ES2244297A1 - Immune regulation based on the targeting of early activation molecules - Google Patents

Abstract

Improvements introduced in invention patent number 200300252 corresponding to a new immune regulation strategy based on the inducible molecule during CD69 leukocyte activation. Improvements made to invention patent PV200300252 corresponding to the use of an effective amount of an early activation molecule, which is an anti-CD69 antagonist antibody molecule selected from a humanized anti-CD69 antibody molecule, an antibody molecule human anti-CD69, a chimeric anti-CD69 antibody molecule and a deimmunized anti-CD69 antibody molecule for the manufacture of a medicament for the treatment of a subject who needs, or may benefit from, an increased immune response, wherein said antagonist decreases the signaling by the early activation molecule or reduces the interaction of the early activation molecule with a ligand of said early activation molecule. (Machine-translation by Google Translate, not legally binding)

Description

Improvements introduced in the invention patent nº 200300252 corresponding to: "A new strategy of immune regulation based on the inducible molecule during leukocyte activation DC69 ".

Object of the invention

The present specification refers to a Addition patent related to a new regulation strategy immune based on the inducible molecule during activation CD69 leukocyte, in which the inhibitory role of an early activation molecule belonging to the family of lectins type C and whose gene is located in the gene region of the NK complex, CD69, in immune reactions by regulation of the synthesis of TGF-? 1.

Also in this invention it is shown that the use of monoclonal antibodies specific for CD69, It constitutes an effective therapy in tumors induced in mice.

The role of early activation molecules as modulators of the immune response suggests the use of these molecules as targets both for the stimulation of the immune response in cancer therapies, infections, immunodeficiencies or vaccination, as for the control of responses  exacerbated, in the case of chronic inflammation, autoimmunity, allergy or transplants

Field of the Invention

The manipulation of activation molecules early as immune regulators can boost the development of new treatments for the adjustment of the immune response, including negative regulation in the case of diseases autoimmune and allergies, or positive in cases of formation of tumors, vaccination and immunodeficiency therapies.

The development of these methods is based on the finding of mechanisms to employ the activation molecules Early as therapeutic targets. Therefore, you must ascribe to the pharmacological sector for application in the sector sanitary.

Background of the invention

The pharmacological treatment of immune diseases is a field of intense study, including in this category autoimmune diseases, which affect approximately 5% of the world population (O'Shea et al ., 2002), transplant rejection, allergy, atherosclerosis , infectious diseases and tumor processes that are a major cause of mortality in industrialized countries. Therefore, the regulation of the immune response is crucial in the treatment of the most important causes of death and morbidity in our society.

CD69 is a molecule that expresses itself rapidly and transiently during leukocyte activation after an immune challenge (Cebrián et al ., 1988; Hara et al ., 1986; Testi et al ., 1994). CD69 is expressed in all hematopoietic lineages except erythrocytes, and although it is detected in vivo in some subtypes of T and B lymphocytes in peripheral lymphoid tissues (Sánchez-Mateos et al ., 1989), its expression is much more intense and persistent in infiltrates leukocytes of various chronic inflammatory diseases such as rheumatoid arthritis and chronic viral hepatitis (García-Monzón et al ., 1990; Laffón et al ., 1991), in leukocytes responsible for transplant rejection (Mampaso et al ., 1993), leukocytes involved in the allergic response (Hartnell et al ., 1993), immune cells in atherosclerotic lesions (Caspar-Bauguil et al ., 1998), lymphocytes that infiltrate tumors (Coventry et al ., 1996) or during persistent infections (Zajac et al ., 1998). There is evidence that CD69 is involved in the activation of bone marrow derived cells (Cebrián et al ., 1988; Testi et al ., 1994).

However, hematopoietic development and maturation of T lymphocytes are almost normal in CD69-deficient mice under physiological conditions (Lauzurica et al ., 2000) .CD69, along with two other proteins, type C lectin induced during activation ( AICL) and the lectin type transcript (LLT-1), are part of the NK complex (Hamann et al ., 1997; Boles et al ., 1999). CD69 protein expression increases early after cell activation (Eichler et al ., 2001).

Autoimmune diseases are characterized by abnormal and persistent activation of leukocytes that contribute to the pathogenesis of the disease.

It is well established that cytokines they play an essential role in the pathogenesis of diseases autoimmune (Falcone and Sarvetnick, 1999). Therefore, the Elimination of these cytokines is the reference strategy in the new treatments of autoimmune diseases. By For example, tumor necrosis factor (TNF) seems to be as the first link in the inflammatory cascade, and anti-therapy TNF has been used effectively in the treatment of arthritis rheumatoid (AR) (Feldmann, 2002). In addition, other cytokines have a anti-inflammatory effect

In this context, the administration of TGF-? 1 has a beneficial effect on some autoimmune diseases (Prud'homme and Piccirillo, 2000), inhibiting the production of proinflammatory cytokines in arthritis (Kuruvilla et al ., 1991), allergic encephalomyelitis, 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", which implies the combined action of humoral and cellular mechanisms (Dighe, 1994; Kaplan, 1998; Shankaran, 2001; Smyth and Godfrey, 2000; Smyth et al ., 2000; Van den Broek, 1996).

The action of cytokines, directed, at least in part, to regulate the immunogenicity of tumor cells, it is crucial to the tumor suppressor function of the system immune.

Advanced tumor cells and metastases inhibit the expression of MHC class I molecules, which makes them targets that can be destroyed by NK cells In addition, NK cells secrete cytokines, such as TGF-? And IFN-?, Which stimulate differentiation of CD4 + T cells to cells Th1 helpers or helper. In this sense, the IFN-? Helps prevent the formation of tumors, since mice deficient for this cytokine develop spontaneous neoplastic diseases (Dighe, 1994; Kaplan, 1998).

In addition, TGF-? Plays an immunosuppressive role, thereby inhibiting the antitumor immune response by regulating the production of proinflammatory cytokines (Letterio and Roberts, 1998), which is dependent on the cellular stage and cytokines present in the microenvironment. mobile. TGF-? Has been implicated in the modulation of anti-tumor effector mechanisms by altering the activation, proliferation and cytotoxicity of immune cells, macrophages, NK cells and T lymphocytes (Gorelik and Flavell, 2000; Kehrl et al ., 1986).

It has been recently described that blocking TGF-? induced signaling increases the efficacy of antitumor immunity, as it facilitates the expansion of tumor-specific CD8 + T lymphocytes (Gorelik and Flavell, 2001). There are other cytokines involved in regulation of recruitment, proliferation, differentiation and survival of the anti-tumor effector cells.

Chemokine MCP-1, which is produced by many cell types, activates cell recruitment and mice deficient for MCP-1 have deficiencies in T-lymphocyte-dependent responses (Allavena et al ., 1994; Carr et al ., 1994; Gu et al ., 1997).

Therefore, cytokines play roles essential in the induction of antitumor immunity, and the blocking them may constitute an effective therapy in different human and murine models (Gill, 1995).

The fibrogenic effect and other negative effects TGF-? may limit your employment immunotherapeutic in humans (Border and Ruoshlati, 1992).

TGF-? Is involved in various pathological conditions and its systemic use has an oncogenic risk, which may explain the high number of tumors observed in patients treated with cyclosporine A (CsA), which is a drug that increases the systemic production of TGF. -? (Red et al ., 1999). Therefore, localized regulation of TGF-? Can result in an effective treatment of chronic inflammatory diseases, avoiding the negative consequences and side effects of the generalized increase in the expression of this cytokine.

Description of the invention

The Addition Patent relating to a new immune regulation strategy based on the molecule inducible during CD69 leukocyte action corresponding to Invention Patent number 200300252 is based, in part, on the discovery that the modulation of CD69 can be used to modulate the immune response of a subject and thus treat a variety of disorders of immune origin.

CD69 antagonists and eliminators are useful, as described later. CD69 and nucleic acids that code for them are referred to here as "molecules of early activation. "

According to this, in one aspect, the invention characterizes methods of treating a subject: e.g., a subject that is affected by a disorder characterized by a response unwanted immune. The disorder can be characterized by a response considered "normal" or for an immune response increased when compared to what is normally observed in a subject.

Examples of such disorders are indicated here, and include acute or chronic inflammatory disorders and disorders immune, e.g., autoimmune disorders.

In some examples, the subject has a inflammatory disease, e.g., an acute inflammatory disease or chronicle. Examples of such diseases include, but are not limited to: acute lung damage, respiratory distress syndrome acute, arthritis (e.g. AIC), asthma, bronchitis, cystic fibrosis, Encephalomyelitis, hepatitis (e.g. chronic viral hepatitis), inflammatory bowel disease, multiple sclerosis, damage from reperfusion (e.g. myocardial), nephritis, pancreatitis, psoriasis, arterial occlusion (e.g. of retina), stroke, systemic lupus erythematosus, transplant (e.g. rejection of host transplantation, e.g., transplanted tissue such as hematopoietic stem cells or an organ, e.g., a kidney, or response against the host caused by the transplant, e.g., graft versus host disease), light-induced damage ultraviolet, and / or vasculitis. The inflammatory disease can be acute or chronic, and is preferably mediated by leukocytes. In some examples, inflammatory disease is associated with the chronic inflammation, e.g. inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.

In one aspect, the invention characterizes methods. of treatment of a subject, e.g. a subject that has a disorder characterized by an unwanted immune response. The disorder is can characterize by an immune response that is increased regarding what is normally seen in a subject. Examples of such  disorders are indicated here and include inflammatory disorders acute or chronic, and immune disorders, e.g. disorders Autoimmune The method includes administering an effective amount of a cell eliminator that expresses an activation molecule early to the subject, e.g., a cell expressing CD69.

Examples of cell scavengers that express the early activation molecule include those described herein, but are not limited to, antibody molecules that bind to CD69 and remove cells that express CD69, eg a human anti-CD69 antibody analogous to the anti-antibody. Mouse CD69 2.3 (Esplugues et al .) Or any anti-CD69 antibody from the literature that can act as a remover of cells expressing CD69 (or an antibody molecule based thereon, eg, an antibody fragment or a chimera antibody, humanized or disinmunogenic), an antibody molecule that binds to the epitope to which such an antibody binds, an antibody molecule that competes for binding with such an antibody, antibody molecules that bind to AICL and remove cells that express AICL, eg an anti-AICL antibody, eg, any anti-AICL antibody from the literature that can act as an eliminator of cells expressing AICL (or an antibody molecule based thereon, eg, an antibody fragment or a chimera antibody, humanized or deimmunogenic), an antibody molecule that binds to the epitope to which such an antibody binds, an antibody molecule that competes for binding with such an antibody; antibody molecules that bind to LLT1 and remove cells that express LLT1, eg any anti-LLT1 antibody from the literature that can act as an eliminator of cells that express LLT1 (or an antibody molecule based on it, eg, an antibody fragment or a chimera antibody, humanized or deimmunogenic), an antibody molecule that binds to the epitope to which such antibody binds, an antibody molecule that competes for binding with such an antibody; or an antibody molecule identified by some method described here. In one example, an anti-early eliminator anti-molecule molecule antibody (eg, an anti-CD69 scavenger antibody, an anti-AICL scavenger antibody molecule, or an anti-LLT1 scavenger antibody molecule) can recruit an immune cell (eg, via an Fc receptor or by recruitment of the complement system) and inactivate or lyse the target cell, eg, the cell that expresses the early activation molecule. In another example, the early activation molecule eliminator can activate the classical complement pathway that leads to the removal of the target cell, (eg, the cell expressing CD69, AICL, and / or LLT1). In another example, the eliminator is an early-activating anti-molecule antibody that is bound to a therapeutic agent, eg, a protein, drug or isotope, which can inactivate or destroy the target cell, eg the cell expressing CD69, AICL, and / or LLT1.

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

In a preferred example, the method includes administer one or more cell scavengers that express a early activation molecule in combination with a second therapeutic agent, e.g. a therapeutic agent or an agent for treat inflammation The second agent can be an antibody or A different agent.

The subject may be a mammal, e.g. a primate, preferably a superior primate, e.g. a human.

In some examples, the cell eliminator that express the early activation molecule can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, by route transdermal, or by inhalation or by administration intracavitary), topically, or by application to the membranes mucous membranes, such as the nose, throat or bronchial tubes. In some cases, the early activation molecule eliminator can be administered before or during an immune response secondary in the subject.

In a preferred example, the eliminator of the Early activation molecule is administered repeatedly. For example, the eliminator can be administered 2, 4, 6 or more times. The administration can be repeated until the improvement in the Subject condition is seen or expected. The administration can be at a frequency of one every 5 to 7 days, 14 to 30 or 30 to 60 days. The administration of other compounds may be more frequent. For example, a small molecule can be administered One, two, three, four or more times a day.

In another aspect, the invention characterizes methods of treating a subject, e.g., a subject who needs or a subject that could benefit from, an immune response increased The subject may have a disorder characterized by a decreased immune response, e.g., an immune response that is reduced compared to what is seen in the absence of said disorder, e.g. the subject could have an immunodeficiency. In other examples, the subject does not have a diminished immune response or compromised, but would benefit from an immune response increased For example, the subject could have a disorder characterized by unwanted cells or tissues, or characterized by an abnormal cell proliferation, as it happens in the Cancer. The method can also be used to increase the immune response in a subject, e.g. an immune response to a antigen (e.g. a vaccine). The method includes the administration of one or more antagonists of the early activation molecule (e.g., a CD69 antagonist, an AICL antagonist, a LLT1 antagonist, or combinations thereof) to the subject.

Examples of molecule antagonists of Early activation include, but is not limited to, antisense of early activation molecule (e.g., CD69 antisense, AICL, or LLT1), RNAi to interfere with the messenger for molecule early activation, an antagonistic antibody molecule for anti-CD69, an antagonist antibody molecule for anti-AICL, an antibody molecule antagonist for anti-LLT1, and other compounds identified by some method described here, e.g., compounds that interact with one or more of CD69, AICL, and LLT1 and decrease the TGF-? expression by a cell and small molecules that bind to an early activation molecule and antagonize your activity. Examples of small molecules include, but are not limited to peptides, peptidomimetics (e.g. peptoids), amino acids, amino acid analogs, polynucleotides, analogs of polynucleotides, nucleotides, nucleotide analogs, compounds organic and inorganic (including heteroorganic compounds and organometallic) with molecular weight less than about 5000 g / mol, and salts, esters and other pharmacologically acceptable forms of such compounds. Examples of other molecule antagonists of early activation include, but are not limited to: antagonists of the CD69 receptor (CD69R), polypeptides, peptides, peptidomimetics, AICL receptor antagonists (AICLR), polypeptides, peptides, peptidomimetics, receptor antagonists of LLT1 (LLT1R), polypeptides, peptides, peptidomimetics, forms soluble CD69R, soluble forms of AICL-R, forms  soluble LLT1-R fusion proteins antagonists of CD69R, AICL-R, or LLT1-R, e.g., antagonist fusion proteins of a receptor of an early activation polypeptide with whey proteins (e.g. CD69R fusion proteins with immunoglobulins, or CD69R with human serum albumin proteins of fusion of AICL-R with immunoglobulins, or of AICL-R with human serum albumin proteins fusion of LLT1-R with immunoglobulins, or of LLT1-R with human serum albumin) or other forms of antagonist fusion proteins of a receptor of a early activation polypeptide designed to increase life serum mean and / or multivalence.

Examples of antagonistic antibody molecules of early activation anti-polypeptide include, but are not limited to, antibody molecules that bind a early activation polypeptide and interfere with the binding of the early activation polypeptide and a polypeptide that binds to an early activation protein and antibody molecules that bind to an early activation polypeptide and decrease the early activation polypeptide expression on the surface of the cell, e.g., by internalization of the polypeptide of early activation Antibody molecules anti-CD69 antagonists include, but are not limited to a, antibody molecules that bind CD69 and interfere with binding from CD69 to a CD69 binding partner, and antibody molecules that  bind to CD69 and decrease CD69 expression on the surface of the cell. Examples of antagonistic antibody molecules anti-CD69 include an antibody human anti-CD69, e.g., an antibody human anti-CD69 antibody analog mouse anti-CD69 2.2 or any antibody anti-CD69, e.g., an anti-CD69 known in the literature that can act as an antagonist (or a antibody molecule based on, e.g., a fragment of antibody, or a chimera, humanized or disinmunogenic antibody) or an antibody molecule that binds to the epitope bound by such antibody, or a molecule that competes for binding with such antibody, or an antibody molecule that binds 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. Anti-AICL antagonist antibody molecules include, but are not limited to, antibody molecules that bind to AICL and interfere with the binding of AICL to a binding partner to AICL, and antibody molecules that bind to AICL and decrease the AICL expression on the cell surface. Examples of a anti-AICL antagonist antibody molecule include a human anti-AICL antibody, e.g., a anti-AICL antibody, e.g., an antibody anti-AICL known in the literature that can act as an antagonist (or an antibody molecule based on the, e.g., an antibody fragment, or a chimera antibody, humanized or disinmunogenic) or an antibody molecule that is binds to the epitope bound by such antibody, or a molecule that competes by binding with such an antibody, or an antibody molecule that binds or interferes with the binding of another antibody or ligand to one or more of the amino acid residues from 37 to 149 of the human AICL.  Anti-LLT1 antagonist antibody molecules include, but are not limited to, antibody molecules that bind LLT1 and interfere with the binding of LLT1 to a binding partner to LLT1 and antibody molecules that bind to LLT1 and decrease the expression of LLT1 on the cell surface. Examples of anti-LLT1 antibody molecule antagonists include any anti-LLT1 antibody, e.g., a anti-CD69 known in the literature you can act as an antagonist (or an antibody molecule based on the, e.g., an antibody fragment, or a chimera antibody, humanized or disinmunogenic) or an antibody molecule that is binds to the epitope bound by such antibody, or a molecule that competes by binding with such an antibody, or an antibody molecule that binds or interferes with the binding of another antibody, receptor, or binding to one or more of amino acid residues 76 to 132 of the LLT1 human.

In some examples, the subject has a disease characterized by unwanted cell proliferation malignant or non-malignant, e.g. Cancer. Examples of cancer include, but they 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., lymphocytic leukemia chronic B, e.g., chronic B lymphocytic leukemia that does not have immunoglobulin mutations), lymphomas, liver cancer, lung cancer (e.g. small cell and non-cell small), melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin cancer, testicular cancer, thyroid cancer, and cancer renal, as well as other carcinomas and sarcomas. In an example, the Cancer is a tumor that does not express CD69, AICL, and / or LLT1. In other example, cancer is a chronic B lymphocytic leukemia without mutation in immunoglobulins and the subject is given a antagonist of an early activation molecule, e.g., a AICL antagonist.

In a preferred example, the method includes the administration of an antagonist of an activation molecule early in combination with a secondary agent, e.g. with one or more therapeutic agents, e.g., a therapeutic agent or agent for the treatment of unwanted cell proliferation. The second agent can be an antibody or not. Therapeutic agents include, for example, one or more chemotherapeutic agents, radioisotopes, or cytotoxins. Examples of chemotherapeutic agents include taxol, cytochalasin B, gramicidin D, mitomycin, ethoxide, tenopoxide, vincristine, vinblastine, colchicine, busulfan, cisplatin, doxorubicin, claunorubicin, dihydroxy anthracin-dione, the mitoxantrone, mitamycin, chlorambucil, gencitabine, actinomycin, procaine, tetracaine, lidocaine, propanolol, puromycin, maitansinoids and analogues or counterparts of these. Additional therapeutic agents include, but are not limited to antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbacin), agents alkylating agents (e.g., mechlorethamine, thioepa-chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine-platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (before daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maitansinoids). Radioisotopes can include alpha, beta and / or gamma emitters. Examples of radioisotopes they are: 212 Bi, 213 Bi, 131 I, 211 At, 186 Re, 90 Y and 117 Lu. The antagonist of the activation molecule Early can be administered before, during or after treatment with the other therapeutic agent.

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

In another example, the subject has a disorder non-pathological hyperproliferative, e.g., fibrosis. The mess Fibrotic may be associated with one or more of: trauma, surgery, infection, environmental pollution, alcohol, tobacco and other toxins. He disorder may be associated with, e.g., one or more of: repair of hypertrophic wounds (e.g., hypertrophic scars and keloids), renal fibrosis (mesangial cell proliferation), fibrosis of liver (liver stellar cell proliferation). Examples of Fibrotic disorders include: keloids, burns, scars hypertrophic or other skin disorders (e.g., scleroderma local or systemic, e.g., systemic sclerosis with scleroderma), liver cirrhosis, renal fibrosis (e.g., related to diabetes or hypertension), surgical adhesions (e.g., after surgery gastrointestinal or neurosurgery adhesions), transplants vascular, idiopathic pulmonary fibrosis, induced fibrosis radiation, asbestos-related fibrosis (e.g., brown lung or black), fibrosis associated with viral hepatitis, degeneration macular, vitreal and retinal retinopathy, and fibrosis associated with acute respiratory syndrome Fibrosis can be acute or chronic. Examples of acute fibrosis include: accidental wounds, infections, surgery, burns, radiation-induced fibrosis and Chemotherapy-induced fibrosis. Chronic fibrosis can be associate with e.g. viral infections, diabetes and hypertension.

The subject may be a mammal, e.g. a primate, preferably a superior primate, e.g. a human.

In some examples, the antagonist of the early activation molecule can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or by intracavitary administration), topically, or by application to mucous membranes, such as the nose, the throat or bronchi

In a preferred example, the antagonist of the Early activation molecule is administered repeatedly. For example, the CD69 antagonist can be administered 2, 4, 6 or more times. Administration may be repeated until improvement in the condition of the subject is seen or expected. The administration can  be at a frequency of one every 5 to 7 days, 14 to 30 or 30 to 60 days. The administration of other compounds may be more frequent. For example, a small molecule can be administered One, two, three, four or more times a day.

In another aspect, the invention characterizes methods of potentiating the immune response to an antigen, e.g. a vaccine. The method includes administration to a subject of a antigen e.g., a vaccine, and / or a DNA encoding a antigen, and an early activation molecule antagonist, so as to enhance the immune response to the antigen by part of the subject.

Examples of antagonists of the molecule Early activation include those described here.

In some examples, the antagonist of the early activation molecule is an antibody molecule early activation anti-molecule, e.g., a anti-CD69 antibody molecule, a molecule of anti-AICL antibody, an antibody molecule anti-LLT1, (e.g., an antibody anti-CD69 antagonist, an antibody anti-AICL antagonist, an antibody anti-LLT1 antagonist described here).

Examples of antigen doses that can be used include 100 mg of antigen for primary immunization and, at less 21 days later, 100 mg of antigen in an immunization high school. Expected titles should be greater at least for Th1 isotypes, such as IgG2a, IgG2b, and IgG3 from mouse, or similar human isotypes.

Examples of vaccines may include, vaccines of cancer (e.g., vaccines against melanoma, sarcoma, cancer lung, prostate cancer, colorectal cancer, pancreatic cancer, or liver cancer), vaccines against HIV, vaccines against hepatitis (e.g., type A, B, or C), vaccines against malaria, herpes vaccines, papilloma vaccines (e.g., HPV-6, 11, 16, 18, 31, 33 and / or 45), vaccines against the flu and vaccines against smallpox.

The antagonist of the activation molecule The subject can be administered early, before, during or after administration of the antigen and / or nucleic acid encoding for the antigen

In another aspect, the invention characterizes methods of treatment or prevention of a cancer that expresses a early activation molecule in a subject, e.g., a cancer that express CD69, AICL, and / or LLT1 in a subject. The method includes the administration of a cell eliminator that expresses the early activation molecule and / or a molecule antagonist of early activation to the subject.

Examples of cancers expressing early activation molecules include: lymphomas (eg, T-cell lymphomas (eg, cutaneous T-cell lymphoma (Berti et al ., 1991. J. Invest. Dermatol. 96: 718-23); lymphomas of peripheral T cells (Dorfman et al . 2002, Hum. Pathol. 33: 330-4)), B-cell lymphomas, non-Hodgkin lymphomas (eg, Hodgkin T or B lymphomas (Erlanson et al ., 1998. Eur. J Haematol. 60: 125-32)), lymphatic leukemia (eg, chronic T or B lymphatic leukemia (Damle et al . 2002, Blood 99: 4087-93), eg, chronic B lymphatic leukemia without 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).

Examples of cell scavengers that express early activation molecules include those described here, but they are not limited to them, such as antibody molecules that bind to an early activation molecule and eliminate cells that express the early activation molecule. Examples of a eliminator of a cell that expresses an activation molecule Early include, but are not limited to, antibody molecules that bind to CD69 and remove cells that express CD69, e.g., a human anti-CD69 antibody analogous to antibody mouse anti-CD69 2.3 or any antibody human anti-CD69 e.g., an antibody known in the bibliography that can act as a remover of the cells that express CD69 (or an antibody molecule based on it, e.g., a antibody fragment, or a chimeric, humanized or disinmunogenic), an antibody molecule that binds to the epitope bound by such antibody, a competing antibody molecule by binding with such antibody, antibody molecules that are bind to AICL and eliminate cells that express AICL, e.g., any human anti-AICL antibody, e.g., an antibody known in the literature that can act as a eliminator of  cells that express AICL (or an antibody molecule based on he, e.g., an antibody fragment, or a chimeric antibody, humanized or disinmunogenic), an antibody molecule that is binds to the epitope bound by such antibody, an antibody molecule which competes for binding with such antibody, molecules of antibody that binds LLT1 and eliminates cells that express LLT1, e.g., any human anti-LLT1 antibody, e.g., an antibody known in the literature that can act as Eliminator of cells expressing LLT1 (or a molecule of antibody based on it, e.g., an antibody fragment, or a chimeric, humanized or disinmunogenic antibody), a molecule of antibody that binds to the epitope bound by such antibody, a antibody molecule that competes for binding with such antibody, or an antibody molecule identified by a described method here. In one example, a molecule of eliminating antibody early activation anti-polypeptide can recruit an immune cell (e.g., via an Fc receptor or by recruitment of the complement system) and inactivate or destroy the target cell, e.g., the cell that expresses the polypeptide of early activation In another example, the antibody molecule early activation anti-polypeptide eliminator can activate the classic complement pathway, leading to cell lysis target (e.g., the cell expressing CD69, AICL, or LLT1). In other For example, the eliminator is an anti-antibody molecule early activation polypeptide coupled to a therapeutic agent, e.g., a protein, drug or isotope, which can inactivate or destroy the target cell, e.g., the cell that expresses the early activation polypeptide.

