Note: Descriptions are shown in the official language in which they were submitted.
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TOLERANCE TO NATURAL ANTIBODY ANTIGENS
The invention relates to the induction of tolerance in graft recipients,
particularly
S xenograft recipients.
Background of the Invention
Increasing success in organ transplantation has been achieved during the last
decade. One consequence of this success is a severe shortage of organ donors;
while the
number of donors has remained relatively unchanged, the need for organs has
continued
to rise. Currently, there are more than 33,000 Americans waiting for organ
transplants,
but only about 4,800 organs donated each year. Because of this growing gap,
xenogeneic organ transplantation is an increasingly important area of
interest.
Size, availability, and ease of genetic manipulation. have made the pig one of
the beat studied organ donor species for xenotransplantation. (Sachs. D.FI. (
1992 )
"MHC-Homozygous Miniature Swine" in Svs~ine u.s Models in Biomedical Research,
Swindle. M.M. et al. (eds.) (Iowa State University Press, Ames. Iowa. 1992)
p.3;
Cooper, D.K.C. et al. "The Pig as Potential Organ Donor for Man" in
~'enotransplantation, Cooper, D.K.C. et al. (cds.) (Springer-Verlag,
Heidelberg,
Germany, 1991 ) p. 481 ).
Xenogeneic natural antibody-mediated hyperacute rejection is a very
significant
barrier to xenotransplantation (Platt, J.L and Bach, F.H. ( 1991 )
Transplantation 52:937).
Overcoming this barrier is important to the long-term success of pig-to-
primate
xenotransplantation. Recent studies have demonstrated that a predominant
epitope on
porcine cells recognized by human natural antibodies is a carbohydrate that
includes a
tewninal galactose residue in the conformation of the galactosyl a( 1, 3)
galactose
disaccharide structure (Neethling, F.A. et al. ( 1994) Transplantation 57:959;
Ye, Y. et
al. (1994) Transplantation 58:330; Sandrin, M.S. et al. (1993) Proc. Natl.
Acad. Sci.
USA 90:11391; Good, A.H. et al. (1992) Transplant. Proc. 24:559).
Immunopathologic
analysis of tissue samples from organs undergoing hyperacute rejection reveals
the
presence of recipient natural antibodies and complement components along the
endothelial surfaces of blood vessels (Leventhal, J.R. et al. (1993)
Transplantation
55:857; Leventhal, J.R. et al. (1993) Transplantation 56:1; Platt, J.L. et al.
(1991)
Transplantation 52:214; Platt, J.L. et al. (1991) Transplantation 52:1037).
When
recipient natural antibodies are depleted by organ perfusion, hyperacute
rejection is
delayed or does not occur.
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Summary of the Invention
The inventors have discovered that an antigen, e.g., a carbohydrate, which
reacts
with natural antibodies can be used to induce tolerance, in a recipient, to
the antigen,
thereby inhibiting hyperacute rejection of a graft which includes the antigen.
The
inhibition of or reduction in natural antibodies which are reactive with the
antigen can
prolong acceptance, by the recipient, of a graft which includes the antigen,
e.g., a
carbohydrate antigen.
Accordingly, the invention features, a method of promoting, in a recipient
mammal of a first species, tolerance to an antigen, e.g., a carbohydrate
moiety, or to a
graft which produces or displays the antigen. Preferably, the first species is
one which
does not produce or display the antigen, e.g., a carbohydrate moiety, on or in
its cells,
tissues, or organs. By way of example. the recipient mammal can be a human or
an Old
World primate, e.g., a baboon. (e.g.. Papio anubis) or cynomolgus money
(Macaca
fascicularis).
The method includes:
providing to the recipient mammal a tolerance-inducing antigen, e.g., a
carbohydrate moiety, thereby inducing tolerance to the antigen or to a graft
which
produces or displays the antigen. Although not wishing to be bound by theory,
the
inventors believe the antigen, e.g., a carbohydrate moiety, mediates the
deletion of
immune cells which would give rise to antigen-. e.g., carbohydrate moiety-
reactive
antibodies.
In preferred embodiments the subject is a human and the antigen is one which
is
not produced or displayed by humans, e.g., a swine antigen.
In preferred embodiments, the antigen, e.g., a carbohydrate moiety, is
produced
by or displayed on a modified cell of the recipient, wherein the cell has been
modif ed to
produce or display the antigen. The cell can be modified in vivo (in the
recipient's
body), e.g., by in vivo gene therapy or by in vivo treatment with an agent
which
modifies the cell, or ex vivo (removed from the recipient's body). The cell
can be
modified by inserting into the cell a nucleic acid which encodes the antigen,
(or
otherwise promotes the production or display of the antigen) such that the
cell produces
or displays the antigen. The cell can be modified to produce or display a
carbohydrate
moiety by inserting into the cell a nucleic acid encoding a protein which
promotes, e.g.,
catalyzes, the formation of the carbohydrate moiety. The encoded protein can
be an
enzyme which results in the formation of a carbohydrate moiety on the surface
of the
cell. In particularly preferred embodiments the encoded protein forms the
moiety by the
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addition of a terminal sugar residue to a pre-existing sugar residue on a cell
surface
molecule.
The cell can be modified to produce or display the antigen, e.g., a
carbohydrate
moiety, by forming the antigen, e.g., a carbohydrate moiety, on the surface of
a cell of
S the recipient mammal, e.g., by contacting the cell with a protein, e.g., an
enzyme, which
results in the formation the antigen, e.g., a carbohydrate moiety, on the
surface of the
cell or by adhering or attaching the antigen to the cell. In particularly
preferred
embodiments the protein forms the moiety by the addition of a terminal sugar
residue to
a pre-existing sugar residue on a cell surface molecule.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal. The donor can be, for example, a
species
which normally produces or displays the antigen, e.g., a carbohydrate moiety,
on its
cells, tissues, or organs. By way of example the donor can be a swine, e.g., a
miniature
swine, or a New World primate, e.g., a squirrel monkey (Suirr~iri Sciureus).
Preferably,
the graft expresses a major histocompatibility complex (MHC) antigen. The
graft can be
an organ. e.g., a heart, liver, or kidney, or skin, or a preparation of
hematopoietic stem
cells, e.g., a bone marrow preparation. In particularly preferred embodiments
the
recipient is a human and the graft is from a swine, e.g.. a miniature swine.
In preferred embodiments the cell is removed from the recipient, modified so
as
to allow it to produce or display the antigen, e.g., a carbohydrate, and
implanted in the
recipient.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing antigen. e.g., a carbohydrate, inactivating immune system
cells, e.g.,
xenoreactive immune cells, e.g, carbohydrate moiety-reactive immune cells, of
the
recipient.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing antigen, e.g., a carbohydrate, inactivating antibodies,
e.g.,
xenoreactive antibodies, e.g, carbohydrate moiety-reactive antibodies, of the
recipient.
In preferred embodiments the method inhibits hyperacute rejection
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen, e.g., a carbohydrate moiety. The
second
antigen can be produced by or displayed on a modified cell of the recipient.
The
modified cell can be the same cell which produces or displays the first
antigen or it can
be a different cell. Generally, methods described herein for providing antigen
to the
recipient can be used to provide the second antigen to the recipient.
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In another aspect, the invention features, a method of promoting, in a
recipient
mammal of a first species, tolerance to the galactosyl a(1, 3) galactose
moiety, or to a
graft which produces or displays the galactosyl a(1, 3) galactose moiety.
Preferably,
the first species is one which does not possess UDP galactose:~i-D-galactosyl-
1,4-N-
S acetyl-D-glucosaminide a(1,3)galactosyltransferase (al,3GT) activity or
which does not
produce or display galactosyl a(I, 3) galactose moieties on its cells,
tissues, or organs.
By way of example, the recipient mammal can be a human or an Old World
primate,
e.g., a baboon, (e.g., Papio anubis) or cynomolgus money
(Macaca_rascicularis).
The method includes:
providing to the recipient mammal a tolerance-inducing galactosyl a( 1, 3)
galactose moiety thereby inducing tolerance to the galactosyl a( 1, 3 )
galactose moiety or
to a graft which produces or displays the galactosyl a(1, 3) galactose moiety.
Although
not wishing to be bound by theory, the inventors believe the galactosyl a( 1.
3) galactose
moiety mediates the deletion of immune cells which would give rise to
galactosvl a( 1,
3) galactose moiety-reactive antibodies,
In preferred embodiments the galactosyl a( 1. 3) galactose moiety is produced
or
displayed on a modified cell of the recipient, wherein modified means the cell
has been
modified to produce or display the galactosyl a( I , 3) galactose moiety. The
modification can be performed in vivo but is preferably performed ex vivo.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal. The donor can be, for example. a
species
which normally produces or displays the galactosyl a(1, 3) galactose moiety,
on its
cells. tissues, or organs. By way of example the donor can be a swine, e.g., a
miniature
swine, or a New World primate, e.g., a squirrel monkey (Saimiri sciureus).
Preferably,
the graft expresses a major histocompatibility complex (MHC) antigen. The
graft can be
an organ, e.g., a heart, liver, or kidney, or skin, or a preparation of
hematopoietic stem
cells, e.g., a bone marrow preparation. In particularly preferred embodiments
the
recipient is a human and the graft is from a swine, e.g., a miniature swine.
In preferred embodiments, the method includes: inactivating immune system
cells, e.g., xenoreactive immune cells, e.g, galactosyl a(1, 3) galactose
moiety-reactive
immune cells, of the recipient, preferably prior to providing the tolerance-
inducing
galactosyl a(l, 3) galactose moiety.
In preferred embodiments, the method includes: inactivating antibodies, e.g.,
xenoreactive antibodies, e.g, carbohydrate moiety-reactive antibodies, of the
recipient,
preferably prior to providing the tolerance-inducing antigen, e.g., a
carbohydrate.
In preferred embodiments the method inhibits hyperacute rejection.
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In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen, e.g., a carbohydrate moiety. The
second
antigen can be produced by or displayed on a modified cell of the recipient.
The
modified cell can be the same cell which produces or displays the galactosyl
a(1, 3)
galactose moiety or it can be a different cell. Generally, methods described
herein for
providing antigen to the recipient can be used to provide the second antigen
to the
recipient.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal of a first species, tolerance to the galactosyl a( 1, 3) galactose
moiety or to a
graft which produces or displays the galactosyl a( 1, 3) galactose moiety by
providing a
cell from the recipient mammal which produces or displays the galactosyl a( I
, 3)
galactose moiety. Preferably, the first species is one which does not possess
UDP
galactose:ø-D-galactosyl-1,4-N-acetyl-D-glucosaminide
a(1,3)galactosyltransferase (cx
1.3GT) activity or which does not produce or display galactosv) a( 1, 3)
galactose
moieties on its cells, tissues, or organs, and can be, by wav of example. a
human or an
Old World primate. e.g., a baboon, (e.g.. Papio anubis) or cynomolgus money
(Afacaca
rascicularis).
The method includes:
providing a cell from the recipient mammal which produces or displays the
galactosyl a(I, 3) galactose moiety (wherein the cell has been modified to
produce or
display a a( 1, 3 ) galactose moiety); and
preferably, allowing the recipient mammalian cell to produce or display the
galactosyl a(I, 3) galactose moiety in the recipient mammal,
thereby inducing tolerance to the galactosyl a( 1, 3 ) galactose moiety or to
a graft which
includes the galactosyl a(1, 3) galactose moiety.
The modification can be performed in vivo but is preferably performed ex vivo.
In preferred embodiments: the cell is modified to produce or display the
galactosyl a(l, 3) galactose moiety by inserting into the cell a nucleic acid
encoding a
protein which promotes, e.g., catalyzes, the formation of the galactosyl a(1,
3) galactose
moiety.
In preferred embodiments: the cell is modified to produce or display the
galactosyl a(1, 3) galactose moiety by forming the galactosyl a(1, 3)
galactose moiety
on the surface of a cell of the recipient mammal, e.g., by contacting the cell
with a
protein, e.g., an enzyme which results in the formation an galactosyl a(1,3)
galactose
moiety on the surface of the cell. In particularly preferred embodiments the
moiety is
formed by the addition of a terminal galactosyl residue to a galactosyl
residue, e.g., to a
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galactosyl residue linked to N-acetylglucosaminyl residue, on the surface of
the recipient
cell, by contacting the cell with an a(1,3)galactosyltransferase, e.g., (3-D-
galactosyl-1,4-
N-acetyl-D-glucosaminide a{1,3)galactosyltransferase.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal. The donor can be, for example, a
species
which normally produces or displays the galactosyl a(1, 3) galactose moiety,
on its
cells, tissues, or organs. By way of example the donor can be a swine, e.g., a
miniature
swine, or a New World primate, e.g., a squirrel monkey (Saimiri sciureus).
Preferably,
the graft expresses a major histocompatibility complex (MHC) antigen. The
graft can be
an organ, e.g., a heart, liver, or kidney, or skin, or a preparation of
hematopoietic stem
cells. e.g., a bone marrow preparation. In particularly preferred embodiments
the
recipient is a human and the graft is from a swine. e.g.. a miniature swine.
In preferred embodiments, the method includes: inactivating immune system
cells. e.g.. xenoreactive immune cells, e.g, galactosyl a(1, 3) galactose
moiety-reactive
immune cells. of the recipient. preferably prior to providing the recipient
cell which
produce or displays the galactosyl a(1, 3) galactose moiety.
In preferred embodiments, the method includes: inactivating antibodies. e.g.,
xenoreactive antibodies, e.g, galactosyl a(l, 3) galactose-reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell which produce or
displays the
galactosyl a(I, 3) galactose moiety.
In preferred embodiments the method inhibits hyperacute rejection.
In preferred emodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen, e.g., a carbohydrate moiety. The
antigen
can be produced by or displayed on a modified cell of the recipient. The
modified cell
can be the same cell which produces or displays the galactosyl a(1,3)
galactose moiety
or it can be a different cell. Generally, methods described herein for
providing antigen
to the recipient can be used to provide the second antigen to the recipient.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal of a first species, tolerance to the galactosyl a(I, 3) galactose
moiety or to a
graft which produces or displays the galactosyl a(l, 3) galactose moiety by
providing a
cell from the recipient mammal, into which cell has been inserted a nucleic
acid
encoding a protein which promotes, e.g., catalyzes, the formation of the
galactosyl a(I,
3) galactose moiety. Preferably, the first species is one which does not
possess UDP
galactose:(3-D-galactosyl-1,4-N-acetyl-D-glucosaminide
a(1,3)galactosyltransferase (a
1,3GT) activity or which does not produce or display galactosyl a(1, 3)
galactose
moieties on its cells, tissues, or organs, and can be, by way of example, a
human or an
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Old World primate, e.g., a baboon, (e.g., Papio anubis) or cynomolgus money
(Macaca
fascicularis).
The method includes:
providing a cell from the recipient mammal, into which cell has been inserted
a
nucleic acid encoding a protein which promotes, e.g., catalyzes, the formation
of the
galactosyl a(I, 3) galactose moiety; and
preferably, allowing the recipient mammalian cell to produce or display the
galactosyl a(1, 3) galactose moiety in the recipient mammal,
thereby inducing tolerance to the galactosyl a( l, 3) galactose moiety or to a
graft which
produce or displays the galactosyl a(1, 3) galactose moiety.
Insertion of the nucleic acid can be done in vivo but is preferably done ex
vivo.
In preferred embodiments: the nucleic acid encodes a protein which promotes
the addition of a terminal galactosyl residue to a galactosyl residue, e.g.,
to a galactosyl
residue linked to N-acetylglucosaminyl residue: the nucleic acid encodes a
mammalian,
e.g., a vertebrate, e.g., porcine or murine a( 1,3 )galactosyltransferase; the
nucleic acid
encodes a New World primate, e.g., a squirrel monkey, a( 1,3
)galactosyltransferase: the
nucleic acid encodes an a( 1,3)galactosyltransferase, e.g., UDP galactose: (3-
D-
galactosyl-1,4-N-acetyl-D-glucosaminide a( 1,3)galactosyltransferase.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal. The donor can be, for example, a
species
which normally produces or displays the galactosyl a(l, 3) galactose moiety on
its
cells, tissues, or organs, or a species which possesses
a(1,3)galactosyltransferase
activity. By way of example the donor can be a swine, e.g., a miniature swine,
or a
New World primate, e.g., a squirrel monkey (Saimiri sciureus). Preferably, the
graft
expresses a major histocompatibility complex (MHC) antigen. The graft can be
an
organ, e.g., a heart, liver, or kidney, or skin, or a preparation of
hematopoietic stem cells,
e.g., a bone marrow preparation. In particularly preferred embodiments the
recipient is a
human and the graft is from a swine, e.g., a miniature swine.
The recipient cell can be any cell suitable for production or display of the
galactosyl a(1, 3) galactose moiety, e.g., a hematopoietic cell. Hematopoietic
stem
cells, e.g., bone marrow cells, which are capable of developing into mature
myeloid
and/or lymphoid cells, are particularly preferred. It is possible that later
stage cells can
be used, since the transgene(a(1,3)galactosyltransferase) should modify
endogenous
proteins causing them to be recognized as self. Stem cells derived from the
cord blood
of the recipient can be used in methods of the invention. Other cells suitable
for use in
the invention include peripheral blood cells. Suitable cells are those which
can produce
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_g_
or display the galactosyl a(l, 3) galactose moiety and tolerize the animal.
Although not
wishing to be bound by theory, the inventors believe that suitable recipient
cells are cells
which produce or display the galactosyl a( I , 3 ) galactose moiety such that
the moiety
can interact with immune cells at an early stage in their development.
Although not
S wishing to be bound by theory, this is believed to allow deletion of cells
which would
give rise to galactosyl a(1, 3) galactose moiety reactive antibodies. Suitable
cells are
those which result in tolerace as opposed to an immune response.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
the nucleic acid into the recipient mammal cell and administering the
recipient
mammalian cell to the recipient mammal.
In preferred embodiments, the method includes: inactivating immune system
cells, e.g., xenoreactive immune cells, e.g, galactosyl a(1, 3) galactose
moiety-reactive
immune cells, of the recipient, preferably prior to providing the recipient
cell
I S In preferred embodiments, the method includes: inactivating antibodies,
e.g.,
xenoreactive antibodies, c.g, galactosyl a(1, 3) galactose-reactive
antibodies, ofthe
recipient, preferably prior to providing the recipient cell.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-galactosyl a{I, 3) galactose antibody. For
example, anti-
galactosyl (a 1, 3 ) galactose epitope antibody activity can be inactivated
prior to the
introduction or formation in the recipient of a recipient cell which produce
or displays
galactosyl a(I, 3) galactose moieties. Thus, in preferred embodiments, the
method
includes one or more of: administering anti-idiotypic antibodies (e.g.,
recombinant.
monoclonal, polyclonal, chimeric, single chain, or humanized antibodies), or
fragments
thereof, specific for an anti-galactosyl a(1, 3) galactose antibody; depleting
natural
antibodies from the blood of the recipient, e.g., by hemoperfusing an organ,
e.g., a liver
or kidney, obtained from a mammal of the donor species or by contacting the
blood of
the recipient with galactosyl a(I, 3) galactose moieties coupled to an
insoluble substrate;
administering to the recipient drugs which inactivate natural antibodies,
e.g.,
deoxyspergualin (DSG) (Bristol); or administering to the recipient anti-IgM
antibodies.
In preferred embodiments the method inhibits hyperacute rejection.
In preferred emodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen, e.g., a carbohydrate moiety. The
second
antigen can be produced by or displayed on a modified cell of the recipient.
The
modified cell can be the same cell which produces or displays the galactosyl
a(I,3)
galactose moiety or it can be a different cell. Generally, methods described
herein for
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providing antigen to the recipient can be used to provide the second antigen
to the
recipient.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal of a first species, tolerance to the galactosyl a(1, 3) galactose
moiety or to a
graft which produce or displays the galactosyl a(1, 3) galactose moiety by
forming the
galactosyl a(I, 3) galactose moiety on the surface of a cell of the recipient
mammal.
Preferably, the first species is one which does not possess UDP galactose:~i-D-
galactosyl-I,4-N-acetyl-D-glucosaminide a(1,3)galactosyltransferase (aI.3GT)
activity
or which does not produce or display galactosyl a( I , 3) galactose moieties
on its cells.
tissues, or organs and can be, by way of example, a human or an Old World
primate.
e.g., a baboon, (e.g., Papio anuhis) or cynomolgus money
(Macaca.fa.scicularis).
