Methods of treating viral hepatitis   

This application is a continuation of U.S. patent application Ser. No. 10/770,639, filed Feb. 2, 2004, which claims the benefit of priority to ES200302587, filed on Nov. 5, 2003, and ES200300252, filed Jan. 31, 2003, each of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to the use of members of the NK gene complex family of type II oligomeric signal transmitting receptor proteins containing a C-type lectin-binding domain to modulate a subject\'s immune response.

BACKGROUND OF THE INVENTION

Immune-mediated diseases are an emerging field of pharmacological treatment that includes autoimmune diseases, which affect approximately 5% of the population (O\'Shea et al., 2002), graft rejection, allergy, atherosclerosis, infectious diseases and tumoral processes that are one major cause of mortality in industrialized countries. Therefore, the regulation of the immune response is key for the treatment of the major causes of morbidity and mortality in our society.

CD69 is expressed early and transiently following leukocyte activation after an immune challenge (Cebrian et al., 1988; Hara et al., 1986; Testi et al., 1994) in all hematopoietic subsets except erythrocytes. Although, CD69 is detected in vivo on small subsets of T and B cells in peripheral lymphoid tissues (Sanchez-Mateos et al., 1989), CD69 is persistently expressed in leukocyte infiltrates of several chronic inflammatory diseases such as rheumatoid arthritis, and viral-chronic hepatitis (García-Monzón et al., 1990; Laffon et al., 1991), in leukocytes responsible for graft rejection (Mampaso et al., 1993), in leukocytes involved in the allergic response (Hartnell et al., 1993), in immune cells at the atherosclerotic lesion (Caspar-Bauguil et al., 1998), in tumor infiltrating lymphocytes (Coventry et al., 1996), or upon persistent infection (Zajac et al., 1998). Several reports suggest that CD69 is involved in activation of bone marrow-derived cells (Cebrian et al., 1988; Testi et al., 1994). Nevertheless, nearly normal hematopoietic cell development and T cell maturation occur in CD69−/− mice under physiological conditions (Lauzurica et al., 2000).

CD69, together with two other proteins, activation-induced C-type lectin (AICL) and Lectin-like transcript (LLT1), form part of the NK complex. (Hamann et al., 1997; Boles et al., 1999). Expression of all three of these proteins, CD69, AICL and LLT1, is upregulated at early time points of cellular activation. (Eichler et al., 2001).

Autoimmune diseases are characterized by the presence of abnormal and persistent leukocyte activation that contributes to disease pathogenesis. It is well established that cytokines have essential roles in the pathogenesis of autoimmune diseases (Falcone and Sarvetnick, 1999). Therefore, the targeting of these cytokines is currently the preferred strategy in the new treatments for autoimmune disease. In this regard, tumor necrosis factor (TNF) appears to be at the top of the pro-inflammatory cascade, and anti-TNF therapy has been revealed as a useful tool for the treatment of RA (Feldmann, 2002). In addition, other cytokines have demonstrated an opposite anti-inflammatory effect. Thus, administration of TGF-β1 has beneficial effects in several autoimmune diseases (Prud\'homme and Piccirillo, 2000), depressing pro-inflammatory cytokine production in arthritis (Kuruvilla et al., 1991), allergic encephalomyelitis (Chen et al., 1994), colitis (Powrie et al., 1996), and autoimmune diabetes (Piccirillo et al., 1998).

The immune response against tumors behaves as “an effective extrinsic tumor-suppressor system” that involves the combined action of humoral and cellular mechanisms, in which lymphocytes and cytokines prevent primary tumor development (Dighe A S, 1994; Kaplan D H, 1998; Shankaran V, 2001; Smyth and Godfrey, 2000; Smyth et al., 2000; van den Broek M E, 1996). Cytokine action, at least in part directed at regulating tumor cell immunogenicity, is critical for the tumor suppressor function of the immune system. Advanced tumors and metastases down-regulate class I MHC expression, becoming susceptible to NK cell killing, and therefore, NK cells recognize and destroy cancer cells. In addition, NK cells secrete cytokines, such as TGF-β and IFN-γ that drive differentiation of activated CD4 T cells into Thi helper effector cells. In this regard, IFNγ helps to prevent tumor formation, as increased spontaneous neoplasic diseases are developed in IFNRγR−/− mice (Dighe A S, 1994; Kaplan D H, 1998). In addition, TGF-β has an important immuno-suppressive role, thus inhibiting anti-tumor immune response. TGF-β prevents and/or suppresses immune responses, regulating production of proinflammatory cytokines, reviewed in (Letterio and Roberts, 1998) and the inhibitory effect of TGF-β in the cytokine network is dependent on both cell stage and cytokine milieu. TGF-β has been implicated in the modulation of anti-tumor effector mechanisms by altering activation, proliferation and cytotoxicity of immune cells, macrophages, NK and T cells (Gorelik and Flavell, 2000; Kehrl et al., 1986). Recently, it has been reported that blockade of TGF-β signaling in T cells enhances anti-tumor immunity by facilitating an expansion of tumor specific CD8+ T cells (Gorelik and Flavell, 2001). In addition, other cytokines are important regulators of recruitment, proliferation, differentiation and survival of anti-tumor effector cells. MCP-1 produced by many cells activates cell recruitment, and animals that lack MCP-1 show diminished T cell responses (Allavena et al., 1994; Carr et al., 1994; Gu et al., 1997). Therefore, cytokines have essential roles in inducing anti-tumor immunity, and targeting cytokines provides effective therapy in human and several murine models (Gill P S, 1995).

However, the fibrogenic and other detrimental effects of TGF-β1 may limit its immunotherapeutic use in humans (Border and Ruoslahti, 1992). TGF-β has been implicated in several pathologic conditions and its systemic use has oncogenic potential, which may explain the increased number of tumors seen in patients treated with cyclosporin, a drug which enhances systemic TGF-β production (Hojo et al., 1999). Therefore, the local regulation of TGF-β1 should result in an effective treatment of chronic inflammatory diseases by avoiding the detrimental consequences of systemic TGF-β1.

Hence, CD69, AICL, and LLT1 may represent targets for immunotherapy in diseases whereby leukocytes express high levels of these activation markers. It follows that it would be desirable to develop specific antibodies to target such molecules for use in human therapy, e.g., in treating diseases associated with the presence of activated cells expressing one or more of these antigens.

Although murine monoclonal antibodies (mAbs) are relatively easy to produce, and some mouse mAbs to anti-human CD69 are now available, there are many restrictions for their therapeutic use in humans, due to their immunogenicity and to the resulting reduction in their efficacy and safety (Jakobovits, 1995). These restrictions could be overcome by the use of fully human mAbs, which would allow their repeated administration without immunogenic and/or allergic responses. Thus, the development of transgenic mice strains, engineered with unrearranged human immunoglobulin (Ig) genes, has become a good strategy for the generation of specific human mAbs.

SUMMARY

The invention is based, in part, on the discovery that modulation of CD69, AICL and/or LLT1 can be used to modulate a subject\'s immune response and, thus can be used to treat a variety of disorders. CD69, AICL and LLT1 agonists, CD69, AICL and LLT1 antagonists and depletors of CD69-, AICL- and LLT1-expressing cells are all useful, as is described below. CD69, AICL and LLT1 and the nucleic acids encoding them are referred to herein as “early activation molecules”.

Accordingly, in one aspect, the invention features methods of treating a subject, e.g., a subject having a disorder characterized by an unwanted immune response. The disorder can be characterized by a “normal” response, or by an immune response that is increased as compared with what is normally seen in a subject. Examples of such disorders are provided herein and include acute or chronic inflammatory disorders, and immune disorders, e.g., autoimmune disorders. The method includes administering an effective amount of one or more early activation molecule agonist to the subject. (Unless other wise indicated, the use of the term agonist, or antagonist, can refer to an agent which exerts an effect at the level of nucleic acid, protein, or both.)

Examples of early activation molecule agonists include, but are not limited to: CD69, AICL, and LLT1 polypeptides, peptides, peptidomimetics or functional fragments thereof; nucleic acids which encode a CD69, an AICL or a LLT1 polypeptide or functional fragments thereof, agents which increase the expression of endogenous CD69, AICL or LLT1, e.g., an agent which increases transcription of CD69, AICL or LLT1 (e.g., an agent which binds to an early activation molecule promoter and increases transcription), small molecules which bind to an early activation molecule and agonize its activity, an agonist anti-CD69 antibody molecule, an agonist anti-AICL antibody molecule and an agonist anti-LLT1 antibody molecule. Examples of small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Examples of other early activation molecule agonists include, but are not limited to: agonist CD69 receptor (CD69R) polypeptides, peptides, peptidomimetics, agonist AICL receptor (AICL-R) polypeptides, peptides and peptidomimetics, agonist LLT1 receptor (LLT1-R) polypeptides, peptides and peptidomimetics, soluble forms of CD69R, soluble forms of AICL-R, soluble forms of LLT1-R, agonist CD69R, AICL-R, or LLT1-R fusion proteins, e.g., an early activation protein receptor-serum protein fusion protein (e.g., CD69R-immunoglobulin fusion proteins, CD69R-human serum albumin fusion proteins, AICL-R-immunoglobulin fusion proteins, AICL-R-human serum albumin fusion proteins, LLT1-R-immunoglobulin fusion proteins, LLT1-R-human serum albumin fusion proteins), or other forms of agonist early activation protein receptor fusion proteins designed to increase serum half-life and/or multivalency. Preferred agonists are anti-early activation protein antibody molecules (e.g., anti-CD69 antibody molecules, anti-AICL antibody molecules and anti-LLT1 antibody molecules), early activation proteins receptor polypeptides, e.g., CD69R fragments, AICL-R fragments, LLT1-R fragments, CD69R fusion proteins, AICL-R fusion proteins, and LLT1-R fusion proteins. Antibody molecules which increase an early activation molecule activity include, but are not limited to, antibody molecules which interact with, e.g., bind to, an early activation polypeptide and result in a change in interaction, e.g., binding activity, of the early activation polypeptide with an early activation polypeptide binding partner (e.g., the antibody molecule can affect the conformation of an epitope of the early activation polypeptide, thereby resulting in increased interaction, e.g., binding, by an early activation polypeptide binding partner to the epitope), an antibody which binds an early activation polypeptide and increases clustering of the protein (e.g., an IgM which binds an early activation polypeptide), and any CD69 antibody, AICL antibody or anti-LLT1 antibody, e.g., an art known antibody, which can act as an agonist.

In a preferred embodiment, the antibody is a human monoclonal antibody that specifically targets an early activation molecule, e.g., hAIM-29 specific for the human CD69 antigen.

In some embodiments, the subject has an inflammatory disease, e.g., an acute or chronic inflammatory disease. Examples of inflammatory diseases include, but are not limited to: acute lung injury, acute respiratory distress syndrome, arthritis (e.g., CIA), asthma, bronchitis, cystic fibrosis, autoimmune diabetes, encephalomyelitis, hepatitis (e.g., viral chronic hepatitis), inflammatory bowel disease, multiple sclerosis, reperfusion injury (e.g., myocardial), nephritis, pancreatitis, psoriasis, artery occlusion (e.g., retinal), stroke, systemic lupus erythematosus, transplantation (e.g., host mediated rejection of transplant, e.g., transplanted tissue such as hematopoietic stem cells or an organ, e.g., a kidney, or graft mediated host response, e.g., graft v. host disease), ultraviolet light induced injury, and/or vasculitis. The inflammatory disease may be acute or chronic, and is preferably mediated by leukocytes. In some embodiments, the inflammatory disease is associated with chronic inflammation, e.g., the disease is inflammatory bowel disease, such as Crohn\'s disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.

In a preferred embodiment, the method includes administering one or more early activation molecule agonist in combination with a second therapeutic agent, e.g., a therapeutic agent or agent for treating inflammation. The second agent can be an antibody or an agent other than an antibody.

The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human.

In some embodiments, the early activation molecule agonist can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes. In some embodiments, the early activation molecule agonist can be administered prior to or during a secondary immune response in the subject.

In a preferred embodiment, the early activation molecule agonist is administered on a repeated basis. E.g., the agonist can be administered 2, 4, 6, or more times. Administration can be repeated until improvement in subject condition is seen or expected. Administration of an antibody can be can be at a frequency of about once, every 5 to 7 days, 14 to 30 or 30 to 60 days. Administration of other compounds can be administered more frequently. For example, a small molecule can be administered one, two, three, four or more times a day.

In another aspect, the invention features methods of treating a subject, e.g., a subject having a disorder characterized by an unwanted immune response. The disorder can be characterized by an immune response that is increased as compared with what is normally seen in a subject. Examples of such disorders are provided herein and include acute or chronic inflammatory disorders, and immune disorders, e.g., autoimmune disorders. The method includes administering an effective amount of a depletor of an early activation polypeptide-expressing cell to the subject, e.g., a cell expressing one or more of CD69, AICL and LLT1.

Examples of early activation molecule depletors include, those described herein, but are not limited to, antibody molecules which bind CD69 and deplete CD69 expressing cells, e.g., an anti-human CD69 antibody analogous to the anti-mouse CD69 antibody 2.3 or any CD69 antibody, e.g., an art known antibody which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; antibody molecules which bind AICL and deplete AICL expressing cells, e.g., any AICL antibody, e.g., an art known antibody, which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; antibody molecules which bind LLT1 and deplete LLT1 expressing cells, e.g., any LLT1 antibody, e.g., an art known antibody which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; or an antibody molecule identified by a method described herein. In a preferred embodiment, the anti-human CD69 antibody is a human monoclonal antibody, e.g., hAIM-29. In another embodiment, a depleting anti-early activation polypeptide antibody molecule (e.g., a depleting anti-CD69 antibody molecule, a depleting anti-AICL antibody molecule or a depleting anti-LLT1 antibody molecule) can recruit an immune cell (e.g., by way of an Fc receptor or by recruitment of the complement system) and inactivate or kill the target cell, e.g., the early activation polypeptide-expressing cell. Thus, in another embodiment, a depleting anti-early activation polypeptide antibody can activate the classical complement pathway that leads to the lysis of the target cell (e.g., the CD69-, AICL-, and/or LLT1-expressing cell). In another embodiment, the depletor is an anti-early activation polypeptide antibody molecule which is coupled to a therapeutic, e.g., a protein, drug or isotope, which can inactivate or kill the target cell, e.g., the CD69-, AICL- and/or LLT1-expressing cell.

In some embodiments, the subject has an inflammatory disease, e.g., an acute or chronic inflammatory disease. Examples of inflammatory diseases include, but are not limited to: acute lung injury, acute respiratory distress syndrome, arthritis (e.g., CIA), asthma, bronchitis, cystic fibrosis, autoimmune diabetes, encephalomyelitis, hepatitis (e.g., viral chronic hepatitis), inflammatory bowel disease, multiple sclerosis, reperfusion injury (e.g., myocardial), nephritis, pancreatitis, psoriasis, artery occlusion (e.g., retinal), stroke, systemic lupus erythematosus, transplantation (e.g., host mediated rejection of transplant, e.g., transplanted tissue such as hematopoietic stem cells or an organ, e.g., a kidney, or graft mediated host response, e.g., graft v. host disease), ultraviolet light induced injury, and/or vasculitis. The inflammatory disease may be acute or chronic, and is preferably mediated by leukocytes. In some embodiments, the inflammatory disease is associated with chronic inflammation, e.g., the disease is inflammatory bowel disease, such as Crohn\'s disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.

In a preferred embodiment, the method includes administering one or more depletor of early activation polypeptide-expressing cells in combination with a second therapeutic agent, e.g., a therapeutic agent or agent for treating inflammation. The second agent can be an antibody or an agent other than an antibody.

The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human.

In some embodiments, the early activation molecule depletor can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes. In some embodiments, the early activation molecule depletor can be administered prior to or during a secondary immune response in the subject.