In a preferred example, the method includes the administration of a cell eliminator expressing a early activation molecule in combination with an agent secondary, e.g., with one or more therapeutic agents, e.g., a therapeutic agent or agent for the treatment of proliferation unwanted cell phone The second agent may be an antibody or not. Therapeutic agents include, for example, one or more agents. chemotherapeutic agents, radioisotopes, or cytotoxins. Examples of agents chemotherapeutic agents include taxol, cytochalasin B, the Gramicidin D, mitomycin, ethoxide, tenopoxide, vincristine, vinblastine, colchicine, busulfan, cisplatin, doxorubicin, claunorubicin, dihydroxy anthracin-dione, the mitoxantrone, mitamycin, chlorambucil, gencitabine, actinomycin, procaine, tetracaine, lidocaine, propanolol, puromycin, maitansinoids and analogues or counterparts of these. Additional therapeutic agents include, but are not limited to antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbacin), agents alkylating agents (e.g., mechlorethamine, thioepa-chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine-platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (before daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maitansinoids). Radioisotopes can include alpha, beta and / or gamma emitters. Examples of radioisotopes they are: 212 Bi, 213 Bi, 131 I, 211 At, 186 Re, 90 Y and 117 Lu. The activator molecule eliminator Early can be administered before, during or after treatment with the other therapeutic agent.

The subject may be a mammal, e.g. a primate, preferably a superior primate, e.g. a human (e.g., a patient who has a cancer that expresses a molecule of early activation).

In some examples, the cell eliminator that express the early activation molecule can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, by route transdermal, or by inhalation or by administration intracavitary), topically, or by application to the membranes mucous membranes, such as the nose, throat or bronchial tubes.

In a preferred example, the eliminator of the Early activation molecule is administered repeatedly. For example, the CD69 eliminator can be administered 2, 4, 6 or more times. Administration may be repeated until improvement in the condition of the subject is seen or expected. The administration can  be at a frequency of one every 5 to 7 days, 14 to 30 or 30 to 60 days. The administration of other compounds may be more frequent. For example, a small molecule can be administered One, two, three, four or more times a day.

In another aspect, the invention characterizes a method for the identification of compounds that modulate a early activation molecule, e.g., CD69 antagonists, AICL, or LLT1 or cell eliminators expressing CD69, AICL, or LLT1 in this way modulate an immune response. The method includes: contact a cell that expresses a molecule of early activation with a compound (e.g., a compound that interacts with, e.g., binds to, the activation molecule early) and determine the effect of the compound on the expression or Activity of the early activation molecule. The expression of an early activation molecule can be determined, e.g., by the determination of the expression of a nucleic acid encoding for an early activation polypeptide, by the expression of said polypeptide, and / or by expression of the polypeptide of early activation on the surface of a cell.

In a preferred example, the method includes: contact of an early activation polypeptide, e.g., CD69, AICL, or LLT1, or a soluble form of the activation polypeptide early (e.g., a soluble form of CD69, e.g., a polypeptide of CD69 lacking the transmembrane and / or cytoplasmic region, a soluble form of AICL, e.g., an AICL polypeptide that lacks the transmembrane and / or cytoplasmic region, a soluble form of LLT1, e.g., an LLT1 polypeptide lacking the region transmembrane and / or cytoplasmic) with a compound, and evaluate the ability of the compound to interact with, join, the early activation polypeptide or soluble form of early activation polypeptide.

A compound that reduces the expression of an early activation molecule and / or activity can be identified as a compound that increases the immune response. In a preferred example, the activity of the decreasing early activation molecule is the expression of TGF-? And / or the inhibition of the formation of the dimer of the early activation molecule, eg, formation of dimers of CD69, AICL or LLT1. In one example, a compound can be evaluated in its ability to decrease the expression of the early activation molecule (eg, the expression of the nucleic acid, the polypeptide, and / or the expression on the cell surface) and / or the activity cell of the early activation molecule, eg, TGF-? expression, in the absence or presence of the compound. Preferably, the compound interacts with, eg, binds to the early activation molecule. Possible compounds include, eg, small molecules (eg, small molecules described herein), peptides, fusion polypeptides (eg, CD69R fusion protein, AICL-R fusion protein, or LLT1-R fusion protein described here), antibody molecules and nucleic acid molecules. Examples of antibody molecules include, but are not limited to, antibody molecules or intrabodies that bind to an early activation molecule and interfere with the binding of the early activation molecule and an early activation molecule binding polypeptide and antibody molecules that bind to an early activation polypeptide and decrease the level of the early activation polypeptide on the cell surface, eg, by internalization. Examples of anti-CD69 antibodies include, but are not limited to, antibody molecules that bind to CD69 in interfering with the binding of CD69 to a CD69 binding partner eg, a human anti-CD69 antibody, eg, an anti antibody -CD69 human analogous to anti-CD69 2.2 antibody. or any human anti-CD69, eg, an antibody described in the literature, which can act as an antagonist (or antibody molecules based on it), an antibody molecule that binds to the epitope to which such antibodies bind, a molecule of antibody that competes with the binding of such antibodies, an antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of the amino acid residues Glu 140, Aspl71, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69, or another antibody molecule described herein. Examples of anti-AICL antibodies include a human anti-AICL antibody, eg, any anti-AICL, eg, an antibody described in the literature, which can act as an antagonist (or antibody molecules based on it), an antibody molecule that binds to the epitope to which such antibodies bind, an antibody molecule that competes with the binding of such antibodies, an antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of amino acid residues 37 to 149 of the human AICL, or other antibody molecule described herein. Examples of anti-LLT1 antibodies include a human anti-LLT1 antibody, eg, any anti-LLT1, eg, an antibody described in the literature, which can act as an antagonist (or antibody molecules based on it), an antibody molecule that binds to the epitope to which such antibodies bind, an antibody molecule that competes with the binding of such antibodies, an antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of amino acid residues 76 to 132 of human LLT1, or other antibody molecule described
here.

In some examples, the cell-based assay measures an activity of an early activation molecule, e.g., decreased TGF-? expression, activation decreased MAPK, lower Ca 2+ signaling and / or formation of dimers of CD69, AICL or reduced LLT1. The compounds that decrease the expression of TGF-? and / or the dimer formation can be selected as antagonists of the early activation molecule.

In other examples, the cell-based assay measures the modulation, e.g., a decrease, of the expression of a early activation molecule. For example, the expression of a early activation polypeptide or acid expression nucleic (e.g., mRNA or cDNA) for CD69, in the presence of a compound can be evaluated and compared to the expression of the early activation molecule in the cell before the compound administration. A decrease in the level of expression is any statistically significant decrease in the expression levels of the early activation molecule. In other cases, the cell-based assay measures modulation, e.g., the decrease in the level of activation polypeptides early on the surface of the cell, e.g., compared to early activation polypeptide levels on the surface of the cell before administration of the compound.

In one example, a compound that inhibits the expression and / or activity of an early activation molecule, e.g., the expression of TGF-?, from the cell, is selected, e.g., selected for further search, e.g., by administration of the compound to a subject, e.g., in a model animal, e.g., in an animal model of cell proliferation not desired (e.g., an animal model for cancer), an animal model for immunodeficiency, and / or an animal model to measure efficacy of a vaccine

In another aspect, the invention characterizes a method of identifying a compound that modulates the immune response. The method includes: the search for a compound that interacts with, e.g., binds to, an early activation polypeptide or a nucleic acid encoding an activation polypeptide early (e.g., the regulatory region of an activation gene early), and determine the effect of the compound on the expression and / or the activity of the early activation molecule, e.g., the TGF-? expression and / or the formation of dimers, e.g., the formation of dimers by CD69, AICL or LLT1.

In one example, the interaction between the compound and the early activation polypeptide is evaluated in vitro , eg, using an isolated early activation polypeptide, eg, a soluble form of CD69, AICL and / or LLT1 eg, a soluble form of CD69 , AICL and / or LLT1 described here. The early activation polypeptide and / or the compound may be in solution (eg, a micelle) or attached to a solid support, eg, a column, microsphere, well or plate or to a chip (eg, a microarray ). In some examples, the method is repeated one or more times like that, eg, a library of compounds to be tested can be evaluated.

In another example, the interaction between the compound and the early activation polypeptide is evaluated using a cell based assay.

In a preferred example, a compound that interacts with, e.g., binds to an activation molecule early, it joins with a cell that expresses the molecule of early activation to determine the effect of the compound on the expression and / or activity of the early activation molecule.

In another example, the method includes the identification of a compound that interacts with a molecule of early activation and decreases the expression and / or activity of the early activation molecule, e.g., decreases the expression of TGF-? MAPK activation, signaling by Ca 2+ and / or the formation of dimers, e.g., the formation of dimers of CD69, AICL or LLT1, in order to identify the compound as a candidate to increase the immune response. The expression of an early activation molecule can be determined, e.g., determining the expression of a nucleic acid that codes for the early activation polypeptide, by expression thereof, and / or by expression of the early activation polypeptide in the cell surface In a preferred example, the activity of the early activation molecule that is reduced is the expression of TGF-? and / or dimer formation. In a For example, a compound can be evaluated in its ability to decrease the expression and / or cellular activity of the molecule of early activation, e.g. expression of TGF-?, In the absence or 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 here), peptides, fusion polypeptides (e.g., the fusion protein of CD69R, the AICL-R fusion protein, or the LLT1-R fusion protein described here), antibody molecules and nucleic acid molecules. Examples of antibody molecules include, but are not limited to, molecules of antibody or intrabodies that bind to a polypeptide of early activation and interfere with the binding of the polypeptide of early activation and a polypeptide binding polypeptide of early activation and antibody molecules that bind to early activation polypeptide and reduce its level of expression on the surface of the cell. Examples of antibody molecules anti-CD69 include, but are not limited to, molecules of antibody that bind CD69 and interfere with the binding of CD69 to a CD69 binding partner, e.g., an antibody human anti-CD69, e.g., an antibody human anti-CD69 antibody analog anti-CD69 2.2. or any human anti-CD69, e.g. an antibody human anti-AICL described in the literature that can act as an antagonist (or an antibody based molecule therein, e.g., a chimeric, immunized, humanized antibody or a antibody fragment), an antibody molecule that binds to the epitope to which such antibodies bind, a molecule of antibody that competes with the binding of such antibodies, a antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of the residues amino acids Glu 140, Asp171, Glu 180, Glu 185, Glu 187, Phe 175, Met 184, Leu 190, Glu 185 and Lys188 of human CD69, or another molecule of antibody described here. Examples of antibody molecules anti-AICL include, e.g., an antibody anti-human AICL, e.g. an antibody human anti-AICL described in the literature that can act as an antagonist (or an antibody based molecule therein, e.g., a chimeric, deimmunized, humanized antibody or a antibody fragment), an antibody molecule that binds to the epitope to which such antibodies bind, a molecule of antibody that competes with the binding of such antibodies, a antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of the residues amino acids 37 to 149 of the human AICL, or other antibody molecule described here. Examples of antibody molecules anti-LLT1 include, e.g., an antibody human anti-LLT1, e.g. an antibody human anti-LLT1 described in the literature that can act as an antagonist (or an antibody based molecule therein, e.g., a chimeric, deimmunized, humanized antibody or a antibody fragment), an antibody molecule that binds to the epitope to which such antibodies bind, a molecule of antibody that competes with the binding of such antibodies, a antibody molecule that binds or interferes with the binding of another antibody, receptor or ligand to one or more of one of the residues amino acids 76 to 132 of the human AICL, or other antibody molecule described here.

In some examples, the cell-based assay measures an activity of the early activation molecule, e.g., decreased TGF-? expression, activation decreased MAPK, lower Ca 2+ signaling and / or formation of reduced dimers, e.g., dimers of CD69, AICL or LLT1. The compounds that decrease the expression of TGF-? And / or dimer formation can be selected as antagonists of the activation molecule early

In other examples, the cell-based assay measures the modulation, e.g., a decrease, of the expression of the early activation molecule. For example, the expression of a early activation polypeptide or acid expression nucleic (e.g., mRNA or cDNA) for the activation molecule early, in the presence of a compound can be evaluated and compared to the expression of the early activation molecule in the cell before administration of the compound. A decrease at the level of expression is any decrease statistically significant in the expression levels of the molecule of early activation In other examples, the cell-based assay measures modulation, e.g., reduced polypeptide levels of early activation on the cell surface, e.g., compared with the cell surface levels before the compound administration.

In one example, a compound that inhibits the expression of an early activation molecule and / or activity cell of an early activation molecule, e.g., expression of TGF-?, is selected, e.g., selected for further search, e.g., by administration of compound a a subject, e.g., in an animal model, e.g., in an animal model of unwanted cell proliferation (e.g., an animal model for cancer), an animal model for immunodeficiency, and / or a model animal to measure the effectiveness of a vaccine.

In another aspect, the invention characterizes a method of identifying a compound that modulates the response immune. This method includes: providing a compound that modulates the expression of the early activation molecule and / or its activity, and administer the compound to a subject, e.g., an animal model.

In one example, the compound increases the expression of the early activation molecule and / or increases an activity of the early activation molecule, e.g., the expression of TGF-?, And / or increases the formation of dimers (e.g., the formation of dimers by CD69, AICL or LLT1), and the compound is administered to a subject, e.g., an animal model, e.g., an animal model for an inflammatory disease (e.g., acute or chronic 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 for collagen-induced arthritis.

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

The compound can be an identified compound by a method described here or they may be other compounds that are knows that they interact with the early activation molecule.

In another aspect, the invention characterizes, a method of making a decision, e.g. a medical decision or financial The method includes: generating or receiving data or information in the treatment of a subject for a disorder described here; and use the data or information to take the decision, e.g., select between a first result and a second Outcome.

In a preferred example, the data comes from the response to the administration of an agonist, antagonist, or early activation molecule eliminator (e.g. a antagonist, or eliminator of CD69, AICL, or LLT1).

In a preferred example, the decision is made comparing the data with a reference standard and taking the decision based on the relationship of the data with the reference. By For example, the data can be a value or other term for the response of a subject, and if the value or other term has a pre-selected relationship with the reference standard, e.g., if the value or term of the data is greater than the standard of reference, a first result is selected, and if the data is less than a reference standard, a second is selected Outcome. A result can be produced by giving or not a service or  treatment, or paying or not for all or part of the service or treatment.

In a preferred example, the first result suggests or gives a first course of medical treatment, e.g. any of the treatments described here, e.g., a additional administration of an antagonist or eliminator of the early activation molecule, and the second course suggests that the Treatment should not be given.

In a preferred example, the first result includes or results in the authorization or transfer of funds to the payment of a service or treatment given to the subject and the second result includes or results in rejection of payment for a service or treatment given to the subject. For example, an entity, e.g., a hospital, government entity, or insurance company or other entity that pays for or reimburses medical costs, you can use the result of a method described here to determine if a group, e.g., a different group to the patient subject, will pay for the services or treatment given to the patient. For example, a first entity, e.g., an insurance company, can use the result to determine if it provides financing to, or in representation of a patient, e.g., if he will reimburse a third party group, e.g., a seller of products or services, a hospital, doctor, or other health professional, for a service or treatment given to a patient. For example, a first entity, e.g., an insurance company, can use the result of a method described here to determine if it continues, stops, or enrolls to an individual in an insurance plan or program, e.g., a program or health insurance or life insurance plan.

In another aspect, the invention characterizes a method of providing a database, e.g., a database useful for setting a reference value referred to here or for other one or more subjects under evaluation. The method includes: generating or receive data, e.g., response to the administration of a antagonist or eliminator of the early activation molecule (e.g., an antagonist or eliminator of CD69, AICL, or LLT1) in the treatment of a disorder, e.g., a disorder described herein; and Enter the data in the database.

In a preferred example, one or more of, a indicator (e.g., a value) for the disease status of a patient and a patient identifier are entered in the database.

In a preferred example, the database includes a plurality of entries, each of which includes one or more of: data (data on management response of antagonists or eliminators of the activation molecule early in the treatment of a disorder, e.g. a disorder described here); an indicator for the disease status of a patient and a patient identifier.

In another aspect, the invention characterizes a method for the evaluation of a patient that includes: comparison of patient data (data on the response of the administration of an antagonist and eliminator of the molecule of early activation in the treatment of a disorder, e.g. a disorder described here), with data from a database described here.

In another aspect, the invention characterizes a method to build a reference standard that includes: inclusion of data from a database described here In the standard. For example, you can take values from the base of data, perform mathematical operations with them and use the result as a reference to reach a standard of reference, e.g., taking the mean of a plurality of values selected from the database and use the average as the standard.

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

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

In one example, the antigen is homologous to animal, e.g., the antigen is of the same species as the species of the animal to which it is administered, e.g. the antigen is an antigen murine administered to mouse. In another example, the antigen is heterologous, e.g., the antigen is of a different species than the species of the animal to which it is administered, e.g., the antigen is a Human antigen administered to a mouse.

In another example, the method also includes the isolation of an animal's immune cell, e.g., to produce monoclonal antibodies.

The invention is based, in part, on the discovery that the modulation of any of the molecules Early activation, CD69, AICL, and LLT1 plays a role in the immune reactivity This effect can be mediated through the regulation of the synthesis of TGF-? 1. CD69, AICL, and LLT1 are structurally similar receptors located in the NK gene complex and that can be expressed in most of cells of hematopoietic origin. The three proteins have at minus a lectin domain type C with sequence similarity significant. The levels of CD69, AICL, and LLT1 increase significantly at an early stage after cell activation, unlike other lectin-like proteins associated with the NK gene complex.

We have focused on two different models of disease in mice to study the role of CD69 in the regulation of the immune response. First, collagen-induced arthritis (AIC) is an experimental model of inflammatory joint disease, especially rheumatoid arthritis (RA) (Feldmann et al ., 1996). The AIC allows the study of autoimmunity processes and can be extended to other diseases in which the autoimmune response is exacerbated. It has been found that CD69 plays an inhibitory role through the regulation of TGF-? In the AIC model. Second, tumor models constitute the opposite strategy, where the goal is to enhance the immune response to suppress tumors and metastases. Therefore, this model constitutes an experimental system to test possible therapies that can be extended to infectious diseases and immunodeficiency syndromes to restore an effective immune response. The possible use of CD69 in this context has also been analyzed in mouse-induced tumor and metastasis models. It has been found that specific monoclonal antibodies against CD69 give an effective therapy in mice with immunocompetent (wt) and immunocompromised negative MHC-I (SCID & RAG1 - / -) tumors. As with CD69, AICL and LLT1 are expressed at low levels in leukocytes and are strongly and transiently induced after activation. In addition, it has been reported that AICL increases in certain cancers, such as B-CLL in lines with non-mutated immunoglobulins (Klein et al ., 2001 J. Exp. Med. 11: 1625; Rosenwald et al ., 2001 J Exp. Med. 11: 1639). This may result in increased expression of TGF-?, Thus producing an immunosuppressive microenvironment in subjects who have this disorder.

The elucidation of CD69 paper as a negative modulator of immune reactivity, gives a new e interesting approach to cancer therapy, inflammation chronic, and other diseases mediated by the immune system (immunodeficiencies, autoimmune diseases, transplants allogeneic, and others like these) by modulation of CD69, AICL, and / or LLT1. In addition, and since CD69, AICL, and LLT1 are expressed selectively in activated leukocytes that infiltrate tissues inflamed or tumors, the pharmacological stimulation of the synthesis of TGF-? 1 through CD69, AICL, and / or LLT1 it can be located in the target tissues, thus avoiding negative consequences of the systemic increase of TGF-? 1. On the other hand, CD69, AICL, and / or LLT1 are suitable markers for the identification of leukocytes activated tissues. The elimination of these subpopulations by specific reagents of these activation molecules Early can inhibit the immune and inflammatory response. By therefore, the antagonism of an early activation molecule e.g., inhibiting the secretion of TGF-?, can allow an enhanced local immune response thanks to increase in cell recruitment and survival immune (which would be adequate to increase the response to  vaccines, immunodeficiency therapy, etc.).

The description of immunoregulatory molecules that enable a local and selective activation or inhibition of the TGF-? synthesis is therefore a new approach to the treatment of diseases mediated by immune system. In addition, early activation molecules can be used as a specific marker of the leukocyte population that is actively producing cytokines pro-inflammatory sites of inflammation. The elimination of this population through the manipulation of CD69, AICL, and / or LLT1, would allow the control of the effectors that are they derive or are controlled by them. These new possible approaches through inducible leukocyte receptors CD69, AICL, and LLT1 are described here.

Description of the drawings

To complement the description that is being performing and in order to help a better understanding of the characteristics of the invention, is accompanied herein descriptive, as an integral part of it, a set of planes in which with an illustrative and non-limiting nature, it has been represented the following:

Figure one

Exacerbated development of AIC in mice deficient for CD69

A, Comparison of control mice treated with Freund's complete adjuvant (CFA) with images representative of the most severe cases of AIC in CD69 + / + and CD69 - / - mice (center panels and to the right, respectively ). B, incidence of arthritis (expressed as a percentage of diseased mice), and severity of clinical symptoms in CD69 + / + (med), CD69 +/- (({115}) mice, and CD69 ^ - / -} (\ blacksquare), evaluated as described in the Methods section. The results represent the arithmetic mean ± standard deviation (of) of three independent experiments (18 mice / group / experiment). * P <0.01 vs. CD69 + / + (Student's t- test).

Figure 2

A. representative sections of the histopathology of the complete legs of control mice (CFA, left panel, 100 x), and CD69 + / + mice (central panel, 100 x) and CD69 - / -} (right panel, 60 x) immunized with IIC. The arrowheads indicate erosions in cartilage and bone, and the arrows complete leukocyte infiltrates and pannus. B. evaluation of inflammation, cartilage damage, joint pannus formation and bone erosion in the front legs of CD69 + / + (\ boxempty) and CD69 - / - (\ blacksquare) mice, as described described in the Methods section. The results correspond to the arithmetic mean ± of the percentage of joints circumscribed to each severity group in three independent experiments (8 mice / group / experiment). * P <0.01 vs. CD69 + / + (Mann-Whitney U test).

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Figure 3

Increased specific immune response against IIC in mice CD69 - / -

A, comparison of the larger size of the spleens of the CD69 - / - mice with respect to the CD69 + / + mice. Horizontal bar = 1 cm. B, weight of the spleens and number of cells (arithmetic mean ± of) of control mice (treated with CFA) and CD69 + / + (\ boxempty) and CD69 - / - (immunized) (\ blacksquare) with IIC (AIC). * P <0.01, compared to immunized control mice (Student's t test). C, proliferation of spleen (Sp) and lymphoid (LN) nodule cells of CD69 + / + (\ boxempty) and CD69 - / - (\ blacksquare) mice immunized with IIC, stimulated with different concentrations of IIC inactivated (x axis). The data reflect the arithmetic mean ± of incorporation of 3 H-TdR in three independent experiments. *, P <0.01 vs. CD69 + / + (Mann-Whitney U test). D, increase in specific Thl response-dependent isotypes against IIC in CD69 - / - mice. Levels of IgG1 and IgG2b antibodies (1/20000 dilution of serum), IgG2c and IgG3 (1/5000 dilution), IgM and IgA (1/200 dilution) in sera from control mice (CFA) or CD69 + / +} (\ boxempty) and CD69 <- / -} (\ blacksquare) immunized with IIC at the time of sacrifice (day 50). The data correspond to the arithmetic mean of the absorbance units at 495 nm in three independent experiments (10 mice / group / experiment). * P <0.01 vs. CD69 + / + (Student's t- test).

Figure 4

Local mRNA analysis for cytokines in CD69 - / - mice immunized with IIC

Analysis of the mRNA of the hind legs of CD69 + / + (\ boxempty) and CD69 - / - (\ blacksquare) mice. Each lane corresponds to the mRNA homogenate of 6 mice per group, and each bar represents similar experiments (6 mice / group / experiment).
ment). The results are expressed in arbitrary densitometry units based on the expression of L-32 in each sample (arithmetic mean ± of four independent experiments). * P <0.01 vs. CD69 + / + (Mann-Whitney U test).

Figure 5

Local analysis of cytokines in limbs and leukocytes synoviums of CD69 - / - mice immunized with IIC

A, Quantitative real-time RT-PCR analysis of the mRNA of the hind legs of CD69 + / + (\ boxempty) and CD69 - / - (\ blacksquare) mice. Each bar represents the arithmetic mean of 12 mice per group in two independent experiments. The results for each cytokine are normalized with respect to the expression of GAPDH in each sample. * P <0.01 vs. control antibody (Mann-Whitney U test). B, Levels of total and active TGF-? 1, IL-1?, TNF-? And RANTES in synovial washes of CD69 + / + mice (\ boxempty) and CD69 - / -} (\ blacksquare) in which AIC has been induced. The data correspond to the arithmetic mean ± of three independent experiments (6 mice / group / experiment). * P <0.01 vs. CD69 + / + (Mann-Whitney U test). C, quantitative real-time RT-PCR analysis of mRNAs from purified subgroups of synovial cells of CD69 + / + (\ boxempty) and CD69 - / - (\ blacksquare) mice. Each bar represents the arithmetic mean of 12 mice per group in two independent experiments. The results for each cytokine are normalized with respect to the expression of GAPDH in each sample. * P <0.01 vs. control antibody (Mann-Whitney U test).

Figure 6

Effect of a blocking antibody 25 of TGF-? In control and deficient mice for CD69

A, left panel: CD69 + / + mice were treated with the anti-TGF-? Blocking antibody (\ blacksquare), an isotype control IgG1 (\ ding {115}) or carrier (\ ding {116 }) from the 30th day 21 (second immunization with IIC) until the moment of measurement. The swelling of the paw was measured with a precision gauge and is expressed as the mm of inflammation with respect to day 21. The results are represented as the arithmetic mean ± of 12 mice per group in two independent experiments. Right panel: quantitative real-time RT-PCR analysis of the mRNA of the legs of the mice treated with an isotype control (\ boxempty) or with anti-TGF-? (\ Blacksquare). Each bar represents the arithmetic mean of 12 mice per group in two independent experiments. The results for each cytokine are normalized with respect to the expression of GAPDH in each sample. * P <0.01 vs. control antibody (Mann-Whitney U test). B, CD69 - / - mice were treated as indicated in A and the swelling of the paw (left panel) and mRNA (right panel) was determined as previously described.

