U.S. patent application number 12/711160 was filed with the patent office on 2010-09-23 for isolation and identification of t cells.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Jingwu Z. Zang.
Application Number | 20100239548 12/711160 |
Document ID | / |
Family ID | 31715870 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100239548 |
Kind Code |
A1 |
Zang; Jingwu Z. |
September 23, 2010 |
Isolation and Identification of T Cells
Abstract
The present invention relates to improved autologous T cell
vaccines and improved methods for their production. The invention
is also directed to methods for treating autoimmune diseases such
as multiple sclerosis or rheumatoid arthritis using autologous T
cell vaccines. The invention further directed to the diagnosis of T
associated diseases.
Inventors: |
Zang; Jingwu Z.; (Shanghai,
CN) |
Correspondence
Address: |
ARNOLD & PORTER LLP;ATTN: IP DOCKETING DEPT.
555 TWELFTH STREET, N.W.
WASHINGTON
DC
20004-1206
US
|
Assignee: |
Baylor College of Medicine
Houston
TX
|
Family ID: |
31715870 |
Appl. No.: |
12/711160 |
Filed: |
February 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10524300 |
Aug 29, 2005 |
7695713 |
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PCT/US03/24548 |
Aug 6, 2003 |
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12711160 |
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60402521 |
Aug 8, 2002 |
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Current U.S.
Class: |
424/93.71 ;
435/325; 435/6.14; 435/7.24 |
Current CPC
Class: |
C12N 5/0636 20130101;
A61K 2039/5158 20130101; A61K 39/0008 20130101; A61P 37/02
20180101; G01N 33/564 20130101; A61P 9/00 20180101; A61P 25/28
20180101; A61P 37/04 20180101; A61P 37/00 20180101; G01N 2800/285
20130101; A61K 2035/122 20130101; A61P 25/00 20180101; A61P 37/06
20180101; A61P 19/02 20180101; G01N 33/505 20130101 |
Class at
Publication: |
424/93.71 ;
435/7.24; 435/325; 435/6 |
International
Class: |
A61K 35/12 20060101
A61K035/12; G01N 33/53 20060101 G01N033/53; C12N 5/0783 20100101
C12N005/0783; C12Q 1/68 20060101 C12Q001/68; A61P 37/02 20060101
A61P037/02 |
Claims
1. A method for isolating one or more T cells specific for an
antigen of interest, comprising: (a) incubating a sample comprising
T cells with said antigen or a derivative thereof; (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. and TNF.alpha. IL5 IL-10 and IL-13.
2. The method of claim 1, wherein said antigen is a
self-antigen.
3. The method of claim 2, wherein said self-antigen 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 said antigen is an immunodominant
epitope of a self-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, 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.
7. The method of claim 6, wherein one or more of said antibodies is
fluorescently labeled.
8. The method of claim 7, wherein said T cell is selected by
fluorescent activated cell sorting.
9. The method of claim 6, wherein said first antibody is conjugated
to a magnetic microbead.
10. The method of claim 9, wherein said T cell is selected by
magnetic activated cell sorting
11. The method of claim 1, wherein said antigen is incubated with
said sample for 1 to 7 days.
12. The method of claim 1, wherein said antigen is incubated with
said sample for less than 1 day.
13. The method of claim 12, wherein said antigen is incubated with
said sample for less than 16 hours.
14. The method of claim 13, wherein said antigen is incubated with
said sample for less than 12 hours.
15. The method of claim 14, wherein said antigen is incubated with
said sample for less than 8 hours.
16. The method of claim 15, wherein said antigen is incubated with
said sample for less than 4 hours.
17. The method of claim 16, wherein said antigen is incubated with
said sample for less than 2 hours.
18. A T cell isolated by the method of claim 1.
19. The method of claim 1 wherein said isolated T cells are
T.sub.H1 or T.sub.H2 T cells or a combination thereof.
20. A method for quantifying the number of T cells in a sample,
wherein said T cells are specific for one or more antigens of
interest, comprising: (a) incubating a sample comprising T cells
with said antigen or a derivative thereof; (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. and TNF.alpha. IL5 IL-10 and IL-13; and (c) determining
the number of T cells selected by step (b).
21. A method for diagnosing an autoimmune disease in a patient,
comprising: (a) incubating a sample derived from said patient
comprising T cells with one or more autoantigens involved in said
disease; (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. and TNF.alpha. IL5 IL-10
and IL-13.
22. A method for monitoring an autoimmune disease in a patient,
comprising: (a) incubating a sample derived from said patient
comprising T cells with 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
11-2 IFN.gamma. and TNF.alpha. 115 IL-10 and IL-13; and (c)
determining the number of autoreactive T cells selected by step
(a).
