U.S. patent application number 10/541115 was filed with the patent office on 2006-09-14 for isolation and identification of cross-reactive t cells.
Invention is credited to Jingwu Z. Zang.
Application Number | 20060204495 10/541115 |
Document ID | / |
Family ID | 32713173 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060204495 |
Kind Code |
A1 |
Zang; Jingwu Z. |
September 14, 2006 |
Isolation and identification of cross-reactive t cells
Abstract
Cross-reactive T cells recognizing both MBP.sub.93-105 and
HHV-6.sub.1-13 peptides represent a significant subset of T cells
with some degree of TCR degeneracy. It appears that the recognition
of the cross-reactive T cells has a less stringent requirement for
the flanking residues of the two peptides. In contrast, these
flanking residues are critical for the T cell recognition of
mono-specific T cells. The association between HHV-6 and
autoreactive immune responses to MBP indicates that cross-reactive
T cells, peptides from the V-D-J region of the T cell receptor from
autoreactive T cells, and antiviral agents may prevent or treat
MS.
Inventors: |
Zang; Jingwu Z.; (Missouri,
TX) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT
2941 FAIRVIEW PARK DR, SUITE 200
FALLS CHURCH
VA
22042-2924
US
|
Family ID: |
32713173 |
Appl. No.: |
10/541115 |
Filed: |
December 23, 2003 |
PCT Filed: |
December 23, 2003 |
PCT NO: |
PCT/US03/41284 |
371 Date: |
April 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60437369 |
Dec 31, 2002 |
|
|
|
Current U.S.
Class: |
424/140.1 ;
435/372 |
Current CPC
Class: |
G01N 33/564 20130101;
A61K 39/0008 20130101; A61K 2039/5158 20130101; A61P 37/04
20180101 |
Class at
Publication: |
424/140.1 ;
435/372 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method for isolating one or more T cells that cross-react with
a self-antigen and a foreign antigen comprising: (a) incubating a
sample comprising T cells with an antigen that comprises an epitope
present in the self-antigen and the foreign antigen, and wherein
said sample optionally comprises one or more autoantigens; and (b)
isolating the one or more cross-reactive T cells by cloning or
direct expansion, wherein the self-antigen is myelin basic protein,
and wherein the foreign antigen is human herpesvirus-6 U24.
2. The method of claim 1, wherein the self-antigen comprises a
sequence of residues 96-102 of myelin basic protein or residues
93-105 of myelin basic protein, and wherein the foreign antigen
comprises a sequence of residues 4-10 of human herpesvirus-6 U24,
residues 1-13 of human herpesvirus-6 U24.
3. The method of claim 1, wherein the autoantigen is selected from
the group consisting of myelin basic protein, proteolipid protein,
myelin oligodendrocyte glycoprotein, collagen type II peptides,
heat shock protein, MAGE, PSA, CA125, GAD protein, and tumor
associated antigen.
4. The method of claim 1, wherein the autoantigen comprises an
immunodominant epitope of a member selected from the group
consisting of myelin basic protein, proteolipid protein, myelin
oligodendrocyte glycoprotein, collagen type II peptides, heat shock
protein, MAGE, PSA, CA125, GAD protein, and tumor associated
antigen.
5. The method of claim 4, wherein said immunodominant epitope is
selected from the group consisting of residues 83-99 of myelin
basic protein and residues 151-170 of myelin basic protein.
6. The method of claim 1, further comprising selecting one or more
T cells that express one or more first markers selected from the
group consisting of CD69, CD4, CD25, CD36 and HLADR and one or more
second markers selected from the group consisting of IL-2,
IFN.gamma., TNF.alpha., IL5, IL-10 and IL-13.
7. The method of claim 6, wherein the self-antigen comprises a
sequence of residues 96-102 of myelin basic protein or residues
93-105 of myelin basic protein and wherein the foreign antigen
comprises a sequence of residues 4-10 of human herpesvirus-6 U24,
residues 1-13 of human herpesvirus-6 U24.
8. The method of claim 6, wherein the autoantigen is selected from
the group consisting of myelin basic protein, proteolipid protein,
myelin oligodendrocyte glycoprotein, collagen type II peptides,
heat shock protein, MAGE, PSA, CA125, GAD protein, and tumor
associated antigen.
9. (canceled)
10. (canceled)
11. The method of claim 7, wherein the cells expressing said first
and said second markers are selected using antibodies to said first
and second markers respectively, or optionally a bi-specific
antibody which binds both first and second markers in combination
with an antibody which binds said second marker.
12. The method of claim 11, wherein one or more of said antibodies
is fluorescently labeled and wherein said T cell is selected by
fluorescent activated cell sorting.
13. (canceled)
14. The method of claim 11, wherein said first antibody is
conjugated to a magnetic microbead and wherein said T cell is
selected by magnetic activated cell sorting.
15. (canceled)
16. A composition comprising one or more T cells that cross-react
with a self antigen and a foreign antigen, wherein the self-antigen
is myelin basic protein, wherein the foreign antigen is human
herpesvirus-6 U24 and wherein the cross-reacting T cells are
enriched with respect to other T cells that react with the
self-antigen.
17. The composition of claim 16, wherein the self-antigen comprises
a sequence of residues 96-102 of myelin basic protein or residues
93-105 of myelin basic protein, and wherein the foreign antigen
comprises a sequence of residues 4-10 of human herpesvirus-6 U24,
residues 1-13 of human herpesvirus-6 U24.
18. A method for quantifying the number of T cells in a sample that
cross-react with an self-antigen and a foreign antigen comprising:
(a) incubating a sample comprising T cells with an antigen that
comprises an epitope present in the self-antigen and the foreign
antigen, and wherein said sample optionally comprises one or more
autoantigens; (b) selecting one or more T cells that express one or
more first markers selected from the group consisting of CD69, CD4,
CD25, CD36 and HLADR and one or more second markers selected from
the group consisting of IL-2, IFN.gamma., TNF.alpha., IL5, IL-10
and IL-13; and (c) determining the number of T cells selected by
step (b). wherein the self-antigen is myelin basic protein, and
wherein the foreign antigen is human herpesvirus-6 U24.
19. A method for diagnosing an autoimmune disease in a patient,
comprising: (a) quantifying the number of cross-reactive T cells
according to the method of claim 18; and (b) comparing the number
of cross-reactive T cells and optionally other autoreactive T cells
to a control.
20. A method for monitoring an autoimmune disease in a patient,
comprising: (a) quantifying the number of cross-reactive T cells
according to the method of claim 18; and (b) comparing the number
of cross-reactive T cells and optionally other autoreactive T cells
to a control. (c)
21. A method for treating an autoimmune disease in a patient,
comprising administering the composition of claim 16 to a patient
in need thereof.
22. A method for producing the composition of claim 16, comprising:
(a) incubating a sample derived from said patient comprising T
cells with an antigen that comprises an epitope present in a
self-antigen and a foreign antigen, wherein said sample optionally
comprises one or more autoantigens; (b) selecting one or more T
cells that express one or more first markers selected from the
group consisting of CD69, CD4, CD25, CD36 and HLADR and one or more
second markers selected from the group consisting of IL-2,
IFN.gamma., TNF.alpha., IL5, IL-10 and IL-13; and (c) inactivating
the T cells selected by step (b), wherein the self-antigen is
myelin basic protein, and wherein the foreign antigen is human
herpesvirus-6 U24.
23. The method of claim 22 further comprising expanding the number
of T cells selected in step (b).
24. (canceled)
25. A method for isolating a nucleic acid encoding a T cell
receptor, or a portion thereof, wherein said T cell receptor is
specific for a self-antigen and a foreign antigen, comprising: (a)
isolating one or more T cells according to the method of claim 6;
and (b) amplifying the nucleic acid encoding said T cell receptor
from a T cell isolated by step (a) using at least one first primer
specific for the variable region of the T cell receptor gene and a
second primer specific for the constant region of the T cell
receptor gene,
26. (canceled)
27. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
diagnosis and treatment of autoimmune disease, such as multiple
sclerosis (MS). More particularly, it concerns the isolation of
cross-reactive T cells that are specific for self-antigens and
foreign antigens. In addition, the present invention concerns the
use of cross-reactive T cells for the diagnosis and treatment of
autoimmune disease, such as MS.
BACKGROUND
[0002] Intercellular recognition complexes formed by T cell
receptors (TCR) on cytotoxic T lymphocytes or T helper cells and
MHC/peptide complexes on antigen presenting cells (APC) are a
common recognition component in a diverse set of cell-cell
encounters that activate T cells both during the development of the
repertoire of T cells within an individual organism (positive
selection; negative selection; peripheral survival) and during the
control (T helper) and effector stages (T killer) of an adaptive
immune response.
[0003] In the adaptive immune response, antigens are recognized by
hypervariable molecules, such as antibodies or TCRs, which are
expressed with sufficiently diverse structures to be able to
recognize any antigen. Antibodies can bind to any part of the
surface of an antigen. TCRs, however, are restricted to binding to
short peptides bound to class I or class II molecules of the major
histocompatibility complex (MIC) on the surface of APCs. TCR
recognition of a peptide/MHC complex triggers activation (clonal
expansion) of the T cell.
[0004] TCRs are heterodimers composed of two chains which can be
.alpha..beta. (alpha-beta) or .gamma..delta. (gamma-delta). The
structure of TCRs is very similar to that of immunoglobulins (Ig).
