U.S. patent application number 10/199995 was filed with the patent office on 2003-04-10 for mhc conjugates useful in ameliorating autoimmunity.
This patent application is currently assigned to Anergen, Inc. a wholly-owned subsidiary of Corixa Corporation. Invention is credited to Clark, Brian R., Lerch, Bernard L., Sharma, Somesh D..
Application Number | 20030068363 10/199995 |
Document ID | / |
Family ID | 27498815 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068363 |
Kind Code |
A1 |
Clark, Brian R. ; et
al. |
April 10, 2003 |
MHC conjugates useful in ameliorating autoimmunity
Abstract
The present invention is directed to complexes consisting
essentially of an isolated MHC component and an autoantigenic
peptide associated with the antigen binding site of the MHC
component. These complexes are useful in treating autoimmune
disease.
Inventors: |
Clark, Brian R.; (Redwood
City, CA) ; Sharma, Somesh D.; (Los Altos, CA)
; Lerch, Bernard L.; (Palo Alto, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Anergen, Inc. a wholly-owned
subsidiary of Corixa Corporation
Seattle
WA
98104
|
Family ID: |
27498815 |
Appl. No.: |
10/199995 |
Filed: |
July 19, 2002 |
Related U.S. Patent Documents
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Application
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10199995 |
Jul 19, 2002 |
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09602807 |
Jun 23, 2000 |
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6451314 |
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09602807 |
Jun 23, 2000 |
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08462351 |
Jun 5, 1995 |
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6106840 |
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08462351 |
Jun 5, 1995 |
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07869293 |
Apr 14, 1992 |
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5468481 |
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07869293 |
Apr 14, 1992 |
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07690840 |
Apr 23, 1991 |
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5260422 |
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07690840 |
Apr 23, 1991 |
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07576084 |
Aug 30, 1990 |
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5130297 |
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07576084 |
Aug 30, 1990 |
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07210594 |
Jun 23, 1988 |
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Current U.S.
Class: |
424/450 ;
424/185.1; 530/350 |
Current CPC
Class: |
A61K 2123/00 20130101;
A61K 39/0008 20130101; A61K 51/1234 20130101; A61K 2039/605
20130101; C07K 14/705 20130101; C07K 14/70571 20130101; C07K 14/78
20130101; C07K 2319/00 20130101; C07K 14/4713 20130101; A61K 47/646
20170801; Y10S 530/868 20130101; A61P 37/06 20180101; A61K 47/6425
20170801; C07K 14/70539 20130101 |
Class at
Publication: |
424/450 ;
424/185.1; 530/350 |
International
Class: |
A61K 039/00; C07K
014/74; A61K 009/127 |
Claims
What is claimed is:
1. A substantially pure MHC-peptide complex consisting essentially
of an autoantigenic peptide and an isolated MHC component having an
antigen binding site, wherein the autoantigenic peptide is
associated with the antigen binding site.
2. A complex of claim 1 wherein the peptide is noncovalently
associated with the antigen binding site.
3. A complex of claim 1 wherein the MHC component is soluble.
4. A complex of claim 1 wherein the peptide is between about 8 to
about 15 amino acids.
5. A complex of claim 1 wherein an epitope on the peptide is
recognized by an autoreactive T cell associated with multiple
sclerosis.
6. A complex of claim 1 wherein the MHC component is Class II
MHC.
7. A complex of claim 1 wherein the MHC component is isolated from
spleen cells.
8. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the MHC-peptide complex of claim 1.
9. A pharmaceutical composition of claim 8 wherein the complex is
inside a liposome.
10. A pharmaceutical composition of claim 8 wherein the complex is
substantially free of carbohydrate moities.
11. A pharmaceutical composition of claim 8 wherein the
concentration of the complex is between about 0.02% and about 1% by
weight.
12. A pharmaceutical composition of claim 8 wherein the
pharmaceutically acceptable carrier is phosphate buffered
saline.
13. A method of inducing anergy in autoreactive T cells in a
mammal, the method comprising administering to the mammal a
therapeutically effective dose of a complex consisting essentially
of an autoantigenic peptide and an isolated MHC component having an
antigen binding site, wherein the autoantigenic peptide is
associated with the antigen binding site.
14. A method of claim 13 wherein the MHC component is Class II
MHC.
15. A method of claim 13 wherein the complex is embedded in a lipid
membrane.
16. A method of claim 13 wherein the T cells are associated with
rheumatoid arthritis or multiple sclerosis.
17. A method of treating autoimmune disease in a mammal, the method
comprising administering to the mammal a therapeutically effective
dose of the pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a purified MHC-peptide
complex consisting essentially of an autoantigenic peptide and an
isolated MHC component having an antigen binding site, wherein the
autoantigenic peptide is associated with the antigen binding
site.
18. A method of claim 17 wherein the mammal is a mouse.
19. A method of claim 18 wherein the effective dose is between
about 50 .mu.g and about 300 .mu.g of the complex.
20. A method of claim 17 wherein the pharmaceutical composition is
administered intravenously.
21. A method of claim 20 wherein the effective dose is between
about 3 mg MHC-peptide complex per kg body weight and about 15 mg
MHC-peptide complex per kg body weight.
22. A method of claim 17 wherein the autoimmune disease is
rheumatoid arthritis or multiple sclerosis.
23. A method for preparing a complex consisting essentially of an
autoantigenic peptide and an isolated MHC component having an
antigen binding site, the method comprising: isolating the MHC
component from a cell producing the component; contacting the MHC
component with the peptide such that the peptide is coupled to the
antigen binding site; and removing excess peptide not coupled to
the antigen binding site.
24. A method of claim 23 wherein the step of removing excess
peptide is carried out by dialysis.
25. A method of claim 23 further comprising the step of dialyzing
the complex in the presence of lipids to form liposomes.
26. A method of claim 23 wherein the peptide is nocovalently bound
to the antigen binding site.
27. A method of claim 23 wherein the MHC component is a Class II
glycoprotein of the major histocompatibility complex.
28. A method of claim 23 wherein the peptide comprises amino acids
1-14 of MBP.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of applicants'
copending U.S. application Ser. No. 07/690,840, filed Apr. 23,
1991, which is a continuation-in-part of U.S. application Ser. No.
07/576,084, filed Aug. 30, 1990, which is a continuation of U.S.
application Ser. No. 07/210,594, filed Jun. 23, 1988, now
abandoned, which are incorporated herein by reference. The
application is related to U.S. Ser. No. 07/367,751 filed Jun. 21,
1989, U.S. application Ser. No. 07/635,840 filed Dec. 12, 1990,
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Technical Field
[0003] The invention relates to the methods and compositions for
the modulation of T cell function in the treatment of for example,
autoimmune diseases, allergic responses, transplant rejection, and
other immunological disorders. In particular, it concerns complexes
which target helper T cells by using a complex of the major
histocompatibility complex (MHC) glycoproteins with peptides
representing fragments of antigens associated with such diseases.
These complexes can be further conjugated to radioisotopes or other
labels for diagnostic purposes, or to toxins or other substances
which render the complexes therapeutically useful.
BACKGROUND OF THE INVENTION
[0004] A number of pathological responses involving unwanted T cell
activation are known. For instance, a number of allergic diseases,
have been associated with particular MHC alleles or suspected of
having an autoimmune component.
[0005] Other deleterious T cell-mediated responses include the
destruction of foreign cells that are purposely introduced into the
body as grafts or transplants from allogeneic hosts. This process,
known as "allograft rejection," involves the interaction of host T
cells with foreign MHC molecules. Quite often, a broad range of MHC
alleles are involved in the response of the host to an
allograft.
[0006] Autoimmune disease is a particulalry important class of
deleterious immune response. In autoimmune diseases, self-tolerance
is lost and the immune system attacks "self" tissue as if it were a
foreign target. More than 30 autoimmune diseases are presently
known; these include many which have received much public
attention, including myasthenia gravis (MG) and multiple sclerosis
(MS).
[0007] A crude approach to treating autoimmune disease and other
immunopathologies is general immunosuppression. This has the
obvious disadvantage of crippling the ability of the subject to
respond to real foreign materials to which it needs to mount an
immune response. An only slightly more sophisticated approach
relies on the removal of antibodies or immune complexes involving
the target tissue. This also has adverse side effects, and is
difficult to accomplish. The invention approach, described in
detail below, relies on a "clonotypic" reagent--i.e., a reagent
which attacks only the cells of the immune system which are
responsive to the autoantigen.
[0008] In the general paradigm now considered to describe the
immune response, specific antigens presented result in a clonal
expansion, as first proposed by Burnet in 1959. According to this
scenario, a particular subject will have hundreds of thousands of T
and B cells each bearing receptors that bind to different antigenic
determinants. Upon exposure to an antigen, the antigen selectively
binds to cells bearing the appropriate receptors for the antigenic
determinants it contains, ignoring the others. The binding results
in a cloned population of thousands of daughter cells, each of
which is marked by the same receptor. A clonotypic reagent affects
only a subset of the T and B cells which are appropriate for the
antigen of interest. In the case of the invention compositions, the
antigenic determinant is usually that associated with an autoimmune
disease.
[0009] The clonotypic reagent compositions of the invention are
specifically designed to target T-helper cells which represent the
clones specific for the antigenic determinant(s) of the tissue
which is affected by the autoimmune disease. T-helper cells
recognize a determinant only in association with an MHC protein;
the complexes of the invention therefore include an effective
portion of the MHC protein.
[0010] There have, recently, been some related approaches which
attempt to interdict the immune response to specific antigens. For
example, the autoantigen thyroglobulin has been conjugated to ricin
A and the conjugate was shown to suppress specifically the in vitro
antibody response of lymphocytes which normally respond to this
antigen. It was suggested that such immunotoxins would specifically
delete autoantibody-secreting lymphocyte clones (Rennie, D. P., et
al., Lancet (Dec. 10, 1983) 1338-1339). Diener, E., et.al., Science
(1986) 231:148-150 suggested the construction of compounds which
cause antigen-specific suppression of lymphocyte function by
conjugating daunomycin to the hapten (in this case, of ovalbumin)
using an acid-sensitive spacer. The conjugate caused
hapten-specific inhibition of antibody secretion by B lymphocytes
in vitro and in vivo. A conjugate of daunomycin (with an
acid-sensitive spacer) to a monoclonal antibody-specific to T cells
also eliminated the response by T-lymphocytes to concanavalin A.
Steerz, R. K. M., et al., J. Immunol. (1985) 134:841-846 utilized
radiation as the toxic element in a toxin conjugate. Rats were
administered a radioactively labeled, purified receptor from
electric fish, prior to injection with cold receptor. Injection
with this receptor is a standard procedure to induce experimental
autoimmune myasthenia gravis (EAMG). Control rats that received
preinjection only either of cold receptor or radiolabeled albumin,
prior to administration of receptor to induce the disease develop
the symptoms of EAMG; those pretreated with radioactively-labeled
receptor showed reduced symptoms. It was surmised that the labeled,
and therefore destructive, receptor selectively eliminated
immunocompetent cells. Similar work utilizing a ricin/receptor
conjugate for pretreatment was reported by Killen, J. A., et al.,
J. Immunol. (1984) 133:2549-2553.
[0011] A less specific approach which results in the destruction of
T cells in general is treatment with an IL-2/toxin conjugate as
reported by Hixson, J. R., Medical Tribune (Jan. 28, 1988) 4-5. In
a converse, but related, approach Liu, M. A., et al., Science
(1988) 239:395-397, report a method to "link up" cytotoxic T cells
with a desired target, regardless of the cytotoxic T cell
specificity. In this approach, antibody specific to the universal
cytotoxic T-lymphocytes to destroy human melanoma cells when
melanocyte-stimulating hormone was the hormone used.
[0012] The current model of immunity postulates that antigens
mobilize an immune response, at least in part, by being ingested by
an antigen-presenting cell (APC) which contains on its surface a
Class II glycoprotein encoded by a gene in the major
histocompatibility complex (MHC). The antigen is then presented to
a specific T helper cell in the context of the surface bound MHC
glycoprotein, and by interaction of the antigen specific T cell
receptor with the antigen -MHC complex, the T helper cell is
stimulated to mediate the antigen-specific immune response,
including induction of cytotoxic T cell function, induction of B
cell function, and secretion of a number of factors aiding and
abetting this response.
[0013] The involvement of the MHC Class II proteins in autoimmune
disease has been shown in animal models. Administration of
antibodies to either MHC Class II proteins themselves or antibodies
to agents that induce expression of the MHC Class II genes
interferes with development of the autoimmune condition in these
model systems. The role of helper T cells has also been
demonstrated in these models by counteracting the autoimmune system
using anti-CD4 monoclonal antibodies; CD4 is the characteristic
helper T cell receptor (Shizuru, J. A. et al., Science (1988)
240:659-662).
[0014] Recent experiments have shown that, under certain
circumstances, anergy or nonresponsiveness can be induced in
autoreactive lymphocytes (see, Schwartz, Cell (1989) 1073-1081,
which is incorporated herein by reference). In vitro experiments
suggest that antigen presentation by MHC Class II molecules in the
absence of an unknown co-stimulatory signal induces a state of
proliferative non-responsiveness in syngeneic T cells (Quill et
al., J. Immunol. (1987) 138:3704-3712, which is incorporated herein
by reference). These reports, however, provide no clear evidence
that induction of anergy in vivo is possible or that autoimmune
disease can be effectively treated in this manner.
SUMMARY OF THE INVENTION
[0015] The present invention is directed to methods and
compositions that can be used to identify and inhibit those aspects
of the immune system which are responsible for undesirable
autoimmunity. The invention compositions and methods are designed
to target helper T cells which recognize a particular antigen in
association with a glycoprotein encoded by the MHC. The invention
complexes effectively substitute for the antigen-presenting cell
and cause non-responsiveness in autoreactive T-lymphocytes and
other cells of the immune system.
[0016] The invention provides forms of an autoantigen which
interact with the immune system, in a manner analogous to those
initiated by the autoantigen itself to cause the autoimmune
reaction. Compositions of the present invention are purified two
component complexes of (1) an effective portion of the MHC-encoded
antigen-presenting glycoprotein; and (2) an effective portion of
the antigen. These two components may be bound covalently or by
noncovalent association. Evidence from both in vitro and in vivo
experiments establishes that such complexes induce clonal anergy in
syngeneic T cells.
[0017] In other aspects, the invention is directed to
pharmaceutical compositions wherein the complexes of the invention
are active ingredients. The compositions can be used to
down-regulate parts of the immune system reactive with a particular
self-antigen associated with an autoimmune disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a diagram of a typical complex of the
invention.
[0019] FIG. 2 shows the 3-dimensional structure of the human HLA-A2
antigen (Class I).
[0020] FIG. 3 shows a diagrammatic representation of the active
portion of a modified Class II MHC-encoded glycoprotein.
[0021] FIG. 4 shows preferred second generation MHC protein
designs.
[0022] FIG. 5 is a diagram of a planar membrane bilayer including
the MHC glycoprotein, mimicking the surface of the antigen
presenting cell.
[0023] FIG. 6 shows the amino acid sequence and encoding MRNA for
the alpha subunit of acetylcholine receptor protein.
[0024] FIG. 7 shows the amino acid sequence of myelin basic
protein.
[0025] FIG. 8 shows the nucleotide sequence encoding the
I-A.sup.b-alpha chain.
