Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-10-13
2001-07-17
Carlson, Karen Cochrane (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C514S002600, C514S008100, C530S350000
Reexamination Certificate
active
06262244
ABSTRACT:
This invention relates to DNA and cDNA sequences which are normally transcribed in natural killer (NK) cells or in some T cells, vectors comprising the DNA or cDNA sequences and the proteins encoded by the DNA or cDNA sequences. The DNA sequences can be detected by employing a combination of differential hybridisation and cDNA subtraction methodology.
Natural killer (NK) cells are present in the peripheral blood in a state capable of lysing NK sensitive targets in a reaction that is not restricted by proteins of the major histocompatibility complex. These cells typically account for about 5% of peripheral blood lymphocytes and they usually possess the large granular lymphocyte (LGL) morphology. In human systems NK activity is usually defined as the ability to lyse cells of the promyeloid leukaemia, K562. In many cases stimulation of NK cells with interleukin 2 (IL-2) will confer on them lymphokine activated killer (LAK) activity, the ability to lyse a broader range of tumour target cells. Many ongoing studies are attempting to use cells with LAK activity in anti-tumour therapy.
From mRNA isolated from human NK cells it has been possible to generate a cDNA library from which has been isolated four related novel genes which constitute a new mammalian gene family, hereinafter designated NKG2. In sequence homology searches using standard selectivity criteria and the Intelligenetics software program to search the SwissProt and PIR databases, significant homologies were detected only with members of the type II transmembrane proteins with C-type animal lectin domains, and not with any other sequences. Although the overall sequence homology is low, they share homology throughout a stretch of approximately 120 amino acids with the most striking feature being the presence of six invariant cysteine residues at fixed positions. In the known transmembrane proteins, this region corresponds to a carbohydrate binding domain which enables the proteins to bind carbohydrates in a Ca
++
and pH-dependent manner (Drickamer 1988 J Biol Chem 263, 9557). It is therefore believed that the novel gene family of the present invention also codes for transmembrane proteins having a carbohydrate bonding domain.
The invention provides the genes coding for the extracellular part of four specific NKG2 transmembrane proteins, as well as for the complete transmembrane proteins. The invention also provides the corresponding proteins, hereinafter designated NKG2-A, -B, -C and -D.
A murine molecule belonging to the gene family of type II membrane proteins is described by Yokoyama (Yokoyama et al 1989 J. Immunol. 143). The murine Ly-49 alloantigen is recognised by the Al monoclonal antibody, and is a type II membrane protein, the expression of which is confined almost exclusively to a subset of mouse NK cells. The gene is found to map to the same position on mouse chromosome six as the gene for NK1.1, a commonly used murine NK cell marker also expressed almost exclusively on NK cells. Ly-49 is expressed on only about 20% of NK1.1 positive cells and is distinct from NK1.1.
A further molecule first identified by a special monoclonal antibody is described by Chambers (Chambers et al 1989 J Exp Med 169, 1373). This molecule corresponds to the antigen NKR-P1 and is expressed almost exclusively on rat NK cells. The reaction of this antibody with its target antigen delivers a transmembrane signal as evidenced by its ability to mediate reverse antibody-dependent cellular cytotoxicity of an appropriate Fc receptor positive target cell. The corresponding cDNA encodes a type II membrane protein of 223 amino acids (Giorda et al 1990 Science 249, 1298). Although the Ly-49 and NKR-P1 antigen genes have similarities with NKG2, they display very limited amino acid sequence homology, the homology with the NKG2-A protein sequence being 33% for Ly49 and 23% for NKRP1. It is accordingly most unlikely that NKG2 is simply the human equivalent of these murine genes (in which case an amino acid homology of approx. 70% or more would be expected), but rather represents a novel gene family.
The novel genes are preferentially expressed in NK cells and T cells of mammals, preferably of primates and most preferably of humans. By using a standard method such as Northern blot technique the mRNA corresponding to the genes of the invention can be detected in some T cells, but cannot be detected in B cells, on EBV-transformed B cell lines (EBV=Epstein-Barr virus) or other cells.