Figure 7

CD69 activation induces secretion of TGF-? In splenocytes and synovial leukocytes mouse with AIC

A, the expression of CD69 (thick line, left panel) in splenocytes stimulated with concanavalin-A was studied. An isotype control (thin line, left panel) is also represented. The splenocytes were purified in different subgroups and treated with an antibody against mouse CD69 (black) or an isotype control (box), subsequently incubated with a crosslinker. Total and active levels of TGF-? (Panels on the right) were determined. B, the expression of CD11b and CD69 (central panel), or isotype controls (left panel) in mouse synovial cells with AIC were determined, which were subsequently treated as in A and the total and active levels of
TGF-? (Panels on the right). The results in A and B are expressed as the arithmetic mean + SD of four independent experiments. *, p <0.01 versus control antibody (Mann Whitney U test).

Figure 8

CD69 crosslinking induces production of TGF-? In human synovial leukocytes and in stable human T cell transfectants

A, the expression of CD69 (thick line, left panel) in human synovial leukocytes of patients with rheumatoid arthritis was studied. An isotype control (thin line, left panel) is also represented. These cells were treated with an antibody against CD69 (TP 1/8) (?) Or an isotype control (?) In the presence (XL) or absence (-) of a cross-linking secondary antibody, and levels were determined of total and active TGF-? (right panel). * P <0.01 vs. control antibody (Mann-Whitney U test). These data are representative of similar results obtained with synovial leukocytes from patients with reactive arthritis or ankylosing spondylitis. B. Jurkat human T leukemia line (thin line, left panel), and stable CD69 transfectants in this line (thick line, left panel) were stained for CD69. The cross-linking of CD69 mediates the production of TGF-? 1, but not TNF (right panel). The Jurkat T lymphoblast line (JK) (?) And the stable transfectant expressing CD69 (JK-CD69) (?) Were treated for 24 hours with cross-linked anti-CD69 antibody. The results in A and B are expressed as the arithmetic mean ± SD of four independent experiments. *, p <0.01 with respect to the parental cell line JK (Mann-Whitney U test).

Figure 9

Increased antitumor activity in CD69 - / - mice

10 4 tumor cells were injected RMA-S (A) or RMA (C) intraperitoneally. It was estimated tumor growth by daily weight monitoring body and ascites fluid development. They were observed similar results when injected subcutaneously (B) 10 5 RMA-S cells (A, n = 9; B, n = 6; C, n = 9). (D) NK cells and T lymphocytes were removed from mice CD69 - / - before intraperitoneal inoculation of 10 4 RMA-S cells. The elimination treatment of NK cells was carried out by injection of control diluent or 100 \ mul / injection of an anti-Asialo GM1 antiserum to days -1 (before the injection of tumor cells) and days +2 and +4 after inoculation of tumor cells (n = 7). He T lymphocyte removal treatment was carried out by injection of an isotype control or 100 µl / injection of a anti-CD4 antibody (GK1.5) at days - 1 (before injection of tumor cells) and at +2 and +4 days after inoculation of tumor cells (n = 7). Results are displayed representative of two independent experiments. The symbols gaps correspond to CD69 + / + control mice and the symbols stuffed to CD69 - / - mice. (E) Photographs of the lungs of control mice and CD69 - / - two weeks after administration of 10 4 RM-1 cells intravenously (part higher). A representative mouse of 10 per group is shown. Be determined the number of lung metastases and each symbol It represents an individual mouse. The hollow symbols correspond to CD69 + / + control mice and mice filled symbols CD69 - / -. Results corresponding to two are shown independent experiments

Figure 10

Exacerbated antitumor response in mice RAG-deficient

10 6 were inoculated intraperitoneally RMA-S cells. After 72h, the cells were examined unfractionated peritoneal of CD69 + / + mice and CD69 - / - - RAG2 - / - (A, B). (A) Profiles of flow cytometry (size vs. complexity analysis). It shows a representative mouse of 10 / group. (B) Comparison profiles obtained by means of a Coulter Multisizer cell counter. The Results shown correspond to two independent experiments (n = 8).

Figure eleven

Increase in cytotoxicity and lymphocyte recruitment in CD69 - / - mice

10 4 were inoculated intraperitoneally RMA-S (A) or RM-1 (B) cells. After 72 h, unfractionated peritoneal cells were examined for CD69 + / + and CD69 - / - mice (A, B) and the cytotoxicity with respect to YAC target cells. Two were used animals per experimental group (arithmetic mean ± d.e). The results represent three experiments performed so identical The hollow symbols correspond to purified NK cells of CD69 + / + control mice and filled symbols to mice CD69 - / -. (C, D, E) Splenic and peritoneal accumulation of lymphocytes in CD69 - / - mice in response to cells Tumor 10 5 RM-1 cells or inoculated PBS only (n = 7 / group). Peritoneal washes were collected after 3 or 6 days, and the total leukocyte numbers were determined (x10-6). (C) The cells collected from the washed in a Coulter Multisizer cell counter, and it they compared the profiles to the times indicated after the tumor inoculation Comparison profiles obtained by means of a Coulter Multisizer cell counter. The results shown correspond to three independent experiments in which they used 2/2, 2/2 and 4/4, control mice (dashed line) / CD69 - / - mice (solid line). (E) Comparison of the largest Spleen size of CD69 - / - mice and control (8 weeks of age) three days after intraperitoneal inoculation of 10 5 RM-1 cells. Spleens are shown representative of various independent experiments.

Figure 12

Attenuation of spontaneous cell death in lymphocytes of CD69 - / - mice

Peritoneal cells of mice without treatment (A) was put in culture in medium and the cell survival by propidium iodide (IP) staining. The data represent the percentage of IP + cells (mean arithmetic ± d.e.). (B, C) Activity analysis intracellular caspase-3 of spleen cells (B) and NK purified (C) from CD69 + / + mice (left panel) and CD69 ^ - / -} (right panel) after three days post-inoculation of 10 5 cells RM-1 Caspase-3 activation is determined by using the fluorogenic substrate PhiPhiLux-G1D2. The staining of PhiPhiLux in FL-1 vs. cell size The data correspond to an experiment (n = 6).

Figure 13

Cytokine expression in CD69 - / - mice

(A) Relative levels of cytokine and chemokine mRNA in peritoneal cells after three days of intraperitoneal post-inoculation of 10 5 RM-1 cells. The results are expressed in arbitrary units of densitometry standardized with respect to the expression of GAPDH or L32 in each sample. Five animals were used per experimental group, and the results correspond to four independent experiments. (B) MCP-1 chemokine levels in peritoneal cells from control and CD69 - / - mice treated with thioglycolate, and stimulated with LPS. The data correspond to the arithmetic mean ± of (n = 4 / group) of a representative experiment of three performed. * P <0.004.

Figure 14

Increase of the antitumor response in normal mice by TGF-? blockade

Survival profile of control mice and CD69 - / - to which 10 4 cells have been injected RMA-S (day 0) and have been treated with the antibody anti-TGF-? 1D11 (500 \ mug / inoculation) or PBS at -3, -1 days (before inoculation of tumor cells), and +1, maintaining the treatment once one week after tumor inoculation (n = 4). The results They correspond to two independent experiments.

Figure fifteen

Activation of CD69 induces TGF-? 1

CD3 + T cells were seeded without stimulation on anti-CD3 immobilized 96-well plates. (A) Expression of CD69 after activation with anti-CD3 for 24h, determined by cytometry of flow by an antibody against mouse CD69 conjugated to FITC (thick line). The dotted line corresponds to the control of isotype (B) Mouse purified T lymphocytes were treated resuspended in serum-free medium for 72h with the antibody anti-CD69 2.2 or an isotype control (IgG1) and a goat serum against mouse IgG (crosslinker). Where I know indicates, the cells were treated with the PD98059 inhibitor. Subsequently, the secretion of TGF-? By ELISA. The data represents the arithmetic mean ± d.e. of wells in duplicate and three independent experiments (C) Cellular mortality was determined  by staining with trypan blue after 48 h of culture. The data represent the arithmetic mean ± d.e. of wells by triplicate. (D) Activation of Erk-1 and Erk-2 by CD69. T lymphocytes were treated at 4 ° C mouse preactivated with anti-CD3 with an antibody anti-CD69 or an isotype control and were lysed after 5, 10, 15, 25 and 60 min incubation at 37 ° C with an antibody crosslinker The lysates were subjected to PAGE / SDS, were transferred to nitrocellulose paper and experiments were performed Western blot with a polyclonal antibody that recognizes Erk-1 and Erk-2 phosphorylated (panel higher). Lower panel, total Erk levels determined by Western blot with a monoclonal antibody against Erk. The position relative of Erk-1 and Erk-2 is denoted by arrows The relative levels of phosphorylation of Erk-1 and Erk-2 with regarding total Erk levels in arbitrary units of densitometry CD69 + / + mice (\ boxempty) and CD69 ^ - / -} (\ blacksquare).

Figure 16

Antitumor therapeutic activity of antibodies against CD69 in normal and RAG deficient mice

Survival profiles of control mice and SCID treated with antibodies against CD69 or isotype controls. 10 4 cells were intraperitoneally inoculated RMA-S in control mice (A, B) or 10 6 cells in SCID mice. (C) Mice were treated as indicated. previously with anti-CD69 or isotype control (A, B, C) and tumor growth was observed daily (up to 20 post-inoculation weeks) by body weight monitoring and fluid development ascites (A, n = 9; B, n = 6; C, n = 9). The results correspond to two independent experiments

Figure 17

Antibodies against CD69 affect the early stage of tumor growth in the peritoneum

The peritoneal cells of control mice (A) or RAG1 - / - three days after Intraperitoneal injection of 10 6 RMA-S cells. Mice were treated one day before inoculation of the 500 µg / mouse tumor cells of an antibody against CD69 (solid line) or an isotype control (dashed line). The cells collected from peritoneal lavage were analyzed by a Coulter Multisizer cell counter and profiles were compared obtained (n = 8 / group). The data correspond to two experiments independent.

Figure 18

Antibody response to DNP-KLH in WT mice and deficient for CD69

It was immunized with 100 µg of DNP-KLH per mouse at days 0 (primary immunization) and day 21 (secondary immunization) sc at the base of the tail or ip, using either Alum or CFA as adjuvants. The mice were bled at day 7 ( a ), and at day 28 ( b ). ( a ) IgM levels in primary immunization (serum dilution 1: 2000). ( b ) levels of IgM (1: 2000), IgG1 (1: 20,000), IgG2c (1: 10,000), IgG2b (1: 10,000), and IgG3 (1: 10,000) in secondary immunization. The results are expressed as the arithmetic mean ± of 4 mice / group / experiment analyzed in two independent experiments. *, p <0.01 with respect to the same treatment in WT mice (Mann-Whitney U test).

Figure 19

Anti-CD69 2.2 decreases the expression of CD69 in thymocytes

Thymocytes of DBA / 1 mice treated (right panel) or not with anti-CD69 2.2 antibody in vivo were isolated and stained for cytometry with the FITC-labeled mouse anti-CD69 antibody H1.2F3 and an APC-labeled CD3 antibody . A representative experiment of five performed is shown.

Figure twenty

Effect of anti-CD69 2.2 treatment on AIC

DBA / 1 mice were treated on days 20 and 28 with the anti-CD69 blocking antibody 2.2 (\ ding {115}) or with an isotype control antibody (\ blacksquare). Leg swelling was measured with a caliber of precision and is expressed as mm of inflammation with respect to the day 21. The results are expressed as the arithmetic mean ± SD of  12 mice / group in two independent experiments. *, p <0.01 vs control antibody (Mann Whitney U test).

Figure twenty-one

The anti-CD69 2.3 eliminates the thymocytes that express CD69

Thymocytes of treated DBA / 1 mice (right panel) or not (left panel) with anti-CD69  2.3 were isolated and stained for flow cytometry with the anti-CD69 mouse antibody H1.2F3 labeled FITC and an antibody against CD3 labeled with APC. It shows a Representative experiment of five performed.

Figure 22

Effect of treatment with anti-CD69 2.3 on AIC

DBA / 1 mice were treated on days 20 and 28 with the anti-CD69 eliminating antibody 2.3 (\ ding {115}) or with the isotype control IgG2a (\ blacksquare). The swelling of the leg was measured with a precision caliber and expressed as mm of inflammation compared to day 21. The results they are expressed as the arithmetic mean ± SD of 12 mice / group in two independent experiments. *, p <0.01 vs antibody control (Mann Whitney U test).

Preferred embodiments of the invention

This patent demonstrates the inhibitory role of CD69 in immune reactions, using an experimental model of collagen-induced murine arthritis (AIC). The rats deficient for CD69 develop a higher incidence and severity of the AIC, with an increase in T and B immune responses against type II collagen (IIC). TGF? 1 and TGF? 2, which they have a protective function in the AIC, they showed levels reduced in inflammatory foci of CD69 - / - mice, correlating with an increase in proinflammatory cytokines IL-1? And RANTES. In this sense, the injection local of a neutralizing antibody against TGF-? In normal mice in which it has been AIC induced also increases the severity of it, as well as proinflammatory cytokine levels in mice CD69 + / +, but not in CD69 - / - mice. Besides, the CD69 activation induces the production of Total and active TGF-? In T lymphocytes activated, mouse synovial cells or mononucleated cells of synoviums of human chronic arthropathies, which express CD69, and in stable transfectants of the Jurkat T line for human CD69, but not in the parental line CD69 negative. So, CD69 is a modulator negative autoimmune reactivity and inflammation through of the synthesis of TGFβ, a cytokine that, in turn, decreases the production of different mediators pro-inflammatory

The inhibitory role of CD69 in vivo is also demonstrated by analyzing the susceptibility of CD69-deficient mice to tumor induction. CD69-deficient mice in which tumors have developed exhibited a strong regression of tumor growth and increased survival compared to normal mice. This potent antitumor response correlates with a deficient production of TGF-?, An increase in the levels of inflammatory cytokines and chemokine MCP-1, and is associated with an increase in the recruitment of effector lymphocytes and a decrease in cell apoptosis. This resistance against tumor growth depends on T lymphocytes and NK cells, and persists in CD69 - / - RAG1 - / - immunodeficient mice. In line with these results, blocking TGF-? In control mice increases the antitumor response, establishing a direct relationship between the decrease in TGF-? Synthesis found in CD69 - / - mice and the improvement. in the antitumor response. On the other hand, the activation of CD69 induces the activation of the ERK kinase and the production of TGF-? By T lymphocytes. The pharmacological blockade of ERK by the PD98059 inhibitor inhibits the production of TGF-?, Demonstrating the importance of CD69-mediated signaling in TGF-? regulation.

This patent also demonstrates that in vivo treatment of normal and immunodeficient mice (SCID and RAG-negative strains) with the CD69 2.2 antagonistic antibody increases the antitumor response. Therefore, we demonstrate that in vivo treatment with anti-CD69 antibodies is a new approach for enhancing the immune response in the manipulation of tumor immunity that can be extended to infectious diseases and immunodeficiency syndromes to recover the immune response. In vivo treatment with anti-CD69 antibodies causes different effects depending on the antibody: antagonist 2.2 increases the immune response by increasing the severity of AIC and tumor rejection, while depleting 2.3 eliminates CD69 + activated effector leukocytes, which causes an attenuation of the AIC. In addition, this reagent can be used in the direct elimination of tumors that are CD69 +.

As CD69, AICL and LLT1 are expressed at low leukocyte levels and are strongly and transiently induced. Therefore, AICL and LLT1, such as CD69, may regulate the release of TGF-? by leukocytes.

The role of early activation molecules as negative modulators of the immune response introduces the possibility of using these molecules as targets in the therapy of chronic inflammation and other immune diseases. Given the CD69, AICL and LLT1 are expressed exclusively in leukocytes activated that infiltrate inflamed tissues, stimulation pharmacological synthesis of TGF-? a through CD69, AICL and LLT1 can have a local effect, avoiding, therefore, the negative effects of the systemic increase of TGF-?.

The filed patent contains procedures for the modulation of the immune response through the use of CD69, AICL and LLT1 as targets through various protocols.

A specific goal is the modulation of the immune response through an early activation molecule using substances that inhibit or block signaling through said early activation molecule, including those that inhibit the expression of the molecule, block the binding of the early activation molecule to its receptors or ligands, or reduce the expression on the cell surface of the early activation polypeptide, and which are identified as antagonists of the early activation molecule . The third objective is the immune regulation through the eradication of cells that express the early activation molecule, using substances that eliminate CD69, AICL and / or LLT1 positive cells, and that are identified as eliminators of the early activation molecule .

Other specific objectives of this patent they are constituted by the methods to discover, identify or evaluate substances that are antagonists or cell scavengers positive for CD69, AICL and / or LLT1. These methods include measurement of intracellular calcium, MAPK activation, production of TGF-?, Induction of apoptosis mediators and cell lysis Some of these substances are identified specifically, such as antibody molecules against early activation polypeptides or direct substances or indirectly derived from them. They are also the subject of this patent the ligand / s or receiver / s putative / s of these early activation molecules identified by employment of affinity chromatography using constructions as substrate related to the early activation molecule, or those whose antagonistic or eliminating activity in the activation molecule early be identified by any of the methods indicated in this invention.

Another specific point of this patent is the use of substances that can modulate the immune response directly or indirectly through an activation molecule Early in the treatment of immune diseases. As shown In the examples, autoimmune or tumor processes are influenced by the presence or absence of activation molecules early, and treatment with substances that act through said receivers. These results can be used to treat other diseases mediated by the immune system, such as:

?

Autoimmune diseases, including rheumatoid arthritis and other types of arthritis or chronic or acute arthropathies with immune component, lupus systemic erythematosus, scleroderma, Sjögren's syndrome, diabetes autoimmune, thyroiditis, and other immune diseases tissue specific, such as psoriasis. They are also contemplated in this group various neurological diseases, such as sclerosis multiple, myasthenia gravis, etc., and gastrointestinal, such as the Crohn's disease, colitis, celiac syndrome, hepatitis and others diseases mediated by the immune system.

?

Cardiovascular diseases, including atherosclerosis, cardiomyopathies, rheumatic fevers, endocarditis, vasculitis, and other cardiovascular diseases with immune component

?

Respiratory diseases, such as emphysema, airway infections, and others.

?

Allergic processes and reactions of hypersensitivity (types I, II, III and IV), including asthma, rhinitis, and other reactions of this type with component immune

?

Transplant or rejection of grafts, comprising organ transplantation, tissue grafts, and blood transfusions. It also includes the disease of graft vs. host during bone marrow transplantation.

?

Oncological diseases, such as leukemia, lymphoproliferative disorders, solid tumors and all The types of cancer.

?

Infectious diseases, including those produced by bacteria, viruses, fungi or parasites, septic shock, and other immunopathological reactions to Infectious agents.

?

Diseases of congenital and acquired immunodeficiency, and syndromes of immunotherapy by radiotherapy and chemotherapy.

?

Degenerative processes like neurodegeneration, involving immunocompetent cells, such as the microglia

?

Administration, control of antigen, used to manipulate the nature of the response antigen-specific.

?

Gene therapy.

?

Some processes mediated by the immune system can be enhanced by manipulating a early activation molecule, such as immunosuppression, which You can apply in various contexts, including those previously indicated, or immunoactivators that can be used in vaccination, as well as DNA injection protection immunity coding for microbial or tumor antigens, or others processes for the treatment of diseases mediated by immune system, including those indicated above.

Since TGF-? And others mediators of signaling by CD69, AICL and LLT1 have a pleiotropic effect, all previously suggested diseases can account for the effect described for these molecules of early activation in immune regulation, and the objectives specific are not limited to the following predictions, which are based on the examples given below: the effect TGF-? immunosuppressant may be useful for the treatment of immune diseases in which the immune system activation is exacerbated, such as autoimmune diseases, transplant rejection and other previous list. The approach using antagonists would follow a opposite path, being used in the treatment of diseases or processes that require the enhancement of the immune system, such as tumor rejection, infection treatment, or its Vaccination prevention. The elimination strategy would eradicate CD69, AICL and / or LLT1 positive cells, which are those activated locally in response to stimulation antigenic and mediate the immune response. Therefore, the expected consequence would be the improvement of associated diseases with exacerbation of the immune response, such as diseases autoimmune, transplant rejection and other list previous.

Early Activation Polypeptides

The terms "activation proteins early "and" early activation polypeptides "were refers to CD69, AICL, LLT1, and its fragments. The terms "protein" and "polypeptide" are interchangeable.

CD69

As used here, "CD69", also known as a "very early activation" protein, "molecule of activation induction "and" gp34 / 28 ", refers to CD69 of mammal, preferably human CD69 protein. Agree, the term "human CD69" refers to a polypeptide that has or is homologous to (e.g., at least about 85%, 90%, 95% identical to) an amino acid sequence shown previously SEQ ID NO 2 [Lopez-Cabrera, 1993 # 208], or that is coded by: (a) a nucleic acid sequence encoding human CD69 (e.g., a nucleic acid sequence encoding CD69 human according to SEQ ID NO 1 [Lopez-Cabrera, 1993 # 208]; (b) a degenerate nucleic acid sequence to a natural human CD69 sequence; (c) a nucleic acid of homologous sequence to (e.g., at least about 85%, 90%, 95% identical to) the nucleic acid sequence for natural CD69; or (d)  a nucleic acid sequence that hybridizes with any of the nucleic acid sequences indicated above under astringent conditions, e.g., highly astringent conditions. A preferred CD69 is a natural variant or allele of CD69.

Anti-polypeptide antibody molecules of early activation

An "early activation anti-polypeptide antibody molecule" is an antibody molecule that interacts (eg, binds to) early activation polypeptide, preferably to a human early activation protein. Preferably, the early activation anti-polypeptide antibody molecule binds to the extracellular domain of the early activation polypeptide (eg, an epitope of the early activation polypeptide located outside the cell). In some aspects, the early activation anti-polypeptide antibody molecule may be an anti-CD69 antibody molecule, eg, a human anti-CD69 antibody molecule. Examples of monoclonal antibodies against human CD69 include, but are not limited to a human anti-CD69 antibody molecule analogous to mouse anti-CD69 antibody 2.2, a human anti-CD69 antibody molecule analogous to mouse anti-CD69 antibody 2.3 or a human anti-CD69 antibody, eg, an anti-CD69 antibody known in the literature, which can act as an antagonist or eliminator of CD69 or antibody molecules that have epitopes that overlap with the epitope of such antibody, or that compete with such binding antibody. Examples of human anti-CD69 antibodies known in the literature include: TP1 / 8, TP1 / 22, TP 1/28, TP 1/33, TP 1/55 (as described, eg, in [Cebrian, 1988 # 170] ); CH / 4, CH / 1, CH / 2, FAB / 1 (as described, eg, in [Sanchez-Mateos, 1991 # 126]); L78, MLR3, FN61, FN50 (as described, eg, in Schwarting, R. et al . (Eds) Leukocyte Typing IV, Springer-Verlag, New York, 1989, p. 428); MLR3 (as described, eg, in [Court, 1981]); EA-1 (as described, eg, in Hara, T (1986) J. Exp. Med. 164: 1988-2005); Leu 23 (as described, eg, in [Lanier, 1988]); and C1.18, E16.5 (as described, eg, in [Gerosa, 1991]). Examples of antibody molecules that can be used in the invention include but are not limited to: an antibody molecule that interacts with, eg, binds to, an epitope that includes one or more residues of the neck region of a CD69 polypeptide. (eg, one or more residues of 62-84 of human CD69); an antibody molecule that interacts with, eg, binds to one or more residues of the NK domain (or carbohydrate recognition domain, CRD) of a CD69 polypeptide (eg, one or more residues of residues 82 to 199 of CD69 human); an antibody molecule that interacts with, eg, binds to, an epitope that includes one or more residues of the intracellular domain of a CD69 polypeptide (eg, one or more 1-40 residues of human CD69); an antibody molecule that interacts with, eg, binds to an epitope, to which when it binds modulates, eg, increases or decreases, the interaction of cytoplasmic and / or transmembrane CD69 regions with an effector, whose activity can be determined by the methods described here (eg, TGF-? production, MAPK activation, or Ca2 + signaling); an antibody molecule that interacts with, eg, binds to, an epitope (eg, a conformational or linear epitope), which is modulated when the antibody is bound, eg, increases or decreases the formation of CD69 dimers (eg, an epitope that includes the Cys68 residue of human CD69 or a residue located near Cys68); an antibody molecule that can modulate the cellular expression of CD69, eg, an antibody molecule that negatively modulates the expression of CD69 in the cell membrane; an antibody molecule that can modulate the binding of a ligand or CD69 receptor, eg, an antibody that can inhibit, competitively inhibit, or enhance the binding of a ligand or receptor to CD69; an antibody molecule that interacts with, eg, binds to, or that can inhibit or enhance the binding of a ligand or receptor to one or more 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 one example, the early activation anti-polypeptide antibody molecule binds to all or part of the epitope of an antibody described herein, eg, a human anti-CD69 antibody known in the literature, can act as an antagonist or a scavenger, or a human early activation anti-polypeptide antibody analogous to a mouse early activation anti-polypeptide antibody, eg, a mouse anti-CD69 antibody (eg, antibody 2.2, 2.3, or H1.2F3 (the latter described, eg , in Yokoyama et al . (1988) J. Immunol. 141: 369-376) An anti-CD69 antibody can bind to one or more residues of the epitopes described in [Sanchez-Mateos, 1991 # 126] or compete for binding to an antibody that binds to one of the described epitopes The early activation anti-polypeptide antibody molecule can inhibit, eg, competitively inhibit, the binding of an antibody described herein, eg, an anti-activation polypeptide antibody early human met or in the literature as described herein against a human early activation polypeptide. An early activation anti-polypeptide antibody molecule can bind to an epitope, eg, a conformational or linear epitope, so that when they interact, the binding of one of the antibodies described herein is prevented, eg, an anti-activation polypeptide antibody. Early human known in the literature as described here against human CD69. The epitope may be very close or functionally associated, eg, an overlapping or adjacent epitope in a linear or conformational sequence, to one of those recognized by an antibody described herein, eg, a human early activation anti-polypeptide antibody known in the literature as those described here.