[0026] FIG. 9 shows the nucleotide sequence encoding the
I-A.sup.b-beta chain.
[0027] FIG. 10 presents a list of the DQ/DR haplotypes in humans
and their associations with autoimmune diseases.
[0028] FIG. 11 shows a protocol suitable for the utilization of the
complexes of the invention for the diagnosis and/or treatment of an
autoimmune disease.
[0029] FIG. 12 shows a scheme for the preparation of I-A.sup.k
containing NP-40 soluble membrane extracts.
[0030] FIG. 13A is a scheme for the affinity purification of
10-2.16 monoclonal antibody and its coupling to CNBr activated
Sepharose 4B.
[0031] FIG. 13B is a copy of a gel showing the purity of 10-2.16
monoclonal antibody purified by the scheme in FIG. 13A.
[0032] FIG. 14 shows a scheme for the purification of
I-A.sup.k.
[0033] FIG. 15 is a polyacrylamide gel showing the purity of
I-A.sup.k purified by the scheme in FIG. 14.
[0034] FIG. 16 is a bar graph showing the results of eight studies
on the inhibition of proliferation by a complex containing
I-A.sup.k and MBP (1-13).
[0035] FIG. 17 is a graph showing the development of EAE in mice
resulting from immunization with MBP (1-13).
[0036] FIG. 18 is a graph showing the adoptive transfer of EAE by T
cell clone 4R3.4, obtained from B10A(4R) strain of mice following
immunization with MBP(1-11).
[0037] FIGS. 19A-C show treatment of passively induced EAE in mice
using PBS alone (19a), I-A.sup.g/MBP1-14 (a non-encephalitogenic
peptide) (19b), and I-A.sup.g/MBp91-103 (19c).
[0038] FIG. 20 shows that complexes of the invention exist as
aggregates because they pass through the column with the void
volume and thus have a molecular weight greater than 600,000.
[0039] FIG. 21 shows that complexes of the invention (MHC
II:AChR.alpha. 100-116) were effective in treating MG in rats.
[0040] FIGS. 22A and 22B show that cell death follows the induction
of anergy in T cells.
[0041] FIGS. 23A and 23B show that the molar concentration of
complex of the invention required to induce anergy is much less
than that of peptide alone.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0042] The present invention provides complexes which can be used
to modulate T cell function. For instance, the complexes can be
used to inhibit a deleterious T cel-mediated immune response, such
as allergic responses, allograft rejection, and autoimmune
diseases. In addition, the complexes of the invention can also be
used as vaccines and thus, promote T cell responses.
[0043] The invention complexes contain at least two components: a
peptide which represents an autoantigen or other antigenic sequence
with the relevant effect on the immune system and an effective
portion of the MHC-encoded glycoprotein involved in antigen
presentation. An effective portion of an MHC glycoprotein-is one
which comprises the antigen binding sites and sequences necessary
for recognition of the MHC-peptide complex by the appropriate T
cell receptor. The MHC component can be either a Class I or a CLass
II molecule. The association between the peptide antigen and the
antigen binding sites of the MHC protein can be by covalent or by
noncovalent bonding.
[0044] In other embodiments the complexes may also contain an
effector component which is generally a toxin or a label. The
effector portion may be conjugated to either the MHC-encoded
glycoprotein or to the autoantigenic peptide. Complexes containing
an effector component are disclosed and claimed in copending
application U.S. Ser. No. 07/367,751 filed Jun. 21, 1989,
supra.
[0045] Each of the components of the system is described separately
below; followed by description of the methods by which these
complexes can be prepared, evaluated and employed.
[0046] The MHC-Derived Component
[0047] The glycoproteins encoded by the MHC have been extensively
studied in both the human and murine systems. In general, they have
been classified as Class I glycoproteins, found on the surfaces of
all cells and primarily recognized by cytotoxic T cells; and Class
II which are found on the surfaces of several cells, including
accessory cells such as macrophages, and are involved in
presentation of antigens to helper T cells. Some of the
histocompatibility proteins have been isolated and characterized.
For a general review of MHC glycoprotein structure and function,
see Fundamental Immunology, 2d Ed., W. E. Paul, ed., Ravens Press
N.Y. 1989, which is incorporated herein by reference. The term
"isolated MHC component" as used herein refers to an MHC
glycoprotein or an effective portion of an MHC glycoprotein (i.e.,
one comprising an antigen binding site or sites and sequences
necessary for recognition by the appropriate T cell receptor) which
is in other than its native state, for example, not associated with
the cell membrane of a cell that normally expresses MHC. As
described in detail below, the MHC component may be recombinantly
produced, solubilized from the appropriate cell source or
associated with a liposome.
[0048] Methods for purifying the murine I-A (Class II)
histocompatibility proteins have been disclosed by Turkewitz, A.
P., et al., Molecular Immunology (1983) 20:1139-1147, which is
incorporated herein by reference. These methods, which are also
suitable for Class I molecules, involve preparation of a soluble
membrane extract from cells containing the desired MHC molecule
using nonionic detergents, such as NP-40, Tween 80 and the like.
The MHC molecules are then purified by affinity chromatography,
using a column containing antibodies raised against the desired MHC
molecule. Use of 0.02% Tween-80 in the elution buffer is helpful to
eliminate aggregation of the purified molecules.
[0049] The isolated antigens encoded by the I-A and I-E subregions
have been shown to consist of two noncovalently bonded peptide
chains: an alpha chain of 32-38 kd and a beta chain of 26-29 kd. A
third, invariant, 31 kd peptide is noncovalently associated with
these two peptides, but it is not polymorphic and does not appear
to be a component of the antigens on the cell surface (Sekaly, R.
P., J. Exp. Med. (1986) 164:1490-1504, which is incorporated herein
by reference). The alpha and beta chains of seven allelic variants
of the I-A region have been cloned and sequenced (Estees, "T cell
Clones", 3-19).
[0050] The human Class I proteins have also been studied. The MHC
of humans (HLA) on chromosome 6 has three loci, HLA-, HLA-B, and
HLA-C, the first two of which have a large number of alleles
encoding alloantigens. These are found to consist of a 44 kd
subunit and a 12 kd beta.sub.2-microglobulin subunit which is
common to all antigenic specificities. Isolation of these
detergent-soluble HLA antigens was described by Springer, T. A., et
al., Proc. Natl. Acad. Sci. USA (1976) 73:2481-2485; Clementson, K.
J., et al., in "Membrane Proteins" Azzi, A., ed; Bjorkman, P.,
Ph.D. Thesis Harvard (1984) all of which are incorporated herein by
reference.
[0051] Further work has resulted in a detailed picture of the 3-D
structure of HLA-A2, a Class I human, antigen. (Bjorkman, P. J., et
al., Nature (1987) 329:506-512, 512-518 which is incorporated
herein by reference). In this picture, the
.beta..sub.2-microglobulin protein and alpha.sub.3 segment of the
heavy chain are associated; the alpha.sub.1 and alpha.sub.2 regions
of the heavy chain appear to form antigen-binding sites to which
the peptide is bound (Science (1987) 238:613-614, which is
incorporated herein by reference) Bjorkman, P. J. et al. Nature
(supra). Soluble HLA-A2 can be purified after papain digestion of
plasma membranes from the homozygous human lymphoblastoid cell line
J-Y as described by Turner, M. J. et al., J. Biol. Chem. (1977)
252:7555-7567, all of which are incorporated herein by reference.
Papain cleaves the 44 kd chain close to the transmembrane region
yielding a molecule comprised of alpha.sub.1, alpha.sub.2,
alpha.sub.3, and .beta..sub.2 microglobulin. A representation of
the deduced three dimensional structure of the Class I HLA-A2
antigen is shown in FIG. 2.
[0052] While the three dimensional structure of Class II MHC
antigens is not known in such detail, it is thought that Class II
glycoproteins have a domain structure, including an antigen binding
site, similar to that of Class I. It is formed from the N-terminal
domain portions of two class II chains which extend from the
membrane bilayer. The N-terminal portion of one chain has two
domains of homology with the alpha.sub.1 and alpha.sub.2 regions of
the MHC Class I antigen sequence. Cloning of the Class II genes (as
described by Estees supra) permits manipulation of the Class II MHC
binding domains for example, as described below.
[0053] The MHC glycoprotein portions of the complexes of the
invention, then, can be obtained by isolation from lymphocytes and
screened for the ability to bind the desired peptide antigen. The
lymphocytes are from the species of individual which will be
treated with the complexes. For example, they may be isolated from
human B cells from an individual suffering from the targeted
autoimmune disease, which have been immortalized by transformation
with a replication deficient Epstein-Barr virus, utilizing
techniques known in the art.
[0054] MHC glycoproteins have been isolated from a multiplicity of
cells using a variety of techniques including solubilization by
treatment with papain, by treatment with 3M KC1, and by treatment
with detergent. In a preferred method detergent extraction of Class
II protein from lymphocytes followed by affinity purification is
used. Detergent can then be removed by dialysis or selective
binding beads, e.g., Bio Beads.
[0055] Alternatively, the amino acid sequence of each of a number
of Class II proteins are known, and the genes have been cloned,
therefore, the proteins can be made using recombinant methods. In a
first generation synthetic MHC protein, the heavy (alpha) and light
(beta) chains are synthesized using a carboxy terminal truncation
which effects the deletion of the hydrophobic domain, and the
carboxy termini can be arbitrarily chosen to facilitate the
conjugation of toxins or label. For example, in the MHC protein
shown in FIG. 3, lysine residues are introduced. In addition,
cysteine residues near the carboxy termini are included to provide
a means to form disulfide linkage of the chains; the synthetic gene
can also include restriction sites to aid in insertion into
expression vectors and in manipulating the gene sequence to encode
analogs. The alpha and beta chains are then inserted into
expression vectors, expressed separately in an appropriate host,
such as E. coli, yeast, or other suitable cells, and the
recombinant proteins obtained are recombined in the presence of the
peptide antigen.
[0056] As the availability of the gene permits ready manipulation
of the sequence, a second generation of preferred construction
includes hybrid Class I and Class II features, as illustrated in
FIG. 4, wherein the alpha.sub.1 and betas domains of Class II MHC
are linked through a flexible portion that permits intramolecular
dimerization between these domains resulting in an edge-to-edge
beta sheet contact. The beta.sub.1 segment is then fused to the
alpha.sub.2 domain of Class I with beta.sub.2 microglobulin
coexpressed to stabilize the complex. The transmembrane and
intracellular domains of the Class I gene can also be included but
there may be no point in doing so unless liposomes are used to
transport the complex. A simpler version includes only the
alpha.sub.1 and beta.sub.1 domains with a C-terminal lysine for
toxin conjugation (FIG. 4).
[0057] Construction of expression vectors and recombinant
production from the appropriate DNA sequences are performed by
methods known in the art per se. Expression can be in procaryotic
or eucaryotic systems. Procaryotes most frequently are represented
by various strains of E. coli. However, other microbial strains may
also be used, such as bacilli, for example Bacillus subtilis,
various species of Pseudomonas, or other bacterial strains. In such
procaryotic systems, plasmid vectors which contain replication
sites and control sequences derived from a species compatible with
the host are used. For example, E. coli is typically transformed
using derivatives of pBR322, a plasmid derived from an E. coli
species by Bolivar et al., Gene (1977) 2:95. Commonly used
procaryotic control sequences, which are defined herein to include
promoters for transcription initiation, optionally with an
operator, along with ribosome binding site sequences, including
such commonly used promoters as the beta-lactamase (penicillinase)
and lactose (lac) promoter systems (Change et al., Nature (1977)
198:1056) and the tryptophan (trp) promoter system (Goeddel et al.,
Nucleic Acids Res. (1980) 8:4057) and the lambda-derived P.sub.L
promoter and N-gene ribosome binding site (Shimatake et al., Nature
(1981) 292:128). Any available promoter system compatible with
procaryotes can be used. All references cited herein whether supra
or infra, are hereby incorporated herein by reference.
[0058] The expression systems useful in the eucaryotic hosts
comprise promoters derived from appropriate eucaryotic genes. A
class of promoters useful in yeast, for example, include promoters
for synthesis of glycolytic enzymes, including those for
3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980)
255:2073). Other promoters include those from the enolase gene
(Holland, M. J., et al. J. Biol. Chem. (1981) 256:1385) or the Leu2
gene obtained from YEp13 (Broach, J., et al., Gene (1978)
8:121).
[0059] Suitable mammalian promoters include the early and late
promoters from SV40 (Fiers, et al., Nature (1978) 273:113) or other
viral promoters such as those derived from polyoma, adenovirus II,
bovine papilloma virus or avian sarcoma viruses. Suitable viral and
mammalian enhancers are cited above.
[0060] The expression system is constructed from the foregoing
control elements operably linked to the MHC sequences using
standard methods, employing standard ligation and restriction
techniques which are well understood in the art. Isolated plasmids,
DNA sequences, or synthesized oligonucleotides are cleaved,
tailored, and relegated in the form desired.
[0061] Site specific DNA cleavage is performed by treating with the
suitable restriction enzyme (or enzymes) under conditions which are
generally understood in the art, and the particulars of which are
specified by the manufacturer or these commercially available
restriction enzymes. See, e.g., New England Biolabs, Product
Catalog. In general, about 1 ug of plasmid or DNA sequence is
cleaved by one unit of enzyme in about 20 ul of buffer solution; in
the examples herein, typically, an excess of restriction enzyme is
used to insure complete digestion of the DNA substrate. Incubation
times of about 1 hr to 2 hr at about 37.degree. C. are workable,
although variations can be tolerated. After each incubation,
protein is removed by extraction with phenol/chloroform, and may be
followed by ether extraction, and the nucleic acid recovered from
aqueous fractions by precipitation with ethanol followed by running
over a Sephadex G-50 spin column. If desired, size separation of
the cleaved fragments may be performed by polyacrylamide gel or
agarose gel electrophoresis using standard techniques. A general
description of size separation is found in Methods in Enzymology
(1980) 65:499-560.
[0062] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) using
incubation times of about 15 to 25 min at 20 to 25.degree. C. in 50
Mm Tris Ph 7.6, 50 Mm NaCl, 6 mM MgCl.sub.2, 6 Mm DTT and 5-10 uM
dNTPs. The Klenow fragment fills in a 5' sticky ends but chews back
protruding 3' single strands, even through the four dNTPS, are
present. If desired, selective repair can be performed by supplying
only one of the, or selected, dNTPs within the limitations dictated
by the nature of the sticky ends. After treatment with Klenow, the
mixture is extracted with phenol/chloroform and ethanol
precipitated followed by running over a Sephadex G-50 spin
column.
[0063] Synthetic oligonucleotides are prepared using commercially
available automated oligonucleotide synthesizers. Kinasing of
single strands prior to annealing or for labeling is achieved using
an excess, e.g., approximately 10 units of polynucleotide kinase to
0.1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM
MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles
.sup.32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
[0064] Ligations are performed in 15-30 ul volumes under the
following standard conditions and temperatures: 20 mM Tris-HCl pH
7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaCl,
and either 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at
0.degree. C. (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6
(Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt end"
ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 ug/ml total DNA concentrations (5-100 nM total
end concentration). Intermolecular blunt end ligations (usually
employing a 10-30 fold molar excess of linkers) are performed at 1
uM total ends concentration.
