Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-10-21
2003-03-04
Wehbe', Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S455000, C536S023100, C424S093200
Reexamination Certificate
active
06528304
ABSTRACT:
The present invention relates to eukaryotic cells which express at least one HLA-G isoform on their surface and to their uses, in particular for obtaining a medicament for modulating the cytolytic activity of NK cells in pathologies in which these NK cells are activated or inhibited and as a model system for use, in particular, in a method for screening antineoplastic substances.
The present invention also relates to transgenic animals which specifically express at least one HLA-G isoform.
The antigens of the major histocompatibility complex (MHC) divide into several classes, i.e. class I antigens (HLA-A, HLA-B and HLA-C), which exhibit
3
globular domains (&agr;1, &agr;2 and &agr;3) and whose &agr;3 domain is associated with &bgr;2 microglobulin, the class II antigens (HLA-DP, HLA-DQ and HLA-DR) and the class III antigens (complement).
In addition to the abovementioned antigens, the class I antigens include other antigens known as non-classical class I antigens, in particular the HLA-E, HLA-F and HLA-G antigens; this latter antigen, in particular, is expressed by the extravillous trophoblasts of the normal human placenta.
The sequence of the HLA-G gene (HLA-6.0 gene) has been described by Geraghty et al., (Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149): it comprises 4396 base pairs and exhibits an intron/exon organization which is homologous with that of the HLA-A, -B and -C genes. More precisely, this gene comprises 8 exons, 7 introns and an untranslated 3′ end; the 8 exons correspond, respectively, to: exon 1: signal sequence, exon 2: &agr;1 extracellular domain, exon 3: &agr;2 extracellular domain, exon 4: &agr;3 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and untranslated 3′ region (Geraghty et al., loc. cit.; Ellis et al.,
J. Immunol.,
1990, 144, 731-735; Kirszenbaum M. et al.,
Oncogeny of hematopoiesis. Aplastic anemia
Eds. E. Gluckman, L. Coulombel, Inserm Symposium/John Libbey Eurotext Ltd). However, the HLA-G gene differs from the other class I genes in that the in-frame translation termination codon is located in the second codon of exon 6; as a consequence, the cytoplasmic region of the protein encoded by this HLA-6.0 gene is considerably shorter than that of the cytoplasmic regions of the HLA-A, -B and -C proteins.
These HLA-G antigens are mainly expressed by the cytotrophoblastic cells of the placenta and are regarded as playing a role in the protection of the foetus (no rejection by the mother). Furthermore, to the extent that the HLA-G gene is monomorphic, it may also be involved in the growth or function of the placental cells (Kovats et al., Science, 1990, 248, 220-223).
Other studies dealing with this non-classical class I antigen (Ishitani et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3947-3951) have shown that the primary transcript of the HLA-G gene can be spliced in several ways and produces at least 3 distinct mature mRNAs: the primary transcript of HLA-G gives one complete copy of 1200 bp (G1), one fragment of 900 bp (G2) and one fragment of 600 bp (G3).
The G1 transcript does not contain exon 7 and corresponds to the sequence described by Ellis et al. (loc. cit.), i.e. it encodes a protein which comprises a leader sequence, three external domains, a transmembrane region and a cytoplasmic sequence. The G2 mRNA does not contain exon 3, i.e. it encodes a protein in which the &agr;1 and &agr;3 domains are linked directly; the G3 mRNA contains neither exon 3 nor exon 4, i.e. it encodes a protein in which the &agr;1 domain and the transmembrane sequence are linked directly.
The splicing which prevails for obtaining the HLA-G2 antigen entails an adenine (A) (originating from the domain encoding &agr;1) being linked to an AC sequence (derived from the domain encoding &agr;
3),
leading to the creation of an AAC (asparagine) codon in place of the GAC (aspartic acid) codon which is encountered at the beginning of the sequence which encodes the &agr;3 domain in HLA-G1.
The splicing which is generated for obtaining HLA-G3 does not entail the formation of a new codon in the splicing zone.
