Transcripts of the MHC class I HLA-G gene and their applications

• US 6,291,659 B1
• Filed: 10/27/1997
• Issued: 09/18/2001
• Est. Priority Date: 03/18/1994
• Status: Expired due to Term

• Current Assignee: Commissariat a L'Energie Atomique (AES Corporation)
• Original Assignee: Commissariat a L'Energie Atomique (AES Corporation)
• Inventors: Gluckman, Eliane, Moreau, Philippe, Carosella, Edgardo Delfino, Kirszenbaum, Marek
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/958,316
• Time in Patent Office: 1,422 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules

Abstract

Transcripts of the major histocompatibility complex (MHC) Class I HLA-G gene which are present in foetal trophoblasts and/or in adult circulating mono-nuclear cells, as well as their applications. Either the said transcripts comprise, in succession in the 5′ to 3′ direction: a fragment encoding the signal peptide (exon 1), a fragment encoding the α1 domain (exon 2), a fragment encoding the α2 domain (exon 3), a fragment encoding the transmembrane TM domain (exon 5), a fragment encoding the cytoplasmic domain (exon 6) and the 3′ untranslated fragment (exon 8), which sequence is designated HLA-G4, or the said transcripts comprise intron 4.

Claims

1. A cDNA sequence derived from an mRNA of the human MHC HLA-G gene, characterized in that it comprises, in succession in the 5′

to 3′

direction;

a sequence encoding the signal peptide (exon

     1),

a sequence encoding the α

1 domain (exon

     2),

a sequence encoding the α

2 domain (exon

     3),

a sequence encoding the transmembrane TM domain (exon

     5),

a sequence encoding the cytoplasmic domain (exon

     6), and

the 3′

untranslated sequence (exon

     8), said cDNA not containing a sequence encoding the α

3 domain.

4. Transcription product of the human MHC HLA-G cDNA of claim 1, characterized in that it includes, proceeding from the 5′

end;

a sequence encoding the signal peptide (exon

     1),

a sequence encoding the α

1 domain (exon

     2),

a sequence encoding the α

2 domain (exon

     3),

a sequence encoding the transmembrane TM domain (exon

     5), and

a 0.43 kb sequence encoding the cytoplasmic domain (exon

     6) of HLA-G.

18. Procedure for selecting and enriching undifferentiated hematopoietic cells, characterized in that it comprises:

(a) withdrawing a sample selected from the group consisting peripheral blood, umbilical cord blood and bone marrow,

(b) bringing the sample into contact with anti-CD34 antibodies,

(c) separating the cell complexes formed which contain a CD34 antigen-anti-CD34 antibody,

(d) carrying out an in-situ RT PCR on the cells obtained in step (c) in the presence of sequence fragments of claim 1 as primers which are labeled with a fluorochrome,

(e) separating the fluorescent cells which are obtained, and

(f) selecting pluripotent, immature nonfluorescent cells and obtaining undifferentiated hematopoietic cells.

19. Procedure for detecting cells which express a transcript of the human MHC HLA-G gene comprising:

(a) bringing blood cells into contact with a pair of primers that are sequence fragments of the cDNA of claim 1 which are labeled, and

(b) performing an RT PCR using said primers, and

(c) separating the labeled cells by any appropriate means and detecting cells which express a transcript of the human MHC HLA-G gene.

20. Procedure for detecting adult circulating mononuclear cells expressing a transcript with intron 4, characterized in that it comprises the in-situ implementation of an RT PCR, by:

(a) bringing blood cells into contact with a pair of labeled primers which are sequence fragments according to claim 1,

(b) selecting transcripts comprising intron 4, by hybridization with a probe having the sequence of SEQ ID NO;

10 or 11; and

(c) separating the labeled cells by any appropriate means and detecting adult circulating mononuclear cells.