In a preferred embodiment, early activation molecule depletor is administered on a repeated basis. E.g., the depletor can be administered 2, 4, 6, or more times. Administration can be repeated until improvement in subject condition is seen or expected. Administration of an antibody can be can be at a frequency of about once, every 5 to 7 days, 14 to 30 or 30 to 60 days. Administration of other compounds can be administered more frequently. For example, a small molecule can be administered one, two, three, four or more times a day.

In another aspect, the invention features, methods of treating a subject, e.g., a subject in need of, or a subject that would benefit from, an increased or augmented immune response. The subject can have a disorder characterized by a decreased immune response, e.g., an immune response that is decreased as compared to what is seen in the absence of a disorder, e.g., the subject could have an immunodeficiency. In other embodiments, the subject does not have a decreased or compromised immune response but would never the less benefit from an increased or augmented immune response. For example, the subject could have a disorder characterized by unwanted cells or tissue or characterized by unwanted cell proliferation, such as cancer. The method can also be used to increase or augment an immune response in a subject, e.g., an immune response to an antigen (e.g., a vaccine). The method includes administering one or more early activation molecule antagonist (e.g., a CD69 antagonist, an AICL antagonist, an LLT1 antagonist or combinations thereof) to the subject.

Examples of early activation molecule antagonists include, but are not limited to, antisense of an early activation molecule (e.g., antisense of CD69, AICL or LLT1), RNAi targeted to an early activation molecule, an antagonist anti-CD69 antibody molecule, an antagonist anti-AICL antibody molecule, an antagonist anti-LLT1 antibody molecule, and other compounds identified by a method described herein, e.g., compounds that interact with one or more of CD69, AICL and LLT1 and decrease TGF-θ expression in a cell, and small molecules which bind to an early activation molecule and antagonize its activity. Examples of small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Examples of other early activation molecule antagonist include, but are not limited to: antagonist CD69 receptor (CD69R) polypeptides, peptides, peptidomimetics; antagonist AICL receptor (AICL-R) polypeptides, peptides, peptidomimetics; antagonist LLT1 receptor (LLT1-R) polypeptides, peptides, peptidomimetics; soluble forms of CD69R; soluble forms of AICL-R; soluble forms of LLT1-R; antagonist CD69R, AICL-R or LLT1-R fusion proteins, e.g., early activation polypeptide-serum protein fusion protein (e.g., CD69R-immunoglobulin fusion proteins, CD69R-human serum albumin fusion proteins, AICL-R-immunoglobulin fusion proteins, AICL-R-human serum albumin fusion proteins, LLT1-R-immunoglobulin fusion proteins, LLT1-R-human serum albumin fusion proteins), or other forms of antagonist early activation polypeptide receptor fusion proteins designed to increase serum half-life and/or multivalency. Antagonist anti-early activation polypeptide antibody molecules include, but are not limited to, antibody molecules that bind an early activation polypeptide and interfere with binding of the early activation polypeptide and an early activation polypeptide binding polypeptide and antibody molecules that bind an early activation polypeptide and decrease the level of the early activation polypeptide on a cell surface, e.g., by internalization of the early activation polypeptide. Antagonist anti-CD69 antibody molecules include, but are not limited to, antibody molecules which bind CD69 and interfere with binding of CD69 to a CD69 binding partner and antibody molecules which bind CD69 and decrease the level of CD69 on a cell surface. Examples of antagonist anti-CD69 antibody molecules include an anti-human CD69 antibody, e.g., an anti-human CD69 antibody analogous to the anti-mouse CD69 antibody 2.2 or an anti-CD69 antibody, e.g., an art known antibody, which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69. In a preferred embodiment, the anti-human CD69 antibody is a human monoclonal antibody, e.g., hAIM-29. Antagonist anti-AICL antibody molecules include, but are not limited to, antibody molecules which bind AICL and interfere with binding of AICL to an AICL binding partner and antibody molecules which bind AICL and decrease the level of AICL on a cell surface. Examples of antagonist anti-AICL antibody molecules include an anti-human AICL antibody, e.g., an anti-AICL antibody, e.g., an art known antibody, which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 37 to 149 of human AICL. Antagonist anti-LLT1 antibody molecules include, but are not limited to, antibody molecules which bind LLT1 and interfere with binding of LLT1 to a LLT1 binding partner and antibody molecules which bind LLT1 and decrease the level of LLT1 on a cell surface. Examples of antagonist anti-LLT1 antibody molecules include an anti-human LLT1 antibody, e.g., an anti-LLT1 antibody, e.g., an art known antibody, which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 76 to 132 of human LLT1.

In some embodiments, the subject has a disease characterized by unwanted malignant or non-malignant cellular proliferation, e.g., cancer. Examples of cancers include, but are not limited to, biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; leukemias (e.g., B-cell chronic lymphocytic leukemia, e.g., B-cell chronic lymphocytic leukemia lacking immunoglobulin mutations), lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. In one embodiment, the cancer is not a CD69, AICL and/or LLT1 expressing cancer. In another embodiment, the cancer is B-cell chronic lymphocytic leukemia lacking immunoglobulin mutation and the subject is administered an early activation molecule antagonist, e.g., an AICL antagonist.

In a preferred embodiment, the method includes administering an early activation molecule antagonist in combination with a second agent, e.g., with one or more therapeutic agents, e.g., a therapeutic agent or agent for treating unwanted cell proliferation. The second agent can be an antibody or a non-antibody agent. Therapeutic agents include, for example, one or more of a chemotherapeutic agent, a radioisotope, and a cytotoxin. Examples of chemotherapeutic agents include taxol, cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, chlorambucil, gemcitabine, actinomycin, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids and analogs or homologs thereof. Additional therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioisotopes can include alpha, beta and/or gamma emitters. Examples of radioisotopes include 212Bi, 213Bi, 131I, 211At, 186Re, 90Y and 117Lu. The early activation molecule antagonist can be administered prior to, concurrent with or after administration of the therapeutic agents.

In one embodiment, the subject has an immunodeficiency, e.g., an acquired immunodeficiency (e.g., AIDS), a hereditary immunodeficiency, or an immunodeficiency induced or caused by exposure to an environmental or medicinal agent (e.g., a chemotherapeutic agent or radiation treatment).

In another embodiment, the subject has a non-pathologic hyperproliferative disorder, e.g., fibrosis. The fibrotic disorder can be associated with one or more of: trauma, surgery, infection, environmental pollutants, tobacco, alcohol or other toxins. The disorder can be associated with, e.g., one or more of: overabundant wound repair (e.g., hypertrophic scars and keloids), renal fibrosis (proliferation of mesangial cells), liver fibrosis (proliferation of liver stellate cells). Examples of fibrotic disorders include: keloids, burns, hypertrophic scars or other skin disorders (e.g., local or systemic scleroderma, e.g., scleroderma systemic sclerosis), liver cirrhosis, kidney fibrosis (e.g., related to diabetes and/or hypertension), surgical adhesions (e.g., from gastrointestinal surgery or neurosurgery adhesions), vascular grafts, indiopathic pulmonary fibrosis, radiation-induced fibrosis, asbestos-related fibrosis (e.g., black or brown lung), fibrosis associated with viral hepatitis, macular degeneration, retinal and vitreal retinopathy, and fibrosis associated with acute respiratory syndrome. The fibrosis can be acute or chronic fibrosis. Examples of acute fibrosis include: accidental injury, infections, surgery, burns, radiation-induced fibrosis and chemotherapy induced fibrosis. Chronic fibrosis can be associated, e.g., with viral infection, diabetes, and hypertension.

The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having an inflammatory disorder).

In some embodiments, the early activation molecule antagonist can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes. In a preferred embodiment the early activation molecule antagonist is administered on a repeated basis. E.g., the antagonist can be administered 2, 4, 6, or more times. Administration can be repeated until improvement in subject condition is seen or expected. Administration of an antibody can be can be at a frequency of about once, every 5 to 7 days, 14 to 30 or 30 to 60 days. Administration of other compounds can be administered more frequently. For example, a small molecule can be administered one, two, three, four or more times a day.

In another aspect, the invention features methods of enhancing the immune response to an antigen, e.g., a vaccine. The method includes administering to a subject an antigen, e.g., a vaccine, and/or DNA encoding an antigen, and an early activation molecule antagonist, to thereby enhance immune response to the antigen by the subject.

Examples of early activation molecule antagonists include those described herein.

In some embodiments, the early activation molecule antagonist is an anti-early activation polypeptide antibody molecule, e.g., an anti-CD69 antibody molecule, an anti-AICL antibody molecule, an anti-LLT1 antibody molecule (e.g., an antagonist anti-CD69 antibody molecule, an antagonist anti-AICL antibody molecule or an antagonist anti-LLT1 antibody molecule described herein). In a preferred embodiment, the anti-CD69 antibody is a human monoclonal antibody, e.g., hAIM-29.

Examples of doses of antigen that can be used include 100 μg of antigen for primary immunization and, at least 21 days later, 100 μg of antigen in a secondary immunization. The expected titers should be higher at least for Thi-type isotypes such as mouse IgG2a, IgG2b, and IgG3 or their analogous human isotypes.

Examples of vaccines can include, cancer vaccines (e.g., melanoma vaccines, sarcoma vaccines, breast cancer vaccines, prostate cancer vaccines, colorectal cancer vaccines, pancreatic cancer vaccines, and liver cancer vaccines), HIV vaccines, hepatitis vaccines (e.g., type A, B or C), malaria vaccines, herpes vaccines, papilloma vaccines (e.g., HPV-6, 11, 16, 18, 31, 33 and/or 45) and small pox vaccines.

The early activation molecule antagonist can be administered to the subject, prior to, concurrent with or after the administration of the antigen and/or a nucleic acid encoding the antigen.

In another aspect, the invention features methods of treating or preventing an early activation molecule-expressing cancer in a subject, e.g., a CD69-, AICL- and/or LLT1-expressing cancer in a subject. The method includes administering an early activation molecule depletor and/or an early activation molecule antagonist to the subject.

Examples of early activation molecule -expressing cancers include: lymphomas (e.g., T-cell lymphomas (e.g., peripheral T-cell lymphomas (Dorfman et al. 2002, Hum. Pathol. 33:330-4) e.g., cutaneous T cell lymphoma (Berti et al., 1991. J. Invest. Dermatol. 96:718-23)), B-cell lymphomas, non-Hodgkin\'s lymphomas (e.g., T- or B-cell non-Hodgkin\'s lymphoma (Erlanson et al., 1998. Eur. J. Haematol. 60:125-32)), and lymphocytic leukemias (e.g., T- or B-cell chronic lymphocytic leukemia (Damle et al. 2002, Blood 99:4087-93), e.g., B-cell chronic lymphocytic leukemia lacking immunoglobulin mutations (Klein et al., 2001 J. Exp. Med. 11:1625; Rosenwald et al., 2001 J. Exp. Med. 11:1639)) and other tumors of hematopoietic origin (Tassone et al. 1996. Tissue Antigens 48:65-8).

Early activation molecule depletors include, those described herein, but are not limited to, antibody molecules which bind an early activation molecule and deplete early activation molecule -expressing cells. Examples of early activation molecule depletors include, but are not limited to, antibody molecules which bind CD69 and deplete CD69-expressing cells, e.g., an anti-human CD69 antibody analogous to the anti-mouse CD69 antibody 2.3 or any CD69 antibody, e.g., an art known antibody which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; antibody molecules which bind AICL and deplete AICL -expressing cells, e.g., any AICL antibody, e.g., an art known antibody which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; antibody molecules which bind LLT1 and deplete LLT1-expressing cells, e.g., any LLT1 antibody, e.g., an art known antibody which can act as a depletor (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody), an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody; or an antibody molecule identified by a method described herein. In a preferred embodiment, the anti-human CD69 antibody is a human monoclonal antibody, e.g., hAIM-29. In another embodiment, a depleting anti-early activation polypeptide antibody molecule can recruit an immune cell (e.g., by way of an Fc receptor or by recruitment of the complement system) and inactivate or kill the target cell, e.g., the early activation polypeptide-expressing cell. Thus, in another embodiment, a depleting anti-early activation polypeptide antibody molecule can activate the classical complement pathway that leads to the lysis of the target cell (e.g., the CD69-expressing cell). In another embodiment, the depletor is an anti-early activation polypeptide antibody molecule which is coupled to a therapeutic, e.g., a protein, drug or isotope, which can inactivate or kill the target cell, e.g., the early activation polypeptide expressing cell.

In a preferred embodiment, the method includes administering an early activation molecule depletor in combination with a second agent, e.g., with one or more therapeutic agents, e.g., a therapeutic agent or agent for treating cancer. The second agent can be an antibody or a non-antibody agent. Therapeutic agents include, for example, one or more of a chemotherapeutic agent, a radioisotope, and a cytotoxin. Examples of chemotherapeutic agents include taxol, cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, chlorambucil, gemcitabine, actinomycin, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids and analogs or homologs thereof. Additional therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioisotopes can include alpha, beta and/or gamma emitters. Examples of radioisotopes include 212Bi, 213Bi, 131I, 211At, 186Re, 90Y and 117Lu. The early activation molecule depletor can be administered prior to, concurrent with or after administration of the therapeutic agents.

The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having an early activation polypeptide-expressing cancer).

In some embodiments, the early activation molecule depletor can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes.

In a preferred embodiment the early activation molecule depletor is administered on a repeated basis. E.g., the depletor can be administered 2, 4, 6, or more times. Administration can be repeated until improvement in subject condition is seen or expected. Administration of an antibody can be can be at a frequency of about once, every 5 to 7 days, 14 to 30 or 30 to 60 days. Administration of other compounds can be administered more frequently. For example, a small molecule can be administered one, two, three, four or more times a day.

The invention further includes compositions including monoclonal antibodies that bind an epitope presented by the amino acid of SEQ ID NO:2, or a fragment thereof. The epitope can be presented on the extracellular region of the protein, for example, but is an epitope other than that recognized by the antibody of TP1/55. In one embodiment, the monoclonal antibody can be a human antibody and possess an isotype of IgM/λ. The human monoclonal antibody can include, for example, hAIM-29, which is specifically targeted to human CD69 antigens. In a further aspect, the invention includes monoclonal antibodies that recognize native forms of CD69 molecules expressed, or over-expressed, on activated peripheral blood mononuclear cells (PBMC), or other such activated cells, e.g., inflammatory cyokines and cells producing autoimmune antibodies. Such antibodies do not recognize, however, naïve immune cells lacking CD69 expression. In one embodiment, monoclonal antobodies of the invention can cause an increase in intracellular calcium concentration when binding such cells. Such monoclonal antibodies can also enhance the phorbyl myristate acetate (PMA)-induced proliferative activities of PBMC, or other activated cell, expressing, or over-expressing, CD69 surface antigen.

Also included in the invention are kits for detecting an early activation molecule in a biolgical sample. The sample is preferably from a human subject believed to possess a disorder or condition characterized by an unwanted immune response. The biological sample can include, for example, biological fluid, e.g., blood or other suitable fluid, or tissue. In one embodiment, the kit can include a container having monoclonal antibodies contemplated by the invention, and instructions for using the monoclonal antibody for the purpose of binding to an early activation molecule to form an immunological complex, and detecting the complex such that the presence or absence of the complex correlates to the presence or absence of an early activation molecule in the biological sample. In another embodiment the kit can include hAIM-29, which is specific to human CD69 antigens, as the monoclonal antibody.

The invention also includes methods for treating a subject having a disorder or condition characterized by an unwanted immune response, whereby an effective amount of at least one monoclonal antibody contemplated by the instant invention is administered, alone or conjugated to a second therapeutic agent, to a subject in need thereof. In one embodiment the monoclonal antibody can be a human antibody specific against human CD69, e.g., hAIM-29. The second therapeutic agent can include, by way of example, any of the chemotherapeutic agents, radioisotopes, or cytotoxins previously described herein.

