U.S. patent application number 11/726708 was filed with the patent office on 2008-03-13 for recombinant mhc molecules useful for manipulation of antigen-specific t-cells.
Invention is credited to Gregory G. Burrows, Arthur A. Vandenbark.
Application Number | 20080064859 11/726708 |
Document ID | / |
Family ID | 26744627 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064859 |
Kind Code |
A1 |
Vandenbark; Arthur A. ; et
al. |
March 13, 2008 |
Recombinant MHC molecules useful for manipulation of
antigen-specific T-cells
Abstract
Two-domain MHC polypeptides useful for manipulation of
antigen-specific T-cells are disclosed. These polypeptides include
MHC class II-based molecules that comprise covalently linked
.beta.1 and .alpha.1 domains, and MHC class I-based molecules that
comprise covalently linked .alpha.1 and .alpha.2 domains. These
polypeptides may also include covalently linked antigenic
determinants, toxic moieties, and/or detectable labels. The
disclosed polypeptides can be used to target antigen-specific
T-cells, and are useful, among other things, to detect and purify
antigen-specific T-cells, to induce or activate T-cells, and to
treat conditions mediated by antigen-specific T-cells.
Inventors: |
Vandenbark; Arthur A.;
(Portland, OR) ; Burrows; Gregory G.; (Portland,
OR) |
Correspondence
Address: |
Jeffrey J. King, Esq.;BLACK LOWE & GRAHAM PLLC
Suite 4800
701 Fifth Avenue
Seattle
WA
98104
US
|
Family ID: |
26744627 |
Appl. No.: |
11/726708 |
Filed: |
March 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10941152 |
Sep 14, 2004 |
7265218 |
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11726708 |
Mar 21, 2007 |
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09858580 |
May 15, 2001 |
6815171 |
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10941152 |
Sep 14, 2004 |
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09153586 |
Sep 15, 1998 |
6270772 |
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09858580 |
May 15, 2001 |
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60064552 |
Sep 16, 1997 |
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60064555 |
Oct 10, 1997 |
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Current U.S.
Class: |
530/402 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 37/02 20180101; G01N 33/56977 20130101; A61P 37/04 20180101;
C07K 14/70539 20130101; C07K 2319/00 20130101; G01N 33/56972
20130101; A61P 43/00 20180101 |
Class at
Publication: |
530/402 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 43/00 20060101 A61P043/00 |
Claims
1-35. (canceled)
35. A single chain class II MHC molecule comprising: a
peptide-binding groove and covalently linked in sequence: 1) a
class II beta chain, 2) a single chain linker, and 3) a class II
alpha chain, wherein the chain of both 1) and 3) lack a
transmembrane domain; and further wherein the single chain class II
MHC molecule is empty.
36. The MHC molecule of claim 35, wherein the MHC molecule is
soluble.
37. The MHC molecule of claim 35, wherein the chain of 1) comprises
a beta domain and the chain of 3) comprises an alpha 1 domain.
38. The MHC molecule of claim 35, wherein the single chain linker
is linked between the carboxyl terminus of the beta chain and the
amino terminus of the alpha chain.
39. The MHC molecule of claim 35, wherein the beta and alpha chains
are each independently selected from the group consisting of IE,
IA, DR, DQ and DP proteins.
40. The MHC molecule of claim 35, wherein the MHC molecule is
modified to carry a detectable tag.
41. A multivalent MHC complex comprising two or more linked MHC
molecules of claim 35.
42. A MHC complex of claim 41, wherein the MHC molecules are linked
to immunoglobulin domains.
43. A MHC complex of claim 41, wherein the MHC complex is modified
to carry a detectable tag.
44. A single chain MHC class II-peptide complex comprising: a
peptide-binding groove; covalently linked in sequence: 1) a class
II beta chain, 2) a single chain linker, and 3) a class II alpha
chain, wherein the chain of both 1) and 3) lack a transmembrane
domain; and a presenting peptide being covalently linked to the MHC
molecule and non-covalently bound to the peptide binding groove of
the MHC molecule.
45. The MHC complex of claim 44, wherein the complex is
soluble.
46. The MHC complex of claim 44, wherein the chains of 1) and 3)
comprise a beta 1 domain and alpha1 domain, respectively.
47. The MHC complex of claim 44, wherein the MHC class II molecule
comprises the presenting peptide covalently linked to the .beta.
chain.
48. The MHC complex of claim 44, wherein a presenting peptide
linker sequence is interposed between the presenting peptide and
the MHC molecule.
49. The MHC complex of claim 44, wherein the beta and alpha chains
are each independently selected from the group consisting of IE,
IA, DR, DQ and DP proteins.
50. The MHC complex of claim 44, wherein the MHC molecule is
modified to carry a detectable tag.
51. A multivalent MHC complex comprising two or more linked MHC
molecules of claim 44.
52. The MHC complex of claim 51, wherein the MHC molecules are
linked to immunoglobulin domains.
53. The MHC complex of claim 51, wherein the MHC complex is
modified to carry a detectable tag.
54. A loaded single chain MHC class II-peptide complex comprising:
a peptide-binding groove; covalently linked in sequence: 1) a class
II beta chain, 2) a single chain linker, and 3) a class II alpha
chain, wherein the chain of both 1) and 3) lack a transmembrane
domain; and a presenting peptide being non-covalently linked to the
MHC molecule and non-covalently bound to the peptide binding groove
of the MHC molecule, wherein the loaded single chain MHC class
II-peptide complex can be recognized by a CD4+ T cell.
55. The MHC complex of claim 54, wherein the MHC molecule is
soluble.
56. The MHC complex of claim 54, wherein the chain of 1) comprises
a beta 1 domain and the chain of 3) comprises an alpha 1
domain.
57. The MHC complex of claim 54, wherein the single chain linker is
linked between the carboxyl terminus of the beta chain and the
amino terminus of the alpha chain.
58. The MHC complex of claim 54, wherein the beta and alpha chains
are each independently selected from the group consisting of IE,
IA, DR, DQ and DP proteins.
59. The MHC complex of claim 54, wherein the MHC molecule is
modified to carry a detectable tag.
60. A multivalent MHC complex comprising two or more linked MHC
complexes of claim 54.
61. The multivalent MHC complex of claim 60, wherein the MHC
complexes are linked to immunoglobulin domains.
62. A multivalent MHC complex of claim 60, wherein the MHC complex
is modified to carry a detectable tag.
Description
PRIORITY CLAIM
[0001] This is a continuation of U.S. patent application Ser. No.
09/858,580, filed May 15, 2001, which is a continuation of U.S.
patent application Ser. No. 09/153,586, filed Sep. 15, 1998, which
issued as U.S. Pat. No. 6,270,772, which claims the benefit of U.S.
Provisional Application No. 60/064,552, filed Sep. 16, 1997, and
U.S. Provisional Application No. 60/064,555, filed Oct. 10, 1997,
all of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The initiation of an immune response against a specific
antigen in mammals is brought about by the presentation of that
antigen to T-cells. An antigen is presented to T-cells in the
context of a major histocompatibility (MHC) complex. MHC complexes
are located on the surface of antigen presenting cells (APCs); the
3-dimensional structure of MHCs includes a groove or cleft into
which the presented antigen fits. When an appropriate receptor on a
T-cell interacts with the MHC/antigen complex on an APC in the
presence of necessary co-stimulatory signals, the T-cell is
stimulated, triggering various aspects of the well characterized
cascade of immune system activation events, including induction of
cytotoxic T-cell function, induction of B-cell function and
stimulation of cytokine production.
[0003] There are two basic classes of MHC molecules in mammals, MHC
class I and MHC II. Both classes are large protein complexes formed
by association of two separate proteins. Each class includes
trans-membrane domains that anchor the complex into the cell
membrane. MHC class I molecules are formed from two non-covalently
associated proteins, the .alpha. chain and .alpha.2-microglobulin.
The .alpha. chain comprises three distinct domains, .alpha.1,
.alpha.2 and .alpha.3. The three dimensional structure of the
.alpha.1 and .alpha.2 domains forms the groove into which antigens
fit for presentation to T-cells. The .alpha..alpha.3 domain is a
trans-membrane Ig-fold like domain that anchors the .alpha. chain
into the cell membrane of the APC. MHC class I complexes, when
associated with antigen (and in the presence of appropriate
co-stimulatory signals) stimulate CD8 cytotoxic T-cells, which
function to kill any cell which they specifically recognize.
[0004] The two proteins which associate non-covalently to form MHC
class II molecules are termed the .alpha. and .beta. chains. The
.alpha. chain comprises .alpha.1 and .alpha.2 domains, and the
.beta. chain comprises .beta.1 and .beta.2 domains. The cleft into
which the antigen fits is formed by the interaction of the .alpha.1
and .beta.1 domains. The .alpha.2 and .beta.2 domains are
trans-membrane Ig-fold like domains that anchors the .alpha. and
.beta. chains into the cell membrane of the APC. MHC class II
complexes, when associated with antigen (and in the presence of
appropriate co-stimulatory signals) stimulate CD4 T-cells. The
primary functions of CD4 T-cells are to initiate the inflammatory
response and to regulate other cells in the immune system.
[0005] The genes encoding the various proteins that constitute the
MHC complexes have been extensively studied in humans and other
mammals. In humans, MHC molecules (with the exception of class I
.beta.2-microglobulin) are encoded in the HLA region, which is
located on chromosome 6 and constitutes over 100 genes. There are 3
class I MHC .alpha. protein loci, termed HLA-A, -B and -C. There
are also 3 pairs of class II MHC .alpha. and .beta. chain loci,
termed HLA-DR(A and B), HLA-DP(A and B), and HLA-DQ(A and B). In
rats, the class I .alpha. gene is termed RT1.A, while the class II
genes are termed RT1.B.alpha. and RT1.B.beta.. More detailed
background information on the structure, function and genetics of
MHC complexes can be found in Immunobiology: The Immune System in
Health and Disease by Janeway and Travers, Current Biology
Ltd./Garland Publishing, Inc. (1997) (ISBN 0-8153-2818-4), and in
Bodmer et al. (1994) "Nomenclature for factors of the HLA system"
Tissue Antigens vol. 44, pages 1-18 (with periodic updates).
[0006] The key role that MHC complexes play in triggering immune
recognition has led to the development of methods by which these
complexes are used to modulate the immune response. For example,
activated T-cells which recognize "self" antigens (autoantigens)
are known to play a key role in autoimmune diseases (such as
rheumatoid arthritis and multiple sclerosis). Building on the
observation that isolated MHC class II molecules (loaded with the
appropriate antigen) can substitute for APCs carrying the MHC class
II complex and can bind to antigen-specific T-cells, a number of
researchers have proposed that isolated MHC/antigen complexes may
be used to treat autoimmune disorders. Thus U.S. Pat. Nos.
5,194,425 (Sharma et al.) and 5,284,935 (Clark et al.) disclose the
use of isolated MHC class II complexes loaded with a specified
autoantigen and conjugated to a toxin to eliminate T-cells that are
specifically immunoreactive with autoantigens. In another context,
it has been shown that the interaction of isolated MHC II/antigen
complexes with T-cells, in the absence of co-stimulatory factors,
induces a state of non-responsiveness known as anergy. (Quill et
al., J. Immunol., 138:3704-3712 (1987)). Following this
observation, Sharma et al. (U.S. Pat. Nos. 5,468,481 and 5,130,297)
and Clarke et al. (U.S. Pat. No. 5,260,422) have suggested that
such isolated MHC II/antigen complexes may be administered
therapeutically to anergize T-cell lines which specifically respond
to particular autoantigenic peptides.
[0007] Methods for using isolated MHC complexes in the detection,
quantification and purification of T-cells which recognize
particular antigens have been studied for use in diagnostic and
therapeutic applications. By way of example, early detection of
T-cells specific for a particular autoantigen would facilitate the
early selection of appropriate treatment regimes. The ability to
purify antigen-specific T-cells would also be of great value in
adoptive immunotherapy. Adoptive immunotherapy involves the removal
of T-cells from a cancer patient, expansion of the T-cells in vitro
and then reintroduction of the cells to the patient (see U.S. Pat.
No. 4,690,915; Rosenberg et al. New Engl. J. Med. 319:1676-1680
(1988)). Isolation and expansion of cancer specific T-cells with
inflammatory properties would increase the specificity and
effectiveness of such an approach.
[0008] To date, however, attempts to detect, quantify or purify
antigen specific T-cells using isolated MHC/antigen complexes have
not met with widespread success because, among other reasons,
binding between the T-cells and such isolated complexes is
transient and hence the T-cell/MHC/antigen complex is unstable. In
an attempt to address these problems, Altman et al. (Science 274,
94-96 (1996) and U.S. Pat. No. 5,635,363) have proposed the use of
large, covalently linked multimeric structures of MHC/antigen
complexes to stabilize this interaction by simultaneously binding
to multiple T-cell receptors on a target T-cell.
[0009] Although the concept of using isolated MHC/antigen complexes
in therapeutic and diagnostic applications holds great promise, a
major drawback to the various methods reported to date is that the
complexes are large and consequently difficult to produce and to
work with. While the complexes can be isolated from lymphocytes by
detergent extraction, such procedures are inefficient and yield
only small amounts of protein. The cloning of the genes encoding
the various MHC complex subunits has facilitated the production of
large quantities of the individual subunits through expression in
prokaryotic cells, but the assembly of the individual subunits into
MHC complexes having the appropriate conformational structure has
proven difficult.
SUMMARY OF THE INVENTION
[0010] This invention is founded on the discovery that mammalian
MHC function can be mimicked through the use of recombinant
polypeptides that include only those domains of MHC molecules that
define the antigen binding cleft. These molecules are useful to
detect, quantify and purify antigen-specific T-cells. The molecules
provided herein may also be used in clinical and laboratory
applications to detect, quantify and purify antigen-specific
T-cells, induce anergy in T-cells, as well as to stimulate T-cells,
and to treat diseases mediated by antigen-specific T-cells.
[0011] By way of example, while Altman et al. (U.S. Pat. No.
5,635,363) contemplate the use of multimers of MHC class II
complexes comprising .alpha.1, .alpha.2, .beta.1 and .beta.2
domains and associated peptide antigens, to bind to and purify
antigen-specific T-cells from a mixture, the present inventors have
discovered that such antigen-specific T-cell binding can be
accomplished with a much simpler monomeric molecule comprising, in
the case of class II MHC molecules, only the .alpha.1 and .beta.1
domains in covalent linkage (and in association with an antigenic
determinant). For convenience, such MHC class II polypeptides are
hereinafter referred to as ".beta.1.alpha.1". Equivalent molecules
derived from MHC class I molecules are also provided by this
invention. Such molecules comprise the .alpha.1 and .alpha.2
domains of class I molecules in covalent linkage and in association
with an antigenic determinant. Such MHC class I polypeptides are
referred to as ".alpha.1.alpha.2". These two domain molecules may
be readily produced by recombinant expression in prokaryotic or
eukaryotic cells, and readily purified in large quantities.
Moreover, these molecules may easily be loaded with any desired
peptide antigen, making production of a repertoire of MHC molecules
with different T-cell specificities a simple task.
[0012] It is shown that, despite lacking the trans-membrane Ig fold
domains that are part of intact MHC molecule, these two domain MHC
molecules refold in a manner that is structurally analogous to
"whole" MHC molecules, and bind peptide antigens to form stable
MHC/antigen complexes. Moreover, these two domain MHC/epitope
complexes bind T-cells in an epitope-specific manner, and inhibit
epitope-specific T-cell proliferation in vitro. In addition,
administration of .beta.1.alpha.1 molecules loaded with the myelin
basic protein (MBP) epitope comprising amino acids 69-89 of MBP to
rats is shown to both suppress the onset of and treat experimental
autoimmune encephalomyelitis (EAE) in rats. Thus, the two domain
MHC molecules display powerful and epitope-specific effects on
T-cell activation both in vitro and in vivo. As a result, the
disclosed MHC molecules are useful in a wide range of both in vivo
and in vitro applications.
[0013] Various formulations of these two domain molecules are
provided by the invention. In their most basic form, the two domain
MHC class II molecules comprise .alpha.1 and .beta.1 domains of a
mammalian MHC class II molecule wherein the amino terminus of the
.alpha.1 domain is covalently linked to the carboxy terminus of the
.beta.1 domain and wherein the polypeptide does not include the
.alpha.2 or .beta.2 domains. The two domain MHC class I molecules
comprise an .alpha.1 and .alpha.2 domains of a mammalian class I
molecule, wherein the amino terminus of the .alpha.2 domain is
covalently linked to the carboxy terminus of the .alpha.1 domain,
and wherein the polypeptide does not include an MHC class I
.alpha.3 domain. For most applications, these molecules are
associated, by covalent or non-covalent interaction, with an
antigenic determinant, such as a peptide antigen. In certain
embodiments, the peptide antigen is covalently linked to the amino
terminus of the .beta.1 domain of the class II molecules, or the
.alpha.1 domain of the class I molecules. The two domain molecules
may also comprise a detectable marker, such as a fluorescent label
or a toxic moiety, such as ricin A.
[0014] The invention also provides nucleic acid molecules that
encode the two domain MHC molecules, as well as expression vectors
that may be conveniently used to express these molecules. In
particular embodiments, the nucleic acid molecules include
sequences that encode the antigenic peptide as well as the two
domain MHC molecule. For example, one such nucleic acid molecule
may be represented by the formula Pr-P-B-A, wherein Pr is a
promoter sequence operably linked to P (a sequence encoding the
peptide antigen), B is the class I .alpha.1 or the class II .beta.1
domain, and A is the class I .alpha.2 domain or the class II
.alpha.1 domain. In these nucleic acid molecules, P, B and A
comprise a single open reading frame, such that the peptide and the
two MHC domains are expressed as a single polypeptide chain.
