Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-20
2002-09-24
Carlson, Karen Cochrane (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S069100, C435S320100, C530S350000, C530S300000
Reexamination Certificate
active
06455683
ABSTRACT:
FIELD OF THE INVENTION
The invention is generally in the field of infection, inflammation and allergy. More specifically, the present invention concerns three novel DNA molecules encoding three polypeptides, which may be useful in controlling and modulating activation and differentiation of lymphoid cells. The present invention also concerns expression vectors comprising the genes, host cells comprising the expression vectors, proteins produced by the genes, methods for producing the proteins, and methods of using the genes and proteins.
BACKGROUND OF THE INVENTION
Natural killer (NK) cells are lymphocytes that participate in innate immune response against certain bacteria, parasites, and viruses. (Lanier, L. L., (1998)
Annu. Rev. Immunol
. 16:359-393). NK cells express a lectin-like receptor superfamily of type II transmembrane proteins (amino terminus intracellular). Their extracellular domains have structural features of C-type lectins. (Ryan, J. C., et al., (1997)
Immunol. Rev
. 155:79-89). The superfamily consists of several families including Ly-49 (in mice and rats), NKR-P1 (in mice, rats, and humans), NKG2 (in humans and rats), and CD94 (in humans). These proteins are encoded by a single genetic region called the NK gene complex (NKC) which are located on human chromosome 12, mouse chromosome 6 and rat chromosome 4. Different receptors, even within the same family, have been shown to activate or to inhibit NK cell functions. (Vely, F., et al., (1997)
J. Immunol
. 159:2075-2077). In many cases, the different activities mediated by individual receptors have been linked to the different structures of these receptors in their cytoplasmic domain and in their transmembrane domain.
For example, the murine Ly-49D and Ly-49H, and the human NKG2C, which possess a positively charged residue (arginine orlysine) within their transmembrane domain, have been shown to activate NK cells by associating with DAP12 membrane adapter protein. (Smith, K. M., et al., (1998)
J. Immunol
. 161:7-10; Lanier, L. L., et al., (1998)
Immunity
8:693-701). The DAP12 contains a negatively charged residue (aspartic acid) in its transmembrane region and an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. Upon cross-linking of CD94/NKG2C, tyrosine residues in ITAM of DAP12 become phosphorylated and recruit tyrosine kinases, such as ZAP-70 or Syk.
On the other hand, the murine Ly-49A that lacks charged residues in its transmembrane region and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain has been demonstrated to inhibit NK cytotoxicity. (Nakamura, M. C., et al., (1997)
J. Exp. Med
. 185:673-684). The inhibitory activity is mediated by cytoplasmic tyrosine phosphatase, SHP-1, which is recruited by the ITIM domain of Ly-49A. The tyrosine phosphatase SHP-1 can dephosphorylate the adjacent adapter proteins and kinases, resulting in the termination of activation signals.
Genes located in NKC also encode other C-type lectins such as CD69 and the recently identified receptor AICL. (Lopez-Cabrera, M., et al., (1993)
J. Exp. Med
. 178:537-547; Hamann, J., et al., (1997)
Immunogenetics
45:295-300). Unlike the restricted expression of other NK cell receptors, both CD69 and AICL are widely expressed on hematopoietic cells including lymphocytes, monocytes and granulcytes. They are not expressed on resting, but are rapidly induced upon activation. CD69 is known as the earliest activation marker of lymphocytes. Anti-CD69 mAb can induce activation and cytokine production of T, B and NK cells, though CD69 lacks a charged residue in its intracellular domain. (Testi, R., et al., (1994)
Immunol Today
. 15:479-483).
Because of the diverse biological activities of members of the lectin-like receptor superfamily and their close relationship to immune cell functions, those skilled in the art are interested in identifying novel members of this family. The identification and study of novel genes and proteins may lead to a better understanding of the mechanisms underlying immune cell functions, and will permit those skilled in the art to regulate or control immune reactions or diseases.
SUMMARY OF THE INVENTION
The present invention includes a novel “CLAX” protein (C-type Lectin, Activation eXpressed) shown in SEQ ID NO:2 (
FIG. 2A
) and a nucleic acid sequence (SEQ ID NO:1) encoding said CLAX protein. Additionally encompassed within the invention are nucleic acid sequences encoding homologues to said CLAX protein (
FIGS. 2B
,
2
C and
2
D). The homologues are referred to herein as clone 7B (nucleic acid sequence shown in SEQ ID NO:3; amino acid sequence shown in SEQ ID NO:4); clone 2I (nucleic acid sequence shown in SEQ ID NO:5; amino acid sequence shown in SEQ ID NO:6); and clone 4A (nucleic acid sequence shown in SEQ ID NO:7; amino acid sequence shown in SEQ ID NO:8). The nucleotide sequences of the isolated cDNA's are disclosed herein along with the deduced amino acid sequences. The cDNA genes of the above clones have been deposited on Jan. 19, 1999 with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and given the Accession Numbers ATCC 203599 (HuCLAX-7B) (clone 7B); ATCC 203601 (HuCLAX-2I) (clone 2I); and ATCC 203600 (HuCLAX-4A) (clone 4A).
The present inventors sequenced the clones encoding the novel CLAX protein homologues and determined the primary sequences of the deduced proteins. The nucleic acid and amino acid sequences of the novel CLAX protein disclosed herein were determined from the sequenced clones. The novel CLAX protein exhibits sequence identity to the known sequence of human CD69.
The CLAX protein of the present invention can be produced by: (1) inserting the cDNA of a disclosed CLAX into an appropriate expression vector; (2) transfecting the expression vector into an appropriate transfection host(s); (3) growing the transfected host(s) in appropriate culture media; and (4) purifying the protein from the culture media.
The present invention therefore provides a purified and isolated nucleic acid molecule, preferably a DNA molecule, having a sequence which codes for CLAX protein, or an oligonucleotide fragment of the nucleic acid molecule which is unique to a CLAX protein of the present invention. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:1 (FIG.
2
A). In another preferred embodiment, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:3 (FIG.
2
B). In still another preferred embodiment the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:5 (FIG.
2
C). In still another preferred embodiment of the present invention the purified and isolated nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO:7 (FIG.
2
D).
The invention also contemplates a double stranded nucleic acid molecule comprising a nucleic acid molecule of the invention or an oligonucleotide fragment thereof hydrogen bonded to a complementary nucleotide base sequence.
The terms “isolated and purified nucleic acid” and “substantially pure nucleic acid”, e.g., substantially pure DNA, refer to a nucleic acid molecule which is one or both of the following: (1) not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or (2) which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantial
Chen Xiaorong
Davis Patricia M.
Kiener Peter A.
Yang Guchen
Bristol--Myers Squibb Company
Carlson Karen Cochrane
Robinson Hope A.
Sher Audrey F.
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