In another aspect, the invention features a method for identifying a compound which modulates an early activation molecule, e.g., a CD69, an AICL or an LLT1 agonist, a CD69, an AICL, or an LLT1 antagonist or a CD69, an AICL or an LLT1 depletor, to thereby modulate an immune response. The method includes: contacting a cell which expresses an early activation molecule with a compound (e.g., a compound which interacts with, e.g., binds to, the early activation molecule), and determining the effect of the compound on expression of the early activation molecule and/or an early activation molecule activity. Expression of an early activation molecule can be determined, e.g., by determining expression of a nucleic acid encoding an early activation polypeptide, expression of an early activation polypeptide, and/or expression of an early activation polypeptide on the surface of a cell.

In a preferred embodiment, the method includes: contacting an early activation polypeptide, e.g., CD69, AICL, or LLT1, or a soluble form of the early activation polypeptide (e.g., a soluble form of CD69, e.g., a CD69 polypeptide which lacks the transmembrane and/or cytoplasmic region of CD69; a soluble form of AICL, e.g., a AICL polypeptide which lacks the transmembrane and/or cytoplasmic region of AICL; a soluble form of LLT1, e.g., a soluble form of LLT1 which lacks the transmembrane and/or cytoplasmic region of LLT1), with a compound, and evaluating the ability of the compound to interact with, e.g., bind to, the early activation polypeptide or soluble early activation polypeptide.

A compound which increases an early activation molecule expression and/or activity (e.g., an early activation molecule agonist) can be identified as a compound which reduces immune response. In a preferred embodiment, the early activation molecule activity which is increased is expression of TGF-θ. In one embodiment, a compound can be evaluated for the ability to increase an early activation molecule expression and/or activity, by methods known in the art, including evaluating the early activation molecule expression and/or an early activation molecule activity of the cell, e.g., expression of TGF-θ, in the absence and presence of the compound. Preferably, the compound interacts with, e.g., binds to, the early activation molecule. Possible compounds include, e.g., small molecules, e.g., small molecules described herein, peptides, fusion proteins, e.g., CD69R fusion proteins, AICL-R fusion protein, and LLT1-R fusion proteins such as those described herein, antibody molecules, and nucleic acid molecules. Antibody molecules which increase an early activation molecule activity include, but are not limited to, antibody molecules, or intrabodies which interact with, e.g., bind to, the early activation polypeptide and result in a change in interaction, e.g., binding activity, of an early activation polypeptide binding partner (e.g., the antibody, intrabody or fragment thereof can affect the conformation of an epitope of the early activation polypeptide, thereby resulting in increased interaction, e.g., binding, by an early activation polypeptide binding partner to the epitope), an antibody molecule which binds the early activation polypeptide and increasing clustering of the polypeptide (e.g., an IgM which binds an early activation polypeptide), and any CD69, AICL or LLT1 antibody molecule, e.g., an art known antibody which can act as an agonist. In some embodiments, the cell-based assay measures an activity of the early activation polypeptide, e.g., increased expression of TGF-θ, MAPK activation, increased Ca2+ signalling and/or increased dimer formation of an early activation polypeptide such as CD69, AICL or LLT1. Compounds which increase expression of TGF-θ and/or dimer formation can be selected as early activation molecule agonists. In other embodiments, the cell based assay measures modulation, e.g., an increase, of an early activation molecule expression. For example, an early activation polypeptide expression or nucleic acid (e.g., mRNA or cDNA) expression, in the presence of the compound can be evaluated and compared to expression of the early activation molecule in the cell prior to administration of the compound. An increase in the expression level is any statistically significant increase in an early activation molecule expression levels. Compounds selected can be confirmed as an agonist. In one embodiment, a compound which increases an early activation molecule expression and/or increases an early activation molecule activity, e.g., expression of TGF-θ, of the cell is selected, e.g., selected for further screening, e.g., by administering the compound to a subject, e.g., an animal model, e.g., an animal model for an inflammatory disease (e.g., chronic or acute inflammatory disease), and/or an animal model for an immune disorder, e.g., an autoimmune disorder. An example of an animal model for an inflammatory disease includes a mouse model of collagen-induced arthritis.

A compound which reduces an early activation molecule expression and/or activity can be identified as a compound which increases the immune response. In a preferred embodiment, the early activation molecule activity which is decreased is expression of TGF-i3 and/or inhibition dimer formation of the early activation polypeptide, e.g., dimer formation of CD69, AICL, or LLT1. In one embodiment, a compound can be evaluated for the ability to decrease expression of an early activation molecule and/or activity, by methods known in the art, including evaluating expression of the early activation molecule (e.g., nucleic acid, polypeptide and/or cell surface expression) and/or an early activation molecule activity of the cell, e.g., expression of TGF-i3, in the absence and presence of the compound. Preferably, the compound interacts with, e.g., binds to, the early activation molecule. Possible compounds include, e.g., small molecules (e.g., small molecules described herein), peptides, fusion polypeptides (e.g., a CD69R fusion protein, an AICL-R fusion protein or an LLT1-R fusion protein described herein), antibody molecules, and nucleic acid molecules. Examples of antibody molecules include, but are not limited to, antibody molecules, or intrabodies which bind an early activation polypeptide and interfere with binding of the early activation polypeptide and an early activation binding polypeptide and antibody molecules which bind to an early activation polypeptide and decrease the level of the early activation polypeptide on a cell surface, e.g., by internalization. Examples of anti-CD69 antibody molecules include, e.g., an anti-human CD69 antibody, e.g., an anti-human CD69 antibody analogous to an anti-mouse CD69 antibody 2.2 or an anti-human CD69 antibody, e.g., an art known antibody, which can act as an antagonist (or antibody molecules based thereon), an antibody molecule which binds the epitope bound by such antibodies, an antibody molecule which competes for binding with such antibodies, an antibody molecule which binds or interferes with binding of another antibody, receptor or ligand to one or more of amino acid residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69, or other antibody described herein. Examples of anti-AICL antibody molecules include an anti-human AICL antibody, e.g., an anti-AICL antibody, e.g., an art known antibody which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 37 to 149 of human AICL, or other antibody described herein. Examples of anti-LLT1 antibody molecules include an anti-human LLT1 antibody, e.g., an anti-LLT1 antibody, e.g., an art known antibody which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 76 to 132 of human LLT1, or other antibody molecule described herein. In some embodiments, the cell-based assay measures an activity of an early activation molecule, e.g., decreased expression of TGF-θ, decreased MAPK, decreased Ca2+ signaling and/or decreased dimer formation such as decreased CD69, AICL or LLT1 dimer formation. Compounds which decrease expression of TGF-θ and/or dimer formation can be selected as early activation molecule antagonists. In other embodiments, the cell based assay measures modulation, e.g., a decrease, of an early activation molecule expression. For example, early activation polypeptide expression or nucleic acid (e.g., mRNA or cDNA) expression, in the presence of the compound can be evaluated and compared to expression of the early activation molecule in the cell prior to administration of the compound. A decrease in the expression level is any statistically significant decrease in an early activation molecule expression levels. In other embodiments, the cell based assay measures modulation, e.g., decreased levels, of the early activation polypeptide on the cell surface, e.g., as compared to the levels of the early activation polypeptide on the cell surface prior to administration of the compound.

In one embodiment, a compound which inhibits expression of an early activation molecule and/or inhibits an early activation molecule activity, e.g., expression of TGF-θ, of the cell is selected, e.g., selected for further screening, e.g., by administering the compound to a subject, e.g., an animal model, e.g., an animal model for unwanted cellular proliferation (e.g., an animal model for cancer), an animal model for an immunodeficiency, and/or an animal model of vaccine efficacy.

In another aspect, the invention features a method of identifying a compound which modulates immune response. The method includes: screening for a compound that interacts with, e.g., binds to, an early activation polypeptide or an early activation nucleic acid (e.g., the regulatory region for an early activation gene), and determining the effect of the compound on expression of the early activation molecule and/or an early activation molecule activity, e.g., TGF-θ expression and/or dimer formation, e.g., CD69, AICL or LLT1 dimer formation. In one embodiment, the interaction between the compound and the early activation polypeptide is evaluated in vitro, e.g., using an isolated early activation polypeptide, e.g., a soluble form of an early activation polypeptide, e.g., a soluble CD69, a soluble AICL or a soluble LLT1 described herein. The early activation polypeptide and/or compound can be in solution (e.g., a micelle) or bound to a solid support, e.g., a column, bead, well or dish, or a chip (e.g., a microarray). In some embodiments, the method is repeated one or more times such that, e.g., a library of test compounds can be evaluated.

In another embodiment, the interaction between the compound and an early activation molecule is evaluated using a cell-based assay.

In a preferred embodiment, a compound which interacts with, e.g., binds to, the early activation molecule, is contacted with a cell which expresses the early activation molecule to determine the effect of the compound on expression of the early activation molecule and/or activity.

In one embodiment, the method includes identifying a compound which interacts with the early activation molecule and increases expression of the early activation molecule and/or an early activation molecule activity, e.g., increases TGF-θ expression, MAPK activation, increased Ca2+ signalling and/or dimer formation to thereby identify the compound as a candidate for decreasing immune response. In a preferred embodiment, the early activation molecule activity which is increased is expression of TGF-θ. In one embodiment, a compound can be evaluated for the ability to increase expression of an early activation molecule and/or activity, by methods known in the art, including evaluating expression of an early activation molecule and/or an early activation molecule activity of the cell, e.g., expression of TGF-θ, in the absence and presence of the compound. Possible compounds include, e.g., small organic molecules (e.g., small molecules described herein), peptides, fusion peptides (e.g., a CD69R fusion protein, an AICL-R fusion protein, or a LLT1-R fusion protein, e.g., as described herein), antibody molecules, and nucleic acid molecules. Antibodies which increase an early activation molecule activity include, but are not limited to, antibody molecules or intrabodies which interact with, e.g., bind to, an early activation polypeptide and result in a change in interaction, e.g., binding activity, of an early activation polypeptide binding partner (e.g., the antibody molecule or intrabody can affect the conformation of an epitope of the early activation polypeptide, thereby resulting in increased interaction, e.g., binding, by an early activation polypeptide binding partner to the epitope), an antibody molecule which binds an early activation polypeptide and increases clustering of the early activation polypeptide (e.g., an IgM which binds an early activation polypeptide), and any CD69 antibody molecule, AICL antibody molecule or LLT1 antibody molecule, e.g., from an art known antibody which can act as an agonist. In some embodiments, the cell-based assay measures an activity of an early activation molecule, e.g., increased expression of TGF-θ, increased MAPK, increased Ca2+ signalling and/or increased dimer formation of the early activation polypeptide. Compounds which increase expression of TGF-θ, increased MAPK, increased Ca2+ signalling and/or CD69 dimer formation can be selected as early activation molecule agonists. In other embodiments, the cell based assay measures modulation, e.g., an increase, of expression of an early activation molecule. For example, an early activation polypeptide expression or nucleic acid (e.g., mRNA or cDNA) expression, in the presence of the compound can be evaluated and compared to expression the early activation molecule in the cell prior to administration of the compound. An increase in the expression level is any statistically significant increase in the expression levels of an early activation molecule.

In one embodiment, a compound which increased expression of an early activation molecule and/or increases an early activation molecule activity, e.g., expression of TGF-θ, of the cell is selected, e.g., selected for further screening, e.g., by administering the compound to a subject, e.g., an animal model, e.g., an animal model for an inflammatory disease (e.g., chronic or acute inflammatory disease), and/or an animal model for an immune disorder, e.g., an autoimmune disorder. An example of an animal model for an inflammatory disease includes a mouse model of collagen-induced arthritis.

In another embodiment, the method includes identifying a compound which interacts with an early activation molecule and decreases expression of the early activation molecule and/or an early activation molecule activity, e.g., decreases TGF-θ expression, decreases MAPK, decreased Ca2+ signalling and/or dimer formation, e.g., CD69, AICL or LLT1 dimer formation, to thereby identify the compound as a candidate for increasing immune response. Expression of an early activation molecule can be determined, e.g., by determining expression of a nucleic acid encoding an early activation polypeptide, expression of an early activation polypeptide, and/or expression of an early activation polypeptide on the surface of a cell. In a preferred embodiment, the early activation molecule activity which is decreased is expression of TGF-θ. In one embodiment, a compound can be evaluated for the ability to decrease expression of the early activation molecule and/or activity, by methods known in the art, including evaluating expression of the early activation molecule and/or an early activation molecule activity of the cell, e.g., expression of TGF-θ, in the absence and presence of the compound. Possible compounds include, e.g., small molecules (e.g., small molecules described herein), peptides, fusion polypeptides (e.g., CD69R fusion polypeptides, AICL-R fusion polypeptides, and LLT1-R fusion polypeptides, e.g., as described herein), antibody molecules, and nucleic acid molecules. Examples of antibodies include, but are not limited to, antibody molecules or intrabodies which bind an early activation polypeptide and interfere with binding of the early activation polypeptide and an early activation binding polypeptide and antibody molecules which bind to an early activation polypeptide and decrease the level of the early activation polypeptide on the cell surface. Examples of anti-CD69 antibody molecules include, e.g., an anti-human CD69 antibody molecule, e.g., an anti-human CD69 antibody molecule analogous to an anti-mouse CD69 antibody 2.2, or an anti-human CD69 antibody molecule, e.g., an art known antibody, an antibody molecule which binds the epitope bound by such antibodies, an antibody molecule which competes for binding with such antibodies, an antibody molecule which interferes with binding to one or more of amino acid residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69, or other antibody described herein. Anti-AICL antibody molecules include, e.g., an anti-human AICL antibody, e.g., an art known anti-AICL antibody which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 37 to 149 of human AICL, or other antibody described herein. Anti-LLT1 antibody molecules include, e.g., an anti-human LLT1 antibody, e.g., an anti-LLT1 antibody, e.g., an art known antibody which can act as an antagonist (or an antibody molecule based thereon, e.g., a fragment, chimeric, humanized, or deimmunized antibody) or an antibody molecule which binds the epitope bound by such an antibody, an antibody molecule which competes for binding with such an antibody, or an antibody molecule which binds to or interferes with the binding of another antibody, receptor or ligand to one or more of amino acid residues of 76 to 132 of human LLT1, or an antibody molecule described herein. In some embodiments, the cell-based assay measures an activity of the early activation molecule, e.g., decreased expression of TGF-θ, decreased MAPK decreased Ca2+ signalling and/or decreased dimer formation, e.g., CD69, AICL or LLT1 dimer formation. Compounds which decrease expression of TGF-θ and/or dimer formation can be selected as early activation molecule antagonists. In other embodiments, the cell based assay measures modulation, e.g., a decrease, of expression of an early activation molecule. For example, the early activation polypeptide expression or nucleic acid (e.g., mRNA or cDNA) expression, in the presence of the compound can be evaluated and compared to expression of the early activation molecule in the cell prior to administration of the compound. A decrease in the expression level is any statistically significant decrease in expression levels of the early activation molecule. In other embodiments, the cell based assay measures modulation, e.g., decreased levels, of the early activation polypeptide on the cell surface, e.g., as compared to the levels of the early activation polypeptide on the cell surface prior to administration of the compound.

In one embodiment, a compound which inhibits expression of an early activation molecule and/or inhibits an early activation molecule activity, e.g., expression of TGF-θ, of the cell is selected, e.g., selected for further screening, e.g., by administering the compound to a subject, e.g., an animal model, e.g., an animal model for unwanted cellular proliferation (e.g., an animal model for cancer), an animal model for immunodeficiency and/or an animal model of vaccine efficacy.

In another aspect, the invention features a method of identifying a compound which modulates immune response. The method includes: providing a compound which modulates expression of an early activation molecule and/or an early activation molecule activity, and administering the compound to a subject, e.g., an animal model.

