U.S. patent application number 14/184060 was filed with the patent office on 2014-10-02 for hla peptide therapy.
This patent application is currently assigned to THE UNIVERSITY OF BIRMINGHAM. The applicant listed for this patent is THE UNIVERSITY OF BIRMINGHAM. Invention is credited to Simon BALL, Bernard MAILLERE.
Application Number | 20140295550 14/184060 |
Document ID | / |
Family ID | 36637126 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140295550 |
Kind Code |
A1 |
BALL; Simon ; et
al. |
October 2, 2014 |
HLA PEPTIDE THERAPY
Abstract
The invention provides polypeptides derived from a major
histocompatibility complex (MHC) class I human leukocyte antigen
(HLA), such as HLA-A2, and derivatives or analogues thereof. The
polypeptides, derivatives and analogues can be used to treat or
prevent allosensitisation, such as the treatment or prevention of
allograft rejection.
Inventors: |
BALL; Simon; (Birmingham,
GB) ; MAILLERE; Bernard; (Gif Sur Yvette,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF BIRMINGHAM |
Birmingham |
|
GB |
|
|
Assignee: |
THE UNIVERSITY OF
BIRMINGHAM
Birmingham
GB
|
Family ID: |
36637126 |
Appl. No.: |
14/184060 |
Filed: |
February 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12299801 |
Mar 10, 2010 |
8715680 |
|
|
PCT/GB2007/001690 |
May 9, 2007 |
|
|
|
14184060 |
|
|
|
|
Current U.S.
Class: |
435/375 |
Current CPC
Class: |
A61K 35/17 20130101;
A61K 2035/122 20130101; A61K 39/001 20130101; A61K 38/00 20130101;
C07K 14/70539 20130101; A61P 37/06 20180101 |
Class at
Publication: |
435/375 |
International
Class: |
A61K 35/14 20060101
A61K035/14 |
Foreign Application Data
Date |
Code |
Application Number |
May 9, 2006 |
GB |
0609121.9 |
Claims
1-42. (canceled)
43. An in vitro method of stimulating T cells, the method
comprising contacting the T cells with: a polypeptide derived from
the transmembrane domain of a MHC class I HLA; under conditions
which allow stimulation of the T cells and thereby stimulating the
T cells, wherein said T cells are in a sample from a transplant
patient and the method is conducted to determine whether there is
an immune system component to graft rejection in said patient.
44. An in vitro method according to claim 43 wherein said
polypeptide is between 15 and 30 amino acids in length and
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 53, 54, 55, 56, 64, and 65, or an amino acid
sequence having greater than 90% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 53, 54, 55, 56,
64, and 65.
45. An in vitro method according to claim 44 wherein said
polypeptide comprises a sequence having greater than 90% identity
to the amino acid sequence selected from the group consisting of
SEQ ID NOs: 53, 54, 55, 56, 64, and 65, based on conservative
sequence modifications.
46. An in vitro method according to claim 45 wherein the sequence
identity is greater than 92% or 95%.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Pat. No. 8,715,680,
issued on May 6, 2014, which is a national stage filing under 35
U.S.C. 371 of International Patent Application No.
PCT/GB2007/001690, filed May 9, 2007, which claims the benefit of
British Patent Application No. 0609121.9, filed May 9, 2006, the
contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide therapy, and
particularly although not exclusively, to the use of peptide
immunotherapy to treat or prevent allograft rejection. The
invention extends to various constructs, and methods of using such
constructs in treating transplant patients, for example, patients
suffering from end stage renal failure (ESRF) who require a kidney
transplant.
BACKGROUND OF THE INVENTION
[0003] The results of renal transplantation are good in the short
and medium term. However, in the long term, kidneys are
consistently lost as a consequence of chronic allograft
(transplant) nephropathy. This is largely a consequence of two
ongoing phenomena:--(i) chronic rejection; and (ii) calcineurin
inhibitor nephrotoxicity. Both of these phenomena interact with a
pre-existing determinant of outcome, i.e. chronic damage to the
renal parenchyma established prior to and early post
transplantation. Furthermore, the requirement for long term
immunosuppression in transplant recipients has adverse
consequences, such as increased susceptibility to infection and to
malignancy.
[0004] The benefits of transplantation as treatment for patients
suffering from ESRF are manifest both in quality of life and
enhanced survival. However, a long wait to be transplanted can be
frustrating for the individual and materially affect long-term
outcome. A variety of factors determine the waiting time, but
particularly important is the presence in the transplant recipient
of antibodies that exhibit immunospecificity against polymorphic
molecules known as human leukocyte antigens (HLA), present on
potential donor organs. Anti-HLA antibodies produced by the
transplant recipient can cause a very rapid onset or `hyperacute`
rejection of the transplant organ, and their presence must
therefore be determined prior to transplantation. The potential
recipient is then excluded from receiving a transplant bearing
relevant HLA, and the patient must wait for an organ bearing HLA
antigens to which antibodies are not produced. Anti-HLA antibodies
may be stimulated by pregnancy, blood transfusion and
transplantation. The use of erythropoetin has reduced transfusion,
and enhanced HLA matching through organ sharing has reduced the
stimulation of antibody synthesis by transplantation.
[0005] Nevertheless, the production of anti-HLA antibodies, i.e.
"HLA sensitisation", remains a significant problem for
transplantation. This is particularly evident in patients who have
long-standing ESRF, often from a young age, who have heavy
cumulative exposure to allogeneic (i.e. foreign HLA). The formation
of affinity-matured class switched anti-HLA antibodies by B
lymphocytes requires the presence of T cell help. The presence of T
cell help for antibody production implies the engagement of HLA by
T cell receptors through the indirect pathway. CD4+ T lymphocytes
can recognise allogeneic HLA through conventional mechanisms of
uptake by autologous antigen presenting cells, processing to
peptide and presentation in the context of self-MHC class II. This
is called the indirect pathway of allorecognition. As well as its
role in antibody formation, T cells are also thought to play a
particularly important role in chronic rejection. The direct
pathway of allorecognition is the cross-reaction of T cell receptor
specific for self-MHC and nominal exogenous peptide on allogeneic
MHC (with associated peptide). This is thought to be particularly
important in acute rejection. The inventor of the present invention
therefore considered that treatment to minimise, prevent or
completely abolish the indirect pathway of allorecognition could be
of considerable value both prior to and after receiving a
transplant in order to reduce the likelihood of rejection and the
synthesis of anti-HLA antibodies.
[0006] A long-term goal of immunological research in
transplantation has been to develop antigen specific modulation of
the immune response that would render non-specific
immunosuppression unnecessary. Although a complete abrogation of
the requirement for immunosuppression may be unrealistic, the
inventor realised that any gain in specificity would be
welcome.
[0007] Non-antigen specific immunosuppression seems to be of
limited value n modulating chronic rejection and anti-HLA antibody
synthesis. However, while the inventor does not wish to be bound by
any hypothesis, they believe that antigen specific reduction or
inhibition of indirect presentation could diminish chronic
rejection and HLA antibody synthesis. By analogy with evidence in
the field of allergy, the inventor speculates that treatments based
on fragments of antigen, i.e. peptides, could prove beneficial.
SUMMARY OF THE INVENTION
[0008] Therefore, the inventor set out to develop a peptide
immunotherapy technique that could modulate the indirect pathway of
allorecognition in humans. They believe that this has the potential
for antigen specific modulation of alloimmune responses. In order
to test their hypothesis, they studied indirect allorecognition in
patients in whom there was evidence of a specific indirect
allogeneic response. The inventor therefore chose to investigate
patients who had made an anti-HLA antibody of known specificity,
since this implied the presence of a specific indirect
alloresponse. The inventor based his studies on a common and
problematic MHC class I antigen molecule, HLA-A2, which is
illustrated in FIG. 1.
[0009] The inventor wanted to understand exactly which parts of
this antigen molecule stimulate T lymphocytes through the indirect
pathway, and could therefore help anti-HLA-A2 antibody synthesis in
these patients. Hence, using bioinformatics, a series of 60
overlapping 15mer peptides was designed, which corresponded to
various regions of the HLA-A2 molecule, and which formed the basis
of a so-called `epitope map`. This `map` of responses to HLA-A2 was
thought likely to differ between individuals, determined by
genetically controlled elements, such as HLA-DR and the nature of
any prior exposure to HLA-A2, i.e. sensitisation. Surprisingly, of
the 60 overlapping peptides that were designed, 7 peptides could
not be synthesised due to technical difficulties in the synthesis
procedure. While the inventor does not wish to be bound by any
hypothesis, they believe that this was a consequence of the extreme
hydrophobicity of the 7 peptides not synthesised.
[0010] The inventor studied the biochemistry of the 53 peptides,
designated p1-p53, that were synthesised from HLA-A2, as listed in
FIG. 3 in order to see which were most likely to act as stimulators
through the indirect pathway of allorecognition by binding to MHC
class II molecules, in particular HLA-DR. Using an ELISA based
system, the inventor measured the binding affinity of these 53
overlapping peptides to a range of purified MHC class II molecules.
The various MHC class II molecules that were tested included: DR1,
DR3, DR4, DR7, DR11, DR13, DR15, DR51, DR52, DR53, DP0401, and
DP0402. The inventor was surprised to find that peptides frown
several locations along the HLA-A2 molecule exhibited promiscuous
binding to MHC class II. The inventor then carried out in vitro
investigations using 30 of the peptides to stimulate peripheral
blood mononuclear cells (PBMC) from 27 transplant-listed patients
with known antibody sensitisation histories. Some patients tested
had been pre-sensitised to HLA-A2, and therefore did produce
anti-HLA-A2 antibodies, other patients had not been pre-sensitised
to HLA-A2 but to other HLA antigens, and others had no anti-HLA-A2
antibodies. The subjects were also stratified according to their
own expression of HLA-A2. Hence, the inventor hoped to
systematically determine peptide epitopes to which an indirect
alloimmune response had been made. These would form candidate
therapeutic peptides for treatment of patients, to reverse and
prevent sensitisation to HLA-A2. In order to count the number of
patient's cells that made a response, the inventor used a technique
to detect cytokine production from a single cell known as the
".gamma.-interferon elispot".
[0011] To his surprise, the inventor found that peptides that were
derived from, and corresponded to certain regions of the HLA-A2
molecule are likely to have a beneficial effect on transplant
patients. To his greater surprise, the inventor found that peptides
derived from the .alpha.3 domain and/or transmembrane domains of
HLA-A2 are likely to have a beneficial effect on transplant
patients. Hence, as a result of these data, the inventor believes
that they are the first to discover and report a first medical use
for HLA-A2 derived peptides or derivatives or analogues
thereof.
[0012] The inventor chose HLA-A2 as a target antigen for their
investigations, as this is the most common type in man (about 50%).
They hypothesised that inhibition of T-cell help in the anti-HLA-A2
specific B-cell activation pathway would reduce the synthesis of
anti-HLA-A2 antibodies. Hence, the inventor believes that the use
of a peptide derived from HLA-A2 or a derivative or an analogue
thereof will have a very wide range of therapeutic uses. For
example, such therapeutic uses include treating medical conditions
characterised by allosensitisation, such as in patients requiring
repeated platelet transfusion.
[0013] The inventor has also shown that many of the polypeptides
derived from HLA-A2 are also found in other MHC class I HLA
proteins, particularly HLA-B.
[0014] Therefore, the invention provides a polypeptide consisting
of less than 30 contiguous amino acids from the .alpha.3 domain
and/or transmembrane domain of a MHC class I human leukocyte
antigen (HLA), or a derivative or analogue thereof.
[0015] The invention also provides: [0016] a nucleic acid molecule
encoding a polypeptide, derivative or analogue according to the
invention; [0017] a recombinant vector containing a nucleic acid
molecule of the invention; [0018] a host cell comprising a
recombinant vector of the invention; [0019] a pharmaceutical
composition comprising a therapeutically effective amount of a
polypeptide, derivative or analogue of the invention or a nucleic
acid molecule of the invention, and optionally a pharmaceutically
acceptable vehicle; [0020] a process for making a pharmaceutical
composition comprising combining a therapeutically effective amount
of a polypeptide, derivative or analogue of the invention or a
nucleic acid molecule of and a pharmaceutically acceptable vehicle;
[0021] a polypeptide derived from a MHC class I HLA, or a
derivative or analogue thereof, for use as a medicament; [0022] a
nucleic acid molecule encoding a polypeptide derived from a MHC
class I HLA, or a derivative or analogue thereof, or a nucleic acid
molecule that hybridizes to a nucleic acid molecule encoding a
polypeptide derived from a MHC class I HLA, or a derivative or
analogue thereof, or its complement under stringent conditions, for
use as a medicament; [0023] use of: [0024] (a) at least one
polypeptide derived from a MHC class I HLA, or a derivative or
analogue thereof; [0025] (b) at least one nucleic acid molecule
encoding a polypeptide, derivative or analogue of (a); or [0026]
(c) at least one nucleic acid molecule that hybridizes to a nucleic
acid molecule of (b) or its complement under stringent conditions;
[0027] for the manufacture of a medicament for the treatment or
prevention of a condition characterised by allosensitisation.
[0028] a method of treating or preventing a condition characterised
by allosensitisation, the method comprising administering to a
subject in need of such treatment, a therapeutically effective
amount of: [0029] (a) at least one polypeptide derived from a MHC
class I HLA, or a derivative or analogue thereof; [0030] (b) at
least one nucleic acid molecule encoding a polypeptide, derivative
or analogue of (a); or [0031] (c) at least one nucleic acid
molecule that hybridizes to a nucleic acid molecule of (b) or its
complement under stringent conditions; and [0032] an in vitro
method of stimulating T cells, the method comprising contacting the
T cells with: [0033] (a) a polypeptide derived from a MHC class I
HLA, or a derivative or analogue thereof; [0034] (b) a nucleic acid
molecule encoding a polypeptide, derivative or analogue of (a); or
[0035] (c) a nucleic acid molecule that hybridizes to a nucleic
acid molecule of (b) or its complement under stringent conditions;
[0036] under conditions which allow stimulation of the T cells and
thereby stimulating the T cells.
DESCRIPTION OF THE FIGURES
[0037] FIG. 1 shows a 3D structure representation of the
extracellular portion of the human MHC class I, HLA-A2, showing
.alpha.1, .alpha.2, .alpha.3, and transmembrane domains, and a
.beta.2 microglobulin bound thereto.
[0038] FIG. 2 shows a schematic representation of the extracellular
portion of HLA-A2, showing amino acid and DNA sequences of the
.alpha.1, .alpha.2, .alpha.3, and transmembrane domains.
[0039] FIG. 3 shows a Table listing 53 peptides (p1 to p53)
covering regions of HLA-A2, their molecular weight, and their
actual sequence. The 53 peptides were used in Examples 1 and 2.
[0040] FIG. 4 shows data resulting from binding affinity studies in
Example 1 of the 53 peptides shown in FIG. 2 to a range of purified
MHC II molecules DR1, DR3, DR4, DR7, DR11, DR13, DR15, DR51-DRB5,
DR52-DRB4, and DR53-DRB3
[0041] FIG. 5 shows a schematic representation of the extracellular
portion of the human class MHC class I. HLA-A2, and peptide p39
(residues 192-206 of HLA-A2).
[0042] FIG. 6 shows a schematic representation of the extracellular
portion of the human class MHC class I, HLA-A2, and peptides p50
(residues 268-282 of HLA-A2) and p51 (residues 270-284 of
HLA-A2).
[0043] FIG. 7 shows a schematic representation of the extracellular
portion of the human class MHC class I, HLA-A2, and peptides p52
(residues 280-294 of HLA-A2) and p53 (residues 282-296 of
HLA-A2).
[0044] FIG. 8 shows a bar graph showing Elispot count data of
reactive cells/500,000 PBMCs in a single patient for the peptides
studied in Example 1.
[0045] FIG. 9 shows data resulting from the binding affinity
studies in Example 1 of the 53 peptides shown in FIG. 2 to a range
of purified MHC II molecules DR1, DR3, DR4, DR7, DR1, DR13, DR15,
DR51-DRB5, DR52-DRB4, and DR53-DRB3. The IC50 expressed in nM were
evaluated from at least three independent experiments for each of
53 different peptides. Biotinylated reference peptides were good
binders to the HLA-DR molecules and exhibited the following IC50:
HA 306-318 (PKYVKQNTLKLAT) for HLA-DRB1*0101 (1 nM; pH 6),
HLADRB1*0401 (22 nM; pH 6), HLA-DRB1*1101 (19 nM; pH 5) and
HLA-DRB5*0101 (8 nM: pH 5.5); YKL (AAYAAAKAAAIAA) for HLADRB1*0701
(6 nM; pH 5); MT 2-16 (AKTIAYDEEARRGLE) for DRB1*0301 (303 nM; pH
4.5); B1 21-36 (TERVRLVTRHIYNREE) for HLA-DRB1*1301 (131 nM; pH
4.5); A3 152-166 (EAEQLRAYLDGTGVE) for HLA-DRB1*1501 (59 nM; pH
4.5); LOL 191-210 (ESWGAVWRIDTPDKLTGPFT) for HLA-DRB3*0101 (20 nM;
pH 5.5) and E2/E168 (AGDLLAIETDKATI) for HLA-DRB4*0101 (27 nM; pH
5). * means adjacent pairs of peptides used in combination in
subsequent functional assays.
[0046] FIG. 10 shows the age, sex, tissue type, transplantation,
transfusion and sensitisation history of study subjects in Example
2. The different patient groups are defined on the basis of the
presence of HLA-A2 in the subject and their production of
antibodies to HLA-A2 or to other HLA. Group 1: HLA-A2 negative with
antibodies to HLA-A2; Group 2: HLA-A2 negative with antibodies to
none-A2 HLA; Group 3: HLA-A2 negative with no history of anti HLA
antibody formation; Group 4: HLA-A2 positive with antibodies to
none-A2 HLA; Group 5: HLA-A2 positive with no history of anti HLA
antibody formation.
[0047] FIG. 11 shows a bar graph showing Elispot count data of
reactive cells/500,000 PBMCs in a single patient for the peptides
studied in Example 2. The mean number of responding cells per well
(5.times.10.sup.5 PBMCs) is shown for one subject cultured in the
absence or presence of increasing concentrations (1, 10 & 50
.mu.gml.sup.-1) of antibody against MHC class II (Tu39. Becton
Dickinson, Oxford, United Kingdom). Antibody against HLA-DR (L243
Becton Dickinson) at 50 .mu.gml.sup.-1 was added at the highest
concentration of anti-MHC class II. PBMC's were cultured in the
presence of peptide (p39. P50/51 or p52/53) at 20 .mu.ml.sup.-1,
PPD at 10 .mu.gml.sup.-1, the anti-CD3 positive control supplied
with the .gamma.-interferon elispot plate (Mabtech) or medium
alone.
[0048] FIG. 12 shows proliferation of T cells in response to some
of the peptides in FIG. 2. Mean .gamma.-interferon elispot
frequencies per 5.times.10.sup.5 PBMCs (y-axis) for each peptide or
peptide pair (x-axis) are shown for individuals who made a response
significantly greater than background. 21 different peptides or
pairs of peptides were studied at a concentration of 20
.mu.gml.sup.-1 (shown) or 4 .mu.gml.sup.-1. Positive controls were
ppd at 10 .mu.gml.sup.-1 or tetanus toxoid at 1 .mu.gml.sup.-1 or
in some later experiments anti-CD3 supplied with the
.gamma.-interferon elispot plate (Mabtech). The HLA-DR types of the
responders are shown in parentheses.
[0049] FIG. 13 shows a schematic representation of the peptide p39
(residues 92-206 of HLA-A2). It also shows the difference(s)
between p39 and the corresponding sequence in various HLA-B
alleles.
[0050] FIG. 14 shows a schematic representation of the peptide
p.sup.40 (residues 202-216 of HLA-A2). It also shows the
difference(s) between p39 and the corresponding sequence in
HLA-B.
[0051] FIG. 15 shows a schematic representation of the peptides p50
(residues 268-282 of HLA-A2) and p51 (residues 270-284 of HLA-A2).
It also shows the difference(s) between p39 and the corresponding
sequence in various HLA-B alleles.
[0052] FIG. 16 shows a schematic representation of the peptides p52
(residues 280-294 of HLA-A2) and p53 (residues 282-296 of HLA-A2).
It also shows the difference(s) between p39 and the corresponding
sequence in various HLA-B alleles.
DESCRIPTION OF THE SEQUENCES
[0053] SEQ ID NO: 1 shows the full-length amino acid sequence of
human MHC class I antigen, HLA-A2, (i.e. the HLA*020101
allele).
[0054] SEQ ID NO: 2 shows the mature version of the amino acid
sequence of human MHC class I antigen, HLA-A2, (i.e. the HLA*020101
allele). In other words, SEQ ID NO: 2 shows the sequence of HLA-A2
without its signal sequence. SEQ ID NO: 2 corresponds to residues
25-365 of SEQ ID NO: 1.
[0055] SEQ ID NO: 3 shows the nucleic acid sequence encoding the
full length human MHC class I antigen, HLA-A2, (i.e. the HLA*020101
allele) shown in SEQ ID NO: 1.
[0056] SEQ ID NO: 4 shows the amino acid sequence of p1 in FIG.
2.
[0057] SEQ ID NO: 5 shows the amino acid sequence of p2 in FIG.
2.
[0058] SEQ ID NO: 6 shows the amino acid sequence of p3 in FIG.
2.