In a preferred example, the interaction, e.g., binding, between an antibody molecule early activation anti-polypeptide and a Early activation polypeptide occurs with high affinity (e.g., affinity constant of at least 3 x 10 7 M -1, preferably, between 3 x 10 8 M -1 and 3 x 10 10, or about 3 x 10 9 M -1) and specificity. Preferably, the anti-polypeptide antibody molecule of early activation modulates the immune response, e.g., acts as a antagonist or eliminator of the activation polypeptide early

As used here, "specific binding" is refers to the property of the binding agent, preferably the antibody, to: (1) bind to an early activation polypeptide, e.g., a human early activation polypeptide protein, with an affinity of at least 1 x 10 7 M -1, and (2) preferably binding to the early activation polypeptide, e.g., the human early activation polypeptide protein, with a affinity that is at least twice, 50 times, 100 times, 1000 times, or greater than its binding affinity to a non-specific antigen (e.g., BSA, casein) other than the activation polypeptide early

As can be seen in the present invention, many types of anti-polypeptide antibody molecules early activation, e.g., antibodies, or fragments thereof that bind to the antigen, are useful in the methods of this invention. The antibody molecules can be several isotypes, including: IgG (e.g., IgG1, IgG2 (e.g., IgG2a, IgG2b), IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. A molecule of Preferred antibody is an IgG isotype. Antibody molecules they can be full length (e.g., an IgG1 or IgG4 antibody) or they may include only an antigen binding fragment (e.g., a Fab, F (ab ') 2, Fv or a single chain of an Fv fragment). The antibody is preferably an antibody molecule designed, e.g., a humanized antibody.

The antibodies, or other agents described here, can be evaluated for their ability to act as antagonists or CD69 eliminators.

A polypeptide eliminating antibody from early activation is one that, after administration to an animal, alone or conjugated to a toxin, reduces the absolute number of cells positive for early activation polypeptide, e.g., cells  tumor CD69 +, AICL + and / or LLT1 +, e.g., CD69 + leukocytes, AICL + and / or LLT1 + present in chronic inflammatory infiltrates. Usually, it will act through the direct lysis of these cells (in contrast to, e.g., a transient retention of cells in a  organic compartment, such as bone marrow or liver).

An anti-polypeptide antibody of early antagonist activation is one that, after administration to animal, induces CD69 + cells to decrease their production of the TGF-? cytokine, e.g., by blocking the endogenous signaling induced by the binding of CD69 to one or more of their natural ligands or receptors; (e.g., by blocking the interaction of CD69 with its natural ligands or receptors) to which we refer to here as "a blocking antagonist." Other early activation polypeptide antagonists include, eg, "an antagonist that reduces expression", which decreases the expression level of the early activation polypeptide in the cell surface, e.g., by binding to a polypeptide of activation and internalization of said polypeptide.

Several in vitro methods can be used, in conjunction with in vivo assays in the whole animal, to assess the ability of candidates for their activity as depleting or antagonistic. An in vitro assay may not be indicative of its activity in vivo in all cases, but may be useful in search strategies.

Eliminators of early activation molecules

An in vitro early activation polypeptide eliminating antibody is an antibody that, after incubation with cells expressing the early activation polypeptide, reduces the absolute number of such cells. This can be done through direct lysis, eg, via complement-dependent mechanisms, Fc receptor or conjugated toxins. Thus, an eliminator of an early activation polypeptide can lyse cells that express the early activation polypeptide in in vitro death assays . The isotype of the early activation anti-polypeptide antibody can play an important role in the possible function as a scavenger, via a complement-based mechanism. If an antibody is capable of binding complement, it will probably behave like a scavenger. Most natural antibodies have a high affinity for complement and will thus be eliminators. This is less likely in the case of mouse IgG1 and human IgG4, which have low complement affinity. In addition, antibodies can be designed to reduce or increase their complement affinity. An early mouse anti-polypeptide antibody (generated in mouse or other species) can kill mouse cells expressing the early activation polypeptide in vitro in the presence of complement. Target cells expressing early activation polypeptides useful for testing such antibodies include, eg, murine leukemias, eg, murine thymoma EL-4, for CD69. For example, an antibody generated in mice or other species against human CD69 that is a killer, can kill human cells expressing CD69, eg, rheumatoid arthritis synovial fluid T cells expressing CD69, leukemia expressing CD69, or stable transfectants of CD69, eg, Jurkat lymphoblastic leukemia cells stably transfected to express CD69. This antibody will not kill (or will do so at a much lower level) control cells that do not express CD69, eg, the non-transfected Jurkat parental line, which does not express CD69. Thus, the ability of an antibody to function as a scavenger can be tested in vitro for its ability to lyse target cells that express the early activation polypeptide and control cells that do not express said receptor. A candidate for eliminator will lyse the cells positive for the early activation polypeptide but not the control cells that do not express the early activation polypeptide (or lysate significantly less). In one example, cell lysis can be monitored in an assay in which the target (and control) cells are labeled with 51 Cr and incubated with the antibody in the presence of rabbit complement, typically 2 h at 37 ° C. After that the supernatant is recovered and the level of cell lysis is evaluated by the amount of 51 Cr released to the supernatant. The level of lysis is indicated by the release of 51 Cr by the lysed cells.

Anti-activation molecule antagonists early

An in vitro antagonist early activation anti-polypeptide antibody is one that, after incubation with CD69 + cells, decreases its production of the TGF-? Cytokine, eg, by blocking the endogenous signaling induced by the binding of CD69 to one or more of its natural ligands or receptors, present in the in vitro culture system. Thus, one can perform a search for antagonists in vitro by incubating an antagonist candidate with a cell expressing the early activation polypeptide and evaluating its effect on the production of TGF-?, Eg, compared to a control cell, eg, a cell. which does not express the early activation polypeptide. For example, a mouse anti-CD69 antibody can be evaluated in its ability to behave as an in vitro antagonist by analyzing its ability to inhibit or reduce the production of TGF-? By concanavalin A-activated splenocytes, which are CD69 +. A human anti-CD69 antibody can be evaluated for its ability to mediate the production of TGF-? By Jurkat cells stably transfected with CD69. An antagonist will inhibit the production of TGF-? By 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 may also be an antagonist that decreases the expression of the early activation molecule, eg, an antagonist early activation anti-polypeptide antibody that decreases the membrane expression of the antigen against which it is directed. An in vitro antagonist can be detected by incubating the candidate antagonist with a cell that expresses the early activation protein and evaluating whether this early activation protein decreases its expression or even disappears from the cell surface. For example, an early activation anti-polypeptide mouse antibody can be evaluated for its ability to decrease the expression of an early activation polypeptide using a reagent capable of detecting the presence of the anti-early mouse anti-molecule molecule antibody, eg, a polyclonal anti-mouse reagent. If the reagent does not detect the candidate on the cell surface, then the antagonist can be identified as an antagonist that reduces the expression of the early activation molecule in the cell membrane. An antagonist should not act as a eliminator.

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

one)

block the binding of the polypeptide from pure early activation to its purified receptor or ligand, or to partially purified preparations of membrane fractions of  cells that respond to the early activation polypeptide, e.g., expressing a receptor for an activation polypeptide early, or to whole cells that respond to the polypeptide of early activation, e.g., expressing a receptor of a early activation polypeptide;

2)

block the junction of a cell that express the early activation polypeptide to the receptor of the purified early activation polypeptide;

3)

block the junction of a cell that expresses the early activation polypeptide to a cell that express the early activation polypeptide receptor (e.g., cells that respond to the early activation polypeptide) or to partially purified preparations of such fractions of membrane of cells that respond to the activation polypeptide early, e.g., that express a receptor for the polypeptide of early activation e.g., the antagonist candidate would block the binding of a CD69 ligand molecule to a stable transfectant of Human CD69 of Jurkat cells (but not to the parental line CD69-);

4)

block the binding of an antibody monoclonal agonist to a cell that expresses the polypeptide of early activation and thus inhibit the production of TGF-? By the agonist antibody.

5)

Decrease the levels of a polypeptide of early activation on the cell surface, e.g., by the binding to an early activation polypeptide and internalization of the polypeptide.

Antibody molecules

As used here, the term molecule of antibody, refers to a molecule that includes a number enough of complementarity determining regions (CDRs), preferably 6, presented in an arrangement that allows binding of the CDRs to the known antigen. Thus, the term includes complete antibodies (including natural antibodies and designed by Molecular Biology), and fragments binding to natural or designed antibody antigen. The term includes various types of antibodies or antibody molecules, including monospecific, monoclonal, recombinant, human, not humans, e.g. murine. Chain antibodies are also included simple, intrabody and bivalent antibodies. I also know include chimeric antibody molecules, with a different CDR grafted, humanized, disinmunogenic, as well as others that have designed to reduce immunogenicity, e.g., those with CDRs derived from a non-human source, e.g., from a non-animal human as the mouse, and / or derivatives of partial generation or totally random sequences, e.g. using a method of phage selection. Such non-human fragments can be inserted into human, humanized, or other molecules provisions that make them less antigenic when administered To a human.

Thus, an antibody molecule may have: CDRs from a non-human source, eg, from a non-human antibody, eg, from a mouse immunoglobulin or other non-human immunoglobulin, from a consensus sequence, or from a sequence generated by selection of phages, or any other method to generate diversity; and having an arrangement that is less antigenic in a human than non-human structure, eg, in the case of CDRs of a non-human immunoglobulin, less antigenic than the non-human structure from which non-human CDRs were taken. The structure of the immunoglobulin can, eg, be human, non-humanized human, eg, a mouse, modified structure to reduce antigenicity in humans, or a synthetic structure, eg, a consensus sequence or a method of generating in vitro diversity .

Preferred antibody molecules may include at least one, and preferably two, variable regions of the heavy chain (VH) or their antigen binding fragments, and at least one or preferably two variable regions of the light chain (VL) or their antigen binding fragments. The VH and VL regions are subdivided into regions of hypervariability, called "complementarity determining regions" (CDR), interspaced with regions that are more conserved, called "structural regions" (FR). The extent of structural regions and CDRs has been precisely defined (see, eg, Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al . (1987) J. Mol. Biol. 196: 901-917). Preferably, each VH and VL of an antibody molecule is composed of three CDRs and four FRs, arranged from the amino-terminal to the carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs can come from different sources.

The VH or VL chain of an antibody molecule it can include all or part of a constant region of the chain light or heavy In one example, the antibody molecule is a tetramer of two heavy and two light chains of immunoglobulins, where heavy and light chains are interconnected by, e.g., disulfide bridges The constant region of the heavy chain is It consists of three domain, CH1, CH2 and CH3. The constant region of the Light chain consists of a domain, CL. The variable region of heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of the antibodies typically mediate the binding of the molecule of antibody to host tissues or factors, including several cell types of the immune system (e.g., effector cells) and the first component (C1q) of the classical system path complement. Antibody molecules may include IgA, IgG, IgE, IgD, IgM (as well as all its subtypes), where the chains Lightweight can be of the kappa or lambda type.

As discussed above, the "antigen binding fragments" of an antibody molecule are within the term antibody molecule. An antigen-binding fragment, as used herein, may refer to a portion of an antibody that specifically binds to the early activation polypeptide (eg, the human early activation polypeptide). Examples of binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists 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 CDRs with sufficient structure to specifically bind, eg, an antigen-binding portion of a variable region.

Antibody fragments can also produced by chemical methods, e.g., by breaking an antibody intact with a protease, such as pepsin or papain, or, optionally, treating the digested product with an agent reducer. Alternatively, useful fragments can be produced using host cells transformed with truncated genes from heavy and / or light chains.

An antigen-binding portion of a variable region of the light chain and an antigen-binding portion of a variable region of the heavy chain, eg, the two domains of the Fv fragment, VL and VH, can be linked, using recombinant methods , by a synthetic union that allows them to constitute a simple protein chain in which the pair of VL and VH regions form monovalent molecules (known as single chain Fv (scFv); see, eg, 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 included within the term "antigen binding fragment" of an antibody. These antibody fragments are obtained using known conventional techniques, and the fragments are studied for their usefulness in the same manner as intact antibodies.

The term "antibody or molecule of monospecific antibody "refers to an antibody or molecule of antibody showing a single binding specificity and affinity for a particular target, e.g., an epitope. That term includes a "monoclonal antibody" or a "composition of monoclonal antibody. "

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

Monoclonal, chimeric and humanized, which have been modified by, e.g., destruction, addition, or substitution of 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  destruction of the constant region; (ii) by replacement of the constant region with another constant region, e.g. a region constant that increases the half-life, stability or affinity of the antibody, or a constant region of another species or class of antibody; or (iii) by the modification of one or more amino acids of the constant region to alter, for example, the number of sites glycosylation, effector cell function, binding to Fc receptors (FcR), complement fixation, transport to through the placenta, among others.

In one example, the constant region of the antibody can be replaced by another constant region of, eg, a different species. This replacement can be performed 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 heavy or light chain gene, respectively, by the operational binding of nucleic acids encoding VH or VL to other acids. nucleic encoding the constant regions of heavy or light chains. The sequences of the genes of the constant regions of human heavy and light chains are known. Preferably, the constant region is human, but the constant region of other species, eg, rodents (eg, mouse or rat), primate, camel, rabbit, can also be used. The constant regions of these species are known (see eg, Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US Department of Health and Human Services, NIH Publication No. 91-3242). Methods for altering the constant region of an antibody are known. Antibodies with impaired function, eg, affinity altered by an effector ligand, such as the FcR in a cell or the C1q component of the complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue ( see, eg, EP 388,151 Al, US 5,624,821 and US 5,648,260, the contents of which are incorporated herein by reference). Similar types of alterations have been described, so that if applied to murine or other species immunoglobulins, they would reduce or eliminate these functions.

Antibody Polypeptide Activation Production early

Immunogens useful for the generation of early activation anti-polypeptide antibody molecules include cells expressing the early activation polypeptide, membrane preparations of cells expressing the early activation polypeptide, micelles or vesicles with the early activation polypeptide, the polypeptide early activation or fragments thereof, eg, the isolated or purified early activation polypeptide, eg, the human early activation polypeptide, eg, the biochemically isolated early activation polypeptide or a portion thereof, or the early activation polypeptide or any of its recombinantly produced fragments, eg, the extracellular or intracellular domain of the early activation polypeptide; or any fusion protein containing any of these fragments of the early activation polypeptide. The antigen is preferably a human antigen. The soluble early activation polypeptide can be obtained, eg, by recombinant production of the early activation polypeptide in soluble form (eg, an early activation polypeptide that lacks all or 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 the generation of anti-CD69 antibodies are described in Sanchez-Mateos et al . (1991) Eur. J. Immunol . 21: 2317-2325, whose content is expressly incorporated by reference. These techniques, as well as others known in the literature, can be used to generate antibodies to CD69.

CD69 fragments that include residues 62-84 per portion of them of SEQ ID NO: 2, may be used to make antibodies against neck regions of each of the early activation polypeptides, respectively; e.g., the fragments can be used as immunogens or be used to characterize the specificity of a antibody. Similarly, fragments of CD69, including residues 82 to 199, or portions thereof of SEQ ID NO: 2; CD69 fragments that include residues 62 to 199 for portions of them of SEQ ID NO: 2 can be used to make a antibody against the extracellular region of the protein early activation; CD69 fragments that include residues of 1 40 or portions thereof of SEQ ID NO: 2 can be used to make an antibody against an intracellular region of the protein of early activation

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

Antibodies that only bind to one protein native early activation, only to the denatured or protein of early activation in any non-native way, or that join Both forms are within the invention. Antibodies with Linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified with antibodies that bind to native early activation protein but not to the denatured form.

As described below in more detail, the antibody molecules (preferably, monoclonal antibodies from different organisms, e.g. rodents, sheep, humans), against The early activation polypeptide can be produced using recognized methods in the field. Once the antibody is obtains, can be examined for suitability for use in the invention, e.g., determining whether the antibody behaves as antagonist or eliminator.

Monoclonal antibodies can be produced by a variety of techniques, including conventional methodology for the generation of monoclonal antibodies, eg, 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, NY. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, eg, viral or oncogenic transformation of B lymphocytes. The preferred animal system for preparing hybridomas is the murine system. The production of hybridomas in mice is a well established procedure. Immunization protocols and techniques for the isolation of splenites immunized by fusion are known. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known.

An antibody can be obtained from a non-human origin and the variable regions, and in particular the CDRs, can be sequenced and used to make antibody molecules, eg, humanized antibodies, useful in the invention. The location of the CDRs and structural waste should be determined (see, eg, Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US 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 known methods. Once murine antibodies are obtained, the variable regions can be sequenced. The location of the CDRs and the structural residues determined (see Kabat, EA, et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US 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 variable regions of the heavy and light chains can, optionally, be linked to their corresponding constant regions.

Mouse antibodies early activation anti-polypeptide can be sequenced using known techniques.

Antibody molecules or humanized immunoglobulins or with the CDR inserted can be produced by replacement or insertion of the CDR, where one, two or all of the CDRs of an immunoglobulin chain can be replaced. See, eg, US Patent 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 US 5,225,539, the contents of which are expressly incorporated herein by reference.

Winter describes a CDR insertion method which can be used to prepare humanized antibodies in the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), whose contents They are expressly incorporated by reference. All CDRs of a particular human antibody can be replaced by at least a portion of a non-human CDR or only some of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for humanized antibody binding to the default antigen.

Humanized antibodies can be generated by replacing the Fv variable region sequences that are not directly involved in antigen binding, with equivalent sequences from the human Fv variable regions. General methods for the generation of humanized antibodies are indicated in Morrison, SL, 1985, Science 229: 1202-1207, by Oi et al ., 1986, BioTechniques 4: 214, and by Queen et al . US 5, 585, 089, US 5,693,761 and US 5,693,762, the contents of which are incorporated herein by reference). These methods include the isolation, manipulation, and expression of nucleic acid sequences encoding all or part of the Fv variable regions of immunoglobulins of at least one heavy or light chain. The origin of such nucleic acids is known and, eg, can be obtained from a hybridoma that produces an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody or a fragment thereof can then be cloned into an appropriate expression vector.

Humanized antibodies in which they have been substituted, destroyed or added specific amino acids are like this within this invention. In particular, humanized antibodies preferred have amino acid substitutions in the region structurally, so that they improve antigen binding. By example, a series of accepting structural waste selected from the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. The Preferred locations of substitutions include residues of amino acids adjacent to the CDR, or that are capable of interacting with a CDR (see, e.g., US 5,585,089). The criteria for Select donor amino acids are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, whose Contents are incorporated here by reference. The structure acceptor can be a consensus sequence or sequence of a mature human antibody structure. As used here, the term "consensus sequence" refers to the sequence formed of the most frequent amino acids (or nucleotides) that they are 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 of the consensus sequence is occupied by the most frequent amino acid in Such position in the family. If two amino acids occur in such position with equal frequency, any of them can be Include in the consensus sequence. A "consensus structure" is refers to the structural region in the consensus sequence of the immunoglobulin

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

Antibody molecule early activation anti-polypeptide can be also modify in another way to decrease its immunogenicity, e.g., by specific deletion of epitopes recognized by the human T cell or "deimmunization" by the methods described in WO 98/52976 and WO 00/34317, the contents of which are specifically incorporated here by reference.

In some examples, the antibody molecule early activation anti-polypeptide includes a variable region of heavy or light chain that has one, two, or preferably, three CDRs substantially identical to the CDR of the variable region of a light or heavy chain non-human early activation anti-polypeptide, respectively. In a preferred example, the modified antibody or the antigen binding fragment thereof includes all six CDRs of the same anti-activation polypeptide early non-human, e.g., any antibody molecule human anti-CD69 used in the literature, e.g., the panel of human anti-CD69 antibody molecules described in [Sanchez-Mateos, 1991 # 126].

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

Monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice with human immunoglobulin genes, rather than in the mouse system. Such transgenic mice may include mice that carry a portion of the heavy and light chain genes of human immunoglobulins. For example, in the HuMAb® mouse, from Medarex and GenMab, the mouse genes for generating antibodies are inactivated and replaced by many of the key sequences for non-recombinant human antibody genes encoding the heavy and light chains. Another available mouse is Abenoix's XenoMouse®, which has approximately 80o of the human antibody heavy chain genes and a significant amount of the human light chain genes. Other transgenic mice are available to generate human antibodies that contain entire regions of the constant and variable genes found in the corresponding human natural immunoglobulin loci. These mice, also referred to as Kirin TC ™ mice are from Medarex. Kirin TC ™ mice are "transchromosomal," meaning that mouse genes to create antibodies have been inactivated and replaced by human chromosomes that contain all human genes for antibody generation, including all kinds of heavy chains that code for all isotypes (IgG1-4, IgA1-2, IgD, IgM and IgE). Also from Medarex, it is the KM® mouse, a strain that results from the crossing of HuMAb and Kirin-TC mice, so that it combines its characteristics. KM, like the Kirin TC mouse, retains the ability to produce all kinds of human isotypes. The splenocytes of these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes of a human protein (see, eg, 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, LL et al . 1994 Nature Genet . 7: 13-21; Morrison, SL 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).

Chimeric antibodies, including chimeric immunoglobulin chains, can be produced by known recombinant DNA techniques. For example, a gene that codes for the Fc constant region of a mouse monoclonal antibody molecule (or other species) is digested with a restriction enzyme to remove the region that codes for mouse Fc, and the equivalent portion of the mouse. gene coding for the 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 . US 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. Na tl Cancer Inst . 80: 1553-1559).

Also included within the term are species generated in vitro , where all or part of the CDRs, are generated by a non-immune selection, eg, an in vitro phage sample, protein chip, or any other method in which the candidate sequences they can be measured in their ability to bind the antigen. For example, combinatorial antibody visualization methods have been developed to identify and isolate antibody fragments that have a specific specificity for an antigen, and can be used to produce an antibody molecule.

Once displayed on a display system (e.g., a filamentous phage), the antibody library is screened with the antigen, or a peptide fragment thereof, to identify and isolate groups that express an antibody with specificity for this antigen. The nucleic acid that encodes for this selected antibody it can be recovered from the system of visualization (e.g., of the phage genome) and subcloned into others Expression vectors by standard recombinant DNA techniques.

Specific antibodies with high affinities for a surface protein can be generated according to known methods, eg, methods that involve searching in libraries (Ladner, RC, et al ., US Patent 5,233,409; Ladner, RC, et al ., US Patent 5,403,484). In addition, the methods of these libraries can be used in searches to obtain binding determinants that are mimetic to the structural determinants of the antibodies.

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

In one example, a peptide library different is expressed by a population of display groups to form a peptide display library. Ideally, the display groups comprise a system that allows the sampling of large number of different peptide libraries, a rapid separation after each round of affinity separation, and a easy isolation of the gene that codes for the peptide of the purified groups. Peptide analysis libraries can be in e.g. prokaryotic organisms or viruses, which may amplify quickly, are relatively easy to manipulate, and  They can allow the creation of a large number of clones. Groups of Preferred visualizations include, for example, bacteria vegetative, bacterial spores, and, preferably, viruses bacterial (especially DNA viruses). However, this The invention also contemplates the use of eukaryotic cells, including yeasts and their spores, as display groups potential. Antibody molecules anti-early activation polypeptide useful in the present invention may be recombinant antibodies produced in transformed host cells, e.g., with DNA coding for the heavy and light chains of an antibody wanted. Recombinant antibody molecules can be produced  by known molecular engineering techniques. For example, recombinant antibodies can be produced by cloning a sequence of nucleotides, e.g., a cDNA or genomic DNA sequence, which code for light or heavy immunoglobulin chains of the  desired antibody from a hybridoma cell that produces a antibody useful in this invention. The nucleotide sequence that encodes for these polypeptides is then inserted into vectors of expression, so that both genes are operatively linked to its own transcriptional and translational control sequences. The expression vector and expression control sequences are chosen for being compatible with the host cell used. Typically, both genes are inserted into the same vector of expression. Prokaryotic host cells or eukaryotic

Expression in eukaryotic host cells is preferred because such cells are more suitable than prokaryotic cells for the proper assembly and secretion of a correctly folded and immunologically active antibody. However, any antibody produced that is inactive due to improper folding can be renatured according to 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 host cells produce intact portions of antibodies, such as light or heavy chain dimers, which are also antibody homologs, according to the present invention.

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

Antibody molecule conjugates

The antibody molecules of the invention they can be conjugated, covalently or noncovalently, with other structures, e.g., therapeutic agents or signals, e.g., toxins (e.g., proteins, (e.g., diphtheria or ricin) or chemical toxins), therapeutic isotopes, or other therapeutic structures.

According to this, an antibody molecule early activation anti-polypeptide can be derivatized or bound to another functional molecule (e.g., another peptide or protein). The antibodies and the antibody portions of the invention include derivatized or modified forms of any form of the antibodies described herein, including the molecules of immunoadhesion. For example, an antibody or portion of antibody of the invention can be functionally linked (by chemical binding, genetic fusion, non-covalent or other association form) to one or more 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 the association of a antibody or a portion of antibody with another molecule (such as the main streptavidin region or a tail of polyhistidine).

A type of derivatized antibody is produced by crosslinking of two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).

Suitable crosslinking agents are those which are heterobifunctional, having two reactive groups distinct separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidil suberate). Such Links are available by Pierce Chemical Company, Rockford, IL.