[0065] In vector construction employing "vector fragments," the
vector fragment is commonly treated with bacterial alkaline
phosphatase (BAP) in order to remove the 5' phosphate and prevent
religation of the vector. BAP digestions are conducted at pH 8 in
approximately 150 mM Tris, in the presence of Na.sup.+ and
Mg.sup.+2 using about 1 unit of BAP per ug of vector at 60.degree.
C. for about 1 hr. In order to recover the nucleic acid fragments,
the preparation is extracted with phenol/chloroform and ethanol
precipitated and desalted by application to a Sephadex G-50 spin
column. Alternatively, religation can be prevented in vectors which
have been double digested by additional restriction enzyme
digestion of the unwanted fragments.
[0066] For portions of vectors derived from cDNA or genomic DNA
which require sequence modifications, site specific primer directed
mutagenesis can be used. This is conducted using a primer synthetic
oligonucleotide complementary to a single stranded phage DNA to be
mutagenized except for limited mismatching, representing the
desired mutation. Briefly, the synthetic oligonucleotide is used as
a primer to direct synthesis of a stand complementary to the phage,
and the resulting double-stranded DNA is transformed into a
phage-supporting host bacterium. Cultures of the transformed
bacteria are plated in top agar, permitting plaque formation from
single cells which harbor the phage.
[0067] Theoretically, 50% of the new plaques will contain the phage
having, as a single strand, the mutated form; 50% will have the
original sequence. The resulting plaques are hybridized with
kinased synthetic primer at a temperature which permits
hybridization of an exact match, but at which the mismatches with
the original strand are sufficient to prevent hybridization.
Plaques which hybridize with the probe are then picked, cultured,
and the DNA recovered.
[0068] In the proteins of the invention, however, a synthetic gene
is conveniently employed. The gene design can include restriction
sites which permit easy manipulation of the gene to replace coding
sequence portions with these encoding analogs.
[0069] Correct ligations for plasmid construction can be confirmed
by first transforming E. coli strain MM294 obtained from E. coli
Genetic Stock Center, CGSC #6135, or other suitable host with the
ligation mixture. Successful transformants are selected by
ampicillin, tetracycline or other antibiotic resistance or using
other markers depending on the mode of plasmid construction, as is
understood in the art. Plasmid from the transformants are then
prepared according to the method of Clewell, D. B., et al., Proc.
Natl. Acad. Sci. USA (1969) 62:1159, optionally following
chloramphenicol amplification (Clewell, D. B., J. Bacteriol. (1972)
110:667). The isolated DNA is analyzed by restriction and/or
sequenced by the dideoxy method of Sanger, F., et al., Proc. Natl.
Acad. Sci. USA (1977) 74:5463 as further described by Messing, et
al., Nucleic Acids Res. (1981) 9:309, or by the method of Maxam, et
al., Methods in Enzymology (1980) 65:499.
[0070] The constructed vector is then transformed into a suitable
host for production of the protein. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride, as
described by Cohen, S. N., Proc. Natl. Acad. Sci. USA (1972)
69:2110, or the RbCl method described in Maniatis, et al.,
Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor
Press, p. 254 is used for procaryotes or other cells which contain
substantial cell wall barriers. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, Virology (1978) 52:546 or electroporation is
preferred. Transformations into yeast are carried out according to
the method of Van Solingen, P., et al., J. Bacter. (1977) 130:946
and Hsiao, C. L., et al., Proc. Natl. Acad. Sci. USA (1979)
76:3829.
[0071] The transformed cells are then cultured under conditions
favoring expression of the MHC sequence and the recombinantly
produced protein recovered from the culture.
[0072] Antigenic Peptides
[0073] The autoantigenic proteins or tissues for a number of
autoimmune diseases are known. For example, in experimentally
induced autoimmune diseases, antigens involved in pathogenesis have
been characterized: in arthritis in rat and mouse, native type-II
collagen is identified in collagen-induced arthritis, and
mycobacterial heat shock protein in adjuvant arthritis (Stuart et
al. (1984), Ann. Rev. Immunol. 2:199-218; van Eden et al. (1988),
Nature 331:171-173.); thyroglobulin has been identified in
experimental allergic thyroiditis (EAT) in mouse (Maron et al.
(1988), J. Exp. Med. 152:1115-1120); acetyl choline receptor (AChR)
in experimental allergic myasthenia gravis (EAMG) (Lindstrom et al.
(1988), Adv. Immunol. 42:233-284); and myelin basic protein (MBP)
and proteolipid protein (PLP) in experimental allergic
encephalomyelitis (EAE) in mouse and rat (See Acha-Orbea et al.,
supra). In addition, for example, target antigens have been
identified in humans: type-II collagen in human rheumatoid
arthritis (Holoshitz et al. (1986), Lancet ii:305-309); and acetyl
choline receptor in myasthenia gravis (Lindstrom et al. (1988),
supra) all of the above are incorporated herein by reference.
[0074] It is believed that the presentation of antigen by the MHC
glycoprotein on the surface of antigen-presenting cells (APCs)
occurs subsequent to the hydrolysis of antigenic proteins into
smaller peptide units. The location of these smaller segments
within the antigenic protein can be determined empirically. These
segments are thought to be 8-15 residues in length, and contain
both the agretope (recognized by the MHC molecule) and the epitope
(recognized by T cell receptor on the T-helper cell). The epitope
itself is a contiguous or non-contiguous sequence of 5-6 amino
acids which recognizes the antigen-specific receptor of T-helper
cells. The agretope is a continuous or non-contiguous sequence
which is responsible for the association of the peptide with the
MHC glycoproteins.
[0075] The empirical process of determining the relevant 8-15 amino
acid subunits is illustrated using the alpha subunit of the
acetylcholine receptor of skeletal muscle. In myasthenia gravis
(MG) an autoimmune response is directed to a region of this
subunit. A loss of the acetyl choline receptors on the postsynaptic
membrane of the neuromuscular junction causes the MG symptoms.
[0076] In MG, autoantibodies against the alpha subunit of the
acetylcholine receptor (AChR) are associated with the autoimmune
response directed at the AChR. Eighty five percent of MG patients
have autoantibodies reactive with the alpha subunit. Of these, 60%
have antibodies that bind to a peptide segment of the alpha subunit
called the main immunogenic region (MIR) which is located between
residues 60 and 80 (Tzartos and Lindstrom, Proc. Natl. Acad. Sci.
USA (1980) 77:755). The peptide segments recognized by autoreactive
human T cells also are located on the alpha subunit (Hohfield, et
al., Proc. Natl. Acad. Sci. USA (1987). The epitopes recognized by
these T cells lie between residues 1-30, 125-147, 169-181, 257-271
and 351-368. In addition, in humans the AChR peptides 195-212 and
257-269 have been partially characterized as epitopes in myasthenia
gravis patients of the HLA-DR5 and HLA-DR3, DQw2 MHC haplotypes,
respectively (See Acha-Orbea (1989), supra).
[0077] The peptides carrying agretopes permitting presentation of
the epitopes associated with alpha subunit of this receptor are
readily determined. For example, determination of the appropriate
peptides in a mouse model is carried out as follows. Strains of
mice which, when immunized with Torpedo californicus AChR develop a
disease with many of the features of human myasthenia gravis, are
used as model. MHC Class II glycoproteins are isolated from spleen
cells of mice of this strain using lectin and monoclonal antibody
affinity supports. The purified MHC Class II proteins are
incorporated into phospholipid vesicles by detergent dialysis. The
resultant vesicles are then allowed to fuse to clean glass cover
slips to produce on each a planar lipid bilayer containing MHC
molecules as shown in FIG. 5 (Brian and McConnell, Proc. Natl.
Acad. Sci. USA (1984) 81:6159, which is incorporated herein by
reference).
[0078] One cover slip containing MHC Class II molecules embedded in
the adherent planar lipid membrane is placed in each well of
several 24-well culture plates. Each one of the approximately 40
overlapping 20-residue synthetic peptides corresponding to the
alpha subunit sequence and containing one or more radiolabeled
amino acid residues (prepared as described below) is placed in a
well with cover slip and PBS and allowed to incubate several days.
The extent of binding of peptide in the MHC Class II glycoprotein
antigen binding site is measured by the amount of radio-activity
incorporated into the MHC Class II-planar lipid membrane on the
cover slip versus planar lipid membrane alone. Specific
incorporation of radioactivity indicates that the bound peptide
contains an agretope (MHC Class II peptide binding site) of one of
the several species of MHC Class II molecules present in the planar
lipid membrane. In this way, the set of agretopes for the alpha
subunit of AChR is defined for the mouse strain that displays the
symptoms of MG upon immunization with AChR or purified alpha
subunit.
[0079] Next, each of the alpha subunit synthetic peptide segments
that contain an agretope is again incorporated into the antigen
binding site of isolated MHC Class II proteins embedded in planar
lipid membranes on cover slips. One cover slip is added to each
well of a 24-well culture plate, and spleen cells from mice
immunized against AChR (and from which strain the adherent MHC
Class II proteins were isolated) are added to each well. T cell
hybridoma proliferation, as measured by tritiated thymidine uptake
into DNA, indicates that the MHC Class II protein-bound peptide
contains both an agretope and an epitope for binding to the T cell.
Activation of T cell clones is determined by measuring IL-3
production (see, Quill et al., supra).
[0080] The Dupont apparatus and technique for rapid multiple
peptide synthesis (RAMPS) is used to synthesize the members of a
set of overlapping (10 residue overlap), 20-residue peptides from
the alpha subunit of Torpedo californicus AChR. The sequence of
this peptide is known and is shown in FIG. 6. One or more
radioactive amino acids is incorporated into each synthetic
peptide. The pentafluorphenyl active esters of side
chain-protected, FMOC amino acids are used to synthesize the
peptides, applying standard stepwise solid phase peptide synthetic
methods, followed by standard side chain deprotection and
simultaneous release of the peptide amide from the solid
support.
[0081] Alternatively the overlapping sequences which include the
putative segments of 8-15 amino acids of the antigenic protein,
such as acetylcholine receptor protein, can be synthesized on the
method of Geysen, H. M., et al. J. Immun. Meth. (1987) 102:274,
which is incorporated herein by reference. The synthesized radio
labeled peptides are tested by incubating them individually (on the
plates) with purified MHC proteins which have been formulated into
lipid membrane bilayers as above.
[0082] In multiple sclerosis (MS), which results in the destruction
of the myelin sheath in the central nervous system, myelin basic
protein (MBP), the major protein component of myelin is the
principal autoantigen. Pertinent segments of the MBP protein are
also determined empirically, using a strain of mice which develops
experimental allergic encephalitis (EAG) when immunized with bovine
myelin basic protein, the sequence of MBP is shown in FIG. 7.
[0083] Systemic lupus erythematosus (SLE) has a complex
systemology, but results from an autoimmune response to red blood
cells. Peptides which are the antigenic effectors of this disease
are found in the proteins on the surface of red blood cells.
[0084] Rheumatoid arthritis (RA) is a chronic inflammatory disease
resulting from an immune response to proteins found in the synovial
fluid.
[0085] Insulin-dependent diabetes mellitus (IDDM) results from
autoimmune attack on the beta cells within the Islets of Langerhans
which are responsible for secretion of insulin. Circulating
antibodies to Islets cells surface antigens and to insulin are
known to precede IDDM. Critical peptides in eliciting the immune
response in IDDM are believed to be portions of the insulin
sequence and the beta cell membrane surface proteins.
[0086] The relevant antigenic peptide subunits, as they are
relatively short, can readily by synthesized using standard
automated methods for peptide synthesis. In the alternative, they
can be made recombinantly using isolated or synthetic DNA
sequences; though this is not the most efficient approach for
peptides of this length.
[0087] Thus, in summary, a set of labeled test peptides is
prepared, and those which bind to MHC in planar lipid membranes
containing MHC proteins are shown to contain the agretope.
[0088] The identified peptides are then prepared by conventional
solid phase synthesis and the subset which contain epitopes for the
disease-inducing helper T cell clones is determined by incubation
of the candidate peptides with murine antigen-presenting cells
(APC) (or with isolated MHC complex) and spleen or lymph node T
cells from mice immunized with the full length protein. Successful
candidates will stimulate T cell proliferation in this system. This
second, smaller, subset represents the suitable peptide
component.
[0089] Formation of the Complex
[0090] The elements of the complex can be associated by standard
means known in the art. The antigenic peptides can be associated
noncovalently with the pocket portion of the MHC protein by, for
example, mixing the two components. They can also be covalently
bound using standard procedures by, for example, photo affinity
labelling, (see e.g., Hall et al., Biochemistry 24:5702-5711
(1985), which is incorporated herein by reference).
[0091] For example, the AChR peptide 195-215, which has been
characterized as an epitope in MG in humans and in mice, may be
connected to the N-terminal antigen binding site of a polypeptide
derived from an MHC antigen associated with MG. The amino acid
sequence of the AChR peptide in one letter amino acid code is:
[0092] DTPYLDITYHFIMQRIPLYFV
[0093] An oligonucleotide which encodes the peptide is synthesized
using the known codons for the amino acid, preferably those codons
which have preferred utilization in the organism which is to be
used for expression are utilized in designing the oligonucleotide.
Preferred codon utilizations for a variety of organisms and types
of cells are known in the art. If, for example, expression is to be
in E. coli, a suitable oligonucleotide sequence encoding AChR
195-215 could be:
[0094] 5' GAC ACC CCG TAC CTG GAC ATC ACC TAC CAC TTC ATC ATG CAG
CGT ATC CCG CTG TAC TTC CTG 3'.
[0095] This sequence may then be incorporated into a sequence
encoding the peptides derived from the MHC antigen, utilizing
techniques known in the art. The incorporation site will be such
that, when the molecule is expressed and folded, the AChR peptide
antigen will be available as an epitope for the target T cells.
[0096] In one protocol, the AChR 195-215 peptide is attached to the
N-terminal end of the appropriate MHC molecule. If the recombinant
complex is to be used in mice, for example, the AChR peptide may be
incorporated into a sequence encoding either the I-A.sup.b-alpha or
I-A.sup.b-beta chain. The sequences encoding these chains are
known, and are shown in FIG. 8 (alpha chain), and FIG. 9 (beta
chain); also shown in the figures are restriction enzyme sites and
significant domains of the chains. If the AChR peptide is to be
incorporated into the beta chain, for example, the oligonucleotide
may be inserted as a replacement for the leader peptide. Methods of
replacing sequences within polynucleotides are known in the art,
examples of which are described in the section on the construction
of plasmids.
[0097] A similar protocol may be used for incorporation of the AChR
peptide into a sequence encoding a peptide derived from the
appropriate human HLA antigen. For example, in humans, the
haplotype DR2W2 is associated with MG. Hence, the AChR peptide may
be incorporated into, for example, a sequence encoding a beta-chain
of a DR2 allele. The structural basis in the DR subregion for the
major serological specificities DR1-9 are known, as are the
sequences encoding the HLA-DR-beta chains from a number of DR
haplotypes. See, for e.g., Bell et al. (1987), Proc. Natl. Acad.
Sci. USA 84:6234-6238 which are incorporated herein by
reference.