[0015] In vitro, the two domain MHC molecules may be used to detect
and quantify T-cells, and regulate T-cell function. Thus, such
molecules loaded with a selected antigen may be used to detect,
monitor and quantify the population of a T-cells that are specific
for that antigen. The ability to do this is beneficial in a number
of clinical settings, such as monitoring the number of tumor
antigen-specific T-cells in blood removed from a cancer patient, or
the number of self-antigen specific T-cells in blood removed from a
patient suffering from an autoimmune disease. In these contexts,
the disclosed molecules are powerful tools for monitoring the
progress of a particular therapy. In addition to monitoring and
quantifying antigen-specific T-cells, the disclosed molecules may
also be used to purify such cells for adoptive immunotherapy. Thus,
the disclosed MHC molecules loaded with a tumor antigen may be used
to purify tumor-antigen specific T-cells from a cancer patient.
These cells may then be expanded in vitro before being returned to
the patient as part of a cancer treatment. When conjugated with a
toxic moiety, the two domain molecules may be used to kill T-cells
having a particular antigen specificity. Alternatively, the
molecules may also be used to induce anergy in such T-cells.
[0016] The two domain molecules may also be used in vivo to target
specified antigen-specific T-cells. By way of example, a
.beta.1.alpha.1 molecule loaded with a portion of myelin basic
protein (MBP) and administered to patients suffering from multiple
sclerosis may be used to induce anergy in MBP-specific T-cells,
thus alleviating the disease symptoms. Alternatively, such
molecules may be conjugated with a toxic moiety to more directly
kill the disease-causing T-cells.
[0017] These and other aspects of the invention are described in
more detail in the following sections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A shows the sequences of the prototypical
.beta.1.alpha.1 cassette without an antigen coding region. Unique
NcoI, PstI, and XhoI restriction sites are in bold. The end of the
.beta.1 domain and start of the .alpha.1 domain are indicated. FIG.
1B shows the sequence of an in-frame antigenic peptide/linker
insertion sequence that can be incorporated into the expression
cassette at the insertion site shown () in FIG. 1A. This sequence
includes the rat MBP 72-89 antigen, a flexible linker with an
embedded thrombin cleavage site, and a unique SpeI restriction site
that can be used for facile exchange of the antigen coding region.
Example 2 below discusses the use of the equivalent peptide from
Guinea pig, which has a serine in place of the threonine residue in
the MBP 72-89 sequence. FIGS. 1C and 1D show exemplary Nco1/SpeI
fragments that can be inserted into the expression cassette in
place of the MBP-72-89 antigen coding region. FIG. 1C includes the
MBP 55-69 antigen, FIG. 1D includes the CM-2 antigen.
[0019] FIGS. 2A and B show the structure-based design of the
.beta.1.alpha.1 molecule. A. Rat class II RT1.B, loaded with the
encephalitogenic MBP-69-89 peptide. B. The single-chain
.beta.1.alpha.1 molecule, loaded with MBP-69-89.
[0020] FIGS. 3 A and B show direct detection of antigen-specific
.beta.1.alpha.1/polypeptide molecules binding rat T cells. The A1 T
cell hybridoma (BV8S2 TCR+) and the CM-2 cell line (BV8S2 TCR-)
were incubated 17 hours at 4.degree. C. with various
.beta.1.alpha.1 constructs, washed, stained for 15 min with OX6-PE
(.alpha.-RT1.B) or a PE-isotype control and then analyzed by FACS.
Background expression of I-A on the CM-2 line was blocked with
unlabeled OX-6. A. Histogram showing staining of the A1 hybridoma.
B. Histogram showing staining of the CM-2 cell line.
[0021] FIG. 4 is a graph showing binding of A488 conjugated
.beta.1.alpha.1/polypeptide molecules to rat BV8S2 TCR.
.beta.1.alpha.1 molecules were conjugated with Alexa-488 dye,
loaded with MBP-69-89, incubated with the A1 T cell hybridomas
(BV8S2 TCR+) for 3 hours at 4.degree. C. and then analyzed by FACS.
A488-.beta.1.alpha.1(empty) and A488-.beta.1.alpha.1/MBP-69-89, as
indicated.
[0022] FIG. 5 is a bar graph showing that the
.beta.1.alpha.1/MBP-69-89 complex blocks antigen specific
proliferation in an IL-2 reversible manner. Short-term T cell lines
selected with MBP-69-89 peptide from lymph node cells from rats
immunized 12 days earlier with Gp-MBP/CFA were pre-treated for 24
hours with .beta.1.alpha.1 constructs, washed, and then used in
proliferation assays in which the cells were cultured with and
without 20 Units/ml IL-2. Cells were incubated for three days, the
last 18 hr in the presence of [.sup.3H]thymidine (0.5 .mu.Ci/10
.mu.l/well). Values indicated are the mean CPM .+-.SEM. Background
was 210 CPM. Column a. Control proliferation assay without IL-2.
Column b. 20 .mu.M .beta.1.alpha.1/MBP-55-69 pretreatment. Column
c. 10 nM .beta.1.alpha.1/MBP-69-89 pretreatment. Column d. 10 nM
.beta.1.alpha.1/MBP-69-89 plus IL-2 during the proliferation assay.
A single representative experiment is shown; the experiment was
done twice. *indicates significant (p<0.001) inhibition with
.beta.1.alpha.1/MBP-69-89 versus control cultures.
[0023] FIGS. 6A-D are graphs showing clinical protection from
experimental autoimmune encephalomyelitis with the
.beta.1.alpha.1/MBP-69-89 complex. Groups of Lewis rats (n=6) were
injected with 25 .mu.g of Gp-MBP/CFA to induce clinical EAE. On
days 3, 7, 9, 11, and 14 after disease induction rats were given
.beta.1.alpha.1/peptide complex, peptide alone, or were left
untreated, as indicated. A. No treatment, or 2 .mu.g MBP-69-89
peptide alone, as indicated. B. 300 .mu.g of
.beta.1.alpha.1/(empty) complex in saline. C. 300 .mu.g of
.beta.1.alpha.1/CM-2 complex in saline. D. 30 .mu.g of
.beta.1.alpha.1/MBP-69-89 complex in saline. Daily body weight
(grams, right-hand y-axis) is plotted for the 300 .mu.g
.beta.1.alpha.1/peptide complex treatments. A single representative
experiment is shown; the experiment was done three times. Values
indicate mean clinical score .+-.SEM on each day of clinical
disease. 30 .mu.g of complex is equivalent to 2 .mu.g of free
peptide.
[0024] FIG. 7 is a graph showing treatment of established EAE with
.beta.1.alpha.1/MBP-69-89 complex. Groups of Lewis rats (n=6) were
injected with 25 .mu.g of Gp-MBP/CFA to induce clinical EAE. On the
day of onset of clinical signs (day 11), day 13, and day 15, rats
were given 300 .mu.g of .beta.1.alpha.1/MBP-69-89 complex.
(indicated by arrows) or were left untreated. A single
representative experiment is shown; the experiment was done twice.
Values indicate mean clinical score .+-.SEM on each day of clinical
disease.
[0025] FIGS. 8A and B are graphs showing that the
.beta.1.alpha.1/MBP-69-89 complex specifically inhibits the DTH
response to MBP 69-89. A. Change in ear thickness 24 hrs after
challenge with PPD. B. Change in ear thickness 24 hrs after
challenge with MBP-69-89. Values indicate mean score .+-.SEM.
*Indicates significant difference between control and treated
(p=0.01). A single representative experiment is shown; the
experiment was done twice.
[0026] FIG. 9 is a graph showing that T cell responses to MBP-69-89
were inhibited in Lewis rats treated with 300 .mu.g
.beta.1.alpha.1/MBP-69-89 complex. Lymph node cells were collected
from control and treated rats after recovery of controls from EAE
(day 17) and stimulated with optimal concentrations of Gp-MBP,
Gp-MBP-69-89 peptide, or PPD. *Indicates significant difference
between control and treated (*p<0.05; **p<0.001). Note
inhibition with Gp MBP and MBP-69-89 peptide but not to PPD in
treated rats.
[0027] FIG. 10A-C shows the amino acid sequences of exemplary (A)
human (DRA and DRB1 0101), (B) mouse (I-EK) and (C) rat (RT1.B)
.beta.1 and .alpha.1 domains (the initiating methione and glycine
sequences in the rat sequence were included in a construct for
translation initiation reasons).
[0028] FIG. 11 shows the amino acid sequences of exemplary .alpha.1
and .alpha.2 domains derived from human MHC class I B*5301.
SEQUENCE LISTING
[0029] The sequence listing appended hereto includes sequences as
follows:
[0030] Seq. I.D. No. 1: the nucleic acid of a single chain
.beta.1.alpha.1 expression cassette.
[0031] Seq. I.D. No. 2: the amino acid sequence encoded by the
construct shown in Seq. I.D. No. 1.
[0032] Seq. I. D No. 3: the nucleic acid sequence of an
antigen/linker insert suitable for insertion into the expression
cassette shown in Seq. I.D. No. 1.
[0033] Seq. I.D. No. 4: the amino acid sequence encoded by the
sequence shown in Seq. I.D. no. 3.
[0034] Seq. I.D. Nos. 5 and 7: alternative antigen encoding
sequences for the expression cassette and, Seq. I.D. Nos. 6 and 8,
the antigen sequences encoded by the sequences shown in Seq. I.D.
Nos. 5 and 7, respectively.
[0035] Seq. I.D. Nos. 9-20 and 28-29 show PCR primers use to
amplify components of the .beta.1.alpha.1 expression cassette.
[0036] Seq. I.D. No. 21 shows the exemplary .alpha.1 and .alpha.2
domains depicted in FIG. 11.
[0037] Seq. I.D. Nos. 22-24 show the exemplary .beta.1 and .alpha.1
domains depicted in FIG. 10.
[0038] Seq. I.D. Nos. 25-27 and 30 show peptides sequences used in
various aspects of the invention.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0039] In order to facilitate review of the various embodiments of
the invention, the following definitions of terms and explanations
of abbreviations are provided:
[0040] Isolated: An "isolated" nucleic acid has been substantially
separated or purified away from other nucleic acid sequences in the
cell of the organism in which the nucleic acid naturally occurs,
i.e., other chromosomal and extrachromosomal DNA and RNA. The term
"isolated" thus encompasses nucleic acids purified by standard
nucleic acid purification methods. The term also embraces nucleic
acids prepared by recombinant expression in a host cell as well as
chemically synthesized nucleic acids.
[0041] cDNA (complementary DNA): a piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences which
determine transcription. cDNA is synthesized in the laboratory by
reverse transcription from messenger RNA extracted from cells.
[0042] ORF (open reading frame): a series of nucleotide triplets
(codons) coding for amino acids without any termination codons.
These sequences are usually translatable into a polypeptide.
[0043] Probes and primers: Nucleic acid probes and primers may
readily be prepared based on the nucleic acids provided by this
invention. A probe comprises an isolated nucleic acid attached to a
detectable label or reporter molecule. Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed, e.g., in Sambrook
et al. (1989) and Ausubel et al. (1987).
[0044] Primers are short nucleic acids, preferably DNA
oligonucleotides 15 nucleotides or more in length. Primers may be
annealed to a complementary target DNA strand by nucleic acid
hybridization to form a hybrid between the primer and the target
DNA strand, and then extended along the target DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification of a
nucleic acid sequence, e.g., by the polymerase chain reaction (PCR)
or other nucleic-acid amplification methods known in the art.
[0045] Methods for preparing and using probes and primers are
described, for example, in Sambrook et al. (1989), Ausubel et al.
(1987), and Innis et al., (1990. PCR primer pairs can be derived
from a known sequence, for example, by using computer programs
intended for that purpose such as Primer (Version 0.5,.COPYRGT.
1991, Whitehead Institute for Biomedical Research, Cambridge,
Mass.).
[0046] Purified: the term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified recombinant MHC protein preparation is one in
which the recombinant MHC protein is more pure than the protein in
its originating environment within a cell. A preparation of a
recombinant MHC protein is typically purified such that the
recombinant MHC protein represents at least 50% of the total
protein content of the preparation. However, more highly purified
preparations may be required for certain applications. For example,
for such applications, preparations in which the MHC protein
comprises at least 75% or at least 90% of the total protein content
may be employed.
[0047] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter effects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein coding regions, the open reading frames are
aligned.
[0048] Recombinant: A recombinant nucleic acid or polypeptide is
one that has a sequence that is not naturally occurring or has a
sequence that is made by an artificial combination of two or more
otherwise separated segments of sequence. This artificial
combination is often accomplished by chemical synthesis or, more
commonly, by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques.
[0049] Mammal: This term includes both human and non-human mammals.
Similarly, the term "patient" includes both human and veterinary
subjects.
[0050] .beta.1.alpha.1 polypeptide: a recombinant polypeptide
comprising the .alpha.1 and .beta.1 domains of a MHC class II
molecule in covalent linkage. To ensure appropriate conformation,
the orientation of such a polypeptide is such that the carboxy
terminus of the .beta.1 domain is covalently linked to the amino
terminus of the .alpha.1 domain.
[0051] .beta.1.alpha.1 gene: a recombinant nucleic acid sequence
including a promoter region operably linked to a nucleic acid
sequence encoding a .beta.1.alpha.1 polypeptide.
[0052] .alpha.1.alpha.2 polypeptide: a polypeptide comprising the
.alpha.1 and .alpha.2 domains of a MHC class I molecule in covalent
linkage. The orientation of such a polypeptide is such that the
carboxy terminus of the .alpha.1 domain is covalently linked to the
amino terminus of the .alpha.2 domain. An .alpha.1.alpha.2
polypeptide comprises less than the whole class I .alpha. chain,
and usually omits most or all of the .alpha.3 domain of the .alpha.
chain.
[0053] .alpha.1.alpha.2 gene: a recombinant nucleic acid sequence
including a promoter region operably linked to a nucleic acid
sequence encoding an .alpha.1.alpha.2 polypeptide.
[0054] Domain: a domain of a polypeptide or protein is a discrete
part of an amino acid sequence that can be equated with a
particular function. For example, the .alpha. and .beta.
polypeptides that constitute a MHC class II molecule are each
recognized as having two domains, .alpha.1, .alpha.2 and .beta.1,
.beta.2, respectively. Similarly, the .alpha. chain of MHC class I
molecules is recognized as having three domains, .alpha.1, .alpha.2
and .alpha.3. The various domains in each of these molecules are
typically joined by linking amino acid sequences. When selecting
the sequence of a particular domain for inclusion in a recombinant
molecule, it is preferable that the entire domain be included; to
ensure that this is done, the domain sequence may be extended to
include part of the linker, or even part of the adjacent domain.
For example, when selecting the .alpha.1 domain of HLA-DR A, the
selected sequence will generally extend from amino acid residue
number 1 of the .alpha. chain, through the entire .alpha.1 domain
and will include all or part of the linker sequence located at
about amino acid residues 76-90 (at the carboxy terminus of the
.alpha.1 domain, between the .alpha.1 and .alpha.2 domains).
However, the precise number of amino acids in the various MHC
molecule domains varies depending on the species of mammal, as well
as between classes of genes within a species. Rather than a precise
structural definition based on the number of amino acids, it is the
maintenance of domain function that is important when selecting the
amino acid sequence of a particular domain. Moreover, one of skill
in the art will appreciate that domain function may also be
maintained if somewhat less than the entire amino acid sequence of
the selected domain is utilized. For example, a number of amino
acids at either the amino or carboxy terminii of the .alpha.1
domain may be omitted without affecting domain function. Typically
however, the number of amino acids omitted from either terminus of
the domain sequence will be no greater than 10, and more typically
no greater than 5. The functional activity of a particular selected
domain may be assessed in the context of the two-domain MHC
polypeptides provided by this invention (i.e., the class II
.beta.1.alpha.1 or class I .alpha.1.alpha.2 polypeptides) using the
antigen-specific T-cell proliferation assay as described in detail
below. For example, to test a particular .beta.1 domain, it will be
linked to a functional .alpha.1 domain so as to produce a
.beta.1.alpha.1 molecule and then tested in the described assay. A
biologically active .beta.1.alpha.1 or .alpha.1.alpha.2 polypeptide
will inhibit antigen-specific T cell proliferation by at least
about 50%, thus indicating that the component domains are
functional. Typically, such polypeptides will inhibit T-cell
proliferation in this assay system by at least 75% and sometimes by
greater than about 90%.
[0055] Sequence identity: the similarity between amino acid
sequences is expressed in terms of the similarity between the
sequences, otherwise referred to as sequence identity. Sequence
identity is frequently measured in terms of percentage identity (or
similarity or homology); the higher the percentage, the more
similar the two sequences are. Variants of MHC domain polypeptides
will possess a relatively high degree of sequence identity when
aligned using standard methods. (An "MHC domain polypeptide" refers
to an .alpha.1 or .beta.1 domain of an MHC class II polypeptide or
an .alpha.1 or .alpha.2 domain of an MHC class I polypeptide).
[0056] Methods of alignment of sequences for comparison are well
known in the art. Altschul et al. (1994) presents a detailed
consideration of sequence alignment methods and homology
calculations. The NCBI Basic Local Alignment Search Tool (BLAST)
(Altschul et al., 1990) is available from several sources,
including the National Center for Biotechnology Information (NCBI,
Bethesda, Md.) and on the Internet, for use in connection with the
sequence analysis programs blastp, blastn, blastx, tblastn and
tblastx. It can be accessed at the NCBI website.
A description of how to determine sequence identity using this
program is available at the NCBI website.