In one embodiment, the compound is a compound which increases expression of an early activation molecule and/or an early activation molecule activity, e.g., increases expression of TGF-θ and/or increases dimer formation (e.g., CD69, AICL or LLT1 dimer formation), and the compound is administered to a subject, e.g., an animal model for an inflammatory disease (e.g., chronic or acute inflammatory disease), and/or an animal model for an immune disorder, e.g., an autoimmune disorder, and compounds which reduce one or more symptoms of the disorder are selected. An example of an animal model for an inflammatory disease includes a mouse model of collagen-induced arthritis.

In another embodiment, the compound is a compound which decreases expression of an early activation molecule and/or an early activation molecule activity, e.g., decreases expression of TGF-θ and/or decreases dimer formation (e.g., CD69, AICL or LLT1 dimer formation), and the compound is administered to a subject, e.g., an animal model for unwanted cellular proliferation (e.g., an animal model for cancer), an animal model for immunodeficiency and/or an animal model for vaccine efficacy, and compounds which reduce one or more symptoms of the disorder are selected.

The compound can be a compound identified by a method described herein or can be other compounds known to interact with an early activation molecule.

In another aspect, the invention features, a method of making a decision, e.g., a medical or financial decision. The method includes: generating or receiving data or information on the treatment of a subject for a disorder described herein; and using the data or information to make the decision, e.g., selecting between a first outcome and a second outcome.

In a preferred embodiment, the data is response to an administration of an early activation molecule agonist, antagonist or depletor (e.g., a CD69, AICL or LLT1 agonist, antagonist or depletor).

In a preferred embodiment, the decision is made by comparing the data to a reference standard and making the decision based on the relationship of the data to the reference. For example, the data can be a value or other term for subject response, and if the value or other term has a preselected relationship to the reference standard, e.g., if the value or term in the data is greater than a reference standard, selecting a first outcome and if the data is less than a reference standard selecting a second outcome. An outcome can be providing or not providing service or treatment, or paying for or not paying for all or part of a service or treatment.

In a preferred embodiment, the first outcome is suggesting or providing a first course of medical treatment, e.g., any treatment described herein, e.g., an additional administration of an early activation molecule agonist, antagonist, or depletor, and the second course is suggesting that the treatment not be given or not providing the treatment.

In a preferred embodiment the first outcome includes or results in the authorization or transfer of funds to pay for a service or treatment provided to a subject and the second outcome includes or results in the refusal to pay for a service or treatment provided to a subject. For example, an entity, e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses, can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services or treatment provided to the patient. For example, a first entity, e.g., an insurance company, can use the outcome to determine whether to provide financial payment to, or on behalf of, a patient, e.g., whether to reimburse a third party, e.g., a vendor of goods or services, a hospital, physician, or other care-giver, for a service or treatment provided to a patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to continue, discontinue, enroll an individual in an insurance plan or program, e.g., a health insurance or life insurance plan or program.

In another aspect, the invention features a method of providing a database, e.g., a database useful for establishing a reference value referred to herein or for otherwise evaluating one or more subjects. The method includes: generating or receiving data, e.g., response to administration of an early activation molecule agonist, antagonist, or depletor (e.g., a CD69, an AICL, or a LLT1 agonist, antagonist or depletor) in the treatment of a disorder, e.g., a disorder described herein; and entering the data into the database.

In a preferred embodiment one or more of, an indicator (e.g., a value) for the disease state of a patient and a patient identifier are entered into the database.

In a preferred embodiment the database includes a plurality of entries, each one of which includes one or more of: data (data on response to administration of an early activation molecule agonist, antagonist, or depletor in the treatment of a disorder, e.g., a disorder described herein); an indicator for the disease state of a patient and a patient identifier.

In another aspect, the invention features a method of evaluating a patient which includes: comparing data from the patient (data on response to administration of an early activation molecule agonist, antagonist, or depletor in the treatment of a disorder, e.g., a disorder described herein, with data from a database described herein.

In another aspect, the invention features a method of constructing a reference standard which includes: including data from a database described herein in the standard. For example, one can take values from a database, perform a mathematical operation on them and use the result as a reference in arriving at a reference standard, e.g., taking an average of a plurality of values selected from the database and use the average as the standard.

In another aspect, the invention features a method of enhancing the production of an antibody to an antigen. The method includes administering an antigen to an animal; and administering an early activation molecule antagonist to the animal.

The animal can be, e.g., a mammal, e.g., a mouse, rat, goat, sheep, pig, cow or horse.

In one embodiment, the antigen is homologous to the animal, e.g., the antigen is from the same species as the species of animal to which it is administered, e.g., the antigen is a murine antigen administered to a mouse. In another embodiment, the antigen is heterologous, e.g., the antigen is from a species other than species of the animal to which it is administered, e.g., the antigen is a human antigen administered to a mouse.

In another embodiment, the method further includes isolating an immune cell from the animal, e.g., to produce a monoclonal antibody.

The invention is based, in part, on the discovery that modulation of any of the early activation molecules CD69, AICL and LLT1 play a role in immune reactivity. While not wishing to be bound by theory, this may be affected through the regulation of TGF-β1 synthesis. CD69, AICL and LLT1 are structurally similar receptors localized to the NK gene complex and expressed in most cells of hematopoietic origin (Boles et al., 1999). All three proteins have at least one C-type lectin domain with significant sequence similarity to each other. Upregulated levels of CD69, AICL and LLT1, unlike other lectin-like proteins associated with the NK gene complex, are seen at early time points of cellular activation. (Eichler et al., 2001).

Two different animal models of disease were analysed to determine the role of CD69 in the regulation of the immune response. First, the inhibitory role of CD69 and targeting of CD69 was evaluated in murine collagen-induced arthritis (CIA). CIA is a widely accepted experimental model of inflammatory joint disease, mainly rheumatoid arthritis (RA) (Feldmann et al., 1996). CIA provides a model for the study of autoimmunity and can be extended to other diseases where the immune response against self is exacerbated. It was found that CD69 plays an inhibitory role through the regulation of TGF-θ in the CIA. The second model, a tumor model, represents the opposite approach, where the goal is to enhance the immune response in order to suppress tumor growth and metastasis. Therefore, it constitutes an experimental system to test effective therapy that can be extended to infectious diseases and immunodeficiency syndromes in reestablishing an efficacious immune response. The use of CD69 in this strategy has been analysed in murine induced tumor and metastasis models that have lost expression of MHC class I genes. It was found that monoclonal antibodies (mAbs) specific for CD69 provide effective therapy in immune competent (wt) and immune compromised (SCID&RAG1−/−) tumor-bearing mice. Like CD69, AICL and LLT1 are expressed at low levels in leukocytes, and are strongly and transiently upregulated. (Homann et al., 1997; Eichler et al., 2001). In addition, the AICL has been shown to be upregulated in certain cancers, namely B-CLL in unmutated immunoglobulin lines (Klein et al., 2001 J. Exp. Med. 11:1625; Rosenwald et al., 2001 J. Exp. Med. 11:1639). This can result in increased expression of TGF-θ, thereby producing an immunosuppressive microenvironment in subjects having this disorder. The elucidation of the role of CD69 as a negative modulator of immune reactivity provides for interesting and novel approaches for the therapy of cancer, chronic inflammation and other immune-mediated diseases (immunodeficiencies, autoimmune diseases, allograft transplants, and the like) by targeting CD69, AICL and/or LLT1. In addition, since CD69, AICL and LLT1 are selectively expressed by activated leukocytes infiltrating inflamed tissues and mediating tumor responses, the pharmacological stimulation of TGFβ1 synthesis through CD69, AICL and/or LLT1 can have a localized effect, thus avoiding the detrimental consequences of a systemic TGF-β1 up-regulation or delivery. Moreover, CD69, AICL and LLT1 are suitable markers for activated tissue leukocytes. Depletion of this activated subset using specific early activation molecule reagents can inhibit the active immune/inflammatory response. Therefore, the antagonism of an early activation molecule, e.g., by inhibiting TGF-θ release, may allow a locally enhanced immune response due to an increased recruitment and survival of highly activated immune effector cells (suitable for an enhanced response to vaccines, immunodeficiency therapy, etc).

The description of immunoregulatory molecules that enable a selective and local activation or inhibition of TGF-β1 synthesis is therefore a novel approach to the treatment of immune-mediated diseases. In addition, early activation molecules can be used as a specific marker of the leukocyte population that is actively producing pro-inflammatory cytokines at inflammatory sites. The depletion of this deleterious population through targeting of CD69, AICL and/or LLT1, would allow the control of the downstream pro-inflammatory effectors. These new possible approaches through the inducible leukocyte receptors CD69, AICL and LLT1 are described herein.

The details of one or more embodiments of the invention have been set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

FIG. 1. Exacerbated CIA in CD69−/− mice. A, CFA treated control mice (left panels) are compared with representative pictures of the most severe cases of CIA in CD69+/+ mice and CD69−/− mice (middle and right panels, respectively).B, incidence of arthritis (as percentage of diseased mice), and severity of clinical signs in CD69+/+ (), CD69+/− (▴), and CD69−/− (▪) mice evaluated as described in Methods. Results correspond to arithmetic mean±s.d. from three separate experiments (18 mice/group/experiment). *, P<0.01 versus CD69+/+ (Student\'s t test).

FIG. 2. A. Representative section of joint histopathology on whole paws of control (CFA, left, 100×), and CII-immunized CD69+/+ (middle panel, 100×) and CD69−/− (right panel, 60×) mice. Arrowheads indicate cartilage and bone erosions, and arrows indicate leukocyte infiltrates and pannus. B. scoring of inflammation, cartilage damage, pannus formation, and bone erosion of paws from CD69+/+ (□) and CD69−/− (▪) mice as described in Methods. Results are expressed as the arithmetic mean±s.d. of the percentage of joints ascribed to each severity group from three independent experiments (8 mice/group/experiment) *, P<0.01 vs. CD69+/+ (Mann-Whitney U test).

FIG. 3. Enhanced CII-specific immune response in CD69−/− mice. a, enlarged spleens of CD69−/− compared to CD69+/+ mice. Horizontal bar=1 cm. b, spleen weight and cell number (arithmetic mean±s.d.) from CFA control and CII-immunized (CIA) CD69+/+ (□) and CD69−/− (▪) mice are shown. *, P<0.01, compared with WT immunized mice (Student\'s t test). c, proliferation of spleen (Sp) and lymph node (LN) cells from CII-immunized CD69+/+ (□) and CD69−/− (▪) mice stimulated with different concentrations of inactivated CII (x-axis). Data correspond to the arithmetic mean±s.d. of 3H-TdR uptake in three independent experiments. *, P<0.01 versus CD69+/+ (Mann-Whitney U test). d, Increment of CII-specific Th1-dependent isotypes in CD69−/− mice. CII-specific IgG1 and IgG2b (1/20000 serum dilution), IgG2c and IgG3 (1/5000 serum dilution), and IgM and IgA (1/200 serum dilution) antibody levels in sera collected at sacrifice (50 d) of CFA-treated control or CII-immunized (CIA) CD69+/+ (□) and CD69−/− (▪) mice. Data are represented as the arithmetic mean±s.d. of absorbance units at 495 nm in three separate experiments (10 mice/group/experiment). *, P<0.01 versus CD69+/+ (Student\'s t test).

FIG. 4. Local cytokine mRNA analysis in CII-immunized CD69−/− mice. mRNA analysis of CIA CD69+/+ (□) and CD69−/− (▪) mouse hind paws. Each lane corresponds to the mRNA pool of 6 mice per group, and each bar represents similar experiments (6 mice/group/experiment). Results are expressed in arbitrary densitometric units normalized for L-32 expression in each sample (arithmetic mean±s.d. of four separate experiments). *, P<0.01 versus CD69+/+ (Mann-Whitney U test).

FIG. 5. Local cytokine analysis in joints and purified leukocyte synovial cells from CII-immunized CD69−/− mice. A, Real-time quantitative RT-PCR analysis of mRNA from CD69+/+ (□) and CD69−/− (▪) mouse hind paws. Each bar represents the arithmetic mean±s.d. of 12 mice per group in two independent experiments. Results for each cytokine are normalized to GAPDH expression measured in parallel in each sample. *, P<0.01 versus CD69+/+ (Mann-Whitney U test). B, Levels of active and total TGF-β1, IL-1β, TNF-α, and RANTES in synovial washouts from CIA CD69+/+ (□) and CD69−/− (▪) mice. Data correspond to the arithmetic mean±s.d. of three independent experiments (6 mice/group/experiment). *, P<0.01 versus CD69+/+ (Mann-Whitney U test). C, quantitative RT-PCR analysis of mRNA from CD69+/+ (□) and CD69−/− (▪) purified subsets of synovial cells. Each bar represents the arithmetic mean±s.d. of 12 mice per group in two independent experiments. Results of each cytokine are normalized to GAPDH expression measured in parallel in each sample. *, P<0.01 versus CD69+/+ (Mann-Whitney U test).

FIG. 6. Effect of a blocking anti-TGF-β in WT and CD69-deficient mice. a, left panel: CD69+/+ mice were treated from day 21 (2nd immunization) with the blocking anti-TGF-β (□), the isotype control IgG1 (▴), or the carrier (▾). Paw inflammation measured with a precision caliper is expressed as mm of inflammation with respect to day 21. Results express the arithmetic mean±s.d. of 12 mice per group in two independent experiments. Right panel: analysis by quantitative real-time RT-PCR of mRNA from paws treated with isotype control antibody (□) or with anti-TGF-β(▪). Each bar represents the arithmetic mean±s.d. of 12 mice per group in two independent experiments. Results of each cytokine are normalized to GAPDH expression measured in parallel in each sample. *, P<0.01 versus control antibody (Mann-Whitney U test). b, CD69−/− mice were treated as in a, paw inflammation measured (left panel) and mRNA determined (right panel) as above.

FIG. 7. CD69 engagement induces TGF-β1 secretion in mouse splenocytes and synovial leukocytes from CIA mice A, Con-A stimulated splenocytes were stained for CD69 (thick line, left panel) and an isotype-matched antibody (dotted line, left panel). Activated splenocytes were then purified in different subsets and treated with an anti-mouse CD69 (▪), or an isotype-matched control antibody (□), and a cross-linking secondary antibody was added. Active and total TGF-β1 was determined (right panels). B, mouse synovial cells from CIA mice were stained for CD11b and CD69 (middle panel), or isotype control Abs (left panel). Synovial cells were treated as in A, and active and total TGF-β1 determined (right panels). Results in A and B are expressed as the arithmetic mean±SD of four independent experiments. *, p<0.01 versus control antibody (Mann Whittney U test).

FIG. 8. CD69 crosslinking induces TGFθ1 production in human synovial leukocytes and in human CD69 T cell stable trasfectants. A, human synovial leukocytes from patients with rheumatoid arthritis were stained for CD69 (thick line, left panel), and isotype-matched antibody (dotted line, left panel). Synovial leukocytes were treated with anti-human CD69(▪), or an isotype-matched control antibody (□) in the presence (XL) or absence (−) of a cross-linking secondary Ab, and active and total TGF-β1 determined (right panel). *, P<0.01 versus control antibody (Mann-Whitney U test). These results are representative of similar data obtained on synovial leukocytes from patients with reactive arthritis or ankylosing spondylitis. B, Jurkat human T cell leukemic cell line (dotted line, left panel), and CD69 stable transfectants (thick line, left panel) were stained for CD69. Production of TGFθ1 but not TNF-I by CD69 engagement (right panel). The human T lymphoblastoid cell line Jurkat (JK) (η) and the stable transfectant expressing CD69 (JK-CD69)(▪) were treated for 24 hours with cross linked anti-CD69 antibody. Results in A and B are expressed as the arithmetic mean±SD of four independent experiments. *, p<0.01 respect to the parental JK cell line (Mann Whittney U test).