[0098] As demonstrated above, the autoimmune antigen peptide and
the MHC component may be linked via peptide linkages. However,
other modes of linkage are obvious to those of skill in the art,
and could include, for example, attachment via carbohydrate groups
on the glycoproteins, including, e.g., the carbohydrate moieties of
the alpha- and/or beta-chains.
[0099] Assessment of the Complex
[0100] The complexes of the invention can be assayed using an in
vitro system or using an in vivo model. In the in vitro system, the
complex is incubated with peripheral blood T cells from subjects
immunized with, or showing immunity to, the protein or antigen
responsible for the condition associated with the peptide of the
complex. The successful complexes will induce anergy in syngeneic T
cells and prevent proliferation of the T cells even upon
stimulation with additional antigen.
[0101] In the in vivo system, T cells that proliferate in response
to the isolated epitope or to the full length antigen in the
presence of APC are cloned. The clones are injected into
histocompatible animals which have not been immunized in order to
induce the autoimmune disease. Symptoms related to the relevant
complex should ameliorate or eliminate the symptoms of the
disease.
[0102] Either of the types of complexes, i.e., with or without the
effector component, may be used. In one mode the treatment is
two-fold. The individual is treated with the complex of MHC-encoded
antigen-presenting glycoprotein containing an effective portion of
the antigen to down-regulate the immune system. Further
down-regulation is achieved by treatment with the three component
complex with includes the MHC-encoded antigen-presenting
glycoprotein, an effective portion of antigen which is specific for
the autoimmune disease being treated, and an effector component. In
addition, panels of complexes may be used for treatment. For
example, if it is suspected that more than one peptide of an
antigen is involved in the autoimmune response, and/or if it is
suspected that more than one antigen is involved, the individual
may be treated with several complexes selected from a panel
containing the effective portion of the appropriate MHC-encoded
antigen-presenting polypeptides, and effective portions of
antigens; these may be with or without effector components.
[0103] Administration of a labeled complex permits identification
of those portions of the immune system involved in the disease, in
diagnostic applications.
[0104] Selection of the MHC Complexes for Therapy and/or
Diagnosis
[0105] In order to select the MHC complexes of the invention which
are to be used in the diagnosis or treatment of an individual for
an autoimmune disease, the type of MHC antigens which are involved
in the presentation of the autoantigen are identified.
[0106] Specific autoimmune dysfunctions are correlated with
specific MHC types. A list of the DQ/DR haplotypes in humans and
their associations with autoimmune diseases are shown in FIG. 10.
Methods for identifying which alleles, and subsequently which MHC
encoded polypeptides, are associated with an autoimmune disease are
known in the art. A method described in EP 286447 is suitable. In
this method several steps are followed. First, the association
between an MHC antigen and the autoimmune disease is determined
based upon genetic studies. The methods for carrying out these
studies are known to those skilled in the art, and information on
all known HLA disease associations in humans is maintained in the
HLA and Disease Registry in Copenhagen. The locus encoding the
polypeptide associated with the disease is the one that would bear
the strongest association with the disease (See FIG. 10).
[0107] Second, specific alleles encoding the disease associated
with MHC antigen/polypeptide are identified. In the identification
of the alleles, it is assumed that the susceptibility allele is
dominant. Identification of the allele is accomplished by
determining the strong positive association of a specific subtype
with the disease. This may be accomplished in a number of ways, all
of which are known to those skilled in the art. E.g., subtyping may
be accomplished by mixed lymphocyte response (MLR) typing and by
primed lymphocyte testing (PLT). Both methods are described in Weir
and Blackwell, eds., Handbook of Experimental Immunology, which is
incorporated herein by reference. It may also be accomplished by
analyzing DNA restriction fragment length polymorphism (RFLP) using
DNA probes that are specific for the MHC locus being examined.
E.g., Nepom (1986), Annals N.Y. Acad. Sci. 475, 1. Methods for
preparing probes for the MHC loci are known to those skilled in the
art. See, e.g., Gregersen et al. (1986), Proc. Natl. Acad. Sci. USA
79:5966; Weissman et al. in Medicine in Transition: the Centennial
of the University of Illinois College of Medicine (E. P. Cohen, ed.
1981) all of which are incorporated herein by reference.
[0108] The most complete identification of subtypes conferring
disease susceptibility is accomplished by sequencing of genomic DNA
of the locus, or cDNA to mRNA encoded within the locus. The DNA
which is sequenced includes the section encoding the hypervariable
regions of the MHC encoded polypeptide. Techniques for identifying
specifically desired DNA with a probe, for amplification of the
desired region are known in the art, and include, for example, the
polymerase chain reaction (PCR) technique.
[0109] Once the allele which confers susceptibility to the specific
autoimmune disease is identified, the polypeptide encoded within
the allele is also identifiable, i.e., the polypeptide sequence may
be deduced from the sequence of DNA within the allele encoding it.
The MHC antigen complexes of the invention used for diagnosis
and/or therapy are derived from the effective portion of the MHC
antigen associated with the autoimmune disease state and from an
autoimmune antigen associated with the same disease state.
[0110] As an example, over 90% of rheumatoid arthritis patients
have a haplotype of DR4(Dw4), DR4(Dw14) or DR1 (See FIG. 10). It is
also known that a target antigen in human rheumatoid arthritis is
type-II collagen. Hence, the complexes of the invention used for
treatment or diagnosis of an individual with rheumatoid arthritis
would include those containing a polypeptide derived from the
DR4(Dw4), DR1 and/or DR4(Dw14) which is capable of antigen
presentation for disease induction, or incapable of antigen
presention for disease suppression, complexed with an effective
portion of type-II collagen.
[0111] A protocol which may be suitable for the utilization of the
complexes of the invention for the diagnosis and/or treatment of an
autoimmune disease is depicted in FIG. 11. Briefly, an individual
having (or susceptible to) an autoimmune disease is identified, and
the autoimmune dysfunction is identified. Identification may be by
symptomology and/or an examination of family histories. The
individual's MHC type is determined by one or more of several
methods known in the art, including, for example, cell typing by
MLR, by serologic assay, and by DNA analysis (including RFLP and
PCR techniques). The individuals T cells are examined in vitro, to
determine the autopeptide(s) recognized by autoreactive T cells;
this is accomplished utilizing labeled complexes of the invention,
described supra, which are of the formula X.sup.1 MHC.sup.2
peptide, wherein X is a label moiety. After it is determined which
complexes target the T cells, the individual is treated with
complexes of the invention which are able to suppress the specific
autoreactive T cell replication and/or those which kill the
autoreactive T cells; these are complexes of the type MHC.sup.2
peptide, and, X.sup.1 MHC.sup.2 peptide (wherein X is a moiety
capable of killing the T cell), respectively. Therapy (as
determined by the autoreactive T cells remaining) is monitored with
T cell binding studies using the labeled complexes of the
invention, described supra.
[0112] As used herein, the term "individual" encompasses all
mammals and all vertebrates which possess basically equivalent MHC
systems.
[0113] Model Systems for in vivo Testing
[0114] The following are model systems for autoimmune diseases
which can be used to evaluate the effects of the complexes of the
invention on these conditions.
[0115] Systemic Lupus Erythematosus (SLE)
[0116] F.sub.1 hybrids of autoimmune New Zealand black (NZB) mice
and the phenotypically normal New Zealand White (NZW) mouse strain
develop severe systemic autoimmune disease, more fulminant than
that found in the parental NZB strain. These mice manifest several
immune abnormalities, including antibodies to nuclear antigens and
subsequent development of a fatal, immune complex-mediated
glomerulonephritis with female predominance, remarkably similar to
SLE in humans. Knight, et al., J. Exp. Med. (1978) 147:1653, which
is incorporated hereby by reference.
[0117] In both the human and murine forms of the disease, a strong
association with MHC gene products has been reported. HLA-DR2 and
HLA-DR3 individuals are at a higher risk than the general
population to develop SLE (Reinertsen, et al., N. Engl. J. Med
(1970) 299:515), while in NZB/W F.sub.1 mice (H-2.sup.d/u), a gene
linked to the h-2.sup.u haplotype derived from the NZW parent
contributes to the development of the lupus-like nephritis.
[0118] The effect of the invention complex can be measured by
survival rates and by the progress of development of the symptoms,
such as proteinuria and appearance of anti-DNA antibodies.
[0119] Proteinuria is measured calorimetrically by the use of
Uristix (Miles Laboratories, Inc., Elkhart, Ind.), giving an
approximation of proteinuria as follows: trace, 10 mg/dl; 1+, 30
mg/dl; 100 mg/dl; 3+, 300 mg/dl; and 4+, 1000 mg/dl. The
development of high grade proteinuria is significantly delayed by
treatment of the mice with complex.
[0120] The presence of anti-DNA specific antibodies in NZB/W
F.sub.1 mice is determined by using a modification of a linked
immunosorbent assay (ELISA) described by Zouali and Stollar, J.
Immunol. Methods (1986) 90:105 which is incorporated herein by
reference.
Myasthenia Gravis (MG)
[0121] Myasthenia gravis is one of several human autoimmune
diseases linked to HLA-D. Safenberg, et al., Tissue Antigens (1978)
12:136; McDevitt, et al., Arth. Rheum. (1977) 20:59 which are
incorporated herein by reference. In MG, antibodies to the acetyl
choline receptors (AcChoR) impair neuromuscular transmission by
mediating loss of AcChoR in the postsynaptic membrane.
[0122] SJL/J female mice are a model system for human MG. In these
animals, experimental autoimmune myasthenia gravis (EAMG) is
induced by immunizing the mice with soluble AcChoR protein from
another species. Susceptibility to EAMG is linked in part to the
MHC and has been mapped to the region within H-2. Christadoss, et
al., J. Immunol. (1979) 123:2540.
[0123] AcChoR protein is purified from Torpedo californica and
assayed according to the method of Waldor, et al., Proc. Natl.
Acad. Sci. (USA) (1983) 80:27.13, incorporated by reference.
Emulsified AcChoR, 15 ug in complete Freund adjuvant, is injected
intradermally among six sites on the back, the hind foot pads, and
the base of the tail. Animals are reimmunized with this same
regimen 4 weeks later.
[0124] Evaluation can be made by measurement of anti-AcChoR
antibodies, Anti-AcChoR antibody levels are measured by a
microliter ELISA assay as described in Waldor, et al., supra. The
standard reagent volume is 50 ul per well. Reagents are usually
incubated in the wells for 2 hr at RT. Five ug of AcChoR diluted in
bicarbonate buffer, pH 9.6, is added to each well. After incubation
with AcChoR, the plates are rinsed four times with a wash solution
consisting of phosphate-buffer saline containing 0.05% Tween and
0.05% NaN.sub.3. Mouse sera are diluted in 0.01M PBS (pH 7.2), 1.5
mfr MgCl.sub.2, 2.0 mM 2-mercaptoethanol, 0.05% Tween-80, 0.05%
NaN.sub.3 (P-Tween buffer) and incubated on the plate. After the
plate is washed, beta-galactosidase-conjugated sheep anti-mouse
antibody diluted in P-Tween buffer is added to each well. After a
final washing, the enzyme substrate, p-nitrophenyl-galctopyranoside
is added to the plate, and the degree of substrate catalysis is
determined from the absorbance at 405 nm after 1 hr.
[0125] Anti-AcChoR antibodies are expected to be present in the
immunized with AcChoR mice as compared to nonimmunized mice.
Treatment with complex is expected to significantly reduce the
titer of anti-AcChoR antibodies in the immunized mice.
[0126] The effect of treatment with complex on clinical EAMG can
also be assessed. Myasthenia symptoms include a characteristic
hunched posture with drooping of the head and neck, exaggerated
arching of the back, splayed limbs, abnormal walking, and
difficulty in righting. Mild symptoms are present after a standard
stress test, and should be ameliorated by administration of complex
after a period of time after which antibody titer has fallen.
Rheumatoid Arthritis (RA)
[0127] In humans, susceptibility to rheumatoid arthritis is
associated with HLA D/DR. The immune response in mice to native
type II collagen has been used to establish an experimental model
for arthritis with a number of histological and pathological
features resembling human RA. Susceptibility to collagen-induced
arthritis (CIA) in mice has been mapped to the H-2 I region,
particularly the I-A subregion. Huse, et al., Fed. Proc. (1984)
43:1820.
[0128] Mice from a susceptible strain, DBA-1 are caused to have CIA
by treatment of the mice with native type II collagen, using the
technique described in Wooley and Luthra, J. Immunol. (1985)
134:2366, incorporated herein by reference.
[0129] In another model, adjuvant arthritis in rats is an
experimental model for human arthritis, and a prototype of
autoimmune arthritis triggered by bacterial antigens, Holoschitz,
et al., Prospects of Immunology (CRC Press) (1986); Pearson
Arthritis Rheum. (1964) 7:80. The disease the result of a
cell-mediated immune response, as evidenced by its transmissibility
by a clone of T cells which were reactive against the adjuvant
(MT); the target self-antigen in the disease, based upon studies
with the same cloned cells, appears to be part(s) of a proteoglycan
molecule of cartilage.
[0130] Adjuvant disease in rats is produced as described by
Pearson, supra, i.e., by a single injection of Freund's adjuvant
(killed tubercle bacilli or chemical fractions of it, mineral oil,
and an emulsifying agent) given into several depot sites,
preferably intracutaneously or into a paw or the base of the tail.
The adjuvant is given in the absence of other antigens.
[0131] The effect of complex treatment of manifestations of the
disease are monitored. These manifestations are histopathological,
and include an acute and subacute synovitis with proliferation of
synovial lining cells, predominantly a mononuclear infiltration of
the articular and particular tissues, the invasion of bone and
articular cartilage by connective tissue pannus, and periosteal new
bone formation, especially adjacent to affected joints. In severe
or chronic cases, destructive changes occur, as do fibrous or bony
ankylosis. These histopathological symptoms are expected to appear
in control animals at about 12 days after sensitization to the
Freund's adjuvant.
Insulin Dependent Diabetes Mellitus (IDDM)
[0132] IDDM is observed as a consequence of the selective
destruction of insulin-secreting cells within the Islets of
Langerhans of the pancreas. Involvement of the immune system in
this disease is suggested by morphologic evidence of early
infiltration of the Islets by mononuclear cells, by the detection
of anti-islet cell antibodies, by the high frequency of HLA-DR3 and
-DR4 alleles in IDDM populations, and by clinical associations
between IDDM and various autoimmune diseases. An animal model for
spontaneous IDDM and thyroiditis has been developed in the BB rat.
As in humans, the rat disease is controlled in part by the genes
encoding the MHC antigens, is characterized by islet infiltration,
and is associated with the presence of anti-islet antibodies. The
I-E equivalent Class II MHC antigens appear to be involved in
manifestation of the autoimmune diseases in the BB rat. Biotard, et
al., Proc. Natl. Acad. Sci. USA (1985) 82:6627.
[0133] In morphologic evaluation, insulitis is characterized by the
presence of mononuclear inflammatory cells within the islets.
Thyroiditis is characterized by focal interstitial lymphocytic
infiltrate within the thyroid gland, as a minimum criterion. Most
severe cases show diffuse extensive lymphocytic infiltrates,
disruption of acini, fibrosis, and focal Hurthle call change. See
Biotard et al., supra.
[0134] Treatment of the BB rats with complex of the invention is
expected to ameliorate or prevent the manifestation of the clinical
and morphological symptoms associated with IDDM and
thyroiditis.