[0059] SEQ ID NO: 7 shows the amino acid sequence of p4 in FIG.
2.
[0060] SEQ ID NO: 8 shows the amino acid sequence of p5 in FIG.
2.
[0061] SEQ ID NO: 9 shows the amino acid sequence of p6 in FIG.
2.
[0062] SEQ ID NO: 10 shows the amino acid sequence of p7 in FIG.
2.
[0063] SEQ ID NO: 11 shows the amino acid sequence of p8 in FIG.
2.
[0064] SEQ ID NO; 12 shows the amino acid sequence of p9 in FIG.
2.
[0065] SEQ ID NO: 13 shows the amino acid sequence of p10 in FIG.
2.
[0066] SEQ ID NO: 14 shows the amino acid sequence of p11 in FIG.
2.
[0067] SEQ ID NO: 15 shows the amino acid sequence of p12 in FIG.
2.
[0068] SEQ ID NO: 16 shows the amino acid sequence of p13 in FIG.
2.
[0069] SEQ ID NO: 17 shows the amino acid sequence of p14 in FIG.
2.
[0070] SEQ ID NO. 18 shows the amino acid sequence of p15 in FIG.
2.
[0071] SEQ ID NO: 19 shows the amino acid sequence of p16 in FIG.
2.
[0072] SEQ ID NO: 20 shows the amino acid sequence of p17 in FIG.
2.
[0073] SEQ ID NO: 21 shows the amino acid sequence of p18 in FIG.
2.
[0074] SEQ ID NO: 22 shows the amino acid sequence of p19 in FIG.
2.
[0075] SEQ ID NO: 23 shows the amino acid sequence of p20 in FIG.
2.
[0076] SEQ ID NO: 24 shows the amino acid sequence of p21 in FIG.
2.
[0077] SEQ ID NO: 25 shows the amino acid sequence of p22 in FIG.
2.
[0078] SEQ ID NO: 26 shows the amino acid sequence of p23 in FIG.
2.
[0079] SEQ ID NO: 27 shows the amino acid sequence of p24 in FIG.
2.
[0080] SEQ ID NO: 28 shows the amino acid sequence of p25 in FIG.
2.
[0081] SEQ ID NO: 29 shows the amino acid sequence of p26 in FIG.
2.
[0082] SEQ ID NO: 30 shows the amino acid sequence of p27 in FIG.
2.
[0083] SEQ ID NO: 31 shows the amino acid sequence of p28 in FIG.
2.
[0084] SEQ ID NO: 32 shows the amino acid sequence of p29 in FIG.
2.
[0085] SEQ ID NO: 33 shows the amino acid sequence of p30 in FIG.
2.
[0086] SEQ ID NO: 34 shows the amino acid sequence of p31 in FIG.
2.
[0087] SEQ ID NO: 35 shows the amino acid sequence of p32 in FIG.
2.
[0088] SEQ ID NO: 36 shows the amino acid sequence of p33 in FIG.
2.
[0089] SEQ ID NO: 37 shows the amino acid sequence of p34 in FIG.
2.
[0090] SEQ ID NO: 38 shows the amino acid sequence of p35 in FIG.
2.
[0091] SEQ ID NO: 39 shows the amino acid sequence of p36 in FIG.
2.
[0092] SEQ ID NO: 40 shows the amino acid sequence of p37 in FIG.
2.
[0093] SEQ ID NO: 41 shows the amino acid sequence of p38 in FIG.
2.
[0094] SEQ ID NO: 42 shows the amino acid sequence of p39 in FIG.
2.
[0095] SEQ ID NO: 43 shows the amino acid sequence of p40 in FIG.
2.
[0096] SEQ ID NO: 44 shows the amino acid sequence of p41 in FIG.
2.
[0097] SEQ ID NO: 45 shows the amino acid sequence of p.sup.42 in
FIG. 2.
[0098] SEQ ID NO: 46 shows the amino acid sequence of p.sup.43 in
FIG. 2.
[0099] SEQ ID NO: 47 shows the amino acid sequence of p44 in FIG.
2.
[0100] SEQ ID NO: 48 shows the amino acid sequence of p45 in FIG.
2.
[0101] SEQ ID NO: 49 shows the amino acid sequence of p46 in FIG.
2.
[0102] SEQ ID NO: 50 shows the amino acid sequence of p47 in FIG.
2.
[0103] SEQ ID NO: 51 shows the amino acid sequence of p48 in FIG.
2.
[0104] SEQ ID NO: 52 shows the amino acid sequence of p49 in FIG.
2.
[0105] SEQ ID NO: 53 shows the amino acid sequence of p50 in FIG.
2.
[0106] SEQ ID NO: 54 shows the amino acid sequence of p51 in FIG.
2.
[0107] SEQ ID NO: 55 shows the amino acid sequence of p52 in FIG.
2.
[0108] SEQ ID NO: 56 shows the amino acid sequence of p53 in FIG.
2.
[0109] SEQ ID NO: 57 shows the amino acid sequence of p50/51
(overlapping amino acids 268-282 and 270-284 SEQ ID NO: 2).
[0110] SEQ ID NO: 58 shows the amino acid sequence of p52/53
(overlapping amino acids 280-294 and 282-296 of SEQ ID NO: 2).
[0111] SEQ ID NO: 59 shows the amino acid sequence of p45/46
(overlapping amino acids 239-253 & 241-256 of SEQ ID NO:
1).
[0112] SEQ ID NO: 60 shows the amino acid sequence of p39 analogue
1.
[0113] SEQ ID NO: 61 shows the amino acid sequence of p39 analogue
2.
[0114] SEQ ID NO: 62 shows the amino acid sequence of p39 analogue
3.
[0115] SEQ ID NO: 63 shows the amino acid sequence of p40
analogue.
[0116] SEQ ID NO: 64 shows the amino acid sequence of p50/51
analogue 1.
[0117] SEQ ID NO: 65 shows the amino acid sequence of p52/53
analogue 1.
[0118] SEQ ID NO: 66 shows the amino acid sequence of p50/51
analogue 2.
[0119] SEQ ID NO: 67 shows the amino acid sequence of p50/51
analogue 3.
[0120] SEQ ID NO: 68 shows the amino acid sequence of p50/51
analogue 4.
[0121] SEQ ID NO: 69 shows the amino acid sequence of p52/53
analogue 2.
[0122] SEQ ID NO: 70 shows the amino acid sequence of p52/53
analogue 3.
[0123] SEQ ID NO: 71 shows the amino acid sequence of p52/53
analogue 4.
[0124] SEQ ID NO: 72 shows the amino acid sequence of p52/53
analogue S.
[0125] SEQ ID NO: 73 shows the amino acid sequence of p45/46
analogue 1.
[0126] SEQ ID NO: 74 shows the amino acid sequence of p45/46
analogue 2.
[0127] SEQ ID NO: 75 shows the nucleic acid sequence encoding
p1.
[0128] SEQ ID NO: 76 shows the nucleic acid sequence encoding
p2.
[0129] SEQ ID NO: 77 shows the nucleic acid sequence encoding
p30.
[0130] SEQ ID NO: 78 shows the nucleic acid sequence encoding
p39.
[0131] SEQ ID NO: 79 shows the nucleic acid sequence encoding
p40.
[0132] SEQ ID NO: 80 shows the nucleic acid sequence encoding
p50.
[0133] SEQ ID NO: 81 shows the nucleic acid sequence encoding
p51.
[0134] SEQ ID NO: 82 shows the nucleic acid sequence encoding
p52.
[0135] SEQ ID NO: 83 shows the nucleic acid sequence encoding
p53.
[0136] SEQ ID NO: 84 shows the nucleic acid sequence encoding
p45.
[0137] SEQ ID NO: 85 shows the nucleic acid sequence encoding
p46.
[0138] SEQ ID NO: 86 shows the nucleic acid sequence encoding
p50/51
[0139] SEQ ID NO: 87 shows the nucleic acid sequence encoding
p52/53
[0140] SEQ ID NO: 88 shows the nucleic acid sequence encoding
p45/46
[0141] SEQ ID NO: 89 shows the nucleic acid sequence encoding
p20
[0142] SEQ ID NO: 90 shows the nucleic acid sequence encoding
p21.
[0143] SEQ ID NO: 91 shows the nucleic acid sequence encoding p39
analogue 1.
[0144] SEQ ID NO: 92 shows the nucleic acid sequence encoding
p50/51 analogue 1.
[0145] SEQ ID NO: 93 shows the nucleic acid sequence encoding
p52/53 analogue 1.
[0146] SEQ ID NO: 94 shows the nucleic acid sequence encoding p40
analogue.
[0147] SEQ ID NO: 95 shows the nucleic acid sequence encoding
p45/46 analogue 1.
[0148] SEQ ID NO: 96 shows the nucleic acid sequence encoding
p45/46 analogue 2.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides
[0149] The invention provides a polypeptide consisting of less than
30 contiguous amino acids from the .alpha.3 domain and/or
transmembrane domain of a major histocompatibility complex (MHC)
class I human leukocyte antigen (HLA), or a derivative or analogue
thereof. The invention also concerns the use of polypeptides
derived from a MHC class I HLA, or a derivative or analogue
thereof, as a medicament.
[0150] By the term "a MHC class I HLA" used herein, we refer to any
gene product (for example, a protein as identified as SEQ ID NO:
1), defined as a MHC class I HLA in the 14.sup.th International HLA
& Immunogenetics Workshop, 2005. The gene product may comprise
at least 50% identity with a gene product encoded by a human MHC
class I HLA allele (for example, as identified as SEQ ID NO: 3)
and/or human HLA homologues from other species, or a variant or
functional fragment thereof. More preferably, the MHC class I HLA
gene product has at least 60%, preferably 70%, preferably 80%,
preferably 90%, preferably 95%, and most preferably 99% identity
with products encoded by human MHC class I HLA alleles and/or human
MHC class I HLA homologues from other species, or a variant or
functional fragment thereof. The HLA may be HLA-A, HLA-B or
HLA-C.
[0151] The inventor based his investigations on the most common MHC
I class I antigen. HLA-A2. Sequences for MHC class I antigen,
HLA-A2, are known in the art, and may be found in publicly
available databases, such as NCBI, e.g. at:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=375
18361
[0152] For example, the full-length protein sequence of human MHC
class I antigen. HLA-A2, (i.e. the HLA*020101 allele) is identified
as SEQ ID NO: 1. The mature protein sequence of human MHC class I
antigen. HLA-A2, (i.e. the HLA*020101 allele) is identified as SEQ
ID NO: 2. Furthermore, the nucleic acid sequence encoding human MHC
class I antigen, HLA-A2, (i.e. the HLA*020101 allele) may be
identified as SEQ ID NO: 3.
[0153] The nucleic acid sequence identified by SEQ ID NO: 3
comprises 8 exons, and 7 introns. These may be defined as
follows:--exon 1: 200 bp-272 bp; intron 1: 273 bp-402 bp; exon 2:
403 bp-672 bp; intron 2: 673 bp-913 bp; exon 3: 914 bp-1189 bp;
intron 3: 1190 bp-1789 bp; exon 4: 1790 bp-2065 bp; intron 4: 2066
bp-2164 bp; exon 5: 2165 bp-2281 bp; intron 5: 2282 bp-2719 bp;
exon 6: 2720 bp-2752 bp; intron 6: 2753 bp-2894 bp; exon 7: 2895
bp-2942 bp; intron 7: 2943 bp-3111 bp; and exon 8: 3112 bp-3116
bp.
[0154] In preferred embodiments, the HLA is HLA-A2. By the term
"HLA-A2" used herein, we refer to any gene product (for example, a
protein as identified as SEQ ID NO: 1 or 2), defined as HLA-A2 in
the 14.sup.th International HLA & Immunogenetics Workshop,
2005. The gene product may comprise at least 50% identity with a
gene product encoded by a human HLA-A2 allele (for example, as
identified as SEQ ID NO: 3) and/or human HLA-A2 homologues from
other species, or a variant or functional fragment thereof. More
preferably, the HLA-A2 gene product has at least 60%, preferably
70%, preferably 80%, preferably 90%, preferably 95%, and most
preferably 99% identity with products encoded by human HLA-A2
alleles and/or human HLA-A2 homologues from other species, or a
variant or functional fragment thereof.
[0155] In preferred embodiments, the HLA has the amino acid
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
[0156] By the term "derived from a MHC class I HLA", we mean a
polypeptide, derivative or analogue, which comprises an amino acid
sequence forming a MHC class I antigen. HLA, and which is a
derivative, analogue or modification thereof. In preferred
embodiments, a polypeptide derived from a MHC class HLA is a
fragment or truncation of a MHC class I HLA. Surprisingly, peptides
or polypeptides, or derivatives or analogues derived from HLA have
been shown to exhibit therapeutic activity, and in particular, have
been shown to be useful for preventing or minimising allograft
failure or rejection.
[0157] By the term "derived from HLA-A2", we mean a polypeptide,
derivative or analogue, which comprises an amino acid sequence
forming the MHC class I antigen, HLA-A2, and which is a derivative,
analogue or modification thereof. In preferred embodiments, a
polypeptide derived from HLA-A2 is a fragment or a truncation of
HLA-A2. Surprisingly, peptides or polypeptides, or derivatives or
analogues derived from HLA-A2 have been shown to exhibit
therapeutic activity, and in particular, have been shown to be
useful for preventing or minimising allograft failure or rejection.
All of SEQ ID NOs: 1, 2 and 4 to 73 are polypeptides that have been
derived from HLA-A2.
[0158] By the term "derivative or analogue thereof", we mean that
the amino acid residues of the polypeptide derived from the MHC
class I HLA protein may be replaced by residues (whether natural
amino acids, non-natural amino acids or amino acid mimics) with
similar side chains or peptide backbone properties. Additionally,
the terminals of such peptides may be protected by N- or C-terminal
protecting groups with similar properties to acetyl or amide
groups.
[0159] Similarly, by the term "derivative or analogue thereof", we
mean that the amino acid residues of the polypeptide derived from
the HLA-A2 protein may be replaced by residues (whether natural
amino acids, non-natural amino acids or amino acid mimics) with
similar side chains or peptide backbone properties. Additionally,
the terminals of such peptides may be protected by N- or C-terminal
protecting groups with similar properties to acetyl or amide
groups.
[0160] Derivatives and analogues can be formed by making one or
more mutations to the polypeptide sequence derived from a MHC class
I HLA protein (or HLA-A2). The mutations can be substitutions,
deletions or insertions of amino acids. A derivative or an analogue
may differ from the polypeptide by at least 1, but less than 5, 10,
20, 50, 100, 150, 200 or 250 amino acids from the sequences shown
in SEQ ID NO: 1 or 2. Examples of derivatives and analogues include
all of the truncations, analogues, variants, fragments and
alloantigens discussed in more detail below.
[0161] A derivative or an analogue of a polypeptide, such as a
derivative or analogue of a polypeptide derived from a MHC class I
antigen or derived from HLA-A2, binds to a MHC class II HLA and
activates a T cell specific for the polypeptide.
[0162] In other words, a derivative or an analogue of a polypeptide
will bind to a MHC class HLA and activate a T cell bearing a T cell
receptor that is specific for the polypeptide. For instance, a
derivative or an analogue of a polypeptide derived from HLA-A2 will
bind to a MHC class II HLA and activate a T cell bearing a receptor
that is specific for the polypeptide derived from HLA-A2. A T cell
is specific for a polypeptide or bears a T cell receptor that is
specific for the polypeptide if it is selected or cloned by
exposure to the polypeptide. Methods for selection or cloning of T
cells are well known in the art. For instance, a suitable method is
described in Lamb et al., J. Exp. Biol., 1983; 157: 1434-1447.
[0163] A MHC class I ELA comprises five domains, namely .alpha.1
domain, an .alpha.2 domain, an .alpha.3 domain, a transmembrane
domain and a cytosolic domain.
[0164] It will be appreciated from SEQ ID NO: 1 that full-length
human class I histocompatibility molecule, HLA-A2 protein, consists
of 365 amino acid residues. It will be appreciated from SEQ ID NO:
2 that mature human class I histocompatibility molecule. HLA-A2
protein, consists of 341 amino acid residues. FIG. 1 shows a
schematic representation of the extracellular portion of HLA-A2. It
can be seen that HLA-A2 comprises an .alpha.1 domain, an .alpha.2
domain, an .alpha.3 domain, and also a transmembrane domain and a
cytosolic domain, the latter two domains not being shown in the
Figure. It will be appreciated that the .alpha.1 and .alpha.2
domains define a substantially polymorphic region of the HLA-A2
molecule, and that the .alpha.3 domain and the transmembrane domain
define a substantially non-polymorphic region of the HLA-A2
molecule. Furthermore, a molecule of beta-2-microglobulin
(.beta..sub.2m) binds to the junction of the .alpha.1 and .alpha.2
domains, and to the .alpha.3 domain by non-covalent interactions.
Not shown in FIG. 1 is the presence of a short peptide bound
non-covalently in the groove between the alpha helices of .alpha.1
and .alpha.2 domains. It will be appreciated that the combination
of the peptide and adjacent portions of alpha helices makes up the
epitope seen by CD8+ T cells. This discussion concerning the
structure and function of the five domains in HLA-A2 also applies
to other MHC class I HLAs, such as HLA-B or HLA-C.
[0165] In some embodiments, the invention may use polypeptides,
derivatives or analogues thereof which are derived substantially
from the whole MHC class I HLA. In some embodiments, the invention
may use polypeptides, derivatives or analogues thereof, which are
derived from substantially the whole HLA-A2 protein molecule,
substantially as set out defined by SEQ ID NO: 1 or 2. Hence, the
MHC class I HLA or the HLA-A2 molecule used in the invention may
comprise the .alpha.1 domain, the .alpha.2 domain, the .alpha.3
domain, the transmembrane domain, and the cytosolic domain. The
entire HLA-A2 molecule comprises 365 amino acids (SEQ ID NO:
1).
[0166] However, the inventor has demonstrated that polypeptides, or
derivatives or analogues derived from the HLA-A2 protein molecule
(i.e. less than 365 amino acids) may also be used in accordance
with the invention. Hence, the polypeptide, derivative or analogue
thereof used in accordance with the invention may comprise a
truncation of the entire MHC class I HLA. Preferably, the
polypeptide, derivative or analogue thereof used in accordance with
the invention comprises a truncation of the entire HLA-A2 protein.
The discussion below concerning truncations focuses on truncations
of HLA-A2. However, such discussion also applies to other MHC class
I HLAs, such as HLA-B or HLA-C
[0167] By the term "truncation", we mean a polypeptide which
corresponds to a region or fragment of the HLA-A2 protein, but
which is reduced in size by removal of amino acids. The reduction
of amino acids may be by removal of residues from the C- or
N-terminal of the peptide, or may be by deletion of one of more
amino acids from within the core of the HLA-A2 molecule (i.e. amino
acids 2-364 of SEQ ID NO: 1). For example, the peptide may comprise
a deletion of 5, 10, 15, 20, or 25 amino acid residues from the
whole HLA-A2 molecule. More preferably, the peptide may comprise a
deletion of 50, 75, 100, 125, 150, 200, 225, or 150 amino acid
residues from the whole HLA-A2 molecule.
[0168] Suitably, the polypeptide or derivative or analogue thereof
derived from HLA-A2 comprises less than about 100 amino acids, more
suitably, less than about 75 amino acids, and even more suitably,
less than 50 amino acids. It is preferred that the polypeptide,
derivative or analogue thereof derived from HLA-A2 comprises less
than 30 amino acids, and more preferably, less than 20 amino acids,
and most preferably, about 15 amino acids. The inventor believes
that reducing the size of HLA-A2 that still shows a therapeutic
effect would help therapeutic delivery to a subject being
treated.
[0169] Since T lymphocytes recognise proteins on the basis of their
primary sequence as short peptides bound to MHC molecules, the
inventor believes that peptides, derivatives or analogues according
to the invention may be used to specifically desensitise T cell
responses, without the risk of exposure to `whole` antigen. This is
particularly advantageous because the whole antigen or at least
large parts of the antigen with secondary and/or tertiary structure
may itself cause sensitisation. Furthermore, another clinical
benefit observed in such so-called peptide therapy is that
peptides, derivatives or analogues thereof which are derived from a
MHC class I HLA, such as HLA-A2, may induce regulatory T cells that
can exert an antigen specific, dominant negative effect on the
immune system. Another significant advantage of the use of peptides
is that they can be easily delivered without the induction of a
danger signal, which would promote productive immunity in the
subject being treated.
[0170] Hence, the polypeptide or derivative or analogue thereof
derived from MHC class I HLA protein may be derived from a domain
independently selected from a group of domains consisting of the
.alpha.1 domain; the .alpha.2 domain; the .alpha.3 domain; the
transmembrane domain; the cytosolic domain; or any combination
thereof. For example, the polypeptide or analogue may be derived
from the .alpha.1 domain and/or the .alpha.2 domain of HLA-A2.
Preferably, the polypeptide or analogue may be derived from the
.alpha.3 domain and/or the transmembrane domain of a MHC class I
HLA.
[0171] The invention specifically provides a polypeptide consisting
of less than 30 contiguous amino acids from the .alpha.3 domain
and/or transmembrane domain of a MHC class I HLA, or a derivative
or analogue thereof. In preferred embodiments, the polypeptide
consists of less than 20 contiguous amino acids or about 15
contiguous amino acids from the .alpha.3 domain and/or
transmembrane domain of a MHC class I HLA. In another preferred
embodiment, the derivative or analogue has a sequence identity of
greater than 65% sequence identity to at least 9 contiguous amino
acids in the polypeptide. Specific sequences provided by the
invention (SEQ ID NOs: 42, 43, 48, 49 and 53 to 74) are discussed
in more detail below.