Useful detectable agents with which a antibody or portion thereof of the invention may be derivatized (or labeled) to include fluorescent components, various enzymes, prosthetic groups, luminescent materials or bioluminescent, metal atoms with fluorescence emission, e.g., europium (Eu), and other lanthanides, and radioactive materials (described below). Examples of agents detectable by fluorescence include fluorescein, isothiocyanate fluorescein, rhodamine, chloride 5-dimethylamine-1-naphthalenesulfonyl, phycoerythrin, and others of the same type. An antibody can be also derivatized with detectable enzymes, such as the alkaline phosphatase, horseradish peroxidase, β-galactosidase, the acetyl-choline esterase, glucose oxidase and others of the same type. When an antibody is derivatized with a detectable enzyme, it is detected by the addition of reagents that the enzyme uses as substrates to generate a product of detectable reaction For example, when the agent detectable from the horseradish peroxidase is present, the addition of peroxide hydrogen and diaminobenzidine leads to a reaction product colored that is detectable. An antibody can also be derivatized with a prosthetic group (e.g., streptavidin / biotin and avidin / biotin). For example, an antibody can be derivatized with biotin and detected through measurement indirect binding of avidin or streptavidin. Examples of suitable fluorescence materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a luminescent material is luminol; and examples of bioluminescent materials are luciferase, Luciferin and Aecuorin.

An anti-polypeptide antibody of early activation or a fragment of it that binds to the antigen it can be conjugated to another molecular entity, typically a brand or a therapeutic agent or structure (e.g., cytotoxic or cytostatic).

A cytotoxin or cytotoxic agent includes any agent that is destructive to the cells with which interacts Examples are taxol, cytochalasin B, the Gramicidin D, ethidium bromide, emetine, mitomycin, ethoxide, tenopoxide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin-dione, the mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propanolol, puromycin, maitansinoids, e.g., maitansinol (see US Patent No. 5,208,020), CC-1065 (see US Patent Nos. 5, 475, 092, 5, 585, 499, 5,846,545) and their analogues or counterparts. Therapeutic agents include, but are not limited to, antimetabolites, (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbacin), agents alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melfalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin); anthracyclines (e.g., daunorubicin (before daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and antramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maitansinoids).

An anti-early polypeptide antibody or an antigen binding fragment thereof can be conjugated to another molecular entity, eg, a structure that modulates immunogenicity and / or half-life. In one example, the molecular entity is polyethylene glycol (PEG) or derivatives thereof. PEGylation is a chemical conjugation method that can reduce potential immunogenicity and / or extend the half-life. Several methods of PEGylation of an antibody are known. See, eg, Bhandra et al . (2002) Pharmazie 57 (1): 5-29.

Other binding reagents to an activation polypeptide early

A polypeptide binding reagent is defined early activation as an agent that interacts (binds) with the early activation polypeptide, preferably of origin human. The interaction occurs preferably with high affinity (with a binding constant of at least 10 7 M -1, preferably between 10 8 and 10 10 M -1) and specificity Reagents binding to an activation polypeptide early they can be antagonists or eliminators (depleting) of CD69. As examples of polypeptide binding reagents of early activation antibodies against the early activation polypeptide (as reviewed above), as well as molecules of small molecular size or peptidomimetics

Useful agents in this invention include agents. mimetics of the early activation polypeptide. These agents, which include peptides, semi-peptide compounds or not peptides (as organic molecules of small molecular size), they are inhibitors of the activity of the activation polypeptide early

In an optimal incarnation, the agent is part of a combinatorial library, for example a peptide collection or organic molecules, or part of a product library natural Under these circumstances, a group of test compounds may include 10, 10 2, 10 3, 10 4, 10 5, 10 6, 10 7 or 10 8 compounds, which share characteristics structural or functional

In its current form, this invention includes libraries of early activation polypeptide binding agents. The genesis of combinatorial libraries is well characterized in the literature and has been thoroughly reviewed (for example, see: EM Gordon et al ., J. Med. Chem . (1994) 37: 1385-1401; DeWitt, SH; Czarnik, AW Acc. Chem. Res . (1996) 29: 114; Armstrong, RW; Combs, AP; Tempest, PA; Brown, SD; Keating, TA Acc. Chem. Res . (1996) 29: 123; Ellman, JA Acc. Chem. Res . (1996) 29: 132; Gordon, EM; Gallop, MA; Patel, DV 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; US Patents 5,359,115, 5,362,899, and 5,288,514; PCT Publication No. WO92 / 10092, WO93 / 09668, WO91 / 07087, WO93 / 20242, WO94 / 08051).

The libraries of compounds of this invention can be prepared according to different methods, some of which have been previously characterized. For example, a strategy called division can be implemented as follows: functionalized polymer substrate microspheres are placed in different reaction vessels; there is a wide variety of polymeric substrates suitable for solid phase peptide synthesis, and some are commercially available (as examples, see M. Bodansky "Principies of Peptide Synthesis", 2nd edition (1993), Springer-Verlag, Berlin). A solution containing an activated amino acid is added to each aliquot of microspheres, and the reactions are carried out, resulting in a series of immobilized amino acids, each in a reaction vessel. The aliquots of derivatized microspheres are washed and collected ("recombination"), and this set is divided again, placing each aliquot in a new reaction vessel, adding a new activated amino acid to each aliquot. This cycle is repeated until peptides of the desired length are achieved. The amino acids added in each cycle are randomly selected, or they can be selected to obtain a "targeted" library, that is, in which certain parts of the inhibitor are selected by a non-random method, for example, to select inhibitors with homology or structural similarity with a known peptide capable of interacting with an antibody, such as the antigen binding site of anti-idiotypic antibodies. Thus, a wide variety of non-peptide peptidomimetic compounds can be obtained.
dicos

This division strategy produces a library of peptides, some of them inhibitors, which can be used to prepare a library of test compounds of the invention. In another illustrative example, a library of diversomers is generated according to the method of Hobbs DeWitt et al . ( Proc. Natl. Acad. Sci . USA. 90: 6909 (1993)). Other methods of synthesis, such as that of Houghten's "tea bag" (see Houghten et al ., Nature 354: 84-86 (1991) can be used to generate libraries of compounds of the subject invention.

Subsequently, screenings can be carried out to determine which members of the library have a desirable activity, and if so, identify the active substance. Combinatorial analysis methods of library screening have been described (see Gordon et al ., J. Med. Chem., Supra ). Soluble compound libraries can be identified by affinity chromatography with an appropriate receptor to isolate ligands for the receptor, followed by the identification of ligands isolated by conventional techniques, such as mass spectrometry, NMR and the like. The immobilized compounds can be identified by contacting them with a soluble receptor, preferably coupled to a label (fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds and the like) that can be detected by indicating ligand binding. Alternatively, immobilized compounds can be selectively released, allowing diffusion through a membrane to interact with a receptor. Representative examples for screening the libraries of the invention are described.

Thus, the interaction of the compounds of this invention with the activation polypeptide early testing the ability of each compound to interact with it, for example, by incubating the compound under investigation with the early activation polypeptide and a lysate in a container of suitable reaction, such as a standard 96-well plate. In this situation, the activity of each individual compound can be determined, using a well or well without the control compound tested. After incubation, the activity of each Compound can be determined in each well. Therefore, they can determine the activities of a plurality of compounds in parallel.

Thus, the binding of large amounts of different compounds can be determined simultaneously. For example, the compounds can be synthesized in solid resin microspheres following a pattern "a microsphere-a compound"; The compounds can be immobilized in the resin through a photolabile bridge. Subsequently, the spheres (100,000 or more) can be combined into yeast and pulverized cells in the form of "nano-drops", such that each drop includes a single sphere (and therefore a compound). Exposure of the nano-drops to UV light results in the release of the compounds from the drops, resulting in a method that allows rapid screening of large libraries.

Combinatorial libraries of compounds can be synthesized with "tags" that encode the identity of each member of the library (see WC Still et al ., US Patent No. 5,565,324 and PCT Publication No. WO 94/08051 and WO 95/28640). In general, this method includes the use of inert, but easily detectable, markers that bind to solid support or compounds. When an active compound is found (by one of the techniques described above), the identity of said compound is determined by the identification of the accompanying label. This method of labeling allows the synthesis of large libraries of compounds that can be identified even at very low levels. Such a labeling scheme may be useful (for example, in the "nano-drop" screening system), to identify compounds released from microspheres.

Compound libraries must contain the minus 30 compounds, and it is preferable that they contain 100 to 500, although they should not exceed 10 7 -10 9.

Search or screening trials

This invention provides methods (also referred to as screening assays) to identify modulators, that is, candidate compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecular molecules or other drugs) that bind to the protein. of early activation or nucleic acids encoding it and have a stimulatory or inhibitory effect on, for example, the expression or activity of the early activation polypeptide. Compounds identified in this way can be used to modulate the activity of the target gene products (early activation polypeptide genes) in a therapeutic protocol, to develop their biological function or to identify compounds that block normal interactions of the target gene.

Thus, this invention includes assays to identify candidate compounds that are substrates of the early activation protein or polypeptide, or a biologically active portion thereof. On the other hand, this invention includes assays for the screening of candidate compounds or that can bind or modulate an activity of the early activation protein or polypeptide, or a biologically active portion thereof, for example, modulators of signaling by the polypeptide. Early activation

Assays of early activation protein activity in the presence of inhibitors can be used to determine the nature of the signaling activity. The assay may include additional controls and steps to ensure that the activity observed comes from the early activation polypeptide, for example, control samples, such as samples produced from cells transformed with a control vector instead of a vector that overexpresses the DNA of the polypeptide. Early activation The activity can also be observed in chromatographic fractions to determine, for example, if the observed activity and the early activation polypeptide copurify. Additional trials and conditions have been described, see Yamaoka et al . (1998) J. Biol Chem 273: 11895-11901, and Thien-Khai et al . (1997) J. Biol Chem 272: 31315-31320.

The compounds tested in the present invention can be obtained using any of the numerous methods of obtaining combinatorial libraries described in the literature, including: biological libraries; Peptoid libraries (libraries of molecules that have peptide functionality, but with a non-peptide skeleton that is resistant to enzymatic degradation but nonetheless remains bioactive; see Zuckermann, RN et al . (1994) J. Med. Chem . 37: 2678-85); libraries of solid phase or in spatially manipulable solution in parallel; synthetic libraries that require convolution; the "one-compound one-sphere"method; and synthetic libraries using affinity chromatography selection. The biological and peptoid approaches are limited to peptide libraries, while the other four approaches can be applied to libraries of peptides, non-peptide oligomers or molecules of small molecular size (Lam (1997) Anticancer Drug Des . 12: 145) .

Examples of methods for the genesis of molecular libraries can be found in the literature, for example in: DeWitt et al . (1993) Proc. Natl Acad. Sci. USA . 90: 6909; Erb et al . (1994) Proc. Natl Acad. Sci. USA 91: 11422; Zuckermann et al . (1994). J. Med. Chem . 37: 2678; Cho et al . (1993) Science 261: 1303; Carrell et al . (1994) Angew. Chem. Int. Ed. Engl . 33: 2059; Carell et al . (1994) Angew. Chem. Int. Ed. Engl . 33: 2061; and Gallop et al . (1994) J. Med. Chem . 37: 1233.

Compound libraries can be presented in solution (see Houghten (1992) Biotechniques 13: 412-421), or in microspheres (Lam (1991) Nature 354: 82-84), biochips (Fodor (1993) Nature 364: 555- 556), bacteria (Ladner, US Patents No. 5,223,409), spores (Ladner US Patents No. 5,223,409), plasmids (Culi et al . (1992) Proc Natl Acad Sci USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al . (1990) Proc. Natl. Acad. Sci . 87: 6378-6382; Felici (1991) J. Mol Biol . 22: 301-310; Ladner supra .).

An essay is defined as that test based in cells in which a cell that expresses the protein of early activation or a biologically active portion thereof is contacted with a compound and the capacity is determined thereof to modulate the activity of the activation polypeptide early The determination of the capacity of the tested compound to modulate the activity of the early activation polypeptide it can be done by monitoring, for example, the perception of environmental stimuli, such as a polypeptide ligand of early activation, or of biological messengers, such as TGF-?. The cell can be mammalian, by example, human.

The capacity of the compound to modulate the binding of the activation polypeptide early to the same or another compound, for example, a substrate of the early activation polypeptide. This can be achieved coupling the compound to a radioisotope (for example, substrate) or an enzymatic "tag", such that the union of the compound to the early activation polypeptide can be detected at through marking, in a complex. Alternatively, the early activation polypeptide can be coupled to a radioisotope or enzymatic "tag" to monitor the ability of a compound to modulate the binding of the polypeptide of early activation to a substrate of the activation polypeptide Early in a complex. For example, the compounds (can be early activation polypeptide substrates) can be labeled with 125 I, 35 S, 14 C or 3 H, both direct and indirectly, and the radioisotope is detected by direct counting of radio broadcast or scintillation counting. Alternatively, the compounds can be enzymatically labeled with, for example, peroxidase, alkaline phosphatase or luciferase, detecting the labeling enzymatic by determining the conversion of a appropriate product substrate.

The ability of a compound (for example, a substrate of the early activation polypeptide) to interact with the early activation polypeptide, with or without the labeling of any of the interacting agents can also be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with the early activation polypeptide without labeling any of the parts (compound or early activation polypeptide) (McConnell, HM et al . (1992) Science 257: 1906-1912). Used in this context, a microphysiometer (for example, Cytosensor) is an analytical instrument that measures the speed with which the cell acidifies its surroundings using a potentiometric sensor stimulated by light ( light-addresable potentiometric sensor, LAPS ). Changes in this acidification rate can be used as an indication of the interaction between a compound and the early activation polypeptide.

Also included is an essay that does not include employ cells in which the early activation protein or a biologically active portion of it is contacted with a compound, evaluating its ability to bind to it. In these assays, the biologically active portions of the protein Early-activated agents preferably include fragments that participate in interactions with different molecules of the early activation polypeptide, for example, fragments with high surface probabilities

The soluble or membrane-associated forms of isolated proteins (CD69, AICL, or LLT1 proteins, or portions biologically active thereof) can be used in cell-free assays included in this invention. When use forms associated with the protein membrane, it is it is advisable to use a solubilizing agent, such as detergents not ionics such as n-octylglucoside n-dodecylglucoside, n-dodecylltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecipoli (ethylene glycol ether) n, sulfonate 3 - [(3-colamidopropyl) dimethylamine] -1-propane (CHAPS), sulphonate 3 - [(3-colamidopropyl) dimethylamine] -2-hydroxy-l-propane  (CHAPSO), or sulfonate of N-dodecyl = N, N-dimethyl-3-ammonium-1-propane.

Cellless assays involve preparation of a reaction mixture with the target protein and compound tested under such conditions and for long enough to allow the interaction and union of the two components, forming a  complex that can be removed and / or detected.

The interaction between two molecules can also be detected using techniques such as fluorescent energy transfer (FET) (see, for example, Lakowicz et al ., US Patent No. 5,631,169; Stavrianopoulos, et al ., US Patent No. 4,868,103). A fluorescent label in the first molecule (donor) is selected, so that the fluorescent energy emitted by the brand will be absorbed by a second fluorescent marker present in the second molecule (acceptor), which in turn emits fluorescence when excited by the absorbed energy Alternatively, the donor protein can simply use the natural fluorescent energy of tryptophan residues. Fluorescent markers of different emission wavelength are chosen, so that the donor's emission can be unequivocally identified from that of the acceptor. Since the efficiency of fluorescent energy transfer depends on the distance that separates the molecules, the spatial relationship between them can be evaluated. In a situation where there is union between the molecules, the fluorescent emission of the acceptor must be maximum. Thus, the FET due to the binding of two molecules can be measured through standard fluorescence detection techniques (such as a fluorimeter).

The detection of the binding of CD69 to a target molecule can also be performed using biomolecular interaction analysis ( Biomolecular Interaction Analysis, BIA ) (see Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al . (1995) Curr. Opin. Struct. Biol . 5: 699-705). Surface plasmon resonance (SPR ) or "BIA" surface resonance detects biospecific interactions in real time, without labeling any of the participating molecules (for example, BIAcore). Changes in the mass of the interaction surface (indication of an interaction) cause alterations in the index of refraction of light near the surface (the phenomenon of SRP), resulting in a detectable signal that can be used as an indicator of a real-time reaction between biological molecules.

On the other hand, the target gene product or the investigated compound can be anchored in a solid phase, and can detected at the end of the reaction. Preferably, the target gene product and the compound under investigation (which is not immobilized) can be marked, either directly or indirectly with  detectable markers such as those described previously.

It may be of interest to immobilize the protein of early activation, an antibody against the polypeptide of early activation or its target molecule to facilitate the separation of complex forms of uncomplexed one or both proteins, as well as to facilitate the automation of the assay. The binding of the tested compounds to the activation polypeptide early, or the binding of said early activation polypeptide with a target molecule in the presence or absence of the compound tested can be made in any suitable container to contain reagents Examples of such containers include plates multiwell, test tubes or microcentrifuge. In a embodiment of these examples, a protein of fusion that allows the union of the molecule to a matrix. By example, fusion proteins of the type can be adsorbed glutathione-S-transferase / polypeptide early activation or glutathione-S-transferase / protein target to sepharose microspheres with glutathione (Sigma Chemical, St. Louis, MO), or to multiwell plates derivatized with glutathione, which are combined with the investigated compound or polypeptide of early activation and non-adsorbed target protein or polypeptide of early activation, said mixture being incubated under conditions favorable for complex formation (including conditions physiological ionic strength and pH). After incubation, the matrix is washed to remove unbound components and formation of complexes is determined directly or indirectly as it has been described above, or the complexes can be dissociated from the matrix and level of binding or activity of the polypeptide of early activation can be determined by techniques conventional.

Other techniques to immobilize or a polypeptide of early activation or a target molecule in matrices includes the conjugation of biotin and streptavidin. Activation protein Early biotinylated or other target molecules can be prepared to from biotin-NHS (N-hydroxy-succinimide) using techniques described in the literature (for example, kit biotinylation, Pierce Chemicals, Rockford, IL), and immobilized in 96-well plates coated with streptavidin.

To perform this test, the component does not immobilized is added on the surface that has been coated with The anchored component. After finishing the reaction, the components in excess (washing), under conditions in which guarantees the maintenance of immobilized complexes. The Detection of them can be done in different ways: when the non-immobilized component is previously marked, the surface anchored marking detection indicates formation of complexes. When the non-frozen component is not marked,  indirect labeling can be used to detect complexes immobilized, for example, using a labeled antibody specific immobilized component (the antibody, in turn, it can be directly or indirectly marked, in the latter case, using a labeled anti-Ig antibody).

This test is performed using antibodies reagents against early activation protein or a molecule target, but that do not interfere with the binding of the polypeptide of early activation to its target molecule. Such antibodies can derivatize into wells, and the unbound target, or the Early activation polypeptide, can be captured in the wells by conjugation with antibodies. As methods for detect these complexes, we can cite the immunodetection of complexes, using antibodies reactive against the polypeptide of early activation, or the target molecule, as well as assays enzymatic based on the detection of an enzymatic activity associated with the early activation polypeptide or its target.

Alternatively, cellless assays can be performed in liquid phase. In such assays, reaction products are separated from non-reactive compounds by conventional techniques that include: differential centrifugation (see Rivas, G., and Minton, AP, (1993) Trends Biochem Sci 18: 284-7); chromatography (molecular exclusion, phonic exchange); electrophoresis (see Ausubel, F. et al ., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology , J. Wiley: New York). Such techniques are common in any laboratory (Heegaard, NH, (1998) J Mol Recognit 11: 141-8; Hage, DS, and Tweed, SA (1997) J. Chromatogr. B. Biomed. Sci. Appl . 699: 499 -525). Fluorescent energy transfer (FET) can also be used to detect binding without purifying the complexes.

Preferably, this test includes the contact of the early activation molecule or a biologically part active thereof with a known compound that binds to early activation polypeptide forming a complex, and adding a second compound from which its capacity is determined to bind preferentially to the early activation polypeptide or to a biologically active part thereof, or its modulating activity of the activity of a target molecule, a known compound being used as reference.

The target gene products of this invention may, in vivo , interact with one or more cellular or extracellular macromolecules, such as proteins, which, for discussion purposes, will be referred to as "ligands." Those compounds that block these interactions may be useful in regulating the activity of the target gene products. Such compounds include, but are not limited to: molecules such as antibodies, peptides and molecules of small molecular size. Preferred target gene products are the early activation polypeptide genes identified herein. An alternative aspect of this invention includes methods for determining the ability of the studied compound to modulate the activity of the early activation protein through the modulation of a molecule activated by it, for example, the activity of an effector molecule on a molecule. target, or the binding of an effector to an appropriate target. The reference test in this case is the determination of TGF-? Activity.

To identify compounds that interfere with the interaction between the target gene products and their ligands cellular or extracellular, a reaction mixture is prepared containing the target gene product and its ligand, under conditions adequate and for sufficient time for the formation of complex. To test an inhibitory agent, the reaction mixture it is incubated in the presence or absence of it, and may be present in the reaction mixture from the first moment or be added some time after incubation of the target gene and its cellular or extracellular ligand. Control reaction mixtures are incubate in the absence of the compound or with a placebo. It is detected then the formation of complexes between the target gene and the ligand.  The formation of complexes in the control reaction but not in the case in which the reaction includes the compound tested indicates that the Compound interferes with the interaction of the target gene and the ligand. In addition, the formation of complexes in the mixture can be compared reaction reaction containing the test compound and the normal product of the target gene with the formation of complexes in the mixture of reaction containing the tested compound and mutant products of the target gene, which is important in those cases where it is desirable to identify compounds that disrupt the interactions of mutant target genes but not normal ones.

These tests can be performed in formats homogeneous or heterogeneous, which include the anchoring of good, the gene target or else the ligand to a solid phase, and the detection of solid phase anchored complexes at the end of the reaction. In homogeneous tests, the reaction is carried out in liquid phase at full. Both in one and another situation, the order of addition of the reagents can be varied to obtain different information on the target complexes of the trial. For example, the compounds tested that interfere with the interaction between target products and their ligands, for example by competition, can be identified by carrying out the reaction in the presence of substances tested. Alternatively, the compounds tested that undo preformed complexes, for example, compounds with high binding constants that displace one of the components of the complex, can be tested by adding the compound tested to the reaction mixture after complexes have formed. The test formats are briefly described below.

In a heterogeneous test system, or the target gene product, either the cellular or extracellular ligand is anchor in a solid phase (for example, titration plates), while the non-anchored reagent is marked directly or indirectly. The anchored reagent can be immobilized by covalent or non-covalent junctions. Alternatively, you can use a specific antibody for the reagent, which acts as bridge between the substrate and the reagent for binding to the phase solid.

To perform the assay, the reagent ligand immobilized is applied on the reaction surface in the presence or absence of the compound tested. Upon completion of the reaction, reagents that have not reacted in are washed conditions such that the complexes formed remain immobilized on the surface. When the reagent does not Fixed assets are previously marked, marking detection surface immobilized indicates the formation of complexes. When the non-immobilized reagent is not marked, it can be employ an indirect label for the detection of complexes in the reaction surface, for example, using an antibody specific for the labeled non-immobilized reagent (which may, at in turn, be marked directly or indirectly, in this case by an anti-Ig antibody). Depending on order of addition of reaction components, compounds tested can be grouped into those that inhibit the formation of complexes or those that previously undo the complexes formed.

Alternatively, the reaction can be performed. in the liquid phase, in the presence or absence of the compound tested, separating the reaction products from the reagents not reacted, subsequently detecting the complexes by, for example, the use of specific antibodies for one of the binding components immobilized to anchor any complex formed in solution, and a specific labeled antibody for the another ligand to detect anchored complexes. Again and depending on the order of addition of the reagents to the phase liquid, can distinguish between compounds that inhibit the complex formation and those that undo complexes previously formed.

As an alternative to that presented above, homogeneous tests can be used. For example, preformed complexes of the target gene product and its cellular or intracellular ligands, in which one or the other are labeled, can be prepared, but the signal generated by the labeling is shielded by the formation of the complex (US Pat. Nos. 4,109,496, which uses this approach for immunoassays). The addition of a substance that competes and displaces one of the components of the preformed complex will result in an increase in the signal. In this way you can identify substances that block the interaction of the target gene product and its
ligands

The early activation protein can be used as bait in double or triple hybrid assays (see, for example, US Patents No. 5,283,317; Zervos et al . (1993) Celi 72: 223-232; Madura et al . (1993) J Biol. Chem . 268: 12046-12054; Bartel et al . (1993) Biotechniques 14: 920-924; Iwabuchi et al . (1993) Oncogene 8: 1693-1696; and Brent WO94 / 10300) to identify other proteins that bind or interact with the early activation polypeptide (early activation binding protein polypeptide or early activation polypeptide -bp), and which are involved in the activity of the early activation polypeptide (eg, TGF-? expression). Such early activation polypeptides -bp can be activators or inhibitors of signaling by the early activation polypeptide or its targets, such as, for example, elements activated by signaling induced by the early activation polypeptide.

The double hybrid system is based on the modular nature of most transcription factors, which consist of independent domains of DNA binding and activation. Briefly, the assay uses two different DNA constructs. One of them codes for the early activation polypeptide fused to the DNA binding domain of a known transcription factor (eg, GAL-4). The other construct includes a DNA sequence from a library, which codes for an unidentified protein ("prey" or "sample"), fused to a gene that codes for the activation domain of the known transcription factor (alternatively , the early activation polypeptide can be fused to the activation domain). If the "bait" and the "prey" are able to interact in vivo forming a complex dependent on the early activation polypeptide, the DNA binding and transcription factor activation domains are very close, allowing the transcription of a gene reporter (for example, lacZ), which is operationally associated with a factor-sensitive transcription regulatory site. Reporter expression can be detected and cell colonies that contain the functional transcription factor are used to obtain the cloned gene, which codes for the protein that interacts with the early activation polypeptide.

Another aspect includes the identification of modulators of early activation polypeptide expression. For example, a mixture with or without cells is incubated with a candidate compound and the expression of the m-RNA or early activation protein in relation to their expression in the absence of the compound. When m-RNA or protein expression of Early activation is lower (statistically significant) in presence of the compound studied that in its absence, it identifies as an expression inhibitor of m-RNA or early activation protein (it is ie, early activation polypeptide antagonist). He m-RNA or activation protein expression level  early can be determined by known methods for determination of m-RNA levels or protein.

In another section, this invention contemplates the combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-free cell assay, and its ability to modulate the activity of the early activation polypeptide can be confirmed in vivo in animal models such as cancer.