Each chain has two extracellular domains, which are immunoglobulin
folds. The amino-terminal domain is highly variable and called the
variable (V) domain. The domain closest to the membrane is the
constant (C) domain. These two domains are analogous to those of
immunoglobulins, and resemble Fab fragments. The V domain of each
chain has three complementarity determining regions (CDR). Proximal
to the membrane, each TCR chain has a short connecting sequence
with a cysteine residue that forms a disulfide bond between both
chains.
[0005] Genes encoding .alpha..beta. and .gamma..delta. heterodimers
are only expressed in the T-cell lineage. The four TCR loci
(.alpha., .beta., .gamma. and .delta.) have a germ-line
organization very similar to that of Ig. .alpha. and .gamma. chains
are produced by rearrangements of V and J segments whereas .beta.
and .delta. chains are produced by rearrangements of V, D, and J
segments. The gene segments for TCR chains are located on different
chromosomes, except the .delta.-chain gene segments that are
between the V and J gene segments of the ax chain. The location of
.delta.-chain gene segments has a significance: a productive
rearrangement of .alpha.-chain gene segments deletes C genes of the
.delta.-chain, so that in a given cell the .alpha..beta.
heterodimer cannot be co-expressed with the .gamma..delta.
receptor.
[0006] In mice, there are about 100 V.alpha. and 50 J.alpha. genes
segments and only one C.alpha. segment. The .delta.-chain gene
family has about 10 V, 2 D, and 2 J gene segments. The .beta.-chain
gene family has 20-30 V segments and two identical repeats
containing 1 D.beta., 6 J.beta. and 1 C.beta.. Finally, the
.gamma.-chain gene family contains 7 V and 3 different J-C repeats.
In humans the organization is similar to that of mice, but the
number of segments varies.
[0007] The rearrangements of gene segments in .alpha. and .beta.
chains is similar to that of Igs. The .alpha. chain, like the light
chain of Ig is encoded by V, J, and C gene segments. The .beta.
chain, like the heavy chain of Ig, is encoded by V, D, and J gene
segments. Rearrangements of a chain gene segments result in VJ
joining and rearrangements of .beta. chain result in VDJ joining.
After transcription of rearranged genes, RNA processing, and
translation, the .alpha. and .beta. chains are expressed linked by
a disulfide bond in the membrane of T cells.
[0008] TCR gene segments are flanked by recognition signal
sequences (RSS) containing a heptamer and a nonamer with an
intervening sequence of either 12 nucleotides (one turn) or 23
nucleotides (two turn). As in Igs, enzymes encoded by
recombination-activating genes (RAG-1 and RAG-2) are responsible
for the recombination processes. RAG1/2 recognize the RSS and join
V-J and V-D-J segments in the same manner as in Ig rearrangements.
Briefly, these enzymes cut one DNA strand between the gene segment
and the RSS and catalyze the formation of a hairpin in the coding
sequence. The signal sequence is subsequently excised.
[0009] The combinatorial joining of V and J segments in .alpha.
chains and V, D and J segments in .beta. chains produces a large
number of possible molecules, thereby creating a diversity of TCRs.
Diversity is also achieved in TCRs by alternative joining of gene
segments. In contrast to Ig, .beta. and .delta. gene segments can
be joined in alternative ways. RSS flanking gene segments in .beta.
and .delta. gene segments can generate VJ and VDJ in the .beta.
chain, and VJ, VDJ, and VDDJ on the .delta. chain. As in the case
of Ig, diversity is also produced by variability in the joining of
gene segments.
[0010] Hypervariable loops of the TCR known as complementarity
determining regions (CDRs) recognize the composite surface made
from a MHC molecule and a bound peptide. The CDR2 loops of .alpha.
and .beta. contact only the MHC molecule on the surface of APC,
while the CDR1 and CDR3 loops contact both the peptide and MHC
molecule. Compared with Ig, TCRs have more limited diversity in the
CDR1 and CDR2. However, diversity of the CDR3 loops in TCRs is
higher than that of Ig, because TCRs can join more than one D
segment leading to augmented junctional diversity.
[0011] The pathogenesis of a number of autoimmune diseases is
believed to be caused by autoimmune T cell responses to
self-antigens present in the organism. Not all autoreactive T cells
are deleted in the thymus, in contradiction with the clonal
selection paradigm. Those T cells with TCRs for a broad spectrum of
self-antigens represent part of the normal T-cell repertoire and
naturally circulate in the periphery. It is unclear why
autoreactive T cells are allowed, during their evolution, to
undergo differentiation in the thymus and are accommodated in the
periphery. While their physiological role is not understood, these
autoreactive T cells, when activated, present a potential risk in
the induction of autoimmune pathologies. Autoreactive T cells can
also be isolated from normal individuals without the consequences
of autoimmune diseases. It has been established that antigen
recognition of autoreactivity by itself is not sufficient to
mediate the autodestructive process. One of the prerequisites for
autoreactive T cells to be pathogenic is that they must be
activated.
[0012] Autoreactive T cells are implicated in the pathogenesis of
autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid
arthritis (RA). The pathogenesis of autoreactive T cells in MS is
generally held to arise from T cell responses to myelin antigens,
in particular myelin basic protein (MBP). MBP-reactive T cells can
be isolated from patients with MS but also from healthy
individuals. One of the important discrepancies between
myelin-reactive T cells in MS patients and those in normal
individuals is the activation state of the T cells. MBP-reactive T
cells are found to undergo in vivo activation, and occur at a
higher precursor frequency in blood and cerebrospinal fluid in
patients with MS as opposed to control individuals. These
MBP-reactive T cells produce T.sub.H1 cytokines, e.g. IL-2,
TNF.alpha., and .gamma.-interferon (IFN.gamma.), which facilitate
migration of inflammatory cells into the central nervous system and
exacerbate myelin-destructive inflammatory responses in MS.
[0013] Microbial infections, in particular viral infections, may
play an important role in the etiology and pathogenesis of multiple
sclerosis (MS). It has been speculated that MBP-reactive T cells
may be activated in vivo by viral infections in patients with MS
through a mechanism of molecular mimicry, which has been supported
by in vitro studies. A number of viruses, including measles virus,
Epstein-Barr virus and most recently human herpesvirus-6 (HHV-6),
have been implicated as etiologic agents in MS based on
epidemiological evidence, geographic pattern and abnormal immune
response to these viruses. The potential pathologic importance of
certain viral infections in MS is thought to involve direct
neurotropic properties of the virus, causing tissue damage, and
their ability to activate autoimmune responses directed at myelin
antigens, such as MBP, through the mechanism known as molecular
mimicry.
[0014] Myelin-reactive T cells have been shown to be involved in
the pathogenesis of experimental autoimmune encephalomyelitis (EAE)
in animals, which resembles MS. EAE can be induced actively in
susceptible animals by injecting myelin proteins emulsified in an
adjuvant or passively by injecting myelin-reactive T-cell lines and
clones derived from myelin-sensitized animals. When activated in
vitro, very small numbers of myelin-reactive T cells are required
to induce EAE, while 100-fold more resting T cells with the same
reactivity are incapable of mediating EAE.
[0015] EAE has been shown to be prevented and also treated by
vaccination with inactivated myelin-reactive T cells, a procedure
called T-cell vaccination (Ben-Nun et al., Nature, 1981; 292:
60-61). T-cell vaccination induces regulatory immune responses
comprised of anti-idiotypic T cells and anti-ergotypic T cells,
which lead to the depletion of myelin-reactive T cells. By
depleting myelin-reactive T cells, therapeutic effects are observed
in EAE and other experimental autoimmune disease models (Lider et
al, Science, 1988; 239:820-822; Lohse et al., Science, 1989; 244:
820-822).
[0016] Due to the success in autoimmune disease models, T cell
vaccination has recently advanced to clinical trials in patients
with MS. Based on the results in experimental models such as EAE,
it is believed that depletion of autoreactive T cells may improve
the clinical course of MS and other autoimmune diseases.
[0017] In a pilot clinical trial, vaccination with irradiated
autologous MBP-reactive T cell clones elicited CD8.sup.+ cytolytic
T cell responses that specifically recognized and lysed circulating
MBP-reactive T cells (Zhang et al., Science, 1993; 261: 1451-1454,
Medaer et al. Lancet 1995: 346:807-808). Three subcutaneous
inoculations with irradiated MBP-reactive T cell clones resulted in
the depletion of circulating MBP-reactive T cells in patients with
MS.
[0018] In a preliminary clinical trial, circulating MBP-reactive T
cells were depleted in relapsing remitting MS patients and
secondary progressive MS patients (Zhang et al., J Neurol., 2002;
249:212-8), by vaccinating the patients with three subcutaneous
injections of irradiated autologous MBP-reactive T cells. T cell
vaccination was beneficial to each group of patients as measured by
rate of relapse, expanded disability scale score and MRI lesion
activity. However, there was a trend for an accelerated progression
after about twelve months following the last injection. The
significance of the apparent accelerated progression is unknown,
but may be associated with a gradual decline of the immunity
induced by T cell vaccination against MBP-reactive T cells. In
approximately 10-12% of the immunized patients, MBP-reactive T
cells reappeared at about the same time as the accelerated
progression. In some cases, the reappearing MBP-reactive T cells
originated from different clonal populations that were not detected
before vaccination, suggesting that MBP-reactive T cells may
undergo clonal shift or epitope spreading. Clonal shift of
MBP-reactive T cells has been observed in previous studies (Zhang
et al. 1995) and may be associated with the on-going disease
process.