[0172] Similarly, a polypeptide or derivative or analogue thereof
derived from HLA-A2 protein may be derived from a domain
independently selected from a group of HLA-A2 domains consisting of
the .alpha.1 domain; the .alpha.2 domain; the .alpha.3 domain; the
transmembrane domain; the cytosolic domain; or any combination
thereof. For example, the peptide or analogue may be derived from
the .alpha.1 domain and/or the .alpha.2 domain of HLA-A2.
Alternatively, the peptide or analogue may be derived from the
.alpha.3 domain and/or the transmembrane domain of HLA-A2.
[0173] In HLA-A2, the .alpha.1 domain is encoded by exon 2, i.e.
nucleotides 403-672 of SEQ ID NO: 3. Hence, the polypeptide,
derivative or analogue may comprise substantially the amino acid
sequence defined as residues 1-90 of SEQ ID NO: 2, and may be
encoded by nucleotides 403-672 of SEQ ID NO: 3. The .alpha.2 domain
is encoded by exon 3, i.e. nucleotides 914-1189 of SEQ ID NO: 3.
Hence, the polypeptide, derivative or analogue may comprise
substantially the amino acid sequence defined as residues 91-182 of
SEQ ID NO: 2, and may be encoded by nucleotides 914-1189 of SEQ ID
NO: 3. The .alpha.3 domain is encoded by exon 4, i.e. nucleotides
1790-2065 of SEQ ID NO: 3. Hence, the polypeptide, derivative or
analogue may comprise substantially the amino acid sequence defined
as residues 183-274 of SEQ ID NO: 2, and may be encoded by
nucleotides 1790-2065 of SEQ ID NO: 3. The transmembrane domain is
encoded by exon 5, i.e. nucleotides 2165-2281 of SEQ ID NO: 3.
Hence, the polypeptide, derivative or analogue may comprise
substantially the amino acid sequence defined as residues 275-314
of SEQ ID NO: 2, and may be encoded by nucleotides 2165-2281 of SEQ
ID NO: 3.
[0174] Surprisingly, the results of the binding studies discussed
the Examples 1 and 2 revealed a significant number of polypeptides
that bind relatively `promiscuously` to MHC class II. Some of the
peptides are derived from the hypervariable or polymorphic region
of HLA-A2 (i.e. the .alpha.1 & .alpha.2 domains). For example,
peptide p20 (SEQ ID NO: 23) is derived from amino acid residues
105-119 of HLA-A2 (SEQ ID NO: 2), and peptide p21 (SEQ ID NO: 24)
is derived from residues 107-121). Both peptides correspond to a 17
amino acid peptide that has been eluted from DR1. The inventor
observed a positive response in 10 out of 15 patients who had made
anti-HLA-A2. Hence, a single 17 amino acid peptide that binds
promiscuously to MHC class II molecules accounts for much of the
immunogenicity in this region whatever the MHC class II.
[0175] Accordingly, the inventor believes that a single (or two
closely overlapping peptides) may be used in a treatment regime.
Hence, the polypeptide, derivative or analogue used according to
the invention may be derived from a substantially polymorphic
region of the MHC class I HLA molecule, and more preferably, from
the at and/or .alpha.2 domain thereof. Similarly, the polypeptide,
derivative or analogue used according to the invention may be
derived from a substantially polymorphic region of the HLA-A2
molecule, and more preferably, from the .alpha.1 and/or .alpha.2
domain thereof.
[0176] Hence, preferred polypeptides used according to the
invention comprise substantially the amino acid sequence:
(a) HSMRYFFTSVSRPGR (SEQ ID NO: 4). This peptide corresponds to
amino acids 3-17 of HLA-A2 protein (i.e. SEQ ID NO: 2), and is
derived from the .alpha.1 domain of the HLA-A2 molecule. This
peptide is designated p1 when referred to herein, and has a
molecular weight of 1827.1. (b) MRYYFFTSVSRPGRGE (SEQ ID NO: 5).
This peptide corresponds to amino acids 5-19 of HLA-A2 protein
(i.e. SEQ ID NO: 2), and is derived from the .alpha.1 domain of the
HLA-A2 molecule. This peptide is designated p2 when referred to
herein, and has a molecular weight of 1789. (c) HKWEAAHVAEQLRAY
(SEQ ID NO: 33). This peptide corresponds to amino acids 145-159 of
HLA-A2 protein (i.e. SEQ ID NO: 2), and is derived from the
.alpha.2 domain of the HLA-A2 molecule. This peptide is designated
p30 when referred to herein, and has a molecular weight of 1808.
(d) SDWRFLRGYHQYAYD (SEQ ID NO: 23). This peptide corresponds to
amino acids 105-119 of HLA-A2 protein (i.e. SEQ ID NO: 2), and is
derived from the .alpha.2 domain of the HLA-A2 molecule. This
peptide is designated p20 when referred to herein, and has a
molecular weight of 1976.1. (e) WRFLRGYHQYAYDGK (SEQ ID NO: 24).
This peptide corresponds to amino acids 107-121 of HLA-A2 protein
(i.e. SEQ ID NO: 2), and is derived from the .alpha.2 domain of the
HLA-A2 molecule. This peptide is designated p21 when referred to
herein, and has a molecular weight of 1959.2.
[0177] It should be appreciated that each of the peptides (a) to
(e) are derived from the polymorphic region of HLA-A2. However, the
inventor was surprised to find that a significant number of
individuals also respond to peptides derived from elsewhere in the
HLA-A2 molecule, and in particular, regions of HLA-A2 that are of
limited polymorphism, for example, the .alpha.3 and transmembrane
domains. The inventor believes that to date there have been no
reports of immune responses to peptides from at least limited or
substantially non-polymorphic regions of HLA-A2, and in particular,
the .alpha.3 and transmembrane domains of HLA-A2, or any other HLA
molecule, and consequently no report or proposal for their use in
peptide-based therapies. The inventor also believes that using
peptides derived from the non-polymorphic regions of HLA-A2 may
have a wider applicability than those derived from the polymorphic
and therefore unique regions of HLA-A2. Because they are of limited
polymorphism, these peptides act as sites of potential
cross-reactivity between different HLA molecules.
[0178] Accordingly, preferred polypeptides, derivatives or
analogues thereof of the invention are derived from a substantially
limited or non-polymorphic region of the MHC class I HLA molecule,
such as the .alpha.3 and/or transmembrane domain. It is especially
preferred that polypeptides, derivatives or analogues thereof used
according to the invention are derived from a substantially limited
or non-polymorphic region of the HLA-A2 molecule. Preferably, the
polypeptide, derivative or analogue thereof is derived from the
.alpha.3 and/or transmembrane domain of HLA-A2.
[0179] Therefore, most preferred peptides used according to the
invention comprise substantially the amino acid sequence:
(f) HAVSDHEATLRCWAL (SEQ ID NO: 42). This peptide corresponds to
amino acids 192-206 of HLA-A2 protein (i.e. SEQ ID NO: 2), and is
derived from the .alpha.3 domain of the HLA-A2 molecule. This
peptide is designated p39 when referred to herein, and has a
molecular weight of 1708. (g) RCWALSFYPAEITLT (SEQ ID NO: 43). This
peptide corresponds to amino acids 202-216 of HLA-A2 protein (i.e.
SEQ ID NO: 2), and is derived from the .alpha.3 domain of the
HLA-A2 molecule. This peptide is designated p40 when referred to
herein, and has a molecular weight of 1770. (h) KPLTLRWEPSSQPTI
(SEQ ID NO: 53). This peptide corresponds to amino acids 268-282 of
HLA-A2 protein (i.e. SEQ ID NO: 2), and is derived from the
.alpha.3 domain and the transmembrane domain of the HLA-A2
molecule. This peptide is designated p50 when referred to herein,
and has a molecular weight of 1752. (i) LTLRWEPSSQPTIPI (SEQ ID NO:
54). This peptide corresponds to amino acids 270-284 of HLA-A2
protein (i.e. SEQ ID NO: 2), and is derived from the .alpha.3
domain and the transmembrane domain of the HLA-A2 molecule. This
peptide is designated p51 when referred to herein, and has a
molecular weight of 1737. (j) PTIPIVGIIAGLVLF (SEQ ID NO: 55). This
peptide corresponds to amino acids 280-294 of HLA-A2 protein (i.e.
SEQ ID NO: 2), and is derived from the transmembrane domain of the
HLA-A2 molecule. This peptide is designated p52 when referred to
herein, and has a molecular weight of 1522. (k) IPIVGIIAGLVLFGA
(SEQ ID NO: 56). This peptide corresponds to amino acids 282-296 of
HLA-A2 protein (i.e. SEQ ID NO: 2), and is derived from the
transmembrane domain of the HLA-A2 molecule. This peptide is
designated p53 when referred to herein, and has a molecular weight
of 1452. (l) GTFQKWAAVVVPSGQEQR (SEQ ID NO: 48). This peptide
corresponds to amino acids 239-253 of HLA-A2 protein (i.e. SEQ ID
NO: 2), and is derived from the .alpha.3 domain of the HLA-A2
molecule. This peptide is designated p45 when referred to herein,
and has a molecular weight of 1574. (m) FQKWAAVVVPSGQEQR (SEQ ID
NO: 49). This peptide corresponds to amino acids 241-256 of HLA-A2
protein (i.e. SEQ ID NO: 2), and is derived from the .alpha.3
domain of the HLA-A2 molecule. This peptide is designated p46 when
referred to herein, and has a molecular weight of 1829.
[0180] It should be appreciated that each of the peptides (f) to
(m) are derived from the substantially non-polymorphic region of
HLA-A2. The inventor has found that peptides that are derived from
the non-polymorphic region (i.e. .alpha.3 and transmembrane
domains) of HLA-A2 show a surprisingly high frequency of response
in patients that have made antibody to HLA-A2 and significant
responses in some others. The inventor believes that these have not
been previously defined as T cell epitopes, and are important
because they are of limited polymorphism.
[0181] The inventor has also found that use of polypeptides,
derivatives or analogues, which comprise overlapping regions of any
of the preferred peptides disclosed herein have a therapeutic
effect, and show surprising efficacy for treating conditions
characterised by allosensitisation, such as, allograft failure of
rejection. It is therefore preferred that polypeptides, derivatives
or analogues thereof derived from the non-polymorphic regions of a
MHC class I HLA, such as HLA-A2, comprise overlapping sections or
regions.
[0182] Hence, further preferred polypeptides used according to the
invention comprise substantially the amino acid sequence:
(n) KPLTLRWEPSSQPTIPI (SEQ ID NO: 57). This peptide corresponds to
overlapping amino acids 268-282 and 270-284 of HLA-A2 protein (i.e.
SEQ ID NO: 2). This peptide is designated p50/51 when referred to
herein. (o) PTIPIVGIIAGLVLFGA (SEQ ID NO: 58). This peptide
corresponds to overlapping amino acids 280-294 and 282-296 of
HLA-A2 protein (i.e. SEQ ID NO: 2). This peptide is designated
p52/53 when referred to herein. (p) GT TQKWAAVVVPSGQEQR (SEQ ID NO:
59). This peptide corresponds to overlapping amino acids 239-253
& 241-256 of HLA-A2 protein (i.e. SEQ ID NO: 2). This peptide
is designated p45/46 when referred to herein.
[0183] By analogy with anti-HLA antibodies, the inventor believes
that the preferred peptides derived from the .alpha.3 and
transmembrane domains could be referred to as `public` T cell
epitopes. This is relevant to the evolution of spreading immune
responses to different HLA, and in designing peptides or mixtures
thereof having therapeutic potential.
[0184] By way of example, as shown in FIGS. 5 and 13, the sequence
of peptide p39, i.e. SEQ ID NO: 42, which is derived from amino
acids 192-206 of HLA-A2, is also present in the sequence of the
following HLA molecules: HLA-A2, HLA-A25, HLA-A26, HLA-A29,
HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A43, HLA-A66, HLA-A68,
HLA-A69, and HLA-A74. Therefore, upon administration of peptide p39
or a derivative or an analogue thereof to a patient, the inventor
believes that it is capable of modulating the immune response to a
transplant bearing any of these HLA molecules, and is not limited
to HLA-A2 only. It will be appreciated that this is a significant
advantage of using such a peptide, derivative or analogue in
therapy, because of its multiple effects, thereby preventing
rejection or failure of an allograft harbouring a wide range of HLA
antigens.
[0185] Furthermore, similarly, there is only a single variant of
the sequence of p39 (SEQ ID NO: 42) expressed by all other HLA-A
molecules. HLA-A I, HLA-A3, HLA-A11, HLA-A23, HLA-A24, HLA-A30,
HLA-A36, HLA-A80, and most HLA-B molecules, as indicated in FIGS. 5
and 13. This variant is called p39 analogue 1 and is shown in SEQ
ID NO: 60. Accordingly, upon administration of peptide p39 and an
analogue thereof having the sequence of SEQ ID NO: 60 (in which
proline replaces alanine at residue 193 and isoleucine replaces
valine at residue 194), the inventor believes that such a
combination has the potential to modulate the immune response to a
transplant bearing all HLA-A molecules and most HLA-B molecules
also.
[0186] Furthermore, similarly, there is a single variant of the
sequence of p39 (SEQ ID NO: 42) expressed by other HLA-B molecules,
HLA-851, HLA-B52, HLA-B353, HLA-B58, HLA-B78 as indicated in FIG.
13. This variant is called p39 analogue 2 and is shown in SEQ ID
NO: 61. Accordingly, upon administration of peptide p39 and an
analogue thereof having the sequence of SEQ ID NO: 61 (in which
proline replaces alanine at residue 193), the inventor believes
that such a combination has the potential to modulate the immune
response to a transplant bearing a wide range of HLA-A molecules
and some HLA-B molecules also.
[0187] Furthermore, similarly, there is a single variant of the
sequence of p39 (SEQ ID NO: 42) expressed by HLA-B44 as indicated
in FIG. 13. This variant is called p39 analogue 3 and is shown in
SEQ ID NO: 62. Accordingly, upon administration of peptide p39 and
an analogue thereof having the sequence of SEQ ID NO: 62 (in which
proline replaces alanine at residue 193 and valine replaces alanine
at residue 199), the inventor believes that such a combination has
the potential to modulate the immune response to a transplant
bearing a wide range of HLA-A molecules as well as HLA-A-B44.
[0188] By way of a further example, as shown in FIG. 13, the
sequence of peptide p40, i.e. SEQ ID NO: 43, which is derived from
amino acids 202-216 of HLA-A2, is also present in the sequence of
the following HLA molecules: HLA-A2, HLA-A25, HLA-A26, HLA-A29,
HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A43, HLA-A66, HLA-A68,
HLA-A69, HLA-A74, and HLA-A74. Therefore, upon administration of
peptide p40 or a derivative or an analogue thereof to a patient,
the inventor believes that it is capable of modulating the immune
response to a transplant bearing any of these HLA molecules, and is
not limited to HLA-A2 only. It will be appreciated that this is a
significant advantage of using such a peptide, derivative or
analogue in therapy, because of its multiple effects, thereby
preventing rejection or failure of an allograft harbouring a wide
range of HLA antigens.
[0189] Furthermore, similarly, there is a single variant of the
sequence of p40 (SEQ ID NO: 43) expressed by other HLA-A molecules,
HLA-A1, HLA-A3, HLA A11, HLA-A23, HLA-A24, HLA-A30, HLA-A36, as
well as most HLA-B molecules, as indicated in FIG. 14. This variant
is called p40 analogue and is shown in SEQ ID NO: 63. Accordingly,
upon administration of peptide p40 and an analogue thereof having
the sequence of SEQ ID NO: 63 (in which glycine replaces serine at
residue 207), the inventor believes that such a combination has the
potential to modulate the immune response to a transplant bearing a
wide range of HLA-A molecules and HLA-B molecules also.
[0190] Furthermore, as illustrated in FIGS. 6 and 15, peptide p50
(SEQ ID NO: 53), which is derived from amino acids 268-282, and
peptide p51 (SEQ ID NO: 54), which is derived from amino acids
270-284, comprise sequences that are offset by only 2 amino acids
and therefore span a 17 amino acid stretch. Similarly, as
illustrated in FIGS. 7 and 16, peptide p52 (SEQ ID NO: 55), which
is derived from amino acids 280-294, and peptide p53 (SEQ ID NO:
56), which is derived from amino acids 282-296, comprise sequences
that are offset by only 2 amino acids and therefore span a 17 amino
acid stretch. In both cases, the identified peptides are not only
present in HLA-A2, but also in HLA-A25, HLA-A26, HLA-A29, HLA-A31,
HLA-A32, HLA-A33, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74, and
HLA-A80.
[0191] p51 (SEQ ID NO: 54) and p52 (SEQ ID NO: 55) are also present
in HLA-B15, HLA-B18, HLA-B35, HLA-B37, HLA-B45, HLA-B48, HLA-B49,
HLA-B50. HLA-B51, HLA-52, HLA-B53, HLA-B54, HLA-B55, HLA-B58,
HLA-B59, HLA-B73, HLA-B78, HLA-B82 and HLA-B95 (FIG. 14).
[0192] Additionally, analogues of these two sequences in which a
leucine is substituted for a proline at residue 276 (for the 17mer
268-284; SEQ ID NO: 64) or a leucine is substituted for a
phenylanine at 294 (for the 17mer 280-296; SEQ ID NO: 65), would
provide peptide sequences that would be expressed by the majority
of HLA-A molecules in a way analogous to that described for p39,
the only substantial omissions being HLA-A23 & HLA-A24.
TABLE-US-00001 TABLE 1 Additional analogues of p50/51 and p52/53
SEQ Change(s) to arrive at the ID NO: Analogue analogue Present in
HLA 66 p50/51 Lysine at 273 A0320 analogue 2 67 p50/51 Valine at
282 A24 analogue 3 All HLA-B in which p50 is not present 68 p50/51
Valine at 282 A23 analogue 4 Histidine at 283 69 p52/53 Valine at
282 A24 analogue 2 Leucine at 294 70 p52/53 Valine at 282 A23
analogue 3 Histidine at 283 71 p52/53 Alanine at 292 HLA-B 15, 18,
35, 37 analogue 4 Valine at 293 45, 48-55, 58, 59, 73, Leucine at
294 78, 82, 95 Alanine at 295 Valine at 296 72 p52/53 Valine at 282
All other HLA-B analogue 5 Alanine at 292 Valine at 293 Leucine at
294 Alanine at 295 Valine at 296
[0193] Other analogues of p50/51 and p52/53 are described in the
Table 1 below.
[0194] Therefore, upon administration of a combination of
polypeptides p50, p51, 52, and p53, or derivatives or analogues
thereof to a patient, the inventor believes that it is capable of
modulating the immune response to a transplant bearing any of these
HLA molecules.
[0195] As will be apparent, specific combinations of p.sup.39, p40,
p50, p51, p50/51, p52, p53 and/or p52/53 and one or more of their
analogues discussed above have the potential to modulate the immune
response to a transplant bearing all HLA-A molecules as well as all
HLA-B molecules. This has the advantage of using as few
polypeptides as possible to modulate the immune response to a
transplant bearing any HLA-A or HLA-B molecule.
[0196] Hence, it will be appreciated from the foregoing that
preferred polypeptides, derivatives or analogues thereof which are
derived from the non-polymorphic region of HLA-A2 are also
expressed by many other HLA-A molecules, and not just HLA-A2, by
acting as sites of potential cross-reactivity between different HLA
molecules. Hence, surprisingly, the inventor believes that the
benefits of using such peptides in methods according to the
invention, and in particular for uses in treating or preventing
conditions characterised by allosensitisation, and particularly,
allograft rejection, and alloantibody synthesis, may be much wider
than to HLA-A2 antigen alone. Accordingly, the inventor believes
that the implications of their findings are much broader than
solely help for antibody production, but relate to immune
mechanisms of graft rejection per se, and are wider than target
HLA-A2, extending to responses to transplantation antigens in
general.
[0197] Polypeptides use in accordance with the invention can be an
alloantigen, or a polypeptide derived therefrom, or a derivative or
analogue thereof. Preferably, the alloantigen, or polypeptide
derived therefrom, or derivative or analogue thereof may be
independently selected from a group consisting of HLA-A2, HLA-A25,
HLA-A26, HLA-A29, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A43,
HLA-A66, HLA-A68, HLA-A69, HLA-A74, HLA-A1, HLA-A3, HLA-A11,
HLA-A24, HLA-A30, HLA-A36, HLA-A80, HLA-A1, HLA-A3, HLA-A11, and
HLA-A23, or any combination thereof.
[0198] Preferred polypeptides derived from the alloantigen may
include p39 (SEQ ID NO: 42), p50/51 (SEQ ID NO: 57), p52/53 (SEQ ID
NO: 58), p50 (SEQ ID NO: 53), p51 (SEQ ID NO: 54), 52 (SEQ ID NO:
55), and p53 (SEQ ID NO: 56), or derivatives or analogues thereof,
or any combination thereof. It will be appreciated that these
polypeptides are all derived from HLA-A2. Furthermore, efficacious
derivatives or analogues of any of the polypeptides defined herein
may also be used in accordance with the invention. For instance,
corresponding polypeptides from other MCH class I antigens, such as
HLA-B and HLA-C can be used.
