Determination of peptide motifs on MHC molecules

The present invention concerns a method for the determination of allele-specific peptide motifs on molecules of the major histocompatibility complex (MHC) of classes I and II as well as the peptide motifs which are obtainable by the method according to the invention. In addition the use of the peptide motifs according to the invention for the production of a diagnostic or therapeutic agent is disclosed.

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 in the "Catalogue of 
Cell Lines and Hybridomas" of the ATCC (American Type Culture Collection, 
12301 Parklawn Drive, Rockville, Md. 20852) but are not limited to these. 
Preferred examples (in the ATCC nomenclature), include HB82, 117, 166, 54, 
122, 164, 95, 120, 116, 118, 94, 152, 178, 56, 115, 157, 119, 59, 105, 
165, 144, 180, 103, 110, 109, 151 and 104. All antibodies against mouse 
H-2 antigens mentioned in the catalogue can also be used in the invention. 
The immunoprecipitation is particularly preferably carried out by solid 
phase-bound antibodies. Solid phase-bound antibodies can be produced in a 
manner well known to a person skilled in the art for example by coupling 
the antibody to cyanogen bromide-activated Sepharose 4B (Pharmacia LKB). 
Other examples of solid phases to which antibodies can be bound for the 
use according to the invention include agarose, cellulose, Sephadex, 
protein-A-Sepharose and protein-G-Sepharose but are not limited to these. 
The preferred method of immunoprecipitation is adsorption chromatography 
by means of antibodies which are coupled to beads which are manufactured 
from cyanogen bromide-activated Sepharose 4B (see example 1). 
The separation of the peptide mixtures to be determined from MHC molecules 
and other protein components is advantageously carried out by a 
chromatographic method, preferably by reverse phase HPLC. In this 
connection it has proven to be advantageous to carry out the separation in 
a trifluoroacetic acid/H.sub.2 O-trifluoroacetic acid/acetonitrile 
gradient. Other methods which can be used according to the invention to 
separate peptide mixtures from MHC molecules include ion exchange, gel 
filtration, electrofocussing, high performance capillary electrophoresis 
(HPCE) and gel electrophoresis but are not limited to these. Another means 
for carrying out the separation is ultrafiltration in which a membrane 
with a permeability of 3000 or 5000 or 10000 Da is used. The separation is 
preferably carried out by means of HPLC. 
In the chromatographic separation of the peptide mixtures it is possible in 
some cases to isolate a single peptide species. Consequently, step (d) of 
the method according to the invention comprises either the sequencing of a 
peptide mixture by which means a consensus sequence can be determined for 
the peptide motifs which are located on the respective MHC molecule or/and 
sequencing a defined peptide. 
Normal cells, tumour cells as well as cells infected by viruses or other 
pathogens and in vitro cultured cells of humans or animals can be used as 
the starting material for the determination of peptide motifs. Normal 
cells which can be used in the invention include but are not limited to 
fresh cells such as e.g. peripheral blood lymphocytes, cells of the 
spleen, lung, thymus or cells of another tissue which expresses MHC 
molecules. Tumour cell lines used in the invention include the tumour 
cells EL4 and P815 but are also not limited to these. Virally infected 
cells which can be used in the invention include but are not limited to JY 
cells which are human B cells transformed by the Epstein-Barr virus. The 
peptide motifs determined by the method according to the invention 
correspond to the following basic principle: 
a) They have an allele-specific peptide length of 8, 9, 10, or 11 amino 
acids in MHC class I molecules as well as of 8 to 15 amino acids in MHC 
class II molecules, 
b) they have two anchor positions (the term "anchor position" is used when 
a position shows a strong signal for a single amino acid residue or when a 
position is occupied by a few amino acid residues with very closely 
related side chains) of which one anchor position is always located at the 
C-terminal end and is frequently aliphatic and 
c) the peptides are naturally presented on MHC molecules of normal, virally 
infected or otherwise infected cells or cells transfected with genes or 
coated with antigen. 
The sequencing of the self-peptide mixtures from the MHC class I molecules 
H2K.sup.d, H2K.sup.b, H2D.sup.b and HLA-A2 shows a different 
allele-specific peptide motif in each case which is presented by each of 
the class I molecules. The peptides presented by K.sup.d, D.sup.b and A2 
are nonamers whereas the K.sup.b -presented peptides are octamers and the 
corresponding peptide motifs contain two anchor positions which are 
occupied by a single amino acid residue or by a small number of amino acid 
residues with closely related side chains. These anchor positions are not 
located at the same site in the various motifs, they can for instance be 
at position 5 and 9 (D.sup.b) or 2 and 8 (K.sup.d, A2) or 5 and 8 
(K.sup.b). The C-terminal anchor residues of all motifs are hydrophobic 
amino acids. The amino acid residues which are not located at anchor 
positions can be quite variable; some however, are chiefly occupied by 
particular amino acids, for example Pro is often found at position 4 of 
the K.sup.d motif, Tyr at position 3 of the K.sup.b motif and hydrophobic 
residues are predominant at positions 3 of the D.sup.b motif and 6 of the 
A2 motif. A proline anchor residue was at position 2 of H-2L.sup.d. 