The authors of this article have also analysed the different proteins which are expressed: the 3 mRNAs are translated into protein in the 0.221-G cell line.
The authors of this article conclude that HLA-G plays a fundamental role in protecting the foetus with regard to a maternal immune response (induction of immune tolerance). However, it is pointed out that the role of the G3 protein, which does not contain the &agr;3 domain, is not established.
Some of the inventors have recently demonstrated the existence of other spliced forms of HLA-G mRNA: i.e. the HLA-G4 transcript, which does not include exon 4; the HLA-G5 transcript, which includes intron 4 between exons 4 and 5, thereby giving rise to a change in the reading frame during translation of this transcript, in particular to the appearance of a stop codon after amino acid 21 of intron 4; and the HLA-G6 transcript, which possesses intron 4 but which has lost exon 3 (Kirszenbaum M. et al.,
Proc. Natl. Acad. Sci.
USA, 1994, 91, 4209-4213; European Application EP 0 677 582; Kirszenbaum M. et al.,
Human Immunol.,
1995, 43, 237-241; Moreau P. et al.,
Human Immunol.,
1995, 43, 231-236); they have also demonstrated that these different transcripts are expressed in several types of human foetal and adult cells, in particular in lymphocytes (Kirszenbaum M. et al.,
Human Immunol.,
1995, loc. cit.; Moreau P. et al.,
Human Immunol.,
1995, loc. cit.).
There are therefore at least 5 different HLA-G mRNAs which potentially encode 5 isoforms of HLA-G.
Although the foetus can be regarded as being a semiallograft, the foetal cells survive and are not rejected by the mother; it has emerged that the HLA-G molecules which are expressed on the surface of the trophoblasts protect the foetal cells from lysis by the maternal natural killer. (NK) cells (Carosella E. D. et al., C.R. Acad. Sci., 318, 827-830; Carosella E. D. et al.,
Immunol. Today,
1996, 407-409).
Earlier studies have demonstrated that expression of HLA-G molecules on the surface of target cells protects the said target cells from the lytic activity of the NK cells of the decidual layer of the maternal endometrium (Chumbley G. et al.,
Cell Immunol.,
1994, 155, 312-322; Deniz G. et al.,
J. Immunol.,
1994, 152, 4255-4261). It is to be noted that these target cells are obtained by means of transfection with vectors which contain the HLA-G genomic DNA, which is potentially able to generate all the alternative transcripts.
The NK cells express receptors for MHC class I molecules (killer inhibitory receptors or KIR or NKIR for NK inhibitory receptors), which receptors are responsible for inhibiting cytotoxicity when these HLA molecules, acting as ligands, are recognized by these receptors; for example, Pazmany L. et al., (Science, 1996, 274, 792-795) showed that the expression of HLA-G protected LCL 721.221 (B lymphoma cell line) target cells, which were transfected with the HLA-G gene, from lysis. These cells are ordinarily sensitive to NK cells; they furthermore identified the receptors on the NK cells which recognize HLA-G, namely the NKIR1 and NKIR2 receptors, which belong to the immunoglobulin (p58) superfamily and which are able to distinguish between two dimorphic groups of HLA-C molecules; HLA-G could be the natural ligand of the NK cell receptors; thus, some of the inventors have shown that NK cells do not express any HLA-G transcript; this result confirms that the expression products of the HLA-G gene probably play a role in immunotolerance (Teyssier M. et al.,
Nat. Immunol.,
1995, 14, 262-270).
In view of the important role which the HLA-G molecule may play both in pathologies in which the NK cells are particularly active (autoimmune diseases, transplantations) or in which they are, on the other hand, inhibited (abnormal presence of HLA-G molecules, in particular on certain tumours or in viral infections), the invent
Carosella Edgardo Delfino
Dausset Jean
Kirszenbaum Marek
Paul Pascale
Rouas-Freiss Nathalie
Commissariat A l'Energie Atomique
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Wehbe' Anne M.
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