[0199] The inventor also believes that various analogues of any of
the preferred polypeptides may also show efficacy at preventing or
minimising allograft failure or rejection, as shown in the
alignments below:
TABLE-US-00002 p39 (SEQ ID NO: 42) HAVSDHEATLRCWAL (q) p39 analogue
1 (SEQ ID NO: 60) HPISDHEATLRCWAL p50 (SEQ ID NO: 53)
KPLTLRWEPSSQPTI p51 (SEQ ID NO: 54) LTLRWEPSSQPTIPI (r) analogue I
17mer (SEQ ID NO: 64) KPLTLRWELSSQPTIPI p52 (SEQ ID NO: 55)
PTIPIVGIIAGLVLF p53 (SEQ ID NO: 56) IPIVGIIAGLVLFGA (s) analogue I
17mer (SEQ ID NO: 65) PTIPIVGIIAGLVLLGA p40 (SEQ ID NO: 43)
RCWALSFYPAEITLT (T) p40 analogue (SEQ ID NO: 63) RCWALGFYPAEITLT
p45/46 (SEQ ID NO: 59) GTFQKWAAVVVPSGQEQR (u) p45/46 analogue I
(SEQ ID NO: 73) GTFQKWAAVVVPSGEEQR (v) p45/46 analogue 2 (SEQ ID
NO: 74) GTFQKWASVVVPSGQEQR
[0200] Hence, preferred analogues used according to the invention
comprise substantially the amino acid sequence:
(q) HPISDHEATIRCWAL (SEQ ID NO: 60). This analogue is derived from
peptide p39 (SEQ ID NO: 42), and is derived from the .alpha.3
domain of the HLA-A2 molecule, except that the alanine residue is
replaced with a proline residue, and the valine residue is replaced
with an isoleucine residue. This analogue is designated "p39
analogue 1" when referred to herein. (r) KPLTLRWELSSQPTIPI (SEQ ID
NO: 64). This analogue is derived from peptides p50 (SEQ ID NO: 53)
and p51 (SEQ ID NO: 54), and is derived from the .alpha.3 domain
and the transmembrane domain of the HLA-A2 molecule, except that
the proline residue at position 276 is replaced with a leucine
residue. This analogue is designated "p50/p51 analogue 1" when
referred to herein. (s) PTIPIVGIIAGLVLLGA (SEQ ID NO: 65). This
analogue is derived from peptides p52 (SEQ ID NO: 55) and p53 (SEQ
ID NO: 56), and is derived from the transmembrane domain of the
HLA-A2 molecule, except that the phenylalanine residue at 294 is
replaced with a leucine residue. This analogue is designated
"p52/53 analogue 1" when referred to herein.
[0201] (t) RCWALGFYPAEITLT (SEQ ID NO: 63). This analogue is
derived from peptide p40 (i.e. SEQ ID NO: 43), and is derived from
the .alpha.3 domain of the HLA-A2 molecule, except that the serine
residue at position 207 is replaced with a glycine residue. This
analogue is designated "p40 analogue" when referred to herein.
(u) GTFQKWAAVVVPSGEEQR (SEQ ID NO: 73). This analogue is derived
from peptide p45/46 (SEQ ID NO: 59), and is derived from the
.alpha.3 domain of the HLA-A2 molecule, except that the glutamine
residue is replaced with a glutamic acid residue. This analogue is
designated "p45/p46 analogue 1" when referred to herein. (v)
GTFQKWASVVVPSGQEQR (SEQ ID NO; 74). This analogue is derived from
peptide p45/46 (SEQ ID NO: 59), and is derived from the .alpha.3
domain of the HLA-A2 molecule, except that the alanine residue is
replaced with a serine residue. This analogue is designated "p45/46
analogue 2" when referred to herein.
[0202] It will be appreciated that the invention extends to use of
any polypeptide, derivative or analogue thereof derived from a MHC
class I HLA, which comprises substantially the amino acid sequences
of any of the sequences referred to herein, including functional
variants, or fragments thereof. It also will be appreciated that
the invention extends to use of any polypeptide, derivative or
analogue thereof derived from HLA-A2, which comprises substantially
the amino acid sequences of any of the sequences referred to
herein, including functional variants, or fragments thereof. By the
terms "substantially the amino acid/polynucleotide/polypeptide
sequence", "functional variant" and "functional fragment", we mean
that the sequence has at least 40% sequence identity with the amino
acid/polynucleotide/polypeptide sequences of any one of the
sequences referred to herein, for example, 40% identity with the
hla-a2 gene identified as SEQ ID NO: 2, or 40% identity with the
HLA-A2 protein identified as SEQ ID NO: 1 or 2. Amino
acid/polynucleotide/polypeptide sequences with a sequence identity
which is greater than 65%, more preferably, greater than 70%, even
more preferably, greater than 75%, and still more preferably,
greater than 80% sequence identity to any of the sequences referred
to is also envisaged. Preferably, the amino
acid/polynucleotide/polypeptide sequence has 85% identity with any
of the sequences referred to more preferably 90% identity, even
more preferably 92% identity, even more preferably 95% identity,
even more preferably 97% identity, even more preferably 98%
identity and, most preferably, 99% identity with any of the
referred to sequences.
[0203] The percentage identity between two amino
acid/polynucleotide/polypeptide sequences can be measured over any
length of the amino acid/polynucleotide/polypeptide sequences. For
instance, the identity can be measured over the full length of the
sequences. Alternatively, the identity can be measured over parts
of the sequences. Such parts may be at least 350, at least 300), at
least 250, at least 200, at least 250, at least 150, at least 100,
at least 50, at least 30, at least 20, at least 15, at least 10 or
at least 9 contiguous amino acids/nucleotides in length.
[0204] The skilled technician will appreciate how to calculate the
percentage identity between two amino
acid/polynucleotide/polypeptide sequences, for example, as
described in http://wikomics.org/wiki/Percentage_identity. In order
to calculate the percentage identity between two amino
acid/polynucleotide/polypeptide sequences, an alignment of the two
sequences must first be prepared, followed by calculation of the
sequence identity value.
[0205] The percentage identity for two sequences may take different
values depending on:--(i) the method used to align the sequences,
for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in
different programs), or structural alignment from 3D) comparison;
and (ii) the parameters used by the alignment method, for example,
local vs global alignment, the pair-score matrix used (e.g.
BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional
form and constants.
[0206] Having made the alignment, there are many different ways of
calculating percentage identity between the two sequences. For
example, one may divide the number of identities by: (i) the length
of shortest sequence; (ii) the length of alignment; (iii) the mean
length of sequence; (iv) the number of non-gap positions; or (iv)
the number of equivalenced positions excluding overhangs.
Furthermore, it will be appreciated that percentage identity is
also strongly length dependent. Therefore, the shorter a pair of
sequences is, the higher the sequence identity one may expect to
occur by chance.
[0207] Hence, it will be appreciated that the accurate alignment of
protein or DNA sequences is a complex process. The popular multiple
alignment program ClustalW (Thompson et al., 1994, Nucleic Acids
Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids
Research, 24, 4876-4882) is a preferred way for generating multiple
alignments of proteins or DNA in accordance with the invention.
Suitable parameters for ClustalW may be as follows: For DNA
alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and
Matrix=Identity. For protein alignments: Gap Open Penalty=10.0. Gap
Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein
alignments: ENDGAP=-1, and GAPDIST=4. Those skilled in the art will
be aware that it may be necessary to vary these and other
parameters for optimal sequence alignment.
[0208] Preferably, calculation of percentage identities between two
amino acid/polynucleotide/polypeptide sequences is then calculated
from such an alignment as (N/T)*100, where N is the number of
positions at which the sequences share an identical residue, and T
is the total number of positions compared including gaps but
excluding overhangs. Hence, a most preferred method for calculating
percentage identity between two sequences comprises (i) preparing a
sequence alignment using the ClustalW program using a suitable set
of parameters, for example, as set out above; and (ii) inserting
the values of N and T into the following formula: Sequence
Identity=(N/T)*100.
[0209] Derivatives and analogues can also include conservative
substitutions. Conservative substitutions can made according to
Table 2. Amino acids in the same block in the second column and
preferably in the same line in the third column may be substituted
for each other:
TABLE-US-00003 TABLE 2 Conservative substitutions that can be made
in accordance with the invention ALIPHATIC Non-polar GAP ILV
Polar-uncharged CSTM NQ Polar-charged DE KR AROMATIC HFWY
[0210] Modifications can be made to the amino acids in the
polypeptide derived from a MHC class I HLA or derivative or
analogue thereof. It will also be appreciated that modified amino
acids may be substituted into HLA-A2 derived peptides, or
derivatives or analogues thereof with a number of amino acid
variants that may be known to those skilled in the art to form
further preferred derivatives or analogues according to the
invention. Such derivative or analogue peptides will have
anti-allograft rejection activity provided that the modification
does not significantly alter its chemical characteristics. For
instance, hydrogens on the side chain amines of R or K may be
replaced with methylene groups (--NH.sub.2.fwdarw.-NH(Me) or
--N(Me).sub.2). Furthermore, the N-terminal amino group of the
peptides may be protected by reacting with a carboxylic acid and
the C-terminal carboxyl group of the peptide may be protected by
reacting with an amine. Other examples include glycosylation and
phosphorylation.
[0211] Analogues of peptides according to the invention may also
include peptide variants that increase or decrease the peptide's
half-life in vivo. Examples of analogues capable of increasing the
half-life of peptides used according to the invention include
peptoid analogues of the peptides, D-amino acid derivatives of the
peptides, and peptide-peptoid hybrids.
[0212] Polypeptides used according to the invention may be subject
to degradation by a number of means (such as protease activity in
biological systems). Such degradation may limit the bioavailability
of the polypeptides, and hence the ability of the polypeptides to
achieve their biological function. There are wide ranges of
well-established techniques by which polypeptide analogues or
derivatives that have enhanced stability in biological contexts can
be designed and produced. Such polypeptide derivatives may have
improved bioavailability as a result of increased resistance to
protease-mediated degradation. Preferably, a polypeptide derivative
or analogue suitable for use according to the invention is more
protease-resistant than the polypeptide from which it is derived.
Protease-resistance of a polypeptide derivative and the polypeptide
from which it is derived may be evaluated by means of well-known
protein degradation assays. The relative values of protease
resistance for the peptide derivative and peptide may then be
compared.
[0213] Peptoid analogues or derivatives of the polypeptides used in
accordance with the invention may be readily designed from
knowledge of the structure of the polypeptide. Commercially
available software may be used to develop peptoid derivatives
according to well-established protocols.
[0214] Retropeptoids, (in which all amino acids are replaced by
peptoid residues in reversed order), are also able to mimic MHC
class I HLA derived peptides or HLA-A2 derived peptides, or
derivatives or analogues thereof. A retropeptoid is expected to
bind in the opposite direction in the ligand-binding groove, as
compared to a polypeptide or peptoid-peptide hybrid containing one
peptoid residue. As a result, the side chains of the peptoid
residues are able point in the same direction as the side chains in
the original peptide.
[0215] A further embodiment of an analogue of a polypeptide used
according to the invention comprises D-amino acid forms of the
polypeptide. The preparation of polypeptides using D-amino acids
rather than L-amino acids greatly decreases any unwanted breakdown
of such an agent by normal metabolic processes, decreasing the
amounts of agent which needs to be administered, along with the
frequency of its administration.
[0216] The polypeptides, derivative or analogues used in accordance
with the invention may be made synthetically or by recombinant
means. For example, a recombinant polypeptide may be produced by
transfecting cells in culture with an expression vector comprising
a nucleotide sequence encoding the polypeptide operably linked to
suitable control sequences, culturing the cells, extracting and
purifying the polypeptide produced by the cells. Methods for the
recombinant production of polypeptides are well-known in the art
(for example, Sambrook et al. 2001, Molecular Cloning: a laboratory
manual. 3.sup.rd edition, Cold Harbour Laboratory Press).
Nucleic Acid Molecules
[0217] The invention concerns provides nucleic acids encoding a
polypeptide, derivative or analogue discussed above.
[0218] The nucleic acid molecule may encode a polypeptide, which
polypeptide forms a MHC class I HLA domain which is independently
selected from a group of MHC class I HLA domains consisting of the
at domain: the .alpha.2 domain; the .alpha.3 domain; the
transmembrane domain; the cytosolic domain; or any combination
thereof. For example, the nucleic acid molecule may encode the
.alpha.1 domain and/or the .alpha.2 domain of a MHC class I HLA.
Alternatively, the nucleic acid molecule may encode the .alpha.3
domain and/or the transmembrane domain of a MHC class I HLA.
[0219] The nucleic acid molecule may encode a polypeptide, which
polypeptide forms an HLA-A2 domain which is independently selected
from a group of HLA A2 domains consisting of the .alpha.1 domain;
the .alpha.2 domain; the .alpha.3 domain; the transmembrane domain;
the cytosolic domain; or any combination thereof. For example, the
nucleic acid molecule may encode the .alpha.1 domain and/or the
.alpha.2 domain of HLA-A2. Alternatively, the nucleic acid molecule
may encode the .alpha.3 domain and/or the transmembrane domain of
HLA-A2.
[0220] The nucleic acid sequence of the HLA-A2 gene is identified
as SEQ ID NO: 3. Hence, preferably the nucleic acid molecule
comprises the sequence substantially as represented by SEQ ID NO: 3
(i.e. encodes the entire HLA-A2 protein as identified by SEQ ID NO:
1 or the mature HLA-A2 shown in SEQ ID NO: 2). The nucleic acid
sequence of the .alpha.1 domain is represented by nucleotides
403-672 of SEQ ID NO: 3. Hence, the nucleic acid molecule may
comprise the sequence substantially as represented by nucleotides
403-672 of SEQ ID NO: 3. The nucleic acid sequence of the .alpha.2
domain is represented by nucleotides 914-1189 of SEQ ID NO: 3.
Hence, the nucleic acid molecule may comprise the sequence
substantially as represented by nucleotides 914-1189 of SEQ ID NO:
3. The nucleic acid sequence of the .alpha.3 domain is represented
by nucleotides 1790-2065 of SEQ ID NO: 3. Hence, the nucleic acid
molecule may comprise the sequence represented by nucleotides
1790-2065 of SEQ ID NO: 3. The nucleic acid sequence of the
transmembrane domain is represented by nucleotides 2165-2281 of SEQ
ID NO: 3. Hence, the nucleic acid molecule may be identified by
nucleotides 2165-2281 of SEQ ID NO: 3.
[0221] Preferred nucleic acid molecules encode a peptide
independently selected from a group of peptides consisting of: p1;
p2; p20; p21; p30; p39; p40; p50; p51; p.sup.52; p53; p45; p46;
p50/51; p52/53; and p45/46.
[0222] Hence, preferred nucleic acid molecules comprise
substantially the nucleotide sequence:
(a) 5'-CAC TCC ATG AGG TAT TTC TTC ACA TCC GTG TCC CGG CCC GGC
CGC-3' (SEQ ID NO: 75). This nucleic acid molecule encodes peptide
p1. (b) 5'-ATG AGG TAT TTC TTC ACA TCC GTG TCC CGG CCC GGC CGC GGG
GAG-3' (SEQ ID NO: 76). This nucleic acid molecule encodes peptide
p2. (c) 5'-CAC AAG TGG GAG GCG GCC CAT GTG GCG GAG CAG TTG AGA GCC
TA C-3' (SEQ ID NO: 77). This nucleic acid molecule encodes peptide
p30. (d) 5'-CAC GCT GTC TCT GAC CAT GAA GCC ACC CTG AGG TGC TGG GCC
CTG-3' (SEQ ID NO: 78). This nucleic acid molecule encodes peptide
p39. (e) 5'-AGG TGC TGG GCC CTG AGC TTC TAC CCT GCG GAG ATC ACA CTG
ACC-3' (SEQ ID NO: 79). This nucleic acid molecule encodes peptide
p40. (f) 5'-AAG CCC CTC ACC CTG AGA TGG GAG CCG TCT TCC CAG CCC ACC
ATC-3' (SEQ ID NO: 80). This nucleic acid molecule encodes peptide
p50. (g) 5'-CTC ACC CTG AGA TGG GAG CCG TCT TCC CAG CCC ACC ATC CCC
ATC-3' (SEQ ID NO: 81). This nucleic acid molecule encodes peptide
p51. (h) 5'-CCC ACC ATC CCC ATC GTG GGC ATC ATT GCT GGC CTG GTT CTC
TTT-3' (SEQ ID NO: 82). This nucleic acid molecule encodes peptide
p52. (i) 5'-ATC CCC ATC GTG GGC ATC ATT GCT GGC CTG GTT CTC TTT GGA
GCT-3' (SEQ ID NO: 83). This nucleic acid molecule encodes peptide
p53. (j) 5'-GGA ACC TTC CAG AAG TGG GCG GCT GTG GTG GTG CCT TCT GGA
CAG-3' (SEQ ID NO: 84). This nucleic acid molecule encodes peptide
p45. (k) 5'-TTC CAG AAG TGG GCG GCT GTG GTG GTG CCT TCT GGA CAG GAG
CAG AGA-3' (SEQ ID NO): 85). This nucleic acid molecule encodes
peptide p46. (l) 5'-AAG CCC CTC ACC CTG AGA TGG GAG CCG TCT TCC CAG
CCC ACC ATC CCC ATC-3' (SEQ ID NO: 86). This nucleic acid molecule
encodes peptide p50/51. (m) 5'-CCC ACC ATC CCC ATC GTG GGC ATC ATT
GCT GGC CTG GTT CTC TTT GGA GCT-3' (SEQ ID NO: 87). This nucleic
acid molecule encodes peptide p52/53. (n) 5'-GGA ACC TTC CAG AAG
TGG GCG GCT GTG GTG GTG CCT TCT GGA CAG GAG CAG AGA-3' (SEQ ID NO:
88). This nucleic acid molecule encodes peptide p45/46. (o) 5'-TCG
GAC TGG CGC TTC CTC CGC GGG TAC CAC CAG TAC GCC TAC GAC-3' (SEQ ID
NO: 89). This nucleic acid molecule encodes peptide p20. (p) 5'-TGG
CGC TTC CTC CGC GGG TAC CAC CAG TAC GCC TAC GAC GGC AAG-3' (SEQ ID
NO: 90). This nucleic acid molecule encodes peptide p21.
[0223] Furthermore, preferred nucleic acid molecules encode peptide
analogues: p39 analogue 1; p50/51 analogue; p52/53 analogue 1; p40
analogue: p45/P46 analogue 1; or p45/46 analogue 2. Hence,
preferred nucleic acid molecules used comprise substantially the
nucleotide sequence:
(q) 5'-CAC CCC ATC TCT GAC CAT GAG GCC ACC CTG AGG TGC TGG GCC
CTG-3' (SEQ ID NO: 91). This nucleic acid molecule encodes peptide
analogue, p39 analogue 1. (r) 5'-AAG CCC CTC ACC CTG AGA TGG GAG
CCT TCT TCC CAG CCC ACC ATC CCC ATC-3' (SEQ ID NO: 92). This
nucleic acid molecule encodes peptide analogue, p50/51 analogue 1.
(s) 5'-CCC ACC ATC CCC ATC GTG GGC ATC ATT GCT GGC CTG GTT CTC CTT
GGA GCT-3' (SEQ ID NO: 93). This nucleic acid molecule encodes
peptide analogue, p52/53 analogue 1. (t) 5'-AGG TGC TGG GCC CTG GGC
TTC TAC CCT GCG GAG ATC ACA CTG ACC-3' (SEQ ID NO: 94). This
nucleic acid molecule encodes peptide analogue, p40 analogue. (u)
5'-GGA ACC TTC CAG AAG TGG GCG GCT GTG GTG GTG CCT TCT GGA GAG GAG
CAG AGA-3' (SEQ ID NO: 95). This nucleic acid molecule encodes
peptide analogue, p45/46 analogue 1. (v) 5'-GGA ACC TTC CAG AAG TGG
GCG TCT GTG GTG GTG CCT TCT GGA CAG GAG CAG AGA-3' (SEQ ID NO: 96).
This nucleic acid molecule encodes peptide analogue, p45/46
analogue 2.
[0224] The nucleic acid molecule may comprise an isolated or
purified nucleic acid molecule. The nucleic acid molecule may
comprise a DNA sequence. The nucleic acid molecule may further
comprise elements capable of controlling and/or enhancing its
expression. The nucleic acid molecule may be contained within a
suitable vector to form a recombinant vector. The vector may for
example be a plasmid, cosmid or phage.
[0225] Other suitable vectors would be apparent to persons skilled
in the art. By way of further example in this regard we refer to
Sambrook et al., 2001, Molecular Cloning: a laboratory manual,
3.sup.rd edition. Cold Harbour Laboratory Press.
[0226] Such recombinant vectors are highly useful in the delivery
systems for transforming cells with the nucleic acid molecule.
Hence, the invention provides a host cell comprising a vector
comprising any of the nucleic acids disclosed herein. For instance,
the invention provides a host cell comprising a vector comprising
nucleic acid molecule encoding a polypeptide derived from HLA-A2,
or a derivative or analogue thereof.
