Mammalian cell surface antigens; related reagents

This application is a Divisional of 10/326,052, filed Dec. 23, 2002, which is a Continuation of U.S. patent application Ser. No. 09/671,658, filed Sep. 27, 2000, now U.S. Pat. No. 6,525,180, which is a Continuation of U.S. patent application Ser. No. 08/989,362, filed Dec. 12, 1997, now U.S. Pat. No. 6,242,586, which claims benefit of U.S. Provisional Patent Application No. 60/032,846, filed Dec. 13, 1996, each of which are incorporated herein by reference.

The present invention pertains to compositions related to proteins which function in controlling activation and expansion of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate activation, development, differentiation, and function of various cell types, including hematopoietic cells.

The activation of resting T cells is critical to most immune responses and allows these cells to exert their regulatory or effector capabilities. See Paul (ed; 1993) Fundamental Immunology 3d ed., Raven Press, N.Y. Increased adhesion between T cells and antigen presenting cells (APC) or other forms of primary stimuli, e.g., immobilized monoclonal antibodies (mAb), can potentiate the T-cell receptor signals. T-cell activation and T cell expansion depends upon engagement of the T-cell receptor (TCR) and co-stimulatory signals provided by accessory cells. See, e.g., Jenkins and Johnson (1993) Curr. Opin. Immunol. 5:361-367; Bierer and Hahn (1993) Semin. Immunol. 5:249-261; June, et al. (1990) Immunol. Today 11:211-216; and Jenkins (1994) Immunity 1:443-446. A major, and well-studied, co-stimulatory interaction for T cells involves either CD28 or CTLA-4 on T cells with either B7 or B70 (Jenkins (1994) Immunity 1:443-446). Recent studies on CD28 deficient mice (Shahinian, et al. (1993) Science 261:609-612; Green, et al. (1994) Immunity 1:501-508) and CTLA-4 immunoglobulin expressing transgenic mice (Ronchese, et al. (1994) J. Exp. Med. 179:809-817) have revealed deficiencies in some T-cell responses though these mice have normal primary immune responses and normal CTL responses to lymphocytic choriomeningitis virus and vesicular stomatitis virus, As a result, both these studies conclude that other co-stimulatory molecules must be supporting T-cell function. However, identification of these molecules which mediate distinct costimulatory signals has been difficult.

Tumor Necrosis Factor (TNF) is the prototypic member of an emerging family of cytokines that function as prominent mediators of immune regulation and the inflammatory response. These ligands are typically type II membrane proteins, with homology at the carboxy terminus. A proteolytic processed soluble protein often is produced. See, e.g., Smith, et al. (1994) Cell 76-959-962; Armitage (1994) Current Opinion in Immunology 6:407-413; Gruss and Dower (1995) Blood 85:3378-3404; Wiley, et al. (1995) Immunity 3:673-682; and Baker and Reddy (1996) Oncogene 12:1-9. Crucial roles for these family members are evidenced by a number of studies, and they are implicated in regulation of apoptosis, peripheral tolerance, Ig maturation and isotype switching, and general B cell and T cell functions. See, e.g., Thomson (ed. 1994) The cytokine Handbook Academic Press, San Diego, Calif. These imply fundamental roles in immune and developmental networks.

The inability to modulate activation signals prevents control of inappropriate developmental or physiological responses in the immune system. The present invention provides at least one alternative costimulatory molecule, agonists and antagonists of which will be useful in modulating a plethora of immune responses.

The present invention is based, in part, upon the discovery of an antigen which exhibits sequence homology to proteins which act as inducers of apoptosis. In particular, it provides a gene encoding a 316 amino acid protein, designated 499E9, which is expressed on a highly polarized Th1 T cell. Engagement of 499E9 may modulate antigen-specific proliferation and cytokine production by effector cells. 499E9 is a novel cell surface molecule which, when engaged, may either potentiate immune cell expansion or apoptosis. The mouse embodiment is described, enabling mammalian genes, proteins, antibodies, and uses thereof. Functional equivalents exhibiting significant sequence homology are available from other mammalian, e.g., human, and non-mammalian species. Moreover, the receptor of 499E9 can function as its binding partner to stimulate other cells expressing the receptor.