FIG. 9. Increased anti-tumor activity in CD69 −/− mice. Mice were injected intraperitoneally with 104 RMA-S (A) or RMA (C) tumor cells. Mice were observed daily for tumor growth up to 12 weeks by monitoring body weight and development of ascites. Similar results were observed in mice injected subcutaneously with 105 RMA-S (B). (A, n=9; B, n=6; C, n=9). (D) NK or T lymphocytes were depleted from CD69−/− mice before intraperitoneal inoculation with 104 RMA-S tumor cells. Mice were treated for NK cell depletion on day −1 (before tumor challenge), and at days +2, and +4 after tumor inoculation with either control diluent or anti-Asialo GM1 antiserum (100 μl/injection). (D, n=7). Mice were treated for Lymphocyte T CD4+ depletion on day −1 (before tumor challenge), and at days +2, and +4 after tumor inoculation with either GK1.5 anti-CD4 or isotype control mAbs (100 μl/injection). (D, n=7) Results shown are representative of two independent experiments. Open symbols represent wt mice and solid symbols represent CD69−/− mice. (E) Photographs are of lungs from CD69−/− and wt mice 2 weeks after the challenge with 104 RM-1 cells i.v. inoculation (upper). One representative mouse out of 10 per group is shown. Number of lung metastases was counted (lower) and each symbol represents one mouse. Open symbols represent wt mice and solid symbols, CD69−/− mice. Results are representative of two independent experiments.

FIG. 10. Exacerbated anti-tumor response in CD69−/− RAG-deficient mice. Mice were injected intraperitoneally with 10 6 RMA-S tumor cells. Total unfractionated peritoneal cells (A, B) from CD69+/+- and CD69−/−-RAG2−/− mice were examined 72 h after tumor inoculation. (A) Forward and scattered FACS analysis. One representative mouse out of 10 per group is shown. (B) Profiles comparison by Coulter Multisizer cell counter. Results shown are representative of two independent experiments (n=8).

FIG. 11. Increased cytotoxicity and lymphocyte recruitment in CD69−/− mice. Mice received 104 RMA-S cells (A) or RM-1 (B) intraperitoneally. 72 h after tumor inoculation, total unfractionated peritoneal cells (A, B) from CD69−/− and wt mice were analyzed for cytotoxicity against YAC target cells. Two animals were used per experimental group (mean±SE). The results are representative of three experiments identically performed. Different symbols represent NK cells purified from different mice: Open symbols (wt) and solid symbols (CD69−/−). (C, D, E) Peritoneal and splenic accumulation of lymphocytes in CD69−/− mice in response tumor cells. Mice were injected intraperitoneally with PBS or with 105 RM-1 cells (n=7, mice/group). Peritoneal lavage was collected 3 d and 6 d later, and total leukocyte numbers (×10−6) were determined (C). Cells collected from the lavage were analyzed with a Coulter Multisizer cell counter and profiles were compared at the indicated times after tumor inoculation (D). Data are representative from three independent experiments with 2/2, 2/2 and 4/4, wt (dotted lines)/CD69−/− (solid lines) mice. Increased spleens removed from 8 weeks old CD69−/− and wt mice after 3 days of i.p. 105 RM-1 challenge were compared (E). Spleens shown are representative from several independent experiments.

FIG. 12. Attenuation of spontaneous cell death of CD69−/− lymphocytes. Unfractionated peritoneal cells of untreated mice (A) were seeded in culture medium and cell survival was measured by PI staining. The values depict the percentage of PI+ cells (mean±SD). Results are representative of three independent experiments. (B, C) Analysis of intracellular caspase-3 activity of spleen cells (B) and purified NK cells (C) from CD69+/+ (left) and CD69−/− (right) challenge mice (3 days with 105 RM-1). Activation of Caspase-3 was detected by using fluorogenic substrate PhiPhiLux-G1D2. PhiPhiLux staining on FL-1 versus forward scatter channels was displayed. Data are representative of one experiment. (n=6).

FIG. 13. Cytokine expression in CD69−/− mice. (A) Relative levels of cytokine and chemokine mRNA levels in peritoneal cells were analyzed after 3 days of 105 RM-1cells i.p. inoculation. Results are expressed in arbitrary densitometric units normalized for the expression of GAPDH or L32 in each sample. Five animals were used per experimental group and results are representative of four independent experiments. (B) MCP-1 levels in LPS-activated peritoneal cells from thioglycollated-treated CD69−/− and wt mice. Data represent the mean±SE (n=4 for each group) of one experiment representative of three independent experiments. *, P<0.004

FIG. 14. Enhanced anti-tumor activity by blocking anti-TGF-β in wt mice. Survival plot of wt and CD69−/− mice i.p. injected on day 0 with 104 RMA-S cells and treated with either 1D11 anti-TGF-β mAb (500 μg/injection) or PBS on day −3, −1 (before tumor challenge), and at days +1 and every week after tumor inoculation. (n=4). Results shown are representative of two independent experiments.

FIG. 15. CD69 engagement induces TGF-β1. Purified resting CD3+ T cells were added to 96-well plates coated with anti-CD3. A) Expression of CD69 after activation with anti-CD3 were analyzed by staining with FITC-anti-murine CD69 mAb (solid line) after 24 h of culture. Dotted line represent negative control. B) Antibody engagement of CD69 induces TGF-β1 secretion in mouse T lymphocytes. Anti-CD69 (CD69.2.2) or the isotypic control (murine IgG1) was added followed by goat anti-mouse IgG antibody. Inhibition by PD98059 of CD69-induced TGF-β secretion. Cells were incubated in serum-free medium for 72 h and TGF-β1 was determined by ELISA. Data are expressed as mean±SD of replicate wells and are representative of three independent experiments. C) Cultured cells were removed and trypan blue was added. Dead cells (trypan blue positive) were determined after 48 h of culture. Data represent means of triplicate wells (bars±SD). (D) CD69 engagement induces Erk-1 and Erk-2 activation in anti-CD3 stimulated T lymphocytes. Preactivated T cells were coated on ice with anti-CD69 or isotype control mAb and lysed 5, 10, 15, 25 and 60 min after addition of warm (37° C.) goat anti-mouse antibody. Cell lysates were blotted with polyclonal anti-phospho Erk 1/2 (upper) antibody, then with anti-Erk mAb (lower) to confirm equal protein loading. The position of Erk1 and Erk2 is marked by arrows. Relative levels of Erk1 (upper right) and Erk2 (lower right) phosphorylation (phospho-Erk/total Erk, in arbitrary densitometric units) are shown. Control ( ) and CD69−/− (▪).

FIG. 16. Therapeutic anti-tumor activity of anti-CD69 mAbs in wt and RAG-deficient mice. Survival plot of wt and SCID mice treated with anti-CD69 mAbs or isotype control mAb. 104 RMA-S tumor cells were injected intraperitoneally in wt mice (A, B) and 106 RMA-S in SCID mice. (C) Mice were treated as indicated with anti-CD69 or isotype control mAbs (A, B, C) and observed daily for tumor growth up to 20 weeks by monitoring body weight and development of ascites. (A, n=9; B, n=6; C, n=9). Results shown are representative of two independent experiments.

FIG. 17. Anti-CD69 Abs affect early phase of peritoneal tumor growth. Peritoneal cells of wt (A) or RAG1−/− (B) mice were examined at day 3 after i.p. injection of 106 RMA-S tumor cells. Mice were treated one day before tumor inoculation with anti-CD69 (solid lines) or isotype control mAb (dotted lines) (500 μg per mouse). Cells collected from the lavage were analyzed with a Coulter Multisizer cell counter and profiles were compared. (n=8, mice/group). Data are representative from two independent experiments.

FIG. 18 depicts an antibody response to DNP-KLH in wildtype and CD69-deficient mice. The protocol followed for immunization was the injection of 100 μg of DNP-KLH per mice s.c. in the base of the tail or i.p., and using either Alum or CFA as adjuvants. Mice were bled at day 0 (not shown) and day 7 after the first injection (primary immunization). The second injection was administered at day 21, and mice were bled at day 28 (secondary immunization). a, levels of IgM in primary immunization (1:2000 serum dilution), expressed as arithmetic mean±s.d. of 4 mice/group/experiment were analyzed in two independent experiments. b, levels of IgM (1:2000 serum dilution), IgG1(1:20000), IgG2c (1:10000), IgG2b (1:10000), and IgG3 (1:10000) in secondary immunization, expressed as in (a). *, p<0.01 respect to the same treatment in WT mice (Mann-Whitney U test).

FIG. 19. anti-CD69 2.2 antibody down-regulates CD69 expression on thymocytes. Thymocytes from DBA/1 mice treated (right panel) or not (left panel) with 2.2 anti-mouse CD69 mAb were isolated and FACS stained with H1.2F3 anti-mouse CD69 FITC mAb and anti-mouse CD3 APC mAb. One representative experiment of five is shown.

FIG. 20. Effect of treatment with 2.2 anti-CD69 mAb in CIA. DBA/1 mice were treated at days 20 and 28 with the blocking anti-CD69 mAb 2.2(▴), or the isotype control IgG1 mAb (▪). Paw inflammation measured with a precision caliper is expressed as mm of inflammation relative to the day 21 value. Results are expressed as the arithmetic mean±s.d. of 12 mice per group in two independent experiments. *, P<0.01 versus control antibody (Mann-Whitney U test).

FIG. 21. anti-CD69 2.3 antibody depletes CD69 expressing-thymocytes. Thymocytes from DBA/1 mice treated (right panel) or not (left panel) with 2.3 anti-mouse CD69 mAb were isolated and FACS stained with H1.2F3 anti-mouse CD69 FITC mAb and anti-mouse CD3 APC mAb. One representative experiment of five is shown.

FIG. 22. Effect of treatment with 2.3 anti-CD69 mAb in CIA. DBA/1 mice were treated at days 20 and 28 with the depleting anti-CD69 mAb (▴), or the isotype control IgG2a mAb (▪). Paw inflammation measured with a precision caliper is expressed as mm of inflammation relative to the day 21 value. Results are expressed as the arithmetic mean±s.d. of 12 mice per group in two independent experiments. *, P<0.01 versus control antibody (Mann-Whitney U test).

FIG. 23 depicts the amino acid sequence (SEQ ID NO:2) of human CD69 and the nucleic acid sequence (SEQ ID NO:1) encoding human CD69.

FIG. 24 depicts the amino acid sequence (SEQ ID NO:4) of human AICL and the nucleic acid sequence (SEQ ID NO:3) encoding human AICL.

FIG. 25 depicts the amino acid sequence (SEQ ID NO:6) of human LLT1 and the nucleic acid sequence (SEQ ID NO:5) encoding human LLT1.

DETAILED DESCRIPTION

As provided above, an inhibitory role for CD69 in immune reactivity has been demonstrated using an experimental model of murine collagen-induced arthritis (CIA). CD69-deficient mice showed a higher incidence and severity of CIA with exacerbated B and T immune responses to type II collagen (CII). TGFθ1 and TGFθ2, which have a protective role in CIA, were reduced in CD69−/− mice inflammatory foci, correlating with an increase in proinflammatory cytokines interleukin (IL)-1θ and RANTES. Local injection of a blocking anti-TGFβ antibody in CII-challenged wild type mice also increased CIA severity and proinflammatory cytokine mRNA levels in CD69+/+ mice, but not CD69−/− mice. In addition, engagement of CD69 directly induces total and active TGF-β1 production in activated mouse T lymphocytes, mouse synovial cells, human synovial mononuclear cells from chronic arthropathies and Jurkat stable transfectants of human CD69 but not the parental CD69 negative cell line. Thus, CD69 is a negative modulator of autoimmune reactivity and inflammation through the synthesis of TGFθ, a cytokine that in turn down regulates the production of different pro-inflammatory mediators.

Further, an inhibitory role of CD69 was demonstrated in vivo by analyzing the susceptibility of CD69−/− mice to tumors. CD69−/− mice challenged with tumors, showed strongly reduced tumor growth and prolonged survival compared with wildtype mice. This potent anti-tumor response correlates with a diminished production of TGF-β, an increase in inflammatory cytokines and the chemokine MCP-1, and was associated with increased recruitment of lymphoid effector cells and diminished apoptosis. The resistance of CD69−/− mice to tumor growth was NK and T lymphocyte-mediated and persisted in immuno-compromised CD69−/− RAG-negative mice. Moreover, TGF-β blockade in wildtype mice increased the anti-tumor response, directly implicating the diminished production of TGF-β found in CD69−/− mice as a basis of their enhanced anti-tumor immunity. In addition, engagement of CD69 induced extracellular signal-regulated kinase (ERK) activation and TGF-β production by T cells. Blockade of ERK by the specific pharmacological inhibitor PD98059 inhibited TGF-β production, directly linking CD69 signaling with TGF-β regulation.

Furthermore, in vivo treatment with the antagonist 2.2 anti-CD69 mAb was demonstrated to enhance the anti-tumor response in wildtype mice and in immune compromised SCID and RAG-deficient mice. Therefore, in vivo treatment with anti-CD69 monoclonal antibodies was demonstrated as a novel approach for enhancement of the immune response in manipulating tumor immunity that may be extended to infectious diseases and immune deficiency syndromes to restore immune responses.

In vivo treatment with anti-CD69 antibodies results in different effects depending on the anti-CD69 used; the antagonist 2.2 anti-CD69 antibody enhances immune response, resulting in increased CIA severity and tumor rejection. The depleting 2.3 anti-CD69 deletes CD69+ activated effector leukocytes, resulting in attenuated CIA. In addition, this depleting 2.3 anti-CD69 antibody may directly reject CD69+ tumors.

Significantly, a human monoclonal antibody (mAb), designated hAIM-29, has been produced, and specifically recognizes human CD69 on the surface of activated-human leukocytes (Molina et al., 2003). Experiments show that hAIM-29 activates intracellular calcium influx without Ig crosslinking and enhances phorbol myristate acetate (PMA)-induced cell proliferation, in a manner similar to other mouse anti-CD69 antibodies.

Like CD69, AICL and LLT1 are expressed at low levels in leukocytes and are strongly and transiently upregulated (Hamann et al., 1997; Eichler et al., 2001). Therefore, AICL and LLT1, like CD69, may regulate leukocyte TGFθ release.

The role of early activation polypeptides as negative modulators of immune reactivity provides for the treatment of chronic inflammation and other immune-mediated diseases by targeting these polypeptides. In addition, since CD69, AICL and LLT1 are selectively expressed in activated leukocytes infiltrating inflamed tissues, the pharmacological stimulation of TGF-β1 synthesis through CD69, AICL and/or LLT1 may have a localized effect, thus avoiding the detrimental consequences of a systemic TGF-β1 up-regulation or delivery.

The current invention provides procedures for the modulation of the immune response by the targeting of CD69, AICL and/or LLT1 through different procedures.

One specific aim/goal of this invention is the regulation of the immune response based on the targeting of an early activation molecule with substances that promote early activation molecule signaling or interaction with its putative ligand(s) or receptor(s), which are identified as early activation molecule agonists. The second aim is the modulation of immune response through an early activation molecule using substances that inhibit or block early activation molecule signaling, including those that inhibit early activation molecule expression, block early activation molecule binding to its receptors or ligands or induce early activation polypeptide down-modulation from a cell surface, identified as early activation molecule antagonists. The third aim is the immune regulation through the depletion of cells expressing an early activation molecule using substances that deplete CD69+, AICL+ and/or LLT1+ cells, identified as early activation molecule depletors.

Other specific aims of this invention include methods for discovering, identifying, or otherwise evaluating an agent for use as an agonist, antagonist or depletor of CD69+, AICL+ and/or LLT1+ cells. These methods can use as all or part of the process of the evaluation of: Ca2+ signaling, activation of MAPK, production of TGF-β, induction of apoptosis mediators, and cell lysis. In addition, examples of some of these substances are specifically identified herein, such as the use of antibodies anti-early activation polypeptide antibody molecules or substances directly or indirectly derived from these antibodies. Moreover, putative ligands or receptors of an early activation polypeptide identified by the use of affinity chromatography with early activation molecule-related constructs or products, or whose agonist, antagonist or depleting activity on the early activation molecule is identified by any of the methods indicated are aims of this invention.

