below is from USPTO Patent Application 20060003940. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application Ser. No. 60/570,161, filed on May 11, 2004, the contents of which are incorporated by reference in its entirety. BACKGROUND [0002] Control of unwanted immune responses is critical in treating autoimmune diseases, transplant rejection, allergic diseases, and T-cell-derived cancers. The activity of overly aggressive T-cells can be contained by immunosuppression or by induction of immunological tolerance. Apoptosis is believed to be involved in maintaining proper functions of the immune system and removing unwanted cells, such as overly aggressive T-cells (Kabelitz et al. (1993) Immunol Today 14, 338-340; and Raff(1992) Nature 356, 397-399). SUMMARY [0003] This invention relates to T-cell death-inducing epitopes. The epitopes can be used for, among others, selecting compounds that bind to the epitopes. Such compounds are useful in treating diseases involving overly aggressive T-cells. Examples of such diseases include autoimmune diseases, transplant rejection, allergic diseases, and T-cell-derived cancers. [0004] In one aspect, the invention features a three-dimensional conformation of an isolated epitope. Binding of a ligand to the epitope on activated T-cells induces death of the cells. Such an epitope is represented by: X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:1), where (1) [0005] X.sub.1 is Tyr, Trp, His, or Met; [0006] X.sub.2 is Asp; [0007] X.sub.3 is Ser, Phe, Pro, Glu, or His; [0008] X.sub.4 is any amino acid that naturally occurring in animals; and [0009] X.sub.5 is Pro, Tyr, His, or Trp; X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 (SEQ ID NO:2), where (2) [0010] X.sub.6 is Asp; [0011] X.sub.7 is Tyr, Met, Asn, Trp, or Phe; [0012] X.sub.8 is Phe or Leu; [0013] X.sub.9 is Pro; and [0014] X.sub.10 is Glu; or X.sub.11-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO:3), where (3) [0015] X.sub.11 is Pro; [0016] X.sub.12 is Met; [0017] X.sub.13 is Glu or Ser; and [0018] X.sub.14 is Ile. [0019] Any of those epitopes described above can be, e.g., a polypeptide, an interacting region of two polypeptides, a carbohydrate moiety, a glycoprotein, or any conformational, functional equivalent thereof. [0020] In another aspect, the invention features an isolated polypeptide containing X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5, X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10, or X.sub.11-X.sub.12-X.sub.13-X- .sub.14. Binding of a ligand to the polypeptide on activated T-cells induces death of the cells. In one embodiment, the polypeptide contains 4 to 400 amino acids (e.g., any integer between 4 and 400, inclusive). For example, the polypeptide can be X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:1), X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 (SEQ ID NO:2), X.sub.11-X.sub.12-X.sub.13-X.sub.14 (SEQ ID NO:3), or any of SEQ ID NOs:4, 6-18, and 20-22. [0021] An "isolated epitope" or "isolated polypeptide" refers to an epitope or polypeptide substantially free from naturally associated molecules, i.e., it is at least 75% (e.g., any number between 75% and 100%, inclusive) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated epitope or polypeptide of the invention can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods. [0022] In still another aspect, the invention features a novel compound that binds to one of the above-described epitopes. The compound can be any kind of molecule, including antibodies such as monoclonal antibodies. A compound of the invention can be used for detecting an epitope of the invention and for inducing death of activated T-cells. Also within the scope of the invention is a method of producing antibodies. The method involves administering to a subject an effective amount of one of the above-described epitopes (e.g., polypeptides). The antibodies can be used for detecting an epitope of the invention or for inducing death of activated T-cells. [0023] The invention also features a method of identifying a candidate compound (e.g., a monoclonal antibody) for inducing death of activated T-cells. The method involves contacting a test compound with an epitope of the invention and determining whether the test compound binds to the epitope. If the test compound binds to the epitope, it is a candidate for inducing death of activated T-cells. [0024] The invention further features a method of inducing death of activated T-cells by contacting activated T-cells with a compound of the invention. [0025] In yet another aspect, the invention features a pharmaceutical composition containing a pharmaceutically acceptable carrier and (1) an epitope of the invention such as a polypeptide, or (2) a compound that binds to the epitope. [0026] The invention provides compositions and methods for treating diseases involving overly aggressive T-cells such as autoimmune diseases, transplant rejection, allergic diseases, and T-cell-derived cancers. The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will be apparent from the detailed description. DETAILED DESCRIPTION [0027] This invention is based on the unexpected discovery that activated T-cells can be induced to undergo apoptosis and be depleted by engagement of new T-cell death-inducing epitopes. Depletion of activated T-cells are particularly useful for treating conditions associated with an excessive or unwanted T-cell-mediated immune response or T-cell proliferation. For example, depletion of activated T-cells can result in reduction or elimination of undesirable T-cell activity or proliferation related to autoimmune diseases, transplant rejection, allergic diseases, or T-cell-derived cancers. [0028] Accordingly, the invention features a three-dimensional conformation of an isolated epitope. Binding of a ligand to the epitope on activated T-cells induces death of the cells. The epitope is represented by X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5, X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 or X.sub.11-X.sub.12-X.sub.13-X.