Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-11-13
2002-04-16
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S194000, C435S320100, C435S325000, C435S252300
Reexamination Certificate
active
06372898
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of biology, e.g., describing compositions which interact in cell signaling. The invention provides various compositions and methods directed to protein interactions occurring in the signal transduction pathway, e.g., compositions which include variants of human Jak3, a protein exhibiting tyrosine kinase-like structure, and which functions in regulating intracellular signaling. In particular, it provides agonists and/or antagonists of said proteins.
BACKGROUND OF THE INVENTION
Cytokines are molecules that mediate differentiation or other signals typically between the circulating cell components of the mammalian circulatory system. Cytokines function through receptors, many of which have been characterized. See, e.g., Aggarwal and Gutterman (eds. 1991)
Human Cytokines: Handbook for Basic and Clinical Research
, Blackwell, Oxford.
Binding of cytokines to their receptors induces a cascade of intracellular signaling events that results in increased tyrosine phosphorylation of specific target proteins, and initiates a program of altered gene expression and proliferation. See e.g., Leonard and O'Shea (1998)
Annu. Rev. Immunol
. 16:293-322. Cytokine receptors typically lack intrinsic tyrosine kinase activity and signal via non-receptor tyrosine kinases of the Jak family that associate with the cytoplasmic domains of receptor chains. The paradigm for ligand-induced signaling is that trans-phosphorylation and activation of receptor-associated Jak kinases occurs upon ligand dependent receptor subunit aggregation. These activated kinases phosphorylate tyrosine residues of the receptor chain subunits, providing docking sites for src-homology (SH2) domains of signal transducers and activators of transcription (STATs), as well as other signaling molecules, e.g., the adapter molecule Shc. STATs are recruited to the phosphorylated receptor, where they are phosphorylated. This allows them to dimerize, translocate to the nucleus, and stimulate the expression of cytokine-inducible genes. While the general model for cytokine-induced signaling has been elucidated, the molecular details of the association of Jak family members with the cytoplasmic domains of receptor chains are not understood.
The functional receptors for the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15 all utilize the common gamma chain (&ggr;
c
) signaling subunit. The importance of signaling through &ggr;
c
is underscored by the fact that specific mutations in either the &ggr;
c
or the Janus kinase that interacts with it, Jak3, result in phenotypically similar Severe Combined Immunodeficiency (SCID). Infants with SCID suffer from severe infections due to reduced levels of T and natural killer (NK) cells, as well as hypogammaglobulinemia. Current preferred treatment typically requires heterologous bone marrow transplantation to replace lost T and NK cell functions.
The Jak family of kinases consists of four known mammalian homologs, each consisting of 1100-1200 amino acids organized into seven Janus homology (JH) domains, based on sequence similarity among the family members. They do not possess classic SH2 or SH3 domains, and, except for the catalytic domains, exhibit little homology to other protein tyrosine kinases (PTKs).
As the IL-2 signaling complex is essential for T cell proliferation and is critically dependent on Jak3 activity, understanding this interaction would be a benefit in effecting processes involving, e.g., graft rejection, rheumatoid arthritis, and autoimmune or inflammatory diseases. The availability of agonists and antagonists to cytokine receptor signaling such as, e.g., IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15 will be used to modulate these processes. The present invention provides these, as well as other proteins, useful, e.g., in determining the structure and mechanisms of immune regulation in a cell via the tyrosine kinases of the JAK family.
SUMMARY OF THE INVENTION
The present invention provides compositions that serve as an agonist or antagonist for Jak3 proteins. These agonists and antagonists will be useful in modulating T cell proliferation, and may be important in other hematopoietic or immunological function. In certain circumstances, these molecules will also have in vitro or in vivo therapeutic effects.
The present invention is based, in part, upon a structural analysis of a Jak3 mutation from a patient with autosomal severe combined immunodeficiency disease (SCID).
The mutation is a single amino acid substitution, Y100C, located in Janus homology domain 7 (JH7) that prevents kinase-receptor interaction and also results in a loss of IL-2-induced signaling in B-cells.
In particular, this insight leads to recognition of which specific amino acid residues of Jak3 kinase bind to the IL-2 receptor beta (IL-2R&bgr;) and common gamma (&ggr;
c
) chains, respectively, and initiate biochemical signals critical in controlling immune responses. Additionally, this information, leads to recognition of a region around the Jak3 Y100C mutation which has the ability to interact with &ggr;
c
specifically the proline rich box1 region of the IL-2 receptor. Furthermore, a Jak3/Jak1 chimeric composition containing this region mediates IL-2 responses. This invention embraces natural ligands, e.g., specific mutations (muteins) of the natural sequences, fusion proteins, and chemical mimetics. It is also directed to DNAs encoding such variant proteins. Various uses of these different protein or nucleic acid compositions are also provided.
The present invention provides a polypeptide that: comprises at least three non-overlapping fragments of at least 17 contiguous amino acids selected from residues 70-193 of SEQ ID NO: 2 (human Jak3); interferes with the interaction of a human Jak3 with a human &ggr; common receptor chain; and lacks any 8 contiguous amino acid residue fragment of SEQ ID NO: 1 residues 1-69 and 193-1124. Incertain embodiments: the interaction of the Jak3 with the human &ggr;
c
is determined in a binding assay; the total number of residues of the three fragments is between 55 and 123; the polypeptide is selected from: residues 1-193, 70-193, 1-130, 70-130, or 70-256 of human Jak3; or a residue corresponding to position 98, 99, 100, or 102 of the Jake polypeptide is substituted, either singly or in combination.
In other embodiments, the invention provides a isolated polynucleotide encoding the described polypeptide, or its complement, including variants resulting from the degeneracy of the genetic code or point mutations. Preferably, the variants encode the same activity of the polypeptide; or the polynucleotide is operably linked to a replication or transcription sequence. Also provided is a cell transfected with the polynucleotide. Methods are provided, e.g., comprising screening a library comprising such polypeptides for a polypeptide that interferes with the interaction of a Jak3 with a &ggr;
c
, and identifying those polypeptides which do interfere. In certain embodiments, the screening is inside a cell.
Alternatively, the invention provides a polypeptide that: comprises a sequence matching at least 45 out of 53 of residues 263-315 of SEQ ID NO: 2 (human &ggr;
c
); comprises an amino acid residue substitution corresponding to position 266 and/or 269; and interferes with the interaction of human Jak3 with a human &ggr;
c
. Typically: the matching is at least 48 out of 53; the polypeptide comprises sequence corresponding to residues 263-269; or the residue at position 266 or 269 is substituted with a conservative substitution.
Nucleic acid embodiments include, e.g., an isolated polynucleotide encoding the described polypeptide, or its complement, including variants resulting from the degeneracy of the genetic code or point mutations. Typically, the variants encode the same activity of the polypeptide; or the polynucleotide is operably linked to a replication or transcription sequence. Cells transfected with the described polynucleotide are provided.
The invention provides methods, e.g., comprising screening a library comprising
Cacalano Nicholas A.
Johnston James A.
Ching Edwin P.
Monshipouri M.
Prouty Rebecca E.
Schering Corporation
Wang Hugh
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