The method includes:
forming the galactosyl a( 1. 3 ) galactose moiety on the surface of a cell of
the
recipient mammal;
preferably, allowing the recipient mammalian cell to produce or dispiay the
galactosyl a(I, 3) galactose moiety in the recipient mammal,
thereby inducing tolerance to the galactosyl a( 1, 3) galactose moiety.
The formation can be effected in vivo but is preferably effected ex vivo.
In preferred embodiments the galactosyl a(1,3) galactose moiety is formed by
contacting the cell with a protein, e.g., an enzyme which results in the
formation an
galactosyl a(I,3) galactose moiety on the surface ofthe cell.
In preferred embodiments the moiety is formed by the addition of a terminal
galactosyl residue to a galactosyl residue, e.g., to a galactosyl residue
linked to N-
acetylglucosaminyl residue, on the surface of the recipient cell. Addition of
the terminal
residue can be promoted by contacting the recipient cell with a protein which
promotes
the addition of a terminal galactosyl residue. By way of example, the protein
can be: a
protein which promotes the addition of a terminal galactosyl residue to a
galactosyl
residue, e.g., to a galactosyl residue linked to N-acetylglucosaminyl residue;
a
mammalian, e.g., a vertebrate, e.g., porcine or marine
a(1,3)galactosyltransferase; a
New World primate, e.g., a squirrel monkey, a(1,3)galactosyltransferase; an a
(1,3)galactosyltransferase, e.g., UDP galactose: (3-D-galactosyl-1,4-N-acetyl-
D-
glucosaminide a(1,3)galactosyltransferase.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal. The donor can be, for example, a
species
which normally produces or displays the galactosyl a(1, 3) galactose moiety on
its
cells, tissues, or organs, or a species which possesses
a(1,3)galactosyltransferase
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activity. By way of example the donor can be a swine, e.g., a miniature swine,
or a
New World primate, e.g., a squirrel monkey (Saimiri sciureus). Preferably, the
graft
expresses a major histocompatibility complex (MHC) antigen. The graft can be
an
organ, e.g., a heart, liver, or kidney, or skin, or a preparation of
hematopoietic stem cells,
e.g., a bone marrow preparation. In particularly preferred embodiments the
recipient is a
human and the graft is from a swine, e.g., a miniature swine.
In preferred embodiments, the method includes: inactivating immune system
cells, e.g., xenoreactive immune cells, e.g, galactosyl a( 1, 3) galactose
moiety-reactive
immune cells, of the recipient, preferably prior to providing the recipient
cell.
In preferred embodiments, the method includes: inactivating an antibodies,
e.g.,
xenoreactive antibodies, e.g, galactosyl a(1, 3) galactose-reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-galactosyl a( 1, 3) galactose antibody. For
example, anti-
1 S galactosyl a{ 1, 3 ) galactose antibody activity can be inactivated prior
to the introduction
or formation in the recipient of a recipient cell which produce or displays
galactosyl a( 1.
3) galactose moieties. Thus, in preferred embodiments. the method includes one
or
more of: administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal,
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
specific for an anti- galactosyl a(1, 3) galactose epitope antibody; depleting
natural
antibodies from the blood of the recipient, e.g., by hemoperfusing an organ,
e.g., a liver
or kidney, obtained from a mammal of the donor species or by contacting the
blood of
the recipient with galactosyl a( 1, 3 ) galactose moieties coupled to an
insoluble substrate;
administering to the recipient drugs which inactivate natural antibodies,
e.g.,
deoxyspergualin (DSG) (Bristol-Myers Squibb Co., Princeton, NJ); or
administering to
the recipient anti-IgM antibodies.
In preferred embodiments the method inhibits hyperacute rejection.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen, e.g., a carbohydrate moiety. The
second
antigen can be produced by or displayed on a modified cell of the recipient.
The
modified cell can be the same cell which produces or displays the galactosyl
a(I, 3)
galactose moiety or it can be a different cell. Generally, methods discribed
herein for
providing antigen to the recipient can be used to provide the second antigen
to the
recipient.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal, e.g., a human, tolerance to an antigen, e.g., a carbohydrate moiety,
e.g., a
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blood group carbohydrate, from a donor mammal of the same species, wherein the
antigen is not expressed in the recipient but is expressed in the donor.
The method includes:
providing to the recipient mammal a tolerance-inducing antigen, e.g., a
carbohydrate moiety, e.g., a blood group carbohydrate, thereby inducing
tolerance to the
antigen or to a graft which produces or displays the antigen. Although not
wishing to be
bound by theory, the inventors believe the antigen, e.g., a carbohydrate
moiety, mediates
the deletion of immune cells which would give rise to antigen-, e.g.,
carbohydrate
moiety-reactive antibodies.
The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks. or fails to express, an allele which results in production or display
of the antigen.
For example, the antigen can be an antigen which conditions blood type. Human
blood
group carbohydrate epitopes are found at the nonreducing termini of protein-
and lipid
bound oligosaccharides. The genes for numerous enzymes which synthesize blood
group antigen determinants have been cloned. These enzymes act on the Gal~i
1.3/4GIcNAc moieties of N- and O- glycans and glycolipids. The carbohydrate
groups
which characterize the various blood groups are referred to herein as blood
group
antigens. carbohydrates, or moieties. The human blood group A, B, H, Le and 1
epitopes
are synthesized, respectively, by UDP-GaINAc:Fucal,2Ga1 -R a1,3-GaINAc
transferase
(EC 2.4.1.40), UDP-GaINAc:Fucal,2Gal-Ral,3Gal transferase (EC 2.4.1.37), GDP-
Fuc:(~galactosidea2-Fuc-transferase (EC 2.4.1.69), GDP-Fuc:Gal/31,3/4GIcNAc-Ra
4/3Fuc transferase (EC 2.4.1.65), and UDP-GIcNAc:GIcNAc~il.3Ga1(31,4GIcNAc-
R~36-
GIcNAc transferase.
In preferred embodiments the subject is a human and the antigen is a blood
group
A carbohydrate moiety.
In preferred embodiments the subject is a human and the antigen is a blood
group
B carbohydrate moiety.
In preferred embodiments the subject is a human and the antigen is a blood
group
H carbohydrate moiety.
In preferred embodiments the subject is a human and the antigen is a blood
group
Le carbohydrate moiety.
In preferred embodiments the subject is a human and the antigen is a blood
group
I carbohydrate moiety.
In preferred embodiments a recipient cell is modified to express UDP-
GaINAc:Fucal,2Ga1-R a1,3-GaINAc transferase (EC 2.4.1.40), or an enzyme of
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equivalent activity, e.g., by insertion of nucleic acid which encodes the
enzyme into a
cell of the recipient.
In preferred embodiments a recipient cell is modified to express UDP-
GaINAc:Fucal,2Gal-Ral,3Ga1 transferase (EC 2.4.1.37), or an enzyme of
equivalent
activity, e.g., by insertion of nucleic acid which encodes the enzyme into a
cell of the
recipient.
In preferred embodiments a recipient cell is modified to express GDP-Fuc:~3
galactosidea2-Fuc-transferase (EC 2.4.1.69), or an enzyme of equivalent
activity, e.g.,
by insertion of nucleic acid which encodes the enzyme into a cell of the
recipient.
In preferred embodiments a recipient cell is modified to express GDP-Fuc:Gal(3
1,3/4GlcNAc-Ra4/3Fuc transferase (EC 2.4.1.65), or an enzyme of equivalent
activity,
e.g., by insertion of nucleic acid which encodes the enzyme into a cell of the
recipient.
In preferred embodiments a recipient cell is modified to express UDP-
GIcNAc:GIcNAc~il,3Ga1~31,4GlcNAc-R~i6-GIcNAc transferase, or an enzyme of
1 S equivalent activity, e.g., by insertion of nucleic acid which encodes the
enzyme into a
cell of the recipient.
In preferred embodiments, the antigen, c.g., a carbohydrate moiety, is
produced
by or displayed on a modified cell of the recipient, wherein modified means
the cell has
been modified to produce or display the antigen. The cell can be modified in
vivo (in
the recipient's body), e.g., by in vivo gene therapy or by in vivo treatment
with an agent
which modifies the cell, or ex vivo (removed from the recipient's body) by
recombinant
means or by treatment with an agent which modifies the cell. The cell can be
modified
to produce or display an antigen by inserting into the cell a nucleic acid
encoding the
antigen or nucleic acid encoding a protein (or proteins) which promotes, e.g.,
catalyzes,
the formation of the antigen, e.g., a carbohydrate moiety.
The encoded protein can be an enzyme, e.g., a transferase, which results in
the
formation an carbohydrate moiety on the surface of the cell. E.g., the cell
can be
modified to express an enzyme (or enzymes) which promotes the formation of a
blood
group carbohydrate moiety or moieties not produced or displayed by the
recipient, e.g.,
one or more of UDP-GaINAc:Fucal,2Gal -R a1,3-GaINAc transferase {EC 2.4.1.40),
UDP-GaINAc:Fucal,2Gal-Ral,3Gal transferase (EC 2.4.1.37), GDP-
Fuc:(3galactoside
a2-Fuc-transferase (EC 2.4.1.69), GDP-Fuc:Gal(31,3/4GlcNAc-Ra4/3Fuc
transferase
(EC 2.4.1.65), and UDP-GIcNAc:GIcNAc~il,3Gal(31,4GIcNAc-R(36-GIcNAc
transferase. In particularly preferred embodiments the encoded protein forms
the moiety
by the addition of one or more terminal sugar residues to a pre-existing sugar
on a cell
surface molecule.
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The cell can be modified to produce or display the antigen, e.g., a
carbohydrate
moiety, by forming the antigen, e.g., a carbohydrate moiety, on the surface of
a cell of
the recipient mammal, e.g., by contacting the cell with a protein, e.g., an
enzyme, which
results in the formation, e.g., by attachment, of the antigen, e.g., a
carbohydrate moiety,
on the surface of the cell. In particularly preferred embodiments the protein
forms the
moiety by the addition of one or more terminal sugar residues to a pre-
existing sugar
residue on a cell surface molecule.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In preferred embodiments the cell is removed from the recipient, modified so
as
to allow it to produce or display the antigen, e.g., a carbohydrate. and
implanted in the
recipient.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing antigen. e.g., a carbohydrate, inactivating immune system
cells. e.g.,
5 antigen-reactive immune cells, e.g, carbohydrate moiety-reactive immune
cells, of the
recipient.
In preferred embodiments the method inhibits hyperacute rejection.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing antigen, e.g., a carbohydrate, inactivating antibodies,
e.g., antigen-
reactive antibodies, e.g, carbohydrate moiety-reactive antibodies, of the
recipient.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to. a second antigen, e.g., a carbohydrate moiety. The
second
antigen can be produced by or displayed on a modified cell of the recipient.
The
modified cell can be the same cell which produces or displays the first
antigen or it can
be a different cell. Generally, methods discribed herein for providing antigen
to the
recipient can be used to provide the second antigen to the recipient.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal, e.g., a human, tolerance to a blood group A carbohydrate antigen,
e.g., a
terminal N-acetyl-D-galactosamine moiety, or to a graft which produces or
displays a
blood group A carbohydrate, e.g., a terminal N-acetyl-D-galactosamine moiety.
The
blood group sugar can be Type 1 or Type 2. Preferably, the recipient does not
possess
an enzyme which promotes the formation of a blood group A carbohydrate, e.g.,
UDP-
GaINAc:Fucal,2Gal-Ral,3-GaINAc transferase {EC 2.4.1.40) ), or an enzyme of
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equivalent activity, or does not produce or display a group A carbohydrate,
e.g., a
terminal N-acetyl-D-galactosamine moiety on its cells, tissues, or organs.
The method includes:
providing to the recipient mammal a tolerance-inducing blood group A
carbohydrate antigen, e.g., a terminal N-acetyl-D-galactosamine moiety,
thereby
inducing tolerance to the blood group A moiety or to a graft which produce or
displays
that moiety. Although not wishing to be bound by theory, the inventors believe
the
carbohydrate moiety mediates the deletion of immune cells which would give
rise to
blood group A-reactive antibodies.
The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks. or fails to express. an allele which results in production or display
of the antigen.
In preferred embodiments a blood group A carbohydrate moiety is produced or
displayed on a modified cell of the recipient, wherein the cell has been
modified to
I S produce or display the blood group A carbohydrate moiety. The cell can be
modified in
vwo or ex vtvo.
In preferred embodiments: the cell is modified to produce or display a blood
group A carbohydrate moiety by inserting into the cell a nucleic acid which
encodes a
protein which promotes, e.g., catalyzes, the formation of the blood group A
carbohydrate
moiety, e.g., UDP-GaINAc:Fuca l,2Ga1-Ra 1,3-GaINAc transferase (EC 2.4.1.40)
or an
enzyme with equivalent activity.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
nucleic acid into the recipient mammal cell and administering the recipient
mammalian
cell to the recipient mammal.
In preferred embodiments: the cell is modified to produce or display a blood
group A carbohydrate moiety by forming the blood group A carbohydrate moiety
on
the surface of a cell of the recipient mammal, e.g., by contacting the cell
with a protein,
e.g., an enzyme which promotes the formation of the blood group A carbohydrate
moiety on the surface of the cell. In particularly preferred embodiments the
moiety is
formed by the addition of a blood group A carbohydrate , on the surface of the
recipient
cell, by contacting the cell with an enzyme which promotes the synthesis or
attachment
of the moiety, e.g.,UDP-GaINAc:Fucal,2Gal-Ral,3-GaINAc transferase (EC
2.4.1.40)
or an enzyme with equivalent activity.
In preferred embodiments, the method includes inactivating immune system
cells, e.g., antigen-reactive immune cells, e.g, blood group A carbohydrate
moiety-
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reactive immune cells, of the recipient, preferably prior to providing the
recipient cell
which produces or displays a blood group A carbohydrate moiety.
In preferred embodiments, the method includes: inactivating antibodies, e.g.,
' antigen-reactive antibodies, e.g, blood group A carbohydrate -reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell which produces or
displays a
blood group A carbohydrate moiety.
In preferred embodiments the method inhibits hyperacute rejection.
The recipient cell can be any cell suitable for presentation of a blood group
A
carbohydrate moiety, e.g., a hematopoietic cell. Hematopoietic stem cells,
e.g., bone
marrow cells, which are capable of developing into mature myeloid and/or
lymphoid
cells, are particularly preferred. It is possible that later stage cells can
be used. Stem
cells derived from the cord blood of the recipient can be used in methods of
the
invention. Other cells suitable for use in the invention include peripheral
blood cells.
Suitable cells are those which can produce or display the blood group
carbohydrate
I S moiety and tolerize the animal. Although not wishing to be bound by
theory. the
inventors believe that suitable recipient cells are cells which produce or
display the
blood group carbohydrate moiety such that the moiety can interact with immune
cells
at an early stage in their development. Although not wishing to be bound by
theory, this
is believed to allow deletion of cells which would give rise to blood group A
reactive
antibodies. Suitable cells are those which result in tolerate as opposed to an
immune
response.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-blood group A carbohydrate antibody. For example,
anti-
blood group A carbohydrate antibody activity can be inactivated prior to the
introduction
or formation in the recipient of a recipient cell which produces or displays
group A
carbohydrate moieties. Thus, in preferred embodiments, the method includes one
or
more of administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal,
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
specific for an anti-group A carbohydrate antibody; depleting natural
antibodies from
the blood of the recipient, e.g., by hemoperfusing an organ, e.g., a liver or
kidney,
obtained from a mammal of the donor species or by contacting the blood of the
recipient
with blood group A moieties coupled to an insoluble substrate; administering
to the
recipient drugs which inactivate natural antibodies, e.g., deoxyspergualin
(DSG)
(Bristol); or administering to the recipient anti-IgM antibodies.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen. The antigen can be produced by or
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displayed on a modified cell of the recipient. The modified cell can be the
same cell
which produces or displays the first antigen or it can be a different cell.
Generally,
methods discribed herein for providing antigen to the recipient can be used to
provide
the second antigen to the recipient.
In preferred embodiments more than one blood group carbohydrate moiety is
provided to the recipient. The second carbohydrate moiety can be provided by a
method
described herein.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In another aspect, the invention features, a method of promoting, in a
recipient
mammal. e.g., a human, tolerance to a blood group B carbohydrate antigen or to
a graft
which produces or displays a blood group B moiety. The blood group sugar can
be
Type 1 or Type 2. Preferably, the recipient does not possess an enzyme which
promotes
the formation ofa blood group B carbohydrate, e.g., UDP-GaINAc:Fucal,2Gal-Ra
I S l.3Gal transferase (EC 2.4.1.37), or an enzyme of equivalent activity, or
does not
produce or display a group B carbohydrate moiety on its cells, tissues. or
organs.
The method includes:
providing to the recipient mammal a tolerance-inducing blood group B
carbohydrate antigen. thereby inducing tolerance to the blood group B moiety
or to a
graft which includes that moiety. Although not wishing to be bound by theory,
the
inventors believe the carbohydrate moiety mediates the deletion of immune
cells which
would give rise to blood group B-reactive antibodies.
The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks, or fails to express, an allele which results in production or display
of the antigen.
In preferred embodiments a blood group B carbohydrate moiety is produced or
displayed on a modified cell of the recipient, wherein the cell has been
modified to
produce or display the blood group B carbohydrate moiety. The cell can be
modified in
vivo or ex vivo.
In preferred embodiments: the cell is modified to produce or display a blood
group B carbohydrate moiety by inserting into the cell a nucleic acid which
encodes a
protein which promotes, e.g., catalyzes, the formation of the blood group B
carbohydrate
moiety, e.g., UDP-GaINAc:Fucal,2Ga1-Ral,3Gal transferase (EC 2.4.1.37), or an
enzyme of equivalent activity.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
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nucleic acid into the recipient mammal cell and administering the recipient
mammalian
cell to the recipient mammal.
In preferred embodiments: the cell is modified to produce or display a blood
group B carbohydrate moiety by forming the blood group B carbohydrate moiety
on the
surface of a cell of the recipient mammal, e.g., by contacting the cell with a
protein, e.g.,
an enzyme which promotes the formation of the blood group B carbohydrate
moiety on
the surface of the cell. In particularly preferred embodiments the moiety is
formed by
the addition of a blood group B carbohydrate , on the surface of the recipient
cell, by
contacting the cell with an enzyme which promotes the synthesis or attachment
of the
moiety, e.g., UDP-GaINAc:Fucal,2Ga1-Ral,3Ga1 transferase (EC 2.4.1.37), or an
enzyme of equivalent activity.
In prefetTed embodiments. the method includes inactivating immune system
cells, e.g., antigen-reactive immune cells, e.g, blood group B carbohydrate
moiety-
reactive immune cells, of the recipient, preferably prior to providing the
recipient cell
which produces or displays a blood group B carbohydrate moiety.
In preferred embodiments, the method includes: inactivating antibodies, e.g.,
antigen-reactive antibodies. e.g, blood group B carbohydrate -reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell which produces or
displays a
blood group B carbohydrate moiety.
In preferred embodiments the method inhibits hyperacute rejection.
The recipient cell can be any cell suitable for presentation of a blood group
B
carbohydrate moiety, e.g., a hematopoietic cell. Hematopoietic stem cells,
e.g., bone
marrow cells, which are capable of developing into mature myeloid and/or
lymphoid
cells, are particularly preferred. It is possible that later stage cells can
be used. Stem
cells derived from the cord blood of the recipient can be used in methods of
the
invention. Other cells suitable for use in the invention include peripheral
blood cells.