[0057] Variants of MHC domain polypeptides are typically
characterized by possession of at least 50% sequence identity
counted over the full length alignment with the amino acid sequence
of a native MHC domain polypeptide using the NCBI Blast 2.0, gapped
blastp set to default parameters. Proteins with even greater
similarity to the reference sequences will show increasing
percentage identities when assessed by this method, such as at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 90% or at least 95% sequence identity. When less than the
entire sequence is being compared for sequence identity, variants
will typically possess at least 75% sequence identity over short
windows of 10-20 amino acids, and may possess sequence identities
of at least 85% or at least 90% or 95% depending on their
similarity to the reference sequence. Methods for determining
sequence identity over such short windows are described at the NCBI
website. Variants of MHC domain polypeptides also retain the
biological activity of the native polypeptide. For the purposes of
this invention, that activity is conveniently assessed by
incorporating the variant domain in the appropriate .beta.1.alpha.1
or .alpha.1.alpha.2 polypeptide and determining the ability of the
resulting polypeptide to inhibit antigen specific T-cell
proliferation in vitro, as described in detail below.
[0058] Linker sequence: a linker sequence is an amino acid sequence
that covalently links two polypeptide domains. Linker sequences may
be included in the recombinant MHC polypeptides of the present
invention to provide rotational freedom to the linked polypeptide
domains and thereby to promote proper domain folding and inter- and
intra-domain bonding. By way of example, in a recombinant
polypeptide comprising Ag-.beta.1-.alpha.1(where Ag=antigen) linker
sequences may be provided between both the Ag and .beta.1 domains
and between .beta.1 and .alpha.1 domains. Linker sequences, which
are generally between 2 and 25 amino acids in length, are well
known in the art and include the glycine(4)-serine spacer
(GGGGS.times.3) described by Chaudhary et al. (1989).
[0059] Recombinant MHC class I .alpha.1.alpha.2 polypeptides
according to the present invention include a covalent linkage
joining the carboxy terminus of the .alpha.1 domain to the amino
terminus of the .alpha.2 domain. The .alpha.1 and .alpha.2 domains
of native MHC class I .alpha. chains are typically covalently
linked in this orientation by an amino acid linker sequence. This
native linker sequence may be maintained in the recombinant
constructs; alternatively, a recombinant linker sequence may be
introduced between the .alpha.1 and .alpha.2 domains (either in
place of or in addition to the native linker sequence).
[0060] Additional definitions of terms commonly used in molecular
genetics can be found in Benjamin Lewin, Genes V published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0061] The following sections provide detailed guidance on the
design, expression and uses of the recombinant MHC molecules of the
invention. Unless otherwise stated, standard molecular biology,
biochemistry and immunology methods are used in the present
invention unless otherwise described. Such standard methods are
described in Sambrook et al. (1989), Ausubel et al (1987), Innis et
al. (1990) and Harlow and Lane (1988). The following U.S. patents
which relate to conventional formulations of MHC molecules and
their uses are incorporated herein by reference to provide
additional background and technical information relevant to the
present invention: U.S. Pat. Nos. 5,130,297; 5,194,425; 5,260,422;
5,284,935; 5,468,481; 5,595,881; 5,635,363; 5,734,023.
2. Design Of Recombinant MHC Class II .beta.1.alpha.1 Molecules
[0062] The amino acid sequences of mammalian MHC class II .alpha.
and .beta. chain proteins, as well as nucleic acids encoding these
proteins, are well known in the art and available from numerous
sources including GenBank. Exemplary sequences are provided in
Auffray et al. (1984) (human HLA DQ .alpha.); Larhammar et al.
(1983) (human HLA DQ .beta.); Das et al. (1983) (human HLA DR
.alpha.); Tonnelle et al. (1985) (human HLA DR .beta.); Lawrance et
al. (1985) (human HLA DP .alpha.); Kelly et al. (1985) (human HLA
DP .beta.); Syha et al. (1989) (rat RT1.B .alpha.);
Syha-Jedelhauser et al. (1991) (rat RT1.B .beta.); Benoist et al.
(1983) (mouse I-A .alpha.); Estess et al. (1986) (mouse I-A
.beta.).
[0063] The recombinant MHC class II molecules of the present
invention comprise the .beta.1 domain of the MHC class II .beta.
chain covalently linked to the .alpha.1 domain of the MHC class II
.alpha. chain. The .beta.1 and .alpha.1 domains are well defined in
mammalian MHC class II proteins. Typically, the .alpha.1 domain is
regarded as comprising about residues 1-90 of the mature .alpha.
chain. The native peptide linker region between the .alpha.1 and
.alpha.2 domains of the MHC class II protein spans from about amino
acid 76 to about amino acid 93 of the .alpha. chain, depending on
the particular .alpha. chain under consideration. Thus, an .alpha.1
domain may include about amino acid residues 1-90 of the .alpha.
chain, but one of skill in the art will recognize that the
C-terminal cut-off of this domain is not necessarily precisely
defined, and, for example, might occur at any point between amino
acid residues 70-100 of the .alpha. chain. The composition of the
.alpha.1 domain may also vary outside of these parameters depending
on the mammalian species and the particular .alpha. chain in
question. One of skill in the art will appreciate that the precise
numerical parameters of the amino acid sequence are much less
important than the maintenance of domain function.
[0064] Similarly, the .beta.1 domain is typically regarded as
comprising about residues 1-90 of the mature .beta. chain. The
linker region between the .beta.1 and .beta.2 domains of the MHC
class II protein spans from about amino acid 85 to about amino acid
100 of the .beta. chain, depending on the particular .beta. chain
under consideration. Thus, the .beta.1 protein may include about
amino acid residues 1-100, but one of skill in the art will again
recognize that the C-terminal cut-off of this domain is not
necessarily precisely defined, and, for example, might occur at any
point between amino acid residues 75-105 of the .beta. chain. The
composition of the .beta.1 domain may also vary outside of these
parameters depending on the mammalian species and the particular
.beta. chain in question. Again, one of skill in the art will
appreciate that the precise numerical parameters of the amino acid
sequence are much less important than the maintenance of domain
function. Exemplary .beta.1.alpha.1 molecules from human, rat and
mouse are depicted in FIG. 10.
[0065] Nucleic acid molecules encoding these domains may be
produced by standard means, such as amplification by the polymerase
chain reaction (PCR). Standard approaches for designing primers for
amplifying open reading frames encoding these domain may be
employed. Libraries suitable for the amplification of these domains
include, for example, cDNA libraries prepared from the mammalian
species in question; such libraries are available commercially, or
may be prepared by standard methods. Thus, for example, constructs
encoding the .beta.1 and .alpha.1 polypeptides may be produced by
PCR using four primers: primers B1 and B2 corresponding to the 5'
and 3' ends of the .beta.1 coding region, and primers A1 and A2
corresponding to the 5' and 3' ends of the .alpha.1 coding region.
Following PCR amplification of the .alpha.1 and .beta.1 domain
coding regions, these amplified nucleic acid molecules may each be
cloned into standard cloning vectors, or the molecules may be
ligated together and then cloned into a suitable vector. To
facilitate convenient cloning of the two coding regions,
restriction endonuclease recognition sites may be designed into the
PCR primers. For example, primers B2 and A1 may each include a
suitable site such that the amplified fragments may be readily
ligated together following amplification and digestion with the
selected restriction enzyme. In addition, primers B1 and A2 may
each include restriction sites to facilitate cloning into the
polylinker site of the selected vector. Ligation of the two domain
coding regions is performed such that the coding regions are
operably linked, i.e., to maintain the open reading frame. Where
the amplified coding regions are separately cloned, the fragments
may be subsequently released from the cloning vector and gel
purified, preparatory to ligation.
[0066] In certain embodiments, a peptide linker is provided between
the .beta.1 and .alpha.1 domains. Typically, this linker is between
2 and 25 amino acids in length, and serves to provide flexibility
between the domains such that each domain is free to fold into its
native conformation. The linker sequence may conveniently be
provided by designing the PCR primers to encode the linker
sequence. Thus, in the example described above, the linker sequence
may be encoded by one of the B2 or A1 primers, or a combination of
each of these primers.
3. Design Of Recombinant MHC Class I .alpha.1.alpha.2 Molecules
[0067] The amino acid sequences of mammalian MHC class I .alpha.
chain proteins, as well as nucleic acids encoding these proteins,
are well known in the art and available from numerous sources
including GenBank. Exemplary sequences are provided in Browning et
al. (1995) (human HLA-A); Kato et al. (1993) (human HLA-B); Steinle
et al. (1992) (human HLA-C); Walter et al. (1995) (rat Ia); Walter
et al. (1994) (rat Ib); Kress et al. (1983) (mouse H-2-K); Schepart
et al. (1986) (mouse H-2-D); and Moore et al. (1982) (mouse
H-2-1).
[0068] The recombinant MHC class I molecules of the present
invention comprise the .alpha.1 domain of the MHC class I .alpha.
chain covalently linked to the .alpha.2 domain of the MHC class I
.alpha. chain. These two domains are well defined in mammalian MHC
class I proteins. Typically, the .alpha.1 domain is regarded as
comprising about residues 1-90 of the mature .alpha. chain and the
.alpha.2 chain as comprising about amino acid residues 90-180,
although again, the cut-off points are not precisely defined and
will vary between different MHC class I molecules. The boundary
between the .alpha.2 and .alpha.3 domains of the MHC class I
.alpha. protein typically occurs in the region of amino acids
179-183 of the mature .alpha. chain. The composition of the
.alpha.1 and .alpha.2 domains may also vary outside of these
parameters depending on the mammalian species and the particular
.alpha. chain in question. One of skill in the art will appreciate
that the precise numerical parameters of the amino acid sequence
are much less important than the maintenance of domain function. An
exemplary a .alpha.1.alpha.2 molecule is depicted in FIG. 11.
[0069] The .alpha.1.alpha.2 construct may be most conveniently
constructed by amplifying the reading frame encoding the
dual-domain (.alpha.1 and .alpha.2) region between amino acid
number 1 and amino acids 179-183, although one of skill in the art
will appreciate that some variation in these end-points is
possible. Such a molecule includes the native linker region between
the .alpha.1 and .alpha.2 domains, but if desired that linker
region may be removed and replaced with a synthetic linker peptide.
The general considerations for amplifying and cloning the MHC class
I .alpha.1 and .alpha.2 domains apply as discussed above in the
context of the class II .beta.1 and .alpha.1 domains.
4. Genetic Linkage of Antigenic Polypeptide to .beta.1.alpha.1 and
.alpha.1.alpha.2 Molecules
[0070] The class II .beta.1.alpha.1 and class I .alpha.1.alpha.2
polypeptides of the invention are generally used in conjunction
with an antigenic peptide. Any antigenic peptide that is
conventionally associated with class I or class II MHC molecules
and recognized by a T-cell can be used for this purpose. Antigenic
peptides from a number of sources have been characterized in
detail, including antigenic peptides from honey bee venom
allergens, dust mite allergens, toxins produced by bacteria (such
as tetanus toxin) and human tissue antigens involved in autoimmune
diseases. Detailed discussions of such peptides are presented in
U.S. Pat. Nos. 5,595,881, 5,468,481 and 5,284,935. Exemplary
peptides include those identified in the pathogenesis of rheumatoid
arthritis (type II collagen), myasthenia gravis (acetyl choline
receptor), and multiple sclerosis (myelin basic protein).
[0071] As is well known in the art (see for example U.S. Pat. No.
5,468,481) the presentation of antigen in MHC complexes on the
surface of APCs generally does not involve a whole antigenic
peptide. Rather, a peptide located in the groove between the
.beta.1 and .alpha.1 domains (in the case of MHC II) or the
.alpha.1 and .alpha.2 domains (in the case of MHC I) is typically a
small fragment of the whole antigenic peptide. As discussed in
Janeway & Travers (1997), peptides located in the peptide
groove of MHC class I molecules are constrained by the size of the
binding pocket and are typically 8-15 amino acids long, more
typically 8-10 amino acids in length (but see Collins et al., 1994
for possible exceptions). In contrast, peptides located in the
peptide groove of MHC class II molecules are not constrained in
this way and are often much larger, typically at least 13 amino
acids in length. Peptide fragments for loading into MHC molecules
can be prepared by standard means, such as use of synthetic peptide
synthesis machines.
[0072] The .beta.1.alpha.1 and .alpha.1.alpha.2 molecules of the
present invention may be "loaded" with peptide antigen in a number
of ways, including by covalent attachment of the peptide to the MHC
molecule. This may be conveniently achieved by operably linking a
nucleic acid sequence encoding the selected peptide to the 5' end
of the construct encoding the MHC protein such that, in the
expressed peptide, the antigenic peptide domain is linked to the
N-terminus of .beta.1 in the case of .beta.1.alpha.1 molecules and
.alpha.1 in the case of .alpha.1.alpha.2 molecules. One convenient
way of obtaining this result is to incorporate a sequence encoding
the antigen into the PCR primers used to amplify the MHC coding
regions. Typically, a sequence encoding a linker peptide sequence
will be included between the molecules encoding the antigenic
peptide and the MHC polypeptide. As discussed above, the purpose of
such linker peptides is to provide flexibility and permit proper
conformational folding of the peptides. For linking antigens to the
MHC polypeptide, the linker should be sufficiently long to permit
the antigen to fit into the peptide groove of the MHC polypeptide.
Again, this linker may be conveniently incorporated into the PCR
primers. However, as discussed in Example 1 below, it is not
necessary that the antigenic peptide be ligated exactly at the 5'
end of the MHC coding region. For example, the antigenic coding
region may be inserted within the first few (typically within the
first 10) codons of the 5' end of the MHC coding sequence.
[0073] This genetic system for linkage of the antigenic peptide to
the MHC molecule is particularly useful where a number of MHC
molecules with differing antigenic peptides are to be produced. The
described system permits the construction of an expression vector
in which a unique restriction site is included at the 5' end of the
MHC coding region (i.e., at the 5' end of .beta.1 in the case of
.beta.1.alpha.1-encoding constructs and at the 5' end of .alpha.1
in the case of .alpha.1.alpha.2-encoding constructs). In
conjunction with such a construct, a library of antigenic
peptide-encoding sequences is made, with each antigen-coding region
flanked by sites for the selected restriction enzyme. The inclusion
of a particular antigen into the MHC molecule is then performed
simply by (a) releasing the antigen-coding region with the selected
restriction enzyme, (b) cleaving the MHC construct with the same
restriction enzyme, and (c) ligating the antigen coding region into
the MHC construct. In this manner, a large number of
MHC-polypeptide constructs can be made and expressed in a short
period of time.
[0074] An exemplary design of an expression cassette allowing
simple exchange of antigenic peptides in the context of a
.beta.1.alpha.1 molecule is shown in FIG. 1. FIG. 1A shows the
nucleic acid sequence encoding a prototype .beta.1.alpha.1 molecule
derived from rat MHC class II RT1.B, without the presence of the
antigenic peptide. The position of the insertion site for the
peptide and linker between the 5th and 6th (serine and proline)
residues of the .beta.1 domain is indicated by a symbol. In order
to integrate the antigen coding region, a PCR primer comprising the
sequence shown in FIG. 1B joined with additional bases from the
FIG. 1A construct 3' of the insertion site is employed in
conjunction with a PCR primer reading from the 3' end of the
construct shown in FIG. 1A.) Amplification yields a product that
includes the sequence shown in FIG. 1B integrated into the
.beta.1.alpha.1 construct (i.e., with the antigenic peptide and
linker sequences positioned between the codons encoding the 5th and
6th amino acid residues of the .beta.1.alpha.1 sequence). In the
case illustrated, the antigenic peptide is the MBP-72-89
antigen.
[0075] Notably, the MBP-72-89 coding sequence is flanked by unique
Nco I and Spe I restriction enzyme sites. These enzymes can be used
to release the MBP-72-89 coding region and replace it with coding
regions for other antigens, for example those illustrated in FIGS.
1C and 1D.
[0076] The structure of the expressed .beta.1.alpha.1 polypeptide
with covalently attached antigen is illustrated in FIG. 2B; FIG. 2A
shows the secondary structure of the complete RT1B molecule
(including .alpha.1, .alpha.2, .beta.1 and .beta.2 domains).
[0077] Nucleic acid expression vectors including expression
cassettes designed as explained above will be particularly useful
for research purposes. Such vectors will typically include
sequences encoding the dual domain MHC polypeptide (.beta.1.alpha.1
or .alpha.1.alpha.2) with a unique restriction site provided
towards the 5' terminus of the MHC coding region, such that a
sequence encoding an antigenic polypeptide may be conveniently
attached. Such vectors will also typically include a promoter
operably linked to the 5' terminus of the MHC coding region to
provide for high level expression of the sequences.
[0078] .beta.1.alpha.1 and .alpha.1.alpha.2 molecules may also be
expressed and purified without an attached peptide (as described in
section 5 below), in which case they may be referred to as "empty".
The empty MHC molecules may then be loaded with the selected
peptide as described in section 6 below.
5. Expression and Purification of Recombinant .beta.1.alpha.1 and
(.alpha.1.alpha.2 Molecules
[0079] In their most basic form, nucleic acids encoding the MHC
polypeptides of the invention comprise first and second regions,
having a structure A-B wherein, for class I molecules, region A
encodes the class I .alpha.1 domain and region B encodes the class
I .alpha.2 domain. For class II molecules, A encodes the class II
.beta.1 domain and B encodes the class II .alpha.1 domain. Where a
linker sequence is included, the nucleic acid may be represented as
B-L2-A, wherein L2 is a nucleic acid sequence encoding the linker
peptide. Where an antigenic peptide is covalently linked to the MHC
polypeptide, the nucleic acid molecule encoding this complex may be
represented as P-B-A. A second linker sequence may be provided
between the antigenic protein and the region B polypeptide, such
that the coding sequence is represented as P-L2-B-L1-A. In all
instances, the various nucleic acid sequences that comprise the MHC
polypeptide (i.e., L1, L2, B, A and P) are operably linked such
that the elements are situated in a single reading frame.