23. A method for treating an autoimmune, disease in a patient,
comprising: (a) incubating a sample derived from said patient
comprising T cells with 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. and TNF.alpha. IL5 IL-10 and IL-13; (c)
inactivating said selected autoreactive T cells; and (d)
administering said autoreactive T cells inactivated by step (b) to
said patient.
24. A method for producing a composition for the treatment of an
autoimmune disease in a patient, comprising: (a) incubating a
sample derived from said patient comprising T cells with 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. and
TNF.alpha. IL5 IL-10 and IL-13; and (c) inactivating said
autoreactive T cells.
25. The method of claim 23 or 24 further comprising expanding the
number autoreactive T cells selected in step (b).
26. A composition for the treatment of a patient with an autoimmune
disease produced by the method of claim 24 or 25.
27. A method for isolating a nucleic acid encoding a T cell
receptor, or a portion thereof, wherein said T cell receptor is
specific for an antigen of interest, comprising: (a) incubating a
sample comprising T cells with said antigen; (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. and TNF.alpha. IL5 IL-10 and IL-13; and (c) amplifying
said nucleic acid encoding a T cell receptor from a T cell isolated
by step (b) 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.
28. A method for isolating one or more nucleic acids encoding one
or more T cell receptors, or a portion thereof, wherein said one or
more T cell receptors are specific for one or more antigens of
interest, comprising: (a) incubating a sample comprising T cells
with said one or more antigens; (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. and TNF.alpha. IL5 IL-10 and IL-13; and (c) amplifying
said one or more nucleic acids encoding one or more T cell
receptors from T cells isolated by step (b) 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.
29. A method for determining the repertoire of nucleic acids
encoding one or more T cell receptors, or a portion thereof, in a
patient, wherein said one or more T cell receptors are specific for
one or more antigens of interest, comprising: (a) incubating a
sample derived from said patient comprising T cells with said one
or more antigens; (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. and
TNF.alpha. IL5 IL-10 and IL-13; (c) amplifying said one or more
nucleic acids encoding one or more T cell receptors from T cells
isolated by step (b) 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; and
(d) determining the nucleotide sequence of said one or more nucleic
acids amplified by step (c).
30. A method for determining the repertoire of nucleic acids
encoding one or more T cell receptors, or a portion thereof, in an
autoimmune patient, wherein said one or more T cell receptors are
specific for one or more autoantigens of interest, comprising: (a)
incubating a sample derived from said autoimmune patient comprising
T cells with said 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. and TNF.alpha. IL5 IL-10 and IL-13; (c) amplifying said
one or more nucleic acids encoding one or more T cell receptors
from T cells isolated by step (b) 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; and (d) determining the nucleotide sequence of said
one or more nucleic acids amplified by step (c).
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
antigen-specific T cells. In addition, the present invention
concerns the use of antigen-specific T cells for the 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 (MHC) 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 .alpha. 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 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 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, TNT.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] Myelin-reactive T cells have also 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.
[0014] 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).
[0015] 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.
[0016] 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 (Mang et al., Science, 1993; 261: 1451-1454,
Medaer et al. Lancet 1995: 346:807-808). Three subcutaneous
inoculations with irradiated MP-reactive T cell clones resulted in
the depletion of circulating MBP-reactive T cells in patients with
MS.
[0017] 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.
[0018] 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., Immunol., 1994; 152:5581-5592, Hong et al.,
J. Immunol., 1999; 163:3530-3538).
[0019] 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.sym. 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.
[0020] 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.
[0021] 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.
SUMMARY OF THE INVENTION
[0022] The present invention is generally directed to methods of
isolating antigen-specific T cells and more particularly T cells
specific for self or autoantigens. The methods for isolating one or
more T cells specific for an antigen of interest generally comprise
incubating a sample comprising T cells obtained from a patient with
said antigen or a derivative thereof; 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, IFNg,
TNF.alpha., IL5, IL-10 and IL-13.
[0023] The methods of the invention are particularly useful for
isolating autoreactive T cells which play a role in the
pathogenesis of autoimmune diseases.
[0024] 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.
[0025] The methods of the invention also allow the preparation of
autologous T cell vaccines for the treatment of T cell related
autoimmune diseases.
[0026] The methods for vaccine preparation generally involve the
isolation of antigen-specific T cells as described above optionally
followed by subsequent culturing steps which allows the expansion
of the population of isolated antigen-specific T cells.