The present invention provides new DNA or cDNA sequences and novel proteins having the corresponding amino acid sequences, all of these being in isolated pure form. By “isolated pure form” is meant in a form substantially (>90%, preferably >95%) free of other DNA or protein of mammalian origin.
In particular, the invention provides an isolated DNA or cDNA molecule encoding the extracellular part of a transmembrane protein herein designated (a) NKG2-A
f
, (b) NKG2-B
f
, (c) NKG2-C
f
and (d) NKG2-D
f
(f=fragment) translated in natural killer cells or T cells, wherein the molecule contains a DNA or cDNA sequence selected from the group comprising:
a) that part of the DNA sequence shown in SEQ ID NO:1, comprising the nucleotides numbered 459 to 863 inclusive (=the entire sequence shown in SEQ ID NO:3); or
b) that part of the DNA sequence shown in SEQ ID NO:1, comprising the nucleotides numbered 504 to 863 inclusive (=the entire sequence shown in SEQ ID NO:4); or
c) that part of the DNA sequence shown in SEQ ID NO:5 comprising the nucleotides numbered 296 to 700 inclusive (=the entire sequence shown in SEQ ID NO:7); or
d) that part of the DNA sequence shown in SEQ ID NO:8, comprising the nucleotides numbered 585 to 986 inclusive (=the entire sequence shown in SEQ ID NO: 10).
Additionally the invention comprises DNA or cDNA sequences that code for the same amino acid sequence as that coded for by any of a)-d) above; and/or which hybridise under stringent conditions to any of a)-d) above; and/or have a homology of 80 to 100% with any of the DNA sequences a)-d) above. A homology of 90 to 100% is preferred, more preferred is a homology of 95 to 100%, particularly preferred is 98 to 100%.
The activity of the extracellular part of the proteins comprises inter alia the recognition of a ligand on a cell surface, and there is evidence that said ligand is a carbohydrate structure which is for example expressed on cancer cells and virus infected cells (Drickamer 1988, J Biol Chem 263, 9557).
Additionally the invention provides an isolated DNA or cDNA molecule encoding a complete transmembrane protein herein designated aa) NKG2-A, bb) NKG2-B, cc) NKG2-C and dd) NKG2-D which are translated in natural killer cells or T cells, wherein the molecule contains a DNA or cDNA sequence selected from the group comprising:
aa) that part of the DNA sequence shown in SEQ ID NO:1, comprising the nucleotides numbered 165 to 863 inclusive (=the entire sequence shown in SEQ ID NO:11), or
bb) that part of the DNA sequence shown in SEQ ID NO:1, comprising the nucleotides numbered 165 to 863 inclusive; the nucleotides numbered 449 to 502 being deleted (=the entire sequence shown in SEQ ID NO:12); or
cc) that part of the DNA sequence shown in SEQ ID NO:5 comprising the nucleotides numbered 8 to 700 inclusive (=the entire sequence shown in SEQ ID NO:13); or
dd) that part of the DNA sequence shown in SEQ ID NO:8, comprising the nucleotides numbered 339 to 986 inclusive (=the entire sequence shown in SEQ ID NO: 14).
The deletion referred to in (bb) interrupts two codons, TC . . . T (Ser) and GC . . . A (Ala), but there is no effect upon the translation as the codon formed after the deletion, TCA, also codes for serine.
Additionally the invention comprises isolated DNA or cDNA sequences that code for the same amino acid sequence as that coded for by any of aa)-dd) above; and/or which hybridise under stringent conditions to any of aa)-dd) above; and/or have a homology of 80 to 100% with any of the DNA sequences aa)-dd) above. A ho
Bach Fritz H.
Hofer Erhard
Houchins Jeffrey P.
McSherry Cynthia M.
Yabe Toshio
Carlson Karen Cochrane
Kassenoff Melvyn M.
Regents of the University of Minnesota
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