[0135] In another spontaneous model, the NOD mouse strain
(H-2K.sup.dD.sup.b) is a murine model for autoimmune IDDM. The
disease in these animals is characterized by anti-islet cell
antibodies, severe insulitis, and evidence for autoimmune
destruction of the beta-cells. Kanazawa, et al., Diabetologia
(1984) 27:113. The disease can be passively transferred with
lymphocytes and prevented by treatment with cyclosporin-A (Ikehara,
et al., Proc. Natl. Acad. Sci. USA (1985) 82:7743; Mori, et al.),
Diabetologia (1986) 29:244. Untreated animals develop profound
glucose intolerance and ketosis and succumb within weeks of the
onset of the disease. Seventy to ninety percent of female and
20-30% of male animals develop diabetes within the first six months
of life. Breeding studies have defined at least two genetic loci
responsible for disease susceptibility, one of which maps to the
MHC. Characterization of NOD Class II antigens at both the
serologic and molecular level suggest that the susceptibility to
autoimmune disease is linked to I-A.sub.B. Acha-Orbea and McDevitt,
Proc. Natl. Acad. Sci. USA (1907) 84:235.
[0136] Treatment of Female NOD mice with complex is expected to
lengthen the time before the onset of diabetes and/or to ameliorate
or prevent the disease.
[0137] Experimental Allergic Encephalomyelitis (EAE)
[0138] Experimental allergic encephalomyelitis (EAE) is an induced
autoimmune disease of the central nervous system which mimics in
many respects the human disease of multiple sclerosis (MS). The
disease can be induced in many species, including mice and
rats.
[0139] The disease is characterized by the acute onset of
paralysis. Perivascular infiltration by mononuclear cells in the
CNS is observed in both mice and rats. Methods of inducing the
disease, as well as symptomology, are reviewed in Aranson (1985) in
The Autoimmune Diseases (eds. Rose and Mackay, Academic Press,
Inc.) pp. 399-427, and in Acha-Orbea et al. (1989), Ann. Rev. 1 mm.
7:377-405.
[0140] One of the genes mediating susceptibility is localized in
the MHC class II region (Moore et al. (1980), J. Immunol.
124:1815-1820). The best analyzed encephalitogenic protein is
myelin basic protein (MBP), but other encephalitogenic antigens are
found in the brain. The immunogenic epitopes have been mapped (see
Acha-Orbea et al., supra.). In the PL mouse strains (H-2.sup.u) two
encephalitogenic peptides in MBP have been characterized: MBP
peptide p35-47 (MBP 35-47), and acetylated (MBP 1-9).
[0141] The effect of the invention complexes on ameliorating
disease symptoms in individuals in which EAE has been induced can
be measured by survival rates, and by the progress of the
development of symptoms.
[0142] Formulation and Administration
[0143] If the transmembrane region of the MHC subunit is included,
the complexes of the invention are conveniently administered after
being incorporated in lipid monolayers or bilayers. Typically
liposomes are used for this purpose but any form of lipid membrane,
such as planar lipid membranes or the cell membrane of a cell
(e.g., a red blood cell) may be used. The complexes are also
conveniently incorporated into micelles. The data presented in
Example 2, below, shows that MHC-peptide complexes comprising
dimeric MHC molecules exist primarily as aggregates.
[0144] Liposomes can be prepared according to standard methods, as
described below. However, if the transmembrane region is deleted,
the complex can be administered in a manner conventionally used for
peptide-containing pharmaceuticals.
[0145] Administration is systemic and is effected by injection,
preferably intravenous, thus formulations compatible with the
injection route of administration may be used. Suitable
formulations are found in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Philadelphia, Pa., 17th ed. (1985), which is
incorporated herein by reference. A variety of pharmaceutical
compositions comprising complexes of the present invention and
pharmaceutically effective carriers can be prepared. The
pharmaceutical compositions are suitable in a variety of drug
delivery systems. For a brief review of present methods of drug
delivery, see, Langer, Science 249:1527-1533 (1990) which is
incorporated herein by reference.
[0146] In preparing pharmaceutical compositions of the present
invention, it is frequently desirable to modify the complexes of
the present invention to alter their pharmacokinetics and
biodistribution. For a general discussion of pharmacokinetics, see,
Remington's Pharmaceutical Sciences, supra, Chapters 37-39. A
number of methods for altering pharmacokinetics and biodistribution
are known to one of ordinary skill in the art (see, e.g., Langer,
supra). For instance, methods suitable for increasing serum
half-life of the complexes include treatment to remove
carbohydrates which are involved in the elimination of the
complexes from the bloodstream. Preferably, substantially all of
the carbohydrate moieties are removed by the treatment.
Substantially all of the carbohydrate moieties are removed if at
least about 75%, preferably about 90%, and most preferably about
99% of the carbohydrate moieties are removed. Conjugation to
soluble macromolecules, such as proteins, polysaccharides, or
synthetic polymers, such as polyethylene glycol, is also effective.
Other methods include protection of the complexes in vesicles
composed of substances such as proteins, lipids (for example,
liposomes), carbohydrates, or synthetic polymers.
[0147] Liposomes of the present invention typically contain the
MHC-peptide complexes positioned on the surface of the liposome in
such a manner that the complexes are available for interaction with
the T cell receptor. The transmembrane region is usually first
incorporated into the membrane at the time of forming the membrane.
The liposomes can be used to target desired drugs (e.g. toxins or
chemotherapeutic agents) to particular autoreactive T cells.
Alternatively, the complexes embedded in the liposome may be used
to induce anergy in the targeted cells.
[0148] Liposome charge is an important determinant in liposome
clearance from the blood, with negatively charged liposomes being
taken up more rapidly by the reticuloendothelial system (Juliano,
Biochem. Biophys. Res. Commun. 63:651 (1975)) and thus having
shorter half-lives in the bloodstream. Liposomes with prolonged
circulation half-lives are typically desirable for therapeutic and
diagnostic uses. For instance, liposomes which can be maintained
from 8, 12, or up to 24 hours in the bloodstream are particularly
preferred.
[0149] Typically, the liposomes are prepared with about 5-15 mole
percent negatively charged phospholipids, such as
phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
Added negatively charged phospholipids, such as
phosphatidylglycerol, also serve to prevent spontaneous liposome
aggregating, and thus minimize the risk of undersized liposomal
aggregate formation. Membrane-rigidifying agents, such as
sphingomyelin or a saturated neutral phospholipid, at a
concentration of at least about 50 mole percent, and 5-15 mole
percent of monosialylganglioside, may provide increased circulation
of the liposome preparation in the bloodstream, as generally
described in U.S. Pat. No. 4, 837,028, incorporated herein by
reference.
[0150] Additionally, the liposome suspension may include
lipid-protective agents which protect lipids against free-radical
and lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as .alpha.tocopherol and water-soluble
iron-specific chelators, such as ferrioxianine, are preferred.
[0151] A variety of methods are available for preparing liposomes,
as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng.
9:467 (1980), U.S. Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028,
all of which are incorporated herein by reference. One method
produces multilamellar vesicles of heterogeneous sizes. In this
method, the vesicle-forming lipids are dissolved in a suitable
organic solvent or solvent system and dried under vacuum or an
inert gas to form a thin lipid film. If desired, the film may be
redissolved in a suitable solvent, such as tertiary butanol, and
then lyophilized to form a more homogeneous lipid mixture which is
in a more easily hydrated powderlike form. This film is covered
with an aqueous solution of the targeted drug and the targeting
component and allowed to hydrate, typically over a 15-60 minute
period with agitation. The size distribution of the resulting
multilamellar vesicles can be shifted toward smaller sizes by
hydrating the lipids under more vigorous agitation conditions or by
adding solubilizing detergents such as deoxycholate.
[0152] The hydration medium contains the targeted drug at a
concentration which is desired in the interior volume of the
liposomes in the final liposome suspension. Typically the drug
solution contains between 10-100 mg/ml of the complexes in a
buffered saline solution.
[0153] Following liposome preparation, the liposomes may be sized
to achieve a desired size range and relatively narrow distribution
of liposome sizes. One preferred size range is about 0.2-0.4
microns, which allows the liposome suspension to be sterilized by
filtration through a conventional filter, typically a 0.22 micron
filter. The filter sterilization method can be carried out on a
high through-put basis if the liposomes have been sized down to
about 0.2-0.4 microns.
[0154] Several techniques are available for sizing liposome to a
desired size. One sizing method is described in U.S. Pat. No.
4,737,323, incorporated herein by reference. Sonicating a liposome
suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles less
than about 0.05 microns in size. Homogenization is another method
which relies on shearing energy to fragment large liposomes into
smaller ones. In a typical homogenization procedure, multilamellar
vesicles are recirculated through a standard emulsion homogenizer
until selected liposome sizes, typically between about 0.1 and 0.5
microns, are observed. In both methods, the particle size
distribution can be monitored by conventional laser-beam particle
size discrimination.
[0155] Extrusion of liposome through a small-pore polycarbonate
membrane or an asymmetric ceramic membrane is also an effective
method for reducing liposome sizes to a relatively well-defined
size distribution. Typically, the suspension is cycled through the
membrane one or more times until the desired liposome size
distribution is achieved. The liposomes may be extruded through
successively smaller-pore membranes, to achieve a gradual reduction
in liposome size.
[0156] Even under the most efficient encapsulation methods, the
initial sized liposome suspension may contain up to 50% or more
complex in a free (nonencapsulated) form.
[0157] Several methods are available for removing non-entrapped
compound from a liposome suspension. In one method, the liposomes
in the suspension are pelleted by high-speed centrifugation leaving
free compound and very small liposomes in the supernatant. Another
method involves concentrating the suspension by ultrafiltration,
then resuspending the concentrated liposomes in a replacement
medium. Alternatively, gel filtration can be used to separate large
liposome particles from solute molecules.
[0158] Following the above treatment, the liposome suspension is
brought to a desired concentration for use in intravenous
administration. This may involve resuspending the liposomes in a
suitable volume of injection medium, where the liposomes have been
concentrated, for example by centrifugation or ultrafiltration, or
concentrating the suspension, where the drug removal step has
increased total suspension volume. The suspension is then
sterilized by filtration as described above. The liposomes
comprising the MHC-peptide complex may be administered parenterally
or locally in a dose which varies according to, e.g., the manner of
administration, the drug being delivered, the particular disease
being treated, etc.
[0159] Micelles are commonly used in the art to increase solubility
of molecules having nonpolar regions. One of skill will thus
recognize that micelles are useful in compositions of the present
invention. Micelles comprising the complexes of the invention are
prepared according to methods well known in the art (see, e.g.,
Remington's Pharmaceutical Sciences, supra, Chap. 20). Micelles
comprising the complexes of the present invention are typically
prepared using standard surfactants or detergents.
[0160] Micelles are formed by surfactants (molecules that contain a
hydrophobic portion and one or more ionic or otherwise strongly
hydrophilic groups) in aqueous solution. As the concentration of a
solid surfactant increases, its monolayers adsorbed at the
air/water or glass/water interfaces become so tightly packed that
further occupancy requires excessive compression of the surfactant
molecules already in the two monolayers. Further increments in the
amount of dissolved surfactant beyond that concentration cause
amounts equivalent to the new molecules to aggregate into micelles.
This process begins at a characteristic concentration called
"critical micelle concentration".
[0161] The shape of micelles formed in dilute surfactant solutions
is approximately spherical. The polar head groups of the surfactant
molecules are arranged in an outer spherical shell whereas their
hydrocarbon chains are oriented toward the center, forming a
spherical core for the micelle. The hydrocarbon chains are randomly
coiled and entangled and the micellar interior has a nonpolar,
liquid-like character. In the micelles of polyoxyethylated nonionic
detergents, the polyoxyethlene moieties are oriented outward and
permeated by water. This arrangement is energetically favorable
since the hydrophilic head groups are in contact with water and the
hydrocarbon moieties are removed from the aqueous medium and partly
shielded from contact with water by the polar head groups. The
hydrocarbon tails of the surfactant molecules, located in the
interior of the micelle, interact with one another by weak van der
Waals forces.
[0162] The size of a micelle or its aggregation number is governed
largely by geometric factors. The radius of the hydrocarbon core
cannot exceed the length of the extended hydrocarbon chain of the
surfactant molecule. Therefore, increasing the chain length or
ascending homologous series increases the aggregation number of
spherical micelles. For surfactants whose hydrocarbon portion is a
single normal alkyl chain, the maximum aggregation numbers
consistent with spherical shape are approximately 27, 39, 54, 72,
and 92 for C.sub.8, C.sub.10, C.sub.12, C.sub.14 and C.sub.16,
respectively. If the surfactant concentration is increased beyond a
few percent and if electrolytes are added (in the case of ionic
surfactants) or the temperature is raised (in the case of nonionic
surfactants), the micelles increase in size. Under these
conditions, the micelles are too large to remain spherical and
become ellipsoidal, cylindrical or finally lamellar in shape.
[0163] Common surfactants well known to one of skill in the art can
be used in the micelles of the present invention. Suitable
surfactants include sodium laureate, sodium oleate, sodium lauryl
sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and
PLURONIC F-127.RTM. (Wyandotte Chemicals Corp.). Preferred
surfactants are nonionic polyoxyethylene and polyoxypropylene
detergents compatible with IV injection such as, TWEEN-80.RTM.,
PLURONIC F-68.RTM., n-octyl-.beta.-D-glucopyranoside, and the like.
In addition, phospholipids, such as those described for use in the
production of liposomes, may also be used for micelle
formation.
[0164] Since the MHC subunits of the present invention comprise a
lipophilic transmembrane region and a relatively hydrophilic
extracellular domain, mixed micelles are formed in the presence of
common surfactants or phospholipids and the subunits. The mixed
micelles of the present invention may comprise any combination of
the subunits, phospholipids and/or surfactants. Thus, the micelles
may comprise subunits and detergent, subunits in combination with
both phospholipids and detergent, or subunits and phospholipid.
[0165] For pharmaceutical compositions which comprise the complexes
of the present invention, the dose will vary according to, e.g.,
the particular complex, the manner of administration, the
particular disease being treated and its severity, the overall
health and condition of the patient, and the judgment of the
prescribing physician. Dosage levels for murine subjects are
generally between about 10 .mu.g and about 500 .mu.g. A total dose
of between about 50 .mu.g and about 300 .mu.g, is preferred. For
instance, in treatments provided over the course of a disease,
three 25 .mu.g or 100 .mu.g doses are effective. Total dosages
range between about 0.5 and about 25 mg/kg, preferably about 3 to
about 15 mg/kg.
[0166] The pharmaceutical compositions are intended for parenteral,
topical, oral or local administration, such as by aerosol or
transdermally, for prophylactic and/or therapeutic treatment. The
pharmaceutical compositions can be administered in a variety of
unit dosage forms depending upon the method of administration. For
example, unit dosage forms suitable for oral administration include
powder, tablets, pills, and capsules.