[0080] Nucleic acid constructs expressing these MHC polypeptides
may also include regulatory elements such as promoters (Pr),
enhancers and 3' regulatory regions, the selection of which will be
determined based upon the type of cell in which the protein is to
be expressed. When a promoter sequence is operably linked to the
open reading frame, the sequence may be represented as Pr-B-A, or
(if an antigen-coding region is included) Pr-P-B-A, wherein Pr
represents the promoter sequence. The promoter sequence is operably
linked to the P or B components of these sequences, and the B-A or
P-B-A sequences comprise a single open reading frame. The
constructs are introduced into a vector suitable for expressing the
MHC polypeptide in the selected cell type.
[0081] Numerous prokaryotic and eukaryotic systems are known for
the expression and purification of polypeptides. For example,
heterologous polypeptides can be produced in prokaryotic cells by
placing a strong, regulated promoter and an efficient ribosome
binding site upstream of the polypeptide-encoding construct.
Suitable promoter sequences include the beta-lactamase, tryptophan
(trp), `phage T7 and lambda P.sub.L promoters. Methods and plasmid
vectors for producing heterologous proteins in bacteria are
described in Sambrook et al. (1989). Suitable prokaryotic cells for
expression of large amounts of .beta..sub.2m fusion proteins
include Escherichia coli and Bacillus subtilis. Often, proteins
expressed at high levels are found in insoluble inclusion bodies;
methods for extracting proteins from these aggregates are described
by Sambrook et al. (1989) (ch. 17). Recombinant expression of MHC
polypeptides in prokaryotic cells may alternatively be conveniently
obtained using commercial systems designed for optimal expression
and purification of fusion proteins. Such fusion proteins typically
include a protein tag that facilitates purification. Examples of
such systems include: the pMAL protein fusion and purification
system (New England Biolabs, Inc., Beverly, Mass.); the GST gene
fusion system (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.);
and the pTrcHis expression vector system (Invitrogen, Carlsbad,
Calif.). For example, the pMAL expression system utilizes a vector
that adds a maltose binding protein to the expressed protein. The
fusion protein is expressed in E. coli. and the fusion protein is
purified from a crude cell extract using an amylose column. If
necessary, the maltose binding protein domain can be cleaved from
the fusion protein by treatment with a suitable protease, such as
Factor Xa. The maltose binding fragment can then be removed from
the preparation by passage over a second amylose column.
[0082] The MHC polypeptides can also be expressed in eukaryotic
expression systems, including Pichia pastoris, Drosophila,
Baculovirus and Sindbis expression systems produced by Invitrogen
(Carlsbad, Calif.). Eukaryotic cells such as Chinese Hamster ovary
(CHO), monkey kidney (COS), HeLa, Spodoptera frugiperda, and
Saccharomyces cerevisiae may also be used to express the MHC
polypeptides. Regulatory regions suitable for use in these cells
include, for mammalian cells, viral promoters such as those from
CMV, adenovirus and SV40, and for yeast cells, the promoter for
3-phosphoglycerate kinase and alcohol dehydrogenase.
[0083] The transfer of DNA into eukaryotic, in particular human or
other mammalian cells, is now a conventional technique. The vectors
are introduced into the recipient cells as pure DNA (transfection)
by, for example, precipitation with calcium phosphate or strontium
phosphate, electroporation, lipofection, DEAE dextran,
microinjection, protoplast fusion, or microprojectile guns.
Alternatively, the nucleic acid molecules can be introduced by
infection with virus vectors. Systems are developed that use, for
example, retroviruses, adenoviruses, or Herpes virus.
[0084] An MHC polypeptide produced in mammalian cells may be
extracted following release of the protein into the supernatant and
may be purified using an immunoaffinity column prepared using
anti-MHC antibodies. Alternatively, the MHC polypeptide may be
expressed as a chimeric protein with, for example, b-globin.
Antibody to b-globin is thereafter used to purify the chimeric
protein. Corresponding protease cleavage sites engineered between
the b-globin gene and the nucleic acid sequence encoding the MHC
polypeptide are then used to separate the two polypeptide fragments
from one another after translation. One useful expression vector
for generating b-globin chimeric proteins is pSG5 (Stratagene, La
Jolla, Calif.).
[0085] Expression of the MHC polypeptides in prokaryotic cells will
result in polypeptides that are not glycosylated. Glycosylation of
the polypeptides at naturally occurring glycosylation target sites
may be achieved by expression of the polypeptides in suitable
eukaryotic expression systems, such as mammalian cells.
[0086] Purification of the expressed protein is generally performed
in a basic solution (typically around pH 10) containing 6 M urea.
Folding of the purified protein is then achieved by dialysis
against a buffered solution at neutral pH (typically phosphate
buffered saline (PBS) at around pH 7.4).
6. Antigen Loading of Empty .beta.1.alpha.1 and .alpha.1.alpha.2
Molecules
[0087] Where the .beta.1.alpha.1 and I .alpha.1.alpha.2 molecules
are expressed and purified in an empty form (i.e., without attached
antigenic peptide), the antigenic peptide may be loaded into the
molecules using standard methods. Methods for loading of antigenic
peptides into MHC molecules is described in, for example, U.S. Pat.
No. 5,468,481. Such methods include simple co-incubation of the
purified MHC molecule with a purified preparation of the
antigen.
[0088] By way of example, empty .beta.1.alpha.1 molecules (1 mg/ml;
40 uM) may be loaded by incubation with a 10-fold molar excess of
peptide (1 mg/ml; 400 uM) at room temperature, for 24 hours.
Thereafter, excess unbound peptide may be removed by dialysis
against PBS at 4.degree. C. for 24 hours. As is known in the art,
peptide binding to .beta.1.alpha.1 can be quantified by silica gel
thin layer chromatography (TLC) using radiolabeled peptide. Based
on such quantification, the loading may be altered (e.g., by
changing the molar excess of peptide or the time of incubation) to
obtain the desired result.
7. Other Considerations,
[0089] a. Sequence Variants
[0090] While the foregoing discussion uses as examples naturally
occurring MHC class I and class II molecules and the various
domains of these molecules, one of skill in the art will appreciate
that variants of these molecules and domains may be made and
utilized in the same manner as described. Thus, reference herein to
a domain of an MHC polypeptide or molecule (e.g., an MHC class II
.beta.1 domain) includes both naturally occurring forms of the
referenced molecule, as well as molecules that are based on the
amino acid sequence of the naturally occurring form, but which
include one or more amino acid sequence variations. Such variant
polypeptides may also be defined in the degree of amino acid
sequence identity that they share with the naturally occurring
molecule. Typically, MHC domain variants will share at least 80%
sequence identity with the sequence of the naturally occurring MHC
domain. More highly conserved variants will share at least 90% or
at least 95% sequence identity with the naturally occurring
sequence. Variants of MHC domain polypeptides also retain the
biological activity of the naturally occurring polypeptide. For the
purposes of this invention, that activity is conveniently assessed
by incorporating the variant domain in the appropriate
.beta.1.alpha.1 or .alpha.1.alpha.2 polypeptide and determining the
ability of the resulting polypeptide to inhibit antigen specific
T-cell proliferation in vitro, as described in detail below.
[0091] Variant MHC domain polypeptides include proteins that differ
in amino acid sequence from the naturally occurring MHC polypeptide
sequence but which retain the specified biological activity. Such
proteins may be produced by manipulating the nucleotide sequence of
the molecule encoding the domain, for example by site-directed
mutagenesis or the polymerase chain reaction. The simplest
modifications involve the substitution of one or more amino acids
for amino acids having similar biochemical properties. These
so-called conservative substitutions are likely to have minimal
impact on the activity of the resultant protein. Table 1 shows
amino acids which may be substituted for an original amino acid in
a protein and which are regarded as conservative substitutions.
TABLE-US-00001 TABLE 1 Original Residue Conservative Substitutions
Ala ser Asn gln; his Asp glu Cys ser Gln asn Glu asp Gly pro His
asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met leu; ile
Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile;
leu
[0092] More substantial changes in biological function or other
features may be obtained by selecting substitutions that are less
conservative than those in Table 1, i.e., selecting residues that
differ more significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. The substitutions which in general
are expected to produce the greatest changes in protein properties
will be those in which (a) a hydrophilic residue, e.g., seryl or
threonyl, is substituted for (or by) a hydrophobic residue, e.g.,
leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or
proline is substituted for (or by) any other residue; (c) a residue
having an electropositive side chain, e.g., lysyl, arginyl, or
histadyl, is substituted for (or by) an electronegative residue,
e.g., glutamyl or aspartyl; or (d) a residue having a bulky side
chain, e.g., phenylalanine, is substituted for (or by) one not
having a side chain, e.g., glycine. The effects of these amino acid
substitutions or deletions or additions may be assessed through the
use of the described T-cell proliferation assay.
[0093] At the nucleic acid level, one of skill in the art will
appreciate that the naturally occurring nucleic acid sequences that
encode class I and II MHC domains may be employed in the expression
vectors, but that the invention is not limited to such sequences.
Any sequence that encodes a functional MHC domain may be employed,
and the nucleic acid sequence may be adapted to conform with the
codon usage bias of the organism in which the sequence is to be
expressed.
[0094] b. Incorporation of Detectable Markers
[0095] For certain in vivo and in vitro applications, the MHC
molecules of the present invention may be conjugated with a
detectable label. A wide range of detectable labels are known,
including radionuclides (e.g., gamma-emitting sources such as
indium-111), paramagnetic isotopes, fluorescent markers (e.g.,
fluorescein), enzymes (such as alkaline phosphatase), cofactors,
chemiluminescent compounds and bioluminescent compounds. The
binding of such labels to the MHC polypeptides may be achieved
using standard methods. U.S. Pat. No. 5,734,023 contains an
extensive discussion of the labeling of MHC polypeptide derivatives
using such labels. Where the detectable marker is to be covalently
linked to the MHC molecule in a directed manner (i.e., rather than
being randomly attached) it will generally be linked to the C
terminus of the molecule so as to minimize interference with a
peptide antigen linked at the N terminus.
[0096] c. Conjugation of Toxic Moieties
[0097] For certain uses of the disclosed MHC polypeptides,
particularly in vivo therapeutic applications aimed at depleting
certain T-cell populations, the polypeptides may be conjugated with
a toxic moiety. Numerous toxic moieties suitable for disrupting
T-cell function are known, including protein toxins,
chemotherapeutic agents, antibodies to a cytotoxic T-cell surface
molecule, lipases, and radioisotopes emitting "hard" e.g., beta
radiation. Examples of such toxins and methods of conjugating
toxins to MHC molecules are described in U.S. Pat. No. 5,284,935.
Protein toxins include ricin, diphtheria and, Pseudomonas toxin.
Chemotherapeutic agents include doxorubicin, daunorubicin,
methotrexate, cytotoxin, and antisense RNA. Radioisotopes such as
yttrium-90, phosphorus-32, lead-212, iodine-131, or palladium-109
may also be used. Where the toxic moiety is to be covalently linked
to the MHC molecule in a directed manner (i.e., rather than being
randomly attached) it will generally be linked to the C terminus of
the molecule so as to minimize interference with a peptide antigen
linked at the N terminus.
[0098] d. Pharmaceutical Formulations
[0099] For administration to animals, purified MHC polypeptides of
the present invention are generally combined with a
pharmaceutically acceptable carrier. In general, the nature of the
carrier will depend on the particular mode of administration being
employed. For instance, parenteral formulations usually comprise
injectable fluids that include pharmaceutically and physiologically
acceptable fluids such as water, physiological saline, balanced
salt solutions, aqueous dextrose, glycerol or the like as a
vehicle. For solid compositions (e.g., powder, pill, tablet, or
capsule forms), conventional non-toxic solid carriers can include,
for example, pharmaceutical grades of mannitol, lactose, starch, or
magnesium stearate. In addition to biologically-neutral carriers,
pharmaceutical compositions to be administered can contain minor
amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, and pH buffering agents and the
like, for example sodium acetate or sorbitan monolaurate.
[0100] As is known in the art, protein-based pharmaceuticals may be
only inefficiently delivered through ingestion. However, pill-based
forms of pharmaceutical proteins may alternatively be administered
subcutaneously, particularly if formulated in a slow-release
composition. Slow-release formulations may be produced by combining
the target protein with a biocompatible matrix, such as
cholesterol. Another possible method of administering protein
pharmaceuticals is through the use of mini osmotic pumps. As stated
above a biocompatible carrier would also be used in conjunction
with this method of delivery. Additional possible methods of
delivery include deep lung delivery by inhalation (Edwards et al.,
1997; Service, 1997) and trans-dermal delivery (Mitragotri et al.,
1996).
[0101] It is also contemplated that the MHC polypeptides of the
present invention could be delivered to cells in the nucleic acid
form and subsequently translated by the host cell. This could be
done, for example through the use viral vectors or liposomes.
Liposomes could also be used for direct delivery of the
polypeptides.
[0102] The pharmaceutical compositions of the present invention may
be administered by any means that achieve their intended purpose.
Amounts and regimens for the administration of the selected MHC
polypeptides will be determined by the attending clinician.
Effective doses for therapeutic application will vary depending on
the nature and severity of the condition to be treated, the
particular MHC polypeptide selected, the age and condition of the
patient and other clinical factors. Typically, the dose range will
be from about 0.1 ug/kg body weight to about 100 mg/kg body weight.
Other suitable ranges include doses of from about 100 ug/kg to 1
mg/kg body weight. The dosing schedule may vary from once a week to
daily depending on a number of clinical factors, such as the
subject's sensitivity to the protein. Examples of dosing schedules
are 3 ug/kg administered twice a week, three times a week or daily;
a dose of 7 ug/kg twice a week, three times a week or daily; a dose
of 10 ug/kg twice a week, three times a week or daily; or a dose of
30 ug/kg twice a week, three times a week or daily.
8. Exemplary Applications of Recombinant .beta.1.alpha.1 and
.alpha.1.alpha.2 Molecules
[0103] The class II .beta.1.alpha.1 and class I .alpha.1.alpha.2
polypeptides of the present invention are useful for a wide range
of in vitro and in vivo applications. Indeed, as a result of the
biological activities of these polypeptides, they may be used in
numerous application in place of either intact purified MHC
molecules, or antigen presenting cells that express MHC
molecules.
[0104] In vitro applications of the disclosed polypeptides include
the detection, quantification and purification of antigen-specific
T-cells. Methods for using various forms of MHC-derived complexes
for these purposes are well known and are described in, for
example, U.S. Pat. Nos. 5,635,363 and 5,595,881. For such
applications, the disclosed polypeptides may be free in solution or
may be attached to a solid support such as the surface of a plastic
dish, a microtiter plate, a membrane, or beads. Typically, such
surfaces are plastic, nylon or nitrocellulose. Polypeptides in free
solution are useful for applications such as fluorescence activated
sell sorting (FACS). For detection and quantification of
antigen-specific T-cells, the polypeptides are preferably labeled
with a detectable marker, such as a fluorescent marker.
[0105] The T-cells to be detected, quantified or otherwise
manipulated are generally present in a biological sample removed
from a patient. The biological sample is typically blood or lymph,
but may also be tissue samples such as lymph nodes, tumors, joints
etc. It will be appreciated that the precise details of the method
used to manipulate the T-cells in the sample will depend on the
type of manipulation to be performed and the physical form of both
the biological sample and the MHC molecules. However, in general
terms, the .beta.1.alpha.1/peptide complex or
.alpha.1.alpha.2/peptide complex is added to the biological sample,
and the mixture is incubated for sufficient time (e.g., from about
5 minutes up to several hours) to allow binding. Detection and
quantification of T-cells bound to the MHC/peptide complex may be
performed by a number of methods including, where the MHC/peptide
includes a fluorescent label, fluorescence microscopy and FACS.
Standard immunoassays such as ELISA and RIA may also be used to
quantify T-cell--MHC/peptide complexes where the MHC/peptide
complexes are bound to a solid support. Quantification of
antigen-specific T-cell populations will be especially useful in
monitoring the course of a disease. For example, in a multiple
sclerosis patient, the efficacy of a therapy administered to reduce
the number of MBP-reactive T-cells may be monitored using MHC/MBP
antigen complexes to quantify the number of such T-cells present in
the patient. Similarly, the number of anti-tumor T-cells in a
cancer patient may be quantified and tracked over the course of a
therapy using MHC/tumor antigen complexes.
[0106] FACS may also be used to separate T-cell--MHC/peptide
complexes from the biological sample, which may be particularly
useful where a specified population of antigen-specific T-cells is
to be removed from the sample, such as for enrichment purposes.
Where the MHC/peptide complex is bound to magnetic beads, the
binding T-cell population may be purified as described by Miltenyi
et al (1990). By way of example, anti-tumor T-cells in the blood of
a cancer patient may be purified using these methods, expanded in
vitro and returned to the patient as part of an adoptive
immunotherapy treatment.
[0107] A specified antigen-specific T-cell population in the
biological sample may be anergized by incubation of the sample with
MHC/peptide complexes containing the peptide recognized by the
targeted T-cells. Thus, when these complexes bind to the TCR in the
absence of other co-stimulatory molecules, a state of anergy is
induced in the T-cell. Such an approach is useful in situations
where the targeted T-cell population recognizes a self-antigen,
such as in various autoimmune diseases. Alternatively, the targeted
T-cell population may be killed directly by incubation of the
biological sample with an MHC/peptide complex conjugated with a
toxic moiety.