[0227] Recombinant vectors may also include other functional
elements. For instance, recombinant vectors can be designed such
that the vector will autonomously replicate in a cell. In this case
elements that induce nucleic acid replication may be required in
the recombinant vector. Alternatively, the recombinant vector may
be designed such that the vector and recombinant nucleic acid
molecule integrates into the genome of a cell. In this case nucleic
acid sequences, which favour targeted integration (e.g. by
homologous recombination) are desirable. Recombinant vectors may
also comprise DNA coding for genes that may be used as selectable
markers in the cloning process. The recombinant vector may also
further comprise a promoter or regulator to control expression of
the gene as required.
[0228] The nucleic acid molecule may (but not necessarily) be one,
which becomes incorporated in the DNA of cells of the subject being
treated. Undifferentiated cell s may be stably transformed leading
to the production of genetically modified daughter cells (in which
case regulation of expression in the subject may be required e.g.
with specific transcription factors or gene activators).
Alternatively, the delivery system may be designed to favour
unstable or transient transformation of differentiated cells in the
subject being treated. When this is the case, regulation of
expression may be less important because expression of the DNA
molecule will stop when the transformed cells die or stop
expressing the potein (ideally when the required therapeutic effect
has been achieved).
[0229] The delivery system may provide the nucleic acid molecule to
the subject without it being incorporated in a vector. For
instance, the nucleic acid molecule may be incorporated within a
liposome or virus particle. Alternatively a "naked" nucleic acid
molecule may be inserted into a subject's cells by a suitable
means. e.g. direct endocytotic uptake. The nucleic acid molecule
may be transferred to the cells of a subject to be treated by
transfection, infection, microinjection, cell fusion, protoplast
fusion or ballistic bombardment. For example, transfer may be by
ballistic transfection with coated gold particles, liposomes
containing the nucleic acid molecule, viral vectors (e.g.
adenovirus) and means of providing direct nucleic acid uptake (e.g.
endocytosis) by application of the nucleic acid molecule
directly.
[0230] Alternative methods for identifying similar sequences will
be known to those skilled in the art. The invention concerns using
a nucleic acid molecule that hybridizes to a nucleic acid molecule
that encodes a polypeptide derived from a MHC class I HLA, or a
derivative or analogue thereof, or its complement. The invention
preferably concerns using a nucleic acid molecule that hybridizes
to a nucleic acid molecule that encodes a polypeptide derived from
HLA-A2, or a derivative or analogue thereof, or its complement. For
example, a substantially similar nucleotide sequence will be
encoded by a sequence which hybridizes to the sequences shown in
SEQ ID NO: 3 or their complements under stringent conditions. By
stringent conditions, we mean the nucleotide hybridises to
filter-bound DNA or RNA in 3.times. sodium chloride/sodium citrate
(SSC) at approximately 45.degree. C. followed by at least one wash
in 0.2.times.SSC/0.1% SDS at approximately 20-65.degree. C.
[0231] Due to the degeneracy of the genetic code, it is clear that
any nucleic acid sequence could be varied or changed without
substantially affecting the sequence of the protein encoded
thereby, to provide a functional variant thereof. Suitable
nucleotide variants are those having a sequence altered by the
substitution of different codons that encode the same amino acid
within the sequence, thus producing a silent change. Other suitable
variants are those having homologous nucleotide sequences but
comprising all, or portions of, sequence, which are altered by the
substitution of different codons that encode an amino acid with a
side chain of similar biophysical properties to the amino acid it
substitutes, to produce a conservative change. For example small
non-polar, hydrophobic amino acids include glycine, alanine,
leucine, isoleucine, valine, proline, and methionine. Large
non-polar, hydrophobic amino acids include phenylalanine,
tryptophan and tyrosine. The polar neutral amino acids include
serine, threonine, cysteine, asparagine and glutamine. The
positively charged (basic) amino acids include lysine, arginine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. It will therefore be appreciated
which amino acids may be replaced with an amino acid having similar
biophysical properties, and the skilled technician will known the
nucleotide sequences encoding these amino acids. Reference is also
made to Table 2 above.
Therapy
[0232] The invention also concerns using the polypeptides,
derivatives, analogues and nucleic acids described herein in
therapy. In particular, the invention provides a polypeptide
derived from a MHC class I HLA, or a derivative or analogue
thereof, for use as a medicament. The invention also provides a
nucleic acid molecule encoding a polypeptide, derivative or
analogue of the invention, or a nucleic acid molecule that
hybridizes to a nucleic acid molecule encoding a polypeptide,
derivative or analogue of the invention or its complement under
stringent conditions, for use as a medicament. The invention
further provides use of: [0233] (a) at least one polypeptide
derived from a MHC class I HLA, or a derivative or analogue
thereof; [0234] (b) at least one nucleic acid molecule encoding a
polypeptide, derivative or analogue of (a); or [0235] (c) at least
one nucleic acid molecule that hybridizes to a nucleic acid
molecule of (b) or its complement under stringent conditions;
[0236] for the manufacture of a medicament for the treatment or
prevention of a condition characterised by allosensitisation. Any
of the polypeptides, derivatives, analogues and nucleic acids
described above may be used.
[0237] The invention also provides a polypeptide derived from
HLA-A2, or a derivative or analogue thereof for use as a
medicament. The invention also provides use of a polypeptide
derived from HLA-A2, or a derivative or analogue thereof, for the
manufacture of a medicament for the treatment or prevention of a
condition characterised by allosensitisation. The invention further
provides a method of treating or preventing a condition
characterised by allosensitisation, the method comprising
administering to a subject in need of such treatment, a
therapeutically effective amount of a polypeptide derived from
HLA-A2, or a derivative or analogue thereof.
[0238] Accordingly, in a most preferred embodiment, there is
provided a polypeptide derived from the .alpha.3 domain or the
transmembrane domain of HLA-A2, or a derivative or analogue
thereof, for use as a medicament. Furthermore, in another most
preferred embodiment, there is provided use of a polypeptide
derived from the .alpha.3 domain or the transmembrane domain of
HLA-A2, or a derivative or analogue thereof, for the manufacture of
a medicament for the treatment or prevention of a condition
characterised by allosensitisation. It is especially preferred that
the medicament is for the treatment or prevention of allograft
rejection.
[0239] It will be appreciated that a polypeptide derived from
HLA-A2, or a derivative or analogue thereof, used according to the
invention also represent favourable agents capable of being
administered by techniques involving cellular expression of nucleic
acid sequences encoding such molecules. Such methods of cellular
expression are particularly suitable for medical use in which the
therapeutic effects of the HLA-A2 derived peptides or derivatives
or analogues derived therefrom are required over a prolonged
period. Hence, the invention also provides a nucleic acid molecule
encoding a polypeptide derived from HLA-A2, or a derivative or
analogue thereof for use as a medicament.
[0240] Furthermore, the invention also provides use of a nucleic
acid molecule encoding a polypeptide derived from HLA-A2, or a
derivative or analogue thereof for the manufacture of a medicament
for the treatment or prevention of a condition characterised by
allosensitisation. Preferably, the medicament is for the treatment
of prevention of allograft failure or rejection.
[0241] By the term "allosensitisation"; we mean the development of
antibodies to foreign antigens, which results in a number of
disease conditions. Often patients suffering from such conditions
require repeated blood transfusions. However, allosensitisation can
often render patients almost impossible to transfuse, and can be
life-threatening. Hence, conditions that are characterised by
allosensitisation include thalassemia, and blood transfusions
[0242] HLA-A2 is known to be a major factor in transplant organ
rejection, a medical problem in which the inventor was particularly
interested, and accordingly, the inventor has found that HLA-A2
derived peptides or derivatives or analogues thereof is
particularly useful for treating or preventing allograft
rejection
[0243] By the term "allograft", we mean a transplant process
wherein a tissue or organ is taken from one individual (i.e. a
donor) and placed into another genetically non-identical individual
(i.e. a recipient). Both the donor and the recipient are members of
the same species. The term allograft may also be used to describe
the transplanted tissue or organ.
[0244] By the term "tissue", we mean a group of similar cells
specialised to perform similar functions, for example, muscle,
nerve, bone, cartilage, skin, or connective tissue.
[0245] By the term "organ", we mean a bodily structure containing
different tissues that are organized to carry out a specific
function of the body, for example, the heart, lungs, brain, skin,
liver, etc.
[0246] While the inventor does not wish to be bound by any
hypothesis, they believe that use of a polypeptide derived from
HLA-A2 or other MHC class I HLAs, such as HLA-B or HLA-C, or a
derivative or an analogue thereof, will have beneficial effects on
transplant outcome, not only by inhibiting the formation of
anti-HLA antibodies, but also by inhibiting other recognised
pathways of allograft rejection. This may occur not simply by
directly inhibiting the activation of certain effector cells, which
damage allograft or organ transplants, but also by activating
regulatory cells that inhibit these effector cells. Accordingly,
the inventor believes that the surprising findings regarding the
pattern of reactivity to a peptide derived from HLA-A2 or a
derivative or an or analogue thereof suggest that, such peptides or
analogues may be harnessed in a method of treatment or therapy to
prevent conditions characterised by allosensitisation, for example,
transfusion.
[0247] The inventor believes that the surprising findings regarding
the pattern of reactivity to a polypeptide derived from HLA-A2, or
a derivative or analogue thereof also suggest that such peptides or
analogues derived therefrom may be harnessed in a method for
treating or preventing allograft rejection in transplant patients,
not only when there is an organ mismatched for HLA-A2, but also in
the majority of cases of transplantation. The same is true for
polypeptides derived from other MHC class I HLAs or derivatives or
analogues thereof. Hence, it is especially preferred that the
method be used to treat allograft rejection.
[0248] The inventor believes that the identification of such
peptide-based epitopes allows the development of a peptide-based
therapy, which comprises administering to a subject a mixture of
peptides that could be used in a range of individuals with
different MHC class I epitopes.
[0249] Accordingly, it is preferred that the method of the
invention comprises administering to a subject in need of treatment
a therapeutically effective amount of at least one polypeptide, or
derivative or analogue thereof, and preferably, a plurality of
polypeptides, derivatives or analogues thereof, which are derived
from a MHC class I HLA. A combination or mixture of polypeptides
derived from a MHC class I HLA, or derivatives or analogues
thereof, may be administered to the subject. A combination or
mixture of any of the polypeptides discussed above may be
administered.
[0250] Accordingly, it is especially preferred that the method of
the invention comprises administering to a subject in need of
treatment a therapeutically effective amount of at least one
polypeptide, or derivative or analogue thereof, and preferably, a
plurality of polypeptides, derivatives or analogues thereof, which
are derived from HLA-A2. A combination or mixture of polypeptides
derived from HLA-A2, or derivatives or analogues thereof, may be
administered to the subject.
[0251] The at least one polypeptide, derivative or analogue, or
plurality thereof, may be administered to a sensitised patient,
i.e. one who has made anti-HLA antibody. Alternatively, the at
least one polypeptide, derivative or analogue, or mixture thereof,
may be administered to an individual prior to transplantation or
transfusion in order to abrogate sensitisation to HLA. In
considering desensitisation, one may expect peptides p39 (SEQ ID
NO: 42), p50/51 (SEQ ID NO: 57) and p52/53 (SEQ ID NO: 58) to
benefit individuals who do not express these sequences themselves,
i.e. a subject who express HLA-A1, HLA-A3, HLA-A 11, HLA-A23,
HLA-A24, HLA-A30, or HLA-A36, and who subsequently receive (or who
have previously received) mismatched transplants (or transfusions)
expressing HLA-A2, HLA-A25, HLA-A26, HLA-A29, HLA-A31, HLA-A32,
HLA-A33, HLA-A34, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74.
[0252] Hence, it will be appreciated from the foregoing that
preferred polypeptides, derivatives or analogues thereof which are
derived from the non-polymorphic region of HLA-A2 are also
expressed by many other HLA-A molecules, and not just HLA-A2, by
acting as sites of potential cross-reactivity between different HLA
molecules. Hence, surprisingly, the inventor believes that the
benefits of using such peptides in methods according to the
invention, and in particular for uses in treating or preventing
conditions characterised by allosensitisation, and particularly,
allograft rejection, and alloantibody synthesis, may be much wider
than to HLA-A2 antigen alone. Accordingly, the inventor believes
that the implications of their findings are much broader than
solely help for antibody production, but relate to immune
mechanisms of graft rejection per se, and are wider than target
HLA-A2, extending to responses to transplantation antigens in
general.
[0253] Hence, in a further aspect, there is provided use of an
alloantigen, or a polypeptide derived therefrom, or a derivative or
analogue thereof, for the manufacture of a medicament for the
treatment or prevention of a condition characterised by
allosensitisation. Preferably, the medicament is for the treatment
of allograft rejection. Preferably, the alloantigen, or polypeptide
derived therefrom, or derivative or analogue thereof may be
independently selected from a group consisting of HLA-A2, HLA-A25,
HLA-A26, HLA-A29, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A43,
HLA-A66, HLA-A68, HLA-A69, HLA-A74, HLA-A1, HLA-A3, HLA-A11,
HLA-A24, HLA-A30, HLA-A36, HLA-A80, HLA-A1, HLA-A3, HLA-A 11, and
HLA-A23, or any combination thereof. Preferred polypeptides derived
from the alloantigen are discussed above.
[0254] The therapeutically effective amount of the polypeptide
derived from the MHC class I HLA, such as HLA-A2, or a derivative
or analogue thereof, may be administered to the subject after the
subject has been given an allograft, or a transplant, or a
transfusion. However, it is most preferred that it is administered
to the subject before the subject has been given an allograft, or
transplant, or transfusion. Hence, preferably, the medicament is
administered to a subject prior to antigen exposure, for example at
the time of transplant listing or transfusion. Hence, the method
effectively comprises administering a vaccine or so-called
`negative vaccine` composing a therapeutically effective amount of
a polypeptide derived from a MHC class I HLA, or a derivative or
analogue thereof. The method also effectively comprises
administering a vaccine or so-called `negative vaccine` comprising
a therapeutically effective amount of a polypeptide derived from
HLA-A2, or a derivative or analogue thereof.
[0255] Therefore, there is provided a vaccine comprising a
polypeptide derived from a MHC class I HLA, or a derivative or
analogue thereof. There is also provided a vaccine comprising a
polypeptide derived from HLA-A2, or a derivative or analogue
thereof.
[0256] The inventor believes that a course of treatment would be
undertaken with progressively higher doses of the `negative
vaccine`, at approximately weekly intervals (but it may be
administered daily, or every other day, for example), over the
course of approximately 1 to 6 months. The amount of polypeptide,
derivative or analogue administered may be in the region of about 1
.mu.g to 1 mg.
[0257] It will be appreciated that the invention is for the
prevention or treatment of a condition characterised by
allosensitisation, rejection of an allograft, or alloimmunisation
secondary to transfusion. It is common practise to transplant
tissues or organs from a donor subject to a recipient subject.
Hence, the invention extends to the treatment or prevention of
failure or rejection of any tissue, such as muscle, nerve, bone,
bone marrow, cartilage, skin, or connective tissue, or any organ
such as the heart, lungs, brain, skin, liver, etc. or
alloimmunisation secondary to transfusion.
[0258] However, the inventor focussed their research on patients
suffering from end stage renal failure. Hence, it is especially
preferred that the medicament may be used for the treatment or
prevention of rejection of a kidney transplant, or alloimmunisation
secondary to such a kidney transplant, or prior to transfusion.
Furthermore, the subject being treated in the method of invention
may be suffering from kidney transplant rejection. Accordingly, it
is preferred that the allograft or transplant comprises a kidney or
a portion thereof.
[0259] The following discussion concerning monotherapy, combination
therapy, timing of administration, compositions, routes of
administration, release devices and dosages relates to polypeptides
derived from HLA-A2, or derivatives or analogues thereof. However,
it also applies to polypeptides derived from another MHC class I
HLAs such as HLA-B or HLA-C, derivatives or analogues thereof, and
nucleic acids encoding the same.
[0260] It will be appreciated that the polypeptide derived from
HLA-A2 or a derivative or analogue thereof, or nucleic acid
molecule encoding HLA-A2 or a polypeptide, derivative or analogue
thereof, may be used in a monotherapy (i.e. use of a polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule encoding same alone to prevent and/or treat a
allograft failure or rejection). Alternatively, a polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule encoding same may be used as an adjunct, or in
combination with known therapies for treating a condition
characterised by allosensitisation, such as, allograft rejection,
or preventing allosensitisation, such as conventional
immunosuppression with calcineurin inhibitors, TOR inhibitors or
antibodies targeting lymphocyte antigens.
[0261] A therapeutically effective amount of the polypeptide
derived from HLA-A2 or a derivative or analogue thereof, or nucleic
acid molecule encoding polypeptide derived from HLA-A2 or a
derivative or analogue thereof may be administered to the subject
after the subject has been given an allograft or transplant.
Alternatively, the at least one peptide or mixture thereof may be
administered to a subject prophylactically, i.e. to attenuate
responses to HLA.
[0262] While the inventor does not wish to be bound by any
hypothesis, they believe that if the mechanism of attenuation
involves dominant negative regulation, then this may also diminish
the response to other mismatched antigens, for example, when an
HLA-A2 mismatched transplant is implanted. Hence, it is preferred
that a therapeutically effective amount of the polypeptide derived
from HLA-A2 or a derivative or analogue thereof, or nucleic acid
molecule encoding polypeptide derived from HLA-A2 or a derivative
or analogue thereof is administered to the subject before the
subject has been given an allograft or transplant, i.e. similar to
a vaccination.
[0263] The inventor envisages administration to a subject at least
one, and preferably a plurality of peptides derived from HLA-A2 or
derivatives or analogues thereof. They believe that the or each
peptide, derivative or analogue thereof may be used to
down-regulate the response to alloantigen, and therefore improve
the outcome of transplantation and/or permit the desensitisation of
patients who have made anti-HLA antibody.
[0264] The polypeptide derived from HLA-A2 or a derivative or
analogue thereof, or nucleic acid molecule encoding a polypeptide
derived from HLA-A2 or a derivative or analogue thereof may be
combined in compositions having a number of different forms
depending, in particular, on the manner in which the composition is
to be used.
[0265] For example, the composition may be in the form of a powder,
tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol,
spray, micelle, transdermal patch, liposome or any other suitable
form that may be administered to a subject in need of treatment. It
will be appreciated that the vehicle of the composition of the
invention should be one which is well tolerated by the subject to
whom it is given, and preferably enables delivery of the
polypeptide derived from HLA-A2 or a derivative or analogue
thereof, or nucleic acid molecule, encoding polypeptide derived
from HLA-A2 or a derivative or analogue thereof to the immune
system. The vehicle of the composition of the invention should be
one which is well tolerated by the subject to whom it is given, and
preferably enables delivery of the polypeptide derived from HLA-A2
or a derivative or analogue thereof, or nucleic acid molecule
encoding polypeptide derived from HLA-A2 or a derivative or
analogue thereof to the immune system.
[0266] Compositions comprising a polypeptide derived from HLA-A2 or
a derivative or analogue thereof, or nucleic acid molecule encoding
a polypeptide derived from HLA-A2 or a derivative or analogue
thereof used according to the invention may be used in a number of
ways. For instance, oral administration may be required in which
case the polypeptide derived from HLA-A2 or a derivative or
analogue thereof or nucleic acid molecule may be contained within a
composition that may, for example, be ingested orally in the form
of a tablet, capsule or liquid. The composition comprising
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule may be administered by inhalation (e.g.
intranasally). Compositions may be formulated for topical use. For
instance, ointments may be applied to the skin.
[0267] The polypeptide derived from HLA-A2 or a derivative or
analogue thereof or nucleic acid molecule may also be incorporated
within a slow or delayed release device. Such devices may, for
example, be inserted on or under the skin, and the composition may
be released over weeks or even months. Such devices may be
particularly advantageous when long-term treatment with polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule used according to the invention is required and which
would normally require frequent administration (e.g. at least daily
injection).
[0268] However, in a most preferred embodiment, the composition
comprising a polypeptide derived from HLA-A2 or a derivative or
analogue thereof or nucleic acid molecule is administered to a
subject in need of such treatment by injection into the blood
stream. Injections may be intravenous (bolus or infusion) or
subcutaneous (bolus or infusion), or intradermal (bolus or
infusion).
[0269] It will be appreciated that the amount of polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule that is required is determined by its biological
activity and bioavailability which in turn depends on the mode of
administration, the physicochemical properties of the polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule employed and whether the polypeptide derived from
HLA-A2 or a derivative or analogue thereof or nucleic acid molecule
is being used as a monotherapy or in a combined therapy. The
frequency of administration will also be influenced by the
above-mentioned factors and particularly the half-life of
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule within the subject being treated.
[0270] Optimal dosages to be administered may be determined by
those skilled in the art, and will vary with the particular
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule in use, the strength of the preparation,
the mode of administration, and the advancement of the disease
condition. Additional factors depending on the particular subject
being treated will result in a need to adjust dosages, including
subject age, weight, gender, diet, and time of administration.
[0271] Known procedures, such as those conventionally employed by
the pharmaceutical industry (e.g. in vivo experimentation, clinical
trials, etc.), may be used to establish specific formulations of
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule used according to the invention and
precise therapeutic regimes (such as daily doses of the polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule and the frequency of administration).
[0272] Generally, a daily dose of between 0.001 .mu.g/kg of body
weight and 100 .mu.g/kg of body weight of polypeptide derived from
HLA-A2 or a derivative or analogue thereof or nucleic acid molecule
may be used for the prevention and/or treatment of allograft
failure or rejection, depending upon which specific polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule is used. More preferably, the daily dose is between
0.01 g/kg of body weight and 10 .mu.g/kg of body weight, and most
preferably, between approximately 0.01 .mu.g/kg and 1 .mu.g/kg.