[0167] Preferably, the pharmaceutical compositions are administered
intravenously. Thus, this invention provides compositions for
intravenous administration which comprise a solution of the complex
dissolved or suspended in an acceptable carrier, preferably an
aqueous carrier. A variety of aqueous carriers may be used, e.g.,
water, buffered water, 0.4% saline, and the like. For instance,
phosphate buffered saline (PBS) is particularly suitable for
administration of soluble complexes of the present invention. A
preferred formulation is PBS containing 0.02% TWEEN-80. These
compositions may be sterilized by conventional, well-known
sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is, or lyophilized,
the lyophilized preparation being combined with a sterile aqueous
solution prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
[0168] The concentration of the complex can vary widely, i.e., from
less than about 0.05%, usually at or at least about 1% to as much
as 10 to 30% by weight and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected. Preferred concentrations for
intravenous administration are about 0.02% to about 0.1% or more in
PBS.
[0169] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient.
[0170] For aerosol administration, the complexes are preferably
supplied in finely divided form along with a surfactant and
propellant. The surfactant must, of course, be nontoxic, and
preferably soluble in the propellant. Representative of such agents
are the esters or partial esters of fatty acids containing from 6
to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,
stearic, linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride such as, for
example, ethylene glycol, glycerol, erythritol, arabitol, mannitol,
sorbitol, the hexitol anhydrides derived from sorbitol, and the
polyoxyethylene and polyoxypropylene derivatives of these esters.
Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. Liquefied propellants are typically gases at
ambient conditions, and are condensed under pressure. Among
suitable liquefied propellants are the lower alkanes containing up
to 5 carbons, such as butane and propane; and preferably
fluorinated or fluorochlorinated alkanes. Mixtures of the above may
also be employed. In producing the aerosol, a container equipped
with a suitable valve is filled with the appropriate propellant,
containing the finely divided compounds and surfactant. The
ingredients are thus maintained at an elevated pressure until
released by action of the valve.
[0171] The compositions containing the complexes can be
administered for therapeutic, prophylactic, or diagnostic
applications. In therapeutic applications, compositions are
administered to a patient already suffering from a disease, as
described above, in an amount sufficient to cure or at least
partially arrest the symptoms of the disease and its complications.
An amount adequate to accomplish this is defined as
"therapeutically effective dose." Amounts effective for this use
will depend on the severity of the disease and the weight and
general state of the patient. As discussed above, this will
typically be between about 0.5 mg/kg and about 25 mg/kg, preferably
about 3 to about 15 mg/kg.
[0172] In prophylactic applications, compositions containing the
complexes of the invention are administered to a patient
susceptible to or otherwise at risk of a particular disease. Such
an amount is defined to be a "prophylactically effective dose." In
this use, the precise amounts again depend on the patient's state
of health and weight. The doses will generally be in the ranges set
forth above.
[0173] In diagnostic applications, compositions containing the
appropriately complexes or a cocktail thereof are administered to a
patient suspected of having an autoimmune disease state to
determine the presence of autoreactive T cells associated with the
disease. Alternatively, the efficacy of a particular treatment can
be monitored. An amount sufficient to accomplish this is defined to
be a "diagnostically effective dose." In this use, the precise
amounts will depend upon the patient's state of health and the
like, but generally range from 0.01 to 1000 mg per dose, especially
about 10 to about 100 mg per patient.
[0174] Kits can also be supplied for therapeutic or diagnostic
uses. Thus, the subject composition of the present invention may be
provided, usually in a lyophilized form in a container. The
complexes, which may be conjugated to a label or toxin, or
unconjugated, are included in the kits with buffers, such as Tris,
phosphate, carbonate, etc., stabilizers, biocides, inert proteins,
e.g., serum albumin, or the like, and a set of instructions for
use. Generally, these materials will be present in less than about
5% wt. based on the amount of complex and usually present in total
amount of at least about 0.001% wt. based again on the protein
concentration. Frequently, it will be desirable to include an inert
extender or excipient to dilute the active ingredients, where the
excipient may be present in from about 1 to 99% wt. of the total
composition. Where an antibody capable of binding to the complex is
employed in an assay, this will usually be present in a separate
vial. The antibody is typically conjugated to a label and
formulated according to techniques well known in the art.
[0175] The following example illustrates, but does not limit, the
invention.
EXAMPLE 1
Down-Regulation of T cells in vitro with a Complex of Mouse
I-A.sup.k and Rat MBP Peptide
[0176] The efficacy of Class II MHC-peptide complexes in induction
of nonresponsiveness or anergy in T cell clones directed against
epitopes of myelin basic protein (MBP), known to induce
experimental allergic encephalomyelitis in mice, a disease model
which mimics human multiple sclerosis, is shown below.
[0177] T cell clones AJ1.2 and 4R3.4 prepared by immunization of
mice against rat MBP peptide (1-11) and characterized for antigen
specificity were obtained from Dr. Pat Jones of Stanford
University.
[0178] The I-A.sup.k complex with rat NSF peptide was formed
utilizing purified mouse I-A.sup.k and synthetic rat MBP peptide
(1-13), the sequence for which is known (Zamvil et al. (1986),
Nature 324:258260), and which is:
[0179] Ac-ASQKRPSQRHGSK
[0180] Mouse I-A.sup.k was purified by a modified method based upon
Turkewitz et al., supra. Basically, a soluble membrane extract of
cells containing I-A.sup.k was prepared using NP-40. I-A.sup.k from
the extract was purified by affinity chromatography, using a column
containing 10-2.16 antibodies, which had been purified by affinity
chromatography on Protein-A, and which were coupled to CNBr
activated Sepharose 4b. The preparation schemes for the NP-40
soluble membrane extract, for the purification of 10-2.16 Mab and
its coupling to CnBr activated Sepharose 4B, and for the
purification of I-A.sup.k, are shown in FIGS. 12, 13a and 14,
respectively. FIG. 13b is a copy of a polyacrylamide gel showing
the Purity of the purified 10-2.16 antibody. The purity of
I-A.sup.k, as monitored by polyacrylamide gel analysis, is shown in
FIG. 15.
[0181] In order to form the complex of I-A.sup.k and rat MBP
peptide, ten ug of affinity-purified I-A.sup.k in PBS containing 30
mM octyl glucoside and 50-fold molar excess of HPLC-purified MBP
peptide were mixed in a total volume of 125 ul. Samples were
incubated at 37.degree. C. for 16 hours with constant shaking and
were either separated from peptide by G-24 Sephadex desalting for
liposome preparation or, for cell studies, were dialyzed against
PBS followed by RPMI media for 36 hours at 4.degree. C.
[0182] The introduction of the I-A.sup.K-MBP peptide complex into
liposomes was as follows. A lipid solution consisting of
cholesterol:dipalmitoylphosphatidyl choline
(DPPC):dipalmitoylphosphatidy- l ethanolamine-fluorescein (DPPEF)
at a molar ration of 25:75:2 was prepared in chloroform containing
30 mM octyl glucoside (OG). Lipid was dried under vacuum and
preformed I-A.sup.k-peptide complex in PBS containing 17 mM OG was
mixed with dried lipid at a ration of 5:1 (w/w). The mixture was
vortexed for 2-3 minutes, cooled to 4.degree. C., and finally
dialyzed against PBS followed by RPMI media for 36 at 4.degree. C.
In experiments using (125-I)-labeled I-A.sup.k, no fluoresceinated
lipid was included in the lipid mixture, and the incorporation of
I-A.sup.k into liposomes was measured by scintigraphy.
[0183] Planar lipid membranes were prepared on sterile 12-mm glass
coverslips using 50-100 ul of liposomes containing
affinity-purified I-A.sup.k alone or purified I-A.sup.k+MBP(1-13)
by the method of Watts et al. (1985), Proc. Natl. Acad. Sci. USA
82:5480-5484, which is incorporated herein by reference. The
presence of I-A.sup.k in planar membranes was confirmed by
fluorescence microscopy after staining with fluorescent
anti-I-A.sup.k anti-body. No fluorescence above background was
noted upon staining with fluorescent anti-I-A.sup.d.
[0184] AJ1.2 and 4R3.4 cells obtained six to eight days after MBP
peptide stimulation were washed twice, and the 4.times.10.sup.5
cells were added to planar membranes. The plates were incubated for
48-72 hours in 5% CO.sub.2 at 37.degree. C. and then examined
visually for formation of colonies.
[0185] The effects of detergent-solubilized Class II molecules were
examined by culturing 1.times.10.sup.5 AJ1.2 or 4R3.4 cells with
50-100 ul of purified I-A.sup.k alone, purified I-A.sup.k plus
MBP(1-13) and medium alone for five hours at 37.degree. C. in 5%
CO.sub.2. Following this incubation the cells were diluted to 900
ul and tested for their ability to respond to antigen-presenting
cells (APC) and antigen [MBP(1-13)] in a proliferation assay.
Uptake of 3-(4,5-dimethyl-thiazol-2- -7')-2,5 diphenyltetrazolium
bromide (MTT) was used as an indication of cell proliferation.
Although DNA synthesis, usually monitored by .sup.3H-thymidine
uptake, and the activity of mitochondria, measured by MTT uptake,
are different cellular functions, it has been demonstrated that
these two activities, monitored three days after initiation of
stimulation of spleen cell cultures, tracked each other very well
(Molecular Device Application Bulletin Number 011-A, Feb. 9,
1988).
[0186] Data are presented as % suppression of proliferation of
cells incubated with Class I+Ag compared to cells cultured with
medium alone and were calculated by using the formula: 1 ( O . D .
) 570 [ T cells a + Spleen cells + MBP ( 1 - 11 ) ] - ( O . D . )
570 [ T cells b + Spleen cells ] ( O . D . ) 570 [ T cells c +
Spleen cells + MBP ( 1 - 13 ) ] - ( O . D . ) 570 [ T cells c +
Spleen cells ]
[0187] wherein
[0188] T cells.sub.a=T cells preincubated with I-A.sup.k-MBP(1-13)
complex
[0189] T cells.sub.b=T cells preincubated with I-A.sup.kMBP(1-13)
alone
[0190] T cells.sub.c=T cells preincubated with medium.
[0191] Since proliferation of cells cultured in the presence of
Class II MHC alone was generally equal to cells cultured with
medium alone in most studies, this latter number was used in
obtaining % suppression. The Standard Deviation of triplicate wells
was <10% in the majority of experiments.
[0192] Initially two qualitative studies were performed to
determine whether pretreatment with I-A.sup.K+MBP(1-13) will alter
the binding of T cell clones to planar membranes prepared from
liposomes containing I-A.sup.k+MBP(1-13). AJ1.2 cells were used for
these studies because they formed characteristic colonies on planar
membranes in the presence of MBP(1-13) alone, i.e., without antigen
presenting cells (APC). Preincubation of AJ1.2 cells with
I-A.sup.k+MBP(1-13) for five hours inhibited the number of colonies
formed on planar membranes compared to cells incubated with
I-A.sup.k or medium alone. In the second experiment, AJ1.2 cells
were incubated with liposomes containing I-A.sup.k+MBP(1-13) or
with I-A.sup.k alone for five hours and then added to planar
membranes prepared as described above. As noted previously with
detergent-solubilized I-A.sup.k MBP(1-13), culturing of cells with
liposome containing I-A.sup.k+MBP(1-13) reduced the number of
colonies in comparison to cells incubated with liposomes containing
I-A.sup.k alone. Although colonies could not be counted accurately,
clear differences in their number were evident.
[0193] Because these studies did not allow quantitation of the
effects of I-A.sup.k+MBP(1-13) on the function of T cell clones, we
examined the effects of preincubation with this complex on the
proliferation of 4R3.4 or AJ1.2 cells in the presence of APCs and
MBP(1-13). Therefore, 4R3.4 or AJ1.2 cells were preincubated with
50-100 ul of I-A.sup.k+MBP(1-13), I-A.sup.k, or medium alone for
five hours at 37.degree. C. The cells were then diluted to an
appropriate concentration and added to APC. Antigen [MBP(1-13)] was
added to a final concentration ranging from 13.3 um to 53.2 um.
[0194] APC used in the study were prepared from spleens of female
A/J mice. Briefly, spleens were removed and single cell suspensions
were prepared by gentle teasing between the frosted ends of sterile
microscope slides. Red cells were lysed by hypotonic shock. The
remaining cells were washed twice with RPMI containing antibiotics
and incubated with 10 micrograms/ml mitomycin-C for 1 hour at
37.degree. C. Following this incubation, spleen cells were washed
five times with RPMI containing antibiotics, counted, and used as
APC's.
[0195] Following a 72-hour incubation period of the cells with APC
and MBP(1-13), the extent of proliferation was quantitated using
MTT uptake. The results of eight such studies are summarized in
FIG. 16. In studies 1, 3, and 4, 4R3.4 cells were incubated as
above. In study 2, 4R3.4 cells were preincubated with liposomes
containing I-A.sup.k+MBP(1-13) or I-A.sup.k alone. T cells were
then separated from unbound liposomes by centrifugation through a
10% Ficoll solution, washed, and used in proliferation assays.
Studies 5 through 8 were carried out with the clone AJ1.2. Cells
incubated with I-A.sup.k alone proliferated to the same extent as
cells cultured in medium. T cell clones preincubated in this manner
did not proliferate in the absence of APC.
[0196] The data presented above demonstrate that the complex of
Class II MHC+MBP(1-13) induces dramatic nonresponsiveness in T cell
clones specific for MBP(1-11). In addition, the data show that this
complex was immunologically reactive with, and hence bound to the
MBP-stimulated T cell clones.
[0197] Preparation and Stability of Human HLA-DR2-MBP Complexes
[0198] The optimum pH for maximum binding of human MBP(83-102)
peptide to HLA-DR2 was determined to be pH 7. Affinity-purified DR2
from homozygous lymphoblastoid cells was incubated with 10 fold
molar excess of radioiodinated MBP(83-102) peptide at 37.degree. C.
for 48 hours at various pH. The unbound peptide was removed by
dialysis and the amount of bound peptide was calculated from the
silica gel TLC assay. Samples were also analyzed on reduced and
non-reduced polyacrylamide gel. Bands were cut out, counted and the
amount of bound peptide was calculated from the specific
activity.
[0199] The stability of human DR2-MBP(83-102) complex at 4.degree.
C. was also investigated. Complexes of DR2 and 125I-MBP(83-102)
were prepared as described above and stored at 4.degree. C. Every
week an aliquot of 1 .mu.l was applied on a silica gel TLC plate in
triplicate. Plates were run, developed and the percent dissociation
was calculated. Over a period of 42 days, there was no significant
dissociation of this complex.
EXAMPLE 2
EBV Transformation of B Cells From an Individual with an Autoimmune
Dysfunction
[0200] Peripheral blood mononuclear cells (PBMNC) from an
individual with an autoimmune dysfunction are isolated by diluting
whole blood or buffy 1:1 with sterile phosphate buffered saline
(PBS), pH 7.2, layering the suspension on Ficoll-Hypaque, and
centrifuging 20 minutes at 1800-2000 RPM in a table top centrifuge.
PBMNC present in a band at the interface of the Ficoll-Hypaque and
PBS-plasma are harvested with a pipette and washed twice with PBS.
Cells are resuspended at 5.times.10.sup.6 cells/ml in RPMI 1640
containing 10% fetal serum (FBS), plated in a polystyrene flask and
incubated for 1 hour at 37.degree. C. to remove monocytes.
