Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-05-09
1998-05-05
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 75, 436 86, 436 89, 530344, G01N 33531, G01N 3368
Patent
active
057472696
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention concerns a method for the determination of peptide motifs or epitopes on molecules of the major histocompatibility complex (MHC) as well as the peptide motifs which are determined by this means and their use for the production of a diagnostic or therapeutic agent.
The cytotoxic T lymphocytes (CTL) recognize antigenic peptide epitopes in association with MHC-coded molecules. This phenomenon is called MHC restriction (1-5). Crystallography of human MHC class I molecules, HLA-2 and Aw68, revealed a groove which is formed by the .alpha.1 and .alpha.2 domains of the heavy chains (3,6). It is presumed that this groove is the binding site for antigenic peptide epitopes since both crystals contained structures of peptide size which were not compatible with MHC sequences and were located at this groove (6).
It is assumed that these peptides are derived from intracellular proteins and are presented at the cell surface in order to allow the cytotoxic T lymphocytes to check the cells for abnormal properties. MHC-associated peptides which represent T cell epitopes have already been extracted from normal or virally infected cells (2,4,5,7,8). Antigens which are recognized by the MHC class II-restricted T cells can also be mimicked in a corresponding manner by artificial peptides (9) and MHC-associated antigenic peptides were eluted by MHC class II molecules (10). Due to their position at the centre of trimolecular complexes which consist of T cell receptor, peptide and MHC molecule (11), the T cell epitopes are a central point of the specific immune system and thus there is a great need to understand the rules governing their occurrence and for a method of determination (12-15).
The object according to the invention is achieved by a method for the determination of allele-specific peptide motifs on molecules of the major histocompatibility complex (MHC) of classes I or II which is characterized in that
(a) a cell extract is produced by lysing cells which contain MHC molecules,
(b) MHC molecules with the peptide mixtures which are located thereon are separated from the cell extract by immunoprecipitation,
(c) the peptide mixtures are separated from MHC molecules and other protein components,
(d) individual peptides or/and a mixture thereof are sequenced and
(e) the allele-specific peptide motif is derived from the information obtained, in particular from the sequencing of a mixture or from the sequencing of a number of individual peptides.
Peptide motifs are determined by the method according to the invention which comprise the rules by which MHC molecules select and present peptides.
The method according to the invention can be carried out with MHC molecules of class I as well as with MHC molecules of class II, whereby MHC molecules of class I are preferred. H-2K.sup.d, H-K.sup.b, H-2D.sup.b H-2K.sup.k, H-2K.sup.m or HLA-A*0201 or A*0205 molecules are particularly preferred.
When MHC molecules are immunoprecipitated by the method according to the invention, it is advantageous to use antibodies which are specific for the MHC molecules which are desired in each case. Preferred MHC class I molecules for the use according to the invention include but are not limited to the molecules A1, A2, A3, A9, A10, A11, A28, A29, Aw19, B5, B7, B8, B12 to B18, B21, B35 and B37. Preferred MHC class II molecules for the use according to the invention include but are not limited to the molecules DR1, DR2, DR3, DR4, DR5, DRw6, DR7, Dw1, Dw2 and Dw3. For the determination of H-2K.sup.d or H-2D.sup.b molecules, K.sup.d -specific antibodies (25) or D.sup.b -specific antibodies (26) are for example used. Monoclonal antibodies are preferably used, it is however, also possible to use an appropriately purified polyclonal antiserum. Antibodies which can be used according to the invention can be produced de novo by means of standard techniques which are well known to a person skilled in the art. Examples of antibodies which can be used in the invention include all antibodies against HLA antigens, which are mentioned
REFERENCES:
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D.F. Hunt et al. Science, 255, 1261-1263, 1992.
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Falk Kirsten
Jung Gunther
Rammensee Hans-Georg
Rotzschke Olaf
Stevanovic Stefan
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
Saunders David
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