[0273] Daily doses may be given as a single administration (e.g. a
single daily Injection). Alternatively, the polypeptide derived
from HLA-A2 or a derivative or analogue thereof or nucleic acid
molecule used may require administration twice or more times during
a day. As an example, polypeptide derived from HLA-A2 or a
derivative or analogue thereof or nucleic acid molecule may be
administered as two (or more depending upon the severity of the
condition) daily doses of between 0.07 .mu.g and 7000 .mu.g (i.e.
assuming a body weight of 70 kg). A patient receiving treatment may
take a first dose upon waking and then a second dose in the evening
(if on a two dose regime) or at 3 or 4 hourly intervals thereafter.
Alternatively, a slow release device may be used to provide optimal
doses to a patient without the need to administer repeated
doses.
[0274] The invention also provides a pharmaceutical composition
comprising a therapeutically effective amount of a polypeptide,
derivative or analogue of the invention or a nucleic acid molecule
of the invention, and optionally a pharmaceutically acceptable
vehicle. The invention preferably provides a pharmaceutical
composition comprising a therapeutically effective amount of
polypeptide derived from HLA-A2 or a derivative or analogue
thereof, or nucleic acid molecule encoding polypeptide derived from
HLA-A2 or a derivative or analogue thereof, and optionally, a
pharmaceutically acceptable vehicle. The composition can comprise
one or more of any of the polypeptides, derivatives, analogues or
nucleic acids discussed above.
[0275] The invention further provides a process for making a
pharmaceutical composition comprising combining a therapeutically
effective amount of a polypeptide, derivative or analogue of the
invention or a nucleic acid molecule of the invention and a
pharmaceutically acceptable vehicle. The invention preferably
provides a process for making a pharmaceutical composition
comprising combining a therapeutically effective amount of a
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule encoding a polypeptide derived from HLA-A2
or a derivative or analogue thereof, and a pharmaceutically
acceptable vehicle.
[0276] The following discussion concerning amounts, vehicles and
routes of administration relates to polypeptides derived from
HLA-A2, or derivatives or analogues thereof. However, it also
applies to polypeptides derived from other MHC class I HLAs, such
as HLA-B or HLA-C, derivatives or analogues thereof, and nucleic
acids encoding the same.
[0277] The amount of the polypeptide derived from HLA-A2 or a
derivative or analogue thereof or nucleic acid molecule may be from
about 0.01 .mu.g to about 800 .mu.g and preferably, from about 0.01
mg to about 500 .mu.g. It is preferred that the amount of
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule is an amount from about 0.01 mg to about
250 mg, more preferably, about 0.1 mg to about 60 mg, and most
preferably, from about 0.1 mg to about 20 mg. A "therapeutically
effective amount" is any amount of polypeptide derived from HLA-A2
or a derivative or analogue thereof or nucleic acid molecule which,
when administered to a subject provides prevention and/or treatment
of a condition characterised by allosensitisation, such as,
allograft failure or rejection. A "subject" may be a vertebrate,
mammal, or domestic animal, and is preferably, a human being.
[0278] A "pharmaceutically acceptable vehicle" as referred to
herein is any physiological vehicle known to those of ordinary
skill in the art useful in formulating pharmaceutical compositions.
In one embodiment, the pharmaceutically acceptable vehicle may be a
solid and the composition may be in the form of a powder or tablet.
A solid pharmaceutically acceptable vehicle may include one or more
substances which may also act as flavouring agents, lubricants,
solubilisers, suspending agents, fillers, glidants, compression
aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the vehicle is a finely divided
solid that is in admixture with the finely divided active
polypeptide derived from HLA-A2 or a derivative or analogue thereof
or nucleic acid molecule. In tablets, the active polypeptide
derived from HLA-A2 or a derivative or analogue thereof or nucleic
acid molecule is mixed with a vehicle having the necessary
compression properties in suitable proportions and compacted in the
shape and size desired. The powders and tablets preferably contain
up to 99% of the active polypeptide derived from HLA-A2 or a
derivative or analogue thereof or nucleic acid molecule. Suitable
solid vehicles include, for example, calcium phosphate, magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin,
cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins. In another embodiment, the pharmaceutical vehicle may be a
gel and the composition may be in the form of a cream or the
like.
[0279] However, in a preferred embodiment, the pharmaceutical
vehicle is a liquid and the pharmaceutical composition is in the
form of a solution. Liquid vehicles are used in preparing
solutions, suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active polypeptide derived from HLA-A2 or a
derivative or analogue thereof or nucleic acid molecule may be
dissolved or suspended in a pharmaceutically acceptable liquid
vehicle such as water, an organic solvent, a mixture of both or
pharmaceutically acceptable oils or fats. The liquid vehicle can
contain other suitable pharmaceutical additives such as
solubilisers, emulsifiers, buffers, preservatives, sweeteners,
flavouring agents, suspending agents, thickening agents, colours,
viscosity regulators, stabilizers or osmo-regulators. Suitable
examples of liquid vehicles for oral and parenteral administration
include water (partially containing additives as above, e.g.
cellulose derivatives, preferably sodium carboxymethyl cellulose
solution), alcohols (including monohydric alcohols and polyhydric
alcohols, e.g. glycols) and their derivatives, and oils (e.g.
fractionated coconut oil and arachis oil). For parenteral
administration, the vehicle can also be an oily ester such as ethyl
oleate and isopropyl myristate. Sterile liquid vehicles are useful
in sterile liquid form compositions for parenteral administration.
The liquid vehicle for pressurized compositions can be halogenated
hydrocarbon or other pharmaceutically acceptable propellant.
[0280] Liquid pharmaceutical compositions which are sterile
solutions or suspensions can be utilized by for example,
intramuscular, intrathecal, epidural, intraperitoneal, intravenous
and particularly subcutaneous, intracerebral or
intracerebroventricular injection. The polypeptide derived from
HLA-A2 or a derivative or analogue thereof or nucleic acid molecule
may be prepared as a sterile solid composition that may be
dissolved or suspended at the time of administration using sterile
water, saline, or other appropriate sterile injectable medium.
Vehicles are intended to include necessary and inert binders,
suspending agents, lubricants, flavourants, sweeteners,
preservatives, dyes, and coatings.
[0281] The polypeptide derived from HLA-A2 or a derivative or
analogue thereof or nucleic acid molecule may be administered
orally in the form of a sterile solution or suspension containing
other solutes or suspending agents (for example, enough saline or
glucose to make the solution isotonic), bile salts, acacia,
gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of
sorbitol and its anhydrides copolymerized with ethylene oxide) and
the like.
[0282] The polypeptide derived from HLA-A2 or a derivative or
analogue thereof or nucleic acid molecule used according to the
invention can also be administered orally either in liquid or solid
composition form. Compositions suitable for oral administration
include solid forms, such as pills, capsules, granules, tablets,
and powders, and liquid forms, such as solutions, syrups, elixirs,
and suspensions. Forms useful for parenteral administration include
sterile solutions, emulsions, and suspensions.
Agents Increasing HLA Activity
[0283] It will be appreciated that the therapeutic effects of
polypeptides derived from HLA-A2 or derivatives or analogues
thereof may be mediated "indirectly" by agents that increase the
activity of such peptides or analogues. Hence, the present
invention also provides the first medical use of such agents.
[0284] Thus, the invention also provides an agent capable of
increasing the biological activity of a polypeptide derived from
HLA-A2, or a derivative or analogue thereof, for use as a
medicament.
[0285] Agents capable of increasing the biological activity of
HLA-A2 derived peptides or derivatives or analogues thereof may
achieve their effect by a number of means. For instance, such
agents may increase the expression of HLA-A2 derived peptides, or
derivatives or analogues thereof. Alternatively, or additionally,
such agents may increase the half-life of a polypeptide derived
from HLA-A2, or a derivative or analogue thereof in a biological
system, for example, by decreasing turnover of HLA-A2 derived
peptides or derivatives or analogues thereof. Due to their
increased biological activity, a polypeptide derived from HLA-A2,
or a derivative or analogue thereof are of utility as
anti-allograft rejection agents.
In Vitro Methods
[0286] The invention also provides an in vitro method of
stimulating T cells, the method comprising contacting the T cells
with: [0287] (a) a polypeptide derived from a MHC class I HLA, or a
derivative or analogue thereof; [0288] (b) a nucleic acid molecule
encoding a polypeptide, derivative or analogue of (a); or [0289]
(c) a nucleic acid molecule that hybridizes to a nucleic acid
molecule of (b) or its complement under stringent conditions;
[0290] under conditions which allow stimulation of the T cells and
thereby stimulating the T cells.
[0291] Any of the polypeptides, derivatives, analogues or nucleic
acids described above can be used in this method. Suitable
conditions for the stimulation of T cell s are known in the
art.
[0292] This method of the invention has various uses. For instance,
the method could be carried out on a sample of T cells from a
transplant patients undergoing chronic rejection of the graft. A
positive response by the patient's T cells to the HLA-derived
polypeptides could suggest that the chronic rejection involves an
immune system component. A negative response by the patient's T
cells to the HLA-derived polypeptides could suggest that the
chronic rejection is due to non-immune system based mechanism, such
as cyclosporin nephrotoxicity.
[0293] All of the features described herein (including any
accompanying claims, abstract and drawings), and/or all of the
steps of any method or process so disclosed, may be combined with
any of the above aspects in any combination, except combinations
where at least some of such features and/or steps are mutually
exclusive.
[0294] For a better understanding of the invention, and to show how
embodiments of the same may be carried into effect, reference will
now be made to the following Examples.
EXAMPLES
Overview
[0295] The inventor realised that the presence of anti-HLA antibody
contraindicates renal transplantation, and post-transplantation is
associated with allograft failure. Furthermore, the acquisition of
HLA specific antibody implies T cell help through indirect
allorecognition, and this pathway also plays a role in rejection
independent to alloantibody synthesis. The inventor therefore
systematically studied responses to a single MHC class 1 molecule,
HLA A2, which is a common target antigen in sensitised patients on
the organ transplantation waiting list. i.e. patients who have made
HLA-A2 antibodies.
[0296] In both Examples 1 and 2, the inventor designed a series of
60 overlapping 15mer peptides that spanned the primary sequence of
HLA*020101. However, only 53 (p1-p53) could be synthesised using
F-moc technology. The binding affinity of these 53 peptides to 13
different MHC class II molecules was then studied by ELISA. The
inventor found that peptides from several locations along the
HLA-A2 molecule exhibited promiscuous binding to MHC.
[0297] The 30 peptides that bound MHC class II were then used to
stimulate peripheral blood mononuclear cells (PBMC) from 40
transplant-listed patients with known antibody sensitisation
histories. Responses were assessed by .gamma.-interferon elispot,
and the findings are summarised below.
[0298] In Example 3, the inventor compared the sequence of some of
the 15mer peptides from HLA-A2 with sequences in other HLA
polypeptides to find additional sequences of relevance.
Example 1
Materials & Methods
1) In Silico Epitope Prediction
[0299] In order to minimise the numbers of peptides to be screened
in direct MHC-peptide binding assays and to maximise discrimination
in areas likely to be of interest, a systematic approach was
adopted involving an initial computer based evaluation of the
primary amino acid sequence of the HLA-A*0201 molecule, i.e.
HLA-A2. The DNA sequence of HLA-A2 is identified as SEQ ID NO. 3
and the amino acid sequence is identified as SEQ ID NOs: 1 and
2.
[0300] Referring to FIG. 1, there is shown a schematic
representation of the extracellular portion of the human class I
histocompatibility molecule, HLA-A2. The stretches of beta
conformation are represented by broad arrows (pointing N to C
terminal). Regions of alpha helix are shown as helical ribbons. The
pairs of spheres represent disulphide bridges. The molecule of
beta-2-microglobulin (.beta..sub.2m) is bound to the junction of
the .alpha.1 and .alpha.2 domains, and to the .alpha.3 domain by
non-covalent interactions only. Not shown in FIG. 1 is the presence
of a short peptide bound non-covalently in the groove between the
alpha helices of .alpha.1 and .alpha.2 domains. The combination of
peptide and adjacent portions of alpha helices makes up the epitope
seen by CD8+ T cells. The transmembrane and cytosolic portion of
the HLA-A2 molecule are not shown, but it will be appreciated that
the transmembrane portion extends from about amino acid residue 283
onwards.
[0301] The analysis of the primary amino acid sequence of the
HLA-A2 molecule was performed using the widely available algorithm
TEPITOPE (Sturniolo, T., et al., Generation of tissue-specific and
promiscuous HLA ligand databases using DNA microarrays and virtual
HLA class II matrices. Nat Biotechnol, 1999, 17(6): p. 555-61). The
programme was used to identify sequences that share HLA-binding
motifs with previously identified and characterised epitopes.
However, because such in silico approaches may omit epitopes that
do not conform to standard motifs, the inventor conducted
additional literature and database searches in order to identify
all previously reported MHC class II-binding sequences from the
HLA-A2 molecule.
2) Solubility Analysis
[0302] Sequences predicted to bind to MHC class II molecules were
evaluated for solubility using predictive computer-based algorithms
such as that at EXPASY (www.expasy.org/). The inventor believed
that such solubility analysis was an important precursor to
physical binding assays as it allowed identification of any peptide
sequences that contain important, naturally processed, but
nominally insoluble MHC-binding core motifs. Frequently, such
epitopes can be rendered soluble by the addition of various
flanking residues that occur naturally in the source protein
(HLA-A*0201 in this case). This approach avoided time-consuming and
costly synthesis of peptides that would not generate data in
MHC-binding assays, and would therefore have ultimately been of
little use for clinical intervention.
3) Peptide Synthesis
[0303] A series of 60 overlapping 15mer peptides that spanned the
primary sequence of HLA*020101 was designed. However, only 53
(p1-p53) could be synthesised. Hence, a series of 53 synthetic
overlapping 15mers offset by 2 to 5 residues, corresponding to
soluble, MHC class II-binding motifs, were generated as HCl salts
by standard Fmoc chemistry and supplied as lyophilised powder
(NeoMPS SA, Strasbourg, France). Peptides were reconstituted in
10.sup.-4M HCl.
[0304] Referring to FIG. 2, there are shown details of all 53
peptides, designated p1 to p53. FIG. 2 shows the position,
molecular weight and sequence of each peptide.
4) Peptide Binding to Purified MHC Molecules In Vitro
[0305] The peptide-MHC binding assay was a competition assay in
which "query" peptides are titrated into wells containing
immobilised, purified MHC molecules and known concentrations of
biotinylated reference peptides that are known to bind to the
molecule in question with high affinity.
[0306] The MHC molecules for study included: DRB1*0101 (DR1).
DRB1*0301 (DR3), DRB1*0401 (DR4), DRB1*0701 (DR7), DRB1*1101
(DR11), DRB1*1301 (DR13), DRB1*1501 (DR15); DRB3*0101 (DRB3),
HLA-DRB4*0101 (DRB4) and HLA-DRB5*0101 (DRB %);
HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402
(DP402). These were undertaken at CEA-Saclay using techniques that
are well established in his laboratory for the analysis of other
antigens (Texier, C., et al., HLA-DR restricted peptide candidates
for bee venom immunotherapy. J Immunol, 2000, 164(6): p.
3177-84).
[0307] Binding experiments were repeated on three separate
occasions to confirm results. Results were expressed as both nm
IC50 and as the ratio of binding of each peptide compared to a
reference peptide (also screened in the assay) of known high
affinity binding to each particular MHC allele.
[0308] Binding ratios of less than 20 are considered high affinity
binders and those with a ratio of 21-100, moderate binders.
Previous data indicated that important T cell epitopes only rarely
have binding affinities outside of these windows, although there
are exceptions.
[0309] Referring to FIG. 4, there is shown the binding studies data
generated. These data show the ratio of the 50% inhibitory
concentration (IC50) of test peptide compared to the 50% binding
concentration of control peptide, to the MHC class II molecule
under investigation.
5) In Vitro Functional Analysis--Immune Responses of Peripheral
Blood Mononuclear Cells
[0310] The studies described above identified that a wide range of
peptides bound to at least one MHC class II molecule, although a
limited number showed promiscuous binding. On the basis of these
studies, the number of peptides for future study was reduced from
53 to 30. 23 peptides which showed little or no significant binding
to MHC class II, that is the ratio of their binding affinity to
that of control peptide was >100, were not studied further. This
was undertaken in order to limit the amount of blood that was
required from each patient under study.
[0311] The presence of lymphocytes specific for any given peptide
was determined using .gamma. interferon elispot with 500,000
peripheral blood mononuclear cells per well in quadruplicate.
(Briefly, peripheral blood mononuclear cells (PBMCs) were separated
from heparinised whole blood by density centrifugation. These were
washed and resuspended in complete culture medium (containing 10%
AB serum). Cells were cultured with individual peptides in 96 well
.gamma. interferon elispot plates, (Mabtech, Nacka Strand, Sweden)
in 100 ul complete medium, with peptide at a concentration of 4
.mu.g and 20 .mu.g/ml. The frequency of .gamma.-interferon
producing cells was evaluated by ELIspot, as shown in FIG. 7 for a
single example patient. The frequency and specificity of peptide
specific responses was then compared between individuals in the
sensitised and unsensitised groups.
6) Recruitment of Subjects for Study
[0312] Patient subjects were recruited from the University Hospital
Birmingham renal transplant waiting list Patients on dialysis at
University Hospital Birmingham were approached initially, following
receipt of local research ethical committee approval. Subjects who
were currently receiving immunosuppression or who had a haemoglobin
<10 g/dl were excluded. Having obtained fully informed consent,
50 ml of venous blood was obtained at the time of routine
venesection.
[0313] The HLA type determined by standard molecular techniques was
available for each subject from routine clinical assessment by the
West Midlands Blood Transfusion Service Immunogenetics Laboratory.
This laboratory characterised anti-HLA antibody specificities using
standard flow-cytometric based techniques including the use of
monospecific beads, within the context of routine clinical practice
and provided relevant historical information on antibody
specificities for the respective subjects.
Results
[0314] The inventor examined 5 groups of patients summarised in the
Table below.
TABLE-US-00004 TABLE 3 Patient groups Patient anti-HLA Patient HLA
type antibody status A2+ A2- Sensitised with 12/15 anti-HLA A2
(Group1) Sensitised no 0/6 1/5 anti-HLA A2 (Group 2) (Group 3)
Unsensitised 1/6 2/8 (Group 4) (Group 5)
[0315] The presence or absence of antibodies was determined using
standard solid phase, flow cytometric based techniques
(`Luminex.TM.`). The denominator in each group identifies the
number of patients studied and the numerator, the number in each
group that responded to at least 1 peptide.
[0316] In group 1, 10 patients responded to peptides p20/21. This
is from the highly polymorphic region of HLA-A2. Closely
overlapping peptides have been eluted from human B lymphocyte HLA
DR1, i.e. it has been shown to be a `naturally processed` and
presented endogenous protein. A larger peptide containing these
sequences has also been identified as a DR15 restricted epitope in
a patient who rejected an A2 positive kidney.
[0317] In group 1: 10 patients responded to peptides derived from
the .alpha.3 and transmembrane domains that show very limited
polymorphism. These peptides were most commonly p39 (192-206),
p50/51 (268-284) and p52/53 (280-296), although a small number of
other sites also resulted in responses including p40 (202-216) and
p45/46 (239-256). These peptides have not previously been
identified as T cell epitopes. In two of the patients, some of the
.alpha.3/transmembrane peptides were autologous epitopes, for
example patient 1 is A68 positive and therefore shares the
sequences of p39, p52/53 with HLA-A2.
[0318] Referring to FIG. 5, there is shown the sequence of peptide
39. The left hand circle shows the HLA types in which the sequence
of the homologous region is shared with HLA-A2 and the right hand
circle, those HLA types in which the homologous region is different
from HLA-A2, and the two amino acids by which they differ (alanine
to proline at 193 and valine to isoleucine at 194).
[0319] Referring to FIG. 6, there is shown the sequences of
peptides p50 and p51. The left hand circle shows the HLA types in
which the sequence of the homologous region is shared with HLA-A2
and the other circles, those in which the homologous region is
different from HLA-A2, and the amino acids by which they differ,
(proline to leucine at 276, for A1, A3, A11, A30, A36 etc).
[0320] Referring to FIG. 7, there is shown the sequences of
peptides p52 and p53. The left hand circle shows the HLA types in
which the sequence of the homologous region is shared with HLA-A2
and the other circles, those in which the homologous region is
different from HLA-A2, and the amino acids by which they differ,
(phenylalanine to leucine at 294 for A 1, A3, A11, A30, A36
etc).
[0321] In group 2: there was no response.
[0322] In group 3: 1/5 patients made a response to peptides from
the .alpha.3 and transmembrane domains. This again is consistent
with the hypothesis that patients sensitised to other HLA-A, not
HLA-A2 are likely to make responses to shared sequences from the
.alpha.3 and transmembrane domains.
[0323] In group 4: 1/6 patients made a response. This is an auto
response to p39, 50/51, 52/53 and p11. Such a response has also
been observed in 1/6 `non-dialysis` normal controls.
[0324] In group 5: 2/8 made a response p39 or p50/51 & p52/53.
These patients had received previous transfusions and may therefore
have been sensitised at the T lymphocyte level without producing
anti-HLA antibody.