[0108] T-cells may also be activated in an antigen-specific manner
by the polypeptides of the invention. For example, the disclosed
MHC polypeptides loaded with a specified antigen may be adhered at
a high density to a solid surface, such as a plastic dish or a
magnetic bead. Exposure of T-cells to the polypeptides on the solid
surface can stimulate and activate T-cells in an antigen-specific
manner, despite the absence of co-stimulatory molecules. This is
likely attributable to sufficient numbers of TCRs on a T-cell
binding to the MHC/peptide complexes that co-stimulation is
unnecessary for activation.
[0109] In vivo applications of the disclosed polypeptides include
the amelioration of conditions mediated by antigen-specific
T-cells. Such conditions include allergies, transplant rejection
and autoimmune diseases including multiple sclerosis, rheumatoid
arthritis, systemic lupus erythematosus, and insulin-dependent
diabetes mellitus. Other researchers have described various forms
of MHC polypeptides that may be used to treat these conditions and
the methods used in those systems are equally useful with the MHC
polypeptides of the present invention. Exemplary methodologies are
described in U.S. Pat. Nos. 5,130,297, 5,284,935, 5,468,481,
5,734,023 and 5,194,425. By way of example, the MHC/peptide
complexes may be administered to patients in order to induce anergy
in self-reactive T-cell populations, or these T-cell populations
may be treated by administration of MHC/peptide complexes
conjugated with a toxic moiety. The disclosed molecules may also be
used to boost immune response in certain conditions such as cancer
and infectious diseases.
EXAMPLES
[0110] The following Examples illustrate certain aspects of the
invention.
Example 1
Cloning, Expression and In Vitro Folding of .beta.1.alpha.1
Molecules
[0111] A prototypical nucleic acid construct was produced that
encoded a single polypeptide chain with the amino terminus of the
MHC class II .alpha.1 domain genetically linked to the carboxyl
terminus of the MHC class II .beta.1 domain. The sequence of this
prototypical construct, made from the rat RT1B .alpha.- and
.beta.-chain cDNAs is shown in FIG. 1A (Seq. I.D. No. 1).
[0112] RT1B .alpha.1- and .beta.1-domain encoding cDNAs were
prepared by PCR amplification of cloned RT1.B .alpha.- and
.beta.-chain cDNA coding sequences (.alpha.6, .beta.118,
respectively) obtained from Dr. Konrad Reske, Mainz, FRG (Syha et
al., 1989; Syha-Jedelhauser et al., 1991). The primers used to
generate .beta.1 were 5'-AATTCCTCGAGATGGCTCTGCAGACCCC-3' (XhoI 5'
primer) (Seq. I.D. No. 9); 5'-TCTTGACCTCCAAGCCGCCGCAGGGAGGTG-3' (3'
ligation primer) (Seq. I.D. No. 10). The primers used to generate
.alpha.1 were 5'-CGGCGGCTTGGAGGTCAAGACGACATTGAGG-3' (5' ligation
primer) (Seq. I.D. No. 11);
5'-GCCTCGGTACCTTAGTTGACAGCTTGGGTTGAATTTG-3' (KpnI3' primer) (Seq.
I.D. No. 12). Additional primers used were
5'-CAGGGACCATGGGCAGAGACTCCCCA-3' (NcoI5' primer) (Seq. I.D. No.
13); and 5'-GCCTCCTCGAGTTAGTTGACAGCTTGGGTT-3' (XhoI3' primer) (Seq.
I.D. No. 14). Step one involved production of cDNAs encoding the
.beta.1 and .alpha.1 domains. PCR was conducted with Taq polymerase
(Promega, Madison, Wis.) through 28 cycles of denaturation at
94.5.degree. C. for 20 seconds, annealing at 55.degree. C. for 1.5
minutes and extension at 72.degree. C. for 1.5 minutes, using
.beta.118 as template and the XhoI5' primer and 3' ligation primer
as primers and .alpha.6 cDNA as template and the 5' ligation primer
and KpnI3' primer. PCR products were isolated by agarose gel
electrophoresis and purified using Gene-Clean (Bio 101, Inc., La
Jolla, Calif.).
[0113] In step two, these products were mixed together without
additional primers and heat denaturated at 94.5.degree. C. for 5
minutes followed by 2 cycles of denaturation at 94.5.degree. C. for
1 minute, annealing at 60.degree. C. for 2 minutes and extension at
72.degree. C. for 5 minutes. In step three, the annealed, extended
product was heat denaturated at 94.5.degree. C. for 5 minutes and
subjected to 26 cycles of denaturation at 94.5.degree. C. for 20
seconds, annealing at 60.degree. C. for 1 minute and extension at
72.degree. C. for 1 minute, in the presence of the XhoI5' primer
and KpnI3' primer. The final PCR product was isolated by agarose
gel electrophoresis and Gene-Cleaned. This produced a 656 base pair
cDNA encoding the .beta.1.alpha.1 molecule. The cDNA encoding the
.beta.1.alpha.1 molecule was moved into cloning vector pCR2.1
(Invitrogen, Carlsbad, Calif.) using Invitrogen's TA Cloning.RTM.
kit. The cDNA in pCR2.1 was used as template and PCR was conducted
through 28 cycles of denaturation at 94.5.degree. C. for 20
seconds, annealing at 55.degree. C. for 1.5 minutes and extension
at 72.degree. C. for 1.5 minutes, using the NcoI 5' primer and XhoI
3' primer. The PCR products were cleaved with the relevant
restriction enzymes and directionally cloned into pET21d+ (Novagen,
Madison, Wis.; Studier et al., 1990). The constructs were confirmed
by DNA sequencing. The .beta.1.alpha.1 molecule used in these
studies differs from wild-type in that it contains a beta-1 domain
Q12R amino acid substitution.
[0114] For insertion of the peptide/linker cartridge (shown in FIG.
1A), the following approach was used. The 210 bp peptide/linker
cartridge was amplified using the XhoI 5' primer and a primer of
sequence:
5'-GAAATCCCGCGGGGAGCCTCCACCTCCA-GAGCCTCGGGGCACTAGTGAGCCTCCACCTCCGAAGTGCAC-
CACTGGGTTCTCA TCCTGAGTCCTCTGGCTCTTCTGTGGGGAGTCTCTGCCCTCAGTCC-3'
(3'-MBP-72-89/linker ligation primer) (Seq. I.D. No. 15) and the
original full-length .beta.118 cDNA as a template. A 559 bp cDNA
with a 5' overhang for annealing to the peptide/linker cartridge
cDNA was generated using a primer:
5'-GCTCCCCGCGGGATTTCGTGTACCAGTTCAA-3' (5' peptide/linker ligation
primer) (Seq. I.D. No. 16); and the Kpn I3' primer and The 656 bp
.beta.1a1 cDNA as the amplification template. Annealing and
extension of the two cDNAs resulted in the 750 bp full-length
.beta.1a1/MBP-72-89 construct. Modifications at the 5' and 3' ends
of the .beta.1a1 and .beta.1a1/MBP-72-89 cDNAs were made for
subcloning into pET21d+ (Novagen, Madison, Wis.; Studier et al.,
1990) using the NcoI5' primer and the XhoI3' primer. The primers
used to generate the MBP-55-69/linker cartridge were
5'-TATTACCATGGGCAGAGACTCCTCCGGCAAGGATTCGCATCATGCGGCGCGGAC
GACCCACTACGGTGGAGGTGGAGGCTCACTAGTGCCCC-3' (5' MBP-55-69 primer)
(Seq. I.D. No. 17) and 5'-GGGGCACTAGTGAGCCTCCACCTCCACCGTAGTGGGT
CGTCCGCGCCGCATGATGCGAATCCTTGCCGGAGGAGTCTCTGCCCATGGTAAT A-3' (3'
MBP-55-69 primer) (Seq. I.D. No. 18). These were gel purified,
annealed and then cut with NcoI and XhoI for ligation into
.beta.1a1/MBP-72-89 digested with NcoI and XhoI, to produce a
plasmid encoding the .beta.1a1/MBP-55-69 covalent construct. The
primers used to generate the Guinea pig MBP-72-89/linker cartridge
were 5'-TATTACCATGGGCAGAGACTCCCCACAGAAGAGCCAGAGGTCTCAGGATGAGAA
CCCAGTGGTGCACTTCGGAGGTGGAGGCTCACTAGTGCCCC-3' (5' Gp-MBP-72-89
primer) (Seq. I.D. No. 28) and 5'-GGGGCACTAGTGAGCCTCCACCTCCGAAGT
GCACCACTGGGTTCTCATCCTGAGACCTCTGGCTCTTCTGTGGGGAGTCTCTGCC
CATGGTAAT-3' (3'Gp-MBP-72-89 primer) (Seq. I.D. No. 29). These were
gel purified, annealed and then cut with NcoI and XhoI for ligation
into .beta.1a1/MBP-72-89 digested with NcoI and XhoI, to produce a
plasmid encoding the .beta.1a1/Gp-MBP-72-89 covalent construct. The
primers used to generate the CM-2/linker cartridge were
5'-TATTACCATGGG CAGAGACTCCAAACTGGAACTGCAGTCCGCTCTGGAAGAAGCTGAAGCTT
CCCTGGAACACGGAGGTGGAGGCTCACTAGTGCCCC-3' (5'CM-2 primer) (Seq. I.D.
No. 19) and 5'-GGGGCACTAGTGAGCCTCCACCTCCGTGTFCCAGGGAAG
CTTCAGCTTCTTCCAGAGCGGACTGCAGTTCCAGTTTGGAGTCTCTGCCCATGGT AATA-3'
(3'CM-2 primer) (Seq. I.D. No. 20). These were gel purified,
annealed and then cut with NcoI and XhoI for ligation into
.beta.1a1/MBP-72-89 digested with NcoI and XhoI, to produce a
plasmid encoding the .beta.1a1/CM-2 covalent construct.
[0115] Protein expression was tested in a number of different E.
coli strains, including a thioredoxin reductase mutant which allows
disulfide bond formation in the cytoplasm (Derman et al., 1993).
With such a small molecule, it became apparent that the greatest
yield of material could be readily obtained from inclusion bodies,
refolding the protein after solubilization and purification in
buffers containing 6M urea. Accordingly, E. coli strain BL21(DE3)
cells were transformed with the pET21d+ construct containing the
.beta.1.alpha.1-encoding sequence. Bacteria were grown in one liter
cultures to mid-logarithmic phase (OD.sub.600=0.6-0.8) in
Luria-Bertani (LB) broth containing carbenicillin (50 .mu.g/ml) at
37.degree. C. Recombinant protein production was induced by
addition of 0.5 mM isopropyl .beta.-D-thiogalactoside (IPTG). After
incubation for 3 hours, the cells were centrifuged and stored at
-80.degree. C. before processing. All subsequent manipulations of
the cells were at 4.degree. C. The cell pellets were resuspended in
ice-cold PBS, pH 7.4, and sonicated for 4.times.20 seconds with the
cell suspension cooled in a salt/ice/water bath. the cell
suspension was then centrifuged, the supernatant fraction was
poured off, the cell pellet resuspended and washed three times in
PBS and then resuspended in 20 mM ethanolamine/6 M urea, pH 10, for
four hours. After centrifugation, the supernatant containing the
solubilized recombinant protein of interest was collected and
stored at 4.degree. C. until purification. Recombinant
.beta.1.alpha.1 construct was purified and concentrated by FPLC
ion-exchange chromatography using Source 30Q anion-exchange media
(Pharmacia Biotech, Piscataway, N.J.) in an XK26/20 column
(Pharmacia Biotech), using a step gradient with 20 mM
ethanolamine/6 M urea/1 M NaCl, pH 10. The homogeneous peak of the
appropriate size was collected, dialyzed extensively against PBS at
4.degree. C., pH 7.4, and concentrated by centrifugal
ultrafiltration with Centricon-10 membranes (Amicon, Beverly,
Mass.). The dialysis step, which removed the urea from the protein
preparation and reduced the final pH, resulted in spontaneous
re-folding of the expressed protein. For purification to
homogeneity, a finish step used size exclusion chromatography on
Superdex 75 media (Pharmacia Biotech) in an HR16/50 column
(Pharmacia Biotech). The final yield of purified protein varied
between 15 and 30 mg/L of bacterial culture.
[0116] Conformational integrity of the molecules was demonstrated
by the presence of a disulfide bond between cysteines .beta.15 and
.beta.79 as detected on gel shift assay, and the authenticity of
the purified protein was verified using the OX-6 monoclonal
antibody specific for RT1B by Western Blotting (data not shown).
Circular dichroism (CD) reveals that the .beta.1.alpha.1 molecules
have highly ordered secondary structures. The empty .beta.1.alpha.1
molecule contains approximately 30% alpha-helix, 15% beta-strand,
26% beta-turn, and 29% random coil structures. Comparison with the
secondary structures of class II molecules determined by x-ray
crystallography provides strong evidence that the .beta.1.alpha.1
molecules share the beta-sheet platform/anti-parallel alpha-helix
secondary structure common to all class II antigen binding domains.
Furthermore, thermal denaturation revealed a high degree of
cooperatively and stability of the molecules (data not shown).
Example 2
.beta.1.alpha.1 Molecules Bind T Lymphocytes in an Epitope-Specific
Manner
[0117] The .beta.1.alpha.1 molecule produced as described above was
tested for efficacy (T-cell binding specificity) using the
Experimental Autoimmune Encephalomyelitis (EAE) system. EAE is a
paralytic, inflammatory, and sometimes demyelinating disease
mediated by CD4+ T cells specific for central nervous system myelin
components including myelin basic protein (MBP). EAE shares similar
immunological abnormalities with the human demyelinating disease MS
(Paterson, 1981) and has been a useful model for testing
preclinical therapies for the human illness (Weiner et al, 1993;
Vandenbark et al., 1989; Howell et al., 1989; Oksenberg et al.,
1993; Yednock et al, 1992; Jameson et al., 1994; Vandenbark et al.,
1994). In Lewis rats, the dominant encephalitogenic MBP epitope
resides in the 72-89 peptide (Bourdette et al., 1991). Onset of
clinical signs of EAE occurs on day 10-11, and the disease lasts
four to eight days. The majority of invading T lymphocytes are
localized in the CNS during this period.
[0118] Materials and Methods
[0119] Test and control peptides for loading into the purified
.beta.1.alpha.1 molecule were synthesized as follows: Gp-MBP-69-89
peptide (GSLPQKSQRSQDENPVVHF) (Seq. I.D. No. 25), rat-MBP-69-89
peptide (GSLPQKSQRTQDENPVVHF) (Seq. I.D. No. 30), Gp-MBP-55-69
peptide (SGKDSHHAARTTHYG) (Seq. I.D. No. 26), and cardiac myosin
peptide CM-2 (KLELQSALEEAEASLEH) (Seq. I.D. No. 27) (Wegmann et
al., 1994) were prepared by solid-phase techniques (Hashim et al.,
1986). The Gp-MBP peptides are numbered according to the bovine MBP
sequence (Vandenbark et al., 1994; Martenson, 1984). Peptides were
loaded onto .beta.1.alpha.1 at a 1:10 protein:peptide molar ratio,
by mixing at room temperature for 24 hours, after which all
subsequent manipulations were performed at 4.degree. C. Free
peptide was then removed by dialysis or centrifugal ultrafiltration
with Centricon-10 membranes, serially diluting and concentrating
the solution until free peptide concentration was less than 2
.mu.M.
[0120] T-cell lines and the A1 hybridoma were prepared as follows:
Short-term T-lymphocyte lines were selected with MBP-69-89 peptide
from lymph node cells of naive rats or from rats immunized 12 days
earlier with Gp-MBP/CFA as described by Vandenbark et al., 1985)
The rat V.beta.8.2+ T cell hybridoma C14/BW12-12A1 (A1) used in
this study has been described previously by Burrows et al., 1996).
Briefly, the A1 hybridoma was created by fusing an encephalitogenic
LEW(RT1.sup.l) T cell clone specific for Gp-BP-72-89 (White et al.,
1989; Gold et al, 1991) with a TCR (.alpha./.beta.) negative
thymoma, BW5147 (Golding et al., 1985). Wells positive for cell
growth were tested for IL-2 production after stimulation with
antigen in the presence of APCs (irradiated Lewis rat thymocytes)
and then subcloned at limiting dilution. The A1 hybridoma secretes
IL-2 when stimulated in the presence of APCs with whole Gp-BP or
Gp-BP-69-89 peptide, which contains the minimum epitope,
MBP-72-89.
[0121] Two color immunofluorescent analysis was performed on a
FACScan instrument (Becton Dickinson, Mountain View, Calif.) using
CellQuest.TM. software. Quadrants were defined using non-relevant
isotype matched control antibodies. .beta.1.alpha.1 molecules with
and without loaded peptide were incubated with the A1 hybridoma (10
.mu.M .beta.1.alpha.1/peptide) for 17 hours, 4.degree. C., washed
three times, stained with fluorochrome (FITC or PE) conjugated
antibodies specific for rat class II (OX6-PE), and TCR V.beta.8.2
(PharMingen, San Diego, Calif.) for 15 minutes at room temperature,
and analyzed by flow cytometry. The CM-2 cell line was blocked for
one hour with unconjugated OX6, washed and then treated as the A1
hybridoma. Staining media was PBS, 2% fetal bovine serum, 0.01%
azide.