Suitable cells are those which can produce or display the blood group
carbohydrate
moiety and toierize the animal. Although not wishing to be bound by theory,
the
inventors believe that suitable recipient cells are cells which produce or
display the
blood group carbohydrate moiety such that the moiety can interact with immune
cells
at an early stage in their development. Although not wishing to be bound by
theory, this
is believed to allow deletion of cells which would give rise to blood group B
reactive
antibodies. Suitable cells are those which result in tolerace as opposed to an
immune
response.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-blood group B carbohydrate antibody. For example,
anti-
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blood group B carbohydrate antibody activity can be inactivated prior to the
introduction
or formation in the recipient of a recipient cell which produces or displays
group B
carbohydrate moieties. Thus, in preferred embodiments, the method includes one
or
more of: administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal,
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
specific for an anti-group B carbohydrate antibody; depleting natural
antibodies from
the blood of the recipient, e.g., by hemoperfusing an organ, e.g., a liver or
kidney,
obtained from a mammal of the donor species or by contacting the blood of the
recipient
with blood group B moieties coupled to an insoluble substrate; administering
to the
recipient drugs which inactivate natural antibodies, e.g., deoxyspergualin
(DSG)
(Bristol); or administering to the recipient anti-IgM antibodies.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen. The antigen can be produced by or
displayed on a modified cell of the recipient. The modified cell can be the
same cell
I 5 which produces or displays the first antigen or it can be a different
cell. Generally,
methods discribed herein for providing antigen to the recipient can be used to
provide
the second antigen to the recipient.
In preferred embodiments more than one blood group carbohydrate moiety is
provided to the recipient. The second carbohydrate moiety can be provided by a
method
described herein.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In another aspect, the invention features. a method of promoting, in a
recipient
mammal, e.g., a human, tolerance to a blood group H carbohydrate antigen or to
a graft
which produces or displays a blood group H moiety. The blood group sugar can
be
Type I or Type 2. Preferably, the recipient does not possess an enzyme which
promotes
the formation of a blood group H carbohydrate, e.g., GDP-Fuc:(3galactosidea2-
Fuc-
transferase (EC 2.4.1.69), or an enzyme of equivalent activity, or does not
produce or
display a group H carbohydrate moiety on its cells, tissues, or organs.
The method includes:
providing to the recipient mammal a tolerance-inducing blood group H
carbohydrate antigen, thereby inducing tolerance to the blood group H moiety
or to a
graft which produce or displays that moiety. Although not wishing to be bound
by
theory, the inventors believe the carbohydrate moiety mediates the deletion of
immune
3 S cells which would give rise to blood group H-reactive antibodies.
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The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks, or fails to express, an allele which results in production or display
of the antigen.
In preferred embodiments a blood group H carbohydrate moiety is produced or
S displayed on a modified cell of the recipient, wherein the cell has been
modified to
produce or display the blood group H carbohydrate moiety. 'The cell can be
modified in
vwo or ex vtvo.
In preferred embodiments: the cell is modified to produce or display a blood
group H carbohydrate moiety by inserting into the cell a nucleic acid which
encodes a
protein which promotes, e.g., catalyzes, the formation of the blood group H
carbohydrate
moiety, e.g., GDP-Fuc:~igalactosidea2-Fuc-transferase (EC 2.4.1.69), or an
enzyme of
equivalent activity.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
nucleic acid into the recipient mammal cell and administering the recipient
mammalian
cell to the recipient mammal.
In preferred embodiments: the cell is modified to produce or display a blood
group H carbohydrate moiety by forming the blood group H carbohydrate moiety
on the
surface of a cell of the recipient mammal, e.g., by contacting the cell with a
protein, e.g.,
an enzyme which promotes the formation of the blood group H carbohydrate
moiety on
the surface of the cell. In particularly preferred embodiments the moiety is
formed by
the addition of a blood group H carbohydrate . on the surface of the recipient
cell. by
contacting the cell with an enzyme which promotes the synthesis or attachment
of the
moiety, e.g., GDP-Fuc:~igalactosidea2-Fuc-transferase (EC 2.4.1.69), or an
enzyme of
equivalent activity.
In preferred embodiments, the method includes inactivating immune system
cells, e.g., antigen-reactive immune cells, e.g, blood group H carbohydrate
moiety-
reactive immune cells, of the recipient, preferably prior to providing the
recipient cell
which produces or displays a blood group H carbohydrate moiety.
In preferred embodiments, the method includes: inactivating antibodies, e.g.,
antigen-reactive antibodies, e.g, blood group H carbohydrate -reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell which produces or
displays a
blood group H carbohydrate moiety.
In preferred embodiments the method inhibits hyperacute rejection.
The recipient cell can be any cell suitable for presentation of a blood group
H
carbohydrate moiety, e.g., a hematopoietic cell. Hematopoietic stem cells,
e.g., bone
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marrow cells, which are capable of developing into mature myeloid and/or
lymphoid
cells, are particularly preferred. It is possible that later stage cells can
be used. Stem
cells derived from the cord blood of the recipient can be used in methods of
the
invention. Other cells suitable for use in the invention include peripheral
blood cells.
Suitable cells are those which can produce or display the blood group
carbohydrate
moiety and tolerize the animal. Although not wishing to be bound by theory,
the
inventors believe that suitable recipient cells are cells which produce or
display the
blood group carbohydrate moiety such that the moiety can interact with immune
cells
at an early stage in their development. Although not wishing to be bound by
theory, this
is believed to allow deletion of cells which would give rise to blood group H
reactive
antibodies. Suitable cells are those which result in tolerace as opposed to an
immune
response.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-blood group H carbohydrate antibody. For example,
anti-
blood group H carbohydrate antibody activity can be inactivated prior to the
introduction
or formation in the recipient of a recipient cell which produces or displays
group H
carbohydrate moieties. Thus, in preferred embodiments, the method includes one
or
more of: administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal.
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
specific for an anti-group H carbohydrate antibody; depleting natural
antibodies from
the blood of the recipient, e.g., by hemoperfusing an organ, e.g., a liver or
kidney,
obtained from a mammal of the donor species or by contacting the blood of the
recipient
with blood group H moieties coupled to an insoluble substrate; administering
to the
recipient drugs which inactivate natural antibodies, e.g., deoxyspergualin
(DSG)
(Bristol); or administering to the recipient anti-IgM antibodies.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen. The antigen can be produced by or
displayed on a modified cell of the recipient. The modified cell can be the
same cell
which produces or displays the first antigen or it can be a different cell.
Generally,
methods discribed herein for providing antigen to the recipient can be used to
provide
the second antigen to the recipient.
In preferred embodiments more than one blood group carbohydrate moiety is
provided to the recipient. The second carbohydrate moiety can be provided by a
method
described herein.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
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In another aspect, the invention features, a method of promoting, in a
recipient
mammal, e.g., a human, tolerance to a blood group Le carbohydrate antigen or
to a graft
which produces or displays a blood group Le moiety. Preferably, the recipient
does not
possess an enzyme which promotes the formation of a blood group Le
carbohydrate, e.g,
express GDP-Fuc:Gal(31,3/4GIcNAc-Ra4/3Fuc transferase (EC 2.4.1.65), or an
enzyme
of equivalent activity, or does not produce or display a group Le carbohydrate
moiety on
its cells, tissues, or organs.
The method includes:
providing to the recipient mammal a tolerance-inducing blood group Le
carbohydrate antigen, thereby inducing tolerance to the blood group Le moiety
or to a
graft which produce or displays that moiety. Although not wishing to be bound
by
theory, the inventors believe the carbohydrate moiety mediates the deletion of
immune
cells which would give rise to blood group Le-reactive antibodies.
The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks, or fails to express, an allele which results in production or display
of the antigen.
In preferred embodiments a blood group Le carbohydrate moiety is produced or
displayed on a modified cell of the recipient, wherein the cell has been
modified to
produce or display the blood group Le carbohydrate moiety. The cell can be
modified
in vivo or ex vivo.
In preferred embodiments: the cell is modified to produce or display a blood
group Le carbohydrate moiety by inserting into the cell a nucleic acid which
encodes a
protein which promotes, e.g., catalyzes, the formation of the blood group Le
carbohydrate moiety, e.g., GDP-Fuc:Gal(31,3/4GIcNAc-Ra4/3Fuc transferase (EC
2.4.1.65), or an enzyme of equivalent activity.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
nucleic acid into the recipient mammal cell and administering the recipient
mammalian
cell to the recipient mammal.
In preferred embodiments: the cell is modified to produce or display a blood
group Le carbohydrate moiety by forming the blood group Le carbohydrate moiety
on
the surface of a cell of the recipient mammal, e.g., by contacting the cell
with a protein,
e.g., an enzyme which promotes the formation of the blood group Le
carbohydrate
moiety on the surface of the cell. In particularly preferred embodiments the
moiety is
formed by the addition of a blood group Le carbohydrate . on the surface of
the
recipient cell, by contacting the cell with an enzyme which promotes the
synthesis or
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attachment of the moiety, e.g., GDP-Fuc:Gal~il,3/4GlcNAc-Ra4/3Fuc transferase
(EC
2.4.1.65), or an enzyme of equivalent activity.
In preferred embodiments, the method includes inactivating immune system
cells, e.g., antigen-reactive immune cells, e.g, blood group Le carbohydrate
moiety-
reactive immune cells, of the recipient, preferably prior to providing the
recipient cell
which produces or displays a blood group Le carbohydrate moiety.
In preferred embodiments. the method includes: inactivating antibodies, e.g.,
antigen-reactive antibodies, e.g, blood group Le carbohydrate -reactive
antibodies. of the
recipient, preferably prior to providing the recipient cell which produces or
displays a
blood group Le carbohydrate moiety.
In preferred embodiments the method inhibits hyperacute rejection.
The recipient cell can be any cell suitable for presentation of a blood group
Le
carbohydrate moiety, e.g;.. a hematopoietic cell. Hematopoietic stem cells,
e.g.. bone
marrow cells, which are capable of developing; into mature myeloid and/or
lymphoid
cells, are particularly preferred. It is possible that later stage cells can
be used. Stem
cells derived from the cord blood of the recipient can be used in methods of
the
invention. Other cells suitable for use in the invention include peripheral
blood cells.
Suitable cells are those which can produce or display the blood group
carbohydrate
moiety and tolerize the animal. Although not wishing to be bound by theory,
the
inventors believe that suitable recipient cells are cells which produce or
display the
blood group carbohydrate moiety such that the moiety can interact with immune
cells
at an early stage in their development. Although not wishing to be bound by
theory, this
is believed to allow deletion of cells which would give rise to blood group Le
reactive
antibodies. Suitable cells are those which result in tolerace as opposed to an
immune
response.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-blood group Le carbohydrate antibody. For
example, anti-
blood group Le carbohydrate antibody activity can be inactivated prior to the
introduction or formation in the recipient of a recipient cell which produces
or displays
group Le carbohydrate moieties. Thus, in preferred embodiments, the method
includes
one or more of: administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal,
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
specific for an anti-group Le carbohydrate antibody; depleting natural
antibodies from
the blood of the recipient, e.g., by hemoperfusing an organ, e.g., a liver or
kidney,
obtained from a mammal of the donor species or by contacting the blood of the
recipient
with blood group Le moieties coupled to an insoluble substrate; administering
to the
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recipient drugs which inactivate natural antibodies, e.g., deoxyspergualin
(DSG)
(Bristol); or administering to the recipient anti-IgM antibodies.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen. The antigen can be produced by or
displayed on a modified cell of the recipient. The modified cell can be the
same cell
which produces or displays the first antigen or it can be a different cell.
Generally,
methods discribed herein for providing antigen to the recipient can be used to
provide
the second antigen to the recipient.
In preferred embodiments more than one blood group carbohydrate moiety is
provided to the recipient. The second carbohydrate moiety can be provided by a
method
described herein.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In another aspect. the invention features. a method of promoting. in a
recipient
mammal, e.g., a human, tolerance to a blood group I carbohydrate antigen or to
a graft
which produces or displays a blood group 1 moiety. Preferably, the recipient
does not
possess an enzyme which promotes the formation of a blood group I
carbohydrate. e.g,
express UDP-GIcNAc:GIcNAc~il,3Galp1.4G1cNAc-R~i6-GIcNAc transferase, or an
enzyme of equivalent activity, or does not produce or display a group I
carbohydrate
moiety on its cells, tissues, or organs.
The method includes:
providing to the recipient mammal a tolerance-inducing blood group I
carbohydrate antigen, thereby inducing tolerance to the blood group I moiety
or to a
graft which includes that moiety. Although not wishing to be bound by theory,
the
inventors believe the carbohydrate moiety mediates the deletion of immune
cells which
would give rise to blood group I-reactive antibodies.
The donor can be, for example, an animal which has or expresses an allele
which
results in production or display of the antigen and the recipient can be an
animal that
lacks, or fails to express, an allele which results in production or display
of the antigen.
In preferred embodiments a blood group I carbohydrate moiety is produced or
displayed on a modified cell of the recipient. wherein the cell has been
modified to
produce or display the blood group I carbohydrate moiety. The cell can be
modified in
vivo or ex vivo.
In preferred embodiments: the cell is modified to produce or display a blood
group I carbohydrate moiety by inserting into the cell a nucleic acid which
encodes a
protein which promotes, e.g., catalyzes, the formation of the blood group I
carbohydrate
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moiety, e.g., UDP-GIcNAc:GIcNAc(31,3Ga1(31,4G1cNAc-R~36-GIcNAc transferase, or
an enzyme of equivalent activity.
In other preferred embodiments, the providing step of the method includes:
removing the recipient mammalian cell from the recipient mammal prior to
introducing
nucleic acid into the recipient mammal cell and administering the recipient
mammalian
cell to the recipient mammal.
In preferred embodiments: the cell is modified to produce or display a blood
group I carbohydrate moiety by forming the blood group I carbohydrate moiety
on the
surface of a cell of the recipient mammal, e.g., by contacting the cell with a
protein, e.g.,
an enzyme which promotes the formation of the blood group 1 carbohydrate
moiety on
the surface of the cell. In particularly preferred embodiments the moiety is
formed by
the addition of a blood group I carbohydrate , on the surface of the recipient
cell. by
contacting the cell with an enzyme which promotes the synthesis or attachment
of the
moiety, e.g., UDP-GIcNAc:GIcNAc~il,3Ga1~31,4G1cNAc-R~i6-GIcNAc transferase. or
an enzyme of equivalent activity.
In preferred embodiments, the method includes inactivating immune system
cells. e.g., antigen-reactive immune cells. e.g, blood group I carbohydrate
moiety-
reactive immune cells, of the recipient, preferably prior to providing the
recipient cell
which produces or displays a blood group I carbohydrate moiety.
In preferred embodiments, the method includes: inactivating antibodies. e.g.,
antigen-reactive antibodies, e.g, blood group I carbohydrate -reactive
antibodies, of the
recipient, preferably prior to providing the recipient cell which produces or
displays a
blood group I carbohydrate moiety.
In preferred embodiments the method inhibits hyperacute rejection.
The recipient cell can be any cell suitable for presentation of a blood group
I
carbohydrate moiety, e.g., a hematopoietic cell. Hematopoietic stem cells,
e.g., bone
marrow cells, which are capable of developing into mature myeloid and/or
lymphoid
cells, are particularly preferred. It is possible that later stage cells can
be used. Stem
cells derived from the cord blood of the recipient can be used in methods of
the
invention. Other cells suitable for use in the invention include peripheral
blood cells.
Suitable cells are those which can produce or display the blood group
carbohydrate
moiety and tolerize the animal. Although not wishing to be bound by theory,
the
inventors believe that suitable recipient cells are cells which produce or
display the
blood group carbohydrate moiety such that the moiety can interact with immune
cells
at an early stage in their development. Although not wishing to be bound by
theory, this
is believed to allow deletion of cells which would give rise to blood group I
reactive
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antibodies. Suitable cells are those which result in tolerace as opposed to an
immune
response.
In preferred embodiments, the method includes an additional step which
inactivates a recipient anti-blood group I carbohydrate antibody. For example,
anti-
s blood group I carbohydrate antibody activity can be inactivated prior to the
introduction
or formation in the recipient of a recipient cell which produces or displays
group I
carbohydrate moieties. Thus, in preferred embodiments, the method includes one
or
more of: administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal,
polycional. chimeric, single chain, or humanized antibodies), or fragments
thereof.
specific for an anti-group I carbohydrate antibody; depleting natural
antibodies from the
blood of the recipient, e.g., by hemoperfusing an organ, e.g., a liver or
kidney, obtained
from a mammal of the donor species or by contacting the blood of the recipient
with
blood group I moieties coupled to an insoluble substrate; administering to the
recipient
drugs which inactivate natural antibodies, e.g.. deoxyspergualin (DSG)
(Bristol); or
administering to the recipient anti-IgM antibodies.
In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second antigen. The antigen can be produced by or
displayed on a modified cell of the recipient. The modified cell can be the
same cell
which produces or displays the first antigen or it can be a different cell.
Generally.
methods discribed herein for providing antigen to the recipient can be used to
provide
the second antigen to the recipient.
In preferred embodiments more than one blood group carbohydrate moiety is
provided to the recipient. The second carbohydrate moiety can be provided by a
method
described herein.
In preferred embodiments, the method further includes: introducing a graft
from
a donor mammal into the recipient mammal.
In another aspect, the invention features, a method of inactivating recipient
natural antibodies which bind to an antigen which is found on the surface of a
xenograft, e.g., a carbohydrate moiety, e.g., a galactosyl a(1, 3) galactose
moiety. e.g., a
galactosyl a(1, 3) galactose moiety on a graft. and thereby inhibiting
hyperacute
rejection by administering anti-idiotypic antibodies (e.g., recombinant,
monoclonal.
polyclonal, chimeric, single chain, or humanized antibodies), or fragments
thereof,
against the natural antibody.
In preferred embodiments the method further includes implanting the graft.
e.g.,
a kidney, liver, heart, or population of hematopoietic stem cells in the
recipient.
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In preferred embodiments the recipient is a human and the graft is from a
swine,
e.g., a miniature swine.
In preferred embodiments the method inhibits hyperacute rejection.
In another aspect, the invention features, a purified preparation of an anti-
idiotypic monoclonal antibody {e.g., recombinant, monoclonal, poiyclonal,
chimeric,
single chain, or humanized antibody), or fragments thereof, directed against a
natural
antibody which reacts with an antigen, e.g., a carbohydrate, e.g., an
galactosyl a(1, 3)
gaiactose moiety, present on the surface of swine cells, to which antigen
humans make
natural antibodies.
In another aspect, the invention features, a purified preparation of an anti-
idiotypic monoclonal antibody (e.g., recombinant, monoclonal, polyclonal,
chimeric,
single chain. or humanized antibody), or fragments thereof, directed against
an antibody
which reacts with a carbohydrate moiety, an galactosyl a( 1, 3) galactose
moiety.
Methods described herein can also include other steps to promote acceptance of
l S or induce tolerance to the recipient cell or to the graft.
Other preferred embodiments include: the step of, preferably prior to
recipient
cell transplantation, creating hematopoietic space in the recipient. The
reintroduction
into the recipient of engineered or otherwise modified autologous cells can be
optimized
by the creation of hematopoietic space. Hematopoietic space can be created by
the
administration of antibodies or drugs which deplete the bone marrow, e.g., by
administering an inhibitor of cell proliferation, e.g., DSG, or an anti-
metabolite, e.g.
Brequinar, or an anti-T cell antibody, e.g., one or both of an anti-CD4 or
anti-CD8
antibody. Hematopoietic space can also be created by irradiating the recipient
mammal
with low dose, e.g., between about 100 and 400 Rads, whole body irradiation to
deplete
or partially deplete the bone marrow of the recipient. The creation of
hematopoietic
space does not totally ablate the recipients bone marrow but allows for the
production of
mixed chimerism. The need for hematopoietic space can be minimized by the
creation
in the recipient of thymic space..
Other preferred embodiments include: the step of creating thymic space in the
recipient, e.g., by irradiating the thymus of the recipient, e.g., by
administering between
100 and 1,000, more preferably between 300 and 700, e.g., 700 Rads, of thymic
irradiation, or by administering anti-T cell antibodies in sufficient dose to
inactivate
thymocytes. Other methods for the creation of thymic space include: the
administration
of steroids, corticosteroids, Brequinar, or immune suppressant drugs, e.g.,
rapamycin,
cyclosporin, or FK506. Methods of creating thymic space are disclosed in
provisional
U.S. patent application 60/017,099, hereby incorporated by reference. The
methods
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disclosed herein can be combined with the methods disclosed in U.S. patent
application
60/017,099.
In preferred embodiments, the method includes: inactivating immune system
cells, e.g., xenoreactive immune cells, of the recipient. Immune system cells
include
S thymocytes, T cells, B cells, and NK cells.
In other preferred embodiments, the method includes: inactivating T cells.
e.g.,
xenoreactive T cells, of the recipient mammal, e.g., by prior to introducing
recipient
cells or a graft into the recipient mammal, introducing into the recipient
mammal an
antibody capable of binding to T cells of the recipient mammal.
