EDG: Modulators of lymphocyte activation and migration

Abstract

The present invention relates to regulation of lymphocyte activation and migration. More particularly, the present invention is directed to nucleic acids encoding EDG family GPCR proteins, e.g., EDG-1, 2, 3, 4, 5, 6, 7, or 8, which are involved in modulation of lymphocyte activation and migration. The invention further relates to methods for identifying and using agents, including small organic molecules, antibodies, peptides, cyclic peptides, nucleic acids, antisense nucleic acids, sphingolipid analogs, and ribozymes, that modulate lymphocyte activation or migration via modulation of EDG GPCRs and EDG related signal transduction; as well as to the use of expression profiles and compositions in diagnosis and therapy related to lymphocyte activation and suppression, and lymphocyte migration.

1 . A method for identifying a compound that modulates lymphocyte activation, the method comprising the steps of:
 (i) contacting a cell comprising an EDG polypeptide or fragment thereof with the compound, the EDG polypeptide or fragment thereof encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8; and   (ii) determining the chemical or phenotypic effect of the compound upon the cell comprising the EDG polypeptide or fragment thereof, thereby identifying a compound that modulates lymphocyte activation.   
     
     
         2 . The method of  claim 1 , wherein the EDG polypeptide or fragment thereof is encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a sequence of SEQ ID NO:1-4. 
     
     
         3 . The method of  claim 1 , wherein the host cell is a B lymphocyte. 
     
     
         4 . The method of  claim 3 , wherein the host cell is a cultured B lymphocyte. 
     
     
         5 . The method of  claim 4 , wherein the host cell is a BJAB cell. 
     
     
         6 . The method of  claim 1 , wherein the host cell is a T lymphocyte. 
     
     
         7 . The method of  claim 6 , wherein the host cell is primary T lymphocyte. 
     
     
         8 . The method of  claim 6 , wherein the host cell is a cultured T lymphocyte. 
     
     
         9 . The method of  claim 8 , wherein the host cell is a Jurkat cell. 
     
     
         10 . The method of  claim 1 , wherein the chemical or phenotypic effect is determined by measuring CD69 expression, IL-2 production, intracellular Ca 2+  mobilization, or lymphocyte proliferation. 
     
     
         11 . The method of  claim 1 , wherein modulation is inhibition of lymphocyte activation. 
     
     
         12 . The method of  claim 11 , wherein modulation is inhibition of T lymphocyte activation. 
     
     
         13 . The method of  claim 11 , wherein modulation is inhibition of B lymphocyte activation. 
     
     
         14 . The method of  claim 1 , wherein the polypeptide is recombinant. 
     
     
         15 . The method of  claim 1 , wherein the EDG polypeptide is selected from the group consisting of EDG-1, 3, 5, 6, 7, or 8. 
     
     
         16 . The method of  claim 1 , wherein the EDG polypeptide is an EDG-1 polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS:1, 2, 3, and 4. 
     
     
         17 . The method of  claim 1 , wherein the fragment of an EDG polypeptide is a polypeptide that comprises an extracellular domain of an EDG polypeptide. 
     
     
         18 . The method of  claim 1 , wherein the fragment of an EDG polypeptide is a polypeptide that comprises a cytoplasmic domain of an EDG polypeptide. 
     
     
         19 . The method of  claim 17 , wherein the polypeptide further comprises at least one transmembrane domain of an EDG polypeptide. 
     
     
         20 . The method of  claim 1 , wherein the EDG polypeptide or fragment thereof has GPCR activity. 
     
     
         21 . The method of  claim 1 , wherein the compound is an antibody. 
     
     
         22 . The method of  claim 1 , wherein the compound is an antisense molecule. 
     
     
         23 . The method of  claim 1 , wherein the compound is a small organic molecule. 
     
     
         24 . The method of  claim 1 , wherein the compound is a sphingolipid. 
     
     
         25 . The method of  claim 1 , wherein the compound is a sphingolipid analog. 
     
     
         26 . The method of  claim 25 , wherein the compound is a synthetic sphingolipid analog. 
     
     
         27 . The method of  claim 25 , wherein the compound is a naturally occurring sphingolipid analog. 
     
     
         28 . A method for identifying a compound that modulates lymphocyte activation, the method comprising the steps of:
 (i) contacting the compound with an EDG polypeptide or a fragment thereof, the EDG polypeptide or fragment thereof encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8;   (ii) determining the physical effect of the compound upon the EDG polypeptide; and   (iii) determining the chemical or phenotypic effect of the compound upon a cell comprising an EDG polypeptide or fragment thereof, thereby identifying a compound that modulates lymphocyte activation.   
     