Example Patient
[0325] Referring to FIG. 8, there is shown an Elispot count of
peptide reactive cells/500,000 PBMCs. The concentration of peptide
was 20.times.g/ml. The data show that this individual, who has made
high levels of anti-HLA antibody for approximately 10 years, on the
background of previous pregnancy and transfusion, makes high
frequency responses to p20, p39 and a mixture of p52 and p53. Her
tissue type is A1,68; B37,44; C6,7 DR10,11. Hence, the patient
responded to the highly polymorphic region and in particular to a
sequence within the HLA-A2 sequence residues 105-121 covered by p20
& 21, that the inventor demonstrated binds promiscuously to MHC
class II. She also responded to peptides from the .alpha.3 and
transmembrane region of HLA-A2, the properties of which are
described above. Indeed in her case, these responses are
autoreactive because these sequences are shared with HLA-A68. The
positive control response is to purified protein derivative of
mycobacterium tuberculosis (PPD) and the negative control to medium
alone.
SUMMARY
[0326] In summary, the results show that peptides of limited
polymorphism from the .alpha.3 and transmembrane domains frequently
induce an immune response. This is usually allogeneic, but
sometimes autoimmune, in specificity. The association of responses
to these peptides with sensitisation to HLA-A2 implies that they
are relevant to anti-HLA antibody formation. They also constitute
indirectly presented epitopes that are likely to drive chronic
rejection through T cell mediated mechanisms of damage. As these
peptides are so widely expressed, they are ideal targets for
therapeutic desensitisation. Furthermore, peptide analogues
disclosed herein comprising either one or two amino acid
substitutions provide almost complete representation of HLA-A.
[0327] Furthermore, each of the preferred peptides in accordance
with the invention may be expressed by many other HLA-A molecules,
and not just HLA-A2. Hence, the inventor believes that the benefits
of using such peptides in methods according to the invention, and
in particular for uses in treating or preventing allograft failure
or rejection, and/or antibody synthesis via this or other means of
allosensitisation, may be much wider than to HLA-A2 alone.
Accordingly, the inventor believes that the implications of their
findings is much broader than solely antibody production, but may
also relate to immune mechanisms of graft failure or rejection per
se, and are wider than target HLA-A2, extending to responses to
transplantation antigens in general.
[0328] While the inventor does not wish to be bound by any
hypothesis, they believe that a mixture or "cocktail" of peptides,
derivatives or analogues in accordance with the invention may be
used to down-regulate the response to alloantigen, and therefore
improve the outcome of transplantation and/or permit the
desensitisation of patients who have made anti-HLA antibody. The
novelty of the invention lies not only in defining precise peptide
sequences of HLA-A2 (i.e. epitopes), but also in the surprising
finding that these sequences are what might be called `public`;
this is a term that has until now only been applied to B cell
epitopes, because their existence had not been appreciated for T
cells. Hence, this surprising finding allows the use of a mixture
or cocktail containing a limited number of peptides that will have
potentially broad therapeutic benefit.
Example 2
Materials and Methods
Design of Peptides
[0329] 60 overlapping peptides, 15 or 16 amino acids long, were
designed so as to give optimal coverage of HLA-A*020101 for
putative T cell epitopes, using the programme Tepitope (33).
Solubility of peptides was predicted using the programme at
www.expasy.org. Peptides were synthesized (NeoMPS, Strasbourg,
France) using Fmoc chemistry as previously described, but of those
designed, only 53 could be made. Biotinylation of control peptides
was achieved by reaction with biotinyl-6-aminocaproic acid (Fluka
Chimie, St. Quentin Fallavier, France) at the NH.sub.2 terminus of
the molecule. All peptides were purified by reverse-phase HPLC on a
C18 Vydac column, and their quality was assessed by electrospray
mass spectroscopy and analytical HPLC.
Purification of HLA-DR Molecules
[0330] HLA-DR molecules were purified from HLA-homozygous EBV cell
lines by affinity chromatography using the monoclonal anti-DR
antibody L-243, coupled to protein A-Sepharose CL 4B gel (Amersham
Pharmacia Biotech, Orsay, France) as previously described (Texier
et al., J Immunol 2000; 164(6):3177-84).
HLA-DR Peptide Binding Assays
[0331] Binding of peptides to different HLA-DR molecules was
performed in a competition assay as previously described (Texier et
al., Eur J Immunol 2001; 31(6):1837-46). Biotinylated control
peptides that are good binders to the MHC class II molecule under
investigation were used (see FIG. 9) and test peptides titrated
into the binding assay with purified, immobilised MHC class II.
Maximal binding was determined by incubating the biotinylated
peptide with the MHC class II molecule in the absence of
competitor. Data are expressed as the concentration of peptide that
prevented binding of 50% of the labelled peptide (IC50). An IC50
ratio of control peptide to test peptide of <20 was considered
high affinity binding and 20-100 as moderate affinity.
Study Population
[0332] This study was performed with the approval of the South
Birmingham research ethics committee. All subjects were on dialysis
at University Hospital Birmingham or St James Hospital, Leeds.
Blood samples were obtained into heparin from dialysis patients at
the time, of routine venepuncture. HLA genotypes were determined
using standard molecular techniques at the National Blood Service
Laboratory, Birmingham, UK., from which the history of alloantibody
formation for each subject was available, screened by standard
cytotoxicity, flow cytometry and from 2004, solid phase assay.
[0333] Patients were excluded on the basis of anaemia (Hb<10
g/dl) or if they had received immunosuppressive drugs within 3
months of the investigation. Subjects were divided into 5 groups on
the basis of tissue type and history of anti-HLA antibody synthesis
(see Table 4).
TABLE-US-00005 TABLE 4 Patient groups Group 1 HLA-A2 negative with
anti HLA antibodies to HLA-A2 Group 2 HLA-A2 negative with anti HLA
antibodies to none -A2 HLA Group 3 HLA-A2 negative with no history
of anti HLA antibody formation Group 4 HLA-A2 positive with anti
HLA antibodies to none -A2 HLA Group 5 HLA-A2 positive with no
history of anti HLA antibody formation
Enzyme-Linked Immunosorbent Spot Assay
[0334] PBMC's were isolated from peripheral blood by Ficoll
density-gradient centrifugation prior to use. Viable cells were
enumerated by trypan blue exclusion. A .gamma.-interferon elispot
assay was used according to the manufacturer's instructions
(Mabtech, Nacka Strand, Sweden). A total of 5.times.10.sup.5 PBMC's
were added to each well, in a final volume of 100 .mu.l of
`complete medium`: 95% RPMI 1640 medium (Sigma, Poole, UK)/5% human
AB serum (PAA laboratories, Somerset, UK), with L-glutamine and
penicillin/streptomycin (Sigma) along with peptide at a final
concentration of 4 .mu.gml.sup.-1 or 20 .mu.gml.sup.-1. Peptides
were used either singly or for some in pairs if the sequences were
offset by 2 amino acids.
[0335] Negative control wells contained responder PBMC's plus
medium alone. Positive control wells contained PPD (SSI,
Copenhagen, Denmark) at a final concentration of 10 .mu.gml.sup.-1,
tetanus toxoid at (Calbiochem, Merck Biosciences, UK) at 1
.mu.g/ml, or latterly a positive control (anti CD3 based) supplied
by the manufacturer of the elispot assay. PBMC's were cultured at
37.degree. C., 5% CO.sub.2 for 48 hours, then discarded and the
plate washed 6 times with PBS. Plates were then incubated at room
temperature for 2 hours with the one-step detection reagent
(alkaline phosphatase-conjugated detection monoclonal antibody
7-B6-1, prepared by diluting to 1:200 in filtered PBS containing
0.5% fetal calf serum (Sigma). This was then discarded and plates
washed 5 times with PBS. Filtered ready-to-use chromogenic alkaline
phosphatase substrate ((nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (BCIP/NBT-plus))
provided by manufacturer was added at a volume of 100 .mu.l/well.
The plates were allowed to develop and reaction terminated once
spots emerged. Plates were washed extensively under tap water and
then air-dried in darkness for at least 12 hours before analysis.
The number of spots in each well was then counted using an AID
Elispot plate reader (Strassberg, Germany). A positive response to
test peptide was defined if the number of spots per well was
significantly greater than control by ANOVA.
Inhibition of Indirect Alloimmune Responses with Anti-MHC Class II
Antibodies
[0336] The MHC restriction of responses to peptides was
investigated by addition of a mixture of an anti-MHC class II
monoclonal antibody (Tu39, Becton Dickinson, Oxford, United
Kingdom) and an anti-DR monoclonal antibody (L243. Becton
Dickinson) to cell cultures.
Assessment of Proliferation by CFSE
[0337] PBMC's were resuspended at 10.sup.6 cells/ml in warm RPMI at
37.degree. C. to which CFSE (Molecular Probes, Cambridge
Biosciences) was added at a final concentration of 10 .mu.M. This
was incubated at 37.degree. C., 5% CO.sub.2 with intermittent
agitation for 10 minutes, when human AB serum (Sigma) was added to
terminate labelling, and cells were then washed twice. Cells were
resuspended at 2.5.times.10.sup.5/well in 96-well round-bottom
culture plates in 200 .mu.l of complete medium, in the absence or
presence of test peptide at a final concentration of 4
.mu.gml.sup.-1 or 20 .mu.gml.sup.-1 or PPD at 10 .mu.gml.sup.-1,
then cultured at 37.degree. C., 5% CO.sub.2 for 10 days. Cells were
recovered from 3 or more wells, washed twice and stained with
antibody to CD4 (SK3-PerCP, Becton Dickinson) or CD8 (SK1-PerCP,
Becton Dickinson), or with isotype control (IgG1-PerCP, Becton
Dickinson). Samples were analysed using a FACSCalibur flow
cytometer (Becton Dickinson) using Winmdi 2.8 software (Scripps
Research), acquiring information from a total of 50,000 viable
cells.
Results
Peptide Binding Studies Demonstrate Multiple Regions of Promiscuous
Binding to HLA-DR
[0338] A total of 60, 15mer peptides spanning HLA-A2 were designed
and 53 could be made. The other 7 peptides, largely derived from
the transmembrane region, were insoluble and could not therefore be
satisfactorily synthesised. The affinity of peptide binding to
HLA-DR is shown in FIG. 9.
[0339] The data show that several sequences exhibit promiscuous
binding to a range of HLA-DR. These include peptide 20 (105-119),
the closely overlapping peptide 21 (107-121) and peptide 7 (31-45)
which correspond to sequences previously reported from experiments
in which peptide was eluted from MHC class II. It is apparent that
large numbers of different epitopes from HLA-A2 bind to MHC class
II of different specificities (FIG. 9). This is of particular
interest since a composite epitope of self derived MHC peptide
presented in the context of self MHC class II, would be a secure
means by which to define self.
[0340] Peptides 20 and 21 also correspond to the epitope (92-120)
recognised by DR15 restricted T cell clones generated from the
recipient of a failed HLA-A2 mismatched renal transplant.
[0341] On the basis of the relatively common finding of high or
moderate affinity binding to MHIC class II, 30 peptides were
further assessed in functional experiments. A small number of
peptides offset by two amino acids were used as a mix of two
peptides, in order to optimise the utilisation of PBMCs.
.gamma.-Interferon Production in Response to Peptides from
HLA-A2
[0342] The details of subjects recruited are shown in FIG. 10. The
number of cells producing .gamma.-interferon in response to PPD:
143 (15-422)/10.sup.6 PBMCs was significantly lower in patients on
dialysis who had made alloantibody than in (unmatched) healthy
controls 337 (40-885)/10' PBMCs (p=0.007). The medium controls in
these subjects varied between 0-9/10.sup.6 PBMCs and the maximum
response to peptide was 122/10.sup.6 PBMCs.
[0343] The number of patients responding to at least, one peptide,
assessed by .gamma.-interferon elispot is shown in Table 5. The
number of patients responding to at least one HLA-A2 derived
peptide is shown for each patient group. Patients who make
anti-HLA-A2 antibody are significantly more likely than other
patients to make an indirect response to HLA-A2 derived
peptide.
TABLE-US-00006 TABLE 5 Summary of results in each of the 5 groups
Tissue type Antibody A2+ A2- Anti-HLA Group1 with anti-A2 14/18
Anti-HLA Group 4 Group 2 with no anti-A2 1/9 3/8 No anti HLA Group
5 Group 3 1/10 2/10
[0344] In most patients the number of responding cells was highest
with peptide at a concentration of 20 .mu.gml.sup.-1 but in a small
number the optimal concentration was 4 .mu.gml.sup.-1. In FIG. 1
the response to peptide at 20 .mu.gml.sup.-1 is shown for each
individual.
[0345] In subject group 1, 14/18 patients made a response to at
least one peptide from HLA-A2, shown in FIG. 1a. The number of
patients making a response in group I was statistically
significantly greater than in the other groups combined
(p<0.0001) (although compared to group 2 alone the difference
did not reach significance p=0.08 by Fishers exact test). The
association of indirect alloimmune responses to HLA-A2 and the
formation of specific antibody against HLA-A2, is consistent with
animal models of alloantibody formation (Lovegrove et al., J
Immunol 2001:167(8):4338-44).
[0346] A less expected finding was the observation that although 12
`group 1` subjects responded to peptides from the .alpha.1 and
.alpha.2 hypervariable region (most commonly p20 or p21), of these
subjects, 8 also responded to other peptides from the .alpha.3 and
early transmembrane region; and two more responded to these alone.
Responses to peptides from these regions have to our knowledge, not
previously been reported in human or animal studies. These
sequences are of very limited polymorphism, they are expressed by a
wide range of alleles as illustrated in Table 6.
TABLE-US-00007 TABLE 6 Illustration of HLA types sharing sequences
with HLA-A2 in the region of peptides p39, 40, 50, 51, 52, and 53
Sequenced Peptide Site Sequence(s) shared by and P39 192-206
HAVSDHEA A2, 25, A34 TLRCWAL 26, 29, P40 202-216 RCWALSFY 31, 32,
A34, 80 PAEITLT 33, 43, P50 268-282 KPLTLRWE 66, 68, A80 PSSQPTI
69, 74 P51 270-284 LTLRWEPS A80 SQPTIPI P52 280-294 PTIPIVGI A3402
IAGLVLF P53 282-296 IPIVGIIA A3402 GLVLFGA
[0347] Furthermore 3 patients from `group 1` made significant
responses to peptides from the .alpha.3 domain that were shared
between HLA-A2 and self HLA-A68 (subject 1.1 responded to p39 and
p52/53), HLA-A2 and self HLA-A33 (subject 1.10 responded to
p521/53) or HLA-A2 and self HLA-A32 (subject 1.17 responded to p39
and p52/53).
[0348] In subject `group 2`, 3 patients responded to a range of
HLA-A2 derived peptides (results not shown). For example subject
2.1 responded to peptide p30, p40, 45/46, 50/51 & 52/53 present
in a range of different HLA-A (Table 5), including the
hypervariable region peptide p30. A similar pattern of response to
both hypervariable and .alpha.3 and transmembrane region was
observed with subject 2.2 and 2.3. None of these peptides are
unique to HLA-A2 so the findings remain consistent with the history
of antibody production. In patient 2.2 responses were made to
sequences p39 and p52/53 shared with self HLA-A68.
[0349] In `group 3`, 3 subjects made significant responses to
HLA-A2 derived peptides. Subject 3.3 responded to p39, 3.1 to p1/2
and p39 and 3.7 responded to p19 and p52/53. Although these
patients did not make anti-HLA antibody detectable on current or
historic sera, all had been multiply transfused and it is therefore
possible that all had been primed to these peptides without making
a humoral response. In patient `group 4` one patient 4.9, who had
rapidly lost a renal transplant (A3,11) to Banff III rejection 17
months prior to testing, made an autoimmune response to p 39, p
50/51 and p52/53 but also to p20 which is unique to HLA-A2. One
patient, 5.1 from `group 5` also made an autoimmune response to
peptides p39, p50/51 and p52/53. A group of 15 normal controls,
(designated group 6) with no known prior sensitising events were
also tested. One male (6.3) made an immune response to HLA-A2
derived peptides including p39, and p50/51 and p52/53. This
individual was HLA-A32 positive and these responses were therefore
autoimmune. No other normal controls made any detectable
response.
.gamma.-Interferon Production in Response to HLA-A2 Peptides are
Inhibited by Antibody Against MHC Class II
[0350] The class II restriction of responses was inferred from
inhibition of interferon production by antibodies against MHC class
II in 3 individuals. A representative experiment is shown in FIG.
11.
Proliferation of CD4.sup.+ T Cells in Response to HLA-A2
Peptides
[0351] The correspondence of proliferation with .gamma.-interferon
production was studied in the `normal control`: 6.3 who responded
to peptides p39 and p52/53 assessed by elispot. As shown in FIG.
12, there was proliferation of CD4+ cells in the presence of these
peptides but not in the presence of control peptides that did not
stimulate interferon production.
Discussion
[0352] The contribution of indirect allorecognition to rejection is
well established in various experimental models of transplantation
(for example. Benichou et al., J. Exp Med 1992; 175(1):305-8). In
clinical studies, indirect allorecognition has been associated with
chronic rejection of the kidney, heart and lung. The antigens used
in these clinical studies include: freeze-thaw lysed cells,
peptides spanning the .beta.1 domain of HLA-DR and peptides from
the at domain of class I HLA. The indirect alloresponse has been
detected by the production of IL2, cellular proliferation (in
primary or secondary cultures) or cytokine production by
elispot.
[0353] In both experimental and clinical studies the presence of a
single `immunodominant` epitope is frequently reported, but these
findings may be determined by experimental design, such as the use
of secondary cultures to detect responses following preliminary
expansion of T lymphocyte numbers in vitro. In fact multiple
epitopes may be detected using these methods but at different time
points post transplantation. Furthermore in animal models of
transplantation it is apparent that there may be recognition of
multiple epitopes apart from the `proliferatively immunodominant`
epitope; and these responses may include `cryptic self`.
[0354] Use of the elispot technique allows the detection of antigen
specific T lymphocytes present at relatively low frequencies, in
primary culture. It has allowed our assessment of indirect
allorecognition in patients on the renal transplant waiting list.
In particular we analysed the immune responses of patients who make
an alloantibody of known specificity against HLA-A2. The frequency
of responses we have observed are consistent with those seen in
renal transplant recipients stimulated with 20mer peptides derived
from donor HLA-DR similarly assessed by .gamma.-interferon elispot.
These are of the same order of magnitude to frequencies reported
from renal transplant recipients stimulated by donor PBMC lysates
assessed by DNA synthesis in limiting dilution analysis and lung
transplant recipients with bronchiolitis obliterans stimulated by
either one or two at domain peptides (A1, A2, B38 and B44 derived)
also assayed by DNA synthesis in limiting dilution (SivaSai et al.,
Transplantation 1999; 67(8): 1094-8). In this report by Sivasai and
colleagues a 25mer peptide: 60-84 from the .alpha.1 domain of
HLA-A2 was used, but we observed a response to this region in only
one patient.
[0355] Our results demonstrate that although responses to
hypervariable region peptides were relatively common, as expected
from the literature, there was the unexpected finding of responses
to peptides from the .alpha.3 and early transmembrane region. As
illustrated in table 6, peptide sequences such as p39, p40, p50/51
and p52/53 are widely represented in HLA-A and in those in which
these sequences are not present there is commonly a single or dual
amino acid polymorphism that encompasses most other types. These
peptides could therefore be described as `public T cell epitopes`,
the implication of which is that exposure to one HLA molecule can
result in the priming of T lymphocytes that respond to a range of
other HLA-A family members or vice versa. Since these epitopes are
distinct from those recognised by antibody this is one potential
mechanism for the diminished allograft survival in sensitised
patients, irrespective of the detection of donor specific
antibodies. Similarly it suggests a mechanism whereby blood
transfusion could influence anamnestic antibody responses,
irrespective of the presence of a recognised B cell epitope.
[0356] Since sequences identified as `public T cell epitopes` in
HLA-A2 show only very limited polymorphism, it is possible that a
portion of the allogeneic response will cross react with
self-peptide, as has been reported by Benichou and colleagues for
the immune response to MHC class I peptides in mice (Tam et al., J
Immunol 1996; 156(10):3765-71); and this possibility is currently
under investigation. Indeed a number of the responses described in
FIG. 2 are truly autoimmune, since the sequence of the peptide to
which a response is made is shared between HLA-A2 and self. This is
illustrated by patients 1.1, 1.10 and 1.17. It is similar to the
response seen by Lovegrove and colleagues to cryptic self-epitopes,
in a rat model of rejection associated with alloantibody formation
(Lovegrove et al., J Immunol 2001; 167(8):4338-44).
[0357] The finding of `public T cell epitopes` is also relevant to
the mechanism of the blood transfusion effect: the benefit of prior
blood transfusion on renal transplant survival, mostly observed
before the widespread use of calcineurin inhibitors. Experimental
models of the transfusion effect suggest that there is induction of
regulatory T cells specific for indirectly presented alloantigen
(Kishimoto et al., J Am Soc Nephrol 2004; 15(9):2423-8). Although
these generally report donor antigen specific tolerance there are
now models, attempting to mimic the clinical scenario, in which
random blood transfusions induce regulatory cells that protect
against rejection (Bushell el al., Transplantation 2003;
76(3):449-55) apparently without non-specific depression of the
immune response (Bushell et al., J Immunol 2005; 174(6);
3290-).