In preferred embodiments, the method includes: inactivating natural killer
cells,
e.g., xenoreactive NK cells, of the recipient mammal, e.g., by prior to
introducing the
cells or a graft into the recipient mammal, introducing into the recipient
mammal an
antibody capable of binding to natural killer cells of the recipient mammal.
One source of anti-NK antibody is anti-human thymocyte polyclonal anti-serum.
A second anti-mature T cell antibody can be administered as well, which
inactivates T
cells as well as NK cells. Depletion, Inactivation of T cells is advantageous
for both
bone marrow and xenograft survival. Anti-T cell antibodies are present. along
with
anti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses of anti-NK or
anti-T
cell antibody may be preferable. Monoclonal preparations can be used in the
methods of
the invention.
The methods described herein can be combined with methods of inducing
tolerance described in United States Serial Number 08/266,427, filed June 27,
1994, the
contents of which are hereby expressly incorporated by reference. Thus, the
methods
disclosed herein can include administering to the recipient a recipient cell
which
expresses a donor MHC class I gene or a donor MHC class II gene (or both). The
cell
which expresses the donor MHC gene can be the same cell which expresses the
galactosyl a(1, 3) galactose moiety or it can be a different cell.
In preferred embodiments, a short course of help reducing treatment can be
used
to induce tolerance to the recipient cell or the graft. In particular, the
methods described
in United States Serial Number 08/458,720, filed June 1, 1995, the contents of
which are
expressly incorporated herein by reference, can be combined with the methods
described
herein.
In preferred embodiments, a short course of an immunosuppressive agent can be
administered to inhibit T cell activity in the recipient. In particular, the
methods
described in United States Serial Number 08/458,720, filed June l, 1995, the
contents of
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which are expressly incorporated herein by reference, can be combined with the
methods
described herein.
Methods of inducing tolerance by the methods described herein can also be
combined with yet other methods for inducing tolerance, e.g., with: methods
which use
the implantation of donor stem cells to induce tolerance, e.g., the methods
described in
United States Serial No. 08/451,210, filed on May 26, 1995, the contents of
which are
hereby expressly incorporated by reference; methods which use stem cells or
other
tissue from genetically engineered swine, e.g., the genetically engineered
swine in
United States Serial No. 08/292,565, filed August 19, 1994, the contents of
which are
expressly incorporated herein by reference, or in United States Serial No.
08/692, 843,
filed August 2, 1996, the contents of which are expressly incorporated herein
by
reference: methods which use the implantation of a xenogeneic thymic graft to
induce
tolerance. e.g., the methods described in United States Serial No. 08/163,912.
filed on
December 7. 1993, the contents of which are hereby expressly incorporated by
reference; methods of increasing the level of the activity of a tolerance
promoting or
GVHD inhibiting cytokine or decreasing the level of activity of a tolerance
inhibiting or
GVHD promoting cytokine, e.g., the methods described in United States Serial
No.
08/114.072, filed August 30, 1993, the contents of which are hereby expressly
incorporated by reference; methods of using cord blood cells to induce
tolerance. e.g.,
the methods described in United States Serial No. 08/150,739 filed November
10. 1993,
the contents of which are hereby expressly incorporated by reference; methods
of
preventing GVHD, e.g., the methods described in United States Serial No.
08/461.693.
filed June ~, 1995, the contents of which are hereby expressly incorporated by
reference;
with methods of promoting tolerance by enhancing or maintaining thymus
function. e.g.,
the methods described in United States Serial No. 08/297,291, filed August 26,
1994,
the contents of which are hereby expressly incorporated by reference; methods
of
detecting the presence of swine retroviral sequences, e.g., the methods
described in
United States Serial No. 08/572,645, filed December 14, 1995, or a
continuation of U.S.
Serial No. 08/572.645, filed December 13, 1996, the contents of which are
hereby
expressly incorporated by reference; and the methods for inducing tolerance
disclosed
in Sykes and Sachs, PCT/US94/01616, filed February 14, 1994, the contents of
which
are hereby expressly incorporated by reference.
In another aspect, the invention features a method of treating a subject
mammal,
e.g., a human, having a disorder characterized by an unwanted antibody
directed against
an autoantigen. The method includes:
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providing to the mammal a tolerance-inducing autoantigen, e.g., a carbohydrate
moiety, protein, or peptide, thereby inducing tolerance to the autoantigen.
Although not
wishing to be bound by theory, the inventors believe the autoantigen mediates
the
deletion of immune cells which would give rise to autoantigen-reactive
antibodies.
In preferred embodiments the subject is a human and the autoantigen is one
which mediates diabetes, MS, lupus, or arthritisis.
In preferred embodiments, the autoantigen is produced by or displayed on a
modified cell of the subject, wherein the cell has been modified to produce or
display the
autoantigen. The cell can be modified in vivo (in the recipient's body), e.g.,
by in vivo
gene therapy or by in vivo treatment with an agent which modifies the cell. or
ex vivo
(removed from the subject's body). The cell can be modified by inserting into
the cell a
nucleic acid which encodes the autoantigen, (or otherwise promotes the
production or
display of the autoantigen) such that the cell produces or displays the
autoantigen. The
cell can be modified to produce or display a carbohydrate moiety by inserting
into the
1 S cell a nucleic acid encodin6 a protein which promotes, e.g., catalyzes,
the formation of
the carbohydrate moiety. The encoded protein can be an enzyme which results in
the
formation of a carbohydrate moiety on the surface of the cell. In particularly
preferred
embodiments the encoded protein fonms the moiety by the addition of a terminal
sugar
residue to a pre-existing sugar residue on a cell surface molecule.
The cell can be modified to produce or display the autoantigen, e.g., a
protein or
carbohydrate moiety, by forming the autoantigen, e.g., a protein or
carbohydrate moiety,
in or on the surface of a cell of the recipient mammal or attaching the
autoantigen to the
subject cell, e.g., by contacting the cell with a protein, e.g., an enzyme,
which results in
the formation of the autoantigen, e.g., a carbohydrate moiety, on the surface
of the cell
or by adhering or attaching the autoantigen to the cell. In particularly
preferred
embodiments the protein forms the moiety by the addition of a terminal sugar
residue to
a pre-existing sugar residue on a cell surface molecule.
In preferred embodiments the cell is removed from the subject, modified so as
to
allow it to produce or display the autoantigen and implanted in the recipient.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing autoantigen, inactivating immune system cells, e.g.,
autoantigen-
reactive immune cells, of the recipient.
In preferred embodiments, the method includes: preferably prior to providing
the
tolerance-inducing autoantigen inactivating antibodies, e.g., autoantigen
reactive
antibodies, e.g, carbohydrate moiety-reactive antibodies, of the recipient.
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In preferred embodiments the method further includes providing to the
recipient,
and inducing tolerance to, a second autoantigen, e.g., a carbohydrate moiety,
protein, or
peptide. The second autoantigen can be produced by or displayed on a modified
cell of
the recipient. The modified cell can be the same cell which produces or
displays the first
autoantigen or it can be a different cell. Generally, methods described herein
for
providing autoantigen to the recipient can be used to provide the second
autoantigen to
the recipient.
"Antigen" as used herein is a molecule which can be recognized as non-self by
a
recipient immune system and includes proteins and carbohydrates, e.g.,
carbohydrates
found on glycoproteins or glycolipids. Preferred antigens are those which
react with
natural antibodies in humans.
"Forming a galactosyl a( 1,3 ) galactose moiety on the surface of a cell"
refers to a
process which results in the cell presenting a galactosyl a( 1,3) galactose
moiety on its
surface. Forming can include attaching, preferably by a covalent modification,
a
IS galactosyl a(1,3) galactose moiety, or enzymatically forming a galactosyl
a( 1,3)
galactose moiety, on the surface of the cell.
"Galactosyl a(1, 3) galactose epitope", as used herein, refers to epitopes
located
wholly or partially on galactosyl a(I, 3) galactose structures, e.g., those
located wholly
or partially on galactosyl (a1,3) galactose structures of aGal (1-3)(3Ga1 (1-
4)~3GlcNAc
or aGal (1-3)~3Gal (1-4)(3Glc structures.
"Galactosyl a(1, 3) galactose moiety", as used herein, refers to the
galactosyl
a(1,3) galactose structure, e.g., as found in aGal (1-3)~iGal (1-4)(3GlcNAc or
aGal (1-
3)~iGal (1-4)~Glc structures.
"a(1,3)galactosyltransferase, e.g., (3-D-galactosyl-1,4-N-acetyl-D-
glucosaminide
a{1,3)galactosyltransferase activity, as used herein refers to the enzymatic
activity of
forming galactosyl a(1, 3) galactose moieties. Enzyme activity which forms the
B
blood group antigen is not covered by this definition.
"Graft", as used herein, refers to a body part, organ, tissue, or cells.
Grafts may
consist of organs such as liver, kidney, heart or lung; body parts such as
bone or skeletal
matrix; tissue such as skin, intestines, endocrine glands, thymic tissue; or
progenitor
stem cells of various types.
"Inactivation of an antibody (or an antibody response)" refers to a treatment
which reduces the number of antibodies, particularly xenoreactive antibodies,
e.g.,
galactosyl a(1, 3) galactose moiety reactive antibodies. which can bind their
cognate
epitope in a subject. Inactivation includes: removal of antibodies from the
subject. e.g.,
by contacting the blood of the subject with a reagent, e.g., an affinity
matrix, which
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allows removal of antibodies from the blood; inactivating an immune cell which
promotes the formation of the antibody; and inhibiting an antibody by
contacting it with
an anti-idiotypic antibody.
"Inactivation of an immune cell" refers to a treatment which reduces the
number
of active immune cells, e.g., thymocytes, T cells, B cells, or NK cells in a
subject.
Inactivation includes: removal from the blood of the subject; temporarily or
permanently inhibiting an immune cell e.g., a T or B cell by, e.g.,
administering a drug
such as an inhibitor of cell proliferation, e.g., DSG, or an anti-metabolite,
e.g. Brequinar;
temporarily or permanently inhibiting an immune cell by administering an anti-
immune
cell antibody, e.g., an anti-T cell antibody, e.g.. one or both of an anti-CD4
or anti-CDS
antibody, an anti-B cell antibody, or an anti-NK cell antibody.
"Lymph node or thymic T cell", as used herein. refers to T cells which are
resistant to inactivation by traditional methods of T cell inactivation, e.g.,
inactivation
by a single intravenous administration of anti-T cell antibodies. e.g.,
antibodies, e.g.,
ATG preparation.
"MHC antigen", as used herein. refers to a protein product of one or more MHC
genes; the term includes fragments or analogs of products of Ml-iC genes which
can
evoke an immune response in a recipient organism. Examples of MHC antigens
include
the products (and fragments or analogs thereof) of the human MHC genes, i.e.,
the HLA
genes. MHC antigens in swine, e.g., miniature swine. include the products (and
fragments and analogs thereof) of the SLA genes. e.g., the DRB gene.
"Miniature swine", as used herein. refers to a miniature pig which is
preferably
wholly or partially inbred at at least one MHC locus. The coefficient of
inbreeding of
the herd which supplies the miniature swine should be at least , 0.70 and more
preferably
at least 0.82.
"Produces or displays", as used herein, means that the entity, e.g., a cell, a
tissue,
or an organ, provides on its surface, secretes, or otherwise provides, the
moiety. The
moiety is accessible to one or more components of the immune system, e.g.,
antibodies,
or cell-bound receptors, e.g., T cell receptors.
"Recipient cell" as used herein refers to a cell suitable for the tolerizing
expression of the galactosyl a(1, 3) galactose epitope. For example, a
recipient cell can
be a hematopoietic cell, e.g., a bone marrow cell which is capable of
developing into a
mature myeloid and/or lymphoid cell. Stem cells derived from cord blood, bone
marrow, or peripheral blood of the recipient can be used in methods of the
invention.
See U.S. Patent 5,192,553, hereby incorporated by reference, and U.S. Patent
5,004.681,
hereby incorporated by reference.
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"Thymic space", as used herein, is a state created by a treatment that
facilitates
the migration to and/or development in the thymus of donor or engineered
autologous
hematopoietic cells of a type which can delete or inactivate recipient
thymocytes that
recognize donor antigens. It is believed that the effect is mediated by
elimination of
preexisting recipient cells in the thymus.
"Hyperacute rejection", as used herein refers a recipient anti-donor response
which is mediated at least in part by preformed antibodies.
"Moiety" as used herein, refers to all or part of a chemical entity, e.g., a
all or
part of a cabohydrate.
"Tolerance", as used herein, refers to the inhibition of a graft recipient's
immune
response, particularly the hyperacute rejection response, which would
otherwise occur,
e.g., in response to the introduction of an antigen. e.g.. galactosyi a( 1, 3)
galactose
moiety, into the recipient. The term "tolerance" refers not only to complete
immunologic tolerance to an antigen, but to partial immunologic tolerance,
i.c.. a degree
1 S of tolerance to an antigen which is greater than what would be seen if a
method of the
invention were not employed. Tolerance can involve humoral, cellular, or both
humoral
and cellular responses. Tolerance is specific for the antigen, e.g.,
galactosyl a( 1, 3 )
galactose moiety, or epitopes which are located wholly or in pan on that
moiety, and
does not refer to a general state of immunosuppression. Although not wishing
to be
bound by theory, the inventors believe tolerance may be achieved by deletion
of immune
which would otherwise give rise to antigen-reactive, e.g., agalactosyl
a(1,3)galactose-
reactive antibodies.
Removal of xenogeneic natural antibodies using organ perfusion, or more
recently using synthetic galactosyl a(1, 3) galactose columns, has been shown
to delay
the onset of hyperacute rejection. However, due to the continued presence of
natural
antibody-producing B cells, the level of natural antibodies increases after
the first week
of transplantation and can contribute to delayed graft rejection. Methods of
the
invention can be used to manipulate the natural antibody response, e.g., with
gene
therapy, to induce tolerance at the B cell level, thereby promoting acceptance
of graft
tissue.
Miniature swine are an attractive potential donor for clinical
xenotransplantation
because of their physiological similarity to humans and their breeding
characteristics
(Sachs, D. et al. (1994) Path. Biol. 42:217). However, a major obstacle to
clinical
xenotransplantation in discordant species combinations such as swine to
primate is
hyperacute graft rejection mediated by preformed natural antibodies present in
the
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recipient (Galili, U. (1993) Immunol. Today 14:480; Platt, J.L. and Bach, F.H.
(1991)
Transplantation 52:937; Platt, J.L. et al. (I990) Immunol. Today 11:450).
The galactosyl (a1,3) galactose epitope is the major target of human natural
antibodies (reviewed in Galili, U. (1993) Immunol. Today 14:480; Platt, J.L.
and Bach,
S F.H. ( 1991 ) Transplantation 52:937; Platt, J.L. et al. ( 1990) Immunol.
Today 11:450;
Sandrin, M. S. and McKenzie, LF. (1994) Immunol. Rev. 141:169). This
carbohydrate
epitope is synthesized by the addition of a terminal galactosyl residue to a
preexisting
galactose residue linked to N-acetyl-glucosaminyl residue. The reaction is
catalyzed by
the glucosyltransferase UDP galactose:~i-D-galactosyl-1,4-N-acetyl-D-
glucosaminide a
(1,3)galactosyltransferase (al.3GT). In species expressing al,3GT. natural
antibodies
reactive against the galactosyl (a I .3) galactose moiety are absent. The lack
of a 1.3GT
in humans. apes, and Old World primates results in a failure to express the
gaiactosyl a
( 1.3) galactose epitope, making the presence of natural antibodies reactive
to this
epitope permissible. It has been shown in mice, a species that normally
expresses
galactosyl a(1,3) galactose epitope, that disruption of murine al,3GT gene by
embryonic stem cell technology leads to the development of natural antibodies
reactive
against the galactosyl a( 1,3) galactose epitope (Thall, A. et al. ( 1995) J.
Biol. Chenz
270:21437; Thall, A. et al. ( I 996) Transplant. Proc. 28:561 ). Prevention of
the
interaction of natural antibodies with the galactosyl a(1,3) galactose epitope
has been a
major goal in the field of xenotransplantation.
Several different approaches aimed at eliminating the problem of natural
antibody-mediated rejection in xenotransplantation have been attempted.
Manipulation
of the galactosyl a(1,3) galactose epitope on donor organs by various methods
as well as
altering the expression of al,3GT has been attempted (Sandrin, M.S. et al.
(1995) Nat.
Med 1:1261; Rosengard, A.M. et al. (1995) Transplant. Proc. 27:326; Langford,
G.A. et
al. (1994) Transplant. Proc. 26:1400; LaVecchio, J.A. et al. (1995)
Transplantation
60:841 ). A short coming of these approaches has been the failure to
completely abolish
the expression of the epitope. Removal of serum natural antibodies from the
host by
adsorption has been successful in preventing hyperacute rejection but does not
result in
the permanent removal of galactosyl a(I, 3) galactose reactive natural
antibodies, and
therefore is not a long-term solution. Modification of the host humoral system
by
inducing tolerance to the galactosyl a(I, 3) galactose epitope provides a
viable long-
term solution to the problem of galactosyl a(1, 3) galactose reactive natural
antibodies.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
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DETAILED DESCRIPTION
Brief Description of the Drawings:
Figure 1 (Panels A, B, C, D) is a set of graphs which show al,3GalBSA-
inhibition of human natural antibody binding to pig cells. Human serum (20 ul)
was
preincubated with aGaIBSA (Panels A,C) or bovine thyroglobulin (Panels B,D) at
concentrations of 1,000 p.g/ml (b) or 0.1 pg/ml (c) prior to staining of
porcine peripheral
blood mononuclear cells (pPBMCs). The negative control (a) consisted of pPBMC
plus
an equivalent volume of galactosyl a{1, 3) galactose reactive natural antibody-
depleted
human serum (XNA-) while the positive control was 20 pl human serum without
competitor (d). Following incubation with the human serum, the cells were
stained with
either anti-human IgG (Panels A.B) or anti-human IgM (Panels C,D).
Figure '_' (Panels A, B) is a set of graphs which show galactosyl a( 1, 3)
galactose
reactivity of human serum. Serial dilutions ( 1:?. 1:4, 1:8, 1:16, 1:32, 1:64,
1:1''8) of
serum samples from twelve unrelated donors were analyzed for binding, of IgG
(A) or
(IgM) (B) to aGaIBSA. The serum samples are designated by donor number.
Figure 3 (Panels A, B) is a set of graphs which show low expression of
galactosyl a(1, 3) galactose reactive natural antibodies in individuals with
the blood
group B antigen Serially diluted human serum from B and non-B expressing
donors
were analyzed by aGal/BSA ELISA. IgM (Panel A) and IgG (Panel B) binding is
expressed an mean optical density (O.D.) versus serum dilution. Solid bars=A,O
serum;
filled bars=B,AB serum.
Figure 4 (Panels A, B) is a set of graphs which show that human natural
antibodies from unrelated donors express a crossreactive idiotype. binding of
IgG
(Panel A) and IgM (Panel B) to aGal/BSA was evaluated by ELISA in the absence
(filled bars) and in the presence (open bars) of 10784 anti-idiotype reagent.
Percent
inhibition was calculated for each donor.
Figure 5 is a diagram of the LGTA7 and MZGT retroviral vectors.
Figure 6 is a graph of the analysis of anti a(1-3)Gal reactive IgM antibodies
in
mice reconstituted with LGTA7 or Neo transduced bone marrow at 12 weeks post
bone
marrow transplantation by ELISA. All assays were performed using a(1-3)Gal
conjugated to bovine serum albumin to coat ELISA plate wells. In all assays,
background binding observed using serum from normal unreconstituted mice was
subtracted. Similar results were obtained by subtracting the background
binding
observed on Iactosamine coated plates. The background binding observed with
serum
from the LGTA7 transduced group is not a(1-3)Gal specific as shown in Figure
7.
Similar results were observed at 18 weeks.
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Figure 7 is a graph of an analysis of serum antibodies capable of lysing a(1-
3)Gal positive porcine PK-15 in the presence of rabbit complement. P value
between
groups of mice reconstituted with Neo of LGTA7 transduced bone marrow is
shown.
Sera was analyzed 9 weeks post bone marrow transplantation.