     
         29 . A method of modulating lymphocyte activation in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the method of  claim 1 . 
     
     
         30 . The method of  claim 29 , wherein the lymphocyte is a T lymphocyte. 
     
     
         31 . The method of  claim 29 , wherein the lymphocyte is a B lymphocyte. 
     
     
         32 . The method of  claim 29 , wherein the subject is a human. 
     
     
         33 . The method of  claim 29 , wherein the compound is an antibody. 
     
     
         34 . The method of  claim 29 , wherein the compound is an antisense molecule. 
     
     
         35 . The method of  claim 29 , wherein the compound is a small organic molecule. 
     
     
         36 . The method of  claim 29 , wherein the compound is a sphingolipid. 
     
     
         37 . The method of  claim 29 , wherein the compound is a sphingolipid analog. 
     
     
         38 . The method of  claim 37 , wherein the compound is a naturally-occurring sphingolipid analog. 
     
     
         39 . The method of  claim 37 , wherein the compound is a synthetic sphingolipid analog. 
     
     
         40 . The method of  claim 29 , wherein the compound inhibits lymphocyte activation. 
     
     
         41 . The method of  claim 29 , wherein the compound inhibits T lymphocyte activation. 
     
     
         42 . The method of  claim 29 , wherein the compound inhibits B lymphocyte activation. 
     
     
         43 . A method for identifying a compound that modulates lymphocyte migration, the method comprising the steps of:
 (i) contacting a cell comprising an EDG polypeptide or fragment thereof with the compound, the EDG polypeptide or fragment thereof encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8; and   (ii) determining the chemical or phenotypic effect of the compound upon the cell comprising the EDG polypeptide or fragment thereof, thereby identifying a compound that modulates lymphocyte migration.   
     
     
         44 . The method of  claim 43 , wherein the EDG polypeptide or fragment thereof is encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a sequence of SEQ ID NO:1-4. 
     
     
         45 . The method of  claim 43 , wherein the host cell is a B lymphocyte. 
     
     
         46 . The method of  claim 46 , wherein the host cell is a primary B lymphocyte. 
     
     
         47 . The method of  claim 46 , wherein the host cell is a cultured B lymphocyte. 
     
     
         48 . The method of  claim 47 , wherein the host cell is a BJAB cell. 
     
     
         49 . The method of  claim 43 , wherein the host cell is a T lymphocyte. 
     
     
         50 . The method of  claim 49 , wherein the host cell is primary T lymphocyte. 
     
     
         51 . The method of  claim 49 , wherein the host cell is a cultured T lymphocyte. 
     
     
         52 . The method of  claim 51 , wherein the host cell is a Jurkat cell. 
     
     
         53 . The method of  claim 43 , wherein the chemical or phenotypic effect is determined by measuring lymphocyte migration in vitro toward an EDG ligand. 
     
     
         54 . The method of  claim 53 , wherein the EDG ligand is SPP or LPA. 
     
     
         55 . The method of  claim 43 , wherein modulation is inhibition of lymphocyte migration. 
     
     
         56 . The method of  claim 43 , wherein modulation is inhibition of T lymphocyte migration. 
     
     
         57 . The method of  claim 43 , wherein modulation is inhibition of B lymphocyte migration. 
     
     
         58 . The method of  claim 43 , wherein the polypeptide is recombinant. 
     
     
         59 . The method of  claim 43 , wherein the EDG polypeptide is selected from the group consisting of EDG-1, 3, 5, 6, 7, or 8. 
     
     
         60 . The method of  claim 43 , wherein the EDG polypeptide is an EDG-1 polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NOS:1, 2, 3, and 4. 
     
     
         61 . The method of  claim 43 , wherein the fragment of an EDG polypeptide is a polypeptide that comprises an extracellular domain of an EDG polypeptide. 
     
     
         62 . The method of  claim 61 , wherein the polypeptide further comprises at least one transmembrane domain of an EDG polypeptide. 
     
     
         63 . The method of  claim 43 , wherein the EDG polypeptide or fragment thereof has GPCR activity. 
     
     
         64 . The method of  claim 43 , wherein the compound is an antibody. 
     
     
         65 . The method of  claim 43 , wherein the compound is an antisense molecule. 
     
     
         66 . The method of  claim 43 , wherein the compound is a small organic molecule. 
     
     
         67 . The method of  claim 43 , wherein the compound is a sphingolipid. 
     
     
         68 . The method of  claim 43 , wherein the compound is a sphingolipid analog. 
     
     
         69 . The method of  claim 68 , wherein the compound is a synthetic sphingolipid analog. 
     
     
         70 . The method of  claim 68 , wherein the compound is a naturally occurring sphingolipid analog. 
     