[0358] In humans the blood transfusion effect requires blood donor
and recipient to share at least one HLA-DR and a relatively small
number of transfusions for maximum benefit. This is despite the
wide range of alloantigen that recipients may encounter. This could
be accounted for by the induction of self restricted regulatory
cells, specific for allogeneic epitopes that are though common to a
wide range of donors. Whilst these need not necessarily be from the
MHC, the `public T cell epitopes` described above fulfil the
properties necessary to account for much of the available
evidence.
[0359] A second property of many of the peptide epitopes derived
from HLA-A2 is that they bind with some promiscuity to MHC class
II, and correspondingly induce responses in a relatively high
proportion of sensitised patients. This was true both of epitopes
unique to HLA-A2 and of `public T cell epitopes`. This, in part
permitted conclusions to be drawn from our study of only 55
subjects unselected for HLA type. The implications are though wider
with respect to the potential use of such peptides in
desensitisation protocols.
[0360] There has been longstanding interest in the modulation of
rejection by alloantigen derived peptides. These effects are
generally antigen specific which if translated into the clinic
would limit the usefulness of such an approach. Those HLA derived
peptides that have stimulated greatest interest have therefore
impacted upon pathways independent of conventional TCR based
antigen recognition. This is distinct from many animal models of
peptide induced tolerance in which there is evidence of antigen
specificity through the induction of regulatory T cells. The
properties exhibited by epitopes from the .alpha.3 and
trans-membrane domains, that is of being both public and
promiscuous, identify ideal candidates with which to explore
peptide immunotherapy in transplantation. The induction of
regulatory T cell activity has for example been reported in
allergic patients treated with peptide desensitisation
protocols.
[0361] In summary the identification of indirect allo-epitopes from
the .alpha.3 and trans-membrane regions of MHC class I has
important implications for the allogeneic immune response, its
regulation and the development of antigen specific therapy.
Example 3
[0362] The inventor compared the sequence of some of the 15mer
allo-epitopes identified in Examples 1 and 2 with the corresponding
sequences in other HLA polypeptides. The inventor thereby
identified additional allo-epitopes. The results are shown in FIGS.
13 to 16.
Sequence CWU 1
1
1031365PRTHomo sapiens 1Met Ala Val Met Ala Pro Arg Thr Leu Val Leu
Leu Leu Ser Gly Ala 1 5 10 15 Leu Ala Leu Thr Gln Thr Trp Ala Gly
Ser His Ser Met Arg Tyr Phe 20 25 30 Phe Thr Ser Val Ser Arg Pro
Gly Arg Gly Glu Pro Arg Phe Ile Ala 35 40 45 Val Gly Tyr Val Asp
Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala 50 55 60 Ala Ser Gln
Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly 65 70 75 80 Pro
Glu Tyr Trp Asp Gly Glu Thr Arg Lys Val Lys Ala His Ser Gln 85 90
95 Thr His Arg Val Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser
100 105 110 Glu Ala Gly Ser His Thr Val Gln Arg Met Tyr Gly Cys Asp
Val Gly 115 120 125 Ser Asp Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr
Ala Tyr Asp Gly 130 135 140 Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu
Arg Ser Trp Thr Ala Ala 145 150 155 160 Asp Met Ala Ala Gln Thr Thr
Lys His Lys Trp Glu Ala Ala His Val 165 170 175 Ala Glu Gln Leu Arg
Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu 180 185 190 Arg Arg Tyr
Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ala 195 200 205 Pro
Lys Thr His Met Thr His His Ala Val Ser Asp His Glu Ala Thr 210 215
220 Leu Arg Cys Trp Ala Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu Thr
225 230 235 240 Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu
Leu Val Glu 245 250 255 Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys
Trp Ala Ala Val Val 260 265 270 Val Pro Ser Gly Gln Glu Gln Arg Tyr
Thr Cys His Val Gln His Glu 275 280 285 Gly Leu Pro Lys Pro Leu Thr
Leu Arg Trp Glu Pro Ser Ser Gln Pro 290 295 300 Thr Ile Pro Ile Val
Gly Ile Ile Ala Gly Leu Val Leu Phe Gly Ala 305 310 315 320 Val Ile
Thr Gly Ala Val Val Ala Ala Val Met Trp Arg Arg Lys Ser 325 330 335
Ser Asp Arg Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser 340
345 350 Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Val 355 360
365 2341PRTHomo sapiens 2Gly Ser His Ser Met Arg Tyr Phe Phe Thr
Ser Val Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala
Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser
Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 35 40 45 Ala Pro Trp Ile
Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr 50 55 60 Arg Lys
Val Lys Ala His Ser Gln Thr His Arg Val Asp Leu Gly Thr 65 70 75 80
Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val Gln 85
90 95 Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg
Gly 100 105 110 Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala
Leu Lys Glu 115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala
Ala Gln Thr Thr Lys 130 135 140 His Lys Trp Glu Ala Ala His Val Ala
Glu Gln Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Thr Cys Val Glu
Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln
Arg Thr Asp Ala Pro Lys Thr His Met Thr His His 180 185 190 Ala Val
Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe 195 200 205
Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210
215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly
Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly
Gln Glu Gln Arg 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu
Pro Lys Pro Leu Thr Leu 260 265 270 Arg Trp Glu Pro Ser Ser Gln Pro
Thr Ile Pro Ile Val Gly Ile Ile 275 280 285 Ala Gly Leu Val Leu Phe
Gly Ala Val Ile Thr Gly Ala Val Val Ala 290 295 300 Ala Val Met Trp
Arg Arg Lys Ser Ser Asp Arg Lys Gly Gly Ser Tyr 305 310 315 320 Ser
Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu 325 330
335 Thr Ala Cys Lys Val 340 33287DNAHomo sapiens 3ggattcccca
actccgcagt ttcttttctc cctctcccaa cctatgtagg gtccttcttc 60ctggatactc
acgacgcgga cccagttctc actcccatcg ggtgtcgggt ttccagagaa
120gccaatcagt gtcgtcgcgg tcgcggttct aaagtccgca cgcacccacc
gggactcaga 180ttctccccag acgccgagga tggccgtcat ggcgccccga
accctcgtcc tgctactctc 240gggggctctg gccctgaccc agacctgggc
gggtgagtgc ggggtcggga gggaaacggc 300ctctgtgggg agaagcaacg
ggcccgcctg gcgggggcgc aggacccggg aagccgcgcc 360gggaggaggg
tcgggcgggt ctcagccact cctcgtcccc aggctctcac tccatgaggt
420atttcttcac atccgtgtcc cggcccggcc gcggggagcc ccgcttcatc
gcagtgggct 480acgtggacga cacgcagttc gtgcggttcg acagcgacgc
cgcgagccag aggatggagc 540cgcgggcgcc gtggatagag caggagggtc
cggagtattg ggacggggag acacggaaag 600tgaaggccca ctcacagact
caccgagtgg acctggggac cctgcgcggc tactacaacc 660agagcgaggc
cggtgagtga ccccggcccg gggcgcaggt cacgacctct catcccccac
720ggacgggcca ggtcgcccac agtctccggg tccgagatcc gccccgaagc
cgcgggaccc 780cgagaccctt gccccgggag aggcccaggc gcctttaccc
ggtttcattt tcagtttagg 840ccaaaaatcc ccccaggttg gtcggggcgg
ggcggggctc gggggaccgg gctgaccgcg 900gggtccgggc caggttctca
caccgtccag aggatgtatg gctgcgacgt ggggtcggac 960tggcgcttcc
tccgcgggta ccaccagtac gcctacgacg gcaaggatta catcgccctg
1020aaagaggacc tgcgctcttg gaccgcggcg gacatggcag ctcagaccac
caagcacaag 1080tgggaggcgg cccatgtggc ggagcagttg agagcctacc
tggagggcac gtgcgtggag 1140tggctccgca gatacctgga gaacgggaag
gagacgctgc agcgcacggg taccaggggc 1200cacggggcgc ctccctgatc
gcctgtagat ctcccgggct ggcctcccac aaggagggga 1260gacaattggg
accaacacta gaatatcgcc ctccctctgg tcctgaggga gaggaatcct
1320cctgggtttc cagatcctgt accagagagt gactctgagg ttccgccctg
ctctctgaca 1380caattaaggg ataaaatctc tgaaggaatg acgggaagac
gatccctcga atactgatga 1440gtggttccct ttgacacaca caggcagcag
ccttgggccc gtgacttttc ctctcaggcc 1500ttgttctctg cttcacactc
aatgtgtgtg ggggtctgag tccagcactt ctgagtcctt 1560cagcctccac
tcaggtcagg accagaagtc gctgttccct cttcagggac tagaattttc
1620cacggaatag gagattatcc caggtgcctg tgtccaggct ggtgtctggg
ttctgtgctc 1680ccttccccat cccaggtgtc ctgtccattc tcaagatagc
cacatgtgtg ctggaggagt 1740gtcccatgac agatgcaaaa tgcctgaatg
atctgactct tcctgacaga cgcccccaaa 1800acgcatatga ctcaccacgc
tgtctctgac catgaagcca ccctgaggtg ctgggccctg 1860agcttctacc
ctgcggagat cacactgacc tggcagcggg atggggagga ccagacccag
1920gacacggagc tcgtggagac caggcctgca ggggatggaa ccttccagaa
gtgggcggct 1980gtggtggtgc cttctggaca ggagcagaga tacacctgcc
atgtgcagca tgagggtttg 2040cccaagcccc tcaccctgag atggggtaag
gagggagacg ggggtgtcat gtcttttagg 2100gaaagcagga gcctctctga
cctttagcag ggtcagggcc cctcaccttc ccctcttttc 2160ccagagccgt
cttcccagcc caccatcccc atcgtgggca tcattgctgg cctggttctc
2220tttggagctg tgatcactgg agctgtggtc gctgctgtga tgtggaggag
gaagagctca 2280ggtggggaag gggtgaaggg tgggtctgag atttcttgtc
tcactgaggg ttccaagacc 2340caggtagaag tgtgccctgc ctcgttactg
ggaagcacca cccacaatta tgggcctacc 2400cagcctgggc cctgtgtgcc
agcacttact cttttgtaaa gcacctgtta aaatgaagga 2460cagatttatc
accttgatta cagcggtgat gggacctgat cccagcagtc acaagtcaca
2520ggggaaggtc cctgaggacc ttcaggaggg cggttggtcc aggacccaca
cctgctttct 2580tcatgtttcc tgatcccgcc ctgggtctgc agtcacacat
ttctggaaac ttctctgagg 2640tccaagactt ggaggttcct ctaggacctt
aaggccctga ctcctttctg gtatctcaca 2700ggacattttc ttcccacaga
tagaaaagga gggagctact ctcaggctgc aagtaagtat 2760gaaggaggct
gatgcctgag gtccttggga tattgtgttt gggagcccat gggggagctc
2820acccacccca caattcctcc tctagccaca tcttctgtgg gatctgacca
ggttctgttt 2880ttgttctacc ccaggcagtg acagtgccca gggctctgat
gtgtctctca cagcttgtaa 2940aggtgagagc ctggagggcc tgatgtgtgt
tgggtgttgg gcggaacagt ggacacagct 3000gtgctatggg gtttctttcc
attggatgta ttgagcatgc gatgggctgt ttaaagtgtg 3060acccctcact
gtgacagata cgaatttgtt catgaatatt tttttctata gtgtgagaca
3120gctgccttgt gtgggactga gaggcaagag ttgttcctgc ccttcccttt
gtgacttgaa 3180gaaccctgac tttgtttctg caaaggcacc tgcatgtgtc
tgtgttcgtg taggcataat 3240gtgaggaggt ggggagacca ccccaccccc
atgtccacca tgaccct 3287415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4His Ser Met Arg Tyr Phe Phe
Thr Ser Val Ser Arg Pro Gly Arg 1 5 10 15 515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Met
Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly Arg Gly Glu 1 5 10 15
615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg
Phe Ile Ala 1 5 10 15 715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Pro Arg Phe Ile Ala Val Gly
Tyr Val Asp Asp Thr Gln Phe Val 1 5 10 15 815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Ile
Ala Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp 1 5 10 15
915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe
Asp Ser Asp 1 5 10 15 1015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Thr Gln Phe Val Arg Phe Asp
Ser Asp Ala Ala Ser Gln Arg Met 1 5 10 15 1115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Val
Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg 1 5 10 15
1216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu
Gln Glu Gly Pro 1 5 10 15 1315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 13Ala Pro Trp Ile Glu Gln Glu
Gly Pro Glu Tyr Trp Asp Gly Glu 1 5 10 15 1415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Pro
Glu Tyr Trp Asp Gly Glu Thr Arg Lys Val Lys Ala His Ser 1 5 10 15
1515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val
Asp Leu Gly 1 5 10 15 1615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16His Arg Val Asp Leu Gly Thr
Leu Arg Gly Tyr Tyr Asn Gln Ser 1 5 10 15 1715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Val
Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala 1 5 10 15
1815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala
Gly Ser His 1 5 10 15 1915PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Arg Gly Tyr Tyr Asn Gln Ser
Glu Ala Gly Ser His Thr Val Gln 1 5 10 15 2015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20His
Thr Val Gln Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp 1 5 10 15
2116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Val Gln Arg Met Tyr Gly Cys Asp Val Gly Ser Asp
Trp Arg Phe Leu 1 5 10 15 2215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Cys Asp Val Gly Ser Asp Trp
Arg Phe Leu Arg Gly Tyr His Gln 1 5 10 15 2315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Ser
Asp Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp 1 5 10 15
2415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr
Asp Gly Lys 1 5 10 15 2515PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Arg Gly Tyr His Gln Tyr Ala
Tyr Asp Gly Lys Asp Tyr Ile Ala 1 5 10 15 2615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26His
Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 1 5 10 15
2715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys
Glu Asp Leu 1 5 10 15 2815PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Lys Asp Tyr Ile Ala Leu Lys
Glu Asp Leu Arg Ser Trp Thr Ala 1 5 10 15 2915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Ile
Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met 1 5 10 15
3015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala
Ala Gln Thr 1 5 10 15 3115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Arg Ser Trp Thr Ala Ala Asp
Met Ala Ala Gln Thr Thr Lys His 1 5 10 15 3215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Ala
Asp Met Ala Ala Gln Thr Thr Lys His Lys Trp Glu Ala Ala 1 5 10 15
3315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu
Arg Ala Tyr 1 5 10 15 3415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 34Ala His Val Ala Glu Gln Leu
Arg Ala Tyr Leu Glu Gly Thr Cys 1 5 10 15 3515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Glu
Gln Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu 1 5 10 15
3615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu
Arg Arg Tyr 1 5 10 15 3715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 37Thr Cys Val Glu Trp Leu Arg
Arg Tyr Leu Glu Asn Gly Lys Glu 1 5 10 15 3815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Val
Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu 1 5 10 15
3915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln
Arg Thr Asp 1 5 10 15 4015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Glu Thr Leu Gln Arg Thr Asp
Ala Pro Lys Thr His Met Thr His 1 5 10 15 4115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Thr
His Met Thr His His Ala Val Ser Asp His Glu Ala Thr Leu 1 5 10 15
4215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42His Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys
Trp Ala Leu 1 5 10 15 4315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 43Arg
Cys Trp Ala Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu Thr 1 5 10 15
4415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu Thr Trp
Gln Arg Asp 1 5 10 15 4515PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 45Ala Glu Ile Thr Leu Thr Trp
Gln Arg Asp Gly Glu Asp Gln Thr 1 5 10 15 4615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Leu
Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu 1 5 10 15
4715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly
Thr Phe Gln 1 5 10 15 4815PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Gly Thr Phe Gln Lys Trp Ala
Ala Val Val Val Pro Ser Gly Gln 1 5 10 15 4916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Phe
Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 1 5 10
15 5015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg
Tyr Thr Cys 1 5 10 15 5116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Glu Gln Arg Tyr Thr Cys His
Val Gln His Glu Gly Leu Pro Lys Pro 1 5 10 15 5215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Glu
Gly Leu Pro Lys Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser 1 5 10 15
5315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Lys Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser Gln
Pro Thr Ile 1 5 10 15 5415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 54Leu Thr Leu Arg Trp Glu Pro
Ser Ser Gln Pro Thr Ile Pro Ile 1 5 10 15 5515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Pro
Thr Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu Phe 1 5 10 15
5615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu
Phe Gly Ala 1 5 10 15 5717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 57Lys Pro Leu Thr Leu Arg Trp
Glu Pro Ser Ser Gln Pro Thr Ile Pro 1 5 10 15 Ile 5817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Pro
Thr Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu Phe Gly 1 5 10
15 Ala 5918PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Gly Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro
Ser Gly Gln Glu 1 5 10 15 Gln Arg 6015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60His
Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu 1 5 10 15
6115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61His Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys
Trp Ala Leu 1 5 10 15 6215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 62His Pro Val Ser Asp His Glu
Val Thr Leu Arg Cys Trp Ala Leu 1 5 10 15 6315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Arg
Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr 1 5 10 15
6417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Lys Pro Leu Thr Leu Arg Trp Glu Leu Ser Ser Gln
Pro Thr Ile Pro 1 5 10 15 Ile 6517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 65Pro Thr Ile Pro Ile Val
Gly Ile Ile Ala Gly Leu Val Leu Leu Gly 1 5 10 15 Ala
6617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Lys Pro Leu Thr Leu Lys Trp Glu Pro Ser Ser Gln
Pro Thr Ile Pro 1 5 10 15 Ile 6717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 67Lys Pro Leu Thr Leu Arg
Trp Glu Pro Ser Ser Gln Pro Thr Val Pro 1 5 10 15 Ile
6817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Lys Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser Gln
Pro Thr Val His 1 5 10 15 Ile 6917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 69Pro Thr Val Pro Ile Val
Gly Ile Ile Ala Gly Leu Val Leu Leu Gly 1 5 10 15 Ala
7017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Pro Thr Val His Ile Val Gly Ile Ile Ala Gly Leu
Val Leu Phe Gly 1 5 10 15 Ala 7117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 71Pro Thr Ile Pro Ile Val
Gly Ile Ile Ala Gly Leu Ala Val Leu Ala 1 5 10 15 Val
7217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Pro Thr Val Pro Ile Val Gly Ile Ile Ala Gly Leu
Ala Val Leu Ala 1 5 10 15 Val 7318PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 73Gly Thr Phe Gln Lys Trp
Ala Ala Val Val Val Pro Ser Gly Glu Glu 1 5 10 15 Gln Arg
7418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Gly Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro
Ser Gly Gln Glu 1 5 10 15 Gln Arg 7545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 75cactccatga ggtatttctt cacatccgtg tcccggcccg gccgc
457645DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 76atgaggtatt tcttcacatc cgtgtcccgg
cccggccgcg gggag 457745DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 77cacaagtggg
aggcggccca tgtggcggag cagttgagag cctac 457845DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78cacgctgtct ctgaccatga agccaccctg aggtgctggg ccctg
457945DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 79aggtgctggg ccctgagctt ctaccctgcg
gagatcacac tgacc 458045DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 80aagcccctca
ccctgagatg ggagccgtct tcccagccca ccatc 458145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81ctcaccctga gatgggagcc gtcttcccag cccaccatcc ccatc
458245DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 82cccaccatcc ccatcgtggg catcattgct
ggcctggttc tcttt 458345DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 83atccccatcg
tgggcatcat tgctggcctg gttctctttg gagct 458445DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84ggaaccttcc agaagtgggc ggctgtggtg gtgccttctg gacag
458548DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 85ttccagaagt gggcggctgt ggtggtgcct
tctggacagg agcagaga 488651DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 86aagcccctca
ccctgagatg ggagccgtct tcccagccca ccatccccat c 518751DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 87cccaccatcc ccatcgtggg catcattgct ggcctggttc
tctttggagc t 518854DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 88ggaaccttcc agaagtgggc
ggctgtggtg gtgccttctg gacaggagca gaga 548945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 89tcggactggc gcttcctccg cgggtaccac cagtacgcct acgac
459045DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90tggcgcttcc tccgcgggta ccaccagtac
gcctacgacg gcaag 459145DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 91caccccatct
ctgaccatga ggccaccctg aggtgctggg ccctg 459251DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 92aagcccctca ccctgagatg ggagccttct tcccagccca
ccatccccat c 519351DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 93cccaccatcc ccatcgtggg
catcattgct ggcctggttc tccttggagc t 519445DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 94aggtgctggg ccctgggctt ctaccctgcg gagatcacac tgacc
459554DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 95ggaaccttcc agaagtgggc ggctgtggtg
gtgccttctg gagaggagca gaga 549654DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 96ggaaccttcc
agaagtgggc gtctgtggtg gtgccttctg gacaggagca gaga
549713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala
Thr 1 5 10 9813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 98Ala Ala Tyr Ala Ala Ala Lys Ala Ala
Ala Leu Ala Ala 1 5 10 9915PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 99Ala Lys Thr Ile Ala Tyr Asp
Glu Glu Ala Arg Arg Gly Leu Glu 1 5 10 15 10016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Thr
Glu Arg Val Arg Leu Val Thr Arg His Ile Tyr Asn Arg Glu Glu 1 5 10
15 10115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 101Glu Ala Glu Gln Leu Arg Ala Tyr Leu Asp Gly
Thr Gly Val Glu 1 5 10 15 10220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 102Glu Ser Trp Gly Ala Val
Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 1 5 10 15 Gly Pro Phe Thr
20 10314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 103Ala Gly Asp Leu Leu Ala Ile Glu Thr Asp Lys
Ala Thr Ile 1 5 10
* * * * *
References