This invention also includes new agents identified by the screening assays described above. Accordingly, it is within the use of this invention the subsequent use of an agent identified as described herein (for example, an early activation polypeptide modulating agent, an antisense nucleic acid molecule of the early activation polypeptide , a specific antibody against the early activation polypeptide, or any other ligand of the early activation polypeptide) in an appropriate animal model to determine the efficacy, toxicity, side effects or mechanism of action of a treatment with such agents. Moreover, new reagents identified by the screening assays described above can be used for treatments such as those described herein.

Substantially identical or homologous

The substantially identical or homologous term refers to a first amino acid or nucleotide sequence that contains a sufficient number of amino acids or nucleotides identical or equivalent (i.e. with side chains similar, conserved amino acid substitutions, etc.) to a second amino acid or nucleotide sequence, such that the first and the second sequence have similar activities. In the case of antibodies, the second antibody has the same specificity and at least 50% of the affinity shown for the first.

Homology calculations between two sequences can be carried out as follows: the sequences are aligned to make an optimal comparison (it is possible to introduce gaps in one or both sequences for optimal alignment, and the non-homologous sequences can be discarded). Ideally, the length of a reference sequence aligned for sequence comparison is at least 30% of the total sequence, although it is all the better the higher the percentage. Thus, amino acid residues or nucleotides of both chains are compared in corresponding positions. When there is an exact match for both sequences in a given position, then both sequences are identical in that position (the terms "identity" and "homology" are used interchangeably). The percentage of identity between two sequences is a function of the number of identical positions found in both sequences, taking into account the number of gaps whose introduction is required for optimal alignment, as well as their length.

The sequence comparison and the determination of the percentage of homology between two sequences can be performed using mathematical algorithms. Ideally, the Needleman and Wunch algorithm (Needleman and Wunsch (1970), J. Mol. Biol . 48: 444-453) is used, which has been implemented in the GAP program of the GCG software package, using a Blossum matrix 62, either a PAM250 and a " gap weight " of 16, 14, 12, 10, 8, 6, or 4 and a " length weight " of 1, 2, 3, 4, 5 or 6. Another suitable way to calculate The homology percentage is to use the GCG software GAP program, using an NWSgapdna matrix. CMP and a " gap weight " of 40, 50, 60, 70 or 80 and a " length weight " of 1, 2, 3, 4, 5, or 6. The group of reference parameters (which should be used if the researcher not sure of the parameters that should be applied to determine if a molecule is within the homology limitation of the invention) a Blossum 62 matrix is constituted with a gap penalty of 12, an extension of the gap penalty of 4 and a penalty of " frameshift gap " of 5.

As used herein, the term "hybridize under conditions of low, medium, high or very high stringency" describes the conditions of hybridization and washing. Guidelines for performing hybridization reactions can be found in Current Protocols in Molecular Biology , John Wiley & Sons, NY (1989), 6.3.1-6.3.6. This quotation describes methods in aqueous or non-aqueous solution that can be used. The specific hybridization conditions referred to here are the following: 1) low stringency, sodium chloride / citrate (CCS) 6 x at 45 ° C, followed by two washes in 0.2 x CCS, 0.1% SDS at least 50 ° C (the washing temperature can be increased to 55 ° C); 2) medium stringency, 6 x CCS at 45 ° C followed by at least one wash in 0.2 x CCS, 0.1% SDS at 60 ° C; 3) high stringency, 6 x CCS at 45 ° C followed by at least one wash at 0.2 x CCS, 0.1% SDS at 65 ° C, or preferably 4) very high stringency, 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by two washes in 0.2X CCS, 0.1% SDS at 65 ° C, the condition (4) being the reference one unless otherwise specified.

It is understood that the binding polypeptides of this invention may have additional conservative or nonessential substitutions, which do not have a substantial effect on the functionality of the polypeptides. It can be determined whether a specific substitution will be tolerable (will not adversely affect the desired biological properties, such as binding activity) as described by Bowie et al . (1990) Science 247: 1306-1310. A "conserved amino acid substitution" is defined as one in which a residue is replaced by another that has a similar side chain, which is well established in the literature. The amino acid families with similar side chains are: basic side chains (lysine, arginine, histidine), acidic (aspartic and glutamic acids), uncharged but polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched (threonine, valine, isoleucine) and aromatic (tyrosine, phenylalanine, tryptophan, histidine) side chains.

A "non-essential" residue is defined as one that can be altered with respect to the wild type of the binding reagent (antibody or others), without inhibiting, or better yet, without substantially altering the biological activity, while an "essential" amino acid residue is one that results in such changes.

Pharmaceutical compounds

On the other hand, the present invention includes compounds (pharmacologically acceptable), which include an early activation polypeptide binding reagent (early activation anti-polypeptide antibody), as described herein, formulated together with a suitable carrier .

In the category of pharmacologically carriers Acceptable includes all solutions, dispersion media, coatings, antibacterial and antifungal reagents, reagents  isotonic and absorption delay and the like that are physiologically compatible The carrier must be suitable for intramuscular, subcutaneous, parenteral, spinal or epidermal (by injection or infusion). Depending on the route of administration, the active substance (the binding reagent to early activation polypeptide), can be coated in a material that protects it from the action of acids or other natural conditions that could inactivate it.

A "pharmacologically acceptable salt" refers to a salt that retains the desired biological activity of the compound from which it is derived, and that does not cause any unwanted toxicological effects (see Berge, SM, et al . (1977) J. Pharm. Sci 66: 1-19). Such salts include acid or base addition salts. Acid additions include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromhydric, hydroiodhydric, phosphoric and the like, as well as non-toxic organic acids, such as mono- and dicarboxylic aliphatic acids, phenyl alkanoic acids -substituted, hydroxyalkanoic, aromatic, aliphatic and aromatic sulfonic, and the like. Base addition salts include those derived from alkali and alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as non-toxic organic amines, such as N-N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The composition can be presented in various formats, which include, for example, forms of administration solid, semi-solid and liquid, as liquid solutions (injectable or by infusion), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The reference format depends of the mode of administration and therapeutic application, although the more typical are in the form of injectable or infusion solutions, with composition similar to that used for passive immunization of humans with other antibodies. The mode of administration of reference is parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular), optimally, by infusion or injection intravenously (for example, with needleless injection). Another way Optimal is by intramuscular or subcutaneous administration.

The phrases "parenteral administration" or "parenterally administered" designate, in this context, a different modes of administration of enteral administration or topical, usually by injection and that includes, without limitations, intravenous, intramuscular, intraarterial injection or infusion, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural or intrasternal.

Therapeutic compositions must be sterile and stable during manufacturing and storage, and It can be formulated in solution, microemulsion, dispersion, liposomes, and other ordered structures suitable for the preparation of High concentrations of compounds. Solutions can be prepared Sterile injectables incorporating the active substance (antibody or a part thereof) in a suitable amount in a solvent suitable with one or more of the ingredients listed previously, followed by filtration under conditions of sterility. In the case of powders for the preparation of solutions  Sterile injectables, the reference methods are drying to vacuum and drying-frozen, from which you get a powder of the active substance in addition to any other ingredient additional present in the starting solution. You can keep a adequate degree of fluidity using coatings such as lecithin, maintaining the particle size in the case of dispersions and through the use of surfactants. Prolonged absorption of injectable compositions can be achieved including a compound which retards absorption, such as a monostatic salt or jelly.

Early activation polypeptide binding agents can be administered through a wide variety of methods described in the literature, although for many therapeutic applications, the route of reference administration is intravenous injection or infusion. Naturally, the route of administration may vary depending on the achievement of the desired results. In certain situations, the compound may be prepared together with a carrier that prevents rapid release of the compound, such as controlled release formulations including implants, transdermal patches and microencapsulated release systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many examples of such formulations are found in the literature ( Sustained and Controlled Release Drug Delivery Systems , JR Robinson, ed., Marcel Dekker, Inc., New York, 1978).

In some cases, compounds can be administered Pharmaceutical Activation Polypeptide Binding Agents early, alone or in combination with other agents, topically or by transdermal patches. In cases where the agent of union is a molecule of small molecular size you can administer orally In addition, the compounds can be administered parenterally, especially for the treatment of arthritis, psoriatic arthritis, and for direct injection into lesions of the skin. Parenteral therapy is typically intradermal, intraarticular, intramuscular or intravenous. Binding agents to the early activation polypeptide can be applied directly together with a carrier in the form of cream or oil, or in  aerosol form, as well as orally.

The therapeutic compounds can be administered through medical equipment described in the literature. For example, a therapeutic compound of this invention can be administered through a needleless hypodermic injection system, such as described in U.S. Pat. Nos. 5,399,163, 5,383,851, 5, 312, 335, 5, 064, 413, 4, 941, 880, 4, 790, 824, or 4,596,556. Others Implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which includes an implantable pump of microinfusion to administer medication at controlled speed; U.S. Patent No. 4., 486,194, which includes a therapeutic instrument to administer medications through the skin; U.S. Patent Do not. 4,447,233, which includes a drug infusion pump an accurate infusion rate; U.S. Patent No. 4,447,224, which describes an implantable variable flow infusion apparatus for continuous administration; U.S. Patent No. 4,439,196, which describes an osmotic system of medication administration with multi-chamber compartments; and U.S. Patent Do not. 4,475,196, which includes a medication administration system osmotic. These patents are included as a reference. Much others implants and administration systems have been described in the literature.

The administration regimes are adjusted to optimize the desired response (therapeutic or others). For example, you can administer a single bolus, or divided doses over time, or the dose can be increased or reduced according to the demands of the therapeutic situation. The formulation of parenteral compounds in dosage form is especially convenient to facilitate administration and confer uniformity in the administration. The dose unit, as described here, refers to physically discrete units in unit doses for the treatment of the subjects; each unit contains an amount default of the active compound calculated to produce the desired therapeutic effect in association with the carrier Pharmacist required The dose unit specification of This invention is dictated by and is directly dependent on: a) The unique characteristics of the active substance and the effect particular therapeutic to be achieved, and b) The limitations  inherent described to compose an active principle for the sensitivity treatment in individuals.

An exemplary and non-limiting range of antibody or a portion thereof therapeutically or prophylactically effective is between 0.1 and 20 mg / kg, preferably between 1-10 mg / kg Antibody early activation anti-polypeptide will administered by intravenous infusion to at least preferably or equal to 5 mg / min to reach a dose between 1 to 100 mg / m2, preferably between 5 and 50 mg / m2, more optimally, between 7 and 25 mg / m2, over 10 mg / m2. Must highlight that the doses may vary with the type or severity of the therapeutic condition It is to be understood that, for a situation In particular, it is necessary to adjust dosage regimens specific over time to adapt to the needs individual and professional judgment of the person administering or supervises the use of the compounds, and that the ranges of Dosage explained above are used exclusively as an example and are not aimed at limiting the range or practice of the claimed compounds.

The pharmaceutical compounds of this invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a binding reagent. A "therapeutically effective amount" refers to the effective amount, applied in doses and for periods of time required to achieve the desired therapeutic result. A therapeutically effective amount of an early activation polypeptide binding reagent may vary according to factors such as the disease status, age, sex and weight of the individual, as well as the ability of the antibody or a portion thereof to induce A desired response in the individual. A therapeutically effective amount is also defined as that in which any toxic or negative effect of the early activation polypeptide binding reagent is exceeded by the beneficial therapeutic effect. A "therapeutically effective dose" preferably inhibits a quantifiable parameter in relation to untreated subjects. The ability of the compound to inhibit a quantifiable parameter can be measured in an animal model predicting its effectiveness in humans. Alternatively, this property of a compound can be evaluated by examining the inhibitory capacity of the compound, for example, in vitro inhibition, in assays described in the literature. A "prophylactically effective amount" refers to the effective amount, in adequate doses and for a certain period of time, required to achieve a desirable prophylactic result. Typically, and since a prophylactic dose is used in individuals in early stages of disease, or even earlier, the prophylactically effective amount should be less than the therapeutically effective amount.

Applications

Antagonists and polypeptide scavengers of early activation described here or identified by methods described here can be used to treat a subject that has a disorder characterized by an increased immune response or diminished For example, polypeptide antagonists of Early activation can be used to treat a subject that have a disorder characterized by an immune response diminished or to a subject that without having diminished or compromised your immune system can benefit anyway increased immune response. Such disorders include those characterized by unwanted cell proliferation processes, like cancer, or an immunodeficiency. The methods include the administration of a therapeutically effective amount of a early activation polypeptide antagonist to a subject with a syndrome like the one described above. It also can prevent the occurrence of such disorders by administration of a prophylactically active amount of a early activation polypeptide antagonist.

Thus, in some aspects, early activation polypeptide antagonists can be used to treat or prevent unwanted cell proliferation processes such as cancer, by inhibiting growth or direct elimination of the hyperproliferative cell. The early activation polypeptide antagonist may decrease the expression and / or activity of the early activation polypeptide, thereby enhancing immunity against tumors of the subject. For example, in subjects with cancer, eg, lymphatic leukemia, eg, chronic B lymphatic leukemia without immunoglobulin mutations, the expression of AICL increases (Klein et al . (2001) J. Exp. Med. 11: 1625; Rosenwald et al . (2001) J. Exp. Med. 11: 1639). An AICL antagonist, eg, a blocking antagonist or that decreases the expression of AICL, can be used to decrease the expression or activation of AICL, thereby increasing immunity in such subjects. It seems to have been shown that early activation polypeptide antagonism inhibits TGF-? Synthesis. Since TGF-? Can act as an immunosuppressant, inhibition of TGF-? Production allows immune cells to continue to inhibit the growth or proliferation of hyperproliferative cells. In addition, an eliminator of an early activation molecule can be used to treat or prevent tumors that express cancers that express the early activation molecule. For example, an AICL scavenger can be used to inhibit the growth or proliferation of cells that express AICL in subjects who have lymphatic leukemia, eg, chronic B lymphatic leukemia without mutation in immunoglobulins. As used in the text, the expression "inhibit the growth or proliferation" of a hyperproliferative cell (neoplastic cell), refers to the delay, interruption, blockage or arrest of its growth and metastatic capacity, and does not necessarily indicate elimination. Total neoplastic growth. The use of the term "induce death" of the hyperproliferative (neoplastic) cell defines the partial or complete elimination of said cells, and not necessarily the total elimination of neoplastic growth.

The terms "induce", "inhibit", "boost", "raise", "increase", "decrease" and similar ones, which denote differences Quantitative between two states, refer to at least statistically significant differences with respect to untreated cells Such terms are used here to refer, for example, to cell proliferation rates.

The terms "cancer", "hyperproliferative" and "neoplastic" designate those cells with autonomous growth capacity. As an example of These cells can be cited to those found in a abnormal state or condition characterized by cell growth and proliferation very fast. Hyperproliferative situations or Neoplastic can be classified as pathological (characterizing or constituting a disease) and not pathological (deviated from normal behavior, but not associated with a state of disease). This term includes all types of growth cancerous or oncogenic processes, tissues in metastases or cells transformed malignancies, tissues or organs, regardless of Histopathological type or level of invasion. The cells "pathologically hyperproliferative" appear in states of disease characterized by the growth of malignant tumors. On the other hand, as examples of hyperproliferative cells not pathological, the cells involved in the wound repair

Therefore, from a certain point of view, a early activation polypeptide antagonist can be employed to treat or prevent disorders characterized by processes of non-pathological hyperproliferation, such as disorders fibrotic The disorder can be associated with one or more of: trauma, surgery, infection, environmental pollution, tobacco, alcohol or other toxins The disorder can be associated with, by example, one of the following: hyperefficient repair of wounds (e.g., hypertrophic scars, keloids), renal fibrosis (mesangial cell proliferation), or liver fibrosis (proliferation of liver starry cells). Examples of Fibrotic disorders include: keloids, burns, scars hypertrophic or other skin disorders (e.g., scleroderma local or systemic, e.g., systemic sclerosis with scleroderma), liver cirrhosis, renal fibrosis (e.g., related to diabetes or hypertension), surgical adhesions (e.g., after surgery gastrointestinal or neurosurgery adhesions), transplants vascular, idiopathic pulmonary fibrosis, induced fibrosis radiation, asbestos-related fibrosis (e.g., brown lung or black), fibrosis associated with viral hepatitis, degeneration macular, vitreal and retinal retinopathy, and fibrosis associated with acute respiratory syndrome Fibrosis can be acute or chronic. Examples of acute fibrosis include: accidental wounds, infections, surgery, burns, radiation-induced fibrosis and Chemotherapy-induced fibrosis. Chronic fibrosis can be Associate with, e.g., viral infections, diabetes and hypertension.

The terms "cancer" or "neoplasm" designate processes of malignization of different systems of organs, such as those affecting the lungs, breasts, gland thyroid, gastrointestinal tracts and genito-urinary as well as adenocarcinomas that include malignant syndromes like most cancers of colon, renal cell carcinomas, prostate cancer and / or testicular tumors, large cell lung carcinomas, small intestine and esophageal cancer.

The term "carcinoma" is recognized in the  literature and refers to malignancies of epithelial tissue or endocrine, including carcinomas of the respiratory system, gastrointestinal, genitourinary, testicular, breast, prostate, of the endocrine system and melanomas. Some examples are those that are formed from the tissue of the cervix, lung, prostate, breast, head and cervix, colon and ovaries. In the definition of the term, carcinosarcomas are included, such as those that They are composed of carcinomatous and sarcomatous tissue. A "adenocarcinorma" refers to a carcinoma derived from tissue glandular or in which tumor cells form structures Glandular recognizable morphology. The term "sarcoma" It is contemplated in the literature and refers to malignant tumors Mesenchymal tissue derivatives.

Some examples of proliferative disorders and / or differentiation include cancer (for example, carcinomas, sarcomas, metastatic disorders or neoplastic disorders hematopoietic, such as leukemia). A metastatic tumor may arise from a multitude of primary tumor types, including, but not limited to, those of prostate, colon, lung, breast and liver.

Other examples of cancer or situations Neoplastic include the following types of disorders and cited above, but not all existing ones are cited: fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, sarcoma osteogenic, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelium-sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, cancer uterine, head and cervical cancer, skin cancer, brain tumors, squamous cell carcinoma, carcinoma of sebaceous gland, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, tract carcinoma biliary, choriocarcinoma, seminoma, embryonic carcinoma, tumor of Wilm, cervical cancer, testicular cancer, lung carcinoma of small cells, large cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi's sarcoma.

There are additional examples of proliferative disorders, which include neoplastic hematopoietic disorders. In this context, the term "hematopoietic neoplastic disorders" designates diseases that involve hyperplastic / neoplastic cells of hematopoietic origin. A hematopoietic disorder of these characteristics may arise from myeloid, lymphoid or erythroid lineages, or precursors thereof. Typically, these diseases arise as poorly differentiated leukemias, such as acute erythroblastic or megakaryoblastic leukemia. Other additional examples include, but are not limited to: acute promyeloid leukemia ( acute promyeloid leukemia, APML ), acute myeloblastic leukemia ( acute myelogenous leukemia, AML ) and chronic myeloblastic leukemia ( CML , reviewed in Vaickus, L. ( 1991) Crit Rev. in Oncol./Hemotol . 11: 267-97); Lymphoid tumors include, but are not limited to, acute lymphoblastic leukemia ( ALL ) that includes ALL of B and T lineages, chronic lymphocytic leukemia ( chronic lymphocytic leukemia, CLL ), (eg, chronic lymphocytic leukemia T or B , eg, chronic B lymphocytic leukemia with or without immunoglobulin mutation), prolymphocytic leukemia (prolymphocytic leukemia, PLL ), hairy cell leukemia (hairy cell leukemia, HLL) and Waldenstrom macroglobulinemia. Other forms of malignant lymphoma include, but are not limited to: non-Hodgkin lymphomas and their variants, peripheral T lymphocyte lymphomas, adult T-cell leukemia / lymphoma ( adult T cell leukemia / lymphoma, ATL ), cutaneous T-cell lymphomas ( cutaneous T-cell lymphoma, CTCL ), large granular lymphocytic leukemia, LGF , Hodgkin's disease and
Reed-Sternberg.

Activation Polypeptide Antagonists early can be administered in combination with one or more agents therapeutic, such as those indicated in the treatment or prevention of unwanted proliferative processes. These agents therapeutic include, for example, one or more agents chemotherapeutics, radioisotopes and / or cytotoxins. As examples of Chemotherapeutic agents may be mentioned taxol, cytochalasin B, Gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxyanthraindione, mitoxantrone, mitramycin, chlorambucil, gencitabine, actinomycin, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids and the like counterparts of the above. Other therapeutic agents include, but they are not limited to: antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (for example, mechlorethamine, thioepa chlorambucil, CC-1065, melfalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiaminoplatin (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (previously called daunomycin) and doxorubicin), antibiotics (for example, dactinomycin (previously referred to as actinomycin), bleomycin, mitramycin, and antramycin (AMC)), and antimitotic agents (for example, vincristine, vinblastine, taxol and maytansinoids). Radioisotopes can be alpha, beta emitters and / or gamma, and as examples, the following may be included: 212 Bi, 213 Bi, 131 I, 211 At, 186 Re, 90 Y and 117 Lu. The early activation polypeptide antagonist and therapeutic agents can be administered simultaneously or sequentially In addition, polypeptide antagonists of early activation can be co-administered with others modalities of cancer treatment, such as surgery to eliminate a part or all of the cancerous tissue or organ that contains

As used herein, the terms "cytotoxic agent", "anticancer agent" or "agent antitumor "are used interchangeably and refer to agents that have the property of inhibiting growth or proliferation (for example, a cytostatic agent), or to induce death of hyperproliferative cells. Preferably, the agent cytotoxic inhibits the development or progression of neoplasms, particularly solid tumors, soft tissue tumors, lesions metastatic, lymphomas or leukemia.

A "therapeutically active amount" of a  agent, alone or in combination with a cytotoxic agent, refers to to the amount of such agents in effective combination in the growth or proliferation inhibition, or induction of death of hyperproliferative cells, in single administration or multiple to a subject (a patient). Such inhibition of cell growth or death may be reflected in the prolongation of the survival of a subject (patient), beyond what was expected in the absence of such treatment, or any improvement in the subject's prognosis regarding the absence of such treatment.

A "prophylactically active amount" of an agent, alone or in combination with a cytotoxic agent, is refers to the amount of such agents in effective combination in the prevention or delay of the occurrence of recurrence processes of a disorder, for example, a neoplastic disorder.

When administered to patients under treatment against cancer, the activation polypeptide antagonist early can be administered in cocktails that contain other anti-cancer agents, and they can also administered in cocktails intended to treat the effects Side effects of irradiation therapy, such as anti-emetics, radiation protectors, etc.

The eliminators of the cells that express the Early activation polypeptide can be used to treat a subject suffering from a disorder characterized by a response Increased or exacerbated immune. Such disorders include chronic inflammatory disorders and immune disorders, such as autoimmune diseases. The methods include the administration of a therapeutically effective amount of a cell scavenger that express the early activation polypeptide to a subject that suffer from a disorder characterized by an immune response increased

Some inflammatory diseases can be treated using this invention, among which are: acute lung injury, acute respiratory stress syndrome, arthritis (for example, CIA), asthma, bronchitis, cystic fibrosis, hepatitis, inflammatory bowel disease, multiple sclerosis, reperfusion injury (eg, myocardium), nephritis, pancreatitis, psoriasis, occlusion of arteries (eg, retinal), stroke, systemic lupus erythematosus, transplants, ultraviolet light-induced damage, and / or vasculitis. The inflammatory disease can be chronic or acute, and preferably leukocyte-mediated (see reviews by Weissmann et al ., Ann. NY Acad. Sci . 389: 11-24, 1982; Janoff, A., Annu. Rev. Med . 36 : 207-216, 1985; Hart et al ., J. Rheumatol . 16: 1184-1191, 1989; Doring, Am. J. Respir. Crit. Care Med . 150: S114-S117, 1994; Demling, Annu. Rev Med . 46: 193-202, 1995). Diseases with symptoms of chronic inflammation include, but are not limited to: inflammation of intestinal handles, such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.

Activation Polypeptide Antagonists early described here or identified by any of the methods described here can be used to increase the response immune to an antigen, for example a vaccine, or a nucleic acid which codes for antigen. These methods include the administration of an antigen (vaccine) or a DNA encoding for an antigen and an antagonist of the activation polypeptide early, to increase the immune response to the antigen by part of the subject.

Examples of vaccines may include, vaccines against cancer, vaccines against HIV, vaccines against hepatitis, or malaria, or herpes, or papilloma, or flu, or smallpox. Several vaccines have been developed against cancer that could be given with an antagonist of early activation polypeptide described here. Examples of such Cancer vaccines include, but are not limited to, vaccines. autologous tumor cells; tumor cell vaccines allogeneic; gangliosides (e.g., for melanoma or sarcoma); CEA (e.g., for colorectal cancer, or other gastrointestinal tumors, pancreatic or breast cancer); PSA (e.g., for cancer of prostate); MART-1 100, tyrosinase (e.g., for melanoma); p53 (for several tumors that express p53); alpha-fetoprotein (AFP) (e.g., for cancers of liver, such as hepatomas).

The activation polypeptide antagonist early can be administered to the subject before, at the same time or after administration of the antigen.

Patent Examples

To examine the in vivo role of CD69 in autoimmune reactivity and inflammation, the behavior of AIC in CD69-deficient mice of strain B6 was analyzed. An exacerbated form of AIC was observed in CD69-deficient mice that correlated with specific T and B responses against increased type II collagen (IIC), increase in some pro-inflammatory mediators and local decrease in TGF-? Synthesis. Local blocking of TGFβ increased the severity of AIC in WT mice but not in those deficient for CD69. In addition, activation of CD69 in vitro induced the production of TGFβ1. These results strongly suggest that CD69 is a negative regulator of autoimmune reactivity through TGF? Synthesis. The increase in the NK cell-dependent antitumor response in CD69-deficient mice is also described. Deficiency in CD69 results in greater protection and rejection of MHC-I tumor cells compared to control mice. This potent antitumor response is dependent on the tumor microenvironment in the host, correlating with a decrease in the production of TGF-?, An increase in the profiles of inflammatory cytokines and chemokine MCP-1, and is associated with an increase in recruitment of effector lymphoid cells and decreased apoptosis. This increase in antitumor activity is dependent on NK cells and T lymphocytes and persists in CD69 - / -, RAG-negative immunodeficient mice. Moreover, in vivo treatment with anti-TGF-? Antibodies induces potentiation of the antitumor response. On the other hand, the activation of CD69 induces the phosphorylation of ERK and the production of TGF-? In T lymphocytes, a production that is inhibited by the treatment of T lymphocytes with a specific inhibitor of MEK1, a kinase that phosphorylates and activates ERK , which constitutes a link between CD69 signaling and TGF-? production. Evidence is also presented that in vivo treatment with the antagonist antibody against CD69 2.2 improves the antitumor response in normal and immunodeficient mice of SCID and RAG-negative strains induced with MHC class I negative tumors.

