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Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same

a histocompatibility complex and single-chain technology, applied in the field of single-chain class i major histocompatibility complexes, constructs encoding same and methods of generating same, can solve the problems of limited production yield of single-chain mhc-peptide complexes, limited production yield of these single-chain mhc-peptide complexes, and limited production efficiency of recombinant mhc-peptide complexes

Inactive Publication Date: 2004-05-06
REITER YORAM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention relates to methods of generating functional mammalian single chain MHC class I complexes in prokaryotic expression systems and eukaryotic or prokaryotic expression systems. These complexes are capable of presenting specific antigenic peptides to CD8+ T cells and can be used for functional and structural studies, as well as for direct visualization of antigen-specific T cells. The invention also provides a novel human single chain MHC class I polypeptide. The technical effect of the invention is the development of a reliable method for producing large amounts of soluble and functional multimeric MHC-peptide complexes, which are necessary for effective T-cell activation."

Problems solved by technology

However, such studies require a reproducible method for producing large amounts of soluble and functional multimeric MHC-peptide complexes.
The expression of functional MHC complexes in eukaryotic cells suffers from several inherent limitations.
In addition, the production yields of these single-chain MHC-peptide complexes was limited, typically reaching levels of several hundred micrograms per liter of culture supernatant.
One of the limitations in generating and using recombinant MHC-peptide complexes is a relatively low production efficiency.

Method used

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  • Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same
  • Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same
  • Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same

Examples

Experimental program
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Effect test

example 1

[0120] Materials and Methods

[0121] Peptides:

[0122] Cancer cell associated peptides G9-209-2M (IMDQVPFSV SEQ ID NO: 1) and G9-280-9V (YLEPGPVTV SEQ ID NO: 2), both derived from the melanoma antigen gp100 [24-26] were used for MHC binding. These peptides are modified at the MHC anchor positions 2 (in G9-209-2M) and 9 (in G9-280-9V) to improve the binding affinity to HLA-A2 [26]. The TAX 11-19 HTLV-1 peptide (LLFGYPVYV, SEQ ID NO: 3) was used as a control [53].

[0123] Peptides were synthesized by standard fluorenylmethoxycarbonyl chemistry and purified to >95% by reverse phase HPLC. Following purification these peptides were used in the refolding of the single-chain MHC (scMHC)-peptide complexes and formation of scMHC-peptide tetramers further described hereinunder.

[0124] Plasmid Constructs:

[0125] A cDNA construct (SEQ ID NO: 4) encoding a single-chain (sc) MHC polypeptide (SEQ ID NO: 5) which includes the first three extracellular domains of HLA-A2 (HLA-A*0201, amino acids 1-275 SEQ ID...

example 2

[0180] Co-Expression of the scMHC Complex and Peptide

[0181] It will be appreciated that the various scMHC constructs described above and any mammalian scMHC class polypeptides can also be co-expressed in E. coli or any other prokaryotic or eukaryotic expression systems along with the respective binding peptides thereof, thus negating the need for separately providing the binding peptide. Thus according to this method of the present invention, a single construct can be configured so as to express both the scMHC class I polypeptide and respective binding peptide coding sequences in the same cell by utilizing for example two separate promoters or an internal ribosome entry site. Alternatively the cells of the prokaryotic or eukaryotic expression system be co-transformed with two expression constructs each expressing a single coding sequence and each having a specific selection marker.

[0182] Suitable eukaryotic expression systems include, but are not limited to mammalian or insect cell ...

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Abstract

Provided are methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a host cell transformed with expression construct(s) capable of expressing a functional human single chain MHC class I complex capable of presenting specific antigenic peptides restricted to specific CTL clones.

Description

[0001] This is a Divisional of U.S. patent application Ser. No. 09 / 534,966, filed Mar. 27, 2000.FIELD AND BACKGROUND OF THE INVENTION[0002] The present invention relates to methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a functional human single chain MHC class I complex in eukaryotic or prokaryotic expression systems, which complexes are capable of presenting specific antigenic peptides restricted to class I MHC and recognizable by specific CTL clones or CD+8 T-cells. The present invention further relates to a method of generating a functional mammalian single chain MHC class I-peptide complex in eukaryotic, or preferably prokaryotic expression systems, to nucleic acid constructs encoding said single chain MHC class I complexes and to a novel human single chain MHC class I polypeptide.[0003] The major histocompatibility complex (MHC) is a complex of antigens encoded by a group of linked loci, which are collectivel...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/47C07K14/74C07K19/00C12N15/09C12N1/11C12N1/19C12N1/21C12N5/10C12P21/02
CPCC07K14/70539
Inventor REITER, YORAM
Owner REITER YORAM
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