- sub.14. The three-dimensional conformation of X.sub.1-X.sub.2-X.sub.3-X.su- b.4-X.sub.5, X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10, or X.sub.11-X.sub.12-X.sub.13-X.sub.14 can be determined, e.g., using computer modeling programs as described in Duggan et al., (1995) J Med. Chem. 38:3332-41 and Toogood (2002) J Med. Chem. 45: 1543-57. Epitopes of conformational, functional equivalence can be designed in accordance with the three-dimensional conformation of X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.s- ub.5, X.sub.6-X.sub.7-Xs-X.sub.9-X.sub.10, or X.sub.11-X.sub.12-X.sub.13-X- .sub.14, prepared using methods known in the art, and tested for their abilities to be involved in induction of death of activated T-cells by methods such as that described in the example below. See, e.g., Barbas et al. (2001) Phage display. A laboratory manual. CSHL Press; Parmley et al. (1998) Gene 73, 305-318; Scott et al. (1990) Science 249, 386-390; U.S. Patent Application No. 20030049252 A1; WO 03/013603 A1; Osborne (1996) Curr Opin Immunol 8:245-248; Lin et al. (1997) J. Immunol. 158, 598-603; Zhang et al. (1995) Nature 377, 348-350; Lai et al. (1995) Eur J Immunol 25, 3243-3248; Mollereau et al. (1996) J Immunol 156, 3184-3190; and Gribben et al. (1995) Proc Natl Acad Sci USA 92, 811-815. [0029] As used herein, an "activated T-cell" is a T-cell having a higher frequency, rate, or extent of proliferation than that of a non-activated T-cell. "Death" of a cell includes programmed cell death, i.e., apoptosis. "Induction of cell death" by an agent occurs when a population of cells treated with the agent exhibits a higher death rate compared to an untreated cell population. For example, the percentage of in vitro activated T-cells undergoing apoptosis is about doubled when treated with monoclonal antibodies m128-9F9, m152-15A7, or m166-43B6 compared to that of untreated cells, as determined by annexin V staining and FACS analysis (see the example below). [0030] The invention also features an isolated polypeptide containing X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5, X.sub.6-X.sub.7-X.sub.8-X.sub.9-- X.sub.10, or X.sub.1-X.sub.12-X.sub.13-X.sub.14. The polypeptide can be used for identifying compounds that induce death of activated T-cells. Binding of such a compound to the polypeptide expressed on the surface of activated T-cells induces cell death. Further, free polypeptides (i.e., those not expressed on the cell surface) can inhibit unwanted cell death by competing for endogenous death-inducing ligands with the cell-surface polypeptides. The length or sequence of the polypeptide may vary for these uses. A polypeptide of the invention can be obtained, e.g., as an isolated T-cell surface protein, a synthetic polypeptide, or a recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., Glutathione-S-Transferase (GST), 6.times.-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by standard methods. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention. [0031] An epitope of the invention or a polypeptide of the invention can be used to generate antibodies in animals (for production of antibodies) or humans (for treatment of diseases). Methods of making monoclonal and polyclonal antibodies and fragments thereof in animals are known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes intact molecules as well as fragments thereof, such as Fab, F(ab').sub.2, Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et. al. (1989) Nature, 341, 544). These antibodies can be used for detecting the epitope, e.g., in identifying a compound that binds to the epitope (see below). The antibodies that are capable of inducing death of activated T-cells are also useful for treating diseases such as autoimmune diseases, transplant rejection, allergic diseases, or T-cell-derived cancers. In general, an epitope of the invention, e.g., a polypeptide, can be coupled to a carrier protein, such as KLH, mixed with an adjuvant, and injected into a host animal. Antibodies produced in that animal can then be purified by peptide affinity chromatography. Commonly employed host animals include rabbits, mice, guinea pigs, and rats. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Useful human adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum. [0032] Polyclonal antibodies, heterogeneous populations of antibody molecules, are present in the sera of the immunized subjects. Monoclonal antibodies, homogeneous populations of antibodies to a particular antigen, can be prepared using standard hybridoma technology (see, for example, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur J Immunol 6, 511; Kohler et al. (1976) Eur J Immunol 6, 292; and Hammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al. (1975) Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole et al. (1983) Proc Natl Acad Sci USA 80, 2026, and the EBV-hybridoma technique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention may be cultivated in vitro or in vivo. The ability to produce high titers of monoclonal antibodies in vivo makes it a particularly useful method of production. [0033] In addition, techniques developed for the production of "chimeric antibodies" can be used. See, e.g., Morrison et al. (1984) Proc Natl Acad Sci USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage library of single chain Fv antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge. Moreover, antibody fragments can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab').sub.2 fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab').sub.2 fragments. Antibodies can also be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully human antibodies, such as those expressed in transgenic animals are also features of the invention (see, e.g., Green et al. (1994) Nature Genetics 7, 13; and U.S. Pat. Nos. 5,545,806 and 5,569,825). [0034] The invention further features a novel compound that binds to an epitope of the invention and induces death of activated T-cells. Such a compound can be designed, e.g., using computer modeling programs, according to the three-dimensional conformation of the epitope, and synthesized using methods known in the art. It can also be identified by library screening as described below. Continue reading... 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