[0027] The invention inter alia is also directed to T cell vaccines
and pharmaceutical compositions comprising antigen-specific T cells
isolated using the methods of the invention.
[0028] The methods of the invention are also useful for
characterizing T cell receptors and their encoding nucleic
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 demonstrates FACS identification of cells expressing
CD69 and .gamma.IFN (top) and CD69 and TNT.alpha. (bottom) in a
multiple sclerosis patient before stimulation (left), after
stimulation with residues 83-99 of MBP (middle), and after
stimulation with residues 83-99 of MBP (right).
[0030] FIG. 2 demonstrates FACS identification of cells expressing
CD69 and .gamma.IFN (top) and CD69 and TNF.alpha. (bottom) in a
healthy control patient before stimulation (left), after
stimulation with residues 83-99 of MBP (middle), and after
stimulation with residues 83-99 of MBP (right).
DETAILED DESCRIPTION
1. Definitions
[0031] To aid in the understanding of the present invention,
several terms are defined below.
[0032] "Autoantigen" or "self-antigen" as used herein refers to an
antigen or epitope which is native to the mammal which is
immunogenic in said mammal disease is conserved in that species of
mammal and which may be involved in the pathogenesis of
autoimmune.
[0033] "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.
[0034] "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.
[0035] "Immunogenic," 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.
[0036] "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.
[0037] "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 incorporated herein by reference. 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.
[0038] A "Peptide" or "polypeptide" is a linked sequence of amino
acids and may be natural, synthetic, or a modification or
combination of natural and synthetic.
[0039] "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.
[0040] "Probe" means an oligonucleotide, whether natural,
synthetic, or a modification thereof, capable of specifically
binding to a sufficiently complementary nucleotide sequence.
[0041] "T cell mediated disease" means a disease arising as a
result of T cells recognizing self-antigens.
[0042] "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 Antigen-Specific T Cells
[0043] a. Isolation of Monoclonal Antigen-Specific T Cells
[0044] T cells may be activated and expanded in cell culture by
incubation with an antigen target and 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.
[0045] In a first aspect, the present invention is directed to a
method for isolating a T cell that is specific for an antigen of
interest. A sample comprising T cells is incubated with a
particular antigen, which causes the activation of a T cell
specific for the antigen of interest. The sample may be incubated
with the antigen for 1 to 7 days. The sample may also be incubated
with the antigen for less than 1 day. The sample may also be
incubated with the antigen for less than 16 hours. The sample may
also be incubated with the antigen for less than 12 hours. The
sample may also be incubated with the antigen for less than 8
hours. The sample may also be incubated with the antigen for less
than 4 hours. The sample may also be incubated with the antigen for
less than 2 hours.
[0046] A T cell specific for the antigen of interest may then be
isolated by selecting for T cells that express gene products of T
cells activated as described above. Subsets of activated T cells
may be isolated by selecting for T cells with subset-specific gene
products or cell surface markers (e.g., CD4 vs. CD8).
[0047] The antigen of interest includes myelin basic protein (MBP),
proteolipid protein (PLP), myelin oligodendrocyte glycoprotein
(MOG), or a fragment thereof. The antigen of interest may also be
an immunodominant fragment including, but not limited to, residues
83-99 or residue 151-170 of MBP. The antigen of interest may also
be a combination of two or more individual antigens of
interest.
[0048] The antigen or a derivative thereof, used to activate the T
cells may be any immunogen which is capable of eliciting an immune
response to the antigen of interest. The activating antigen may be
the antigen of interest, or a derivative thereof. The activating
antigen may be MBP, PLP, MOG, or a fragment and/or derivative
thereof. The activating antigen may also be an immunodominant
fragment including, but not limited to, residues 83-99 or residue
151-170 of P, or a fragment and/or derivative thereof. The
activating antigen may also be a combination of two or more
individual activating antigens. The activating antigen may be used
one or more times to activate T cells specific for an antigen of
interest.
[0049] The 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. T cells may also be enriched in the sample by using
positive selection, negative selection, or a combination thereof
for expression of gene products of T cells.
[0050] The gene product for identifying or negatively selecting for
activated T cells may be a cell surface marker or cytokine, or a
combination thereof. Cell surface markers for identifying activated
T cells include, but are not limited to, CD69, CD4, CD8, CD25,
HLA-DR, CD28, and CD134. CD69 is an early activation marker found
on B and T lymphocytes, NK cells and granulocytes. CD25 is an IL-2
receptor and is a marker for activated T cells and B cells. CD4 is
a TCR coreceptor and is marker for thymocytes, T.sub.H1- and
T.sub.H2-type cells, monocytes, and macrophages. CD8 is also a TCR
coreceptor and is marker for cytotoxic T cells. CD134 is expressed
only in activated CD4.sup.+ T cells.