[0019] Although T cell vaccination has been demonstrated to be
effective for depleting myelin-reactive T cells and potentially
beneficial for MS patients, there are several problems with the
treatment. T cell vaccine treatment for each patient must be
individualized because the T cell receptors of myelin-reactive T
cells are highly diverse and vary between different MS patients
(Vandevyver et al., Eur. J. Immunol., 1995; 25:958-968,
Wucherpfennig et al., J. Immunol., 1994; 152:5581-5592, Hong et
al., J. Immunol., 1999; 163:3530-3538).
[0020] In addition to being individualized for each patient, up to
8 weeks is required to produce a given T cell vaccine using current
procedures. Production of a T cell vaccine begins with isolating
mononuclear cells from the cerebrospinal fluid (CSFMCs) or
peripheral blood (PBMCs) of a patient. The isolated mononuclear
cells are then cultured with myelin peptides for 7-10 days to
activate myelin-reactive T cells. Cultures are then tested for
specific proliferation to myelin peptides by measuring
[.sup.3H]-thymidine incorporation in the presence of myelin
peptides over a period of 3 days. Cultures testing positive for
specific proliferation to myelin peptides are then serially diluted
to obtain clonal T cell lines or directly expanded. The cells are
then cultured up to 6-8 weeks to expand the T cells. When the final
T cell vaccine product is clonal, the T cells are homogenous with a
single pattern of V.beta.-D.beta.-J.beta. gene usage. Usually,
three to six of the clonal cell lines are combined to produce a
heterogeneous formulation with multiple patterns of
V.beta.-D.beta.-J.beta. gene usage. When the final T cell vaccine
product is produced by direct expansion, the T cells are
heterogeneous with more than one pattern of V.beta.-D.beta.-J.beta.
gene usage.
[0021] The individualized nature of T cell vaccination and the
prolonged cell culture needed for production of each vaccine make
treatment expensive and labor intensive under current
methodologies. The extended time required for cell culture also
creates a significant risk of contamination. Finally, the
likelihood of clonal shift or epitope spreading of myelin-reactive
T cells may require the subsequent production of a T cell vaccine
for each patient with a different pattern of
V.beta.-D.beta.-J.beta. gene usage.
[0022] Therefore, there exists a need to develop improved methods
of isolating T cells with specificity for antigens, such as MBP,
that may be used to produce T cell vaccines for the treatment of
patients with T cell-mediated diseases such as MS. There also
exists a need to develop improved methods for producing T cell
vaccines with a heterogeneous pattern of V.beta.-D.beta.-J.beta.
gene usage to account for clonal shift of autoreactive T cells.
[0023] In addition to vaccines based on inactivated T cells,
vaccines may be based on immunogenic peptides. In EAE,
encephalitogenic MBP-reactive T cells are restricted to very
limited epitopes on MBP. These restrictions in the diversity of the
pathogenic T-cell responses permit specific immune intervention.
Various therapeutic strategies have been designed accordingly to
target the V.beta. region of the TCR in preventing the development
of EAE in sensitized animals. For example, monoclonal antibodies
have been targeted to the V.beta. gene product and peptide vaccines
have been based on the CDR2 region of the responsible V.beta.
gene.
[0024] Some of the studies on EAE have been extended to human
autoimmune diseases. For instance, a peptide corresponding to TCR
V.beta. 5.2 has been used in clinical trials to treat patients with
MS and a V.beta. 14 peptide has been used to vaccinate patients
with RA. U.S. Pat. No. 5,614,192 (Vandenbark) discloses treatment
of autoimmune diseases by the use of immunogenic TCR peptides of 15
to 30 amino acids comprising at least part of CDR2. U.S. Pat. No.
6,303,314 (Zhang) discloses the treatment of autoimmune diseases by
using certain immunogenic TCR peptides in combination with
immunogenic T cell activation marker peptides.
[0025] One area in which vaccination with TCR peptides can be
improved is by determining which, if any, common motifs are found
in the autoreactive TCRs of a patient with an autoimmune disease
such as MS. Such common motifs can be used either as a basis for a
peptide vaccine to activate an anti-idiotypic immune response in MS
patients for the purpose of depleting T cells which have TCRs
comprising said motifs, or as a target for the preparation of
antibodies that can functionally block or directly deplete T cells
which have TCRs comprising said motifs.
[0026] Therefore, it is desirable to determine the amino acid
sequences of common motifs specifically found in the TCRs of
autoreactive T cells from patients with autoimmune diseases. It is
also desirable to be able to readily detect such motifs in a
patient sample by a convenient method, such as PCR. In addition, it
is desirable to use peptides identical to or derived from the
detected motifs to treat patients with the autoimmune disease. It
is also desirable to use antibodies which specifically bind to said
motifs to treat a patient with an autoimmune disease.
[0027] U.S. Pat. No. 6,303,314 (Zhang) discloses such a common
motif found in the TCRs of a subset of V.beta.13.1 T cells, the
"LGRAGLTY motif", which has the amino acid sequence Leu Gly Arg Ala
Gly Leu Thr Tyr (SEQ ED NO: 10), as well as a method for its ready
detection by PCR. This motif is found in some TCRs of some
autoreactive T cells that recognize amino acids 83-99 of MBP
(hereinafter "MBP83-99"). Peptides based on the LGRAGLTY motif can
be used to vaccinate some patients in order to treat or prevent
autoimmune diseases with which V.beta.13.1-LGRAGLTY is associated
(e.g., MS). U.S. Provisional Patent No. 60/386,287 (Zhang)
discloses an additional three motifs present in the TCRs of
MBP-reactive T cells.
[0028] As autoreactive T cells associated with autoimmune diseases,
such as MS, may be activated by foreign antigens that mimic
self-antigens, such as MBP, there remains a need to identify other
TCR sequences, including CDR sequences, that cross-react with a
self-antigen and a foreign antigen mimicking said self-antigen. In
addition, there remains a need to be able to detect TCR sequences,
including CDR sequences, which are expressed by MBP-reactive T
cells that cross-react with a self-antigen and a foreign antigen
mimicking said self-antigen. Finally, there remains a need to
develop treatments for autoimmune diseases associated with TCR
sequences, and more particularly CDR sequences, expressed by
MBP-reactive T cells that cross-react with a self-antigen and a
foreign antigen mimicking said self-antigen.
SUMMARY
[0029] The present invention is generally directed to methods of
isolating cross-reactive T cells and more particularly T cells
specific for a self-antigen and also a foreign antigen. The methods
for isolating a cross-reactive T cell comprises incubating a sample
comprising T cells with an antigen that comprises an epitope
present in the self-antigen and the foreign antigen, and optionally
one or more autoantigens. The T cell may also be isolated by
selecting T cells that express one or more first markers selected
from the group consisting of CD69, CD4, CD25, CD36 and HLADR; and
one or more second markers selected from the group consisting of
IL-2, IFN.gamma., TNF.alpha., IL5, IL-10 and IL-13.
[0030] The methods of the invention are particularly useful for
isolating cross-reactive T cells which play a role in the
pathogenesis of autoimmune diseases.
[0031] The methods of the invention also permit the diagnosis of
autoimmune disease as well as monitoring the progression of the
disease and for monitoring the efficacy of treatments for the
disease.
[0032] The methods of the invention also allow the preparation of
autologous T cell vaccines for the treatment of T cell related
autoimmune diseases.
[0033] The methods for vaccine preparation generally involve the
isolation of cross-reactive T cells as described above optionally
followed by subsequent culturing steps which allows the expansion
of the population of isolated cross-reactive T cells.
[0034] The invention inter alia is also directed to T cell vaccines
and pharmaceutical compositions comprising cross-reactive T cells
isolated using the methods of the invention.
[0035] The methods of the invention are also useful for
characterizing cross-reactive T cell receptors and their encoding
nucleic acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 demonstrates the T cell responses to the
MBP.sub.93-105 and HHV-6 (U24).sub.1-13 peptides in MS patients and
healthy controls. The circles represent the individual precursor
frequency and the bars indicate the mean precursor frequency of the
T cells within the group. The asterisks indicate statistically
significant differences (p<0.05) between the MS group and the
control group.
[0037] FIG. 2 demonstrates cytokine concentrations produced by
mono-specific and cross-reactive T cells after challenge with the
MBP.sub.93-105 and HHV-6 (U24).sub.1-13 peptides. Panel A shows the
the cytokine profile of the T cell lines primed with peptide
MBP.sub.93-105 and challenged with the same peptide in week 2.
Panel B shows the cytokine profile of the cross-reactive T cell
lines primed with peptide MBP.sub.93-105 and challenged with
peptide HHV-6 (U24).sub.1-13 in week 2. Panel C shows the cytokine
profile of the T cell lines primed with peptide HHV-6
(U24).sub.1-13 and challenged with the same peptide in week 2.
Panel D shows the cytokine profile of cross-reactive T cell lines
primed with peptide HHV-6 (1124).sub.1-13 and challenged with
peptide MBP.sub.93-105 in week 2.
[0038] FIG. 3 shows the reactivity pattern of mono-specific and
cross-reactive T cell clones to the MBP.sub.93-105 and HHV-6
(U24).sub.1-13 peptides, or a control peptide (T cell receptor
amino acid sequence: ASSENRASYNEQFFG)(SEQ ID NQ: 7).