The results obtained by the method according to the invention correspond 
very well with the structure of the groove in MHC class I molecules found 
by crystallography (3,6). Different MHC class I alleles differ at this 
groove by the presence of different pockets which is presumably due to the 
fact that the pockets can accomodate different amino acids in each case. 
Thus the allele-specific pockets in the MHC crystals and the side chains 
of the allele-specific anchor residues presumably represent complementary 
structures. 
The present invention in addition concerns the use of the peptide motifs 
according to the invention in a process for the production of a diagnostic 
or therapeutic agent. A possible area of application for the peptide 
motifs is the diagnostic detection of MHC molecules. Since the MHC 
molecules are characterized by their individual specific binding of 
peptides, a binding test can be carried out by means of peptides with a 
marker group in which for example a biotin or a fluorescent group is 
coupled to the peptide as the marker group. Other labels known to a person 
skilled in the art can also be used in the invention. These labels 
include, without being limited thereto, radioactive markers such as e.g. 
.sup.131 I, or .sup.125 I bound to the tyrosine residues of peptides or 
.sup.3 H or .sup.14 C (both of which are incorporated into the peptides 
during their synthesis). Binding of the labels to the peptides can be 
achieved according to methods well known to a person skilled in the art. 
The labelling is preferably carried out at non-anchor positions. The 
correlations between the occurrence of autoimmune diseases and the 
expression of MHC molecules with disease-specific peptide motifs which are 
found in this manner can be utilized diagnostically. Examples of in vitro 
diagnostic uses of the peptide sequences according to the invention 
include, without being limited thereto, measurement of the binding 
specificity of MHC molecules, correlation of the binding specificity of 
MHC molecules with diseases, and determination of the sequence of T cell 
epitopes of unknown origin by incubating suitable cells which express the 
MHC molecules of interest with HPLC fractions of a peptide library 
(mixture of peptides which fit into the motif being examined) and 
determining the peptides recognized by the T cell, followed by a 
chromatographic comparison of the natural T cell epitope with the 
synthetic peptide recognized as the T cell epitope (Nature 348: 252-254 
(1990)). 
The invention in addition concerns the use of the peptide motifs according 
to the invention in a process for the production of a therapeutic agent 
for the therapy of disturbances of the immune system or of tumour 
diseases. In particular the peptide motifs according to the invention can 
be used for intervention in autoimmune diseases (prophylaxis and therapy), 
for example by blocking certain MHC molecules as well as by inducing the 
peptide-specific non-reactivity of T cells. In addition an intervention in 
transplant rejections and graft-versus-host reactions is also possible in 
an analogous manner. In addition the peptides according to the invention 
can be used in vitro and in vivo for the induction or amplification or 
proliferation of T cells directed against tumour cells in particular for 
vaccination against tumour diseases and for the therapy of existing tumour 
diseases in which in particular the so-called graft-versus-leukemia effect 
(Sullivan et al., N. Engl. J. Med. 320: 828-834) can be utilized. The 
peptides according to the invention can also be used to amplify T cell 
responses towards infectious or malignant diseases by employing 
MHC-binding peptides in vivo which are specific for the infectious agent 
or for tumours. Alternatively, T cells can be obtained from animals, their 
number increased in vitro by using peptides and suitable growth 
conditions, including cytokines such as e.g. interleukin 2, interleukin 4 
or interleukin 6, and subsequently returned to the patient. In addition 
the peptides according to the invention can be used to treat all tumours 
which express antigens which can be attacked by T cells including, but not 
being limited to, melanomas, breast cancer, tumours of viral origin such 
as e.g. Burkitt's lymphoma and those tumours which are caused by human 
papilloma virus such as cervical carcinoma and other anogenital tumours. 
Peptides which are derived from T cell receptor molecules or antibody 
molecules can also be utilized for the targetted manipulation of 
immunoregulatory mechanisms, in particular for the control of autoimmune 
diseases and transplant rejections as well as graft-versus-host reactions. 
In vivo uses of the proteins according to the invention for prevention 
include without being limited to their use as peptide vaccines against 
infectious or malignant diseases and use of the information compiled in 
this invention with regard to suitable T cell epitopes for incorporation 
into all other types of vaccines including recombinant vaccines (including 
viruses such as vaccinia or bacteria such as salmonella or mycobacteria) 
and proteins which have been produced by using recombinant bacteria (e.g. 
E. coli) or other cells, including yeast, insect, murine or human cells. 
The dosage or concentrations of the peptides according to the invention can 
be routinely determined by a person skilled in the art. These can be 
expected in vivo to be in a range of 10 .mu.g to 1 g. In vitro 
concentrations can be expected to be in a range of 1 femtomole to 1 
micromole. The in vivo administration includes, but is not limited to, a 
subcutaneous, intramuscular, intraveneous, intradermal and oral route. 
In the therapeutic application, a peptide which corresponds to a peptide 
motif according to the invention is preferably covalently linked at the N- 
or/and C-terminus to lipophilic or amphiphilic groups, in particular 
lipophilic peptide helices. An example of such a group is 
tripalmitoyl-S-glycerylcysteinylserylserine.