Another specific aim of this invention is the use of substances that can modulate the immune response directly or indirectly through an early activation molecule in the treatment of immune-mediated diseases. Autoimmune or tumoral processes are influenced by the absence or presence of early activation molecules and the treatment with substances that act through the early activation molecules. Thus, methods of the invention can be used to treat a number of other immune-mediated diseases, such as:

Autoimmune diseases, including rheumatoid arthritis and other types of chronic or acute arthritis or arthropathies with an immune component, systemic lupus erythematosus, scleroderma, Sjögren syndrome, autoimmune diabetes, thyroiditis, and other organ-specific immune diseases, including psoriasis. Also included are neurologic diseases, including multiple sclerosis, myasthenia gravis, and other neurologic immune-mediated diseases. Also included are gastrointestinal diseases, including Crohn\'s disease, colitis, celiac disease, hepatitis, and other immune-mediated diseases.

Cardiovascular diseases, including atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis, vasculitis, and other immune-mediated cardiovascular diseases.

Respiratory diseases, including emphysema, respiratory airways infections, and other immune-mediated respiratory diseases.

Allergic processes and hypersensitivity reactions (type I, II, III, and IV), including asthma, rhinitis, and other immune-mediated hypersensitivity reactions.

Transplant or graft rejection, including organ transplants, tissue graft, or blood transfusion. Also included is graft versus host disease, e.g., as occurs during bone marrow transplant.

Oncologic diseases, including leukemia, lymphoproliferative diseases, solid tumors and all other types of unwanted cell proliferation including non-malignant and malignant disorders, e.g., all other forms of cancer.

Infectious diseases, including bacteria, virus, fungal or parasitic infections, septic shock syndromes and other immunopathological responses to infectious agents.

Inherited immuno-deficiency diseases and acquired immuno-deficiency syndromes, and immuno-suppression syndromes by radiotherapy and chemotherapy treatments.

Degenerative processes as neurodegenerative processes that implicate immune competent cells as microglia.

Control administration of antigen used to manipulate the nature of antigen-specific response.

Gene therapy

In addition, some immune-mediated processes can be helped by an intervention through an early activation molecule, such as immunosuppression that can be applied to a variety of immune-mediated processes, including those previously indicated, or immuno-enhancers that can be applied to vaccination, as well as protective immunity injecting DNA encoding microbial antigens or tumor antigens, or other processes for the treatment of immune-mediated diseases, including those previously indicated.

Since the TGF-β cytokine and other mediators of CD69, AICL and LLT1 signaling have a pleiotropic effect, all the diseases above suggested may be covered for the described effect of these early activation molecules in immune regulation, and the specific aims are not limited to the following predictions, that are based in the examples indicated below. The agonist strategy through an early activation molecule would predict a higher production of TGF-β, with its consequent effects on immune modulation that have been extensively described (Letterio and Roberts, 1997; Prud\'homme and Piccirillo, 2000). The immunosuppressor effect of TGF-β could be useful for the treatment of immune diseases where the activation of the immune system is exacerbated, such as autoimmune diseases, transplant rejection, and others from the previous list. The antagonist approach would act through the opposite pathway, being used for treatment of diseases or processes that need to enhance the immune system, such as tumor rejection, infection treatment or its prevention as vaccination. The depleting strategy would eliminate CD69+, AICL+ and/or LLT1+ cells, which are the cells locally activated in response to antigenic stimulus and mediate the immune response. Therefore, the expected consequence would be the amelioration of diseases associated with exacerbation of immune response, such as autoimmune diseases, transplant rejection, and others from the previous list.

The terms “early activation proteins” and “early activation polypeptides” refer to CD69, AICL, LLT1 and fragments thereof. The term “protein” and “polypeptide” are used interchangeably herein.

CD69

As used herein, “CD69”, also known as “very early activation” protein, “activation inducer molecule”, and “gp 34/28”, refers to mammalian CD69, preferably human CD69 protein. Accordingly, the term “human CD69” refers to a polypeptide which has or is homologous to (e.g., at least about 85%, 90%, 95% identical to) an amino acid sequence (SEQ ID NO:2) as shown in Lopez-Cabrera (1993) J. Exp. Med. 178 (2), 537-547; or which is encoded by: (a) a nucleic acid sequence encoding a human CD69 (e.g., a nucleic acid sequence (SEQ ID NO:1) encoding human CD69 as provided in Lopez-Cabrera (1993) J. Exp. Med. 178 (2): 537-547) (see FIG. 23); (b) a nucleic acid sequence degenerate to a naturally occurring human CD69 sequence; (c) a nucleic acid sequence homologous to (e.g., at least about 85%, 90%, 95% identical to) the naturally occurring human CD69 nucleic acid sequence; or (d) a nucleic acid sequence that hybridizes to one of the foregoing nucleic acid sequences under stringent conditions, e.g., highly stringent conditions. A preferred CD69 is a naturally occurring variant or allele of CD69.

AICL

As used herein, “AICL”, also known as “activation-induced C-type lectin” refers to mammalian AICL, preferably human AICL protein. Accordingly, the term “human AICL” refers to a polypeptide which has or is homologous to (e.g., at least about 85%, 90%, 95% identical to) an amino acid sequence (SEQ ID NO:4) as shown in Hamann et al. (1997) Immunogenetics 45:295-300; or which is encoded by: (a) a nucleic acid sequence encoding a human AICL (e.g., a nucleic acid sequence (SEQ ID NO:3) encoding human AICL as provided in Hamann et al. (1997) Immunology 45:295-300 (see FIG. 24); (b) a nucleic acid sequence degenerate to a naturally occurring human AICL sequence; (c) a nucleic acid sequence homologous to (e.g., at least about 85%, 90%, 95% identical to) the naturally occurring human AICL nucleic acid sequence; or (d) a nucleic acid sequence that hybridizes to one of the foregoing nucleic acid sequences under stringent conditions, e.g., highly stringent conditions. A preferred AICL is a naturally occurring variant or allele of AICL.

LLT1

As used herein, “LLT1”, also known as “lectin-like transcript” refers to mammalian LLT1, preferably human LLT1 protein. Accordingly, the term “human LLT1” refers to a polypeptide which has or is homologous to (e.g., at least about 85%, 90%, 95% identical to) an amino acid sequence (SEQ ID NO:6) as shown in Boles et al. (1999) Immunogenetics 50:1-7; or which is encoded by: (a) a nucleic acid sequence encoding a human LLT1 (e.g., a nucleic acid sequence (SEQ ID NO:5) encoding human LLT1 as provided in Boles et al. (1999) Immunogenetics 50:1-7 (see FIG. 25); (b) a nucleic acid sequence degenerate to a naturally occurring human LLT1 sequence; (c) a nucleic acid sequence homologous to (e.g., at least about 85%, 90%, 95% identical to) the naturally occurring human LLT1 nucleic acid sequence; or (d) a nucleic acid sequence that hybridizes to one of the foregoing nucleic acid sequences under stringent conditions, e.g., highly stringent conditions. A preferred LLT1 is a naturally occurring variant or allele of LLT1.

An “anti-early activation polypeptide antibody molecule” is an antibody molecule that interacts with (e.g., binds to) an early activation polypeptide, preferably a human early activation protein. Preferably, anti-early activation polypeptide antibody molecules bind to the extracellular domain of the early activation polypeptide (i.e., an epitope of the early activation polypeptide located outside of a cell).

In some aspects, the anti-early activation polypeptide antibody molecule can be an anti-CD69 antibody molecule, e.g., an anti-human CD69 antibody molecule. Examples of monoclonal antibodies to human CD69 include, but are not limited to, an anti-human CD69 antibody molecule analogous to an anti-mouse antibody 2.2, an anti-human CD69 antibody molecule analogous to an anti-mouse CD69 antibody 2.3, or an anti-human CD69 antibody, e.g., an art known antibody, which can act as an agonist, an antagonist or a depletor of CD69, or antibody molecules having epitopes which overlap the epitope of such an antibody, or which compete with such an antibody for binding. Examples of art known anti-human CD69 antibodies include: TP1/8, TP1/22, TP 1/28, TP 1/33, TP 1/55 (as described, e.g., in Cebrian et al. (1988) J. Exp. Med. 168:1621-37); CH/4, CH/1, CH/2, FAB/1 (as described, e.g., in Sánchez-Mateos et al. (1991) Eur. J. Immunol. 21:2317-25); L78, MLR3, FN61, FN50 (as described in, e.g., Schwarting, R. et al. (Eds) Leukocyte Typing IV, Springer-Verlag, New York, 1989, p. 428); MLR3 (as described, e.g., in Corte, G et al. (1981) Eur. J. Immunol. 11:162-164; EA 1 (as described, e.g., in Hara, T (1986) J. Exp. Med. 164:1988-2005); Leu 23 (as described, e.g., in Lanier, L L et al. (1988) J. Exp. Med. 167:1572-1585); and C1.18, E16.5 (as described, e.g., in Gerosa, F (1991) Mol. Immunol. 28:159-168. Non-limiting examples of antibody molecules which can be used in the invention include: hAIM-29; an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the neck of a CD69 polypeptide (e.g., one or more residues from residues 62 to 84 of human CD69); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the NK domain (or carbohydrate recognition domain (CRD)) of a CD69 polypeptide (e.g., one or more residues from residues 82 to 199 of human CD69); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues from the intracellular domain of a CD69 polypeptide (e.g., one or more residues from 1 to 40 of human CD69); an antibody molecule which interacts with, e.g., binds to an epitope, which when bound modulates, e.g., enhances or reduces, the interaction of CD69 cytoplasmic and/or transmembrane regions with a downstream effector, which activity can be determined by the methods described herein (e.g., production of TGF-θ, MAPK activation, or Ca2+ signaling); an antibody molecule which interacts with, e.g., binds, an epitope (e.g., a conformational or linear epitope), which epitope when bound modulates, e.g., enhances or reduces, CD69 dimer formation (e.g., an epitope which includes residue Cys68 of human CD69 or a residue proximally located near Cys68); an antibody molecule which can modulate cell surface expression of CD69, e.g., an antibody molecule which can down-modulate CD69 from a cell surface; an antibody molecule which can modulate binding of a CD69 ligand or receptor, e.g., an antibody which can inhibit, e.g., competitively inhibit, or enhance binding of a ligand or a receptor to CD69; an antibody molecule which interacts with, e.g., binds, or which can inhibit or enhance binding of a ligand or receptor to, one or more of amino acid residues Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69).

In other embodiments, the anti-early activation polypeptide antibody molecule can be an anti-AICL antibody molecule, e.g., an anti-human AICL antibody molecule. Examples of monoclonal antibodies to human AICL include, but are not limited to, an art known anti-human AICL antibody which can act as an agonist, an antagonist or a depletor of AICL, or antibody molecules having epitopes which overlap the epitope of such an antibody, or which compete with such an antibody for binding. Examples of antibody molecules which can be used in the invention include: an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the neck of an AICL polypeptide (e.g., one or more residues from residues 26 to 36 of human AICL); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the NK domain (or carbohydrate recognition domain (CRD)) of an AICL polypeptide (e.g., one or more residues from residues 37 to 149 of human AICL); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues from the intracellular domain of an AICL polypeptide (e.g., one or more residues from 1 to 7 of human AICL); an antibody molecule which interacts with, e.g., binds to an epitope, which when bound modulates, e.g., enhances or reduces, the interaction of AICL cytoplasmic and/or transmembrane regions with a downstream effector, which activity can be determined by the methods described herein (e.g., production of TGF-θ, MAPK activation, or Ca2+ signaling); an antibody molecule which interacts with, e.g., binds, an epitope (e.g., a conformational or linear epitope); an antibody molecule which can modulate cell surface expression of AICL, e.g., an antibody molecule which can down modulate AICL from a cell surface; an antibody molecule which can modulate binding of an AICL ligand or receptor, e.g., an antibody which can inhibit, e.g., competitively inhibit, or enhance binding of a ligand or a receptor to AICL.

In yet other embodiments, the anti-early activation polypeptide antibody molecule can be an anti-LLT1 antibody molecule, e.g., an anti-human LLT1 antibody molecule. Examples of monoclonal antibodies to human LLT1 include, but are not limited to, an art known anti-human LLT1 antibody which can act as an agonist, an antagonist or a depletor of LLT1, or antibody molecules having epitopes which overlap the epitope of such an antibody, or which compete with such an antibody for binding. Examples of antibody molecules which can be used in the invention include: an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the neck of n LLT1 polypeptide (e.g., one or more residues from residues 60 to 75 of human LLT1); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues of the NK domain (or carbohydrate recognition domain (CRD)) of a LLT1 polypeptide (e.g., one or more residues from residues 76 to 132 of human LLT1); an antibody molecule which interacts with, e.g., binds to, an epitope which includes one or more residues from the intracellular domain of a LLT1 polypeptide (e.g., one or more residues from 1 to 30 of human LLT1); an antibody molecule which interacts with, e.g., binds to an epitope, which when bound modulates, e.g., enhances or reduces, the interaction of LLT1 cytoplasmic and/or transmembrane regions with a downstream effector, which activity can be determined by the methods described herein (e.g., production of TGF-θ, MAPK activation, or Ca2+ signaling); an antibody molecule which interacts with, e.g., binds, an epitope (e.g., a conformational or linear epitope), which epitope when bound modulates, e.g., enhances or reduces, LLT1 dimer formation (e.g., an epitope which includes residue Cys63 of human LLT1 or a residue proximally located near Cys63); an antibody molecule which interacts with, e.g., binds, an epitope (e.g., a conformational or linear epitope); an antibody molecule which can modulate cell surface expression of LLT1, e.g., an antibody molecule which can down modulate LLT1 from a cell surface; an antibody molecule which can modulate binding of a LLT1 ligand or a LLT1 receptor, e.g., an antibody which can inhibit, e.g., competitively inhibit, or enhance binding of a ligand or a receptor to LLT1.

In one embodiment, the anti-early activation polypeptide antibody molecule binds all or part of the epitope of an antibody described herein, e.g., an art known anti-human CD69 antibody, an art known anti-human AICL antibody, or an art known anti-LLT1 antibody which can act as an agonist, an antagonist or a depletor, or an anti-human early activation polypeptide antibody analogous to an anti-mouse early activation polypeptide antibody, e.g., an anti-mouse CD69 antibody (e.g., antibody 2.2, 2.3, or H1.2F3 (described, e.g., in Yokoyama et al. (1988) J. Immunol. 141:369-376). An anti-CD69 antibody can bind one or more residues of the epitopes described in Sanchez-Mateos (1991) or compete for binding with an antibody which binds one of the described epitopes. The anti-early activation polypeptide antibody molecule can inhibit, e.g., competitively inhibit, the binding of an antibody described herein, e.g., an art-known anti-human early activation polypeptide antibody described herein, to a human early activation polypeptide. An anti-early activation polypeptide antibody molecule may bind to an epitope, e.g., a conformational or a linear epitope, which epitope when bound prevents binding of an antibody described herein, e.g., an art known anti-human early activation polypeptide antibody described herein. The epitope can be in close proximity spatially or functionally-associated, e.g., an overlapping or adjacent epitope in linear sequence or conformationally to the one recognized by an antibody described herein, e.g., an art known anti-human early activation polypeptide antibody described herein.

In a preferred embodiment, the interaction, e.g., binding, between an anti-early activation polypeptide antibody molecule and an early activation polypeptide occurs with high affinity (e.g., affinity constant of at least about 109 M−1 or better) and specificity. Preferably, the anti-early activation polypeptide antibody molecule modulates immune response, e.g., is an early activation molecule agonist, an early activation molecule antagonist or an early activation molecule depleting antibody.

As used herein, “specific binding” refers to the property of the binding agent, preferably the antibody, to: (1) to bind to an early activation polypeptide, e.g., human early activation polypeptide, with an affinity of at least 1×107 M−1, and (2) preferentially bind to the early activation polypeptide, e.g., human early activation polypeptide, with an affinity that is at least two-fold, 50-fold, 100-fold, 1000-fold, or more greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the early activation polypeptide.