The Galactosyl(a 1 3lGalactose Moiety
Galactosyl a( 1, 3) galactose reactive natural antibodies are important in the
process of hyperacute rejection. The determinants recognized by human anti-pig
natural antibodies appear to be expressed in all tissues. including
lymphocytes . The
terminal galactosyl a( 1. 3) galactose carbohydrate structure, responsible for
most of the
human anti-pig response, is synthesized by all mammals with the exception of
humans
and Old World primates. More than 80% of the xenoreactive natural antibodies
in
. human serum are specifically reactive with galactosyl a(1, 3) galactose.
suggesting that
the majority of this population of human natural antibodies is highly
restricted in terms
of antigen specificity. The removal of galactosyl a( 1. 31 galactose reactive
natural
antibodies from the sera of recipient monkeys by column perfusion eliminates
hyperacute rejection.
The gene encoding the enzyme responsible for the galactosyl a( 1, 3 )
galactose
structure. a(1,3)galactosyltransferase (al,3GT) is non-functional in humans
and Old
World monkeys . The inactivation of al,3GT is estimated to have occurred some
28
million years ago. Neither human nor Old World monkey derived cells are
reactive with
natural antibodies. Conversely, the expression of murine or porcine a 1,3GT in
COS
cells (Old World Monkey) results in the production of the galactosyl a( 1, 3 )
galactose
epitope which is recognized by anti-galactosyl a(1,3) galactose natural
antibodies.
Thus, susceptibility to hyperacute rejection is determined by expression of a
functional
al,3GT rather than simply by phylogenetic distance between animals. This was
demonstrated by transplantation studies between New World and Old World
monkeys.
When the heart of a New World monkey was transplanted into an Old World
monkey,
the graft ceased functioning within one hour. Consistent with natural antibody
mediated hyperacute rejection, immtmopathologic studies revealed the presence
of
immunoglobulin deposits in the graft.
The selective advantage conferred by the loss of a(1,3)galactosyltransferase
and
subsequent anti-galactosyl a(1, 3) galactose antibody production in human and
Old
World primates is not known. The loss of the enzyme activity and the
suppression of
this epitope may be related to the production of anti-galactosyl a(i, 3)
galactose
antibody. There may be a role for these antibodies in protection against the
transmission
of C-type retroviruses from discordant species. According to published
reports,
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galactosyl a(1, 3) galactose reactive natural antibodies comprise
approximately 1% of
the circulating Ig in healthy individuals and thus represent a significant
barrier to
xenotransplantation.
Formation of aalactosyl(a 1,3l~alactose epitopes on Recipient Cells
Genetically eneineerine cells to present aalactosvl al 1. 3) aalactose
moieties
Nucleic acid encoding a protein which promotes the formation of the galactosyl
a( 1, 3 ) galactose epitope can be introduced into the recipient cells by any
method which
allows expression of the nucleic acid at a level and for a period sufficient
to confer
tolerance. These methods include, by way of example, transfection,
electroporation,
particle gun bombardment, and transduction by viral vectors, e.g., by
retroviruses.
Some classical methods for introducing genes in mammalian cells have a limited
efficiency which limits their usefulness in many systems (Hwang, L. H. et al.
( 1984) J.
t~~rol. 50:417). Recombinant retroviruses have therefore been developed as
vehicles for
gene transfer (Eglitis, M. A. et al. ( 1988) Adr. Exp. Med. Biol. 241:19;
Anderson. W.F.
( 1992) Hunr. Gene Ther. 3:1: al-Lebban. Z.S. et al. ( 1990) Exp. Hema~ol.
18:180). The
most straightforward retroviral vector construct is one in which the
structural genes of
the virus are replaced by a single gene which is then transcribed under the
control of
regulatory elements contained in the viral long terminal repeat (LTR) (Blair,
D.G. et al.
( 1980) Proc. Natl. Acad. Sci. USA 77:3504). A variety of single-gene-vector
backbones
have been used, including the Moloney murine leukemia virus (MoMuLV). Derived
from this type of backbone are retroviral vectors into which multiple genes,
e.g., a
selectable marker and a gene of interest both under the control of an internal
promoter,
can be inserted (McLachlin, J.R. et al. (1990) Prog. Nucleic Acid Res. Mol.
Biol. 38:91 ).
Use of efficient packaging cell lines has increased both the efficiency and
spectrum of infectivity of the recombinant virions produced (Miller, A.D. (
1989)
Biotechnigues 7:980). Following transduction with these retroviruses, the most
efficient
expression was observed when "strong" promoters were used to control
transcription of
the introduced genes separately from the viral transcription initiated within
the LTR
(Chang, J.M. et al. ( 1989) Int. J. Cell Cloning 7:264). The major limitation
of this
strategy has been that the second transcriptional unit containing the
transduced gene was
placed within the retroviral transcriptional unit, causing transcriptional
interferences
(Emerman, M. et al. ( 1984) Cell 39:449; Kadesch, T. et al. ( 1986) Mol. Cell
Biol.
6:2593; Cullen, B.R. et al. ( 1984) Nature 307:241 ). Results from different
laboratories
suggest that the outcome of placing promoter elements internal to the LTR is
somewhat
unpredictable; in some cases leading to efficient transcription (carver, R. I.
et al. (1987)
Proc. Natl. Acad. Sci. USA 84:1050) and in other cases resulting in weak or
absent
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expression (Dzierzak, E.A. et al. (1988) Adv. Exp. Med. Biol. 241:41;
Williams, D.A. et
al. ( 1986) Proc. Natl. Acad Sci USA 83:2566). A new type of retroviral
vector, called
the double-copy (DC) vector, has been developed to overcome this problem by
physically separating the viral and non-viral transcription units
(Hantzopoulos, P.A. et
al. (1989) Proc. Natl. Acad Sci. USA 86:3519). In addition, the DC vector
allows
multiple insertions and leads to efficient expression in human lymphocytes.
Such
retroviruses represent the best technology available at present for the
transfer of genes
that may prove to be clinically relevant.
In situ formation of ~alactosyl al l 3) ealactose epitopes
Galactosyl a(1,3) galactose moieties can be added, in situ, to the surface of
recipient cells. For example. recipient cells can be removed from the
recipient and
incubated with an enzyme which promotes the formation of galactosyl a(1,3)
galactose
moieties on the cell. See, e.g., LaTemple et al., 1996. Cancer Res. X6:3069-
3074, which
is hereby incorporated by references. which discloses the use of recombinant
alpha 1,3
galactosyltransferase to synthesize galactosyl a(1,3) galactoside epitopes on
human
cells, in vitro; Josiasse et al., 1990, Eur. J. Biochem. 191:75-83, which is
hereby
incorporated by reference, which describes the production of recombinant
enzyme: and
Hamadeh et al., 1996, Infect- Immun. 64:528-534, which is hereby incorporated
by
reference. which describes the use of bacterial enzymes to form alpha l,3ga1
structures
on human cells.
The invention is further illustrated by the following examples which in no way
should be construed as being further limiting. The contents of all cited
references
(including literature references, issued patents, published patent
applications. and co-
pending patent applications) cited throughout this application are hereby
expressly
incorporated by reference.
EXAMPLES
EXAMPLE 1: THE MAJORITY OF NATURAL ANTIBODIES PRESENT IN
HUMAN SERUM ARE REACTIVE WITH THE GALACTOSYL a(1,3)
GALACTOSE MOIETY
A competitive binding assay was used to demonstrate that the natural
antibodies
recognize the gaiactosyl a(1, 3) galactose moiety.
Human sera were obtained from healthy adult volunteers. Individual samples
were isolated and stored at 4°C for short periods or aliquoted and
stored at -70° C.
Porcine peripheral blood mononuclear cells (pPBMC) were isolated from
heparinized
whole blood obtained from an inbred miniature swine herd and resuspended in
flow
cytometric analysis staining buffer (Hank's Buffered Saline Solution (HBSS),
2.0% fetal
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calf serum, 0.04% sodium azide (Sigma Chemical Co., St. Louis, MO)) to a final
concentration of 1 x 107 pPBMC/ml.
Human serum ( I Sp l ) was pre-incubated with serially diluted aGaIBSA, bovine
thyroglobulin (Sigma Chemical Co., St. Louis, MO) or BSA (Fisher Scientific,
Pittsburgh, PA) at final concentrations of 1 mg/ml to 1 ng/ml for 90 minutes
at 4°C with
gentle rocking. Fifty ~1 of pPBMC (5x105 cells) was incubated with SOpI of
human
serum plus competitor for 90 minutes at 4°C. The positive control
consisted of staining
with human serum alone and the negative control was human serum depleted of
natural
antibodies (XNA-). The cells were processed for all subsequent steps in the
same way as
for direct flow cytometric analysis (DerSimonian, H.M. et al. (1993) J. Exp.
~t~led.
177:1623 ).
When human serum was pre-incubated with aGaIBSA, the binding of natural
antibodies to porcine PBMC, as determined by median fluorescence intensity
(M.F.I.),
was substantially reduced. At an aGaIBSA concentration of 1 mg/ml, a 94%
reduction
of IgM binding to pig cells was observed; with IgG there was a 84% decrease in
binding
(Figure 1 ). With lower concentrations of aGal/BSA, the level of
immunoglobulin
binding increased. Pre-incubation of human antibodies with another galactosyl
a( 1, 3)
galactose containing molecule, bovine thyroglobulin, also had an inhibitory
effect on the
binding of human IgG and human IgM to porcine PBMC. Bovine thyroglobulin at a
concentration of 1 mg/ml reduced the binding of antibodies to porcine cells by
66% for
IgM and 79% for IgG (Figure 1 ). The bovine thyroglobulin molecule contains an
estimated eleven naturally occurring galactosyl a(1, 3) galactose residues
(Thall. A. and
Galili, U. (1990) Biochemistry 29:3959), while the synthetic conjugate,
aGal/BSA, has
from fifteen to thirty-nine aGal moieties per BSA molecule. In addition,
aGal/BSA has
a smaller molecular size (70 kD) than bovine thyroglobulin (300 kD).
Consequently, a
GaIBSA competes more effectively with natural antibodies for binding to pig
cells than
bovine thyroglobulin. No inhibition was seen when human serum was pre-
incubated
with BSA under the same conditions. These observations have implicated
galactosyl a
(I, 3) galactose as the determinant recognized by the majority of xeno-
reactive natural
antibodies, consistent with the observations of other groups (Collins, B.H. et
al. (1995)
J. Immunol. 154:5500; Oriol, R. et al. (1993) Transplantation 56:1433;
Sandrin, M. S. et
al. (1993) Proc. Natl. Acad. Sci. USA 90:11391; Cooper, D.K. et al. (1993)
Transplant.
Immunol. 1:198; Parker, W. et al. (1994) J. Immunol. 153:3791 ).
EXAMPLE 2. DETERMINATION OF THE AMOUNTS OF IgG AND IgM IN
HUMAN SERA
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In order to facilitate the detection of galactosyl a(1, 3) galactose-reactive
antibodies in human serum samples, an ELISA system using aGal/BSA as the
antigen
was used. aGaIBSA (provided by BioTransplant, Inc.) was used to coat 96 well
polystyrene plates (Costar) at a concentration of 10~g/ml in PBS overnight at
4°C.
Plates coated with BSA were included to control for background reactivity to
BSA.
Wells were blocked for 1-2 hours at room temperature with I% BSA in TBS (100
mM
Tris-HCI, pH 7.5, 0.9% NaCI). All plates were washed three times with 0.1%
Tween-20
in TBS (TBS/Tween) using a Skatron plate washer (Skatron Instruments, Inc..
Sterling,
VA.). Serial dilutions of human serum from a single donor were used as the
standard
positive controls. XNA- (see Example 3) was used as the negative control.
Samples
were aliquoted in triplicate and allowed to incubate at room temperature for 1
hour.
After washing three times with TBS/Tween. the plates were incubated with
alkaline
phosphatase conjugated mouse monoclonal anti-human IgG or IgM antibody f Sigma
Chemical Co., St. Louis, MO) for I-2 hours at room temperature in the dark.
The plates
I S were washed and developed with Sigma 104 alkaline phosphatase substrate
tablets in
carbonate buffer (0.2038 MgCl2, 2.28 Na2C03. 2.438. NaHC03 in 1 L. water). The
amount of colored product was measured at 40~ nm using the SLT Lab Instruments
340
ATC ELISA reader. For competitive ELISA, bovine thyroglobulin and aGal/BSA
were
serially diluted ten-fold in TBS to final concentrations of I mg/ml to 1
ng/ml. Equal
volumes of human serum and competitor were combined and incubated for 1 hour
at
4°C. The competition reactions were then aiiquoted to antigen coated
plates and
incubated for 1 hour at room temperature. The plates were washed and incubated
with
alkaline phosphatase conjugated mouse monoclonal anti-human IgG or IgM
antibody
(Sigma Chemical Co., St. Louis, MO) for I hour at room temperature. The wells
were
washed and developed with Sigma 104 alkaline phosphatase substrate. All
samples
were analyzed in triplicate.
To ensure that the galactosyl a(I, 3) galactose epitope was stable under these
conditions, experiments were performed to determine if there was any
detectable
hydrolysis of the galactosyl a(I, 3) galactose carbohydrate moiety in the
presence of
human serum. Although all the assays were earned out at 24°C, it was
found that a
GaIBSA was stable in this ELISA system even when the assay was carried out at
37°C
for up to 24 hours.
The specificity of this ELISA was demonstrated through competition with
bovine thyroglobulin. The binding of serum natural antibodies to aGaIBSA were
completely inhibited by pre-incubation with bovine thyroglobulin. Again,
aGaIBSA
was a more effective competitor than bovine thyroglobulin since it completely
inhibited
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the binding of human IgG and IgM to bovine thyroglobulin at 1 ~g/ml, while a
higher
concentration (100-1.OOO~g/ml) of bovine thyroglobulin was necessary to block
binding
of aGal-reactive natural antibodies to aGal/BSA.
Having determined that this ELISA system could be used to assess the
galactosyl
a(1, 3) galactose-reactive natural antibody levels in human serum, samples
from more
than 95 unrelated donors were assayed in this manner. IgG and IgM
concentrations were
determined by competitive ELISA (All, R. et al. (1985) Mol. Immunol. 22:1415)
using
commercial IgG and IgM (Sigma Chemical Co., St. Louis, MO) as standards. In
the
ELISA results for 12 representative donors, and all others which have been
tested. the
relative IgG and IgM aGal/BSA reactivity varied substantially between donors;
for
these 12 samples there was approximately an eight-fold range in galactosyl a(
1, 3)
galactose reactivity. (Figure 2)
EXAMPLE 3 CHARACTERIZATION OF AFFINITY' PURIFIED NATURAL
ANTIBODIES
Human galactosyl a(I, 3) galactose-reactive XNAs were affinity purified from
human serum using an galactosyl a(1, 3) galactosc column. Beads coupled with
galactosyl a( 1, 3 ) galactose were used to affinity purify galactosyl a( 1, 3
) galactose-
reactive natural antibodies. Columns containing these beads (10 ml) were
washed
extensively with PBS and then loaded with 10 ml human plasma. Samples were
loaded
and allowed to run at a flow rate of 30 ml per hour, followed by washing with
8 column
volumes of PBS. Bound antibodies were eluted with 100 mM sodium citrate, pH 3
and
immediately neutralized with 1 M Tris-HC1, pH 8Ø One ml fractions were
collected
and assayed for total IgG and IgM concentrations as well as for galactosyl
a(1, 3)
galactose specificity. Prior to assaying either the aGaI/BSA or pig cell
reactivity of the
column fractions, any dilution of the flowthrough or concentration of the
eluate was
compensated for relative to the original plasma. That galactosyl a(1, 3)
galactose
column removed all of the detectable galactosyl a(1, 3) galactose-reactive IgG
and IgM
was seen by analysis of the flowthrough and wash fractions. In contrast, the
renatured
eluate fractions had substantial levels of aGal/BSA reactive IgG and IgM. The
flowthrough and wash fractions contained high levels of IgG and IgM which
gradually
decreased throughout the wash fractions and increased only slightly in the
eluate
fractions indicating that all of the aGal/BSA reactivity was present in the
small fraction
of IgG and IgM retained by the column.
To assess whether the majority of the IgG and IgM in the eluate fraction
(XNA+)
were the result of non-specific binding to the column or were indeed
galactosyl a(1, 3)
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galactose-reactive, natural antibodies (XNA) adsorption experiments were
carried out
using porcine erythrocytes. Porcine red blood cells (pRBCs) were chosen for
this
adsorption study as they have cell surface galactosyl a(1, 3) galactose
moieties but lack
Fc receptors which can non-specifically bind IgG. pRBCs were isolated from
S heparinized pig blood following processing with LSM (Organon Teknika,
Durham, NC).
The erythrocytes were taken from the base of the red cell pellet to avoid
contamination
with granulocytes or PBMC. The pRBCs were washed with HBSS and counted to
determine cell number and purity. Natural antibodies (XNA) were isolated from
pooled
human serum by affinity chromatography as previously described (Galili, U. et
al.
( 1987) Proc. Natl. Acad. Sci. USA 84:1369). The XNA' fraction was diluted so
that the
IgM concentration was equal to that of the XNA' sample. 2x 107, 2x 108, 2x I
09, or
2x1010 pRBCs were incubated with 2 ml samples of XNA+ or XNA- for 20 minutes
at
4°C with gentie rocking. The pRBCs were spun down at 1,500 rpm for 10
minutes at
4°C. The total IgG and IgM concentrations as well as the aGaIBSA
reactivity of the
I S supernatants were determined by ELISA for each of the adsorbed samples.
The a
GaIBSA reactivity and the IgG and IgM concentrations were determined pre- and
post-
adsorption. With 2x 1 O 10 pRBC, the adsorbed XNA' underwent a 92% reduction
in a
GaIBSA IgM reactivity and a 70% depletion of total IgM; for IgG, these values
were
62% and 64% respectively. XNA- was adsorbed with pRBCs in parallel; no
reduction in
the immunoglobulin concentration was observed. gaiactosyl a(1, 3) galactose-
reactive
natural antibody affinity purified under these conditions may contain as much
as 30%
non-specific IgM and much lower levels of contaminating IgG.
In order to determine if galactosyl a(1, 3) galactose-reactive natural
antibodies comprise
a constant percentage of total immunoglobulin, the immunoglobulin
concentrations of
the affinity purified galactosyl a(1, 3) galactose-reactive natural antibodies
from ten
serum samples were quantified by competitive ELISA. The concentrations of IgG
natural antibodies ranged from 39 to 153 p.g/ml with a mean of 65.3 ~tg/ml,
while for
IgM XNAs the concentrations ranged from 24 to 63 Itg/ml with a mean of 40.1
~tg/ml.
Table 1 shows the percentage of IgG and IgM natural antibodies in different
individuals.
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TABLE 1
I~G
Plasma SampleTotal IgG (~glml)XNA+ (IgG ~g/ml)Percent XNA+
1 4100 56 1.4%
2 6333 153 2.4%
3225 39 1.2%
6 4808 58 1.2%
7 4117 72 1.7%
8 4060 75 1.8%
9 4200 46 1.1 o
4792 53 1.1 ~o
11 4708 47 1Ø'0
14 3650 59 1.6~0
1 s 3250 51 1.6%
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IQM
Plasma SampleTotal IgM (ftg/ml)XNA+ (IgM pg/ml)Percent XNA+
1 305 17 ~ 5.6%
2 1103 49 4.4%
299 24 8.0%
6 1033 44 4.3%
7 742 39 5.3%
8 1035 63 6.1
9 563 26 4.6%
647 ~ 45 7.0%
1 1 1070 42 3.9%
14 810 37 4.6%
I 5 672 32 4.8%
To determine if the amount of galactosyl a( I. 3) galactose-reactive natural
5 antibodies was related to total IgG or IgM, the immunoglobulin
concentrations of the
purified natural antibodies were compared with the levels of IgG and IgM in
the
original serum. The calculated percentage of galactosyl a(l, 3) galactose-
reactive IgG
ranged from 1.0 to 2.4% with a mean of 1.5% while for IgM it was 3.9 to 8.0%
with a
mean of 5.3%. Based upon this sample, the amount of natural antibodies in
human
10 serum does not appear to represent a constant percentage of total
immunoglobulin. With
each of the serum samples assessed, more IgG XNA+ was isolated than IgM XNAy;
however, the overall percentage of XNA+ immunoglobulin relative to total
immunoglobulin was substantially higher for IgM than for IgG.
Previously it has been estimated that galactosyl a(1, 3) galactose-reactive
IgG
accounts for approximately 1 % of total IgG (Galili, U. et al. ( 1984) J. Exp.
Med.
160:1519: Galili, U. et al. {1985) J. Exp. Med. 162:573); more recently, it
has been
reported that IgM natural antibody comprises 1-4% oftotal IgM (Parker, W. et
al. (1994)
J. Immunol. 153:3791). While the results presented are in agreement with these
estimates, a substantially higher percentage of natural antibodies in some
samples is
reported here. The adsorption studies with porcine red blood cells (pRBC)
suggest that
there may be contaminating non-specific IgM in the affinity purified natural
antibodies
and consequently these values could be an overestimate by 30%. However, the
non-
galactosyl a(1, 3) galactose reactive IgM remaining after adsorption could
also be due to
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a loss of function. Consistent with this, it has been observed that the
natural antibodies
undergo a substantial reduction in ability to bind galactosyl a(l, 3)
galactose following
acid elusion from protein A. As natural antibodies were eluted from the
galactosyl a(l,
3) galactose column under acid conditions, the loss of galactosyl a(1, 3)
galactose
reactivity by some of the immunoglobulin in the eluate fraction would not be
unexpected.
EXAMPLE 4: LOW EXPRESSION OF GALACTOSYL a(I, 3) GALACTOSE XMAS
IN INDIVIDUALS WITH THE BLOOD GROUP B ANTIGEN
The inventors have made the surprising discovery that a moiety on human cells
which is similar to the galactosyl a(I, 3) galactose moiety results in a
significant
reduction of anti-galactosyl a(l, 3) galactose natural antibody in human
serum.
Poly-N-acetyllactosamines in human ervthrocynes carry the ABO-blood group
antigens.
In humans the terminal galactose is first substituted with a( 1,2) fucose,
forming the H
antigen. In B blood group individuals the H antigen is further modified, by an
a
(1,3)galactosyltransferase, by the addition of a(1,3)galactose. This a
( 1,3)galactosyltransferase is different from the a( I ,3
)galactosyltransferase present in
New World primates and swine. It requires the presence of the fucosyl moiety
on the H
antigen. The swine a(1,3)galactosyltransferase does not require a fucose
attached to N-
acetyllactosamine. To detect possible antibody cross reactivity between the
blood group
B antigen and the galactosyl a(1, 3) galactose determinant, the relative level
of
galactosyl a( 1, 3) galactose reactive natural antibodies in human serum from
an equal
number (n=12) of serum samples from the A. B ,AB and O blood groups were
assayed
by direct ELISA. To ensure that the antibody binding detected was due entirely
to the
galactosyl a(1, 3) galactose determinant, each serum sample was also tested
for BSA
reactivity and serum samples exhibiting BSA reactivity (approximately 10% of
the
samples) were eliminated.
The overall gaIactosyl a(1, 3) galactose reactivity of the B antigen
expressing
blood groups (AB,B) when compared with the non-B antigen expressing blood
groups
(A,O) indicated a reduction in the level of natural antibody in the presence
of the B
antigen. See Figure 3. This effect was more striking with IgG than with IgM.
In order
to quantify the observed reduction more precisely, a statistical analysis of
the O.D.
values for each of the serum dilutions was undertaken. The data was analyzed
by
comparing the non-B-expressing donors (A,O) against the B expressing donors
(AB,B)
using the Student's T Test. The results indicated that there was significant
difference
between the combined B and AB groups, when compared with the A and O blood
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groups for IgG (p<0.0002) and IgM (p<0.06) at the three (1/4, 1/8, 1/16) serum
dilutions
assayed. These results support the hypothesis that the human natural antibody
population is regulated by the presence of the B antigen on human blood cells.
EXAMPLE 5: TRANSIENT EFFECT OF GALACTOSYL a(I, 3) GALACTOSE
NATURAL ANTIBODY DEPLETION
In vitro experiments were performed to measure the recipient plasma natural
antibody levels before and after galactosyl a(1, 3) galactose column
perfusion.
Following column perfusion, a porcine kidney was transplanted into a monkey
without
subsequent hyperacute rejection. In all of the cases (n=8), the level of
natural
antibodies in the monkey plasma decreased to background levels after column
perfusion
as measured by flow cy~tometric analysis (i.e.. measuring porcine reactivity)
and ELISA
(measuring galactosyl a(l. 3) galactose reactivity). However, the natural
antibodies
levels rebounded several days later. As demonstrated for one of the ivngest
xenogralt
I S sun~ivors. the level of natural antibodies in the plasma remained low for
only a short
time. By day 15, the galactosyl a(l, 3) galactose reactive natural antibody
returned to
pre-adsorption levels of IgM while the IgG level rose to ten-fold its original
value.
These results not only emphasize the importance of depleting the galactosyl a(
1, 3)
galactose reactive natural antibody_but also demonstrates the importance of
regulating
the natural antibody producing B cell population in xenotransplantation.
EXAMPLE 6. PRODUCTION OF A RETROVIRAL VECTOR FOR DELIVERY OF
a(1,3)GALACTOSYLTRANSFERASE TO CELLS
A 1145 by EcoRI-CacBI restriction cDNA fragment containing the coding region
of porcine a(1,3)galactosyltransferase (al,3GT) from pSa13GT1 (Strahan, K.M.
et al.
( 1995) Immunogenetics 4I :1 O l ) was cloned into pBluescript II KS (-)
(Stratagene) and
then the marine phosphoglycerate kinase (PGK) transcriptional promoter was
inserted
upstream of the a(1,3)galactosyltransferase coding region to construct
PGKaI,3GT.
PGKaI,3GT is then introduced into the 3' LTR of the retrovirus vector N2A
(Armentano, D. et al. (198?) J. Virol. 61:1647; Bordignon, C. et al. (1989)
Proc. Natl.
Acad. Sci. USA 86:6784; Hayashi, H. et al. (1995) Transplant. Proc. 27:179) to
construct a provirus carrying a 1,3GT driven by the PGK transcriptional
promoter (PGK
al,3GTRV). PGKaI,3GTRV is then introduced into the amphotropic retroviral
packaging cell line PA317 (Miller, A.D. and Buttimore, C. (1986) Mol. Cell.
Biol.
6:2895) by transfection to create a virus producer cell line. Following
selection of the
transfected clones with 6418, producer clones are picked and expanded to test
viral titer.
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Cell lines producing high titer helper-free virus are used to prepare
retrovirus stocks (a
1,3GTRV).
To test whether al,3GTRV can transfer functional al,3GT capable of generating
galactosyl a(I, 3) galactose epitopes, COS cells (al,3Gal negative) are
infected with the
S recombinant virus (M.O.I >2) and it is determined whether the al,3Gal
epitope is
expressed on the cell surface proteins by staining with the lectin from
Bandeiraea
simplicifolia (IB4, BS-I isolectin B4) which specifically recognizes the
galactosyl a(1, 3)
galactose epitope for the al,3Gal as well as purified galactosyi a(I, 3)
galactose
reactive human natural antibodies and analyzed by flow cytometry using
standard
methodologies. The levels of galactosyl a(1, 3) galactose epitopes encoded for
by the
transduced enzyme are compared to the level normally present on cells from
swine.
To test the efficacy of transduction with al,3GTRV, RAG-I (R-) deficient mice
which lack mature B and T cells (Mombaerts, P. et al. (1992) Cell 68:869) are
reconstituted with bone marrow from a 1.3GT deficient mice (A-) mice
transduced by an
1 S a 1,3GTRV or a control retrovirus ENJ36 (Eraser, C.C. et al. (1995) J.
Immunol.
154:1587) carrying only the NEO resistance gene. M.O.I are greater than 2 for
all
infections and performed as described previously (Sykcs, M. et al. (1993)
Transplantation 55:197). On day 14 post reconstitution, the mice are
sacrificed and
colony forming units from the spleen (CFU-S) are harvested. Cell suspensions
are
prepared and stained with IB4 lectin or purified galactosyl a(1, 3) galactose
binding
human natural antibody and analyzed by flow cytometry. DNA is prepared from
1/2 of
each colony and analyzed by PCR to determine whether the colony was derived
from A-
bone marrow.
EXAMPLE 7: A MUR.INE MODEL FOR THE INDUCTION OF TOLERANCE TO
CELLS EXPRESSING GALACTOSYL a(I, 3) GALACTOSE MOIETY
The marine system described herein can be used to evaluate a particular
component, e.g., a sequence which encodes an galactosyl a(1, 3) galactose
moiety
forming enzyme, for the ability to promote tolerance to the galactosyl a( 1,
3) galactose
moiety. To test the ability of retrovirally transfected cells to induce
tolerance to the
galactosyl a(1, 3) galactose epitope, a marine host which lacks both (a)
galactosyl a(1,
3) galactose epitopes that cause deletion of developing B cells producing
galactosyl a(I,
3) galactose reactive antibodies, and (b) galactosyl a(l, 3) galactose
reactive natural
antibody capable of rejecting the modified bone marrow cells was developed. a
1,3GT
deficient mice (A-) are crossed with RAG-I (R-) deficient mice which have been
previously shown to lack mature B and T cells (Mombaerts,P. et al. (1992) Cell
68:869)
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to generate al,3GT-/-,RAG-1 -/- (A-R-) mice. In order to construct A-R- mice,
homozygous A- and R- deficient mice are crossed. The resulting F 1 generation
is then
intercrossed to generate A-R- mice at an expected frequency of 1 in 16.
Offspring are
genotyped based on Southern hybridization. The A-R- are then be intercrossed
to
establish a colony. Irradiated A-R- mice are reconstituted with bone marrow
cells from
A- mice transduced with retrovirus carrying a 1,3GT, or a control retrovirus
carrying
only the neomycin resistance gene. The ability of reconstituted mice to
produce
galactosyl a(1, 3) galactose reactive antibodies is used to measure whether
gene therapy
leads to B cell tolerance in this model.
EXAMPLE 8: GENERATION OF PORCINE ANTI-HUMAN ANTI-IDIOTYPIC
ANTIBODIES
An galactosyl a( 1, 3 ) galactose carbohydrate column was used to isolate XNA
(250 fig) from the serum of a single donor which was emulsified with Complete
Freund's Adjuvant (CFA) and used to immunize a miniature swine. This animal
was
repeatedly boosted with purified natural antibodies until a high level of
reactivity
against human immunoglobulin was produced. After the fifth boost. the animal
was
exsanguinated and serum was collected and stored at -20°C. To remove
non-idiotype
specific anti-human antibodies, the sera was passed over a column to which
natural
antibody depleted human Ig had been conjugated. The flowthrough fractions were
then
screened for differential binding to XNA+ or XNA- coated plated by ELISA. The
partially purified flowthrough reacted preferentially with XNA+ suggesting
that a pig
anti-idiotypic reagent was generated which was able to distinguish XNA+ and
the XNA-
fractions of the original donor.
EXAMPLE 9: HUMAN NATURAL ANTIBODY FROM UNRELATED DONORS
EXPRESS A CROSSREACTIVE IDIOTYPE
The ability of the anti-idiotype antisera produced in the example above to
inhibit
serum natural antibody binding to galactosyl a(1, 3) galactose was assessed
using a
competitive ELISA in which sera from four unrelated donors were tested. The
binding
of natural antibody to al,3Gal BSA coated plates was inhibited 20-60% for IgM
and
13-78% for IgG. See Figure 4. These results show that a crossreactive natural
antibody
idiotype is expressed in unrelated individuals. Such cross reactivity is
indicative of
structural relatedness possibly as a result of limited V gene usage in the
human natural
antibody population.
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EXAMPLE 10: PRODUCTION OF MOUSE ANTI-HUMAN ANTI-IDIOTYPIC
ANTIBODIES
The cross reactivity of the porcine anti-idiotype reagent with natural
antibody
from four unrelated individuals indicates the practicality of producing
monoclonal anti-
s idiotype reagents. Anti-idiotype reagents are useful for therapeutic
applications and for
screening natural antibody producing EBV transformed B cells for the
predominating
idiotype.
Mouse anti-human anti-idiotypic monoclonal antibody producing hybridomas
can be generated against human natural antibody that is affinity purified from
plasma or
from EBV transformed clonal B-cells. To purifj' a human antibody fraction from
all
other serum components. an initial affinity purification step is performed
using an anti-
human IgG and IgM column. The purified Ig is then subjected to an galactosyl
a( 1, 3)
galactose column to enrich for galactosyl a( 1. 3) galactose reactive natural
antibodies
(XNA~). The anti-galactosyl a(I. 3) galactose antibody depleted fraction
(h'l'A'I is also
1 S collected. The affinity purified XNA' is then concentrated in a Centricon
column
(Amicon) and quantitated for total IgG and IgM concentrations by competitive
ELISA.
Purified XNA+ and XNA- immunoglobulin are subjected to SDS-PAGE to ensure
purity.
This procedure provides material for experiments including 2-Dimensional
Electrophoresis and the production of anti-idiotypic antibodies.
Mice are immunized and after testing for strong anti-human Ig reactivity, the
mice are sacrificed and spleen cells fused. Hybridoma supernatant is screened
directly
by ELISA on XNA+ and XNA- coated plates. Wells producing reactivity against
the
gaiactosyl a(1, 3) galactose -specific XNAY coated plates. but not to the XNA-
coated
plates, are selected for further analysis. These hybridoma cell lines are
expanded and
cryopreserved. Putative anti-idiotypic monoclonals are also tested for their
ability to
block natural antibody binding to galactosyl a(1, 3) galactose coated ELISA
plates.
EXAMPLE 11: ELIMINATION OF ANTI-a(1-3)GALACTOSE a(1-3)GAL)
REACTIVE ANTIBODIES BY GENE THERAPY
Retroviral gene therapy can be used in a tolerance inducing regimen to
eliminate
production of galactosyl a(1,3)galactose reactive antibodies. This method will
inhibit
graft rejection mediated by a(I-3)Gal reactive antibodies which is important
in
xenotransplantation across discordant barriers. al,3GT mice knockout mice
(GTO)
(Thall et al., 1995, J. Biol. Chem. 270:21437) capable of producing galactosyl
a
(1,3)galactose reactive antibodies (Thall et ai., 1996, Transplantation
Proceedings
28:561) were used as a model system for introducing porcine al,3GT, by
retrovirus
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mediated gene transfer, into bone marrow lymphohematopoietic progenitors on
production of galactosyl a(1,3)galactose reactive antibodies. The
reconstitution of
lethally irradiated GTO mice with porcine al,3GT transduced syngeneic bone
marrow
effectively prevents the development of galactosyl a( 1,3)galactose producing
B cells.
S Retrovirus vectors capable of transferrin~porcine a(1-31GT as a
constitutively
expressed eene into bone marrow derived cells
Two retroviral vectors carrying the gene encoding porcine aGT was constructed,
see Fig. ~. The first vector (LGTA7) is a N2A (Hantzapouious et al., 1989,
PNAS
86:3519) based retroviral vector in which aGT expression is driven by the
murine
phosphoglycerate kinase (PGK) promoter. The second vector (MZGT) is a Macrozen
(Johnson et al., 1989, EMBO J. 8:441 ) based vector in which expression of a
1.3GT is
driven by the myeloproliferative sarcoma virus promoter contained in the 5'LTR
of the
virus. Two different retroviral vector were designed because it is possible
that different
vectors may be more or less effective in allowing a 1.3GT expression. In order
to derive
virus producer cell lines, the above constructs were introduced separately
into AM 1
amphotropic packaging cell lines (Markovitz ei al., 1988. Y'irology 167:400)
and virus
producing lines established as described previously (Fraser et al., 1995, J.
Immunol.
154: i 587). Amphotoropic packaging cells were used in a preclinical primate
xenotransplantation mode.
To test whether the recombinant retroviruses were able to transfer functional
a
1,3GT expression, Vero cells (African Green monkey kidney epithelial cell
lines, a
1,3GT negative) were transduced with LGTA7 or MZGT virus produced in AM 12
cells.
Surface expression of galactosyl a(1,3)galactose epitopes was then analyzed on
selected
clones by staining with FITC labeled lectin from Bandeiraea simplicifolia
(BS=I
isolectin B4) specific for galactosyl a(1,3)gaIactose and analyzed by flow
cytometry. In
all experiments, Vero cells infected with a control retrovirus containing only
the
neomycin resistance gene (NEO) were analyzed in parallel. Vero cells infected
with
LGTA7 expressed galactosyl a(1,3)galactose epitopes on the cell surface at
levels
detectable by flow cytometry. Surface expression of galactosyl a(1,3)galactose
epitopes
was stable and could be detected on the surface of clones after several months
in culture.
No surface expression of galactosyl a(1,3)galactose epitopes was detected on
control
NEO transduced cells. In order to confirm that the staining with IB4 lectin
was indeed a
consequence of galactosyi a(1,3)galactose epitope expression encoded for by
the
introduced transgene, Vero cells transduced with LGTA7 were treated with a-
galactosidase. Treatment of LGTA7 transduced Vero cell clones with a-
galactosidase
specifically reduced galactosyl a(1,3)galactose expression detectable by
staining IB4-
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FITC. Similar results were obtained using MZGT amphotropic retrovirus. These
data
indicate that LGTA7 and MZGT retroviruses are able to transfer expression of
porcine
al,3GT, which in turn catalyzes the addition of galactose epitopes in a a(1-3)
linkage
on the surface of primate cells.
Bone marrow cells from GTO mice treated in vivo for 7 days prior to harvest
with 1 SOmg/kg 5-fluorouracil were transduced by co-cultivation with the LGTA7
virus
producer cells or control lines producing virus containing only the neomycin
resistance
gene as described (Fraser et al., 1995, J. Immunol. 154:1587). After 4 days of
in vitro
culture. transduced bone marrow cells were harvested and lethally irradiated (
10.25Gy)
GTO mice were reconstituted with 106 LGTA7 (group 1, n=4) or Neo transduced
(group
'_', n=3) bone marrow cells. Starting at 3 weeks post bone marrow
transplantation mice
in each group were bled and the presence of a( 1-3)Gal reactive senum
antibodies
IS analyzed by ELISA. As shown in Figure 6, while galactosyl a(1,3)galactose
reactive
serum antibodies were readily detectable in control mice reconstituted with
Neo
transduced bone marrow, mice reconstituted with LGTA7 transduced bone marrow
failed to develop a(1-3)gal reactive antibodies. Serum galactosyl
a(1,3)galactose
reactive antibodies were undetectable in mice reconstituted with LGTA 7
transduced
bone marrow analyzed for at least 18 weeks post bone man;ow transplantation.
To
confirm the results obtained by ELISA, serum from mice in each group was
analyzed for
the presence of antibodies capable of lysing a(1-3)Gal positive porcine PK-15
cells in
the presence of rabbit complement as described (Koren et al., 1994,
Transplantation
Proceedings 26: i 166; and Koren et al., 1994, Transplantation Proceedings
26:1336).
As show in Figure 7 while GTO mice immunized with porcine PBMC, and mice
reconstituted with Neo transduced bone marrow contained serum antibodies
capable of
mediating lysis of PK-15 cells, serum antibodies were not detectable in normal
mice
immunized with porcine PBMC or mice reconstituted with LGTA7 transduced bone
marrow capable of mediating lysis of PK-1 ~ cells. Together, these data
demonstrate that
reconstitution of lethally irradiated GTO mice with porcine al,3GT transduced
syngeneic bone marrow effectively prevents the development of galactosyl a(1-
3)galactose producing B cells.
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EXAMPLE 12: INDUCTION OF TOLERANCE TO GALACTOSYL a(1,3)
GALACTOSE MOIETIES
The following procedure was designed to induce tolerance to a galactosyl a(l,
3)
galactose moiety in a human or Old World primate. It can be used to prepare an
Old
World primate, a baboon (Papio anubis), for receipt of a kidney from a
miniature swine
donor.
The procedure is designed to reduce the anti- galactosyl a(l. 3) galactose
natural antibody (XNA) response of the recipient, by introducing autologous
stem cells
which present galactosyl a(l, 3) galactose moieties into the recipient.
Recipient bone
marrow is aspirated from the iliac crest of the recipient. This provides
autologous cells
for the production of a feeder layer which will be used to culture recipient
stem cells.