[0122] Results
[0123] Epitope-specific binding was evaluated by loading the
.beta.1.alpha.1 molecule with various peptides and incubating
.beta.1.alpha.1/peptide complexes with the A1 hybridoma that
recognizes the MBP-72-89 peptide (Burrows et al., 1997), or with a
cardiac myosin CM-2-specific cell line. As is shown in FIG. 3A, the
.beta.1.alpha.1 construct loaded with MBP-69-89 peptide
(.beta.1.alpha.1/MBP-69-89) specifically bound to the A1 hybridoma,
with a mean fluorescence intensity (MFI) of 0.8.times.10.sup.3
Units, whereas the .beta.1.alpha.1 construct loaded with CM-2
peptide (.beta.1.alpha.1/CM-2) did not stain the hybridoma.
Conversely, .beta.1.alpha.1/CM-2 specifically bound to the CM-2
line, with a MFI of 1.8.times.10.sup.3 Units, whereas the
.beta.1.alpha.1/MBP-69-89 complex did not stain the CM-2 line (FIG.
3B). The .beta.1.alpha.1 construct without exogenously loaded
peptide does not bind to either the A1 hybridoma (FIG. 3A) nor the
CM-2 line (data not shown). Thus, bound epitope directed the
specific binding of the .beta.1.alpha.1/peptide complex.
Example 3
.beta.1.alpha.1 Molecules Conjugated With A Fluorescent Label
[0124] To avoid using a secondary antibody for visualizing the
interaction of .beta.1.alpha.1/peptide molecules with TCR (such as
OX-6, used above), a .beta.1.alpha.1 molecules directly conjugated
with a chromophore was produced. The Alexa-488.TM. dye (A488;
Molecular Probes, Eugene, Oreg.) has a spectra similar to
fluorescein, but produces protein conjugates that are brighter and
more photo-stable than fluorescein conjugates. As is shown in FIG.
4, A488-conjugated .beta.1.alpha.1 (molar ratio dye/protein=1),
when loaded with MBP-69-89, bound to the A1 hybridomas (MCI=300
Units), whereas empty .beta.1.alpha.1 did not.
Example 4
.beta.1.alpha.1 Molecules Inhibit Epitope-Specific T-cell
Proliferation In Vitro
[0125] T-cell proliferation assays were performed to evaluate the
effect of the constructs on T cell activation.
[0126] Materials and Methods
[0127] Proliferation assays were performed in 96-well plates as
described previously (Vandenbark et al., 1985). Briefly,
4.times.10.sup.5 cells in 200 .mu.l/well (for organ stimulation
assays) or 2.times.10.sup.4 T cells and 1.times.10.sup.6 irradiated
APCs (for short-term T cell lines) were incubated in RPMI and 1%
rat serum in triplicate wells with stimulation medium only, Con A,
or antigen with or without supplemental IL-2 (20 Units/ml) at
37.degree. C. in 7% CO.sub.2. The cultures were incubated for three
days, the last 18 hr in the presence of [.sup.3H]thymidine (0.5
.mu.Ci/10 .mu.l/well). The cells were harvested onto glass fiber
filters and [.sup.3H]thymidine uptake assessed by liquid
scintillation. In some experiments, the T cells were pretreated 24
hours with .beta.1.alpha.1 constructs (with and without loaded
peptides), washed, and then used in proliferation assays with and
without IL-2, as above. Mean counts per minute .+-.SD were
calculated from triplicate wells and differences between groups
determined by Student's t-test.
[0128] Results
[0129] A range of concentrations (10 nM to 20 .mu.M) of
peptide-loaded .beta.1.alpha.1 complexes were pre-incubated with an
MBP-69-89 specific T cell line prior to stimulation with the
MBP-69-89 peptide+APC (antigen-presenting cell). As is shown in
FIG. 5, pre-treatment of MBP-69-89 specific T cells with 10 nM
.beta.1.alpha.1/MBP-69-89 complex significantly inhibited
proliferation (>90%), whereas pre-incubation with 20 .mu.M
.beta.1.alpha.1/MBP-55-69 complex produced a nominal (27%) but
insignificant inhibition. Of mechanistic importance, the response
inhibited by the .beta.1.alpha.1/MBP-69-89 complex could be fully
restored by including 20 Units/ml of IL-2 during stimulation of the
T cell line (FIG. 5) suggesting that the T-cells had been rendered
anergic by exposure to the .beta.1.alpha.1/MBP-69-89 complex.
Example 5
Antigen-Loaded .beta.1.alpha.1 Molecules Suppress and Treat EAE
[0130] The .beta.1.alpha.1/MBP-69-89 complex was evaluated for its
ability to suppress the induction, as well as to treat existing
signs of EAE in Lewis rats.
[0131] Materials and Methods
[0132] Female Lewis rats (Harlan Sprague-Dawley, Inc.,
Indianapolis, Ind.), 8-12 weeks of age, were used for clinical
experiments in this study. The rats were housed under germ-free
conditions at the Veterans Affairs Medical Center Animal Care
Facility, Portland, Oreg., according to institutional guidelines.
Active EAE was induced in the rats by subcutaneous injection of 25
.mu.g guinea pig myelin basic protein (GP-MBP) or 200 .mu.g
GP-MBP-69-89 peptide in Freund's complete adjuvant supplemented
with 100 or 400 .mu.g Mycobacterium tuberculosis strain H37Ra
(Difco, Detroit, Mich.), respectively. The clinical disease course
induced by the two emulsions was essentially identical, with the
same day of onset, duration, maximum severity, and cumulative
disease index. The rats were assessed daily for changes in clinical
signs according to the following clinical rating scale: 0, no
signs; 1, limp tail; 2, hind leg weakness, ataxia; 3, paraplegia;
and 4, paraplegia with forelimb weakness, moribund condition. A
cumulative disease score was obtained by summing the daily
disability scores over the course of EAE for each affected rat, and
a mean cumulative disease index (CDI) was calculated for each
experimental group.
[0133] Spinal cord mononuclear cells were isolated by a
discontinuous percol gradient technique and counted as previously
described (Bourdette et al., 1991). The cells were stained with
fluorochrome (FITC or PE) conjugated antibodies specific for rat
CD4, CD8, CD11b, CD45ra, TCR V.beta.8.2 and CD134 (PharMingen, San
Diego, Calif.) for 15 min at room temperature and analyzed by flow
cytometry. The number of positive staining cells per spinal cord
was calculated by multiplying the percent staining by the total
number of cells per spinal cord. Control and
.beta.1.alpha.1/MBP-69-89 protected rats were sacrificed at peak
and recovery of clinical disease, spinal cords were dissected and
fixed in 10% buffered formalin. The spinal cords were
paraffin-embedded and sections were stained with luxol fast
blue-periodic acid schiff-hematoxylin for light microscopy.
[0134] Results
[0135] Intravenous injection (i.v.) of 300 .mu.g of the
.beta.1.alpha.1/MBP-69-89 complex in saline on days 3, 7, 9, 11,
and 14 after injection of MBP or MBP-69-89 peptide in CFA
suppressed the induction of clinical (FIG. 6 and Table 3) and
histological (not shown) signs of EAE. Injection of as little as 30
.mu.g of the .beta.1.alpha.1/MBP-69-89 complex following the same
time course was also effective, completely suppressing EAE in 4 of
6 rats, with only mild signs in the other 2 animals. All of the
control animals that were untreated, that received 2 .mu.g
MBP-69-89 peptide alone (the dose of free peptide contained in 30
.mu.g of the complex), or that received 300 .mu.g of the empty
.beta.1.alpha.1 construct developed a comparable degree of
paralytic EAE (Table 2). Interestingly, injection of 300 .mu.g of a
control .beta.1.alpha.1/CM-2 peptide complex produce a mild (about
30%) suppression of EAE (FIG. 6 and Table 2). In parallel with the
course of disease, animals showed a dramatic loss in body weight
(FIG. 6), whereas animals treated with the
.beta.1.alpha.1/MBP-69-89 complex showed no significant loss of
body weight throughout the course of the experiment. TABLE-US-00002
TABLE 2 Effect of .beta.1.alpha.1/peptide complexes on EAE in Lewis
rats. Maximum Day of Duration Disease Cumulative Treatment of
EAE.sup.a Incidence Onset (days) Score Disease Index
Untreated.sup.b 11/11 12 .+-. 1.sup.c 5 .+-. 1 2.9 .+-. 0.3 10.0
.+-. 2.2 2 .mu.g MBP-69-89 6/6 12 .+-. 1 6 .+-. 1 3.3 .+-. 0.3 11.2
.+-. 1.9 .beta.1.alpha.1/(empty) 5/5 12 .+-. 1 6 .+-. 1 2.9 .+-.
0.6 9.7 .+-. 2.1 300 .mu.g .beta.1.alpha.1/CM-2 5/5 12 .+-. 1 6
.+-. 2 1.9 .+-. 0.8 7.2 .+-. 2.6* 300 .mu.g
.beta.1.alpha.1/MBP-69-89 0/6* -- -- 0 .+-. 0** 0 .+-. 0** 300
.mu.g .beta.1.alpha.1/MBP-69-89 2/6 14 .+-. 0 4 .+-. 0 0.2 .+-.
0.1** 0.7 .+-. 0.3** 30 .mu.g .sup.aEAE was induced with either
Gp-BP/CFA or MBP-69-89/CFA. .sup.bCombined controls from two
experiments. .sup.cValues represent the mean .+-. S.D. *P .ltoreq.
0.05 **P .ltoreq. 0.01
[0136] TABLE-US-00003 TABLE 3 Characterization of infiltrating
spinal cord cells at the peak of EAE in control and
.beta.1.alpha.1/MBP-69-89 protected rats. Spinal cord Total* OX40+
V.beta.8.2+ V.beta.8.2+/OX40+ Protected 200 38 10 5 Control 7500
1750 980 667 *Number of cells/spinal cord .times.10.sup.-3
[0137] To evaluate the effect of the construct on established
disease, Lewis rats were treated with 300 .mu.g of the
.beta.1.alpha.1/MBP-69-89 complex on the first day of disease
onset, with follow-up injections 48 and 96 hours later. EAE in the
control rats progressed to complete hind limb paralysis, whereas no
progression of the disease occurred in any of the treated animals
(FIG. 7). The mild course of EAE (mean cumulative index,
MCI=3.+-.0.13) in the treated group was significantly less than the
severe course of EAE in the control group (MCI=11.2.+-.2.7,
p=0.013), although the duration of disease (6 days) was the same in
both groups.
[0138] Consistent with the complete lack of inflammatory lesions in
spinal cord histological sections (not shown), suppression of EAE
with the .beta.1.alpha.1/MBP-69-89 complex essentially eliminated
the infiltration of activated inflammatory cells into the CNS.
Mononuclear cells were isolated from the spinal cords of control
and protected animals at peak and recovery of clinical disease and
examined by FACS analysis. The total number of mononuclear cells
isolated from spinal cords of control animals at peak of clinical
disease (day 14) was 40-fold higher than from protected animals
evaluated at the same time point (Table 3). Moreover, protected
animals had 72% fewer activated (OX40+), V.beta.8.2+ T cells in the
spinal cord when compared to control animals (Table 3). CD4+ and
CD8+ T cells, macrophages and B cell numbers were also
significantly reduced in protected animals (not shown). The number
of mononuclear cells isolated after recovery from EAE was reduced
4.5-fold in protected animals (0.64.times.10.sup.5 cells/spinal
cord) compared to control animals (2.9.times.10.sup.5 cells/spinal
cord). Protected animals also had 10-fold fewer activated (OX40+),
V.beta.8.2+ T cells in the spinal cord than control animals after
recovery from disease.
[0139] Treatment with .beta.1.alpha.1/MBP-69-89 complex
specifically inhibited the delayed-type hypersensitivity (DTH)
response to MBP-69-89. As shown in FIG. 8A, changes in ear
thickness 24 hours after challenge with PPD were unaffected by in
animals treated with .beta.1.alpha.1 or .beta.1.alpha.1 loaded with
peptides. However, as is shown in FIG. 8B, while animals treated
with .beta.1.alpha.1 alone or complexed with CM-2 had no effect on
the DTH response, animals treated with the
.beta.1.alpha.1/MBP-69-89 complex showed a dramatic inhibition of
the DTH response to MBP-69-89.
[0140] Treatment of EAE with the .beta.1.alpha.1/MBP-69-89 complex
also produced an inhibition of lymph node (LN) T cell responses. As
is shown in FIG. 9, LN cells from rats treated with the suppression
protocol (FIG. 6) were inhibited 2-4 fold in response to MBP or the
MBP-69-89 peptide compared to control rats. This inhibition was
antigen specific, since LN T cell responses to PPD (stimulated by
the CFA injection) were the same in treated and control groups. T
cell responses tested in rats treated after disease onset (FIG. 7)
were also inhibited, in an IL-2 reversible manner. LN cell
responses to MBP and MBP-69-89 peptide were optimal (S.I=4-5X) at
low antigen (Ag) concentrations (4 .mu.g/ml), and could be enhanced
2-fold with additional IL-2. In contrast, responses were inhibited
in treated rats, with optimal LN cell responses (.+-.3X) requiring
higher Ag concentrations (20-50 .mu.g/ml). However, in the presence
of IL-2, responses could be restored to a level comparable to
control rats (S.I.=6-11X) without boosting Ag concentrations.
[0141] Discussion
[0142] The following Examples illustrate the efficacy of the
two-domain MHC molecules. While the experimental details concern
the MHC class II .beta.1.alpha.1 polypeptides, it will be
appreciated that these data fully support application of MHC class
I .alpha.1.alpha.2 polypeptides.
[0143] In the presented Examples, polypeptides comprising the MHC
class II .beta.1 and .alpha.1 domains are described. These
molecules lack the .alpha.2 domain, the .beta.2 domain known to
bind to CD4, and transmembrane and intra-cytoplasmic sequences. The
reduced size and complexity of the .beta.1.alpha.1 construct
permits expression and purification of the molecules from bacterial
inclusion bodies in high yield. The .beta.1.alpha.1 molecules are
shown to refold in a manner that allows binding of allele-specific
peptide epitopes and to have excellent solubility in aqueous
buffers. When complexed with peptide antigen, direct detection of
the .beta.1.alpha.1/peptide complexes to T cells can be visualized
by FACS, with the specificity of binding determined by the peptide
antigen. The .beta.1.alpha.1/69-89 complex exerted powerful and
selective inhibitory effects on T cell activation in vitro and in
vivo. Because of its simplicity, biochemical stability, biological
properties, and structural similarity with human class II homologs,
the .beta.1.alpha.1 construct represents a template for producing a
novel class of TCR ligands.
[0144] Direct binding studies using the A1 hybridoma specific for
MBP-72-89 showed distinct staining with .beta.1.alpha.1/MBP-69-89,
with a 10-fold increase in MFI over background, and was not stained
with .beta.1.alpha.1/CM-2 nor "empty" .beta.1.alpha.1. In a
reciprocal manner, binding studies using a CM-2 specific cell line
showed strong staining with .beta.1.alpha.1/CM-2 and no staining
with .beta.1.alpha.1/MBP-69-89. Thus, bound epitope directed
specific interaction of the .beta.1.alpha.1/peptide complexes.
Identification of antigen-specific T cells has been possible in a
few systems (McHeyzer et al., 1995; MacDonald et al., 1993; Walker
et al., 1995; Reiner et al., 1993), using labeled anti-idiotypic T
cell receptor antibodies as specific markers, but the general
approach of staining specific T cells with their ligand has failed
because soluble peptide-MHC complexes have an inherently fast
dissociation rate from the T cell antigen receptor (Corr et al.,
1995; Matsui et al., 1994; Syulkev et al., 1994). Multimeric
peptide-MHC complexes containing four-domain soluble MHC molecules
have been used to stain antigen-specific T lymphocytes (Altman et
al., 1996), with the ability to bind more than one T cell receptor
(TCR) on a single T cell presumably giving the multimeric molecules
a correspondingly slower dissociation rate. Staining with
.beta.1.alpha.1/peptide complexes, while specific, did take an
incubation period of approximately 10 hours to saturate (data not
shown). The extraordinarily bright staining pattern of the A1
hybridoma with the .beta.1.alpha.1/MBP-69-89 complex, and the CM-2
line with .beta.1.alpha.1/CM-2, coupled with the length of time it
takes to achieve binding saturation, suggests that this molecule
might have a very slow off-rate once bound to the TCR. These
complexes and modified versions of them would be unusually well
suited to directly label antigen-specific T cells for purposes of
quantification and recovery.
[0145] The .beta.1.alpha.1/peptide complex was highly specific in
its ability to bind to and inhibit the function of T cells. In
vitro proliferation of MBP-specific T cells was inhibited >90%
with the .beta.1.alpha.1/MBP-69-89 complex, and in vivo there was a
nearly complete inhibition of clinical and histological EAE.
[0146] The most profound biological activity demonstrated for
.beta.1.alpha.1/MBP-69-89 was its ability to almost totally ablate
the encephalitogenic capacity of MBP-69-89 specific T cells in
vivo. Injection of this complex after initiation of EAE nearly
completely suppressed clinical and histological signs of EAE,
apparently by directly inhibiting the systemic activation of
MBP-69-89 specific T cells, and preventing recruitment of
inflammatory cells into the CNS. Moreover, injection of
.beta.1.alpha.1/MBP-69-89 after onset of clinical signs arrested
disease progression, demonstrating the therapeutic potential of
this molecular construct. Interestingly, the effect of the complex
on already activated T cells was not only to inhibit stimulation,
but also to reduce sensitivity to antigen, with optimal activation
after treatment requiring a 10-fold increase in antigen
concentration.