As can be seen from the disclosure herein, many types of anti-early activation polypeptide antibody molecules, e.g., antibodies, or antigen-binding fragments thereof, are useful in the methods of this invention. The antibody molecules can be of the various isotypes, including: IgG (e.g., IgG1, IgG2 (e.g., IgG2a, IgG2b), IgG3, IgG4), IgM, i.e., IgM/λ, IgA1, IgA2, IgD, or IgE. A preferred antibody molecule is an IgG isotype. The antibody molecules can be full-length (e.g., an IgG1 or IgG4 antibody) or can include only an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment). The antibody is preferably an engineered antibody molecule, e.g., a fully human or a humanized antibody.

Antibodies, or other agents described herein, can be evaluated for the ability to agonize, antagonize or deplete CD69, AICL or LLT1.

A depletor anti-early activation polypeptide antibody is one which, upon administration to an animal, either alone or conjugated to a toxin, reduces the absolute number of early activation polypeptide+cells, e.g. CD69+, AICL+ and/or LLT1+ tumor cells; e.g., CD69+, AICL+ and/or LLT1+ leukocytes at chronic inflammatory infiltrates. It will usually act through direct lysis of these cells (as opposed to, e.g., transient sequestration of cells in an organ compartment such as bone marrow or liver).

An antagonist anti-early activation polypeptide antibody is one which, upon administration to an animal, induces CD69+, AICL+ and/or LLT1+ cells to decrease their production of TGF-beta cytokine, e.g., by blocking endogenous signaling induced by engagement of CD69, AICL and/or LLT1 by one or more of its natural ligands or receptors (e.g. by blocking CD69, AICL or LLT1 interaction with its natural ligands or receptors) referred to herein as “a blocking antagonist”. Other early activation polypeptide antagonists include, e.g., “a down-modulating antagonist” which decreases the level of an early activation polypeptide on the cell surface, e.g., by binding to an early activation polypeptide and internalizing the polypeptide.

An agonist anti-early activation polypeptide antibody is one which, upon administration to an animal, induces CD69+, AICL+ and/or LLT1+ cells to increase their production of TGF-beta cytokine, e.g., by enhancing signaling in the absence of binding to a natural ligand or receptor, or over and above the endogenous signaling induced by engagement of CD69, AICL or LLT1 by one or more of its natural ligands or receptors.

Various in vitro methods can be used, e.g., in conjunction with whole animal assays, to evaluated candidates for activity as a depletor, antagonist, or agonist. An in vitro assay may not be indicative of in vivo activity in every case but never the less can be useful in screening strategies.

Early Activation Molecule Depletors

An in vitro early activation polypeptide depletor antibody is one which, upon incubation with cells expressing the early activation polypeptide, reduces the absolute number of those cells. This can be effected through their direct lysis, e.g. via complement-dependent, Fc-Receptor-dependent, or toxin-dependent mechanisms. Thus, an early activation polypeptide depletor can kill cells expressing the early activation polypeptide in in vitro killing assays. Isotype can play an important role in whether an anti-early activation polypeptide antibody will function as a depletor by way of a complement-based mechanism. If an antibody is able to bind complement, it will probably behave as a depletor. Most naturally occurring antibodies have a relatively high affinity for complement and will thus be depletors. This is less likely in the case of murine IgG1 and human IgG4, which have low affinity for complement. In addition, antibodies can be engineered to reduce or increase affinity for complement. A mouse, or other, anti-mouse early activation polypeptide depletor antibody can kill mouse cells expressing the early activation polypeptide in vitro in the presence of complement. Target early activation polypeptide expressing cells useful for testing such antibodies include, e.g., murine leukemias, e.g., EL4 thymoma cell line for CD69. For example, a mouse, or other, anti-human CD69 depletor antibody, (e.g., hAIM-29 mAb), can kill human CD69-expressing cells, e.g., CD69 expressing human T lymphocytes from rheumatoid arthritis synovial fluid, CD69 expressing human leukemias, or stable transfectants of CD69, e.g., Jurkat leukemia cells which stably express CD69. This antibody will not kill (or will kill at a much lower level) control cells which do not express CD69, e.g., parental Jurkat leukemia cells that do not express CD69. Thus, the ability of an antibody to function as a depletor can be assayed, in vitro, for its ability to lyse target cells expressing an early activation polypeptide and control cells that do not express the early activation polypeptide. A candidate depletor will lyse the cells expressing the early activation polypeptide but not control cells that do not express the early activation polypeptide (or will lyse them to a much lesser extent). In one example, cell lysis can be monitored in an assay in which target (and control) cells are loaded with CR51 and incubated with the antibody in the presence of fresh baby rabbit complement, typically for 2 h at 37° C. Afterwards, supernatant is recovered and the level of cell lysis evaluated by the amount of CR51 released into the supernatant. Lysis is indicated by release of CR51 by lysed cells.

Early Activation Molecule Agonists

An in vitro agonist anti-early activation polypeptide antibody is one which, upon incubation with CD69+, AICL+ and/or LLT1+ cells, increases their production of TGF-beta cytokine, e.g., by enhancing signaling in the absence of binding to a natural ligand or receptor, or by enhancing signaling over and above the endogenous signaling induced by engagement of CD69, AICL or LLT1 by one or more of its natural ligands or receptors, present in the in vitro culture system. Thus, one can perform an in vitro screen for agonists by incubating a candidate agonist with a cell expressing the early activation polypeptide and evaluating its effect on TGF-θ production, e.g., as compared with a control cell, e.g., a cell that does not express the early activation polypeptide. For example, an anti-mouse CD69 antibody can be evaluated for the ability to behave as an in vitro agonist by evaluating its ability to promote TGF-θ production by ConA-activated CD69 expressing splenocytes. An anti-human CD69 antibody, such as hAIM-29 mAb, can be evaluated for the ability to mediate TGF-θ synthesis by Jurkat cells stably transfected with CD69. An agonist will promote TGF-θ production in cells expressing the early activation polypeptide but not in a control line, preferably the parental line that does not express the early activation polypeptide. Agonism may require addition of a second crosslinking antibody which cross links the candidate agonists. An agonist should not act as a depletor.

Early Activation Molecule Antagonist

An in vitro antagonist anti-early activation polypeptide antibody is one which, upon incubation with CD69+, AICL+ and/or LLT1+cells, decreases their production of TGF-θ cytokine, e.g., by blocking endogenous signaling induced by engagement of CD69, AICL or LLT1 by one or more of its natural ligands or receptors, present in the in vitro culture system. Thus, one can perform an in vitro screen for antagonists by incubating a candidate antagonist with a cell expressing the early activation polypeptide and evaluating its effect on TGF-θ production, e.g., as compared with a control cell, e.g., a cell that does not express the early activation polypeptide. For example, an anti-mouse CD69 antibody can be evaluated for the ability to behave as an in vitro antagonist by evaluating its ability to inhibit or reduce TGF-θ production by ConA-activated CD69 expressing splenocytes. An anti-human CD69 antibody, such as hAIM-29 mAb, can be evaluated for the ability to mediate TGF-θ synthesis by Jurkat cells stably transfected with CD69. An antagonist will inhibit TGF-θ production in cells expressing the early activation polypeptide but not in a control line, preferably the parental line that does not express the early activation polypeptide. An in vitro antagonist can also be a down modulating antagonist, e.g., an in vitro down modulating antagonist anti-early activation polypeptide antibody. An in vitro antagonist can be screened for by incubating a candidate antagonist with a cell expressing the early activation polypeptide and evaluating whether the early activation polypeptide disappears from the cell surface. For example, a murine anti-early activation polypeptide antibody can be evaluated for its ability to down modulate an early activation polypeptide by using a reagent capable of detecting the presence of the murine anti-early activation polypeptide antibody, e.g., an anti-mouse polyclonal reagent. If the reagent does not detect the candidate, then the antagonist can be identified as a down modulating antagonist. An antagonist should not act as a depletor.

In addition an antagonist can have one or more of the following properties:

1) it blocks binding of pure early activation polypeptide to its purified receptor or ligand, to partially purified preparations such as membrane fractions from cells, which normally respond to the early activation polypeptide, e.g., which express an early activation polypeptide receptor, or to whole cells which normally respond to the early activation polypeptide, e.g., which express an early activation polypeptide receptor;

2) it blocks the binding of a cell expressing the early activation polypeptide to purified early activation polypeptide receptor;

3) it blocks binding of a cell expressing the early activation polypeptide to a cell expressing early activation polypeptide receptor (e.g., cells which normally respond to the early activation polypeptide) or to partially purified preparations such as membrane fractions from cells, which normally respond to the early activation polypeptide, e.g., which express an early activation polypeptide receptor. E.g., the candidate antagonist blocks binding of preparations containing a putative CD69R to a human CD69 stable transfectant of Jurkat cells (but not with the parental CD69-cell line);

4) it blocks binding of agonist monoclonal antibodies to a cell expressing the early activation polypeptide, thus preventing TGF-θ production by the agonist antibody;

5) it decreases the level of an early activation polypeptide on the cell surface, e.g., by binding to an early activation polypeptide and internalizing the polypeptide.

Antibody Molecules

As used herein, the term antibody molecule, refers to a molecule which includes a sufficient number of complementarity determining regions (CDRs), preferably 6, presented in a framework which allows binding to the cognate antigen of the CDRs. Thus, the term includes, full length antibodies (including naturally occurring antibodies and engineered antibodies), and antigen binding fragments of naturally occurring or engineered antibodies. The term includes various types of antibodies or antibody molecules including monospecific, monoclonal, recombinant, human, non-human, e.g., murine. Also included are single chain antibodies, intrabodies, and bi-valent antibodies. Also included are chimeric, CDR-grafted, humanized, deimmunized, as well as other antibody molecules which have been engineered to reduce immunogenicity, e.g., those having CDRs derived from a non-human source, e.g., derived from an nonhuman animal such as a mouse, and or derived from random or partially random generation of sequences, e.g., a by use of a phage display method. Such non-human can be inserted into human, humanized, or other frameworks which are less antigenic when administered to a human.

Thus, an antibody molecule can have: CDRs from a non-human source, e.g., from a non-human antibody, e.g., from a mouse immunoglobulin or another non-human immunoglobulin, from a consensus sequence, or from a sequence generated by phage display, or any other method of generating diversity; and having a framework that is less antigenic in a human than a non-human framework, e.g., in the case of CDRs from a non-human immunoglobulin, less antigenic than the non-human framework from which the non-human CDRs were taken. The framework of the immunoglobulin can, e.g., be human, humanized non-human, e.g., a mouse, framework modified to decrease antigenicity in humans, or a synthetic framework, e.g., a consensus sequence, or from an in vitro method of generating diversity.

Preferred antibody molecules can include at least one, and preferably two, heavy

(H) chain variable regions (abbreviated herein as VH) or antigen binding fragments thereof, and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL) or antigen binding fragments thereof. The VH and VL regions are subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDRs has been precisely defined (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Preferably, each VH and VL of an antibody molecule is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs can be from different sources.

The VH or VL chain of an antibody molecule can include all or part of a heavy or light chain constant region. In one embodiment, the antibody molecule is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The full length heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody molecule to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibody molecules can include IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains may be of types kappa or lambda.

As discussed above, “antigen-binding fragments” of an antibody molecule are within the term antibody molecule. An antigen-binding fragment, as used herein, can refer to a portion of an antibody which specifically binds to the early activation polypeptide (e.g., human early activation polypeptide). Examples of binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) one or more isolated complementarity determining regions (CDRs) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region.

Antibody fragments may also be produced by chemical methods, e.g., by cleaving an intact antibody with a protease, such as pepsin or papain, and optionally treating the cleaved product with a reducing agent. Alternatively, useful fragments may be produced by using host cells transformed with truncated heavy and/or light chain genes.

An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VL and VH, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term “monospecific antibody or antibody molecule” refers to an antibody or antibody molecule that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a “monoclonal antibody” or “monoclonal antibody composition,”.

The term “recombinant” antibody or antibody molecule, as used herein, refers to antibodies or antibody molecules that are prepared, expressed, created or isolated by recombinant means, such as antibody molecules expressed using a recombinant expression vector transfected into a host cell, antibody molecules isolated from a recombinant, combinatorial antibody library, antibody molecules isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibody molecules prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibody molecules include humanized, CDR grafted, chimeric, deimmunized, in vitro generated (e.g., by phage display) antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.

Monoclonal, chimeric and humanized antibodies, which have been modified by, e.g., deleting, adding, or substituting other portions of the antibody, e.g., the constant region, are also within the scope of the invention. For example, an antibody can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, e.g., a constant region meant to increase half-life, stability or affinity of the antibody, or a constant region from another species or antibody class; or (iii) by modifying one or more amino acids in the constant region to alter, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement fixation, placental transport, among others.

A preferred monoclonal antibody, for example, can involve use of the five-feature BABκ,λ mouse strain, carrying a human Ig transloci in its germ line (Magadán et al., 2002; Nicholson et al., 1999), in which a human mAb, hAIM-29, directed against the early human activation marker CD69, can be generated.

In one embodiment, the constant region of the antibody can be replaced by another constant region from, e.g., a different species. This replacement can be carried out using molecular biology techniques. For example, the nucleic acid encoding the VL or VH region of an antibody can be converted to a full-length light or heavy chain gene, respectively, by operatively linking the VH or VL-encoding nucleic acid to another nucleic acid encoding the light or heavy chain constant regions. The sequences of human light and heavy chain constant region genes are known in the art. Preferably, the constant region is human, but constant regions from other species, e.g., rodent (e.g., mouse or rat), primate, camel, rabbit, can also be used. Constant regions from these species are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the C1q component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar types of alterations could be described, which if applied to immunoglobulins of murine or other species, would reduce or eliminate these functions.

Useful immunogens for making anti-early activation polypeptide antibody molecules include cells which express the early activation polypeptide, membrane preparations from cells expressing the early activation polypeptide, early activation polypeptide containing vesicles or micelles, early activation polypeptides or fragments of the early activation polypeptide, e.g., isolated or purified early activation polypeptide, e.g., a human early activation polypeptide, e.g., a biochemically isolated early activation polypeptide, or a portion thereof, or a recombinantly produced early activation polypeptide or fragments there, e.g., the extracellular or intracellular domain of an early activation polypeptide, or a fusion protein containing an early activation polypeptide or any of the fragments or constructs described herein. The antigen is preferably a human antigen. A soluble early activation polypeptide can be obtained by, e.g., recombinant production of soluble the polypeptide (e.g., an early activation polypeptide lacking all or a part of the transmembrane and/or intracellular domains of the early activation polypeptide), or by lysis of a cell expressing the early activation polypeptide. Techniques for generating antibodies to CD69 are described in Sanchez-Mateos et al. (1991) Eur. J. Immunol. 21:2317-2325, the contents of which is expressly incorporated by reference. These techniques as well as other techniques known in the art can be used to generate antibodies to any of CD69, AICL and LLT1.

Fragments of CD69 which include residues about 62-84 or portions thereof of SEQ ID NO:2, fragments of AICL which include about residues 26 to 36 or portions thereof of SEQ ID NO:4, or fragments of LLT1 which includes about residues 60 to 75 or portions thereof of SEQ ID NO:6, can be used to make antibodies against neck regions of each or the early activation polypeptides, respectively, e.g., the fragments can be used as immunogens or used to characterize the specificity of an antibody. Similarly, fragments of CD69 which include residues 82 to 199, or portions thereof of SEQ ID NO:2, fragments of AICL which include residues 37 to 149, or portions thereof of SEQ ID NO:4, or fragments of LLT1 which include residues 76 to 132, or portions thereof of SEQ ID NO:6, can be used to make an antibody against the NK domain of an early activation protein; fragments of CD69 which include residues 62 to 199 or portions thereof of SEQ ID NO:2, fragments of AICL which include residues 26 to 149 or portions thereof of SEQ ID NO:4, or fragments of LLT1 which include residues 60 to 132 or portions thereof of SEQ ID NO:6, can be used to make an antibody against an extracellular region of the early activation polypeptide protein; fragments of CD69 which include residues about 1 to 40 or portions thereof of SEQ ID NO:2, fragments of AICL which include residues about 1 to 7 of SEQ ID NO:4, or fragments of LLT1 which include residues about 1 to 30 or portions thereof of SEQ ID NO:6, can be used to make an antibody against an intracellular region of the early activation protein.

