Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1998-10-02
2001-07-31
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C930S140000, C424S085100
Reexamination Certificate
active
06268482
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cytokine polypeptides designated as LERK-6 that bind to the hek or elk receptor, the nucleic acids encoding such polypeptides, processes for production of recombinant LERK-6 polypeptides, and pharmaceutical compositions containing such polypeptides.
BACKGROUND OF THE INVENTION
Proteins known as the receptor tyrosine kinases have an intrinsic kinase activity that is activated upon ligand binding. This class of proteins is characterized by conserved structural motifs within the catalytic domains (Hanks et al.,
Science
, 242:42, 1988) and can be subdivided into families based on structural features of the regions N-terminal to the catalytic domain.
Boyd et al. (
J. Biol. Chem
., 267:3262, 1992) purified a cell-surface glycoprotein exhibiting tyrosine kinase activity. The amino acid sequence identified this protein as a member of the eph/elk family, and the protein was thus designated hek (human eph/elk-like kinase). A monoclonal antibody immunoreactive with hek was used to study hek expression on a number of human cell types (Boyd et al., supra). Hek antigen was detected on the human pre-B cell leukemia cell line LK63 (the cell line employed as the immunogen against which the antibody was raised) and the human T-cell leukemia cell line, JM. The Raji B lymphoma cell line showed weak hek antigen expression, and the remaining cell lines tested (both normal and tumor cell lines, among which were hemopoietic cell lines that included pre-B and T-cell lines) were consistently negative. Of the normal and tumor tissue biopsy specimens that were also tested for hek antigen expression, none of the normal tissues was positive and only a very low proportion of hemopoietic tumors was positive.
Expression of hek transcripts in the above-described LK63 and JM cell lines, as well as the human T-cell leukemia cell line HSB-2, has been demonstrated by northern blot analysis (Wicks et al.,
Proc. Natl. Acad. Sci. USA
, 89:1611, 1992). Nucleotide and amino acid sequences for an isolated hek cDNA clone are presented in Wicks et al., supra.
The cell surface protein designated elk is another member of the eph-related tyrosine kinase receptor family of proteins. A partial clone of elk was first discovered in a rat brain cDNA expression library that was screened for proteins expressing tyrosine kinase activity (Letwin et al.,
Oncogene
3:621, 1988). Later, a composite sequence spanning the entire elk coding region was derived from partial clones isolated from a rat brain cDNA library and a rat cerebellar brain library using the partial clone as a probe (Lhotak et al.,
Mol. Cell. Biol
. 11:2496, 1991).
The hek and elk proteins are closely related to a number of other receptor tyrosine kinases, including the hek homologs mek4 and cek4 (Sajjadi et al.
New Biol
. 3:769, 1991); eek (Chan et al.
Oncogene
6:1057, 1991); erk (Chan et al. supra.), eck (Lindberg et al.
Mol. Cell. Biol
. 10:6316, 1990); cek5 (Pasquale, E. B.
Cell Regulation
2:523, 1991); and eph (Hirai et al.
Science
23:1717, 1987). The proteins of this subfamily are related not only in their cytoplasmic domains, but also in their extracellular domains, which are 41 to 68% identical. Interestingly, the tissue distributions of these various receptors are diverse. For example, expression of elk mRNA has been reported to be limited to testis and brain (Lhotak et al., supra), whereas eck is found not only in these same two tissues but in lung, intestine, kidney, spleen, ovary, and skin as well. In addition, most eph-related receptors are primarily expressed in the brain. Due to the homology of the receptros in the eph family, a given ligand for one specific receptor may also bind other receptors.
Those ligands that have been identified for the receptor tyrosine kinases are a diverse group of proteins that affect the growth, differentiation, and survival of cells expressing the receptors. Ligands for hek and elk have been isolated, as discussed in more detail below.
Identification of additional ligands for hek and elk that may exist would prove useful in investigating the nature of cellular processes regulated by signaling through these receptors. If enhancement or inhibition of a particular biological signal mediated through these receptors is desired, it is advantageous to identify each of the proteins that may play a role in transduction of such signals. Further, it is known that certain proteins can bind to receptors without initiating signal transduction, including interleukin-1 receptor antagonist protein (Eisenberg et al.,
Nature
343:341, 1990; Hannum et al.,
Nature
343:336, 1990; and Carter et al.,
Nature
344:633, 1990). Identification of additional proteins that bind hek or elk is also desirable in order to determine whether such proteins function as antagonists.
SUMMARY OF THE INVENTION
The present invention pertains to mammalian LERK-6 as an isolated or homogeneous protein. In addition, the invention is directed to isolated DNAs encoding mammalian LERK-6 and to expression vectors comprising a cDNA encoding mammalian LERK-6. Within the scope of this invention are host cells that have been transfected or transformed with expression vectors that comprise a cDNA encoding LERK-6, and processes for producing LERK-6 by culturing such host cells under conditions conducive to expression of LERK-6.
In addition, LERK-6 can be bound to a solid phase matrix and used to affinity-purify or separate cells that express hek or elk on their cell surface. The invention encompasses separating cells having the hek or elk receptor on the surface thereof from a mixture of cells in solution, comprising contacting the cells in the mixture with a contacting surface having a LERK-6 molecule thereon, and separating the contacting surface and the solution.
DETAILED DESCRIPTION OF THE INVENTION
A cDNA encoding murine LERK-6 has been isolated and is disclosed in SEQ ID NO:1. An exon from the human LERK-6 has been isolated and is disclosed in SEQ ID NO:7. This discovery of a cDNA encoding LERK-6 enables construction of expression vectors comprising cDNAs encoding LERK-6; host cells transfected or transformed with the expression vectors; biologically active LERK-6 as homogeneous proteins; and antibodies immunoreactive with LERK-6.
LERK-6 may be useful in the enhancement, stimulation, proliferation or growth of cells expressing the hek or elk receptor. Since the hek or elk receptor is found in the tissue of the brain and testis, treatment of a variety of conditions associated with tissue damage thereof is possible. Moreover, the ligand and receptor complex may be inveloved in neural growth, development and/or maintenance. While not limited to such, particular uses of the LERK-6 are described infra.
As used herein, the term “LERK-6” refers to a genus of polypeptides that bind and complex independently with hek or elk receptor found on T-cells and brain cells. The term “LERK-6” encompasses polypeptides having the amino acid sequence 1-184 of SEQ ID NO:2, and proteins that are encoded by nucleic acids that contain the nucleic acid sequence of of SEQ ID NO:7. In addition, those LERK-6 polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequenc e 1-184 of SEQ ID NO:2, or the amino acid sequence of SEQ ID NO:8, and which polypeptides are biologically active and bind the hek or elk receptor. In addition, the term “murine LERK-6” refers to biologically active gene products of the DNA of SEQ ID NO:1. Further encompasse d by the term “LERK-6” are the GPI-linked proteins (which include an extracellular region and a C-terminal hydrophobic region), and soluble or truncated proteins that comprise primarily the receptor-binding portion of the protein, retain biological activity and are capable of being secreted. Specific examples of such soluble proteins are those comprising the sequence of amino acids 1 (Ala)-145 (Asn) of SEQ ID NO:2.
The term “hek/elk ” means either hek or elk or both hek and elk. For example, the term “anti-hek/elk antibodies” refers to antibodies again
Henry Janis C.
Immunex Corporation
Jones Simone L.
Mertz Prema
Murphy Joseph F.
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