[0039] FIG. 4 shows serum IgG antibodies specific for the
MBP.sub.93-105 and HHV-6.sub.1-13 peptides in MS patients and
healthy subjects compared to a control peptide (T cell receptor
amino acid sequence: ASSENRASYNEQFFG)(SEQ ID NO: 7).
DETAILED DESCRIPTION
1. Definitions
[0040] To aid in the understanding of the present invention,
several terms are defined below.
[0041] "Autoantigen" or "self-antigen" as used herein refers to an
immunogenic antigen or epitope which is native to a mammal and
which may be involved in the pathogenesis of an autoimmune
disease.
[0042] "CD," "cluster of differentiation" or "common determinant"
as used herein refers to cell surface molecules recognized by
antibodies. Expression of some CDs is specific for cells of a
particular lineage or maturational pathway, and the expression of
others varies according to the state of activation, position, or
differentiation of the same cells.
[0043] "Derived from" or "a derivative thereof," in the context of
nucleotide sequences means that the nucleotide sequence is not
limited to the specific sequence described, but also includes
variations in that sequence, which may include nucleotide
additions, deletions, substitutions, or modifications to the extent
that the variations to the described sequence retain the ability to
hybridize under moderate or highly stringent conditions to the
complement of the described sequence. In the context of peptide or
polypeptide sequences, "derived from" or "a derivative thereof"
means that the peptide or polypeptide is not limited to the
specific sequence described, but also includes variations in that
sequence, which may include amino acid additions, deletions,
substitutions, or modifications to the extent that the variations
in the listed sequence retain the ability to elicit an immune
response to the described sequence.
[0044] "Imunogenic," when used to describe a peptide or
polypeptide, means the peptide or polypeptide is able to induce an
immune response, either T cell mediated, antibody, or both.
[0045] "Immune-related disease" means a disease in which the immune
system is involved in the pathogenesis of the disease. A subset of
immune-related diseases are autoimmune diseases. Autoimmune
diseases include, but are not limited to, rheumatoid arthritis,
myasthenia gravis, multiple sclerosis, psoriasis, systemic lupus
erythematosus, autoimmune thyroiditis (Hashimoto's thyroiditis),
Graves' disease, inflammatory bowel disease, autoimmune
uveoretinitis, polymyositis, and certain types of diabetes. In view
of the present disclosure, one skilled in the art can readily
perceive other autoimmune diseases treatable by the compositions
and methods of the present invention.
[0046] "PCR" means the polymerase chain reaction, for example, as
generally described in U.S. Pat. No. 4,683,202 (issued Jul. 28,
1987 to Mullis), which is hereby incorporated by reference in its
entirety. PCR is an amplification technique wherein selected
oligonucleotides, or primers, may be hybridized to nucleic acid
templates in the presence of a polymerization agent (such as a DNA
or RNA polymerase) and nucleotide triphosphates, whereby extension
products may be formed from the primers. These products may then be
denatured and used as templates in a cycling reaction that
amplifies the number and amount of existing nucleic acids which may
facilitate their subsequent detection. A variety of PCR techniques
are known in the art and may be used in connection with the
disclosure herein.
[0047] "Peptide" or "polypeptide" is a linked sequence of amino
acids and may be natural, synthetic, or a modification or
combination of natural and synthetic.
[0048] "Primer" means an oligonucleotide, whether natural,
synthetic, or a modification thereof, capable of acting as a point
of initiation of nucleotide synthesis sufficiently complementary to
a specific nucleotide sequence on a template molecule.
[0049] "Probe" means an oligonucleotide, whether natural,
synthetic, or a modification thereof, capable of specifically
binding to a sufficiently complementary nucleotide sequence.
[0050] "T cell mediated disease" means a disease arising as a
result of T cells recognizing self-antigens.
[0051] "Treatment" or "treating," when referring to protection of
an animal from a disease, means preventing, suppressing,
repressing, or completely eliminating the disease. Preventing the
disease involves administering a composition of the present
invention to an animal prior to onset of the disease. Suppressing
the disease involves administering a composition of the present
invention to an animal after induction of the disease but before
its clinical appearance. Repressing the disease involves
administering a composition of the present invention to an animal
after clinical appearance of the disease.
2. Isolation of Cross-Reactive T Cells
[0052] T cells may be activated and expanded in cell culture by
incubation with an antigen target and optionally antigen presenting
cells. Once activated, T cells undergo a complex cascade of cell
signaling which leads to the transcription and expression of many
gene products. The invention described herein takes advantage of
gene products specific for activated T cells for the identification
and isolation of T cells with desired antigen specificity.
[0053] In a first aspect, the present invention is directed to a
method for isolating a T cell that cross-reacts with a self-antigen
and a foreign antigen. A sample comprising T cells is incubated
with an antigen that comprises an epitope present in the
self-antigen and the foreign antigen, thereby causing the
activation of a T cell that cross-reacts with the self-antigen and
the foreign antigen. The sample may be incubated with antigen under
conditions as described in U.S. patent Ser. No. 09/952,532 (Zhang)
and U.S. Provisional Patent No. 60/405,521 (Zhang), which are
hereby incorporated by reference in their entirety.
[0054] A cross-reactive T cell may then be isolated by cloning or
direct expansion as described in U.S. patent Ser. No. 09/952,532
(Zhang). A cross-reactive T cell may also be isolated by selecting
for T cells that express gene products of T cells activated as
described in U.S. Provisional Patent No. 60/405,521 (Zhang).
Isolated cross-reactive T cells may have a homogeneous or
heterogeneous pattern of V.beta.-D.beta.-J.beta. gene usage. The
isolated cross-reactive T cells may be enriched by at least 90%,
95%, 98% or 99.5% from whole blood.
[0055] T cells comprising a homogenous pattern of
V.beta.-D.beta.-J.beta. gene usage may be used to formulate a
monoclonal T cell vaccine as described in U.S. Provisional Patent
No. 60/405,521 (Zhang). T cells comprising a heterogeneous pattern
of V.beta.-D.beta.-J.beta. gene usage may be used to formulate a
polyclonal T cell vaccine as described in U.S. Provisional Patent
No. 60/405,521 (Zhang), which may prevent epitope spreading in
vaccinated patients.
[0056] The self-antigen may be myelin basic protein or a fragment,
variant, analog, homolog or derivative thereof. The self-antigen
may also be a fragment of MBP of between 4 and 50 amino acids that
comprises residues 96-102 of myelin basic protein or a fragment,
variant, analog, homolog or derivative thereof. The fragment of MBP
may be 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 amino
acids. The self-antigen may also be a fragment of MBP comprising
amino acids 93-105 or a fragment, variant, analog, homolog or
derivative thereof. The self-antigen may also be a peptide
consisting of amino acids 93-105 of MBP or a fragment, variant,
analog, homolog or derivative thereof.
[0057] The foreign antigen may be human herpesvirus-6 U24 or a
fragment, variant, analog, homolog or derivative thereof. The
foreign may also be a fragment of HHV-6 (U24) of between 4 and 50
amino acids that comprises residues 4-10 of HHV-6 (U24) or a
fragment, variant, analog, homolog or derivative thereof. The
fragment of HHV-6 (U24) may be 4, 6, 8, 10, 12, 15, 20, 25, 30, 35,
40, 45 or 50 amino acids. The foreign antigen may also be a
fragment of HHV-6 (U24) comprising amino acids 1-13 or a fragment,
variant, analog, homolog or derivative thereof. The foreign antigen
may also be a peptide consisting of amino acids 1-13 of HHV-6 (U24)
or a fragment, variant, analog, homolog or derivative thereof.
[0058] HHV-6 has predominant tropism for CD4.sup.+ T cells. It is
the etiologic agent for infantile exanthem. Common to other
herpesviruses, latency of HHV-6 is established after primary
infection and its genomic material can be found in T cells of
healthy adults. HHV-6 infection can re-activate under certain
conditions, such as in immuno-compromised patients. Expression of
HHV-6 virion proteins has been found in oligodendrocytes obtained
from patients with MS. More recent studies have demonstrated higher
titers of antibodies to HHV-6 and cell-free DNA of HHV-6 in sera
and cerebrospinal fluid of MS patients, suggesting re-activation of
HHV-6 in MS while other studies have reported conflicting results.
Interestingly, a viral protein of both variant A and variant B of
HHV-6, designated U24, shares significant amino acid sequence
homology with MBP. There is a stretch of 7 identical amino acids
within residues 4-10 of HHV-6 U24 antigen and residues 96-102 of
human MBP, raising the possibility that MBP-reactive T cells may be
susceptible to activation by the HHV-6 viral antigen sharing the
sequence homology.
[0059] The cross-reactive T cells may be present in any sample
comprising mononuclear cells. The sample may be isolated from the
peripheral blood or cerebral spinal fluid of an MS patient or from
the synovial fluid of a RA patient. T cells from patients with
other autoimmune diseases may be similarly isolated from peripheral
blood and/or tissues involved with the disease. Mononuclear cells
may be enriched in the sample by using centrifugation techniques
known to those in the art including, but not limited to,
Ficoll.RTM. gradients.
[0060] In a second aspect, the present invention is directed to a T
cell that cross-reacts with a self-antigen and foreign antigen
isolated by the method of the first aspect of the present
invention.
[0061] In a third aspect, the present invention is directed to a
method for isolating a T cell that cross-reacts with a self-antigen
and a foreign antigen together with T cells that are specific for
one or more antigens of interest. Similar to the method of the
first aspect of the present invention, a sample comprising T cells
is incubated with an antigen that comprises an epitope present in
the self-antigen and the foreign antigen together with with one or
more other antigens, thereby causing the activation of a T cell
that cross-reacts with the self-antigen and the foreign antigen and
the activation of T cells specific for the one or more other
antigens.
[0062] The one or more other antigens may be autoantigens. The
autoantigen may be myelin basic protein (MBP), proteolipid protein
(PLP), myelin oligodendrocyte glycoprotein (MOG), or a fragment
thereof. The autoantigen may also be an immunodominant fragment
including, but not limited to, residues 83-99 or residue 151-170 of
MBP. The one or more other antigens may also be any immunogen which
is capable of eliciting an immune response to MBP, PLP, MOG, or a
fragment and/or derivative thereof. When the one or more other
antigens are an autoantigen, the activated T cells may be
autoreactive T cells.
[0063] The T cells may have a heterogeneous pattern of
V.beta.-D.beta.-J.beta. gene usage that express different TCRs
which are each specific for the self-antigen or the antigen of
interest. The T cells may also have a heterogeneous pattern of
V.beta.-D.beta.-J.beta. gene usage that express different TCRs
which are specific for more than one antigen of interest. T cells
comprising a heterogeneous pattern of V.beta.-D.beta.-J.beta. gene
usage may be used to formulate a polyclonal T cell vaccine as
described in U.S. Provisional Patent No. 60/405,521 (Zhang), which
may prevent epitope spreading in vaccinated patients.
[0064] In a fourth aspect, the present invention is directed to a T
cell that cross-reacts with a self-antigen and foreign antigen
together with T cells that are specific for one or more antigens of
interest isolated by the method of the third aspect of the present
invention.
3. Quantifying the Number of Antigen-Specific T cells
[0065] In a fifth aspect, the present invention is directed to a
method of determining the relative frequency of cross-reactive T
cells in a sample by determining the number of T cells isolated by
the method of the first or third aspects of the present
invention.
4. Diagnosing an Autoimmune Disease
[0066] In a sixth aspect of the present invention, a patient with
an autoimmune disease may be diagnosed by obtaining a sample from a
patient and isolating cross-reactive T cells by the method of the
first or third aspects of the present invention. The autoimmune
disease may be diagnosed by comparing the level of cross-reactive T
cells and optionally other autoreactive T cells in a patient to a
control. The level of cross-reactive T cells may be determined in
accordance with the method of the fifth aspect of the present
invention.
5. Monitoring the Progress of an Autoimmune Disease
[0067] In a seventh aspect of the present invention, an autoimmune
disease may be monitored by determining the frequency of
cross-reactive T cells and optionally other autoreactive T cells in
a sample from a patient with an autoimmune disease in accordance
with the fifth aspect of the present invention. The severity of
symptoms of the autoimmune disease may correlate with the number of
cross-reactive T cells and optionally other autoreactive T cells.
In addition, an increase in the number of cross-reactive T cells
and optionally other autoreactive T cells in the sample may be used
as an indication to apply treatments intended to minimize the
severity of the symptoms and/or treat the disease before the
symptoms appear.
6. Producing a Vaccine for the Treatment of an Autoimmune
Disease
[0068] In an eighth aspect of the present invention, a composition
may be produced for treating an autoimmune disease by
inactivating-cross-reactive T cells and optionally other
autoreactive T cells which have been isolated (and optionally
expanded in culture as described herein) by the method of the first
or third aspects of the present invention. The cross-reactive T
cells and optionally other autoreactive T cells may be inactivated
using a number of techniques known to those in the art including,
but not limited to, chemical inactivation or irradiation. The
cross-reactive T cells and optionally other autoreactive T cells
may be preserved either before or after inactivation using a number
of techniques known to those in the art including, but not limited
to, cryopreservation. As described below, the composition may be
used as a vaccine to deplete cross-reactive T cells and optionally
other autoreactive T cells in autoimmune patients.
[0069] The composition may be a pharmaceutical composition, which
may be produced using methods well known in the art. Pharmaceutical
compositions used as preclinical and clinical therapeutics in the
treatment of disease or disorders may be produced by those of
skill, employing accepted principles of diagnosis and treatment.
Such principles are known in the art, and are set forth, for
example, in Braunwald et al., eds., Harrison's Principles of
Internal Medicine, 11th Ed., McGraw-Hill, publisher, New York, N.Y.
(1987), which is hereby incorporated by reference in its entirety.
The pharmaceutical composition may be administered to any animal
which may experience the beneficial effects of the composition.
Animals receiving the pharmaceutical composition may be humans or
other mammals.
[0070] a. Vaccine
[0071] In a ninth aspect, the present invention is drawn to a
composition produced by the method of the eighth aspect of the
present invention. The composition may be a vaccine, which may be
used to deplete cross-reactive T cells and optionally autoreactive
T cells in autoimmune patients.
7. Treatment of an Autoimmune Disease
[0072] In a tenth aspect, an autoimmune disease may be treated in
patients with cross-reactive T cells and optionally autoreactive T
cells by administering a composition according to the ninth aspect
of the present invention. The composition may be a vaccine, which
may lead to the depletion of cross-reactive T cells and optionally
autoreactive T cells in the patient.
[0073] A vaccine may comprise autoreactive T cells comprising
homogeneous ("monoclonal") or heterogeneous ("polyclonal") patterns
of V.beta.-D.beta.-J.beta. gene usage. Clinical studies indicate
that autoimmune patients receiving autologous monoclonal T cell
vaccination may show a gradual decline in the immunity against
myelin-reactive T cells. In some cases, the reappearing
autoreactive T cells may originate from different clonal
populations, suggesting that myelin-reactive T cells may undergo
clonal shift or epitope spreading potentially associated with the
ongoing disease process. Clonal shift or epitope spreading may be a
problem in autoimmune diseases mediated by autoreactive T cells. A
vaccine comprising polyclonal autoreactive T cells capable of
depleting multiple populations of autoreactive T cells may avoid
problems with clonal shift or epitope spreading.
[0074] The composition may be a pharmaceutical composition, which
is administered by any means that achieve their intended purpose.
For example, administration may be by parenteral, subcutaneous,
intravenous, intraarterial, intradermal, intramuscular,
intraperitoneal, transdermal, transmucosal, intracerebral,
intrathecal, or intraventricular routes. Alternatively, or
concurrently, administration may be by the oral route. The
pharmaceutical compositions may be administered parenterally by
bolus injection or by gradual perfusion over time.
[0075] The dosage administered may be dependent upon the age, sex,
health, and weight of the recipient, kind of concurrent treatment,
if any, frequency of treatment, and the nature of the effect
desired. The dose ranges for the administration of the
pharmaceutical compositions may be large enough to produce the
desired effect, whereby, for example, autoreactive T cells are
depleted, as measured by the seventh aspect of the present
invention, is achieved, and the autoimmune disease is significantly
prevented, suppressed, or treated. The doses may not be so large as
to cause adverse side effects, such as unwanted cross reactions,
generalized immunosuppression, anaphylactic reactions and the
like.
[0076] The pharmaceutical compositions may further comprise
suitable pharmaceutically acceptable carriers comprising excipients
and auxiliaries which may facilitate processing of the active
compositions into preparations which can be used pharmaceutically.
Additives to the pharmaceutical compositions may include the
inclusion of an adjuvant, such as alum, chitosan, or other
adjuvants known in the art. (See, for example, Warren et al., Ann.
Rev. Immunol. 4:369-388 (1986); Chedid, L., Feder. Proc.
45:2531-2560 (1986), which are hereby incorporated by reference in
their entirety). The pharmaceutical compositions may also further
comprise liposomes to enhance delivery or bioactivity, using
methods and compounds known in the art.
[0077] Suitable formulations for parenteral administration include
aqueous solutions of the inactivated autoreactive T cells, for
example, water-soluble salts in aqueous solution. In addition, oil
suspensions comprising inactivated autoreactive T cells may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. The suspension
may also contain stabilizers.
[0078] The inactivated cross-reactive T cells and optionally
autoreactive T cells may be formulated using conventional
pharmaceutically acceptable parenteral vehicles for administration
by injection. These vehicles may be nontoxic and therapeutic, and a
number of formulations are set forth in Remington's Pharmaceutical
Sciences, (supra). Nonlimiting examples of excipients are water,
saline, Ringer's solution, dextrose solution and Hank's balanced
salt solution. Pharmaceutical compositions may also contain minor
amounts of additives such as substances that maintain isotonicity,
physiological pH, and stability.
[0079] The inactivated cross-reactive T cells and optionally
autoreactive T cells may be formulated at total cell concentrations
including from about 5.times.10.sup.2 cells/ml to about
1.times.10.sup.9 cells/ml. Preferred doses of the inactivated
autoreactive T cells for use in preventing, suppressing, or
treating an autoimmune disease may be in the range of about
2.times.10.sup.6 cells to about 9.times.10.sup.7 cells.
8. Determination of TCR Repertoire
[0080] In an eleventh aspect, the present invention is drawn to a
method of determining the repertoire of nucleic acids encoding one
or more T cell receptors, or a portion thereof, in an autoimmune
patient by amplifying nucleic acids encoding one or more T cell
receptors from T cells isolated by the first or third aspects of
the present invention, wherein said amplification is performed
using a primer pair. The first primer of the primer pair may be an
oligonucleotide of about 15 to 30 nucleotides in length that
hybridizes to a nucleic acid comprising the variable region of the
TCR gene. The second primer of the primer pair may be an
oligonucleotide of about 15 to 30 nucleotides in length that
hybridizes to a nucleic acid comprising the constant region of the
TCR gene. The primer pair may be used to amplify a nucleic acid
that hybridizes to the V.beta.-D.beta.-J.beta. region of the TCR
gene.
[0081] Nucleic acids encoding one or more T cell receptors from T
cells (the "Target Sequence") or a fragment thereof may be
amplified from a sample by the polymerase chain reaction (PCR)
using any particular PCR technique or equipment known in the art.
For example, PCR amplification may follow a procedure wherein a
reaction mixture is prepared that contains the following
ingredients: 5 .mu.L 10.times.PCR buffer II (100 mM Tris-HCl, pH
8.3, 500 mM KCl), 3 .mu.L 25 mM MgCl.sub.2, 1 .mu.L 10 mM DNTP mix,
0.3 .mu.L Taq polymerase (5 U/.mu.L) (AmpliTaq Gold, Perkin Elmer,
Norwalk, Conn.), 30 pmol of a first primer, 30 pmol of a second
primer, and 1 .mu.L of sample DNA. The polymerase may be stable at
temperatures of at least 95.degree. C., have a processivity of
50-60 and have an extension rate of greater than 50 nucleotides per
minute.
[0082] The PCR reaction may be performed with an amplification
profile of 1 min at 95.degree. C. (denaturation), 20 sec at
56.degree. C. (annealing), and 40 sec at 72.degree. C. (extension)
for a total of 40 cycles. Before the first cycle begins, the
reaction mixture may undergo an initial denaturation for a period
of about 5 nmin to 15 min. Similarly, after the final cycle is
complete, the reaction mixture may undergo a final extension for a
period of about 5 min to 10 min. Certain PCR reactions may work
with as few as 15 to 20 cycles or as many as 50 cycles. Depending
upon the specific PCR reaction, longer or shorter incubation times
and higher or lower temperatures for each step of the amplification
profile may be used.
[0083] The sample comprising the Target Sequence, may be a nucleic
acid, such as genomic DNA, cDNA, DNA previously amplified by PCR,
or any other form of DNA. The sample may be isolated, directly or
indirectly, from any animal or human tissue comprising T cells,
such as peripheral blood mononuclear cells (PBMC). Genomic DNA may
be isolated directly from a tissue comprising T cells. cDNA may be
isolated indirectly by reverse transcription of mRNA directly
isolated from a tissue comprising T cells.
[0084] The ability to detect the Target Sequence may be enhanced by
isolating the sample DNA indirectly by amplification of genomic
DNA, cDNA, or any other form of DNA, by a two-step PCR reaction.
For example, a first PCR amplification reaction may be performed to
amplify a preliminary fragment that is larger than, and comprises,
a fragment to which the first and second primers are capable of
selectively binding on opposite strands. A second PCR amplification
reaction may then be performed, using the preliminary fragment as a
template with the first and second primers, to amplify a fragment
comprising the Target Sequence. If either the first or second
primer is used in the first PCR reaction to amplify the preliminary
fragment, the second PCR reaction is "semi-nested." If neither the
first or second primer is used in the first PCR reaction to amplify
the preliminary fragment, the second PCR reaction is "nested."
[0085] In an exemplary two-step PCR reaction, one or more nucleic
acids encoding one or more T cell receptors from T cells may be
amplified by performing a first PCR reaction using a first
preliminary primer that anneals to the V.beta. region of the TCR
gene and a second preliminary primer that anneals to the C.beta.
region of the TCR gene, which amplifies a preliminary fragment that
extends from V.beta. through the V.beta.-D.beta.-J.beta. junction
to C.beta., followed by a second PCR reaction which may be nested
or semi-nested. In light of the present disclosure, the skilled
artisan will be able to select appropriate primers and reaction
conditions for PCR amplification of the Target Sequence.
[0086] After amplification of the Target Sequence, the amplified
product may be detected by a number of procedures. For example, an
aliquot of amplification product may be loaded onto an
electrophoresis gel, to which an electric field is applied to
separate DNA molecules by size. In another method, an aliquot of
amplification product may be loaded onto a gel stained with SYBR
green, ethidium bromide, or another molecule that will bind to DNA
and emit a detectable signal. A dried gel may contain a labeled
oligonucleotide that hybridizes to the Target Sequence, from which
an autoradiograph may be taken by exposing the gel to film.
[0087] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
9. Peptides
[0088] In a twelfth aspect, the present invention is directed to a
peptide comprising about 4 to 50 amino acids in length, comprising
at least 4 contiguous amino acids of the V-D-J sequences shown on
Table 2 (SEQ ID NOS: 1-6), or sequences derived therefrom. The
peptide may also be about 4 to 12 amino acids in length, comprising
either 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous amino acids of SEQ
ID NOS: 1-6, or sequences derived therefrom. The peptide may also
be about 4 to 20 amino acids in length, comprising sequence from
SEQ ID NOS: 1-6, or sequences derived therefrom. The peptide may be
natural, synthetic, or derived therefrom.
[0089] The peptide may be used as an antigen to elicit an immune
response to a peptide or polypeptide comprising sequence from SEQ
ID NOS: 1-6, or sequences derived therefrom. As described in U.S.
Provisional Patent No. 60/386,287 an immune response elicited by
the peptide may be used as a basis for the production of a vaccine,
the prevention and treatment of an autoimmune disease, the
production of antibodies, the purification of antibodies, and in
diagnostic assays.
10. Prevention of MS
[0090] In a thirteenth aspect, an autoimmune disease such as MS may
be prevented in patients infected with HHV-6 by administering an
antiviral agent that eliminates or reduces reactivation of latent
HHV-6 and optionally the composition comprising autoreactive T
cells produced by the method of the eighth aspect of the present
invention or a composition comprising one or more peptides of the
twelfth aspect of the present invention, or a combination thereof.
Antiviral agents are well known in the art, including nucleoside
analogs and interferon.
EXAMPLE 1
Precursor Frequency of Cross-Reactive T Cells in MS Patients
[0091] Twelve MS patients were included in the study. All patients
were characterized as having relapsing-remitting or secondary
progressive MS for more than two years. They were not treated with
immunosuppressive agents or immunomodulatory agents (e.g.
beta-interferon or Glatiramer Acetate) for at least 3 months prior
to the enrollment in the study and throughout the study. Informed
consent was obtained from the patients after explaining the
experimental procedures. Eleven asymptomatic healthy volunteers
were included as control subjects. As has been previously reported,
a higher incidence of serum cell-free HHV-6 viral DNA was detected
in this group of MS patients than that in healthy controls (Table
1).
[0092] The T cell responses to two synthetic peptides containing an
identical core sequence of seven amino acids was measured to
estimate the precursor frequency of cross-reactive T cells in PBMC.
Two 13-mer peptides corresponding to residues 1-13 of HHV-6 U24 and
residues 93-105 of MBP were synthesized and purified to greater
than 95%. The MBP.sub.93-105 and HHV-6 (U24).sub.1-13 peptides
contain the identical core sequence of 7 amino acids with distinct
flanking residues (MBP-IVTPRTPPPSQGK and HHV6-MDRPRTPPPSYSE) (SEQ
ID NO: 8 and SEQ ID NO: 9, respectively).
[0093] Peripheral blood mononuclear cells (PBMC) were seeded at
3.times.10.sup.5 cells per well in 96-well U-bottomed plates in the
presence of the peptides of MBP.sub.93-105 or HHV-6 (U24).sub.1-13
at 30 .mu.g/ml or the whole MBP (40 .mu.g/ml), respectively. A
total of 24 wells were plated for each antigen. Seven days later,
the cultures were tested for the reactivity to both peptides using
AIM-V serum free medium (Gibco BRL, Grand Island, N.Y.) alone as a
control. To this end, each culture was split into three identical
aliquots and tested for the reactivity to MBP and the indicated
peptides in duplicate. Two aliquots were tested for reactivity to
peptide MBP.sub.93-105 and peptide HHV-6 (U24).sub.1-13 at the same
concentration of 30 .mu.g/ml, respectively, in the presence of
irradiated (6,000 rads) autologous PBMC (10.sup.5 cells per well)
as a source of antigen-presenting cells (APC). One aliquot served
as a control (medium alone in the presence of APC). The reactivity
of the end cultures was measured after 72 hours by proliferation
assays as described in Zhang et al., "MHC restricted clonotypic
depletion of human myelin basic protein by T cell vaccination,"
Science 261:1451-1454 (1993), which is hereby incorporated by
reference in its entirety.
[0094] According to the reactivity of the resulting T cells, they
were categorized as mono-specific T cells or cross-reactive T
cells. Mono-specific MBP- or HHV-6 reactive T cells were reactive
only to the peptide originally used to prime the cells.
Mono-specific T cells were defined as reactive to the given peptide
when CPM was greater than 1,000 and exceeded CPM of the control
(medium alone) and of the other peptide by at least three-fold.
Cross-reactive T cells were reactive to both peptides after being
originally primed with only one of the peptides. Cross-reactive T
cells were defined as CPM of both peptides exceeded 1,000 and the
control CPM by at least three-fold. The frequency of
peptide-reactive T cells was then estimated by dividing the number
of positive wells by the total number of PBMC seeded in the initial
culture.
[0095] As shown in FIG. 1, although T cells recognizing the whole
MBP occurred at a similar estimated frequency (0.6.times.10.sup.-6
in PBMC) in MS patients and healthy subjects, the estimated
frequency of T cells specific for the two peptides was
significantly higher in MS patients than that of healthy controls
(0.53.times.10.sup.-6 vs. 0.19.times.10.sup.-6 for peptide
MBP.sub.93-105 and 0.7.times.10.sup.-6 vs. 0.32.times.10.sup.-6 for
peptide HIV-6 (U24).sub.1-13, p<0.05). The estimated frequency
of the cross-reactive T cells primed by peptide HHV-6
(U24).sub.1-13 was 0.30.times.10.sup.6 in MS patients, which
represented greater than 50% of all T cells recognizing peptide
MBP.sub.93-105 in the MS cohort (FIG. 1). T cells reactive to both
peptides were also present but at significantly lower precursor
frequency (0.17.times.10.sup.-6) in healthy controls. Similarly,
the cross-reactive T cells were also detected in cultures primed
with peptide MBP.sub.93-105 in MS patients as well as in control
subjects (0.36.times.10.sup.-6 vs. 0.09.times.10.sup.-6,
p<0.05). Consistent with these observations was the increased
frequency of T cells that were initially primed with the whole MBP
and reacted to peptide HHV-6 (U24).sub.1-13 in patients with MS
compared to the control group (0.33.times.10.sup.-6 vs.
0.05.times.10.sup.-6) but the difference was not statistically
significant (p=0.08). Furthermore, there was no correlation between
the detection of HHV-6 DNA and the specific T cell frequencies.
Taken together, the results suggest that a significant fraction of
T cells recognizing the 93-105 region of MBP could be fully
activated to proliferate by peptide HHV-6 (U24).sub.1-13 that
shares the sequence homology with the MBP peptide.
EXAMPLE 2
Cytokine Profile and Phenotypic Expression
[0096] The cytokine profile was determined for mono-specific and
cross-reactive T cells lines derived from the T cells of Example 1.
T cell lines were cloned under limiting dilution conditions in the
presence of PHA (Sigmha, St. Louis Mo.) and irradiated autologous
PBMC as accessory cells. Briefly, T cells were plated out at 0.3
cell per well under limiting dilution conditions and cultured with
10.sup.5 irradiated autologous PBMC and 5 .mu.g/ml PHA. Cultures
were fed with fresh medium containing 50 IU/ml rIL-2 (Boehringer
Mannheim, Indianapolis, Ind.) every 4 days. After approximately
10-12 days, growth-positive wells became visible and were tested in
proliferation assays for proliferative responses to the peptides. A
total of 67 lines were produced from MS patients and 38 lines from
control subjects.
[0097] The cytokine profile of the resulting T cell lines was
determined quantitatively using ELISA kits (PharMingen, San Diego,
Calif.). Microtiter plates (96-wells, NUNC Maxisorp) were coated
overnight at 4.degree. C. with 1 .mu.g/well of a purified mouse
capturing monoclonal antibody to human cytokine (IL-4, IL-10,
TNF-.alpha., .gamma.-IFN) (PharMingen). Plates were washed and
non-specific binding sites were saturated with 10% (w/v) fetal
bovine serum (FBS) for 1 hour and subsequently washed. Supernatants
and cytokine standards were diluted with PBS and added in duplicate
wells. Plates were incubated at 37.degree. C. for 2 hr and
subsequently washed with PBS-T. Matched biotinylated detecting
antibody were added to each well and incubated at room temperature
for 2 hours. After washing, avidin-conjugated horseradish
peroxidase was added and plates were incubated for 1 hour.
3,3',5,5'-tetramethylbenzidine (TMB, Sigma) was used as a substrate
for color development. Optical density was measured at 450 nm using
an ELISA reader (Bio-Rad Laboratories, Hercules, Calif.) and
cytokine concentrations were quantitated by Microplate computer
software (Bio-rad) using a double eight-point standard curve.
[0098] As illustrated in FIG. 2, overall the T cell lines examined
were predominantly of Th1 phenotype, producing predominantly
.gamma.-IFN, various amounts of TNF-.alpha. but not IL-4 and IL-10,
regardless of their peptide specificity. However, MS-derived T cell
lines specific for peptide MBP.sub.93-105 as well as the
cross-reactive T cell lines initially primed by peptide
MBP.sub.93-105 differed considerably in the production of
.gamma.-IFN and TNF-.alpha. from those generated from control
subjects. In general, the cross-reactive T cell lines exhibited
similar cytokine profile as those specific for the same peptide by
which the cross-reactive T cell lines were initially primed (FIG.
2B vs. 2A and FIG. 2D vs. 2C). All T cell lines obtained expressed
the CD4 phenotype as determined by flow cytometry (data not
shown).
[0099] It is somewhat contradictory that MBP.sub.93-105 specific T
cells derived from MS patients produce less Th1 cytokines than
those from healthy controls. One possibility is that although
patients studied here did not receive any treatment 3 months prior
to the study, some T cell lines were derived from patients that had
previous treatment with beta-interferon (more than 5-9 months prior
to the study) and their cytokine profile may be altered as a
result. Beta-interferon has been shown to have a regulatory effect
on the cytokine profile of T cells and the effect may be sustained
after the termination of the treatment.
EXAMPLE 3
Reactivity Pattern and TCR V Gene Usage of Cross-Reactive T Cell
Clones
[0100] To confirm that the observed reactivity of the T cells
recognizing both peptides of HHV-6 (U24).sub.1-13 and
MBP.sub.93-105 results from monoclonal T cell populations,
cross-reactive T cell lines and mono-specific T cells lines
described in Example 2 were characterized for their reactivity
pattern to the individual peptides. As shown in FIG. 3, the T cell
clones could be categorized, according to their reactivity to the
peptides, into mono-specific T cell clones and cross-reactive T
cell clones. The reactivity of the T cell clones was consistent
with that of the parental T cell lines from which the T cell clones
were generated.
[0101] The T cell clones were subsequently expanded in culture in
an attempt to analyze the TCR V gene usage and CDR3 sequence.
Unfortunately, the majority of the clones (18/24 clones) were lost
during the process of expansion due to prolonged equipment/electric
power failure and closing of the facility as a result of severe
flood in the Houston area during the course of the study and only
six clones survived.
[0102] Total cellular RNA was extracted from the six remaining
independent T cell clones using RNeasy mini kit (QIAGEN, CA). TCR
.beta. chains were amplified by PCR with a set of V.beta.-specific
primers as by Wucherpfennig et al., "Shared human T cell receptor
V.beta. usage to immunodominant regions of myelin basic protein,"
Science 248:1016-1019 (1990) and Zang et al., "Restricted TCR
V.alpha. gene rearrangements in T cells recognizing an
immunodominant peptide of myelin basic protein in DR2 patients with
multiple sclerosis," Intern Immunol 10:991-998 (1998), which are
hereby incorporated by reference in their entirety. Briefly, RNA
was reverse transcribed to first-strand cDNA using an Oligo-dT
primer and the superscript pre-amplification system (Gibco, Md.).
cDNA was amplified in a standard PCR using a set of primers
specific for 24 V.beta. gene families in combination with C.beta.
primer, respectively. For each PCR experiment, C.beta. gene was
amplified simultaneously to control the integrity of TCR cDNA. The
amplification profile used was 1 min at 95.degree. C. for
denaturation, 20 sec at 56.degree. C. for annealing, 40 sec at
72.degree. C. for extension in a total of 35 cycles. The amplified
PCR products were separated on a 1% agarose gel by electrophoresis
and stained with ethidium bromide. The purified PCR products were
directly sequenced with the 17 sequencing kit (Pharmacia, Uppsala,
Sweden).sup.44.
[0103] As shown in Table 2, the six clones (including five
cross-reactive T cell clones) examined were found to express single
TCR BV genes and V-D-J junctional sequences, confirming
monoclonality of the T cell clones.
EXAMPLE 4
Serum Antibody Titers and the Reactivity Pattern to HHV-6 and MBP
Peptides
[0104] We furter addressed whether the increased T cell responses
to both peptides of HHV-6 (U24).sub.1-13 and MBP.sub.93-105 were
consistent with potential B cell sensitization to the peptides in
MS patients as compared to healthy controls. To this end, serum
specimens freshly obtained from the same MS patients and the
control subjects were examined for specific antibody reactivity to
the peptides by ELISA.
[0105] Antibody titers were determined in serum specimens by ELISA.
Briefly, microtiter plates were coated overnight at 4.degree. C.
with the peptides at 5 .mu.g/ml in a carbonate buffer (100 mM, pH
9.5). An irrelevant peptide and MBP.sub.41-59 were used as
reference peptides. Non-specific binding sites were saturated with
FBS-PBS for 1 hour and washed subsequently with PBS-T. Serum
samples were prepared in serial dilutions (1:50, 1:00, 1:500) with
FBS-PBS and 100 .mu.l of each sample were added in duplicate wells.
The rest of the procedure is the same as that described above.
[0106] As illustrated in FIG. 4, the results showed that the
antibody titers for both peptide HHV-6 (U24).sub.1-13 and peptide
MBP.sub.93-105 were elevated at the serum dilutions of 1:50 and
1:100 in MS patients as compared to those detected in control
subjects.