Non-adherent cells are collected, pelleted by centrifugation and
resuspended at 10.times.10.sup.6 cells/ml in Ca.sup.++-Mg.sup.++
free Dulbecco's PBS containing 15% FBS. AET-SRBC (2% v/v) is mixed
1:1 with PBMC, the mixture is centrifuged for 20 min. at
100.times.g, and then incubated on ice for 1 hour. The pellet is
gently resuspended, and the suspension centrifuged through
Ficoll-Hypaque as described earlier. The band which contains B
cells and remaining monocytes is harvested.
[0201] Transformation of B cells is with B95-8 cell line (Walls and
Crawford in Lymphocytes: A Practical Approach (G. G. B. Klaus ed.,
IRL Press)). The B95-8 cells are diluted 1:3 in medium, and
cultured for 5 days at 37.degree. C. The supernatant is harvested,
centrifuged at 250.times.g for 15 minutes, and filtered through a
0.45 micron millipore filter. The EBV is then concentrated by
centrifugation at 10,000 rpm for 2 hours at 4.degree. C., and the
pellet containing the virus is suspended in RPMI 1640 containing
10% FBS, at 1% of the original volume.
[0202] In order to transform B cells, the virus stock is diluted
1:9 with culture medium containing 2.times.10.sup.6 cells. After
the virus is absorbed to the cells for 1-2 hours at 4.degree. C.,
the cells are centrifuged at 250.times.g. The resulting cell pellet
is suspended at approximately 0.7.times.10.sup.6 to
7.0.times.10.sup.6 cells/ml in RPMI 1640 containing 10% FBS.
Transformed cells are cloned using standard methods.
[0203] The transformed B cells made by the procedure are suitable
for the isolation of human MHC glycoproteins.
EXAMPLE 3
Induction of EAE in Mice
[0204] Adoptive transfer of T cell clones AJ1.2 and 4R3.4, as well
as immunization of mice with MBP(1-13) causes mice to develop
EAE.
[0205] EAE was induced with the peptide using the method for
induction of EAE in mice with intact MBP. Briefly, MBP(1-13) was
dissolved in PBS and mixed vigorously with complete Freund's
adjuvant so as to form a thick emulsion. Female A/J mice were
injected with 100 micrograms of this mixture at four sites on the
flank. Twenty-four and 72 hours later, 400 ng of pertussis toxin
was injected intravenously. Mice are observed daily by two
individuals for the development of EAE and mortality. The results
in FIG. 17 show the development of EAE in mice resulting from
immunization with MBP(1-13).
[0206] For the adoptive transfer of EAE, T cell clone 4R3.4,
obtained from B10A(4R) strain of mice following immunization with
MBP(1-11) was used. B10.A(4R) mice were given 350 rad of whole body
radiation and then injected with 400 ng pertussis toxin
intravenously. Two to three hours later 10.times.10.sup.6 4R3.4
cells, stimulated with MBP(1-13) three days previously, were
injected intravenously. These animals were observed twice daily for
signs of EAE and mortality. The results of this study are
summarized in FIG. 18.
EXAMPLE 4
Down-Regulation of EAE by I-A.sup.g-MBP(91-103) Complex
[0207] This example demonstrates that in vivo therapy with
complexes of the present invention results in prevention of
passively induced EAE. In addition, the therapy significantly
lowered mortality and morbidity in treated animals.
[0208] In order to demonstrate that treatment with
I-A.sup.g/MBP(91-103) complex will prevent the development of EAE
following T cell activation, SJL mice were injected with
MBP(91-103) reactive T cell blasts in vivo. Briefly, SJL mice 10-12
weeks of age were immunized with 400 .mu.g of MBP(91-103)
(Ac-FFKNIVTPRPPP-amide, >95% purity) in complete Freund's
adjuvant on the dorsum. After 10-12 days, regional draining lymph
node cells were harvested and cultured in 24 well plates (Falcon)
at a concentration 6.times.10.sup.6 cells/well in a 1.5 mls of RPMI
1640 media containing 10% fetal bovine serum, 1%
penicillin/strepmycin and 50 .mu.g/ml of MBP. Following a 4 day in
vitro stimulation, MBP(91-103) reactive T cell blasts were
harvested via ficoll-hypaque gradient (Hypaque 1077, Sigma, Mo.)
and washed twice in PBS according to standard techniques.
Approximately 1.3-1.5.times.10.sup.7 cells were injected into each
mouse.
[0209] Mice that received encephalitogenic MBP(91-103) reactive T
cells then received either 100 .mu.g of soluble
I-A.sup.g/MBP(91-103) complexes in 100 .mu.l PBS, 100 .mu.g of
I-A.sup.g/MBP(1-14) (a peptide that is not encephalitogenic in SJL
mice) complexes in 100 .mu.l PBS or PBS alone on days 0, 3, and 7
(total dose 300 .mu.g). Animals were observed daily and graded for
clinical signs of EAE: grade 1, loss of tail tone; grade 2, hind
leg weakness; grade 3, hind leg paralysis; grade 4, moribund; grade
5, death. In accordance with the regulations of the animals care
committee, mice that could not feed themselves were sacrificed.
[0210] Only one of the seven mice that received
I-A.sup.g/MBP(91-103) complex developed clinical EAE on day 16
(FIG. 19). In contrast, all four animals that received the
I-A.sup.g/MBP1-14, and six of seven animals that received PBS
developed paralysis. In the former group, the mean onset of disease
was on days 9.7 and in the latter group it was 9.0 with mean
severity of 2.3 and 2.5 respectively.
[0211] The inability of other auto-antigens presented by the
I-A.sup.g allele to inhibit disease induction was also
demonstrated. SJL mice were immunized with the peptide 139-151 of
proteolipoprotein (PLP) in complete Freund's adjuvant to induce
EAE. SJL mice were immunized with the peptide dissolved in PBS and
mixed with complete Freund's adjuvant containing 4 mg/ml
Myobacterium tuberculosis H37Ra in a 1:1 ratio. Animals were
injected with 152 .mu.g of peptide, a dose found to induce EAE in
100% of the animals, subcutaneously in both abdominal flanks. On
the same day, and 48 hours later, all animals were given 400 .mu.g
of pertussis toxin intravenously. Mice were treated with PBS, 15
.mu.g of I-A.sup.g alone or 15 .mu.g of I-A.sup.g plus PLP(139-151)
on days 1, 4, and 7 after immunization as described above.
[0212] As shown in Table 1, animals that received the appropriate,
I-A.sup.g/PLP(139-151) peptide complex were protected from the
severe fulminant paralytic disease induced by the immunization with
peptide in adjuvant. The was no mortality in the I-A.sup.g/PLP
peptide treated group. Although all six animals did develop
paralysis, the mean severity of animals that were paralyzed was 2.2
and the mean day of onset was 10.6. In contrast, all six animals
that received I-A.sup.g complex alone, died with a mean day of
onset of 8.2. Five animals died by day 11 and one animal died on
day 21. Animals that received saline or no treatment had a
mortality of 87% and the average day of onset was 9.2 (p<0.0001
I-A.sup.g/PLP(139-151)
1TABLE 1 Treatment No Animals Mean Day of Onset Received Paralyzed
Severity Mortality Paralysis None or 15 of 15 4.7 87% 8 Saline I-A'
complex 6 of 6 5 100% 7 alone I-A' PLP (139- 6 of 6 2.2 0 10
151)
[0213] Our observations indicate that in vivo therapy with
I-A.sup.g/MBP(91-103) complexes (300 .mu.g) results in the
prevention of passively induced EAE. In addition, therapy with 45
.mu.g of I-A.sup.g/PLP(139-151) significantly lowered the mortality
and morbidity in animals that received this therapy.
[0214] The complexes of the invention were also tested for the
ability to prevent relapse of EAE in mice. In these experiments,
SJL mouse received 1.2.times.10.sup.7 p91-103 reactive T cells ip
on day zero. Initial paralytic signs developed between 8-12 days
and all animals recovered by at least 2 clinical grades by day 22.
The mice received 50 .mu.gm of MHC complex (with the cognate
peptide and without), or PBS iv on days 27, 37, and 47. In
experiment 1 mice were observed for 102 days and in Experiment 2,
they were observed for 112 days. The results indicated that the
complexes with the cognate peptide were significantly more
effective than controls in preventing relapses.
EXAMPLE 5
Down-Regulation of RA by MHCII-HSP (180-188) Complexes
[0215] Lewis rats develop a form of arthritis called adjuvant
arthritis in response to subcutaneous injections of Mycrobacterium
tuberculosis emulsified in incomplete Freund's adjuvant. This model
of arthritis fulfills many of the criteria necessary for evaluating
efficacy of drugs being developed for the treatment of rheumatoid
arthritis. The pathology of the tissue and the infiltration of
monocytic and lymphocytic cells indicate a strong T cell mediated
response. The experiments described here use a technique that
anergizes peptide-specific-T cells that recognize and bind the
peptide-MHC Class II complex. The studies involve in vivo treatment
with the soluble MHC Class II-peptide complex soon after the
induction of the disease. The results show significantly less bone
degeneration in the MHC Class II-peptide treated rats compared to
the group that received saline treatment.
[0216] Lewis rat MHC Class II RT1B and RT1D molecules were affinity
purified from NP-40 extract of splenocyte membranes on OX-6 and
OX-17 monoclonal antibodies coupled to sepharose 4B columns. The
relative yield of RT1B to RT1D was 1:2. MHC Class II molecules were
loaded with the peptide by mixing 56 .mu.g of RT1B and 113 .mu.g of
RT1D molecules with 50-fold molar excess of the heat shock protein
(HSP) peptide, p(180-188), at 37.degree. C. for 48h in a total
volume of 1 ml phosphate buffer pH 7.5 containing 1%
octylglucoside. The unbound peptide was removed by extensive
dialysis of the sample against PBS buffer at 4.degree. C. The final
complex concentration was 170 .mu.g/ml and was free of endotoxin as
tested by Limulus amebocyte lysate procedure as described by
Whittaker Bioproduct, Inc.
[0217] Six male Lewis rats (age 77 days) were injected in both hind
foot pads with 1 mg of Mycobacterium tuberculosis in incomplete
Freund's adjuvant to induce arthritis. Three of the rats were
treated with the MHC Class II+ plus HSP180-188 complex
intravenously on days 1, 4 and 7 after the induction of the
disease. The other three rats were given saline as above. Arthritic
index was determined by the gross appearance and by the following
criteria: 0) no change; 1) slight change in the joints of the
digits; 2) slight to moderate edema of the paw or swelling of more
than two digits; 3) swelling of the paw with slight scabbiness,
moderate curing of toes and nails; 4) severe swelling of the paw,
marked scabbiness, and prominent curing of toes and nails.
[0218] The rats treated with MHC Class II-peptide or saline had
swelling of most of their feet by day 20, with an arthritic index
of 4. It is most likely that the differences in the swelling seen
in some of the rats' feet was a reflection of the amount of MT
injected. However, when radiographs of the feet were taken on day
35 after the induction of the disease, there was a significant
difference between the MHC Class II-peptide treated and the saline
treated groups.
[0219] In a second experiment, 18 animals were treated over 5 weeks
during the course of disease progression. As illustrated in Table
2, animals treated with MHC-peptide of the present invention had a
greatly reduced arthritic index and significantly reduced joint
swelling compared with the saline treated group. Animals received
25 .mu.g of MHC-peptide on days 4, 8, and 12.
2TABLE 2 Reduction of inflammation and severity of disease
Thickness Arthritic Treatment # of Rats (mm .+-. SD) Index Saline 4
10.5 .+-. 0.6 4 .+-. 0 MHC alone 4 9.0 .+-. 0.7* 3.25 .+-. 0.5 MHC
+ 5 7.88 .+-. 1.4* 2.6 .+-. 0.55 HSP (180-188) Normal 5 5.55 .+-.
0.36 00 *Statistically significant compared to saline treatment (p
< 0.05 by student's t-test).
[0220] On day 35, tarsal joints of all animals were measured using
vernier calipers. Measurements represent the sum of the thickness
of both hind feet.
EXAMPLE 6
Increasing Serum Half-Life of the Complexes
[0221] This example presents data showing that various
modifications of the complexes lead to increased serum
half-life.
[0222] The protocol for these studies was generally as follows:
Affinity-purified, soluble MHC molecules were labeled with
.sup.125I by the iodobeads method (Pierce Chemical Co., Rockford,
Ill.). Excess .sup.125I was removed by dialysis against PBS
containing 0.1% neutral detergent. The quality of the labeled
protein was assessed by thin layer chromatography, cellulose
acetate electrophoresis, and polyacrylamide gel electrophoresis.
The MHC glycoprotein was administered by tail vein injection to
mice subjected to Lugol's solution in the drinking water at least
one day before injection. Blood samples were obtained at different
time points. The animals were then sacrificed to obtain organs of
interest. Radioactivity in the blood and organ samples was detected
in a gamma well counter according to standard techniques.
[0223] A. Effect of Asialoletuin on Serum Half-Life
[0224] IA.sup.k was labeled and administered to mice as described
above. The mice were divided into three sets: (1) I-A.sup.k (10
.mu.g i.v.); (2) I-A.sup.k (10 .mu.g i.v.) plus asialoletuin (10 mg
i.v.) plus asialoletuin (100 mg i.p.); and (3) I-A.sup.k (10 .mu.g
i.v.) plus asialoletuin (10 mg i.p.). Blood was drawn at different
time points and the percent of injected dose retained in the blood
was calculated.
[0225] The mean serum half-life for the three sets was as
follows:
[0226] Set 1--3 min.
[0227] Set 2--40 min.
[0228] Set 3--35 min.
[0229] B. Effect of Liposomes on Serum Half-Life
[0230] I-A.sup.g was labeled as described above. The labeled
I-A.sup.g molecules were captured inside liposomes by standard
procedures (see, e.g., Remingtion's, supra). Ten mice were injected
with 10 .mu.g I-A.sup.g, as described above, and divided into four
sets: (1) I-A.sup.g alone; (2) liposomal I-A.sup.g; (3) liposomal
I-A.sup.g coinjected with blank liposomes; and (4) blank liposomes
plus, 10 minutes later, liposomal I-A.sup.g coinjected with blank
liposomes. Blood samples were obtained at different time points
after injection, and the percent of injected dose of I-A.sup.g
retained in the blood was calculated.
[0231] The serum mean half-life of the three sets was as
follows:
[0232] Set 1--2 min.
[0233] Set 2--7 min.
[0234] Set 3--10 min.
[0235] Set 4--60 min.
[0236] C. Effect of Periodate/Cyanoborohydride Treatment on Serium
Half-Life
[0237] I-A.sup.k in a phosphate buffer containing 3 mM
taurodeoxycholate at pH 7.5 was labeled as described above. The
labeled molecules were subjected to periodate oxidation and
cyanoborohydride reduction for 5 or 21 hours at 40C, using 20 mM
sodium periodate and 40 mM cyanoborohydride (final concentrations)
in 0.1 M acetate buffer at pH 5.5. The reaction was quenched by
addition of ethylene glycol (final concentration 0.7%). The treated
I-A.sup.k was purified by dialysis and administered (10 .mu.g i.v.)
to mice, as described above. Nine mice were divided into three
sets: (1) I-A.sup.k (untreated); (2) I-A.sup.k (5 hr. treatment);
and (3) I-A.sup.k (21 hr. treatment). Blood samples were obtained
at different time points and the percent of injected dose of
I-A.sup.k retained in the blood was calculated.