These results indicate that CD69 is a negative regulator of immune reactivity through TGF-? Synthesis. In vivo treatment with anti-CD69 antibodies depends on the antibody used. The antagonist 2.2 enhances the immune response, increasing the severity of the AIC but also increasing the efficiency of tumor rejection, while the eliminator 2.3 eradicates effector leukocytes, improving the clinical parameters of the AIC. In addition, this antibody can directly remove CD69 + tumors.

The following examples illustrate the patent proposal and should not be considered as a limitation of Findings included in this report:

Example 1 CD69 deficient mice develop an exacerbation of the AIC

To explore the role of CD69 in a model of chronic autoimmune arthritis, the incidence and severity of AIC induced by intradermal immunization of CII in CFA in mice of strain B6 have been compared (in which this protocol induces moderate arthritis (Campbell et al ., 2000)), using control animals (CD69 + / +) and CD69 - / - (Lauzurica et al ., 2000). The mice developed an exacerbated form of AIC, showing marked swelling of the leg, ankle joints and interdigitants (Fig. 1A), and an increase in severity and incidence compared to control mice (Fig. 1B). The heterozygous CD69 +/-, which express intermediate levels of CD69 (Lauzurica et al ., 2000), showed aggravation of the AIC, but significantly less than the homozygous CD69 - / - (Fig. 1B).

To further assess arthritis, sections stained with hematoxylin and eosin from the extremities of WT and CD69 - / - mice with different degrees of AIC were analyzed by histological examination. The results of these studies coincided with those of the clinical assessment, with a significantly higher percentage of extremities with severe pathology than were observed in mice deficient for CD69, compared with WT mice (Fig. 2A). The limbs of the CD69 - / - mice showed a higher incidence of inflammatory cell infiltrates, with pannus extending from the marginal areas to the cartilage and through the subchondral bone marrow (Fig. 2A and 2B). In later stages, destruction of articular cartilage and bone was observed, with loss of normal joint morphology. Therefore, these results demonstrate a clear inverse correlation between the severity of the AIC and the expression of CD69, suggesting an inhibitory role in vivo for CD69 in the development of
AIC

Example 2 Enhancement of the specific immune response against IIC in CD69 - / - mice

These results suggest that IIC induces an exacerbated immune response in CD69 deficient mice. Accordingly, the spleen of CD69 - / - mice in which AIC has been induced were larger (about 1.4 times heavier, p <0.001, t Student test) than 12 control mice. weeks of age (Fig. 3A, B). The increase in spleen weight correlates with an increase in the number of cells (Fig. 3B). Next, we determine whether the increase in cellularity was due to any difference in any of the subpopulations of immune cells present in the spleen. The proportion of T, B, macrophage, and NK cells, as well as the CD4 / 8 ratio and naïve / effector-memory cells (CD62L / CD44) and the population of low CD45RB, CD25 +, CD4 + regulatory T cells were analyzed. The proportion of these groups was conserved between WT and CD69 - / - mice both in the absence of stimulation and after immunization with IIC. To determine the lymphocyte activation capacity, the lymphoproliferative response in response to IIC in lymphocytes from the spleen or lymphoid nodules was analyzed. The lymphocytes of CD69 - / - mice showed higher levels of dose-dependent proliferation when compared to control mice (Fig. 3C), indicating a CD69 inhibitory effect on specific proliferation against IIC by of immune cells from a mouse immunized in vivo with CII.

The humoral response against IIC The serum concentration of antibodies against IIC and its Isotypic characterization revealed higher levels of the IgG2c isotype, dependent on the Thl response, and that is the equivalent in the strain of B6 mice to IgG2a and IgG2b (Fig. 3D). I also know demonstrated a significant increase in IgG3, indicating a B lymphocyte hyperresponse of CD69 - / - mice, while the reduction in IgM would suggest an enhanced change to IgG IgG1, dependent on Th2 response, was not significantly different, and IIC did not induce an IgA response serum These results suggest the potentiation of the immune reactivity both T and B in mice deficient for CD69.

Example 3 Mice deficient for CD69 produce less locally TGF-?

The inflammatory process and the development of arthritis involve a wide range of cytokines (Feldmann et al ., 1996; O'Shea et al ., 2002). Since the joints are the most relevant place of cytokine production in arthritis, the effect of CD69 deficiency on the profile of cytokines expressed in the joints of mice suffering from AIC was investigated. Using protection assays against RNase activity, it was shown that the deficiency in CD69 produces an increase in the expression of some inflammatory mediators such as IL-1?, RANTES, MIP-1? And MIP-1? (Fig. 4 ). However, the most significant difference is the reduction in the levels of the anti-inflammatory cytokine TGF-? 1 and TGF-? 2 in CD69 - / - mice (Fig. 4). Quantitative real-time RT-PCR analysis of the mRNA of the hind legs of the mice demonstrated the reduction in TGF-? 1 mRNA present in inflamed joints and the increase in IL-1? And RANTES (Fig. 5A).

Then, the levels of total and active TGFβ1, directly produced by activated leukocytes (Kehr et al ., 1986), and other cytokines in joint tissue washes were analyzed. It was shown that both the total levels of TGF-? And the activated fraction of this cytokine are decreased in mice deficient for CD69 (Fig. 5B), in contrast to the systemic levels thereof, in which it was not observed differences (data not shown). On the other hand, a local increase in proinflammatory cytokines such as IL-1? And RANTES in the affected joints was observed, while TNF-? Levels showed no significant differences (Fig. 5B). Since TGF-? Is considered an anti-inflammatory cytokine that protects in AIC models (Kuruvilla et al ., 1991), these results suggest that deficiency in the production of TGF-? 1 may cause the increase in response Inflammation observed in CD69 - / - mice through increased production of IL-1? and various chemokines, thereby altering the balance between pro and anti-inflammatory cytokines in the synovium (15).

Subsequently, the populations were investigated. Cells responsible for the cytokine expression profile observed in CD69 - / - mice. The purification of synovial populations showed that these cells are in their mostly CD11b + macrophages (66.3 ± 29.4%), with some CD3 + cells (9.6 ± 5.3%), and non-leukocyte cells CD45 <-> (24.1 ± 14.8%). These three populations were isolated, and  it was shown that both the CD11b + and CD3 + populations contributed to the local decrease of TGF-? in mice deficient for CD69. However, it was shown that subpopulation CD11b + caused the subsequent increase in proinflammatory response mediated by IL-1? (Fig. 5C), which gives it a role crucial in the perpetuation of synovitis.

Example 4 TGF-? Block increases severity of AIC in control mice but not deficient in CD69

The protective effects of TGFβ1 in the AIC can explain in itself the deregulation of the balance of some pro-inflammatory cytokines and chemokines (O'Shea et al ., 2002; Kuruvilla et al ., 1991; Brandes et al ., 1991 ). These results would explain the increased leukocyte infiltration and inflammatory responses of mice deficient for CD69. To establish a causal link between TGF-? Levels and the severity of the AIC, a blocking antibody against TGF-? (Dasch et al ., 1989) was injected locally into the legs of the mice with AIC. TGF-? Blockade induced a significant increase in leg inflammation compared to an isotype control or diluent (Fig. 6A). Joint mRNA analysis by quantitative RT-PCR demonstrated a significant induction of IL-1? And RANTES, but not TNF-? Or IL-18 (Fig. 6A). These results demonstrate that blocking TGF-? In control mice with AIC results in a phenotype similar to that observed in CD69 - / - mice. However, local injection of the antibody against TGF-? In CD69-deficient mice with AIC did not increase leg swelling (Fig. 6B), and mRNA analysis showed no appreciable change in cytokine levels. pro-inflammatory studied. These findings reveal that the blockade of TGF-? And CD69 deficiency do not cooperate to aggravate the AIC, suggesting its interdependence.

Example 5 Induction of TGF-? 1 expression by CD69 activation

To analyze the possible CD69 modulator paper in the synthesis of TGF-? 1, the secretion of said cytokine in stimulated mouse splenocytes with Con-A. The cross-linking of CD69 induced a marked increase in TGF-? levels total and active in different subgroups of leukocytes (Fig. 7A), of according to the data found in NK cells and in splenocytes activated with anti-CD3 {Esplugues). In addition, the cross-linking of CD69 induced the production of TGF-? 1 in synovial infiltrate cells of mouse, most of which were CD11b + CD69 + (Fig. 7B). In contrast, no changes in the expression of TNF-?, RANTES or IL-1? after activation of CD69 (data not shown).

To investigate the possible extension of these results to humans, the expression of TGF-? 1 in CD69 + mononuclear cells coming from the joints of patients with diseases inflammatory (Fig. 8A). TP1 / 8 antibody cross-linked anti-CD69 (but not soluble) induced the TGF-? expression without another costimulus (Fig. 8A), while no differences were observed in other cytokines, as TNF-? or IL-6 (data not shown). In these trials, the increase in TGF-? Active correlated with the increase in total cytokine levels (Fig. 7 and 8). These results show that, both in humans (Fig. 8) and mice (Fig. 7), there is a direct link between the activation of CD69 and the TGF-? 1 expression, which could explain the increased levels of TGF-? 1 observed in CD69 + / + mice compared to CD69 - / -.

To establish a direct connection between the cross-linking of CD69 and the production of TGF? 1, we use a Jurkat line stably transfected with human CD69 (JK-CD69). The JK-CD69 line that express CD69 produced significant synthesis of TGF-? Active after cross-linking with anti-CD69, in contrast to the absence of synthesis of TGF-? in the parental line Jurkat negative for CD69 (Fig. 8B). In addition, our data shows that the cross-linking of CD69 did not induce TNF production (Fig. 8B). These results suggest that CD69, a receptor induced during activation, is able to modulate the immune response and the inflammatory reaction through direct secretion of TGF-? 1 in the inflammatory focus.

Example 6 In vivo treatment of AIC with antibodies against CD69

The effect of in vivo treatment with anti-CD69 antibodies has been analyzed using two different antibodies against CD69, monoclonal antibody (mAb) 2.2 and mAb 2.3, in the AIC model in DBA1 mice.

The anti-CD69 2.2 antibody (IgG1) is a non-eliminating antibody. Like IgG1, it is unable to bind complement and does not eliminate CD69-expressing cells in an in vitro assay with Cr 51 (not shown). Furthermore, mAb 2.2 does not induce synthesis of TGF-? In vitro in the absence of cross-linking (Esplugues et al . 2002. J. Exp. Med. 197: 1093; Sancho et al ., 2003. J. Clin. Invest 112: 872).

The effect of mouse 2.2 anti-CD69 antibody was analyzed in vivo in an AIC model in DBA / 1 mice. In vivo treatment with this antibody leads to complete loss of CD69 expression in populations expressing the molecule, such as CD3hi thymocytes (Fig. 19). As shown in the upper right quadrant of the left panel, 14.2% of the thymocytes express CD69. After treatment with mAb 2.2, only 0.9% express CD69 (right panel, upper right quadrant). However, the subset of the CD3hi thymocytes remains constant, since the sum of the upper quadrants in each panel is the same, that is, 20.7% (6.5 + 14.2 for the treatment with isotype control and 19.8 + 0.9 for the treaty) . This shows that mAb 2.2 does not mediate depletion of CD69 + cells in vivo . Deeper studies show that mAb 2.2 reduces the membrane expression of CD69, that is, antagonizes by negative modulation of CD69.

The treatment of DBA / 1 mice in the model of AIC with mAb 2.2 significantly exacerbated the disease when it was administered on days 20 and 28 during the onset of secondary response (Fig. 20), according to our data in the mouse deficient in CD69 (Fig. 1).

The mRNA analyzes of the hind legs by Quantitative RT-PCR in AIC mice treated with Isotype control and with anti-CD69 mAb 2.2 showed  that IL-1β levels were increased (25.0 ± 9 to 58.4 ± 8 units, respectively; p <0.01, U Mann Whitney test) and TGF-? 1 was decreased (56.5 ± 13 to 18.2 ± 8 units, respectively; p <0.01, U Mann Whitney test). The levels of IL-4, TNF, MCP1 and IFN-? MRNA did not vary (n = 12 mice per group in two experiments independent). The results for each cytokine are normalized for the expression of GAPDH measured in parallel in each sample. The results show that the negative modulation of CD69 by the mAb 2.2 leads to the decrease of TGF-? 1 mRNA and to the increase of mRNA for IL-1β, according with the exacerbation of the inflammation observed.

The monoclonal antibody (mAb) 2.3 behaves in vitro as a eliminating antibody. As IgG2a, it binds complement and eliminates cells expressing CD69 in an in vitro Cr 51 release assay (not shown).

The effect of mAb 2.3 was analyzed in vivo in an AIC model in DBA / 1 mice. In vivo treatment with this antibody leads to depletion of CD3hi thymocytes expressing CD69 (Fig. 21). As shown in the upper right quadrant of the left panel, in this experiment 16.7% of the thymocytes expressed CD69. After treatment with mAb 2.3, only 0.1% express CD69 (upper right quadrant, right panel). However, the set of total thymocytes CD3hi, which express CD69, is strongly reduced, since the sums of the upper panels are now different, 24.8% in the control, but only 8.3% in the treated group. This shows that mAb 2.3 has eliminated all cells expressing CD69, rather than performing a functional blockage of the
molecule.

Treatment of induced DBA / 1 mice with AIC with mAb 2.3 significantly reduced the AIC when administered on days 20 and 28 during the start of the response secondary (Fig. 22).

These results show that treatment with an anti-CD69 that produces negative modulation of CD69 may be useful for potentiating certain immune responses. In contrast, the removal of cells expressing CD69 can improve diseases mediated by system activation immune.

Example 7 Mice deficient for CD69 develop a response enhanced antitumor

To assess the possible role of CD69 in vivo in the antitumor response, RMA-S tumor cells were injected intraperitoneally into control and CD69 - / - mice, and tumor progression was analyzed {Fig. 9A). Following the injection of 10 4 RMA-S cells, all control mice developed tumors at five weeks, with a significant increase in body weight observable at 15 days, while only 20% of CD69 ^ mice {- / -} tumors developed, which was verified up to 7 weeks post-inoculation (Fig. 9A). Similar results were obtained when 10 5 RMA-S cells were injected subcutaneously (Fig. 9B), demonstrating that the differences observed are not due to the site of inoculation. In contrast, very subtle differences were observed in the progression of tumors derived from the RMA cell line (H-2 +) between control and CD69 - / - mice (Fig. 9C, and data not shown), suggesting that CD8 + cytotoxic lymphocytes (CTL) play an accessory role in potentiating the antitumor response observed in CD69 - / - mice.

Previous studies have shown that the growth of RMA-S tumors are monitored in vivo by NK cells in normal mice (Smyth et al ., 1998). To investigate the involvement of NK cells in the development of RMA-S tumors in mice deficient for CD69, these cells were removed before tumor induction by treatment with an antiserum against Asialo-GM1. This antiserum was inoculated on days -1 (before tumor induction), +2 and +4, using the diluent as a control. As expected, tumor growth was observed in all animals in which the NK population had been removed, but in none of the controls (Fig. 9D). Mice treated with anti-Asialo-GM1 developed tumors in the first 10 days post-inoculation, data comparable to the earliest tumor induction, observed in C57BL / 6 mice deficient for perforin (Smyth and Johnstone, 2000; Smyth et al . , 1998). The removal of CD4 + lymphocytes in CD69 - / - mice increased tumor development, but less markedly than in mice without NK cells (Fig. 9D). These results indicate that, although NK cells are very important in the rejection of RMA-S tumors in CD69-deficient mice, T lymphocytes also play a role in this process. In accordance with the relevant role of T cells in tumor suppression in CD69 - / - mice, a greater control in the overgrowth of RMA cells was observed after three days of inoculation (data not shown).

The potentiation of antitumor activity observed in CD69 - / - mice was not restricted to RMA-S cells, since it was also observed in the case of inoculation of RM-1 cells, a carcinoma of  C57BL / 6 syngeneic prostate (MHC-I - CD69 -). There was a decrease in the number of tumor metastases in mice deficient for CD69 when 10 4 RM1 cells were injected intravenously, while control mice showed a high number of pulmonary metastases (Fig. 9E).

Example 8 Enhancement of the antitumor response in CD69 - / - mice  RAG-deficient

To analyze the contribution of immunity natural to the potentiation of the antitumor response observed in CD69 deficient mice were injected intraperitoneally RMA-S cells in CD69 - / - mice RAG-deficient and CD69 +/ + RAG-deficient (Fig. 10). Tumor cells are they overgrown in virtually all mice RAG-deficient in response to 10 6 cell RMA-S, while the CD69 - / - RAG-deficient controlled tumor growth almost completely (Fig. 10A, B).

Example 9 Enhancement of the immune response in mice CD69 - / -

It was investigated whether tumor rejection was associated with an increase in the function of NK cells by determining the in vitro lytic activity of the peritoneal cells of CD69 - / - mice and control stimulated with RMA-S cells, using as target the YAC-1 tumor line, susceptible to NK. Unfractionated peritoneal cells from CD69 - / - mice were shown to be more effective in destroying YAC-1 cells than those from control mice (Fig. 11A), showing a correlation between the NK response in vivo and NK cytotoxicity in vitro in CD69 - / - mice. Similar results were obtained by inoculating the mice with RM-1 cell (Fig. 11B). Next, T lymphocytes and peritoneal cells from mice inoculated with RMA-S, RM-1 or uninoculated cells were quantified. The peritoneal NK population was established as positive for DX5 and 2B4 antibodies by flow cytometry. As shown in Fig. 11C and in Table 1, a modest increase in the total number of peritoneal cells was found in CD69 - / - mice compared to control mice, which appeared to be due to an increase in the number and proportion of lymphocytes, specifically NK cells and CD3 + T cells. When RM-1 cells were employed, the total number of cells recruited to the peritoneum was significantly higher in CD69 - / - mice (Fig. 11D,. This reflects an almost threefold increase in NK cell numbers and CD3 + T lymphocytes (Fig. 11) In contrast, the number of CD5 + B220 + B-1 and CD5 + B220 + B-2 lymphocytes was similar in CD69 - / - and control mice, both at baseline conditions and after challenge with RM-1 cells (data not shown). A modest increase in monocyte and granulocyte levels of the peritoneum of CD69 mice was also detected. - (-) (Fig. 11A) In addition, the reduction in cell recruitment observed in control mice on day 6 post-inoculation of tumor cells was not observed in CD69 - / - mice (Fig. 10C) These data demonstrate the existence of a correlation between the number of NK cells and CD3 + T lymphocytes in the peritoneum of CD69 - / - mice and antitumor activity. nto, CD69 seems to regulate the number of NK cells and CD3 + T cells recruited to the tumor formation site.

The spleen is a strongly lymphoid organ influenced by peritoneal inflammation. Agree with this, the spleens of the CD69 - / - mice are much larger comparing with control mice (Fig. 11D). Cell count splenic confirmed these results, but the analysis by Flow cytometry of the different subpopulations demonstrated the existence of similar proportions of B220 + lymphocytes, CD3 + and DX5 +. All of the above suggests a role for CD69 as a regulator of lymphocyte accumulation in The sites of inflammation.

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Example 10 Increased lymphocyte survival of mice CD69 - / -

The expansion and elimination of activated cells is carried out by homeostatic mechanisms in the peripheral circulation (Krammer, 2000). To investigate the mechanism by which lymphocyte accumulation is observed in the spleen and peritoneum of CD69 - / - mice, the survival of lymphoid lineages in these mice was analyzed. When cell viability was investigated in vitro , an increase in cell survival was observed in CD69 - / - mice (Fig. 12A). A significant difference of 19% decrease (p <0.02) in spontaneous apoptosis, determined by DNA content in cell cycle assays (24 h), of spleen lymphocytes from CD69 - / - mice was observed in the that RM-1 cells (23.36 ± 1.73%, n = 9) have been injected with respect to similarly inoculated control mice (28.89 ± 1.18%, n = 9). The use of a caspase-3 activity test confirmed a significant decrease (p <0.0001) in spontaneous apoptosis (48 h) of the spleen lymphocytes of CD69 - / - mice inoculated with RM-1 cells (25.46 \ pm 0.5%, n = 6) vs. control mice (44.56 ± 1.3%, n = 6) (Fig. 12B), differences that were reproduced (p <0.0001) using purified NK cells (DX5 + CD3-) of CD69 - / - mice (35.26 \ pm 1.6%, n = 4) and control (45 ± 1.8%, n = 4). Therefore, it seems that differences in lymphocyte survival may contribute to the increase in spleen size and observable cellularity in the peritoneum of CD69 - / - mice.

Example 11 Reduction in TGF-? E levels increase in proinflammatory factors in mice CD69 - / -

The antitumor response is controlled by a wide range of growth factors and cytokines, and the altered expression of them can have a huge impact on Immune responses Therefore, it has been analyzed whether the enhancement of the antitumor response of mice CD69 - / - is influenced by differences in the levels of cytokines and / or chemokines, which was studied by experiments of RNasa protection. The mRNA analysis of cells peritoneal of CD69 - / - mice in which it has been inoculated RM-1 cells showed a variation in the levels of the cytokine TGF-? with respect to the control. Peritoneal cells of CD69 - / - mice produced less transcripts of TGF-? 1, -? 2 and -? 3 than cells from normal mice. The decreases observed in four independent experiments ranged from 33 ± 5% for TGF-? 1 up to 74 ± 19% for TGF-? 3. The decrease in TGF-? 2 was dependent on sex of the animals, varying from 20% in males to 110% in  females The analysis of other cytokines revealed an increase in IL-12p35 levels (32-68%), IL-1α (27-64%), IL-1? (32% in males, 294% in females) and MCP-1 (133%) in CD69 - / - mice compared to control mice (Fig. 13). They were not observed significant changes in the expression profiles of LT-?, IFN-?, MIF, IL-1Rα, IL-18, eotaxin, MIP-1?, MIP-1? E IP-10 (Fig. 13 and data not shown). So, these results suggest that the immune cells of mice CD69 - / - have an abnormal cytokine expression profile, with defective synthesis of immunosuppressive factors and high production of proinflammatory cytokines and chemokine MCP-1 A more thorough analysis of MCP-1 expression, measuring the secretion of said chemokine by stimulated peritoneal cells with LPS of thioglycolate induced mice. As it is shown in Fig. 13B, the levels of MCP-1 produced by the peritoneal cells of the CD69 - / - mice were almost three times higher than the levels of control mice. These changes can account for the potentiation of the immune response and enrichment in T lymphocytes and NK cells observed in the CD69 - / - mice.

Example 12 Effect of blocking TGF-? On the antitumor response in normal mice

To examine whether the decrease in levels of TGF-? may correspond to the increase in the antitumor response observed in CD69 - / - mice, control mice were treated with a blocking monoclonal antibody of TGF-?. This blockade prevented tumor development compared to the use of an antibody control or diluent (Fig. 14). These results indicate that the TGF-? block in mice in which have inoculated tumor cells results in a phenotype similar to observed in CD69 - / - mice.

Example 13 CD69 induces TGF-? Secretion

It was examined whether signaling by CD69 is involved in the expression of TGF-? In T lymphocytes. Stimulation of purified T cells from spleens of B6 mice with an antibody against CD3 induced high expression of CD69 (Fig. 15A), whose cross-linking resulted in an induction of TGF-? production (Fig. 15B). This effect was not observed using an isotype control, which indicates that cross-linking of CD3 does not induce TGF-? Secretion, as previously described (Chen et al , 2001). The quantification of viable and non-viable cells at 48 hours (Fig. 15C) demonstrated a decrease in lymphocyte survival, which correlates with higher levels of TGF-?.

Stimulation of CD69 in mature transfected cell lines induces phosphorylation of the kinase regulated by extracellular signals (ERK) (Zingoni et al ., 2000). To determine the CD69 signaling cascades that regulate TGF-? Expression, ERK activation was analyzed. The cross-linking of CD69 induced the activation of ERK-1 and ERK-2 in T lymphocytes stimulated with anti-CD3 (Fig. 15D). ERK activation was detected at 5 min, being maximum at 15 min. In addition, a significant reduction in the expression of TGF-? Induced by CD69 was observed when cells were pretreated with the MEK1 inhibitor, PD98059 (Fig. 15B), indicating that ERK activation is important for the production of TGF- ? induced by CD69. These results establish a causal link between CD69 signaling and TGF-? Production via ERK activation, and suggests that CD69 plays a fundamental role in lymphocyte homeostasis.

Example 14 The use of antibodies against CD69 increases the antitumor response in vivo

Different murine antibodies were selected against CD69 to examine the role of this molecule in therapy tumor of mice in which tumors have been induced with cells RMA-S. Intraperitoneal inoculation of 10 5 syngeneic RMA-S cells MHC-I - in C57BL / 6 mice it caused 100% life mortality at development of lethal tumors (Fig. 16A). When several were treated times to these animals with the anti-CD69 antibody 2.2, before and after inoculation of tumor cells, a 80% of the mice survived without evidence of development tumor (Fig. 16A), a trend that was also observed when He treated these animals with a single dose of the antibody (Fig. 16, and data not shown). Similarly, the treatment of mice SCID with the same antibody also inhibited the growth of RMA-S-induced tumors, indicating that some of the antitumor effects persist in the absence of T or B lymphocytes.

Moreover, mice treated with anti-CD69 were able to eliminate cells of the RM-1 line, observing a very number small lung metastases in said mice (1.25 ± 0.9), while mice treated with an isotype control were unable to limit the development of pulmonary metastases induced by these cells, with an average of 21.5 ± observed 5.5 metastases after 14 days from intravenous injection of 10 4 RM-1 cells.