[0147] From a drug engineering and design perspective this
prototypic molecule represents a major breakthrough. The
demonstrated biological efficacy of the .beta.1.alpha.1/MBP-69-89
complex in EAE raises the possibility of using this construct as a
template for engineering human homologs for treatment of autoimmune
diseases such as multiple sclerosis, that likely involves
inflammatory T cells directed at CNS proteins. One candidate
molecule would be HLA-DR2/MBP-84-102, which includes both the
disease-associated class II allele and a known immunodominant
epitope that has been reported to be recognized more frequently in
MS patients than controls. However, because of the complexity of T
cell response to multiple CNS proteins and their component
epitopes, it is likely that a more general therapy may require a
mixture of several MHC/Ag complexes. The precision of inhibition
induced by the novel .beta.1.alpha.1/MBP-69-89 complex reported
herein represents an important first step in the development of
potent and selective human therapeutic reagents. With this new
class of reagent, it may be possible to directly quantify the
frequency and prevalence of T cells specific for suspected target
autoantigens, and then to selectively eliminate them in affected
patients. Through this process of detection and therapy, it may
then be possible for the first time to firmly establish the
pathogenic contribution of each suspected T cell specificity.
[0148] Having illustrated and described the principles of
synthesizing two domain class II .beta.1.alpha.1 and class I
.alpha.1.alpha.2 molecules and the methods of using such molecules,
it will be apparent to one skilled in the art that the invention
can be modified in arrangement and detail without departing from
such principles. We claim all modifications coming within the
spirit and scope of the claims presented herein.
REFERENCES
[0149] Altman, J. D. et al. Phenotypic analysis of antigen-specific
T lymphocytes. Science 274, 94-96 (1996). [0150] Arimilli, S.,
Cardoso, C., Mukku, P., Baichwal, V. & Nag, B. Refolding and
reconstitution of functionally active complexes of human leukocyte
antigen DR2 and myelin basic protein peptide from recombinant alpha
and beta polypeptide chains. Journal of Biological Chemistry
270(2), 971-977 (1995). [0151] Auffray et al. (1984). Isotypic and
allotypic variation of human class II histocompatibility antigen
alpha-chain genes. Nature 308 (5957), 327-333. [0152] Ausubel et
al. (1987). In Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley-Intersciences. [0153] Benoist et
al. (1983). The murine Ia alpha chains, E alpha and A alpha, show a
surprising degree of sequence homology. Proc. Natl. Acad. Sci.
U.S.A. 80 (2), 534-538. [0154] Boniface, J. J. & Davis, M. M.
T-cell recognition of antigen. A process controlled by transient
intermolecular interactions. Annals New York Acad. Sciences. 766,
62-69 (1995). [0155] Bourdette, D. N. et al. Myelin basic protein
specific T cells in the CNS and lymph nodes of rats with EAE are
different. J. Neurosci. Res. 30, 308-315 (1991). [0156] Brown, J.
H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G.
& Strominger, J. L Three dimensional structure of the human
class II histocompatibility antigen HLA-DR1. Nature 364, 33-39
(1993). [0157] Browning et al., (1995). The HLA-A, B, C genotype of
the class I negative cell line Daudi reveals novel HLA-A and -B
alleles. Tissue Antigens 45 (3), 177-187. [0158] Burrows, G. G. et
al. Variation in H-2K.sup.k peptide motif revealed by sequencing
naturally processed peptides from T cell hybridoma class I
molecules. J. Neurosci. Res. 45, 803-811 (1996). [0159] Burrows, G.
G. et al. Multiple Class I Motifs Revealed by Sequencing Naturally
Processed Peptides Eluted from Rat T Cell MHC Molecules. Rapid
Communication. J. Neurosci. Res. 49, 107-116 (1997). [0160]
Cammarota, G. et al. Identification of a CD4 binding site on the
b.sub.2 domain of HLA-DR molecules. Nature 356, 799-801 (1992).
[0161] Caspi, R. R. et al. A new model of autoimmune disease:
Experimental autoimmune uveoretinitis induced in mice with two
different retinal antigens. J. Immunol. 140, 1490-1495 (1988).
[0162] Chaurhary et al. (1989). Nature 339: 394-397. [0163]
Cobbold, S. P., Nash, J. A., Prospero, T. D. & Waldham, H.
Therapy with monoclonal antibodies by elimination of T-cell subsets
in vivo. Nature 312, 548-551 (1988). [0164] Cobbold, S. P., Nash,
J. A., Prospero, T. D. & Waldham, H. Therapy with monoclonal
antibodies by elimination of T-cell subsets in vivo. Nature 312,
548-551 (1988). [0165] Collins et al. Nature 371 (6498): 626-629
(1994). [0166] Corr, M. et al. T cell receptor-MHC class I peptide
interactions: affinity, kinetics, and specificity. Science 265,
946-949 (1995). [0167] Cush, J. J. & Lipsky, P. E. Phenoytpic
analysis of synovial tissue and peripheral blood lymphocytes
isolated from patients with rheumatoid arthritis. Arthritis Rheum.
31, 1230-1238 (1988). [0168] Das et al. (1983). Structure and
nucleotide sequence of the heavy chain gene of HLA-DR. proc. Natl.
Acad. Sci. U.S.A. 80 (12), 3543-3547. [0169] Derman, A. I., Prinz,
W. A., Belin, D. & Beckwith, J. Mutations that allow disulfide
bond formation in the cytoplasm of Escherichia coli. Science 262,
1744-1747 (1993). [0170] Desbarats, J., Freed, J. H., Campbell, P.
A., & Newell, M. K. Fas (CD95) expression and death-mediating
function are induced by CD4 cross-linking on CD4+ T cells. PNAS 93,
11014-11018 (1996). [0171] Edwards et al. Science 276 (5320):
1868-1871 (1997). [0172] Estess et al. (1986). Sequence analysis
and structure-function correlations of murine q, k, u, s, and f
haplotype I-A beta cDNA clones. Proc. Natl. Acad. Sci. U.S.A. 83
(11), 3594-3598. [0173] Ferrin, T. E., Huang, C. C., Jarvis, L. E.
& Langridge, R. The MIDAS display system. J. Mol. Graphics. 6,
13-27 (1988). [0174] Fleury et al., CELL 66, 1037-1049 1991 [0175]
Fremont, et al. Structures of an MHC class II molecule with
covalently bound single peptides. Science 272, 1001-1004 (1996).
[0176] Gold, D. P., H. Offner, D. Sun, S. Wiley, A. A. Vandenbark
and D. B. Wilson. 1991. Analysis of T cell receptor Ochains in
Lewis rats with experimental autoimmune encephalomyelitis:
Conserved complementarity determining region 3. J. Exp. Med.
174:1467. [0177] Golding, H., J. McCluskey, T. I. Munitz, R. N.
Germain, D. H. Margulies and A. Singer. 1985. T-cell recognition of
a chimaeric class II/class I MHC molecule and the role of L3T4.
Nature, 317:425. [0178] Govaerts, A. et al. HLA and multiple
sclerosis: population and family studies. Tissue Antigens 25,
187-199 (1985). [0179] Harlow and Lane (1988). Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, New York. [0180]
Hashim, G. A., Day, E. D., Fredane, L., Intintola, P., and
Carvalho, E. Biological activity of region 65 to 102 of the myelin
basic protein. J. Neurosci. Res. 16, 467-478 (1986). [0181]
Housset, D., Habersetzer-Rochat, C., Astier, J. P. &
Fontecilla-Camps, J. C. Crystal structure of toxin II from the
scorpion Androctonus Australis Hector refined at 1.3 angstroms
resolution. J. Mol. Biol. 238, 88 (1994). [0182] Howell, M. D. et
al. Vaccination against experimental allergic encephalomyelitis
with T-cell receptor peptides. Science 246, 668-670 (1989). [0183]
Huang, B., Yachou, A., Fleury, S., Hendrickson, W. & Sekaly, R.
Analysis of the contact sites on the CD4 molecule with class II MHC
molecule. J. Immunol. 158, 216-225 (1997). [0184] Innis et al.
(1990). PCR Protocols, A Guide to Methods and Applications, Innis
et al. (eds.), Academic Press, Inc., San Diego, Calif. [0185]
Jameson, B. A., McDonnel, J. M., Marini, J. C. & Korngold, R. A
rationally designed CD4 analogue inhibits experimental allergic
encephalomyelitis. Nature 368, 744-746 (1994). [0186] Janeway &
Travers (1997). Immunobiology: the immune system in health and
disease, Current Biology Ltd./Garland Publishing, Inc. New York.
[0187] Kato et al., (1993). Molecular analysis of HLA-B39 subtypes.
Immunogenetics 37 (3), 212-216. [0188] Kelly & Trowsdale.
(1985). Complete nucleotide sequence of a functional HLA-DP beta
gene and the region between the DP beta 1 and DP alpha 1 genes:
comparison of the 5' ends of HLA class II genes Nucleic Acids Res.
13 (5), 1607-1621. [0189] King, J. and Leimmli, U.K. Bacteriophage
T4 tail assembly: structural proteins and their genetic
identification. J. Mol. Biol. 75, 315-337 (1973). [0190] Konig, R.,
Shen, X. & Germain, R. N. Involvement of both major
histocompatibility complex class II .alpha. and .beta. chains in
CD4 function indicates a role for ordered oligomerization in T cell
activation. J. Exp. Med. 182, 779-787 (1995). [0191] Konig, R.,
Huang, L. Y. & Germain, R. MHC class II interaction with CD4
mediated by a region analogous to the MHC class I binding site for
CD8. Nature 356, 796-798 (1992). [0192] Kozono, H., White, J.,
Clements, J., Marrack, P. & Kappler, J. Production of soluble
MHC class II proteins with covalently bound single peptides. Nature
369, 151-154 (1994). [0193] Kress et al., (1983). Alternative RNA
splicing in expression of the H-2K gene. Nature 306 (5943),
602-604. [0194] Larhammar et al. (1983). Exon-intron organization
and complete nucleotide sequence of a human major
histocompatibility antigen DC beta gene. Proc. Natl. Acad. Sci.
U.S.A. 80 (23), 7313-7317. [0195] Lawrence et al. (1985). The
genomic organisation and nucleotide sequence of the HLA-SB(DP)
alpha gene Nucleic Acids Res. 13 (20), 7515-7528. [0196] MacDonald,
H. R., Casanova, J. L., Maryanski, J. L., Cerottini, J. C.
Oligoclonal expansion of major histocompatibility complex class
I-restricted cytolytic T lymphocytes during a primary immune
response in vivo: direct monitoring by flow cytometry and
polymerase chain reaction. J. Exp. Med. 177, 1487-1492 (1993).
[0197] Madden, D. R., Gorga, J. C., Strominger, J. L. & Wiley,
D. C. The structure of HLA-B27 reveals nonamer self-peptides bound
in an extended conformation. Nature 353, 321-325 (1991). [0198]
Martenson, R. E. Myelin basic protein speciation. in Experimental
Allergic Encephalomyelitis: A useful Model for Multiple Sclerosis.
1984. Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 10011.
pages 511-521. [0199] Matsui, K., Boniface, J. J., Steffner, P.,
Reay, P.A., Davis, M. M. Kinetics of T-cell receptor binding to
peptide/I-E.sup.K complexes: correlation of the dissociation rate
with T-cell responsiveness. Proc. Natl. Acad. Sci. U.S.A. 91,
12862-12866 (1994). [0200] Matsui, K. et al. Low affinity
interaction of peptide-MHC complexes with T cell receptors. Science
254, 1788-1791 (1991). [0201] McHeyzer, M. G., Davis, M. M. Antigen
specific development of primary and memory T cells in vivo. Science
268, 106-111 (1995). [0202] Miltenyi et al. Cytometry 11: 231-238
(1990). [0203] Mitragotri et al. Pharmaceutical Research 13 (3):
411-20 (1996). [0204] Moebius, U., Pallai, P., Harrison, S. C.
& Reinherz, E. L. Delineation of an extended surface contact
area on human CD4 involved in class II MHC binding. PNAS 90,
8259-8263 (1993). [0205] Moore et al., (1982). DNA sequence of a
gene encoding a BALF/c mouse Ld transplantation antigen. Science
215 (4533), 679-682. [0206] Nag, B., Deshpande, S. V., Sharma, S.
D., & Clark, B. R. Cloned T cells internalize peptide from
bound complexes of peptide and purified class II major
histocompatibility complex antigen. J. Biol. Chem. 268, 14360-14366
(1993). [0207] Nag, B., Kendrick, T., Arimilli, S., Yu, S. C.,
& Sriram, S. Soluble MHC II-peptide complexes induce
antigen-specific apoptosis in T cells. Cell. Immunol. 170, 25-33
(1996). [0208] Nag, B., Passmore, D., Kendrick, T., Bhayani, H.,
& Sharma, S. D. N-linked oligosaccharides of murine major
histocompatibility complex class II molecule. Role in antigenic
peptide binding, T cell recognition, and clonal nonresponsiveness.
J. Biol. Chem. 267, 22624-22629 (1992). [0209] Nag B., Arimilli S.,
Mukku P. V. & Astafieva, I. Functionally active recombinant
alpha and beta chain-peptide complexes of human major
histocompatibility class II molecules. Journal of Biological
Chemistry 271(17), 10413-10418 (1996). [0210] Nag B. et al.
Stimulation of T cells by antigenic peptide complexed with isolated
chains of major histocompatibility complex class II molecules.
Proceedings of the National Academy of Sciences of the United
States of America 90(4), 1604-1608 (1993). [0211] Nicolle, M. W. et
al. Specific tolerance to an acetylcholine receptor epitope induced
in vitro in myasthenia gravis CD4+ lymphocytes by soluble major
histocompatibility complex class II-peptide complexes. J. Clin
Invest. 93, 1361-1369 (1994). [0212] Oksenberg, J. R. et al.
Selection of T-cell receptor V-D-J gene rearrangements with
specificity for a MBP peptide in brain lesions of MS. Nature 362,
68-70 (1993). [0213] Ota, K. et al. T cell recognition of an
immunodominant myelin basic protein epitope in multiple sclerosis.
Nature 346, 183-187 (1990). [0214] Paterson, P. Y. Multiple
sclerosis: An immunologic reassessment. J. Chron. Dis. 26, 119-125
(1981). [0215] Quill, H. & Schwartz, R. H. Stimulation of
normal inducer T cell clones with antigen presented by purified Ia
molecules in planer lipid membranes: specific induction of a
long-lived state of proliferative nonresponsiveness. J. Immunol.
138, 3704-3712 (1987). [0216] Reiner, S. L., Wang, Z. E., Hatam,
F., Scott, P., Locksley, R. M. TH1 and TH2 cell antigen receptors
in experimental leishmaniasis. Science 259, 1457-1460 (1993).
[0217] Rhode, P. R. et al. Single-chain MHC class II molecules
induce T cell activation and apoptosis. J. Immunol. 157, 4885-4891
(1996). [0218] Sambrook et al. (1989). In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N. Y. [0219] Schepart et
al., (1986). The nucleotide sequence and comparative analysis of
the H-2Dp class I H-2 gene. J. Immunol. 136 (9), 3489-3495. [0220]
Schwartz, R. H. Models of T cell anergy: is there a common
molecular mechanism? J. Exp. Med. 184, 1-8 (1996) [0221] Service et
al. Science 277(5330): 1199-1200 (1997). [0222] Sharma, S. D. et
al. Antigen-specific therapy of experimental allergic
encephalomyelitis by soluble class II major histocompatibility
complex-peptide complexes. PNAS 88:11465-11469 (1991). [0223]
Spack, E. G. et al. Induction of tolerance in experimental
autoimmune myasthenia gravis with solubilized MHC class
II:acetylcholine receptor complexes. J. Autoimmun. 8, 787-807
(1995). [0224] Steinle et al., (1992). Isolation and
characterization of a genomic HLA-Cw6 clone. Tissue Antigens 39(3),
134-137. [0225] Steinman, L. Autoimmune disease. Sci. Am. 269,
106-114 (1993). [0226] Studier, F. W., A. H. Rosenberg, J. J. Dunn,
and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct
expression of cloned genes. Methods Enzymol. 185:60. [0227]
Swanborg, R. H. Autoimmune effector cells. V. A monoclonal antibody
specific for rat helper T lymphocytes inhibits adoptive transfer of
auto-immune encephalomyelitis. J. Immunol. 130, 1503-1505 (1983).
[0228] Syha, J., Henkes, W. & Reske, K. Complete cDNA sequence
coding for the MHC class II RT1.B .alpha.-chain of the Lewis rat.
Nuc. Acids. Res. 17(10), 3985 (1989). [0229] Syha-Jedelhauser, J.,
Wendling, U. & Reske, K. Complete coding nucleotide sequence of
cDNA for the Class II RT1.B .beta.-chain of the Lewis rat. Biochim.
Biophys. Acta 1089, 414-416 (1991). [0230] Sykulev, Y. et al.
Kinetics and affinity of reactions between an antigen-specific T
cell receptor and peptide-MHC complexes. Immunity 1, 15-22 (1994).
[0231] Thompson, D. and Larson, G. Western blots using stained
protein gels. Biotechniques 12, 656-658 (1992). [0232] Tonnell et
al. (1985). Do beta: a new beta chain gene in HLA-D with a distinct
regulation of expression. EMBO J. 4 (11), 2839-2847. [0233]
Vandenbark, A. A., Vainiene, M., Celnik, B., Hashim, G. A.,
Buenafe, A. C. & Offner, H. Definition of encephalitogenic and
immunodominant epitopes of guinea pig myelin basic protein (Gp-BP)
in Lewis rats tolerized neonatally with Gp-BP peptides. J. Immunol.
15, 852-861 (1994). [0234] Vandenbark, A. A., Hashim, G. &
Offner, H. Immunization with a synthetic T-cell receptor V-region
peptide protects against experimental autoimmune encephalomyelitis.
Nature 341, 541-544 (1989). [0235] Vandenbark, A. A., Gill, T.