21. Procedure for specifically separating circulating mononuclear cells, characterized in that it comprises the in-situ implementation of an RT PCR, by:

(a) bringing blood cells into contact with a pair of primers that are sequence fragments of claim 1 which are labeled, and

(b) separating the labeled cells in a suitable manner and separating mononuclear cells.

2. A cDNA sequence derived from mRNA of the human MHC HLA-G gene characterized in that it comprises, in succession in the 5′

to 3′

direction;

a sequence encoding the α

2 domain (exon

     3),

a sequence encoding the transinembrane TM domain (exon

     5),

a sequence encoding the cytoplasmic domain (exon

     6), and

the 3′

untranslated fragment (exon

     8) , said cDNA not containing a sequence encoding the α

3 domain.

3. Sequence according to claim 2, characterized in that it encodes a protein in which the α

2 domain and the transmembrane sequence of HLA-G are directly linked, and consists of SEQ ID NO;

1.

5. Oligonucleotide, characterized in that it consists of SEQ ID NO:

2.

6. Oligonucleotide, characterized in that it consists of SEQ ID NO:

3.

7. Oligonucleotide, characterized in that it consists of SEQ ID NO:

4.

8. Oligonucleotide, characterized in that it consists of SEQ ID NO:

5.

9. Oligonucleotide, characterized in that it consists of SEQ ID NO:

6.

10. Probe characterized in that it consists of SEQ ID NO:

4.

11. Probe, characterized in that it consists of SEQ ID NO:

6.

12. Pair of primers, characterized in that the pair consists of an oligonucleotide of the SEQ ID NO:

2 and an oligonucleotide of the SEQ ID NO;

5.

13. Pair of primers, characterized in that the pair consists of an oligonucleotide of the SEQ ID NO:

3 which is paired with an oligonucletide of the SEQ ID NO;

5.

14. A cDNA sequence, designated HLA-G5 and derived from an mRNA of the human MHC HLA-G gene of adult circulating mononuclear cells, comprising, in succession in the 5′

to 3′

direction;

a sequence encoding the signal peptide (exon

     1),

a sequence encoding the α

1 domain (exon

     2),

a sequence encoding the α

2 domain (exon

     3),

a sequence encoding the α

3 domain (exon

     4),

intron 4,

a sequence encoding the transmenibrane TM domain (exon

     5),

a sequence encoding the cytoplasmic domain (exon

     6), and

the 3′

untranslated sequence (exon

     8), for the preparation of a drug for use as an immunomodulating agent.

16. A sequence according to claim 14, without exon 6 and/or without exon 8.

15. A cDNA sequence, designated HLA-G6 and derived from an mRNA of the human MHC HLA-G gene of adult circulating mononuclear cells, comprising, in succession in the 5′

to 3′

direction;

a sequence encoding the signal peptide (exon

     1),

a sequence encoding the α

1 domain (exon

     2),

a sequence encoding the α

3 domain (exon

     4),

intron 4,

a sequence encoding the transmembrane TM domain (exon

     5),

a sequence encoding the cytoplasmic domain (exon

     6), and

the 3′

untranslated sequence (exon

     8), for the preparation of a drug for use as an immunomodulating agent.

17. Method for obtaining immunomodulating soluble proteins, comprising preparing an expression vector construct comprising a cDNA as defined according to claim 15 and causing expression of said vector construct to form soluble proteins.

22. An isolated cDNA sequence derived from an mRNA of the human MHC HLA-G gene characterized in that it comprises, in succession in the 5′

to 3′

direction;

a sequence encoding the signal peptide (exon

     1),

a sequence encoding the α

1 domain (exon

     2),

a sequence encoding the α

2 domain (exon

     3),

a sequence encoding the transmembrane TM domain (exon

     5),

a sequence encoding the cytoplasmic domain (exon

     6), and

the 3′

untranslated sequence (exon

     8), which sequence is designated HLA-G4 and is present in trophoblasts of the first trimester of gestation as well as in adult circulating mononuclear cells, said cDNA sequence not including a sequence encoding the α

3 domain (exon

     4).