[0238] The serum half-life of the three sets was as follows:
[0239] Set 1--4 min.
[0240] Set 2--7 min.
[0241] Set 3--70 min.
EXAMPLE 7
[0242] The results presented below demonstrate that MHC-peptide
complexes of the invention exist primarily as aggregates in the
absence of detergent.
[0243] An IA.sup.k-P complex labeld with .sup.125I was prepared as
described above. The complex (1.5 mg/ml) was dialysed extensively
against phosphate buffered saline (PBS) to remove detergent and
loaded on a 6 ml b.v. Sephadex-G200 column (fractionation size
5000-600,000). The results presented in FIG. 20 show that the
aggregated complexes pass through the column with the void volume
and thus have a molecular weight greater than 600,000.
[0244] The same dialyzed IA.sup.k complex (1.5 mg/ml) was also
centrifuged and the pellet was counted. To do this, 200 .mu.l of
complex (300 .mu.g) was diluted in 5 ml PBS and centrifuged in a
fixed angle rotor at 100,000.times.g for 60 minutes. The results
are given in Table 3, below.
3TABLE 3 DETECTION OF COMPLEX AGGREGATION BY HIGH SPEED SPIN EXP #
Starting cpm cpm in pellet cpm in sup % aggreg. 1 514,576 317,717
205,797 60.68 2 519,340 321,304 209,108 60.57
[0245] The results of the chromatography and centrifugation
experiments both show that MHC-peptide complexes exist largely in
aggregated or micellar form. These results strongly indicate that
the single subunit complexes of the present invention are also
aggregated or in micellar form, in the absence of detergent.
EXAMPLE 8
[0246] This example demonstrates that administration of soluble MHC
class II-AChR .alpha. peptide 100-116 complexes alter the function
of AChR-reactive T cells and thereby modulate the course of EAMG,
an antibody mediated, but T cell dependent autoimmune disease.
[0247] AchR and AchR Peptides. Electroplax tissue from Torpedo
californica (Pacific Biomarine) was homogenized and the membrane
fraction was detergent solubilized (2% Triton X-100, 100 mM NaCl,
10 mM MOPS, 0.1 mM EDTA, 0.02% NaN.sub.3). AChR was isolated from
the solubilized membranes by affinity chromatography with the
anti-AChR monoclonal antibody mAb 35 and dialized against 1.0%
n-octyl .beta.-D-glucopyranoside (OG)/PBS.
[0248] Torpedo AChR.alpha. subunit peptide 100-116
(YAIVHMTKLLLDYPGKI) was synthesized by solid-phase
9-fluorenylmethoxycarbonyl (FMOC) procedures, using standard
procedures. The peptides were purified by reverse-phase HPLC, and
characterized by HPLC and mass spectroscopy.
[0249] Rat MHC II Purification and Peptide Loading. Rat class II
RT1.B and RT1.D molecules were detergent solubilized (0.5% NP-40,
10 mM Tris HCl pH 8.3, 1 mM PMSF, 0.02% NaN.sub.3) from homogenized
spleens and purified by affinity chromatography with monoclonal
antibodies OX 6 and OX 7. The RT1.B/D mixture was incubated at
37.degree. C. for 24 hours with a 50-fold molar excess of peptide
AChR.alpha. 100-116, followed by 24 hours of dialysis at 4.degree.
C. against 0.1% OG/PBS to remove unbound peptide. Analysis by
nitrocellulose filter binding and TLC (B. Nag et al. (1991) J.
Immunol. Meth. 142: 105) revealed 70-80% of the RT1.B and 90-100%
of the RT1.D bound peptide AChR.alpha. 100-116. This complex is
stable at 4.degree. C. for at least 8 weeks.
[0250] Induction of EAMG and Treatment with Soluble MHC
II:AchR.alpha. 100-116. Male Lewis rats (8-12 weeks old) were
injected in both hind footpads with Tc AchR emulsified in complete
Freund's adjuvant, and intraperitoneally with 600 ng of Pertussis
toxin. Sick rats (clinical stage 1-3) were fed moist chow and teeth
were trimmed weekly.
[0251] On days 1, 4, and 7 post AChR immunization, individual rats
were treated i.v. with saline, 25 .mu.g MHC II alone (MHC II:0), or
25 .mu.g of MHC II complexed with the T.c. AchR.alpha. peptide
100-116 (MHC II:AchR.alpha. 100-116). T cells were purified from
popliteal lymph nodes 9 days after AchR immunization and tested for
proliferation to a panel of antigens.
[0252] T Cell Proliferation Assay. T cells from the peripheral
lymph nodes were isolated by nylon wool chromatography.
2.times.10.sup.5 T cells and 3.times.10.sup.5 irradiated syngeneic
splenocytes were incubated in 0.2 ml of culture medium (RPMI 1640,
10% FBS, 10 mM HEPES, 5.times.10.sup.-5 M 2-ME, 100 U/m.
penicillin, 100 U/ml streptomycin) with peptide or whole antigen
for 3 days at 37.degree. C., 5% CO.sub.2, pulsed with 1 .mu.Ci
.sup.3H-thymidine for 18 hours, harvested, and counted.
[0253] Timecourse of EAMG in Lewis Rats. Immunization of Lewis rats
with T. c. AchR induces anti-AchR antibodies, causing weight loss
and progressive muscle weakness with a predictable timecourse. A
drop in weight in the week following immunization coincides with an
"acute phase" due to infiltration of the neuromuscular junction by
mononuclear cells. A second, sustained weight loss begins at day
33-40, a "chronic phase" accompanied by the clinical symptoms
resembling MG in human patients. Weight changes of individual rats
are shown to demonstrate the range of disease onset, progression,
and time of death (indicated by intersection with the
abscissa).
[0254] In untreated rats, progressive EAMG may be classified into
three stages. In stage 1, anti-AChR antibodies cause endocytosis of
the AchR. Subthreshold levels of AChR at the neuromuscular junction
reduce muscle contractions, particularly in the posterior muscles.
Weak back and hind leg muscles cause a characteristic hunched
posture at rest. Stage 2 is characterized by progressive weakness
that causes frequent rest periods, with the head unsupported due to
weakness in the neck muscles. In stage 3, the diaphragm and
intercostal muscles are weakened, causing labored breathing.
Deterioration of the mandible muscles leads to excessive tooth
growth.
[0255] To test the therapeutic effect of soluble MHC I:AChR.alpha.
100-116, rats were treated after clinical symptoms appeared in the
chronic phase.
[0256] Antigen-Specific Unresponsiveness Induced by Soluble MHC
II:AChR.alpha. 100-116. In saline treated rats, the T cell response
to AChR.alpha. peptide 100-116 equalled 30% of the response to AChR
(.alpha..sub.2.beta..gamma..delta.) (FIG. 21), confirming
literature reports that 100-116 is a major epitope in the T cell
response to AChR. T cells from the MHC:0 treated rat respond to
each antigen at levels similar to T cells from the saline treated
rat.
[0257] The T cell response of MHC II:AChR.alpha. 100-116 treated
rats to whole Tc AChR and AChR.alpha. 100-116 were respectively 22%
and 20% of saline treated rat T cell proliferation levels (FIG.
21). Proliferation of T cells from the MHC II:AChR.alpha. 100-116
treated rat to Pertussis toxin was equivalent to T cells from
saline and MHC II:0 treated rats, indicating that the T cell
inactivation was antigen specific (FIG. 21).
[0258] Therapeutic Effect of Soluble MHC II:AChR.alpha. 100-116.
Randomized groups of rats with clinical stage 1 EAMG (approximately
day 42-56 post-inoculation) were injected i.v. at five weekly
intervals with saline, 25 .mu.g MHC II:HSP 180-188 (MHC II bearing
an irrelevant heat shock peptide), 25 .mu.g MHC II alone (MHC
II:0), or 5 .mu.g AChR.alpha. 100-116 alone (O:AChR.alpha.
100-116). The weight and clinical symptoms were monitored.
[0259] Treatment of rats with clinical stage 1 EAMG with MHC
II:AChR.alpha. 100-116 resulted in a survival frequency of 67% at
140 days post-induction. In comparison, the maximum survival rate
in the control groups was 20% (16.7% saline, 0% Tc AChR.alpha.
100-116 alone, 20% MHC II alone, 20% MHC II:HSP 180-188). The time
course of EAMG for representative rats in each treatment group is
presented in Table 4.
[0260] The four surviving MHC II:AChR.alpha. 100-116 treated rats
exhibited relatively severe EAMG (maxima of 2.0, 2.5, 2.5, and
3.0), followed by remission approximately three weeks after the
first therapeutic injection.
4 TABLE 4 DAYS POST EAMG INDUCTION TREATMENT 61 123 224 MHC
II:AChR.alpha. 100-116 2.5 0.0 0.0 MHC II:HSP 180-188 3.0 3.0 Dead
(day 138) MHC II:0 3.0 Dead (day 66) 0:AChR 100-116 2.5 Dead (day
66) Saline 3.0 Dead (day 82)
[0261] Representative rats from each treatment group are shown 61,
123, and 224 days after EAMG induction by AChR immunization. The
clinical stage of each rat is indicated. By day 123, the MHC
II:AChR.alpha. 100-116 treated rat shows improved mobility and
posture, in contrast to the lone surviving rat treated with MHC:HSP
180-188.
[0262] These results show that soluble MHC II:AChR.alpha. 100-116
complex injected at the start of EAMG induction significantly
reduces the T cell response to the AChR.alpha. peptide 100-116 and
to whole AChR. The effect of the soluble MHC II:AChR.alpha. 100-116
complex is antigen-specific, as the complex does not affect the T
cell response to an unrelated antigen, pertussis toxin. Treatment
with the soluble MHC II:AChR.alpha. 100-116 complex during the
chronic phase of EAMG reduces mortality and clinical symptoms of
the disease.
EXAMPLE 9
[0263] This example demonstrates the ability of complexes of the
invention to induce anergy in human T cells associated with
myasthenia gravis (MG).
[0264] The T cells in this experiment were derived form the thymus
of young onset MG patient. They recognize the residues 138-167 AChR
.alpha.subunit and are restricted by DR4Dw4. In the experiments,
2.times.10.sup.4 T cells were preincubated for varying times with
either complex (DR4:p138-167), DR4 alone, or peptide alone. Antigen
presenting cells (APC's) pulsed with different antigens were then
added and proliferation of the T cells was measured using tritiated
thymidine as described above. The stimulating antigens included
p138-167 and a recombinantly produced AChR .alpha.subunit
polypeptide termed r37-181, which was prepared as described in
Beeson et al., EMBO J 9:2101-2106 (1990) and Beeson et al.,
Biochem. Soc. Trans. 17:219-220 (1989), both of which are
incorporated herein by reference.
[0265] Preincubation of the T cells with complex at 10 .mu.gm/ml
overnight lead to nonresponsiveness in the T cells when
subsequently presented with antigen. Incubation with lower
concentrations and for shorter times were not as effective at
inducing anergy.
[0266] Viability of the anergized cells was tested with Trypan
Blue. T-cells were incubated with media, or complex at 5 .mu.g/ml,
and aliquots withdrawn for counting at various times. Cells were
counted as either living or dead based on trypan blue exclusion in
living cells. The results are presented in FIGS. 22A and 22B. In
the media incubated cells, the total number of cells gradually rose
while the number of dead cells remained the same. In the complex
incubated cells, the total number of cells fell--rapidly during the
first 6 hours and more slowly thereafter. The number of dead cells
gradually rose so that after several days incubation, almost 90% of
the cells were dead. These results show that cell death follows the
induction of anergy in T cells.
[0267] To test the specificity of the interaction of the complexes
with the T cells, T-cells (1.times.10.sup.4) were incubated with
various forms of the complex. These were--the relevant complex
DR4:p138-167, DR4 alone, soluble DR4 and "sham" p138-167 in
equimolar (proportionally) amounts but not bound together and
DR4:MBPp1-14 complex. Sham peptide was used as previous experiments
had shown that soluble p138-167 alone had similar effects to the
complex. Sham p138-167 was a sample of p138-167 subjected to the
same preparation as the complex including dialysis, thus
controlling for the effects of any soluble peptide which may be
released from the complex. After overnight preincubation,
irradiated PBL's (2.times.10.sup.5) and stimulating antigens were
added.
[0268] Irrelevant complexes and peptides did not induce anergy.
Incubation overnight with media and subsequent stimulation with
rec.alpha.37-181 resulted in a good proliferative response.
Preincubation with DR4:p13B-167 substantially reduced subsequent
proliferation. However, preincubation with any of the other
substances had no effect on proliferation compared to preincubation
with media.
[0269] In addition to the effects of preincubating with various
complexes, the effect of adding various antibodies to cell surface
markers, or of adding IL2 0.5%, at the time of preincubation with
DR4:p138-167 was studied. Addition of an anti-TCR Ab
(anti-TAC--786) inhibited the background stimulation caused by the
complex, inhibited any antigen-induced response and partially
inhibited the response to stimulation with IL2 after preincubation.
An anti-DR Ab (L234) had no effects on the inhibition of antigen
induced response induced by preincubation with complex. Combination
of anti-TAC and anti-DR reduced background proliferation and
antigen-induced proliferation but had no effect on IL2-induced
proliferation. Preincubation of the T-cells with complex in the
presence of 0.5% IL2 did not diminish the inhibitory effects of the
complex.
[0270] Similarly, experiments designed to identify any non-specific
effects of the complex on non-DR4, non-AChR T cell lines. Cells
(2.times.10.sup.4) from lines raised against KLH or Tetanus Toxoid
were preincubated overnight with the complex. They were then
stimulated in the with irradiated PBL's (2.times.10.sup.5) and
antigen. Preincubation with the complex did not have any effect on
background proliferation response to specific antigen. Thus the
complex does not appear to have any non-specific stimulatory or
inhibitory effects on unrelated cell lines.
[0271] Finally, FIGS. 23A and 23B show that the molar concentration
of complex required to induce anergy is much less than that of
peptide alone. As can be seen form the figures, preincubation of
T-cells with p138-167 at concentrations above 1.7.times.10.sup.-8
resulted in a decrease in the response to recombinant.
Preincubation with lower concentrations caused an increase in
background proliferation compared to preincubation with media (far
left). Not until concentrations of 1.7.times.10.sup.-5 were reached
did the proliferative response fall to background levels.
[0272] Preincubation of T-cells with DR4:p138-167 at concentrations
greater than 1.7.times.10.sup.-11 inhibited the antigen-induced
proliferative response. At concentrations above 1.7.times.10.sup.-8
the antigen-induced proliferation fell to background levels. The
lowest concentration of the complex used, 1.7.times.10.sup.-11,
resulted in an increase in antigen-induced proliferation, an
increase in background proliferation and an increase in the
proliferative response to IL2.
[0273] Thus, both complex and p138-167 alone cause some
stimulation. At higher concentrations, both inhibit
antigen-specific response. However, the amount of p138-167 required
for comparable inhibition (i.e. to levels of background
stimulation) was approximately 1000.times.(molar) that of
DR4:p138-167.
[0274] The results described in the Examples, above, demonstrate
the ability of the complexes of the present invention to treat
autoimmune disease in vivo. These data in combination with the in
vitro data showing induction of anergy establish the effectiveness
of the claimed complexes. Although the invention has been described
in some detail in these examples for purposes of clarity and
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *