Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W O 94/11011214883S PC-r/US93/10534
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TREATMENT OF AUTOIMMUNE DISEASES . --
BY I~DUCING TOLERAN OE TO .
OE LLS, TISS~S AND ORG~NS ::
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. Thi~ invention relates to ~he treatment of autoimmune -.
di~ease~. More particularly, this invention relates to the
treatment of autoimmune diseases by administering genetically
angineered cell8 which induce tolerance to a particular protein, -`
cell, or organ.
The immune system protects a~ individual from invading
organisms by attacking and d~stroying such invading organi~ms,
while at th~ same time recognizing and tolerating the ~:~
individual's own proteins, cell~, and organs.
In order for the T-cell3 of the immune system to mount an
immune response again~t a given foreign antigen, two types o~
interactions between T-c~ and antigen presenting cells are
required. Th~ first interaction, or "first signal, 1l occurs
between the T-cell antigen receptors of T-cells ~nd the antigen
it~elf, which is c~rried upon the major histocompatibility
molecule complexes of antigen presenting cells, whereby the
T-cells recognize foreign a~ti~en~. ~he second interaction, or
"second signal", Qccurs when the B7 molecule (which is a ~:
co-stimulatory antigen) of antigen presenting cells is recognized
by its cognate receptor, CD28, on the membrane of T-cells. This .
"second si~nal" is the one which ~icnals the T-cells to '~
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` proliferate, and to attack and destroy the cells which express
the foreign antigen.
It has been postulated that autoimmune diseases, such as
Type I diabetes, Gra~es or Hashimotols disease, rh~umatoid
arthritis, autoimmune male and fema~e infertility, myasthenia ~ -
gravis, and multiple sclerosis, are due to the failure cf -~
specific proteins in an individual (sometimes referred to as
"self" proteins) to be recognized as such by the immune system.
Instead, such proteins and the cells whïch express such proteins ~
are recognized as foreign, which results in the activation of -;
autoimmune pathogenic lymphocytes. Once such lymphocytes are ~-
activated, they are continuou~ly pro~uced.
In an autoimmune di6ease, an antigen presenting cell induces ~;
a T cell response against "self" proteins or "self" cells by
providing two signals. The '1first signal" enables the T~cells to
recognize the "self" protein or "self" cell which expresses the
protein as foreign. ThiB signal stimulates T-cells known as CD4
cells. The "~econd signal" is one which signals the T-cells to
proliferate~ and to attack and destroy the cells which express ;~
the "self" protein. The second ~ignal, such as B7, is recognized
by the T-cell6. Upon recognition by the T-cells of B7, the CD4+
cells are induced to produce I~-2, and expand clonally, followed
by attack and destruction of the target cells.
In the absence of the ~econd ~ignal (eg., B7), the T-cells
recognize the antigen di~played upon the antigen presenting cell; ;-~
however, interactions of this T-cell with this antigen does not -
stimulate clonal expanfiion, but in~tead stimulates a long-lasting
tolerance to the antigen, known ~8 anergy.
It is therefore an object of the present invention to treat
or prevent autoimmun~ diseaseR by inducing tolerance to a "self"
protein or "self" cells.
In accordance with an aspe~t of the present invention, there
are provided cells which are genetically engineered with DNA ,
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encoding a plurality of antigens of a cell, tissue, or organ to
which tolerance is to be induced. The genetically engineered
cells are free of co-stimulatory anti~ens, such as B7 or heat
stable antiyen, also known as HSA.
The term "co-stimulatory antigen~' as used herein, means that
upon recognition of such antigen by a cognate receptor, T-c~
are signaled to proliferate, and to attack and destroy cells
which express a foreign antigen.
Applicant has found that when such cells, which include DNA
encoding a plurality o~ antigens of a particular cell, tissue, or
organ, yet are free of co~stimulatory antiyens, such as B7 or
HSA, are administered to an individual, there is induced a T-cell
anergy whereby the target cells, tissues or organs which normally
express such anti~ens are not attacked by the T-cells.
Such a treatment to induce T-cell anergy leads to a ;~
"re-educationr' of the immune system, resulting in the induction
of tolerance to the target cell antigens, whereby the T-cells ~
will not respond when they come in contact with proteins, cells, ~ -
or organs that contain these antigens. `~
In one embodiment the cell6 from which the antigens may be
derived may be any type of cells which may be attacked or
destroyed by T-cells during the course of events in an autoimmune
disease. Such cells include, but are not limited to, pancreatic
beta cells, thyroid follicular cells, cells of synovial joints, -
o~arian cells, testicular cells, muscle cells, and nerve cells.
The antigens may be obtaine~ by obtaining a purified population
of the desired cells from a patient, and then collecting mRNA
from the purified cell~. The m~NA may then be converted to cDNA.
The cDNA may then be clon~d into an appropriate expression
vector(s) which is then transfected into cells obtained from the ~ ~
patient. Appropriate expression vectors are those which may be ~ ;
employed for transfecting DNA into eukaryotic cells. Such ~ ,
vectors iAclude, but are not limited to, eukaryotic vectors, such
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as, for example, yeast vectors and fungal vectors, and viral
vectors, such as, but not limited to, retroviral vectors.
Examples of retroviral vectors which may be employed include, but
are not limited to, those derived from Moloney Murine Leukemia
Virus, Moloney Murine Sarcoma Virus, Rous Sarcoma Virus and '-
Spleen Necrosis Virus.
In one alternative, a single cDNA iibrary may be obtained
for a particular cell, tissue, or organ. This cDNA library may
then be used to engineer cells which may be used for the
treatment of any and all patients.
In one embodiment, the genetically engineered cells which
are free of co-stimulatory antigens such as B7 antigen or HSA are
parenchymal cells; i.e., cells which are contained within an
organ or a gland.
Parenchymal cells which may be genetically engineered
include, but are not limited to, keratinocytes, hepatocytes,
endothelial cells, muscle ceIls, or fibroblasts. In one
embodiment, the parench~mal cells are keratinocytes. The
g~netically engineered cell~ may be obtained from the patient.
The genetically engineered cells may then be administered to
the patient in an amount effective to prevent the onset o~ or to
treat an autoimmune disease in a patient. Depending upon the
severity of the di~ease, the dosage regimen will vary. In one
embodiment, once a patient is diagnosed with the particular
disease, lO8 cells are administered subcutaneously daily for
a~out 2 weeks. Level~ of circulating antibodies directed against
the protein, cell, or orqan are monitored routinely thereafter.
The cells may be a~mini~tered intravenously or injected into the
thymus. i ~
Autoimmune di~ease~ which may be prevented or treated with ` ~:
the genetically engineered cell~ of the present invention
include, but are not limited to, Type I diabetes (wherein the
parenchymal cells are engineered with DNA encoding pancreatic , lr
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~beta cell antigens), Graves or Hashimoto's disease (wherein the
parenchymal cell~ are engineered with DNA encoding thyroid
follicular cell antigens), rheumatoid arthritis ~wherein th~e
parenchymal cells are engineered with DNA encoding antigens from
cells of synovial joints), myasthenia gravis (wherein parenchymal
cells are engineered with DNA encoding antig~ns from muscle
cells), multiple sclerosis (wherein the parenchymal cells are
engineered with DNA encoding antigens from nerve cells), or
autoimmune infertility ~wherein the parenchymal cells are
engineered with DNA encoding antigens from ovarian cells or
testicular cells).
In one illustrative embodiment, keratinocytes are obtained
from a patient with an autoimmune disease, or a patient who may
be susceptible to an autoimmune disease. The genes encoding the ~-
antigens of a target cell (such as, for example, a pancreatic
beta cell) are obtained by collecting mRNA from a purified
preparation of target cells. The mRNA is converted to cDNA,
which is cloned into an appropriate expression vector, such as,
but not limited to, a simian virus 40 vector or a bovine
papilloma ~irus vector. The expression vector may contain a
~electable marker such as, for example, a G418 resistance marker.
The expression vector is then transfected into the
keratinocytes. Transfected keratinocytes which express the !'
target cell protein~ are then selected with an appropriate
selection agent, such as G418. Expression of autoreactive
epitopes may be confirmed with FACS analysis. ~
The selected cells are then cultured in vitro and treated ,~-
with Vitamin D, TNF-~, or gamma-interferon to increase expression ~:
of the MHC antigens of the target cells that display the
processed target cell proteins. Expression of such antigens may -
be confirmed with FACS analysis. The keratinocytes may then be .
a~ministered to a patient in an amount effective for preventing ~1.
the on~et of or for treating an autoimmune disease. Such
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administration may be by intravenous administration or
alternatively, the keratinocytes may be administered directly f
into the thymus. After administration, peripheral blood samples T
may be obtained and guantitated for changes in antibodies
directed against "sel" epitopes. Also, spleen and lymph node -
T-cells may be tested in vitro for auto-reac$ivity against target
cells.