Stromal cell cultures are generated by separating low density bone marrow
cells over a
Ficoll gradient and plating 2x 1 O6 cells per well in a 24-well plate pre-
coated with 1 °~o
gelatin. The cultures are incubated, in 5% COa and 95% humidity, using M 199
medium
containing 10% fetal bovine serum. 10% horse serum and 10'° M
hydrocortisone at 37"C
for one week, and then, at 33°C for two additional weeks. Medium is
demi-depleted at
weekly intervals and supplemented with fresh medium for 3 weeks while a
confluent
stromal cell layer is fotzned.
After preparation of the feeder layer, a second bone marrow aspiration is
performed to provide autologous stem cells for transduction. Transduction of
CD34+
autologous bone marrow cells is performed in the presence of the preformed
stromal
cell culture. CD34+ cells are enriched from the low density Ficoll gradient
fractions of
recipient bone marrow harvested 3 days (day -3) prior to bone marrow
transplantation,
by immunoadsorption using a Ceprate column (CellPro Inc., Bothel, WA). The
bone
marrow cells are then plated at Sx104 cells/ml/well onto the autologous
stromal cell
layer described above. The CD34+ cells are cultured overnight in M 199, 10% FB
S, 10%
horse serum supplemented with 100 ng/ml rhSCF (R & D Systems, Minneapolis.
MN),
100 ng/ml rhIL-3 (Sandoz Pharmaceuticals Co., Basel, Switzerland) at
37°C. Cultures
are exposed to a(1,3)GT expressing retroviral supernatant (4x106 infectious
particles
/ml of amphotropic recombinant virus) for 18 hr. in the presence of 6 p.g/ml
of polybrene
and growth factors. The cells are reexposed to cytokines and virus for a
second time
following the above procedure. (A control transduction experiment is set up
under
identical conditions except that the cells do not receive retrovirus.) On day
0,
transduced adherent and non-adherent populations are harvested and infused
into the
same animal from which the marrow is harvested.
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A non-myeloablative conditioning regimen is used to prepare the recipient for
transplantation of the engineered autologous stem cells. The recipient
receives nonlethaI
total body irradiation of 300 Rads from a 6°Co source on day -3. The
animal is further
treated with thymic irradiation of 700 Rads on day -l, and anti-thymocyte
globulin
(ATG, Upjohn, Kalamazoo, MI), 50 mg/kg, i.v., on days -3, -2, and -1.
Prior to introduction of the galactosyl a(1, 3) galactose expressing recipient
cells
natural antibodies are removed from the recipient's circulation by passing the
recipient's
blood through a galactosyl a(1, 3) galactose affinity column. The galactosyl
a(1, 3)
galactose affinity column is prepared according to the manufacturer's
directions (Alberta
Research Council. Edmonton, CA). The recipient is anesthetized with halothane
and
maintained by general endotracheal intubation anesthesia with monitoring of
blood
pressure. blood oxygen saturation. blood gases and pl-1 throughout the case.
In addition
to an internal jugular vein cutdown. a brachial arten~ indwelling catheter is
placed to
allow for direct blood pressure measurements. A splenectomy may be performed
on the
recipient. The recipient's aorta and vena cava are then exposed and cannulated
using
silastic shunts. The aortic cannula is connected either to the column inlet.
and the circuit
is completed by connecting the recipient vena cava cannula to the column
outlet. Flow
rates are measured using a volume meter. Continuous monitoring by an
anesthesiologist
is required to maintain euvolemia and manage introperative coagulopathy,
anemia. and
hypothermia. The recipient's blood is perfused through the column for sixty
minutes.
The efficacy of the perfusion technique for the removal of natural antibodies
is assayed
by flow cvtometric analysis.
Administration of cyclosporine (Sandoz Pharmaceuticals Co.. Basel,
Switzerland) is given between days 0 and day 28, at a dose of 1 S mg/kg/day,
i.v., to
maintain a plasma level of greater than 300 ng/ml. Recombinant human GM-CSF
(Sandoz Pharmaceuticals Co., Basel, Switzerland) is given subcutaneously from
days 0
through 14 at a dose of 5 pg/kg/day. Ofloxacin is given throughout the
neutropenic
period as prophylaxis against infection, starting on day -3, at a dose of 50
mg/day i.v.
After depletion of anti-galactosyl a(1, 3) galactose natural antibodies from
the
recipient's blood, the engineered recipient cells are introduced into the
recipient. The
recipient is then monitored for the production of galactosyl a(1, 3) galactose
antibodies.
After establishing that the galactosyl a(1, 3) galactose antibodies have
decreased or been
eliminated the porcine bone marrow stem cells and kidney can be transplanted
into the
recipient as is described in Example 12 below.
Human natural antibodies can be detected with the following ELISA assay.
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Nunc Maxisorb plates are coated with 100 ~1/well of Sp,g/mL of aGal (1~3)
~iGal (1-~
4) ~iGlc-X-Y conjugated to BSA (Alberta Research Council, Canada) in carbonate
bi-
carbonate buffer (pH>9.5). These plates are then incubated at 4°C for
overnight.
Coated plates are washed 5-6 times with PBS-Tween-20 (0.5%) and blocked with
200 p.
S L/well of 1 % BSA (Sigma, MO) in PBS-Tween-20 (0.5%). For 1 hour at
37°C. The
plates are either used immediately or frozen at -20°C until used.
Before use, the plates
are washed 5-6 times with PBS-Tween-20 (0.5%) and loaded with 100~t1/well of
graded
doses (0.016%-2%) of baboon or human serum. The plates are then incubated for
1 hour
at 37°C and washed 5-6 times with PBS-Tween-20 (0.5%). Bound antibodies
are
detected using polyclonal donkey anti-human IgG (Accurate, NY) and rabbit anti-
human
igM (Dako, Denmark) conjugated to Horseradish peroxidase (HRP). The plates arc
incubated for 1 hour at 37°C. After the plates are washed 5-6 times
with PBS-Tween-20
(0.5%), color development is achieved by using o-phenylenediamine
dihydrochloride
(OPD. sigma, MO) as a substrate at 0.9 mg/mL in phosphate citrate buffer with
urea
hydrogen peroxidase (Sigma. MO). After 13 minutes of incubation at room
temperature
and in complete darkness. the plates are blocked with 50 uL of 2N H~SO,~ and
absorbance at 490 nm is measured by THERMOmax plate reader (Molecular Devices,
CA).
Mouse natural antibodies reactive with Gal a 1.3 Gal can be detected in an
assay
identical to the above-described assay except for the use of donkey anti-mouse
IgG and
donkey anti-mouse IgM (Acurate. NY) as the detecting antibodies.
EXAMPLE 13: INDUCTION OF TOLERANCE TO A GRAFT WHICH PRESENTS
GALACTOSYL a(1,3) GALACTOSE MOIETIES
The following procedure was designed to lengthen the time an implanted tissue
which displays a galactosyl a(1, 3) galactose moiety survives in a human or
Old World
primate prior to rejection. The tissue can be, e.g., hematopoietic stem cells,
or an organ,
e.g., a liver, a kidney, or a heart. The main strategies are: the elimination
of natural
antibodies which recognize the galactosyl a(1, 3) galactose moiety on the
graft by
implantation in the recipient of galactosyl a(1, 3) galactose presenting
recipient stem
cells (as described in Example 11 above); the reduction of recipient anti-
donor T and
NK cell activity; the transplantation of tolerance-inducing donor bone marrow;
and the
administration of a short course of a help reducing agent at about the time of
introduction of the graft.
Elimination of the anti-ealactosyl all 3) ealactose natural antibody response.
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Recipient natural antibodies which recognize the galactosyl a(1, 3) galactose
moiety are minimized as described in Example 11 above.
Preparation of the recipient for donor stem cells Recipient T and NK cell
activity is inactivated by the administration of anti-T and anti-NK cell
antibodies. Thus,
on the third, second and first day prior to introduction of donor stem cells,
a commercial
preparation (Upjohn, Kalamazoo, MI) of horse anti-human anti-thymocyte
globulin
(ATG) is injected into the recipient ATG eliminates mature T cells and NK
cells that
could promote would otherwise cause rejection of the bone marrow cells used to
induce
tolerance. The recipient is anesthetized, an IV catheter is inserted into the
recipient, and
6 ml of heparinized whole blood are removed before injection. The ATG
preparation is
then injected (50 mg/kg) intravenously. Six ml samples of heparinized whole
blood are
drawn for testing at time points of 30 min.. 24 hours and 48 hours. Blood
samples are
analyzed for the effect of antibody treatment on NK cell activity (testing on
K562
targets) and by flow cytometric analysis for lymphocyte subpopulations,
including CD4,
CDB, CD3, CDllb, and CD16. If mature T cells and NK cells are not sufficiently
inhibited. ATG can be re-administered at later times in the procedure, both
before and
after organ transplantation. Anti-human ATG obtained from any mammalian host
can
also be used, e.g., ATG produced in pigs, although thus far preparations of
pig ATG
have been of lower titer than horse-derived ATG. ATG is superior to anti-NK
monoclonal antibodies, as the latter are generally not lytic to all host NK
cells, while the
polyclonal mixture in ATG is capable of lysing all host NK cells. Anti-NK
monoclonal
antibodies can, however, be used.
The presence of donor antigen in the host thymus during the time when host T
cells are regenerating post-transplant is critical for tolerizing host T
cells. If donor
hematopoietic stem cells are not able to become established in the host thymus
and
induce tolerance before host T cells regenerate repeated doses of anti-
recipient T cell
antibodies may be necessary throughout the non-myeloablative regimen.
Continuous
depletion of host T cells may be required for several weeks.
Sublethal irradiation is administered to the recipient between days -3 and -1
prior
to donor stem cell transplantation to create hematopoietic space. Sublethal
whole body
irradiation is sufficient to permit engraftment with minimal toxic effects to
the recipient.
Whole body radiation (300 Rads) can be administered to nonhuman primate
recipients
from a bilateral cobalt teletherapy unit at 10 Rads/min.
The creation of thymic space is also useful in the induction of tolerance.
Local
irradiation of the thymus (700 Rads) can be used to induce thymic space.
Thymic
irradiation can be administered on the day prior to donor stem cell
administration.
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Administration of porcine donor stem cells To promote long-term survival of
the implanted organ through T-cell and B-cell mediated tolerance, donor bone
marrow
cells are injected into the recipient to form chimeric bone marrow. (As liver
is the major
site of hematopoiesis in the fetus, fetal liver can also serve as an
alternative to bone
marrow as a source of hematopoietic stem cells.) Donor bone marrow cells home
to
appropriate sites of the recipient and grow contiguously with remaining host
cells and
proliferate, forming a chimeric lymphohematopoietic population. The presence
of donor
antigens in the bone marrow allows newly developing B cells, and newly
sensitized T
cells, to recognize antigens of the donor as self, and thereby induces
tolerance for the
implanted organ from the donor. To stabilize the donor BMC, porcine IL-3 and
stem
cell factor (BioTransplant, Inc. Charlestown, MA) can be administered to the
recipient
between days 0 through 14 at 10 pg/l:glday. Bone marrow can be harvested and
injected intravenously (7.5 x 108/kg) as previously described (Pennington et
al., 1988,
Tran.splamation 45:21-26). Should natural antibodies be found to recur before
tolerance
I 5 is induced. and should these antibodies cause damage to the graft, the
protocol can be
modified to permit sufficient time following BMT for humoral tolerance to be
established prior to organ grafting.
To follow chimerism. two color flow cytometry can be used. This assay uses
monoclonal antibodies to distinguish between donor class I major
histocompatibility
antigens and leukocyte common antigens versus recipient class I major
histocompatibility antigens. Chimerism can also be followed by using
quantitative
polymerase chain reaction to amplify porcine specific sequences, thereby
indicatine the
presence of porcine cells.
Cyclosporine (Sandoz Pharmaceuticals Co., Basel, Switzerland) is administered
for about 28 days, beginning at the time of donor cell implantation (or a few
days
before), at a dose of 15 mg/kg/day, i.v., to maintain a plasma level of
greater than 300
ng/ml.
Introduction of the porcine rg aft. After donor stem cells have been
administered
a miniature swine kidney is implanted into the recipient. When an organ graft
is placed
in such a recipient several months after bone marrow chimerism has been
induced.
natural antibody against the donor will have disappeared, and the graft should
be
accepted by both the humoral and the cellular arms of the immune system. Organ
transplantation can be performed sufficiently long following transplant of
hematopoietic
cells, that normal health and immunocompetence will have been restored at the
time of
organ transplantation. The use of xenogeneic donors allows the possibility of
using
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bone marrow cells and organs from the same animal, or from genetically matched
animals.
The approaches described above are designed to synergistically prevent the
problem of transplant rejection. While any of these procedures may aid the
survival of
S an implanted organ, best results are achieved when all steps are used in
combination.
The method of introducing stem cells may be altered, particularly by ( 1 )
increasing the time interval between injecting hematopoietic stem cells and
implanting
the graft; (2) increasing or decreasing the amount of hematopoietic stem cells
injected;
(3) varying the number of hematopoietic stem cell injections; (4) varying the
method of
delivery of hematopoietic stem cells; (S) varying the tissue source of
hematopoietic stem
cells, e.g., a fetal liver cell suspension may be used: or (6) varying the
donor source of
hematopoietic stem cells. Although hematopoietic stem cells derived from the
graft
donor are preferable, hematopoietic stem cells may be obtained from other
individuals,
preferably from inbred donor strains, or from in vitro cell culture.
i S Irradiation of the recipient may make use of: ( 1 ) varying the absorbed
dose of
whole body radiation below the sublethal range; (2) targeting different body
parts (e.g.,
thymus, spleen); (3) varying the rate of irradiation (e.g., 10 Rads/min., IS
Radsimin.); or
(4) varying the time interval between irradiation and transplant of
hematopoietic stem
cells; any time interval between 1 and 14 days can be used, and certain
advantages may
flow from use of a time interval of 4-7 days.
Antibodies introduced prior to hematopoietic cell transplant may be varied by:
( 1 ) using monoclonal antibodies to T cell subsets or NK cells (e.g., anti-
NKH 1 A, as
described by United States Patent No. 4,772,552 to Hercend, et al., hereby
incorporated
by reference); (2} preparing anti-human ATG in other mammalian hosts (e.g.,
monkey,
pig, rabbit, dog); or (3) using anti-monkey ATG prepared in any of the above
mentioned
hosts.
Other Embodiments
The preferred tolerogen for use in methods of the invention is the galactosvl
a(l,
3) galactose moiety. However, as is shown in Example 4 above, other moieties,
e.g., the
blood group B antigen, can induce a degree of tolerance to the galactosyl a(l,
3)
galactose moiety. Thus, any moiety, particularly other carbohydrate moieties,
which are
sufficiently similar in structure to induce tolerance to the galactosyl a(I,
3) galactose
moiety can be used in the methods and compositions described herein. Compounds
can
be screened for use as a tolerogen by testing for cross reactivity with anti-
galactosyl a( 1,
3) galactose antibodies. The ability of a candidate compound to bind the
antibody is
indicative of usefulness as a tolerogen.
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The methods of the invention can also be used to induce tolerance to other
natural antibody antigens, e.g., other carbohydrates which are the target of
natural
antibodies. The moiety to which tolerace is induced can be found as follows.
Human
natural antibodies can be isolated and depleted of galactosyl a( 1, 3 )
galactose moiety
reactive antibodies. The remaining natural antibodies can be tested against a
panel of
antigens. e.g., a panel of carbohydrate moieties, to select antigens for use
in tolerization.
Once the antigen is identified. tolerance can be induced by modifying the
methods
described herein for use with the new antigen.
The methods of the invention are particularly useful for replacing a tissue or
organ afflicted with a neoplastic disorder, particularly a disorder which is
resistant to
normal modes of therapy, e.g., chemotherapy or radiation therapy. In preferred
embodiments: the graft includes tissue from the digestive tract or gut. e.g..
tissue from
the stomach, or bowel tissue, e.g., small intestine, large intestine, or
colon: the graft
replaces a portion of the recipient's digestive system e.g., all or pan of any
of the
I S digestive tract or gut. e.g., the stomach. bowel, e.g., small intestine.
large intestine. or
colon.
As is discussed herein, it is often desirable to expose a graft recipient to
irradiation in order to promote the development of mixed chimerism. Mixed
chimerism
can be induced with less radiation toxicity by fractionating the radiation
dose, i.e., by
delivering the radiation in two or more exposures or sessions. Accordingly, in
any
method of the invention calling for the irradiation of a recipient, e.g., a
primate, e.g., a
human. recipient, of a xenograft the radiation can either be delivered in a
single
exposure. or more preferably, can be fractionated into two or more exposures
or
sessions. The sum of the fractionated dosages is preferably equal, e.g., in
Rads or Gy,
to the radiation dosage which can result in mixed ehimerism when given in a
single
exposure. The fractions are preferably approximately equal in dosage. For
example, a
single dose of 700 Rads can be replaced with, e.g., two fractions of 350 Rads,
or seven
fractions of 100 Rads. Hyperfractionation of the radiation dose can also be
used in
methods of the invention. The fractions can be delivered on the same day, or
can be
separated by intervals of one, two, three, four, five, or more days. Whole
body
irradiation, thymic irradiation, or both, can be fractionated.
Methods of the invention can include recipient splenectomy.
As is discussed herein, contacting the recipient's blood with galactosyl (al,
3)
galactose epitopes can be used to deplete the host of natural antibodies.
Other methods
for depleting or otherwise inactivating natural antibodies can be used with
any of the
methods described herein. For example, drugs which inactivate natural
antibodies. e.g.,
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deoxyspergualin (DSG) (Bristol), or anti-IgM antibodies, can be administered
to the
recipient of an allograft or a xenograft. One or more of, DSG (or similar
drugs), anti-
IgM antibodies, and hemoperfusion, can be used to inactivate recipient natural
antibodies in methods of the invention. DSG at a concentration of 6 mg/kglday,
i.v., has
been found useful in suppressing natural antibody function in pig to
cynomolgus kidney
transplants.
Methods for the inactivation of thymic T cells or thymocytes are also included
in
embodiments of the invention. Some of the methods described herein include the
administration of thymic irradiation to inactivate host thymic-T cells or to
otherwise
diminish the host's thymic-T cell mediated responses to donor antigens. It has
been
discovered that the thymic irradiation called for in xenogeneic methods of the
invention
can be supplemented with, or replaced by, other treatments which diminish
(e.g., by
depleting thymic-T cells and/or down modulating one or more of the T cell
receptor
(TCR), CD4 co-receptor, or CD8 co-receptor) the host's thymic-T cell mediated
response. For example, thymic irradiation can be supplemented with, or
replaced by,
anti-T cell antibodies (e.g., anti-CD4 and/or anti-CD8 monoclonal antibodies)
administered a sufficient number of times, in sufficient dosage, for a
sufficient period of
time, to diminish the host's thymic-T cell mediated response.
For best results, anti-T cell antibodies should be administered repeatedly.
E.g.,
anti-T cell antibodies can be administered one, two, three, or more times
prior to donor
bone marrow transplantation. Typically, a pre-bone marrow transplantation dose
of
antibodies will be given to the patient about 5 days prior to bone marrow
transplantation.
Additional, earlier doses 6, 7, or 8 days prior to bone marrow transplantation
can also be
given. It may be desirable to administer a first treatment then to repeat pre-
bone marrow
administrations every 1-5 days until the patient shows excess antibodies in
the serum
and about 99% depletion of peripheral T cells and then to perform the bone
marrow
transplantation. Anti-T cell antibodies can also be administered one, two,
three, or more
times after donor bone marrow transplantation. Typically, a post-bone marrow
transplant treatment will be given about 2-14 days after bone marrow
transplantation.
The post bone marrow administration can be repeated as many times as needed.
If more
than one administration is given the administrations can be spaced about 1
week apart.
Additional doses can be given if the patient appears to undergo early or
unwanted T cell
recovery. Preferably, anti-T cell antibodies are administered at least once
(and
preferably two, three, or more times) prior to donor bone marrow
transplantation and at
least once (and preferably two, three, or more times) after donor bone marrow
transplantation.