More particularly, the present invention provides a composition of matter selected from: a substantially pure or recombinant 499E9 protein or peptide exhibiting at least about 85% sequence identity over a length of at least about 12 amino acids to SEQ ID NO: 2; a natural sequence 499E9 of SEQ ID NO: 2; or a fusion protein comprising 499E9 sequence. Certain embodiments include a substantially pure or isolated protein comprising a segment exhibiting sequence identity to a corresponding portion of a 499E9, wherein: the homology is at least about 90% identity and the portion is at least about 9 amino acids; the homology is at least about 80% identity and the portion is at least about 17 amino acids; or the homology is at least about 70% identity and the portion is at least about 25 amino acids. Other embodiments include a composition of matter, wherein the: 499E9 comprises a mature sequence of Table 1; or protein or peptide: is from a warm blooded animal selected from a mammal, including a rodent; comprises at least one polypeptide segment of SEQ ID NO: 2; exhibits a plurality of portions exhibiting the identity; is a natural allelic variant of 499E9; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a mammalian 499E9; exhibits a sequence identity at least about 90% over a length of at least about 20 amino acids to a rodent 499E9; exhibits at least two non-overlapping epitopes which are specific for a rodent 499E9; exhibits a sequence identity at least about 90% over a length of at least about 20 amino acids to a rodent 499E9; is glycosylated; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence. Also provided are various compositions, e.g., comprising: a sterile 499E9 protein or peptide; or the 499E9 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration. Fusion proteins are provided, e.g., comprising: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another TNF-ligand protein. Kit embodiments are provided, e.g., comprising such a protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.

Antibody, or binding compound embodiments include those comprising an antigen binding portion from an antibody, which specifically binds to a natural 499E9 protein, wherein: the protein is a rodent protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide comprising sequence of Table 1; is raised against a mature 499E9; is raised to a purified 499E9; is immunoselected; is a polyclonal antibody; binds to a denatured 499E9; exhibits a Kd to antigen of at least 30 μM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. Other embodiments include a kit comprising the binding compound, and: a compartment comprising the binding compound; and/or instructions for use or disposal of reagents in the kit. Other forms include, e.g., a composition comprising: a sterile binding compound; or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration. Such also allow methods of purifying a 499E9 protein or peptide from other materials in a mixture comprising contacting the mixture to such an antibody, and separating bound 499E9 from other materials.

Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding a protein or peptide or fusion protein, wherein: the 499E9 protein is from a mammal, including a rodent; or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; exhibits at least about 80% identity to a natural cDNA encoding the segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a rodent; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding the 499E9 protein; or is a PCR primer, PCR product, or mutagenesis primer. A cell or tissue comprising such a recombinant nucleic acid is also embraced within the invention, e.g., wherein the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a rodent cell; or a human cell. Kit forms include those comprising the nucleic acid, and: a compartment comprising the nucleic acid; a compartment further comprising a 499E9 protein or polypeptide; and/or instructions for use or disposal of reagents in the kit. Other nucleic acid embodiments include those which: hybridize under wash conditions of: 30° C. and less than 2M salt, 45° C. and/or 500 mM salt, or at 55° C. and/or 150 mM salt to SEQ ID NO: 1; or exhibit at least about 85% identity over a stretch of at least about 30 nucleotides, at least 90% and/or the stretch is at least 55 nucleotides, or at least 95% and/or the stretch is at least 75 nucleotides to a rodent 499E9.

The invention further provides methods of modulating physiology or development of a cell or tissue culture cells comprising introducing into the cell an agonist or antagonist of a 499E9. Other methods include modulating the physiology of a cell comprising contacting the cell with: a substantially pure 499E9 or fragment; an antibody or binding partner which specifically binds a 499E9; or c) a nucleic acid encoding a 499E9 or peptide. Preferably, the is a T cell and the modulating of physiology is: apoptosis of the T cell; or activation of the T cell.

The invention further provides a method of treating a patient having an abnormal immune response by administering an effective dose of an antibody or binding partner specific for 499E9; a 499E9 protein or polypeptide; or a nucleic acid encoding a 499E9 peptide. The abnormal immune response is characterized by a T cell immune deficiency; chronic inflammation; or tissue rejection.

All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Outline

I. General

II. Purified 499E9

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