Antibody molecules reactive with, or specific for, any region or domain of CD69, AICL or LLT1, particularly an extracellular region or domain of CD69, AICL or LLT1, can be made and tested for suitability for use in the invention.

Antibodies which bind only a native early activation protein, only denatured or otherwise non-native early activation protein, or which bind both, are within the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured early activation protein.

As described in more detail below, antibody molecules (preferably, monoclonal antibodies from differing organisms, e.g., rodent, sheep, human) against the early activation polypeptide can be produced using art-recognized methods. Once the antibody molecules are obtained, they can be tested for suitability for use in the invention, e.g., to determine if the antibody molecule is an agonist, antagonist, or depleting antibody.

Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). See generally, Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes. The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

An antibody can be obtained from a non-human source and the variable regions, and in particular the CDRs, can be sequenced, and used to make antibody molecules, e.g., humanized antibodies, useful in the invention. The location of the CDRs and framework residues can be determined (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference).

An antibody or an immunoglobulin chain can be humanized by methods known in the art. Once the murine antibodies are obtained, the variable regions can be sequenced. The location of the CDRs and framework residues can be determined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). The light and heavy chain variable regions can, optionally, be ligated to corresponding constant regions.

Murine anti-early activation polypeptide antibodies can be sequenced using art-recognized techniques.

Humanized or CDR-grafted antibody molecules or immunoglobulins can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference.

Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which are expressly incorporated by reference. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Humanized antibodies in which specific amino acids have been substituted, deleted or added, are thus within the invention. In particular, preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. The acceptor framework can be a mature human antibody framework sequence or a consensus sequence. As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

Other techniques for humanizing immunoglobulin chains, including antibodies, are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

The anti-early activation polypeptide antibody molecule may also be modified in other ways to decrease immunogenicity, e.g., by specific deletion of human T cell epitopes or “deimmunization” by the methods disclosed in WO 98/52976 and WO 00/34317, the contents of which are specifically incorporated by reference herein.

In some embodiments, the anti-early activation polypeptide antibody molecule includes a light or a heavy chain variable region having at least one, two and, preferably, three CDRs substantially identical to a CDR from a non-human anti-early activation polypeptide light or heavy chain variable region, respectively. In one preferred embodiment, the modified antibody or antigen-binding fragment thereof includes all six CDRs from the same non-human anti-early activation polypeptide antibody, e.g., an art known anti-human CD69, AICL or LLT1 antibody molecule, e.g., the panel of anti-CD69 antibody molecules described in Sanchez-Mateos (1991) Eur. J. Immunol. 21:2317-2325.

The light or heavy chain immunoglobulin of the modified anti-early activation polypeptide antibody or antigen-binding fragment thereof can further include a light chain or a heavy chain variable framework sequence from a light chain or heavy chain variable framework present in a human or a non-human, e.g., rodent, antibody.

Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Such transgenic mice can include mice that carry a portion of the human immunoglobulin heavy and light chain genes. For example, the five-feature BABκ,λ transgenic mouse strain carries transgenes of human μ heavy and κ and λ light chains in an inactivated endogenous IgH and Igκ background (Magadán et al., 2002; Nicholson et al., 1999). The immunization of these transloci mice allows the generation of an immune response that induces the production and secretion of human IgM (HuIgM) Abs against a wide variety of human and non-human antigens (Ags) (Magadán et al., 2002). Molina et al. (2003) have successfully used these mice to generate a HuIgM mAb, hAIM-29, which specifically binds to the human CD69 antigen.

Similarly, in the HuMAb mouse®, available from Medarex and GenMab, mouse genes for creating antibodies are inactivated and replaced with many of the key gene sequences from unarranged human antibody genes coding for heavy and light chains. Another mouse available is the XenoMouse® from Abgenix which has approximately 80% of the human heavy chain antibody genes and a significant amount of the human light chain genes. Other transgenic mice are available for generating human antibodies which contain complete sets of the variable and constant genes found in the corresponding natural human immunoglobulin loci. These mice, also referred to as Kirin TC mice™ are available from Medarex. The Kirin TC mice™ are “transchromosomic,” meaning that the mouse genes for creating antibodies have been inactivated and have been replaced by the human chromosomes containing all of the human antibody genes, including all heavy chain classes that encode all isotypes (IgG1-4, IgA1-2, IgD, IgM and IgE). Also available from Medarex, is the KM-mouse®, a crossbred mouse that combines the characteristics of the HuMAb-Mouse with Kirin\'s TC Mouse. The KM-mouse, like the Kirin TC mouse, retains the capability to produce all human antibody isotypes. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326). The procedures for the isolation of human mAbs are identical to those used for conventional mouse immunization and hybridoma production (reviewed in Brüggemann and Taussig, 1997; Brüggemann and Neuberger, 1996; Jakobovits, 1995).

Chimeric antibodies, including chimeric immunoglobulin chains, can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

Also within the term are in vitro generated species where all or part of the variable region, e.g., one or more or all CDRs, is generated in a non-immune cell selection, e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen. E.g., combinatorial antibody display methods, have been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce an antibody molecule.

Once displayed on the surface of a display package (e.g., filamentous phage), the antibody library is screened with the antigen, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for the antigen. Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.

Specific antibodies with high affinities for a surface protein can be made according to methods known to those in the art, e.g., methods involving screening of libraries (Ladner, R. C., et al., U.S. Pat. No. 5,233,409; Ladner, R. C., et al., U.S. Pat. No. 5,403,484). Further, the methods of these libraries can be used in screens to obtain binding determinants that are mimetics of the structural determinants of antibodies.

An antigen-binding region can also be obtained by screening various types of combinatorial libraries with a desired binding activity, and to identify the active species, by methods that have been described.

In one embodiment, a variegated peptide library is expressed by a population of display packages to form a peptide display library. Ideally, the display package comprises a system that allows the sampling of very large variegated peptide display libraries, rapid sorting after each affinity separation round, and easy isolation of the peptide-encoding gene from purified display packages. Peptide display libraries can be in, e.g., prokaryotic organisms and viruses, which can be amplified quickly, are relatively easy to manipulate, and which allows the creation of large number of clones. Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses). However, the present invention also contemplates the use of eukaryotic cells, including yeast and their spores, as potential display packages.

Anti-early activation polypeptide antibody molecules useful in the present invention can be recombinant antibodies produced by host cells transformed, e.g., with DNA encoding immunoglobulin light and heavy chains of a desired antibody. Recombinant antibody molecules may be produced by known genetic engineering techniques. For example, recombinant antibodies may be produced by cloning a nucleotide sequence, e.g., a cDNA or genomic DNA sequence, encoding the immunoglobulin light and heavy chains of the desired antibody from a hybridoma cell that produces an antibody useful in this invention. The nucleotide sequence encoding those polypeptides is then inserted into expression vectors so that both genes are operatively linked to their own transcriptional and translational expression control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Typically, both genes are inserted into the same expression vector. Prokaryotic or eukaryotic host cells may be used.

Expression in eukaryotic host cells is preferred because such cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. However, any antibody produced that is inactive due to improper folding may be renaturable according to well known methods (Kim and Baldwin, “Specific Intermediates in the Folding Reactions of Small Proteins and the Mechanism of Protein Folding”, Ann. Rev. Biochem. 51, pp. 459-89 (1982)). It is possible that the host cells will produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which also are antibody homologs according to the present invention.

It will be understood that variations on the above procedure are useful in the present invention. For example, it may be desired to transform a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding the early activation polypeptide, e.g., the constant region may be modified by, for example, deleting specific amino acids. The molecules expressed from such truncated DNA molecules are useful in the methods of this invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are anti-early activation polypeptide antibody and the other heavy and light chain are specific for an antigen other than that early activation polypeptide, or another epitope of that early activation polypeptide.

Antibody molecules of the invention can be conjugated, covalently or non-covalently, to other moieties, e.g., labels or therapeutic agents, e.g., toxins (e.g., protein (e.g., diphtheria or ricin) or chemical toxins), therapeutic isotopes, or other therapeutic moieties.

Accordingly, an anti-early activation polypeptide antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). The antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized (or labeled) to include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms, e.g., europium (Eu), and other lanthanides, and radioactive materials (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin). For example, an antibody may be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin.

An anti-early activation polypeptide antibody or antigen-binding fragment thereof may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

An anti-early activation polypeptide antibody or antigen-binding fragment thereof may be conjugated to another molecular entity, e.g., a moiety which modulates immunogenicity and/or half life. In one embodiment, the molecular entity is polyethylene glycol (PEG) or derivatives thereof. PEGylation is a chemical conjugation method which can reduce potential immunogenicity and/or extends half life. Various methods of PEGylating an antibody are known. See, e.g., Bhandra et al. (2002) Pharmazie 57(1):5-29.

An “early activation polypeptide binding agent” is an agent which interacts with (e.g., binds to) an early activation polypeptide, preferably a human early activation polypeptide. Preferably, the interaction, e.g., binding, occurs with high affinity, e.g., affinity constant of at least 107 M−1, preferably, between 108 M−1 and 1010 M−1, or about 109 M−1, and specificity. Early activation polypeptide binding agents can be CD69, AICL or LLT1 agonists, antagonists or depletors. Examples of early activation polypeptide binding agents include anti-early activation polypeptide antibodies (e.g., antibodies as described above) as well as small molecules or peptidomimetics.

Useful in the methods of this invention are early activation polypeptide mimetic agents. These agents, which include peptides, semi-peptidic compounds or non-peptidic compounds (e.g., small organic molecules), are inhibitors of an early activation molecule activity.

In preferred embodiments, the agent is a member of a combinatorial library, e.g., a peptide or organic combinatorial library, or a natural product library. In a preferred embodiment, a plurality of test compounds, e.g., library members, includes at least 10, 102, 103, 104, 105, 106, 107, or 108 compounds. In a preferred embodiment, the plurality of test compounds, e.g., library members, share a structural or functional characteristic.

In one embodiment, the invention provides libraries of early activation polypeptide binding agents. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e.g., E. M. Gordon et al., J. Med. Chem. (1994) 37:1385-1401; DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res. (1996) 29:114; Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.; Keating, T. A. Acc. Chem. Res. (1996) 29:123; Ellman, J. A. Acc. Chem. Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V. Acc. Chem. Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelle et al. Trends Anal. Chem. (1995) 14:83; Chen et al. J. Am. Chem. Soc. (1994) 116:2661; U.S. Pat. Nos. 5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. WO92/10092, WO93/09668, WO91/07087, WO93/20242, WO94/08051).

Libraries of compounds of the invention can be prepared according to a variety of methods, some of which are known in the art. For example, a “split-pool” strategy can be implemented in the following way: beads of a functionalized polymeric support are placed in a plurality of reaction vessels; a variety of polymeric supports suitable for solid-phase peptide synthesis are known, and some are commercially available (for examples, see, e.g., M. Bodansky “Principles of Peptide Synthesis”, 2nd edition, Springer-Verlag, Berlin (1993)). To each aliquot of beads is added a solution of a different activated amino acid, and the reactions are allow to proceed to yield a plurality of immobilized amino acids, one in each reaction vessel. The aliquots of derivatized beads are then washed, “pooled” (i.e., recombined), and the pool of beads is again divided, with each aliquot being placed in a separate reaction vessel. Another activated amino acid is then added to each aliquot of beads. The cycle of synthesis is repeated until a desired peptide length is obtained. The amino acid residues added at each synthesis cycle can be randomly selected; alternatively, amino acids can be selected to provide a “biased” library, e.g., a library in which certain portions of the inhibitor are selected non-randomly, e.g., to provide an inhibitor having known structural similarity or homology to a known peptide capable of interacting with an antibody, e.g., the an anti-idiotypic antibody antigen-binding site. It will be appreciated that a wide variety of peptidic, peptidomimetic, or non-peptidic compounds can be readily generated in this way.

The “split-pool” strategy results in a library of peptides, e.g., inhibitors, which can be used to prepare a library of test compounds of the invention. In another illustrative synthesis, a “diversomer library” is created by the method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993)). Other synthesis methods, including the “tea-bag” technique of Houghten (see, e.g., Houghten et al., Nature 354:84-86 (1991)) can also be used to synthesize libraries of compounds according to the subject invention.

Libraries of compounds can be screened to determine whether any members of the library have a desired activity, and, if so, to identify the active species. Methods of screening combinatorial libraries have been described (see, e.g., Gordon et al., J Med. Chem., supra). Soluble compound libraries can be screened by affinity chromatography with an appropriate receptor to isolate ligands for the receptor, followed by identification of the isolated ligands by conventional techniques (e.g., mass spectrometry, NMR, and the like). Immobilized compounds can be screened by contacting the compounds with a soluble receptor; preferably, the soluble receptor is conjugated to a label (e.g., fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds, and the like) that can be detected to indicate ligand binding. Alternatively, immobilized compounds can be selectively released and allowed to diffuse through a membrane to interact with a receptor. Exemplary assays useful for screening the libraries of the invention are described below.

In one embodiment, compounds of the invention can be screened for the ability to interact with an early activation polypeptide by assaying the activity of each compound to bind directly to the polypeptide, e.g., by incubating the test compound with an early activation polypeptide and a lysate, in one well of a multiwell plate, such as a standard 96-well microtiter plate. In this embodiment, the activity of each individual compound can be determined. A well or wells having no test compound can be used as a control. After incubation, the activity of each test compound can be determined by assaying each well. Thus, the activities of a plurality of test compounds can be determined in parallel.

In still another embodiment, large numbers of test compounds can be simultaneously tested for binding activity. For example, test compounds can be synthesized on solid resin beads in a “one bead-one compound” synthesis; the compounds can be immobilized on the resin support through a photolabile linker. A plurality of beads (e.g., as many as 100,000 beads or more) can then be combined with yeast cells and sprayed into a plurality of “nano-droplets”, in which each droplet includes a single bead (and, therefore, a single test compound). Exposure of the nano-droplets to UV light then results in cleavage of the compounds from the beads. It will be appreciated that this assay format allows the screening of large libraries of test compounds in a rapid format.

Combinatorial libraries of compounds can be synthesized with “tags” to encode the identity of each member of the library (see, e.g., W. C. Still et al., U.S. Pat. No. 5,565,324 and PCT Publication Nos. WO 94/08051 and WO 95/28640). In general, this method features the use of inert, but readily detectable, tags, that are attached to the solid support or to the compounds. When an active compound is detected (e.g., by one of the techniques described above), the identity of the compound is determined by identification of the unique accompanying tag. This tagging method permits the synthesis of large libraries of compounds which can be identified at very low levels. Such a tagging scheme can be useful, e.g., in the “nano-droplet” screening assay described above, to identify compounds released from the beads.

In preferred embodiments, the libraries of compounds of the invention contain at least 30 compounds, more preferably at least 100 compounds, and still more preferably at least 500 compounds. In preferred embodiments, the libraries of compounds of the invention contain fewer than 109 compounds, more preferably fewer than 108 compounds, and still more preferably fewer than 107 compounds.

The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to early activation proteins or early activation nucleic acids, have a stimulatory or inhibitory effect on, for example, early activation molecule expression or an early activation molecule activity. Compounds thus identified can be used to modulate the activity of target gene products (e.g., early activation molecule genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidate or test compounds that are substrates of an early activation protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of an early activation protein or polypeptide or a biologically active portion thereof, e.g., compounds that modulate signaling of an early activation protein or polypeptide.