& Offner, H. A myelin basic protein specific T lymphocyte line
which mediates EAE. J. Immunol. 135, 223-228 (1985). [0236]
Veillette, A., Bookman, M. A., Horak, E. M. & Bolen, J. B. The
CD4 and CD8 T cell surface antigens are associated with the
internal membrane tyrosine-protein kinase p56.sup.lck. Cell 55,
301-308 (1988). [0237] Walker, P. R., Ohteki, T., Lopez, J. A.,
MacDonald, H. R., Maryanski, J. L. Distinct phenotypes of
antigen-selected CD8 T cells emerge at different stages of an in
vivo immune response.
J. Immunol. 155, 3443-3452 (1995). [0238] Walter et al., (1994).
Sequence, expression, and mapping of rat Mhc class Ib gene.
Immunogenetics 39 (5), 351-354. [0239] Walter et al., (1995).
Genomic organization and sequence of the rat major
histocompatibility complex class Ia gene RT1.Au Immunogenetics 41
(5), 332. [0240] Wegmann K W, Zhao W., Griffin A C., and Hickey W
F. Identification of myocarditogenic peptides derived from cardiac
myosin capable of inducing experimental allergic myocarditis in the
Lewis rat. The utility of a class II binding motif in selecting
self-reactive peptides. J. Immunol. 153(2), 892-900 (1994). [0241]
Weinberg, A. D. et al. Target organ specific upregulation of the
MRC OX-40 marker and selective production of Th1 lymphokine mRNA by
encephalitogenic T helper cells isolated from the spinal cord of
rats with experimental autoimmune encephalomyelitis. J. Immunol.
152, 4712-5721 (1994). [0242] Weinberg, A. D. et al. TGF-.beta.
enhances the in vivo effector function and memory phenotype of
Ag-specific T helper cells in EAE. J. Immunol. 148, 2109-2117
(1992). [0243] Weiner, H. L. et al. Double-blind pilot trial of
oral tolerization with myelin antigens in MS. Science 259,
1321-1324 (1993). [0244] White, J., M. Blackman, J. Bill, J.
Kappler, P. Marrack, D. P. Gold and W. Born. 1989. Two better cell
lines for making hybridomas expressing specific T cell receptors.
J. Immunol. 143:1822. [0245] Yednock, T. A. et al. Prevention of
experimental autoimmune encephalomyelitis by antibodies against
.alpha.4/.beta.1 integrin. Nature 356, 63 (1992). [0246] Zhao, B.,
Carson, M., Ealick, S. E. & Bugg, C. E. Structure of scorpion
toxin variant-3 at 1.2 angstroms resolution. J. Mol. Biol. 227, 239
(1992). [0247] Zinn-Justin, S., Guenneugues, M., Drakopoulou,
Gilquin, B., Vita, C. & Menez, A. Transfer of a beta-hairpin
from the functional site of snake curaremimetic toxins to the
alpha/beta scaffold of scorpion toxins: Three-dimensional solution
structure of the chimeric protein. Biochemistry 35(26): 8535-43
(1996).
Sequence CWU 1
1
30 1 566 DNA Rattus sp. CDS (3)..(560) 1 cc atg ggc aga gac tcc cca
agg gat ttc gtg tac cag ttc aag ggc 47 Met Gly Arg Asp Ser Pro Arg
Asp Phe Val Tyr Gln Phe Lys Gly 1 5 10 15 ctg tgc tac tac acc aac
ggg acg cag cgc ata cgg gat gtg atc aga 95 Leu Cys Tyr Tyr Thr Asn
Gly Thr Gln Arg Ile Arg Asp Val Ile Arg 20 25 30 tac atc tac aac
cag gag gag tac ctg cgc tac gac agc gac gtg ggc 143 Tyr Ile Tyr Asn
Gln Glu Glu Tyr Leu Arg Tyr Asp Ser Asp Val Gly 35 40 45 gag tac
cgc gcg ctg acc gag ctg ggg cgg ccc tca gcc gag tac ttt 191 Glu Tyr
Arg Ala Leu Thr Glu Leu Gly Arg Pro Ser Ala Glu Tyr Phe 50 55 60
aac aag cag tac ctg gag cag acg cgg gcc gag ctg gac acg gtc tgc 239
Asn Lys Gln Tyr Leu Glu Gln Thr Arg Ala Glu Leu Asp Thr Val Cys 65
70 75 aga cac aac tac gag ggg tcg gag gtc cgc acc tcc ctg cgg cgg
ctt 287 Arg His Asn Tyr Glu Gly Ser Glu Val Arg Thr Ser Leu Arg Arg
Leu 80 85 90 95 gga ggt caa gac gac att gag gcc gac cac gta gcc gcc
tat ggt ata 335 Gly Gly Gln Asp Asp Ile Glu Ala Asp His Val Ala Ala
Tyr Gly Ile 100 105 110 aat atg tat cag tat tat gaa tcc aga ggc cag
ttc aca cat gaa ttt 383 Asn Met Tyr Gln Tyr Tyr Glu Ser Arg Gly Gln
Phe Thr His Glu Phe 115 120 125 gat ggt gac gag gaa ttc tat gtg gac
ttg gat aag aag gag acc atc 431 Asp Gly Asp Glu Glu Phe Tyr Val Asp
Leu Asp Lys Lys Glu Thr Ile 130 135 140 tgg agg atc ccc gag ttt gga
cag ctg aca agc ttt gac ccc caa ggt 479 Trp Arg Ile Pro Glu Phe Gly
Gln Leu Thr Ser Phe Asp Pro Gln Gly 145 150 155 gga ctt caa aat ata
gct ata ata aaa cac aat ttg gaa atc ttg atg 527 Gly Leu Gln Asn Ile
Ala Ile Ile Lys His Asn Leu Glu Ile Leu Met 160 165 170 175 aag agg
tca aat tca acc caa gct gtc aac taa ctcgag 566 Lys Arg Ser Asn Ser
Thr Gln Ala Val Asn 180 185 2 185 PRT Rattus sp. 2 Met Gly Arg Asp
Ser Pro Arg Asp Phe Val Tyr Gln Phe Lys Gly Leu 1 5 10 15 Cys Tyr
Tyr Thr Asn Gly Thr Gln Arg Ile Arg Asp Val Ile Arg Tyr 20 25 30
Ile Tyr Asn Gln Glu Glu Tyr Leu Arg Tyr Asp Ser Asp Val Gly Glu 35
40 45 Tyr Arg Ala Leu Thr Glu Leu Gly Arg Pro Ser Ala Glu Tyr Phe
Asn 50 55 60 Lys Gln Tyr Leu Glu Gln Thr Arg Ala Glu Leu Asp Thr
Val Cys Arg 65 70 75 80 His Asn Tyr Glu Gly Ser Glu Val Arg Thr Ser
Leu Arg Arg Leu Gly 85 90 95 Gly Gln Asp Asp Ile Glu Ala Asp His
Val Ala Ala Tyr Gly Ile Asn 100 105 110 Met Tyr Gln Tyr Tyr Glu Ser
Arg Gly Gln Phe Thr His Glu Phe Asp 115 120 125 Gly Asp Glu Glu Phe
Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp 130 135 140 Arg Ile Pro
Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly 145 150 155 160
Leu Gln Asn Ile Ala Ile Ile Lys His Asn Leu Glu Ile Leu Met Lys 165
170 175 Arg Ser Asn Ser Thr Gln Ala Val Asn 180 185 3 113 DNA
Artificial Sequence CDS (3)..(113) Description of Artificial
Sequence antigen/linker insert 3 cc atg ggc aga gac tcc cca cag aag
agc cag agg act cag gat gag 47 Met Gly Arg Asp Ser Pro Gln Lys Ser
Gln Arg Thr Gln Asp Glu 1 5 10 15 aac cca gtg gtg cac ttc gga ggt
gga ggc tca cta gtg ccc cga ggc 95 Asn Pro Val Val His Phe Gly Gly
Gly Gly Ser Leu Val Pro Arg Gly 20 25 30 tct gga ggt gga ggc tcc
113 Ser Gly Gly Gly Gly Ser 35 4 37 PRT Artificial Sequence
Description of Artificial Sequence antigen/linker insert 4 Met Gly
Arg Asp Ser Pro Gln Lys Ser Gln Arg Thr Gln Asp Glu Asn 1 5 10 15
Pro Val Val His Phe Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser 20
25 30 Gly Gly Gly Gly Ser 35 5 83 DNA Artificial Sequence CDS
(3)..(83) Description of Artificial Sequence alternative antigen
encoding sequences for the expression cassette 5 cc atg ggc aga gac
tcc tcc ggc aag gat tcg cat cat gcg gcg cgg 47 Met Gly Arg Asp Ser
Ser Gly Lys Asp Ser His His Ala Ala Arg 1 5 10 15 acg acc cac tac
ggt gga ggt gga ggc tca cta gtg 83 Thr Thr His Tyr Gly Gly Gly Gly
Gly Ser Leu Val 20 25 6 27 PRT Artificial Sequence Description of
Artificial Sequence alternative antigen encoding sequences for the
expression cassette 6 Met Gly Arg Asp Ser Ser Gly Lys Asp Ser His
His Ala Ala Arg Thr 1 5 10 15 Thr His Tyr Gly Gly Gly Gly Gly Ser
Leu Val 20 25 7 89 DNA Artificial Sequence CDS (3)..(89)
Description of Artificial Sequence alternative antigen encoding
sequences for the expression cassette 7 cc atg ggc aga gac tcc aaa
ctg gaa ctg cag tcc gct ctg gaa gaa 47 Met Gly Arg Asp Ser Lys Leu
Glu Leu Gln Ser Ala Leu Glu Glu 1 5 10 15 gct gaa gct tcc ctg gaa
cac gga ggt gga ggc tca cta gtg 89 Ala Glu Ala Ser Leu Glu His Gly
Gly Gly Gly Ser Leu Val 20 25 8 29 PRT Artificial Sequence
Description of Artificial Sequence alternative antigen encoding
sequences for the expression cassette 8 Met Gly Arg Asp Ser Lys Leu
Glu Leu Gln Ser Ala Leu Glu Glu Ala 1 5 10 15 Glu Ala Ser Leu Glu
His Gly Gly Gly Gly Ser Leu Val 20 25 9 28 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 9 aattcctcga
gatggctctg cagacccc 28 10 30 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 10 tcttgacctc caagccgccg cagggaggtg
30 11 31 DNA Artificial Sequence Description of Artificial Sequence
PCR primer 11 cggcggcttg gaggtcaaga cgacattgag g 31 12 37 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
12 gcctcggtac cttagttgac agcttgggtt gaatttg 37 13 26 DNA Artificial
Sequence Description of Artificial Sequence PCR primer 13
cagggaccat gggcagagac tcccca 26 14 30 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 14 gcctcctcga
gttagttgac agcttgggtt 30 15 128 DNA Artificial Sequence Description
of Artificial Sequence PCR primer 15 gaaatcccgc ggggagcctc
cacctccaga gcctcggggc actagtgagc ctccacctcc 60 gaagtgcacc
actgggttct catcctgagt cctctggctc ttctgtgggg agtctctgcc 120 ctcagtcc
128 16 31 DNA Artificial Sequence Description of Artificial
Sequence PCR primer 16 gctccccgcg ggatttcgtg taccagttca a 31 17 92
DNA Artificial Sequence Description of Artificial Sequence PCR
primer 17 tattaccatg ggcagagact cctccggcaa ggattcgcat catgcggcgc
ggacgaccca 60 ctacggtgga ggtggaggct cactagtgcc cc 92 18 92 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
18 ggggcactag tgagcctcca cctccaccgt agtgggtcgt ccgcgccgca
tgatgcgaat 60 ccttgccgga ggagtctctg cccatggtaa ta 92 19 98 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
19 tattaccatg ggcagagact ccaaactgga actgcagtcc gctctggaag
aagctgaagc 60 ttccctggaa cacggaggtg gaggctcact agtgcccc 98 20 98
DNA Artificial Sequence Description of Artificial Sequence PCR
primer 20 ggggcactag tgagcctcca cctccgtgtt ccagggaagc ttcagcttct
tccagagcgg 60 actgcagttc cagtttggag tctctgccca tggtaata 98 21 184
PRT Homo sapiens 21 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ala Met
Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ala Val Gly
Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala
Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Pro Pro Trp Ile Glu Gln
Glu Gly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys
Thr Asn Thr Gln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu
Arg Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile Gln 85 90 95
Arg Met Tyr Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg Gly 100
105 110 His Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn
Glu 115 120 125 Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln
Ile Thr Gln 130 135 140 Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln
Leu Arg Ala Tyr Leu 145 150 155 160 Glu Gly Leu Cys Val Glu Trp Leu
Arg Arg Tyr Leu Glu Asn Gly Lys 165 170 175 Glu Thr Leu Gln Arg Ala
Asp Pro 180 22 174 PRT Homo sapiens 22 Arg Pro Arg Phe Leu Trp Gln
Leu Lys Phe Glu Cys His Phe Phe Asn 1 5 10 15 Gly Thr Glu Arg Val
Arg Leu Leu Glu Arg Cys Ile Tyr Asn Gln Glu 20 25 30 Glu Ser Val
Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 35 40 45 Glu
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu 50 55
60 Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
65 70 75 80 Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val Glu Glu
His Val 85 90 95 Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln
Ser Gly Glu Phe 100 105 110 Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
His Val Asp Met Ala Lys 115 120 125 Lys Glu Thr Val Trp Arg Leu Glu
Glu Phe Gly Arg Phe Ala Ser Phe 130 135 140 Glu Ala Gln Gly Ala Leu
Ala Asn Ile Ala Val Asp Lys Ala Asn Leu 145 150 155 160 Glu Ile Met
Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn 165 170 23 174 PRT Mus
sp. 23 Arg Pro Trp Phe Leu Glu Tyr Cys Lys Ser Glu Cys His Phe Tyr
Asn 1 5 10 15 Gly Thr Gln Arg Val Arg Leu Leu Val Arg Tyr Phe Tyr
Asn Leu Glu 20 25 30 Glu Asn Leu Arg Phe Asp Ser Asp Val Gly Glu
Phe Arg Ala Val Thr 35 40 45 Glu Leu Gly Arg Pro Asp Ala Glu Asn
Trp Asn Ser Gln Pro Glu Phe 50 55 60 Leu Glu Gln Lys Arg Ala Glu
Val Asp Thr Val Cys Arg His Asn Tyr 65 70 75 80 Glu Ile Phe Asp Asn
Phe Leu Val Pro Arg Arg Val Glu Glu His Thr 85 90 95 Ile Ile Gln
Ala Glu Phe Tyr Leu Leu Pro Asp Lys Arg Gly Glu Phe 100 105 110 Met
Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Ile Glu Lys 115 120
125 Ser Glu Thr Ile Trp Arg Leu Glu Glu Phe Ala Lys Phe Ala Ser Phe
130 135 140 Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala
Asn Leu 145 150 155 160 Asp Val Met Lys Glu Arg Ser Asn Asn Thr Pro
Asp Ala Asn 165 170 24 180 PRT Rattus sp. 24 Met Gly Arg Asp Ser
Pro Arg Asp Phe Val Tyr Gln Phe Lys Gly Leu 1 5 10 15 Cys Tyr Tyr
Thr Asn Gly Thr Gln Arg Ile Arg Asp Val Ile Arg Tyr 20 25 30 Ile
Tyr Asn Gln Glu Glu Tyr Leu Arg Tyr Asp Ser Asp Val Gly Glu 35 40
45 Tyr Arg Ala Leu Thr Glu Leu Gly Arg Pro Ser Ala Glu Tyr Trp Asn
50 55 60 Ser Gln Lys Gln Tyr Leu Glu Gln Thr Arg Ala Glu Leu Asp
Thr Val 65 70 75 80 Cys Arg His Asn Tyr Glu Gly Ser Glu Val Arg Thr
Ser Leu Arg Arg 85 90 95 Leu Ala Asp His Val Ala Ala Tyr Gly Ile
Asn Met Tyr Gln Tyr Tyr 100 105 110 Glu Ser Arg Gly Gln Phe Thr His
Glu Phe Asp Gly Asp Glu Glu Phe 115 120 125 Tyr Val Asp Leu Asp Lys
Lys Glu Thr Ile Trp Arg Ile Pro Glu Phe 130 135 140 Gly Gln Leu Thr
Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn Ile Ala 145 150 155 160 Ile
Ile Lys His Asn Leu Glu Ile Leu Met Lys Arg Ser Asn Ser Thr 165 170
175 Gln Ala Val Asn 180 25 19 PRT Artificial Sequence Description
of Artificial Sequence artificial peptide 25 Gly Ser Leu Pro Gln
Lys Ser Gln Arg Ser Gln Asp Glu Asn Pro Val 1 5 10 15 Val His Phe
26 15 PRT Artificial Sequence Description of Artificial Sequence
artificial peptide 26 Ser Gly Lys Asp Ser His His Ala Ala Arg Thr
Thr His Tyr Gly 1 5 10 15 27 17 PRT Artificial Sequence Description
of Artificial Sequence artificial peptide 27 Lys Leu Glu Leu Gln
Ser Ala Leu Glu Glu Ala Glu Ala Ser Leu Glu 1 5 10 15 His 28 95 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
28 tattaccatg ggcagagact ccccacagaa gagccagagg tctcaggatg
agaacccagt 60 ggtgcacttc ggaggtggag gctcactagt gcccc 95 29 94 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
29 ggggcactag tgagcctcca cctccgaagt gcaccactgg gttctcatcc
tgagacctct 60 ggctcttctg tggggagtct ctgcccatgg taat 94 30 19 PRT
Artificial Sequence Description of Artificial Sequence artificial
peptide 30 Gly Ser Leu Pro Gln Lys Ser Gln Arg Thr Gln Asp Glu Asn
Pro Val 1 5 10 15 Val His Phe
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