The genetically engineered cells may.also be useful in the
prevention of transplant rejection diseàse. Thus, in one
embodiment, the cells, ~uch as keratinocytes, may be engineered
with DNA encoding a plurality of antigens from cells or an organ
whi.ch is to be transplanted into a patient. The cells or organ
may be derived from the same species as the host, or may be
derived from a different species. For example, if one desired to
transplant porcine pancreatic beta cells into a human patient in
order to treat Type I diabetes, one may engineer parenchymal
cells wlth DNA encoding a plurality of porcine beta cell
antigens, and administer the engineered parenchymal cells to the
patient prior to the transplantation of the of such cells into a
human patient, and such porcine pancreatic beta cells may be~-;
transplanted into the human patient with a decreased risk of
rejection. It is to be understood, however, that this embodiment -
of the present invention is not to be limited to the prevention
of the rejection of transplanted porcine beta cells into a human
patient.
The invention will now be described with respect to the
following example; however, the scope of the present invention is
not intended to be limited thereby.
Example
A. mRNA extraction from cells to be used in the Production of a
cDNA library
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Total RNA is isolated from pancreatic Beta cells by the
method described in Maniatis, et al., Molecular Cloninq - A
Laboratory ~andbook (1982).
The cells are washed with l0 volumes o~ ice cold phosphate
buffered ~aline without magnesium and calcium ions. The cells
are then pelleted by centrifugation. The cells are then ~
resuspended in l0-20 volumes of RNA extraction buffer (NaCl, ~;
MgC12, Tris, Nonidet, dithiothreitol, and placental RNase ~;
inhibitor). An equal volume of proteinase digestion buffer is
added (Tris, EDTA, NaCl, SDS). The solution is vortexed, and the ~
ly~ate is drawn into a 21 gauge needle and rapidly expelled into ~:
a polypropylene tube. Proteinase K is added, and the solution is
incubated for 30 minutes at 37C. Proteins are extracted with an
equal volume of phenol:chloroform. The a~ueous phase is ;~
collected and 2.5 volumes of ethanol are added. RNA is recovered
by centrifugation. The pellet is then washed with 70% ethanol,
and then dried at room temperature. The pellet is then
redi6solved in Tris buffer with EDTA. MgCl2, dithiothreitol, and
RNase inhibitor are then added, ~olLowed by addition of RNase
free pancreatic ~Na~e I, EDTA, and SDS. The solution is then
extracted with phenol:chloroform. Ice cold ethanol is then added
to the aqueous pha~e, and the RNA is collected by centrifugation.
The pellet is then stored at -70C. -
B. Selection of mRNA
A column of oligo(dT)-cellulose i~ poured and washed with
st~rile water. The column i8 then washed with Tris, NaCl, EDTA, ; ~-
and sodium lauryl ~arcosinate. The RNA is then dissolved in
sterile water, heated, and 2X buffer is added. The solution is
applied to the column. The eluate is collected, heated, and
re-applied to the colu~n. lml fractions are then collected, and
absorbance of each fr~ction i8 read at OD260. PolyA and RNA are
eluted with Tris, EDTA and SDS. 3M ~odium acetate is then add~d 5 ,~
to the eluted RNA, and RNA is recovered by centrifugation. mRNA P`
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is then fractionated by gel electrophoresis to insure that the
majority of the mRNA appears between 500 bases and 8 kb. ¦~
C. Synthesi~ of a DNA and enqineerinq expression vector
Synthesis of first and second strands of cDNA are performed
according to classical methods described in Maniatis, et al.,
Molecular Cloninq - A LaboratorY Handbook (1982). Double
stranded cDNA is blunt-ended and synthetic linkers are added for
insertion into a eukaryotic expression vector known as pSVT7
(Maniatis, 1982). This vector includes an SV40 origin of
replication, a bacteriophage T7 promoter, and a multiple cloning
site. The vector al~o includes a neomycin resistance marker. ~;;
replication, a bacteriophaye T7 promoter, and a multiple cloning
site. The vector includes a neomycin resistance marker.
D Insertion of expre~sion vector into tolerance-inducinq cell
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Keratinocytes are obtained from the patient to be treated.
The ceLls then are propagated in vitro with standard tissue
culture methods. Expression vectors containing the double
stranded cDNA are transfected into the keratinocytes by the
calcium phosphate precipitated DNA method. Cells that have taken
up the expression vector are selected by culture in the presence
of G418.
Æ. Administration of enqineered cells
The engineered cells are expanded such that lO8 cells can be
injected into the patient daily for 2 weeks. Circulating
antibody levels are routinely quantitated in vitro by FITC
analysis or activity directed against the cells from which the
mRNA was obtained. During thi 8 time, peripheral blood leukocytes
are obtained from the patient and tested by a mixed leukocyte
reaction for their ability to respond to the cell from which the
mRNA was obtained.
Within two weeks, the response to the target cells will be
reduced due to the inductlon of tolerance afforded by the
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presentation df the target antigens by the host's antigens
without the co-stimulaltory angtigen. : .
It is to be understood, however, that the scope of the -~
present invention is not to be limited to the specific examples
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanyiny claim~
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