[0051] Cell surface markers for negatively selecting for activated
T cells include, but are not limited to, CD36, CD40, and CD44. CD28
acts as a stimulatory T-cell activation pathway independent of the
T-cell receptor pathway and is expressed on CD4.sup.+ and CD8.sup.+
cells. CD36 is a membrane glycoprotein and is a marker for
platelets, monocytes and endothelial cells. CD40 is a marker for B
cells, macrophages and dendritic cells. CD44 is a marker for
macrophages and other phagocytic cells. Subsets of T cells may be
isolated by using positive selection, negative selection, or a
combination thereof for expression of cell surface gene products of
helper T cells or cytotoxic T cells (e.g., CD4 vs. CD8).
[0052] Cytokines for identifying activated cells of the present
invention include, but are not limited to cytokines produced by
T.sub.H1-type T cells (cell-mediated response) and T.sub.H2-type T
cells (antibody response). Cytokines for identifying activated
T.sub.H1-type T cells include, but are not limited to, IL-2, gamma
interferon (.gamma.IFN) and tissue necrosis factor alpha
(TNF.alpha.). Cytokines for identifying activated T.sub.H2-type T
cells include, but not limited to, IL-4, IL-5, IL-10 and IL-13.
Subsets of T cells may also be isolated by using positive
selection, negative selection, or a combination thereof for
expression of cytokine gene products of helper T cells or cytotoxic
T cells (e.g., .gamma.IFN vs. IL4).
[0053] An activated T.sub.H1-type T cell specific for an antigen of
interest may be isolated by identifying cells that express CD69,
CD4, CD25, IL-2, IFN.gamma., TNF.alpha., or a combination thereof.
An activated T.sub.H1-type T cell specific for an antigen of
interest may also be isolated by identifying cells that express
CD69 and CD4 together with IFN.gamma. or TNF.alpha.. An activated
T.sub.H2-type T cell specific for an antigen of interest may be
isolated by identifying cells that express CD69, CD4, IL-4, IL-5,
IL-10, IL-13, or a combination thereof. A combination of an
activated T.sub.H1-type T cell and a T.sub.H2-type T cell specific
for an antigen of interest may be isolated by identifying cells
that express CD69, CD4, CD25, IL-2, IFN.gamma., TNF.alpha., or a
combination thereof and cells that express CD69, CD4, IL-4, IL-5,
IL-10, IL-13, or a combination thereof.
[0054] The gene products used for positive or negative selection of
the activated T cells of the present invention may be identified by
immunoselection techniques known to those in the art which utilize
antibodies including, but not limited to, fluorescence activated
cell sorting (FACS), magnetic cell sorting, panning, and
chromatography. Immunoselection of two or more markers on activated
T cells may be performed in one or more steps, wherein each step
positively or negatively selects for one or more markers. When
immunoselection of two or more markers is performed in one step
using FACS, the two or more different antibodies may be labeled
with different fluorophores.
[0055] Magnetic cell sorting may be performed using
super-paramagnetic microbeads composed of iron oxide and a
polysaccharide coat. Preferably the microbeads may be approximately
50 nanometers in diameter, and have a volume about one-millionth
that of a typical mammalian cell. The microbeads are preferably
small enough to remain in colloidal suspension, which permits
rapid, efficient binding to cell surface antigens. The microbeads
preferably do not interfere with flow cytometry, are biodegradable,
and have negligible effects on cellular functions. The antibody
coupling to the microbeads may be direct or indirect, via a second
antibody to a ligand such as fluorescein.
[0056] The antibody may be of classes IgG, IgM, IgA, IgD, and IgE,
or fragments or derivatives thereof, including Fab, F(ab').sub.2,
Fd, and single chain antibodies, diabodies, bispecific antibodies,
bifunctional antibodies and derivatives thereof. The antibody may
be a monoclonal antibody, polyclonal antibody, affinity purified
antibody, or mixtures thereof which exhibits sufficient binding
specificity to an epitope of a gene product specific for activated
T cells, or a sequence derived therefrom. The antibody may also be
a chimeric antibody.
[0057] The antibody may be derivatized by the attachment of one or
more chemical, peptide, or polypeptide moieties known in the art
that allow the identification and/or selection of the activated T
cell to which the antibody is bound. The antibody may be conjugated
with a chemical moiety such as a fluorescent dye. An activated T
cell bound by a fluorescently labeled antibody may be isolated
using techniques including, but not limited to, fluorescence
activated cell sorting (FACS). The antibody may also be conjugated
with a magnetic particle, such as a paramagnetic microbead
(Miltenyi Biotec, Germany). An activated T cell bound by a
magnetically labeled antibody may be isolated using techniques
including, but not limited to, magnetic cell sorting.
[0058] For cell-surface expressed gene products, the antibody may
directly bind to the gene product and may be used for cell
selection. For cell-surface gene products expressed at low
concentrations, magnetofluorescent liposomes (Scheffold, et al.
Nature Med 6:107-110, 2000) may be used for cell selection. At low
levels of expression, conventional fluorescently labeled antibodies
may not be sensitive enough to detect the presence of the cell
surface expressed gene product. Fluorophore-containing liposomes
may be conjugated to antibodies with the specificity of interest,
thereby allowing detection of the cell surface expressed gene
product.
[0059] For intracellular gene products, such as cytokines, the
antibody may be used after permeabilizing the cells. Alternatively,
to avoid killing the cells by permeabilization, the intracellular
gene product if it is ultimately secreted from the cell may be
detected as it is secreted through the cell membrane using a
"catch" antibody on the cell surface. The catch antibody may bc a
double antibody that is specific for two different antigens: (i)
the secreted gene product of interest and (ii) a cell surface
protein. The cell surface protein may be any surface marker present
on T cells, in particular, or lymphocytes, in general, (e.g.,
CD45). The catch antibody may first bind to the cell surface
protein and then bind to the intracellular gene product of interest
as it is secreted through the membrane, thereby retaining the gene
product on the cell surface. A labeled antibody specific for the
captured gene product may then be used to bind to the captured gene
product, which allows the selection of the activated T cell (Manz,
et al. Proc. Natl. Acad. Sci. USA 92:1921-1925, 1995, incorporated
herein by reference).
[0060] Certain forms of cytokines are also found expressed at low
concentration on the cell surface. For example, .gamma.IFN is
displayed at a low concentration on the cell surface with kinetics
similar to those of intracellular .gamma.IFN expression
(Assenmacher, et al. Eur J. Immunol, 1996, 26:263-267). For forms
of cytokines expressed on the cell surface, conventional
fluorescently labeled antibodies or fluorophore containing
liposomes may be used for detecting the cytokine of interest. One
of ordinary skill in the art will recognize other techniques for
detecting and selecting extracellular and intracellular gene
products specific for activated T cells.
[0061] The T cells isolated by the present invention may be
enriched by at least 90% from whole blood. The T cells may also be
enriched by at least 95% from whole blood. The T cells may also be
enriched by at least 98% from whole blood. The T cells may also be
isolated at least 99.5% from whole blood.
[0062] b. Isolated Monoclonal Antigen-Specific T Cells
[0063] In a second aspect, the present invention is directed to a T
cell specific for an antigen of interest isolated by the method of
the first aspect of the present invention.
[0064] c. Isolation of Polyclonal Antigen-Specific T Cells
[0065] In a third aspect, the present invention is directed to a
method for isolating T cells that are specific for one or more
antigens of interest. A sample comprising T cells is incubated with
one or more antigens, which cause the activation of T cells
specific for one or more antigens. T cells specific for one or more
antigens may then be isolated as in the first aspect of the present
invention.
[0066] 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 an 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. As described below, 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 which may prevent
epitope spreading in vaccinated patients.
[0067] d. Isolated Polyclonal Antigen-Specific T Cells
[0068] In a fourth aspect, the present invention is directed to T
cells 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
[0069] In a fifth aspect, the present invention is directed to a
method of determining the relative frequency of T cells specific
for one or more antigens of interest 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
[0070] 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 autoreactive 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 autoreactive T
cells in a patient to a control. The level of autoreactive 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
[0071] In a seventh aspect of the present invention, an autoimmune
disease may be monitored by determining the frequency of
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 autoreactive T cells. In addition, an
increase in the number of 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
[0072] In an eighth aspect of the present invention, a composition
may be produced for treating an autoimmune disease by inactivating
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 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 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 autoreactive T cells in autoimmune patients.
[0073] 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 incorporated by reference herein. 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.
[0074] a. Vaccine
[0075] 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 autoreactive T cells in autoimmune patients.
7. Treatment of an Autoimmune Disease
[0076] In a tenth aspect, an autoimmune disease may be treated in
patients with 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
autoreactive T cells in the patient.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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 incorporated herein by reference).
The pharmaceutical compositions may also further comprise liposomes
to enhance delivery or bioactivity, using methods and compounds
known in the art.
[0081] 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.
[0082] The inactivated 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.
[0083] The inactivated 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
[0084] 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.
[0085] 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 in M 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.
[0086] 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 min to 15 min. Similarly, after the final cycle is
complete, the reaction mixture may undergo a final extension for a
period of about 5 mM 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.
[0087] 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.
[0088] 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."
[0089] 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.
[0090] 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.
[0091] 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 at 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.
Example 1
Isolation of Myelin-Reactive T Cells for T Cell Vaccination
1. Preparation of PBMC and the Primary Stimulation
[0092] Fresh blood specimens from MS patients and control patients
were processed within 2 hours of collection. Alternatively,
mononuclear cells may be obtained from the cerebrospinal fluid
(CSFMCs) of MS patients. Peripheral blood mononuclear cells (PBMCs)
were isolated from the whole blood by standard Ficoll gradient
separation method. Specifically, heparinized blood was diluted with
Hanks balanced salt solution (HBSS) (1:1 blood/HBSS) and then
slowly laid over the Ficoll-hypaque solution in a centrifuge tube
and centrifuged for 20 minutes at 1800 rpm, 18.degree. C. to
25.degree. C., with no brake. PBMCs were then washed by adding
excess HBSS and centrifuged at 1700 rpm for 10 minutes at
18.degree. C. to 25.degree. C. Purified PBMCs were washed three
times in RPMI 1640 medium by centrifugation and subsequently
resuspended in AIM V medium (Gibco, Grand Island, N.Y.), Cell
number was counted and cells were plated onto 96-well U-bottomed
culture plates at a density of 200,000 cells/well. All plates were
labeled with patient number and patient initials. The cells were
incubated at 37.degree. C. in the presence of synthetic peptides
listed in Table 1 corresponding to the known immunodominant regions
of three myelin proteins, myelin basic protein (MBP), proteolipid
protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) at a
concentration of 20 .mu.g/ml. Plates were placed in a CO.sub.2
incubator at 37.degree. C. and visually inspected daily. Cells were
cultured for 7-10 days without change of culture medium to
selectively grow antigen-specific T cells.
TABLE-US-00001 TABLE 1 Activating Peptides Myelin Antigen Peptides
Amino Acid Sequences Myelin basic protein, peptide-1 (MBP-1)
ENPVVHFFKNIVTPRTP SEQ ID NO. 1 Myelin basic protein, peptide-2
(MBP-2) SKIFKLGGRDSRSGSPMARR SEQ ID NO. 2 Proteolipid protein,
peptide-3 (PLP-3) LFCGCGHEALTGTEKLIETY SEQ ID NO. 3 Proteolipid
protein, peptide-4 (PLP-4) WTTCQSIAFPSKTSASIGSL SEQ ID NO. 4 Myelin
oligodendrocyte glycoprotein, peptide-6 (MOG-6) GQFRVIGPRHPIRALVG
SEQ ID NO. 5 Myelin oligodendrocyte glycoprotein, peptide-7 (MOG-7)
EVELPCRISPGKNATGMEVGW SEQ ID NO. 6
2. Identification and Selection of Antigen-Specific T Cells
[0093] The cells described above are then selected for the
expression of gene products indicative of activated T cells. See
Section 2(a) above. A Cytokine Catch Reagent (Miltenyi Biotec) (as
described above) is used in order to detect the intracellular
cytokine .gamma.IFN or TNF.alpha. when ultimately excreted from the
cell. Briefly, the Cytokine Catch Reagent (typically a bispecific
antibody which binds to both the activated T cell marker and the
secreted cytokine) is incubated first with the cells at 4-8.degree.
C. in order to bind to the CD45 molecule on the cell surfaces or
other activated T cell surface marker such as CD69. The cells with
the bound Cytokine Catch Reagent are then incubated at 37.degree.
C. for 45 minutes to allow the .gamma.IFN or TNF.alpha. within the
cell to also bind to the Cytokine Catch Reagent as the cytokine is
secreted from inside the cell during this incubation period.
.gamma.IFN or TNF.alpha., now bound to the cell surface by the
Cytokine Catch Reagent which is then detected using an antibody
specific for cytokine of interest conjugated to the fluorochrome
PE.
[0094] The cell surface molecules CD4 and CD69, are detected using
antibodies conjugated to different fluorochromes. The CD4.sup.+
cell population is selected first by gating and then, within this
population, the "double-positive" (CD69 and IFN.gamma. or CD69 and
TNF.alpha.) stained cells are separated by FACS and collected
aseptically.
[0095] The isolated myelin-reactive T cells are then directly
expanded by stimulating with rIL-2, PHA, anti-CD3 or other general
T cell mitogen in the presence of irradiated autologous PBMCs for
7-10 days. Myelin-reactive T cells lines are propagated in culture
until the total cell number reached approximately 20 million.
Example 2
Diagnosis of MS
[0096] Two to 100 ml of blood are collected from the patient and
one or more synthetic peptides are added directly to the whole
blood to prime, or stimulate, the T lymphocytes. The peptides
correspond to the known immunodominant regions of three myelin
proteins, myelin basic protein (MBP), proteolipid protein (PLP),
and myelin oligodendrocyte glycoprotein (MOG). The blood is
incubated with the peptides for 1 to 7 days to activate the
myelin-specific T cells. At the end of this antigen-priming period,
the cells are re-challenged with antigens in a short re-stimulation
assay.
[0097] Myelin peptide-activated T cells are detected by
permeabilizing the cell membrane with a detergent solution, washing
the cells, then incubating with one or more staining antibodies to
detect CD4 or CD69 molecules on the cell surface or IFN.gamma. or
TNF.alpha. intracellularly. The staining antibodies are conjugated
to different fluorochromes so that they fluoresce at different
wavelengths when excited by a 488 nanometer laser by FASC analysis.
The population of CD4.sup.+T cells is selected first by using the
CD4.sup.+ cells for gating and then within this population, the
cells that are immunoreactive with both antibodies (CD69 and
.gamma.IFN or CD69 and INF.alpha.) are identified. This population
of "double-positive" myelin-reactive T cells has been shown to
increase significantly in multiple sclerosis (MS) patients as
compared to healthy controls in a similar study (FIG. 1, MS
patient, FIG. 2, healthy control). Using this method, the number of
myelin-reactive T cells circulating in the blood of a patient may
be determined before, during and after treatment to determine the
effect of an MS therapy on the autoreactive T cell population. The
endpoint may be determined as either the percentage of
double-positive stained cells or as the absolute number of
double-positive stained cells.
Example 3
Diagnosis of MS Using Antibody-Conjugated Liposomes
[0098] Whole blood is obtained from a patient and stimulated with
one or more synthetic peptides as described in Example 2. The blood
is incubated with the peptides for 3 to 16 hours to activate the
myelin-specific T cells. At the end of this antigen-priming period,
the cells may be re-challenged with antigens in a short
re-stimulation assay prior to staining with magnetofluorescent
liposomes conjugated to antibodies against IFN.gamma., TNF.alpha.,
or a combination thereof. The cells are also stained with an
antibody to CD4 and/or CD69. The stained myelin-reactive T cells
are detected as described in Example 2.
Example 4
Determination of TCR Clonal Repertoire
[0099] The T cell receptor (TCR) clonal repertoire represented in a
cell population may be analyzed by isolating out the
double-positive stained cell population by cell sorting as
described in Example 2 and Example 3. DNA is extracted from the
isolated cells and used to perform quantitative polymerase chain
reaction (PCR) assays using oligonucleotide primers specific for 25
known TCR variable beta chain (V.beta.) gene families. This
procedure yields information on the distribution of TCR V.beta.0
gene usage and indicates the clonality of pathogenic T cell
populations. This method may also be used to determine if clonal or
epitopic shifting of the myelin-reactive T cell population is
occurring in an MS patient.
Example 5
The Depletion of Myelin-Reactive T Cells by T Cell Vaccination
[0100] Patients with relapsing-remitting (RR)-MS and
secondary-progressive (SP)-MS received three subcutaneous
injections of irradiated autologous myelin-reactive T cell clones
isolated by direct expansion, with three additional injections 4,
12 and 20 weeks thereafter. Patients were monitored for changes in
the precursor frequency of myelin-reactive T cells, rate of
relapse, expanded disability status score (EDSS) and MRI lesion
activities over a period of 24 months. The results were compared
with pre-vaccination values in a self-paired manner. In addition,
the clinical data of the placebo arms of RR-MS in the
beta-interferon-1a clinical trial (Jacobs et al., 1996) and SP-MS
in a recent beta-IFN-1b study (European Study Group, Lancet,
352:1491-1497 (1998)) were included to provide natural history data
of MS for comparison. The T cell frequency was either undetectable
or substantially declined after vaccination at week 20. The results
confirmed depletion of myelin-reactive T cells by T cell
vaccination in patients with MS.
Example 6
Vaccination of MS Patient Using Autologous Myelin-Reactive T
Cells
[0101] The vaccination protocol is similar to that used in previous
clinical studies (Zhang et al., 1993, Medaer et al., 1995).
Briefly, myelin-reactive T cell clones prepared according to
Example 1 are activated with phytohaemagglutinin (PHA) (4 .mu.g/ml)
in the presence of irradiated PBMCs as a source of accessory cells.
Cells are then cultured for 10 days in RPMI 1640 media supplemented
with 10% heat-inactivated human AB serum and 100 units per mL of
rIL-2. Activated myelin-reactive T cells are subsequently washed
three times with sterile saline to remove residual PHA, rIL-2 and
cell debris and finally resuspended in 2 ml of saline. After
irradiation (10,000 rads, .sup.137Ce source), the cells are
injected subcutaneously on two arms (1 ml/arm). The number of T
cells used for vaccination range from 40.times.10.sup.6 to
80.times.10.sup.6 cells per injection and are chosen by an
extrapolation of T cell doses effective in experimental animals on
the basis of relative skin surface areas (Ben-Nun et al., 1981).
Each patient receives two subcutaneous injections followed by
repeat injections at 4, 12 and 20 weeks.
[0102] Patients are then observed for time to onset of confirmed
progression of disability, EDSS, rate of relapse and MRI lesion
activities. The results are compared with the patient's own
pre-treatment course as well as the placebo aims of two recent
clinical trials in RR-MS and SP-MS patients, which serve as an
estimate of the natural history of MS (Jacobs et al., 3996),
European Study Group, 1998). Time to progression is determined by
an increase of at least 1.0 on the EDSS (Poser et al., 1983)
persisting for at least 2 months. On-study exacerbations are
defined by the appearance of new neurological symptoms or worsening
of pre-existing neurological symptoms lasting for at least 48
hours, accompanied by objective change on neurological examination
(worsening of at least 0.5 point on EDSS). Patients are instructed
to report events between the scheduled regular visits, and are
examined by a neurologist if symptoms suggested an exacerbation.
Safety assessments included adverse events, vital sips and physical
examinations at regular visits. The differences in the clinical
variables in study patients before and after T cell vaccination are
analyzed using the Wilcoxon's rank-sum test.
Example 7
Alteration of Clinical Course of MS After Vaccination
[0103] Patients receive T-cell vaccinations prepared according to
Example 1 with no adverse effects. The mean EDSS declines in
patients with RR-MS over a period of 24 months after vaccination.
By comparison, there is an increase of mean EDSS by 0.61 in the
natural history of RR-MS (tr-56) over the same period of
observation, as was reported in a trial conducted using beta-IFN-1a
trial (Jacobs et al., 1996). In addition, the proportion of the
patients that have either unchanged or improved EDSS is higher than
that of the natural MS history. Few, if any, patients in the
treated RR-MS group progress beyond EDSS of 2.0 within 24 months as
compared to 18% of patients in the natural history of MS.
[0104] In the SP-MS cohort, mean EDSS progresses slower over a
period of 24 months as compared to +0.6 recorded in the natural
history of SP-MS (European Study Group, Lancet 1998;
352:1491-1497). Furthermore, estimation of time to confirmed
progression using the Kaplan-Meier method shows considerable delay
as compared to the natural history of MS patients (20% progression
in 12 months for RR-MS and 9 months for SP-MS) (Jacobs et al., Ann.
Neurol, 1996; 39:285-294, European Study Group, 1998).
Sequence CWU 1
1
6117PRTArtificialamino acids 110 to 126 of human myelin basic
protein 1Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro
Arg Thr1 5 10 15Pro220PRTArtificialamino acids 167 to 186 of human
mylelin basic protein 2Ser Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser
Arg Ser Gly Ser Pro1 5 10 15Met Ala Arg Arg 20320PRTArtificialamino
acids 31 to 50 of human myelin proteolipid protein 3Leu Phe Cys Gly
Cys Gly His Glu Ala Leu Thr Gly Thr Glu Lys Leu1 5 10 15Ile Glu Thr
Tyr 20420PRTArtificialamino acids 181 to 200 of human myelin
proteolipid protein 4Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro Ser
Lys Thr Ser Ala Ser1 5 10 15Ile Gly Ser Leu 20517PRTArtificialamino
acids 1 to 17 of human myelin oligodendrocyte glycoprotein 5Gly Gln
Phe Arg Val Ile Gly Pro Arg His Pro Ile Arg Ala Leu Val1 5 10
15Gly621PRTArtificialamino acids 18 to 38 of human myelin
oligodendrocyte glycoprotein 6Glu Val Glu Leu Pro Cys Arg Ile Ser
Pro Gly Lys Asn Ala Thr Gly1 5 10 15Met Glu Val Gly Trp 20
* * * * *