     
         71 . A method for identifying a compound that modulates lymphocyte migration, the method comprising the steps of:
 (i) contacting the compound with an EDG polypeptide or a fragment thereof, the EDG polypeptide or fragment thereof encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8;   (ii) determining the physical effect of the compound upon the EDG polypeptide; and   (iii) determining the chemical or phenotypic effect of the compound upon a cell comprising an EDG polypeptide or fragment thereof, thereby identifying a compound that modulates T lymphocyte migration.   
     
     
         72 . A method of modulating lymphocyte migration in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the method of  claim 43 . 
     
     
         73 . The method of  claim 72 , wherein the subject is a human. 
     
     
         74 . The method of  claim 72 , wherein the lymphocyte is a T lymphocyte. 
     
     
         75 . The method of  claim 72 , wherein the lymphocyte is a B lymphocyte. 
     
     
         76 . The method of  claim 72 , wherein the compound is an antibody. 
     
     
         77 . The method of  claim 72 , wherein the compound is an antisense molecule. 
     
     
         78 . The method of  claim 72 , wherein the compound is a small organic molecule. 
     
     
         79 . The method of  claim 72 , wherein the compound is a sphingolipid. 
     
     
         80 . The method of  claim 72 , wherein the compound is a sphingolipid analog. 
     
     
         81 . The method of  claim 80 , wherein the compound is a naturally-occurring sphingolipid analog. 
     
     
         82 . The method of  claim 80 , wherein the compound is a synthetic sphingolipid analog. 
     
     
         83 . The method of  claim 72 , wherein the compound inhibits T lymphocyte migration. 
     
     
         84 . The method of  claim 72 , wherein the compound inhibits B lymphocyte migration. 
     
     
         85 . A method of modulating lymphocyte activation or migration in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an EDG polypeptide, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8. 
     
     
         86 . The method of  claim 85 , wherein the EDG polypeptide or fragment thereof is encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a sequence of SEQ ID NO:1-4. 
     
     
         87 . The method of  claim 85 , wherein the EDG polypeptide is selected from the group consisting of EDG-1, 3, 5, 6, 7, and 8. 
     
     
         88 . The method of  claim 85 , wherein the EDG polypeptide is an EDG-1 polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NO:1, 2, 3, and 4. 
     
     
         89 . A method of modulating lymphocyte activation or migration in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a nucleic acid encoding an EDG polypeptide or fragment thereof, wherein the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a polypeptide comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8. 
     
     
         90 . The method of  claim 89 , wherein the EDG nucleic acid or fragment thereof is encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a sequence of SEQ ID NO:1-4. 
     
     
         91 . The method of  claim 89  wherein the EDG nucleic acid is selected from the group consisting of EDG-1, 3, 5, 6, 7, and 8. 
     
     
         92 . The method of  claim 91 , wherein the EDG nucleic acid is an EDG-1 nucleic acid selected from the group consisting of SEQ ID NO:1, 2, 3, and 4. 
     
     
         93 . A method of modulating T lymphocyte migration and activation in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the method of  claim 43 , and administering to the subject a therapeutically effective amount of a compound identified using the method of  claim 1 . 
     
     
         94 . A method of screening for modulators of lymphocyte activation, the methods comprising the steps of:
 (i) transfecting into lymphocytes a cDNA library;   (ii) stimulating T or B cell receptors of the lymphocytes;   (iii) screening for modulation of lymphocyte activation by detecting the level of CD69 cell surface expression via FACS; and   (iv) rescuing cDNAs that modulate lymphocyte activation.   
     
     
         95 . The method of  claim 94 , wherein the lymphocytes are T cells. 
     
     
         96 . The method of  claim 95 , wherein the T cells are cultured T cells. 
     
     
         97 . The method of  claim 95 , wherein the T cells are Jurkat cells. 
     
     
         98 . The method of  claim 94 , wherein cDNAs of the library are operably linked to an inducible promoter. 
     
     
         99 . The method of  claim 98 , wherein the inducible promoter comprises a thymidine kinase promoter and a tetracycline regulatory element 
     
     
         100 . The method of  claim 94 , wherein the library is transfected by retroviral vectors. 
     
     
         101 . The method of  claim 94 , wherein the cDNA library is from a primary lymphocyte organ. 
     
     
         102 . The method of  claim 101 , wherein the primary lymphocyte organ is selected from the group consisting of thymus, spleen, lymph node, and bone marrow. 
     
     
         103 . The method of  claim 94 , wherein the modulation is inhibition of lymphocyte activation. 
     
     
         104 . The method of  claim 103 , wherein the modulation is inhibition of T lymphocyte activation.