Example 15 Anti-CD69 antibodies affect the stages early tumor growth in the peritoneum

The in vivo effect of treatment with anti-CD69 antibodies on tumor growth was examined ex vivo in peritoneal cells removed 3 days after intraperitoneal inoculation of 2 x 10 6 RMA-S cells. Analysis of the peritoneal cells of mice treated with antibody 2.2 or an isotype control showed that treatment with the antibody inhibited the proliferation of tumor cells in the peritoneum (3 x 10 6 in mice treated with the antibody vs. 20 x 10 6 cells in the isotype control). Similar differences were observed when the peritoneal cells of RAG1 - / - mice treated with the anti-CD69 antibody were analyzed; exacerbated growth of RMA-S cells was detected in control mice, but these cells were not found in 75% of mice treated with anti-CD69 (Fig. 17). Therefore, CD69-induced tumor removal occurs during the early immune response, and is effective in the absence of T and B lymphocytes.

Example 16 Antibody response to DNP-KLH in WT mice and deficient for CD69

The possible role of CD69 in adaptive response B has been analyzed. Different immunization protocols were used to evaluate the response. For the study of the humoral response against T-dependent antigens, wt or CD69-deficient mice were treated with 100 µg of DNP-KLH on day 0 and 21, using CFA or Alum adjuvants, and with two immunization routes: ip or intradermal (id) at the base of the tail. This protocol is a review of the protocol used in Lauzurica et al ., (2000) Blood 95: 2312. The antigen dose was increased to 100 µg per injection and the second injection was made on day 21, to allow for the proper development of the germinal center. The results of the experiment are shown in Fig. 18. The presence of Igs of different isotypes specific for DNP was then measured in serum samples of day 7 post-injection (primary immunization) or day 28 post-injection (seven days after second injection, secondary immunization).

In primary immunization only one significant increase in serum of CD69 - / - mice for s.c. administration from CFA-DNP-KLH (p = 0.019, Fig. 18). Therefore, there were no significant differences between WT and CD69 deficient mice for IgM in primary immunization IgG subtype levels were low (1: 2000 serum dilution) as corresponds to the primary response, and revealed no differences (not shown).

In secondary immunization, the i.p. it was the most effective in inducing IgM, without revealing differences significant (Fig. 18). Instead, differences were found relevant to IgG2c in response to CFA-DNP-KLH, regardless of the administration path, although the i.p. was the more effective (Fig. 18). Also IgG2b and IgG3 increased in response to CFA-DNP-KLH i.p. These results suggest that the absence of CD69 favors in some way the collaboration T: B.

As for the adjuvant, they found only significant differences when the CFA was used. This results suggest that an adjuvant that promotes Thl responses, as it is the CFA, allows the detection of the exacerbated response type Thl in the deficient mouse for CD69 more easily than an adjuvant used to increase Th2 responses, such as the Alum.

Also the administration path shows different results, since the i.p. from DNP-KLH gave more effective answers than the immunization s.c.

In addition, the nature of the antigen can play a role in the different immune response, since the DNP-KLH is an external antigen, while the Type II collagen promotes a reaction as an autoantigen. In conclusion, significant similarities have been found using this system and secondary immunization using type II collagen, such as the significant increases in IgG2c, IgG2b and IgG3 CII specific in the mouse deficient for CD69.

Material and methods

Animals The mice were crossed and 1 maintained according to animal safety protocols at the National Center for Biotechnology (Madrid, Spain). All experiments were performed on strain C57BL / 6, except for tumor development experiments on RAG2 - / -, which were performed on animals of the BALB / c strain. Both control and CD69 - / - mice were 6 to 12 weeks old at the time of the experiments. As a control, cage companion mice or animals of exactly the same age were used whose parents were cage mates. RAG1 - / - and RAG2 - / - mice were purchased from Jackson Labs (Bar Harbor, Maine, USA). For the generation of the doubly deficient mice in RAG2 and CD69, the status of the RAG locus was checked through the absence of CD3 + T cells by flow cytometry. The genotyping of the CD69 locus was performed by polymerase chain reaction as previously described (Lauzurica et al ., 2000).

Induction and evaluation of AIC . CFA was prepared by dissolving 100 µg of heat-inactivated M. tuberculosis (strain H37Ra; Difco, Detroit, Michigan, USA) in 20 ml of IFA (Freund's Incomplete Adjuvant). The IIC (Sigma Chemical Co., St. Louis, Missouri, USA) was dissolved at a concentration of 2 mg / ml in 10 mM acetic acid and mixed in a 1: 1 ratio with CFA. The resulting emulsion was injected at the base of the animals' tail and the treatment was repeated on day 21 as previously described (Campbell et al ., 2000; Campbell et al ., 2001). The same protocol was used with the strain of DBA1 mice (Harlan, Madison, Wisconsin, USA), using CFA in which 1 mg / ml of heat-inactivated M. tuberculosis had been dissolved. Control animals were treated with CFA without IIC. The severity of arthritis was monitored by direct examination of the legs with a digital caliper according to the following scale: grade 0, without detestable inflammation; 1, slight redness and erythema; 2, pronounced inflammation; 3, joint stiffness. Each leg was measured separately, obtaining a maximum score of 12 for each animal.

After the sacrifice, they were selected randomly the legs were fixed, decalcified and embedded in paraffin. Subsequently, the sections were stained (5 mm of thickness) with hematoxylin and eosin, and were quantified according with the following scale: 0, absence of inflammation; 1, light thickening of the synovial cell area and / or presence of some inflammatory cells in the periphery; 2, thickening of the boundaries of the synovial zone, infiltration of the area subperipheral and erosions located in the cartilage; 3, infiltrated into the synovial space, pannus, destruction of cartilage and bone erosion.

TGF-? Block . The blocking antibody against TGF-? 1D11.16.8 (mouse hybridoma, IgG1 isotype, purchased from ATCC, Manassas, Virginia, USA), and previously described (Dasch et al ., 1989) was prepared at 0 2 mg / ml . The treatment of the animals was carried out by subcutaneous injection of 25 µl of PBS containing the antibody, an isotype control or nothing, being applied every 2 days from the second injection of IIC (day 21), the inflammation being monitored daily. On the other hand, the effect of blocking TGF-? On tumor induction was performed by administering 0.5 mg of the same antibody against TGF-?, Inoculated on days -3, -1, and +1 and once per week from this, taking as reference (day 0) the day of inoculation of the tumor cells.

Proliferation assays Cells extracted from lymph nodes and spleen (2 x 10 5) were cultured in 200 ml of RPMI 164 0 medium supplemented with 50 µM 2-mercaptoethanol and 10% bovine calf serum, and in the presence of a concentration of 0 to 50 µg / ml denatured IIC (100 ° C, 10 min) for 60 h. Then 1 µCi / well of <3> H-TdR (Amersham, Little Chanfot, England) was added and kept another 12 h, before being collected in fiberglass filters for the determination of metabolic incorporation of ^ {3} H-TdR.

Detection of antibodies against IIC . ELISA experiments were performed for the detection of antibodies against IIC as previously described (Campbell et al ., 1998). Secondary HRP-conjugates of the IgG1, IgG2a, IgG2b, IgG3 or IgM (Southern Biotechnology, Birmingham, Alabama, USA) and IgA (Sigma) isotypes were used.

Isolation of synovial cells . Positive selection of splenocytes was performed using specific antibodies against CD11b, CD3, CD4 and CD8 conjugated with biotin, all from BD-Pharmingen, and biotin-binding Dynabeads (Dynal AS, Oslo, Norway), resulting in 98% of purity Mouse synovial cells were isolated from WT AIC mice, as described for human synovial tissue (Butler et al ., 1997), and further purification was performed as shown above. After positive selection of the synovial cells CD11b {+} and CD3 +, the synovial cells were incubated with biotinylated anti-CD45 and were negatively selected using an excess of magnetic microspheres with avidin. The remaining CD45 negative cells were used for RNA extraction. Synovial fluid leukocytes were obtained from synovial effusions of the inflamed knees of patients with reactive arthritis, ankylosing spondylitis and RA, who met the criteria of the American College of Rheumatology (1987) (27), and were purified with Ficoll-Hypaque ( Pharmacia, Uppsala, Sweden) by density gradient centrifugation.

RNase protection assays (RPA) and real-time quantitative RT-PCR analysis . The joints were homogenized in a Polytron® (Kinematica, Littau, Switzerland) and the total RNA was purified using Ultraspec RNA (Biotecx, Houston, Texas, USA). In tumor assays, RNA was extracted from unfractionated synovial cells. RNase activity protection assays were performed on 2.5-5 µg of RNA using the Riboquant Multiprobe RPA system (Pharmingen, San Diego, California, USA). For RT-PCR, 2 µg of Dnasal-treated RNA was transcribed with the RT MuLV enzyme (Roche Diagnostics Ltd., Lewes, United Kingdom). Real-time PCR was performed in a Lightcycler (Roche) rapid thermocycler system using primers belonging to different exons that generated products of around 200 bp.

Anti CD69 antibodies . Specific antibodies were generated by the fusion of myeloma cells of the NS-1 line with splenocytes from CD69 - / - mice (Lauzurica et al ., 2000), previously immunized with pre-B mouse cells expressing CD69. Said antibodies were purified and used in in vitro cytokine production experiments.

In vivo treatment with anti-CD69 . For the evaluation of the effect of anti-CD69 treatment on AIC, mice were inoculated with 300 µg of mouse anti-CD69 antibodies 2.2 (IgG1) or 2.3 (IgG2a), as a preventive treatment (days 20 and 28) or therapeutic (days 32 and 40) and limb inflammation was monitored. As an isotype control, antibodies 2.8 (IgG1) and 2.22 (IgG2a) were used. For the treatment of tumors, 500 µg of antibody 2.2 (days +4, +8, +12) or 100 µg of 2.3 (-1, +2, +4, +8) were injected intraperitoneally. 500 µg of the 2.2 antibody (days -1 and +6) were injected in the SCID mice, and in the RAG1 - / - 500 µg of 2.2 were injected on the same day as the inoculation of the tumor cells. The inoculation of the antibodies was intraperitoneally in all cases.

Determination of cytokine production in AIC. To determine the levels of cytokines in the joint tissue washes, mice were sacrificed (day 50), the paws were removed with the adjacent synovium of the knee joints according to standard protocols (Lubberts et al ., 2000), and they were incubated for 1 h at room temperature in 200 µl of RPMI1640 culture medium with 0.1% BSA. Subsequently, the levels of active and total TGF-? 1 (Emax ImmunoAssay System; Promega Corp., Madison, Wisconsin, USA), IL-1?, TNF-? And RANTES (OptEIA ELISA Sets) were determined in the culture supernatants ; BD-Pharmingen). For in vitro production of cytokines, mouse splenocytes (pre-stimulated with 5 µg / ml Con-A (Sigma) for 16 h and purified as previously described) were incubated with 10 µg / ml anti-CD69 (clone 2.2) or with an isotype control (IgG1) and 20 µg / ml of goat serum against mouse IgG, specific against the Fc fragment (F (ab ') 2, Jackson Immunoresearch, West Grove, Pennsylvania, USA). After 24 h of culture, the levels of active and total TGF-? 1, IL-1?, TNF-? And RANTES were determined. Finally, the levels of TGF-? 1 in leukocytes (1 x 10 6) obtained from the joints of patients with inflammatory joint diseases (RA, reactive arthritis or ankylosing spondylitis) were determined and treated for 24 h with antibody against human CD69 TP1 / 8 or with an isotype control, in the presence or absence of crosslinker.

Measurement of production of TGF \ beta and MCP-1 by ELISA for the antitumor response. Said measurements were performed on purified CD3 + T lymphocytes from suspensions of lymphoid nodules and spleens of mice of the C57BL / 6 or BALB / c strains. CD3 + T lymphocytes were isolated by selection by incubation with an antibody against MAC-1 followed by two adhesion steps at 4 ° C to petri dishes upholstered with a rabbit antibody against mouse IgG (DAKO, Glostrup, Denmark) . The average purity of this protocol was> 95%, determined by flow cytometry. Subsequently, lymphocytes were resuspended in 200 µl / point of Stem Span medium (Stem Cell Technologies Inc., Vancouver, BC, Canada) without serum, and were stimulated with anti-CD3 (1 µg / ml) immobilized in plaques. 96 well plastic. Anti-CD69 antibody (clone 2.2, IgG1, K, 20 µg / ml) or an isotype control was added. To crosslink, 20 µg / ml of the F (ab ') 2 fragment of a goat-generated antibody against Fc-specific mouse IgG (Jackson Immunoresearch) was used. The MEK1 PD98059 inhibitor (Calbiochem, LaJolla, California, USA) was used at a concentration of 20 µM and added 1 h before the antibody. After 72 h of incubation at 37 ° C and 5% CO2, the supernatants were taken and TGF-? Levels were determined as previously described. For the measurement of MCP-1, 1 ml of thioglycolate (3% w / v, Sigma) was injected intraperitoneally into control mice and CD69 - / -. The mice were sacrificed at 72 h and the leukocyte infiltrate of the peritoneum was recovered by peritoneal washing with 5 ml of RPMI1640 medium + 2% calf serum at 4 ° C. Subsequently, the cells (2 x 10 6) were seeded in 1 ml of complete medium in 24-well plates and stimulated with LPS (1 µg / ml). Supernatants were taken and the production of MCP-1 was measured as previously described.

Cell cultures The cell lines used in this study were: RM1, prostate carcinoma (H-2b), courtesy of Dr. T. Thompson, Baylor College of Medicine, Houston, Texas, USA), YAC-1 (H -2nd) and RMA (H-2b), lymphomas, RMA-S, mutated lymphoma derived from the murine line induced by Rauscher virus, and which is deficient for the MHC class I peptide load, and 300.19, murine pre-B line. All were maintained at 37 ° C with an atmosphere of 5% CO2 in RPMI1640 medium supplemented with 10% heat-inactivated calf serum, 2 mM L-glutamine, 100 U / ml penicillin and 100 µg / ml streptomycin (Gibco-Life Sciences, Gaithersburg, Maryland,
USES).

Tumor control in vivo . Tumor cells (RMA, RMA-S or RM-1 in 200 µP of PBS) were injected subcutaneously or intraperitoneally into control or CD69 - / - mice without prior or depleted antibody treatment, as previously indicated. In experiments of elimination of the NK population, 100 µl of anti-Asialo-GM1 (rabbit serum, Wako Chemicals, Richmond, Virginia, USA) was injected intraperitoneally 1 day before tumor inoculation and on +2 days +4 In CD4 + lymphocyte removal experiments, 100 ml of GK1.5 antibody (anti-CD4) or an isotype control was inoculated at days -1, +2 and +4 with respect to tumor induction. The evolution of the tumors was monitored by daily weighing of the animals (ascites fluid development). For ethical reasons, the animals were sacrificed when the body weight had increased by 25%, which unambiguously corresponded to irreversible tumor growth.

Pulmonary metastasis of RM-1 cells . 10 4 RM1 cells (in 100 µl of PBS) were injected into the tail vein of control mice and CD69 - / -. After 14 days, the animals were sacrificed, the lungs were removed, fixed in 4% paraformaldehyde in PBS and the lung metastases were quantified by a dissecting microscope.

Flow cytometry Cells (10 6) from peritoneal exudate or spleen were pre-incubated with a blocking solution (PBS containing 5% and 15% heat-inactivated calf and rabbit serum respectively, 0.02% sadistic azide and 2.4G2 antibody ), which prevents the binding of antibodies to Fcγ receptors. Subsequently, the cells were incubated for 30 min at 4 ° C with antibodies conjugated with FITC or PE, or biotinylated antibodies followed by streptavidin-FITC or -PE (Southern Biotech). The following antibodies were used: anti-DX5 (DX5), -2B4 (2B4), -CD3 (145-2C11), -B220 / CD45 (RA3-6B2), -CD4 (GK1.5), -CD8 (53.6. 7), -CD5 (53-7.3), -CD11b (M1 / 70) and -CD69 (H1.2F3), all of them from Pharmingen. Finally, the cells were washed with cold PBS and analyzed on a FACScalibur flow cytometer (Becton Dickinson, Mountain View, California, USA), analyzing 104 cells by CellQuest® software (Becton Dickinson). The number of cells collected in the peritoneum was determined by a Coulter Multisizer II system (Beckton Coulter, Fullertone, California, USA).

Obtaining NK cells . It was performed using the CELLection Biotin Binder kit system (Dynal, Oslo, Norway) together with the biotinylated anti-DX5 antibody according to the manufacturer's specifications. Briefly, the cells (1 h at 37 ° C) were incubated on polystyrene culture plates (Becton Dickinson). The non-adhered cells were incubated for 15 min at 4 ° C with a biotinylated antibody (DX5), washed twice and the antibody coated cells were captured with CELLection microspheres (1 x 10 7 microspheres per 1 x 10 6 cells). The purity obtained was always> 95%. NK cells were maintained in complete medium (20% FCS) supplemented with 1000 IU / ml of human rIL-2 (72 h).

51 Cr release test . The direct cytotoxic activity of NK cells was determined by standard 51 Cr release experiments. In all experiments, 5 x 10 3 YAC-1 target cells labeled with Na 2 CrO 4 were mixed with effector cells at the indicated proportions (4 h, 37 ° C). The spontaneous release of 51 Cr was determined by incubating the target cells with medium without cells; Maximum release was determined by incubating the cells with 2.5% Triton X-100. The percentage of specific lysis was calculated as:

% \ \ text {specific lysis} = [(\ text {cpm sample} - \ text {cpm spontaneous}) \ / \ (\ text {cpm maximum} - \ text {cpm spontaneous})] \ times 100

All experiments were performed by tripled, and the spontaneous release of Cr was always < 10%

Cell death trials . Splenocytes (4 x 10 6 / ml) of mice inoculated with RM-1 cells (3 days with 10 5 cells) were cultured in 24-well plates (Costar, Cambridge, Massachusetts, USA), and the cell viability was determined 24 h after the start of the culture. The cell cycle was monitored, the cells were stained with propidium iodide (IP), and apoptosis was determined by flow cytometry on a Coulter XL cytometer (Beckman Coulter). Data are expressed as the arithmetic mean ± of (n = 9). Caspase-3 activity was determined 48 hours after the start of the culture by incubation with the PhiPhiLux-GID2 substrate (OncoImmunin, College Park, Maryland, USA), according to the manufacturer's instructions. Flow cytometric analysis was performed immediately after 60 min incubation. Peritoneal cells (1 x 10 6) of control mice were cultured in 24-well plates and their viability was determined by staining with IP. IP + cells were considered apoptotic.

Western blot T lymphocytes were isolated from C57BL / 6 mice with anti-CD3 (5 µg / ml) immobilized in plastic (37 ° C, 12 h). The cells were collected, washed 2 times and cultured 4 h in serum free medium. Subsequently an antibody against CD69 (IgG1) or an isotype control at 10 µg / ml was added, and incubated 30 min at 4 ° C on ice. The cells were then washed 2 times and a cross-linking antibody (generated in goat against mouse IgG, specific for the Fc fragment, F (ab ') 2, 20 µg / ml) was added. The reaction was stopped at the indicated times by adding cold PBS, after which the cells were centrifuged and lysed in cold lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS w / v, 10 or glycerol, 50 mM DTT, 0.01% bromophenol blue w / v) supplemented with protease and phosphatase inhibitors. After centrifuging the lysates (13000 x g, 20 min, 4 ° C), the supernatants were separated into 12% polyacrylamide gels, transferred to PVDF membrane (BioRad, Hercules, California, USA), and Western blot experiments were performed with the indicated antibodies. The polyclonal anti-phospho-ERK 1/2 antibody was from Calbiochem. The blot signal was removed and reincluded with a monoclonal antibody against ERK 1/2 (Zymed Lab. Inc., San Francisco, California, USA). Secondary peroxidase conjugates (HRP) were used and bands were revealed by ECL.

Cell viability analysis . T lymphocytes were incubated under the same conditions as for TGF-? Production. Cell viability was determined at 24 and 48 h of culture by staining with trypan blue.

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Zingoni , A., G. Palmieri , S. Morrone , M. Carretero , M. Lopez-Botel , M. Piccoli , L. Frati , and A. Santoni . 2000 CD69-triggered ERK activation and functions are negatively regulated by CD94 / NKG2-A inhibitory receptor. Eur. J. Immunol . 30: 644-51.

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Claims (27)

1. Improvements introduced in the patent of invention P200300252 corresponding to the use of a effective amount of a modulator of an activation molecule early, which is an antagonistic antibody molecule anti-CD69 selected from a molecule of humanized anti-CD69 antibody, a molecule of human anti-CD69 antibody, a molecule of chimeric anti-CD69 antibody and a molecule of deimmunized anti-CD69 antibody for manufacture of a medicine intended for the treatment of a subject  that needs, or can benefit from, an immune response increased and said antagonist decreases signaling by early activation molecule or reduces the interaction of the early activation molecule with a ligand of said molecule of early activation 2. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 1 characterized in that said disorder is characterized by a diminished immune response. 3. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 1 characterized in that said disorder is characterized by unwanted tissue or cells or characterized by an unwanted proliferation of cells. 4. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 1 characterized in that said disorder is cancer. 5. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 1 characterized in that said disorder is fibrosis. 6. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 1 characterized in that said disorder is an immunodeficiency. 7. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 6, characterized in that the immunodeficiency is selected from: inherited immunodeficiency disorders, congenital immunodeficiency disorders, immunosuppression syndrome associated with radiotherapy, and syndrome of immunosuppression associated with chemotherapy. 8. Improvements introduced in the patent of invention P200300252 corresponding to the use of a effective amount of an antigen and / or a DNA that codes for antigen and an antagonist of the early activation molecule the what is an antagonist antibody molecule anti-CD69 selected from a molecule of humanized anti-CD69 antibody, a molecule of human anti-CD69 antibody, a molecule of chimeric anti-CD69 antibody and a molecule of deimmunized anti-CD69 antibody for manufacture of a medicine intended to enhance the response immune against said antigen in a subject. 9. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 8 characterized in that said antigen is included in a vaccine. 10. Improvements introduced in the invention patent P200300252 corresponding to the use of an effective amount of a remover which is an anti-CD69 antibody molecule that eliminates CD69 expressing cells, selected from: a humanized anti-CD69 antibody molecule, a human anti-CD69 antibody molecule, an anti-CD69 chimeric antibody molecule and an anti-CD69 immunized antibody molecule for the manufacture of a medicament intended for the treatment of a subject having a disease or condition characterized by a non-immune response desired. 11. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is an acute or chronic inflammatory disorder, or an immune disease. 12. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is an autoimmune disease. 13. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is selected from the group consisting of: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjögren's syndrome, autoimmune diabetes, thyroiditis, and other organ-specific immune diseases, including psoriasis. 14. Improvements in patent P200300252 relating to the use according to claim 10 wherein said disorder is neurological, gastrointestinal, cardiovascular or respiratory. 15. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is a neurological type disorder selected from: multiple sclerosis, myasthenia gravis, and other neurological immune diseases. 16. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is a gastrointestinal disorder selected from the following group: Crohn's disease, colitis, celiac disease, and hepatitis. 17. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is a respiratory type disorder, selected from among: emphysema, respiratory tract infections. 18. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10, characterized in that said disorder is a cardiovascular type disorder selected from the following group: atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis, vasculitis, and other diseases Cardiological immune nature. 19. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is an allergic process or a hypersensitivity reaction (types I, II, III, and IV), including asthma, rhinitis, and other hypersensitivity reactions mediated by the immune system. 20. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder is the rejection of a transplant or graft. 21. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 10 characterized in that said disorder or condition is: acute lung damage, acute respiratory distress syndrome, bronchitis, cystic fibrosis, reperfusion damage, nephritis, pancreatitis, arterial occlusion, cerebrovascular accident, damage induced by ultraviolet light, vasculitis, and sarcoidosis. 22. Improvements introduced in the patent of invention P200300252 corresponding to the use of a effective amount of a cell scavenger that express the early activation molecule, which is a molecule of anti-CD69 antibody cell killer that express CD69 selected from an antibody molecule Humanized anti-CD69, an antibody molecule human anti-CD69, an antibody molecule chimeric anti-CD69 and an antibody molecule deimmunized anti-CD69, for the manufacture of a medication intended for the treatment of a subject who has a cancer expressing CD69. 23. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 22 characterized in that the cancer expressing the early activation molecule is selected from the group consisting of: hematopoietic neoplasms, including diseases that involve cells of hematopoietic origin hyperplastic or neoplastic, such as: lymphomas and lymphatic leukemias. 24. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 22 characterized in that the cancer expressing an early activation molecule is a lymphoma and the lymphoma is selected from the group consisting of: T-cell lymphoma (including peripheral T-cell lymphomas , adult T-cell lymphoma / leukemia (ATL, Adult T cell leukemia / lymphoma ), cutaneous T-cell lymphoma (CTCL, Cutaneous T cell lymphoma ), granular large lymphocyte leukemia (LGF, Large Granular lymphocytic leukemia ), B-cell lymphoma, Hodgkin lymphoma and non-Hodgkin lymphoma. 25. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 22 characterized in that the cancer expressing an early activation molecule is a lymphatic leukemia and the lymphatic leukemia is selected from the group consisting of: poorly differentiated acute leukemia , eg, acute megakaryoblastic leukemia; myeloid disorders, including, but not limited to, acute promyeloid leukemia (APML, Acute ProMyeloid Leukemia ), acute myeloid leukemia (AML, Acute Myelogenous leukemia ), and chronic myelogenous leukemia (CML, Chronic Myelogenous Leukemia ); lymphoid tumors, including, but not limited to, acute lymphoblastic leukemia (ALL, Acute Lymphoblastic Leukemia ), which includes those of the B and T lineage, chronic lymphatic leukemia (CLL, Chronic Lymphocytic Leukemia ), pro-lymphocytic leukemia (PLL, Prolymphocytic Leukemia ), hairy cell leukemia (HLL, hairy cell leukemia), and Waldenstrom 's macroglobulinemia (WM). 26. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 22 characterized in that the cancer expressing the early activation molecule is a non-hematopoietic tumor that can express an early activation molecule in an ectopic manner. 27. Improvements introduced in the invention patent P200300252 corresponding to the use according to claim 25 characterized in that the lymphatic leukemia is a chronic B lymphatic leukemia without immunoglobulin mutations.