EXAMPLE 5
Vaccination of MS Patient Using Autologous Cross-Reactive T
Cells
[0107] The observation in Example 1 that the precursor frequency of
cross-reactive T cells is significantly elevated in MS patients
suggests that these T cells are sensitized in vivo in patients with
MS. The possibility that the immune system is sensitized to the
peptides in MS patients is further strengthened by the increase in
antibody titers for both peptides in serum specimens obtained from
the MS cohort. The findings are consistent with a number of recent
reports indicating the increased replication of HHV-6 in MS
patients as evident by more frequent detection of elevated
cell-free viral DNA of HHV-6 in serum and cerebrospinal fluid
specimens obtained from MS patients. It is likely that active
replication of HHV-6 may be responsible for the observed
sensitization of the immune system to the 93-105 region of MBP in
MS as peptide MBP.sub.93-105 shares an identical sequence with
peptide HHV-6 (U24).sub.1-13.
[0108] Although the T cell recognition of MBP in MS patients is
relatively heterogeneous, some regions of MBP have immunodominant
properties, such as the 84-102/83-99 region and the 151-170 region
of MBP. The immunodominant properties of these regions may be
associated with their binding affinity to HLA-DR (DRB1*1501)
preferentially expressed in the MS population. It has been
demonstrated that peptides corresponding to both the 84-103 and the
144-163 regions have the highest binding affinity to DRB1*1501.
Although the 93-105 region of MBP homologues and the 1-13 region of
HHV-6 (U24) have an overlap of 4 amino acids (PRTP) (SEQ ID NO:11)
with the 83-99 immunodominant region of MBP, the PRTP sequence
alone is not sufficient to form an individual T cell epitope and is
unlikely to represent the immunodominant T cell epitopes. Indeed,
additional experiments revealed no cross-reactivity between the T
cell responses to the MBP.sub.83-99 peptide and the MBP.sub.93-105
peptide when four independent MBP.sub.83-99 specific T cell clones
and six MBP.sub.93-.sub.105 specific T cell clones were examined
(data not shown). However, the possibility exists that the
increased T cell sensitization to MBP in MS patients may be
initially triggered by the HHV-6 antigen that shares the sequence
homology with MBP.sub.93-105 and later spreads to other epitopes of
MBP by the mechanism known as epitope spreading. As the
immunodominant properties of the T cell epitopes are preferentially
associated with the HLA-DR molecules expressed in MS patients
regardless of the sequence of stimulation events, the triggering
epiotpe(s) of MBP, such as MBP.sub.93-105 that does not have high
binding affinity, may not manifest itself as an immunodominant
epitope when the disease is established.
[0109] The findings suggest that the cross-reactive T cells
recognizing both MBP and HHV-6 peptides represent a significant
subset of T cells with some degree of TCR degeneracy. It appears
that the recognition of the cross-reactive T cells has a less
stringent requirement for the flanking residues of the two
peptides. In contrast, these flanking residues are critical for the
T cell recognition of mono-specific T cells. Similar findings have
also been described for the T cell recognition of the 83-99 peptide
of MBP, which demonstrated that the different role of the central
versus flanking residues, allowing some degree of promiscuity in T
cell recognition. The results described herein provide new evidence
indicating the association between HHV-6 and autoreactive immune
responses to MBP, which has particular importance and clinical
relevance because of the potential involvement of the two agents,
as a candidate myelin autoantigen and a suspected etiologic agent,
in the pathogenesis of MS. The results herein indicate that MS may
be prevented or treated in patients infected with HHV-6 by
administering antiviral agents which eliminate or reduce
reactivation of latent HHV-6 and optionally inactivated
autoreactive or cross-reactive T cells or peptides corresponding to
the V-D-J region of autoreactive or cross-reactive T cells, or a
combination thereof. TABLE-US-00001 TABLE 1 Clinical
characteristics and demographic information, serum cell-free HHV-6
DNA and HLA types of MS patients and control subjects Type of
Disease Serum cell-free Subjects Age Sex MS Duration (yrs.) EDSS
HHV-6 DNA HLA-DR genotypes MS-1 44 M RR 5 3.0 - B1*1501; B1*1301;
B3*0101; B5*0101 MS-2 38 F RR 2 1.0 + B1*0405; B1*1301; B3*0101;
B4*0103 MS-3 46 F RR 4 3.0 + B1*1501; B1*0405; B5*0101; B4*0103
MS-4 47 F SP 7 7.0 + B1*1501; B1*1502; B5*0101; B5*0102 MS-5 45 F
RR 4 1.5 + B1*5101; B1*0404; B5*0101; B4*0131 MS-6 60 F SP 11 6.6 -
B1*1301; B1*0802; B3*0101 MS-7 51 F SP 13 3.5 + B1*0103; B1*0802
MS-8 57 F RR 6 1.5 + B1*1501; B1*0404; B5*0101; B4*0131 MS-9 46 F
RR 6 1.5 + B1*1501; B1*1104; B3*0202; B5*0101 MS-10 41 F SP 13 6.0
+ B1*1602; B1*0408; B5*0202; B4*0103 MS-11 31 F RR 4 1.0 + B1*1602;
B1*1303; B3*0101; B5*0202 MS-12 50 F SP 28 7.0 + B1*1101; B1*0701;
B3*0202; B4*0101 NS-1 47 M -- -- -- - B1*1501; B1*0701; B5*0101;
B4*0103 NS-2 49 F -- -- -- - B1*0102; B1*0405; B4*0131 NS-3 35 M --
-- -- - B1*1503; B1*1301; B3*0101 NS-4 32 F -- -- -- + B1*0103;
B1*1402; B3*0101 NS-5 46 F -- -- -- + B1*1101; B1*0701; B3*0202;
B4*0101 NS-6 60 F -- -- -- + B1*1501; B1*0404; B5*0101; B4*0131
NS-7 33 F -- -- -- - B1*0103; B1*1104; B3*0202 NS-8 34 F -- -- -- +
B1*1503; B1*1301; B3*0101; B5*0101 NS-9 43 F -- -- -- + B1*0101;
B1*0405; B4*0131 NS-10 46 F -- -- -- - B1*0301; B1*0802; B3*0101
NS-11 30 F -- -- -- + B1*1501; B1*1402; B3*0101 RR,
relapsing-remitting MS; SP, secondary progressive MS. EDSS,
expanded disability scales score. Cell-free viral DNA for HHV-6 was
detected in serum specimens derived from the subjects using nested
PCR and Southern hybridization with specific primers and
probes.sup.33. The results are expressed as detectable (+) (>5
DNA copies/.mu.l) and undetectable (-).
[0110] TABLE-US-00002 TABLE 2 TCR VDJ region sequence of the
cross-reactive T cell clones SEQ ID NO: Clone BV V-D-J BC 1
MS11C4-1 BV5 YLCASSLVRDSGYTFGSGTRLTVV EDLNK 2 MS11B6-1 BV22
YFCASSENRASYNEQFFGPGTRLTVL EDLKN 3 MS2E1-1 BV13
YFCASSLGRLINSPLHFGNGTRLTVT EDLNK 4 MS2A7-1 BV12
YFCAISEDGNYGYTFGSGTRLTVV EDLNK 5 MS2-E3-1 BV8
YFCASSLRAGGYQYGYTFGSGTRLTVV EDLNK 6 MS9-A5-1 BV2
FYICSASLGMGDIQYFGAGTRLSVL EDLKN
[0111]
Sequence CWU 1
1
11 1 24 PRT Homo sapiens 1 Tyr Leu Cys Ala Ser Ser Leu Val Arg Asp
Ser Gly Tyr Thr Phe Gly 1 5 10 15 Ser Gly Thr Arg Leu Thr Val Val
20 2 26 PRT Homo sapiens 2 Tyr Phe Cys Ala Ser Ser Glu Asn Arg Ala
Ser Tyr Asn Glu Gln Phe 1 5 10 15 Phe Gly Pro Gly Thr Arg Leu Thr
Val Leu 20 25 3 26 PRT Homo sapiens 3 Tyr Phe Cys Ala Ser Ser Leu
Gly Arg Leu Ile Asn Ser Pro Leu His 1 5 10 15 Phe Gly Asn Gly Thr
Arg Leu Thr Val Thr 20 25 4 24 PRT Homo sapiens 4 Tyr Phe Cys Ala
Ile Ser Glu Asp Gly Asn Tyr Gly Tyr Thr Phe Gly 1 5 10 15 Ser Gly
Thr Arg Leu Thr Val Val 20 5 27 PRT Homo sapiens 5 Tyr Phe Cys Ala
Ser Ser Leu Arg Ala Gly Gly Tyr Gln Tyr Gly Tyr 1 5 10 15 Thr Phe
Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 6 25 PRT Homo sapiens 6
Phe Tyr Ile Cys Ser Ala Ser Leu Gly Met Gly Asp Ile Gln Tyr Phe 1 5
10 15 Gly Ala Gly Thr Arg Leu Ser Val Leu 20 25 7 15 PRT Homo
sapiens 7 Ala Ser Ser Glu Asn Arg Ala Ser Tyr Asn Glu Gln Phe Phe
Gly 1 5 10 15 8 13 PRT Homo sapiens 8 Ile Val Thr Pro Arg Thr Pro
Pro Pro Ser Gln Gly Lys 1 5 10 9 13 PRT Homo sapiens 9 Met Asp Arg
Pro Arg Thr Pro Pro Pro Ser Tyr Ser Glu 1 5 10 10 8 PRT Homo
sapiens 10 Leu Gly Arg Ala Gly Leu Thr Tyr 1 5 11 4 PRT Homo
sapiens 11 Pro Arg Thr Pro 1 DM_US\8219926.v1 DM_US\8219926.v1
* * * * *