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Some of the methods herein include the administration of hematopoietic stem
cells (engineered autologous cells or donor cells) to a recipient. In many of
those
methods, hematopoietic stem cells are administered prior to or at the time of
the
implantation of a graft, the primary purpose of the administration of
hematopoietic stem
cells being the induction of tolerance to the graft. The inventors have found
that one or
more subsequent administrations (e.g., a second, third, fourth, fifth, or
further
subsequent administration) of hematopoietic stem cells can be desirable in the
creation
and/or maintenance of tolerance. Thus, the invention also includes methods in
which
hematopoietic stem cells are administered to a recipient, e.g., a primate,
e.g., a human,
which has previously been administered hematopoietic stem cells as part of any
of the
methods referred to herein.
While not wishing to be bound by theory, it is believed that repeated stem
cell
administration may promote chimerism and possibly long-term deletionai
tolerance in
graft recipients. Accordingly, any method referred to herein which includes
the
administration of hematopoietic stem cells can further inciude multiple
administrations
of stem cells. In preferred embodiments: a first and a second administration
of stem
cells are provided prior to the implantation of a graft: a first
administration of stem cells
is provided prior to the implantation of a graft and a second administration
of stem cells
is provided at the time of implantation of the graft. In other preferred
embodiments: a
first administration of stem cells is provided prior to or at the time of
implantation of a
graft and a second administration of stem cells is provided subsequent to the
implantation of a graft. The period between administrations of hematopoietic
stem cells
can be varied. In preferred embodiments a subsequent administration of
hematopoietic
stem cell is provided: at least two days, one week, one month, or six months
after the
previous administration of stem cells; at least two days, one week, one month,
or six
months after the implantation of the graft.
The method can furthei include the step of administering a second or
subsequent
dose of hematopoietic stem cells: when the recipient begins to show signs of
rejection,
e.g., as evidenced by a decline in function of the grafted organ, by a change
in the host
donor specific antibody response. or by a change in the host lymphocyte
response to
donor antigen; when the level of chimerism decreases; or generally, as is
needed to
maintain tolerance or otherwise prolong the acceptance of a graft. Thus,
method of the
invention can be modified to include a further step of determining if a
subject which has
received a one or more administrations of hematopoietic stem cells is in need
of a
subsequent administration of hematopoietic stem cells, and if so,
administering a
subsequent dose of hematopoietic stem cells to the recipient.
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Any of the methods referred to herein can include the administration of
agents,
e.g., 1 S-deoxyspergualin, mycophenolate mofetil, brequinar sodium, or similar
agents,
which inactivate, e.g., inhibit the production, levels, or activity of
antibodies in the
recipient. One or more of these agents can be administered: prior to the
implantation of
donor tissue, e.g., one, two, or three days, or one, two, or three weeks
before
implantatibn of donor tissue; at the time of implantation of donor tissue; or
after
implantation of donor tissue, e.g., one, two, or three days, or one, two or
three weeks
after, implantation of a graft.
The administration of the agent can be initiated: when the recipient begins to
show signs of rejection, e.g., as evidenced by a decline in function of the
grafted organ,
by a change in the host donor specific antibody response, or by a change in
the host
lymphocyte response to donor antigen; when the level of chimerism decreases:
~~~hen
the level of chimerism falls below a predetermined value: or generally, as is
needed to
maintain tolerance or otherwise prolong the acceptance of a graft.
The period over which the agent is administered (or the period over which
clinically effective levels are maintained in the subject) can be long term.
e.g., for six
months or more or a year or more, or short term, e.g.. for less than a year.
more
preferably six months or less, more preferably one month or less, and more
preferably
two weeks or less. The period will generally be at least about one week and
preferably
at least about two weeks in duration. In preferred embodiments the period is
two or
three weeks long.
Preferred embodiments include administration of 15-deoxyspergualin (6
mg/kg/day) for about two weeks beginning on the day of graft implantation.
An anti-CD2 antibody, preferably a monoclonal, e.g., BTI-322, or a monoclonal
directed at a similar or overlapping epitope, can be used in addition to or in
place of any
anti-T cell antibodies (e.g., ATG) in any method referred to herein.
In another aspect, the invention features, a genetically engineered swine
cell,
e.g., a cultured swine cell, a retrovirally transformed swine cell, or a cell
derived from a
transgenic swine. The cell includes a transgene which encodes an intracellular
antibody
which binds to an a(1,3)galactosyltransferase, e.g., ~i-D-galactosyl-1,4-N-
acetyl-D-
glucosaminide a(1,3)galactosyltransferase, and inhibits the ability of the
enzyme to form
a galactosyl a(1,3) galactose moiety on the swine cell. The swine cell can be
from a
full-size swine or from a miniature swine. Preferably, the transgene is
integrated into
the genome of the cell.
In preferred embodiments the transgene encodes: an antibody which is targeted
to the endoplasmic reticulum; a single chain antibody, e.g., a single chain
variable-
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region fragment. (A single chain variable region fragment antibody includes
immunoglobulin heavy and light chain variable region (VH and VL) domains
joined by
a flexible peptide linker.)
In preferred embodiments the genetically engineered swine cell is: a swine
hematopoietic stem cell, e.g., a cord blood hematopoietic stem cell, a bone
marrow
hematopoietic stem cell, or a fetal or neonatal liver or spleen hematopoietic
stem cell;
derived from differentiated blood cells, e.g. a myeloid cell, such as a
megakaryocyte,
monocyte, granulocyte, or an eosinophil; an erythroid cell, such as a red
blood cell. e.g.
a lymphoid cell, such as B lymphocytes and T lymphocytes; derived from a
pluripotent
hematopoietic stem cell, e.g. a hematopoietic precursor, e.g. a burst-forming
units-
crythroid (BFU-E), a colony forming unit-erythroid (CFU-E), a colony forming
unit-
megakaryocyte (CFU-Meg), a colony forming unit-granulocyte-monocyte (CFU-GM).
a
colony forming unit-eosinophil , or a colony forming unit-granulocyte-
erythrocy~te-
megakaryocyte-monocyte (CFU-GEMM); a swine cell other than a hematopoietic
stem
cell, or other blood cell; a swine thymic cell, e.g., a swine thymic stromal
celi: a bone
marrow stromal cell; a swine liver cell; a swine kidney cell; a swine
epithelial ccll: a
swine hematopoietic progenitor cell; a swine muscle cell, e.g., a heart cell;
an
endothelial cell; or a dendritic cell or precursor thereof.
In yet other preferred embodiments the cell is: isolated or derived from
cultured
cells, e.g., a primary culture, e.g., a primary cell culture of hematopoietic
stem cells;
isolated or derived from a transgenic animal.
In another aspect, the invention features. a transgenic swine having cells
which
include a transgene which encodes an intracellular antibody which binds to an
a
(I,3)galactosyltransferase, e.g., (3-D-galactosyl-1,4-N-acetyl-D-glucosaminide
a
(1,3)galactosyltransferase. The transgenic antibody inhibits the ability of
the enzyme to
form a galactosyl a( 1,3) galactose moiety on cells of the transgenic swine.
The
transgenic swine can be a full-size swine or a miniature swine. Preferably,
the transgene
is integrated into the genome of the animal.
In preferred embodiments the transgene encodes: an antibody which is targeted
to the endoplasmic reticulum; a single chain antibody, e.g., a single chain
variable-
region fragment. Transgenic swine (or swine cells) of the invention can be
used as
a source for tissue for grafting into a human recipient, e.g., hematopoietic
cells or other
tissues or organs.
In another aspect, the invention features, a swine organ or a swine tissue,
having
cells which include a transgene which encodes an intracellular antibody which
binds to
an a(1,3)galactosyltransferase, e.g., (3-D-galactosyl-1,4-N-acetyl-D-
glucosaminide a
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(1,3)galactosyltransferase. The transgenic antibody inhibits the ability of
the enzyme to
form a galactosyl a(1,3) galactose moiety on cells ofthe transgenic swine. The
transgenic swine organ or tissue can be a full-size swine or a miniature swine
organ or
tissue. Preferably, the transgene is integrated into the genome of the cells.
In preferred embodiments the transgene encodes: an antibody which is targeted
to the endoplasmic reticulum; a single chain antibody, e.g., a single chain
variable
region fragment.
In preferred embodiments the organ is a heart, lung, kidney, pancreas, or
liver.
In preferred embodiments the tissue is: thymic tissue; islet cells or islets;
stem
cells; bone marrow; endothelial cells: skin; or vascular tissue.
The swine organs and tissues of the invention can be used as a source of
tissue
for grafting into a human recipient, e.g., hematopoietic cells or other
tissues or organs .
Graft tissue which expresses a transgenic anti-a( 1,3)galactosyltransferase
intracellular antibody can be used to improve methods of transplanting
xenogeneic
tissue into a recipient. For example. acceptance of swine, e.g., miniature
swine or full-
size swine, tissue by a human recipient can be prolonged if the porcine tissue
expresses
an antibody, preferably an intracellular antibody, which binds to an a
(1,3)galactosyltransferase, e.g., ~i-D-galactosyl-1,4-N-acetyl-D-glucosaminide
a
(1,3)galactosyltransferase, and thereby reduces the number of galactosyl
a(1,3)
galactose moieties on the surfaces of a graft. Transgenic tissue described
herein can be
used in place of other swine tissue in any of the methods described or
referred to herein.
Genetically engineered swine cells of the invention can be made by methods
known to those skilled in the art, e.g., by retroviral transduction of swine
cells. Methods
for producing transgenic swine of the invention use standard transgenic
technology.
These methods include, e.g., the infection of the zygote or organism by
viruses including
retroviruses; the infection of a tissue with viruses and then reintroducing
the tissue into
an animal; and the introduction of a recombinant nucleic acid molecule into an
embryonic stem cell of a mammal followed by appropriate manipulation of the
embryonic stem cell to produce a transgenic animal.
As used herein, the term "transgene" refers to a nucleic acid sequence
(encoding,
e.g., an antibody, e.g., an intracellular antibody), which is inserted by
artifice into a cell.
The transgene can become part of the genome of an animal which develops in
whole or
in part from that cell. If the transgene is integrated into the genome it
results, by its
insertion, in a change in the nucleic acid sequence of the genome into which
it is
inserted. A transgene can include one or more transcriptional regulatory
sequences and
any other nucleic acid sequences, such as introns, that may be necessary for a
desired
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level or pattern of expression of a selected nucleic acid, all operably linked
to the
selected nucleic acid. The transgene can include an enhancer sequence. The
transgene
is typically introduced into the animal, or an ancestor of the animal, at a
prenatal, e.g., an
embryonic, or earlier, stage. The transgene can include a sequence which
targets the
transgene product to the enopiasmic reticulum.
As used herein, the term "transgenic cell" refers to a cell containing a
transgene.
As used herein, a "transgenic animal" is any animal in which one or more, and
preferably essentially all, of the cells of the animal includes a transgene.
The transgene
is introduced into the cell, directly or indirectly by introduction into a
precursor of the
cell, by way of deliberate genetic manipulation. such as by microinjection or
by
infection with a recombinant virus. The term genetic manipulation does not
include
classical cross-breeding. or in vitro fertilization. but rather is directed to
the introduction
of a recombinant DNA molecule. This molecule may be integrated within a
chromosome. or it may be extrachromosomally replicating DNA.
1 S As used herein, the term "recombinant swine cells" refers to cells derived
from
swine, preferably miniature swine, which have been used as recipients for a
recombinant
vector or other transfer nucleic acid, and include the progeny of the original
cell which
has been transfected or transformed. Recombinant swine cells include cells in
which
transgenes or other nucleic acid vectors have been incorporated into the host
cell's
genome. as well as cells harboring expression vectors which remain autonomous
from
the host cell's genome.
The term "tissue" as used herein, means any biological material that is
capable of
being transplanted and includes organs (especially the internal vital organs
such as the
heart, lung, liver, kidney, pancreas and thyroid). cornea, skin, blood vessels
and other
connective tissue, cells including blood and hematopoietic cells, Islets of
Langerhans,
brain cells and cells from endocrine and other organs and bodily fluids, alt
of which may
be candidate for transplantation.
Production of Intrabodies: Sinele chain variable rel;ion fragment antibodies
Single chain variable region fragment antibodies are particularly preferred
for
use in methods described herein. The first step in the production of
intrabodies of the
invention is the production of monoclonal antibodies specific for the a
(1,3)galactosyltransferase, e.g., ~3-D-galactosyl-1,4-N-acetyl-D-glucosaminide
a
(1,3)galactosyltransferase. These antibodies can be prepared by injection of
the enzyme,
preferably the swine enzyme, into an animal, e.g., a mouse. Antibodies
produced by
individual hybridomas can be tested, in vitro. for the ability to bind an
block a
(1,3)galactosyltransferase activity. The immunoglobulin heavy and light chain
variable
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region (VH and VL) domains from an antibody which inhibits activity are cloned
and
used to prepare a single chain antibody construct. A construct can be
evaluated for in
vivo activity by transfecting it into a swine cell line and determining the
effect of the
antibody on presentation of the galactosyl a(1,3) galactose moiety. A
construct which
reduces presentation of the moiety can be used to construct a transgenic
animal or to
prepare genetically engineered cells.
Genetically Engineered Swine Cells
Transgenic swine cells of the invention can be produced by any methods known
to those in the art. Transgenes can be introduced into cells, e.g., stem
cells, e.g.,
cultured stem cells. by any methods which allows expression of these genes at
a level
and for a period sufficient to promote engraftment or maintenance of the
cells. These
methods include e.g., transfection, electroporation, particle gun bombardment,
and
transduction by viral vectors, e.g., by retroviruses. Transgenic swine cells
can also be
derived from transgenic animals. Recombinant retroviruses are a preferred
delivery
system.
Preparation of Tran~enic Swine
Microinjection of swine oocvtes
In preferred embodiments the transgenic swine of the present invention is
produced by:
i) microinjecting a recombinant nucleic acid molecule into a fertilized swine
egg to
produce a genetically altered swine egg;
ii) implanting the genetically altered swine egg into a host female swine;
iii) maintaining the host female for a time period equal to a substantial
portion of the
gestation period of said swine fetus.
iv) harvesting a transgenic swine having at least one swine cell that has
developed
from the genetically altered mammalian egg, which expresses a human class I
gene.
In general. the use of microinjection protocols in transgenic animal
production is
typically divided into four main phases: (a) preparation of the animals; (b)
recovery and
maintenance in vitro of one or two-celled embryos; (c) microinjection of the
embryos
and (d) reimplantation of embryos into recipient females. The methods used for
producing transgenic livestock, particularly swine, do not differ in principle
from those
used to produce transgenic mice. Compare, for example, Gordon et al. (1983)
Methods
irr Enrymolo~ 101:41 l, and Gordon et al. (1980) PNAS 77:7380 concerning,
generally,
transgenic mice with Hammer et al. ( 1985) Nature 315:680, Hammer et al. (
1986) J
Anim Sci 63:269-278, Wall et al. (1985) Biol Reprod. 32:645-651, Pursel et al.
(1989)
Science 244:1281-1288, Vize et al. (1988) J Cell Science 90:295-300, Muller et
al.
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(1992) Gene 121:263-270, and Velander et al (1992) PNAS 89:12003-12007, each
of
which teach techniques for generating transgenic swine. See also, PCT
Publication WO
90/03432, and PCT Publication WO 92/22646 and references cited therein.
One step of the preparatory phase comprises synchronizing the estrus cycle of
at
least the donor females, and inducing superovulation in the donor females
prior to
mating. Superovulation typically involves administering drugs at an
appropriate stage of
the estrus cycle to stimulate follicular development, followed by treatment
with drugs to
synchronize estrus and initiate ovulation. As described in the example below,
pregnant
mare's serum is typically used to mimic the follicle-stimulating hormone (FSH)
in
combination with human chorionic gonadotropin (hCG) to mimic luteinizing
hormone
(LH). The efficient induction of superovulation in swine depend, as is well
known. on
several variables including the age and weight of the females, and the dose
and timing of
the gonadotropin administration. See for example, Wall et al. ( 1985) Biol.
Reprod
32:645 describing superovulation of pigs. Superovulation increases the
likelihood that a
large number of healthy embryos will be available after mating, and further
allows the
practitioner to control the timing of experiments.
After mating, one or two-cell fertilized eggs from the superovulated females
are
harvested for microinjection. A variety of protocols useful in collecting
el;gs from pigs
are known. For example, in one approach. oviducts of fertilized superovulated
females
can be surgically removed and isolated in a buffer solution/culture medium,
and
fertilized eggs expressed from the isolated oviductal tissues. See, Gordon et
al. ( 1980)
PNAS 77:7380; and Gordon et al. (1983) Methods in Enwmology 101:411.
Alternatively, the oviducts can be cannulated and the fertilized eggs can be
surgically
collected from anesthetized animals by flushing with buffer solution/culture
medium,
thereby eliminating the need to sacrifice the animal. See Hammer et al. (
1985) Nature
315:600. The timing of the embryo harvest after mating of the superovulated
females
can depend on the length of the fertilization process and the time required
for adequate
enlargement of the pronuclei. This temporal waiting period can range from, for
example, up to 48 hours for larger breeds of swine. Fertilized eggs
appropriate for
microinjection, such as one-cell ova containing pronuclei, or two-cell
embryos, can be
readily identified under a dissecting microscope.
The equipment and reagents needed for microinjection of the isolated swine
embryos are similar to that used for the mouse. See, for example, Gordon et
al. (1983)
Methods in Errrymology 101:411; and Gordon et al. (1980) PNAS77:7380,
describing
equipment and reagents for microinjecting embryos. Briefly, fertilized eggs
are
positioned with an egg holder (fabricated from 1 mm glass tubing), which is
attached to
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a micro-manipulator, which is in turn coordinated with a dissecting microscope
optionally fitted with differential interference contrast optics. Where
visualization of
pronuclei is difficult because of optically dense cytoplasmic material, such
as is
generally the case with swine embryos, centrifugation of the embryos can be
earned out
without compromising embryo viability. Wall et al. (1985) Biol. Reprod.
32:645.
Centrifugation will usually be necessary in this method. A recombinant nucleic
acid
molecule of the present invention is provided, typically in linearized form,
by linearizing
the recombinant nucleic acid molecule with at least 1 restriction
endonuclease, with an
end goal being removal of any prokaryotic sequences as well as any unnecessary
flanking sequences. In addition, the recombinant nucleic acid molecule
containine the
tissue specific promoter and the human class I gene may be isolated from the
vector
sequences using 1 or more restriction endonucleases. Techniques for
manipulating and
linearizing recombinant nucleic acid molecules are well known and include the
techniques described in Molecular Clonine: A Laboraton~ Manual. Second
Edition.
Maniatis et al. eds.. Cold Spring Harbor, N.Y. ( 1989).
The linearized recombinant nucleic acid molecule may be microinjected into the
swine egg to produce a genetically altered mammalian egg using well known
techniques.
Typically, the linearized nucleic acid molecule is microinjected directly into
the
pronuclei of the fertilized eggs as has been described by Gordon et al. (1980)
PNAS
77:7380-7384. This leads to the stable chromosomal integration of the
recombinant
nucleic acid molecule in a significant population of the surviving embryos.
See for
example. Brinster et ai. (1985) PNAS 82:4438-4442 and Hammer et al. (1985)
Nature
315:600-603. The microneedles used for injection, like the egg holder, can
also be
pulled from glass tubing. The tip of a microneedle is allowed to fill with
plasmid
suspension by capillary action. By microscopic visualization, the microneedle
is then
inserted into the pronucleus of a cell held by the egg holder, and plasmid
suspension
injected into the pronucleus. If injection is successful, the pronucleus will
generally
swell noticeably. The microneedle is then withdrawn, and cells which survive
the
microinjection (e.g. those which do not lysed) are subsequently used for
implantation in
a host female.
The genetically altered mammalian embryo is then transferred to the oviduct or
uterine horns of the recipient. Microinjected embryos are collected in the
implantation
pipette. the pipette inserted into the surgically exposed oviduct of a
recipient female, and
the microinjected eggs expelled into the oviduct. After withdrawal of the
implantation
3 S pipette, any surgical incision can be closed, and the embryos allowed to
continue
gestation in the foster mother. See, for example, Gordon et al. (1983) Methods
in
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Enzymology 101:411; Gordon et al. (1980) PNAS 77:7390; Hammer et al. (1985)
Nature
315:600; and Wall et al. (1985) Biol. Reprod. 32:645.
The host female mammals containing the implanted genetically altered
mammalian eggs are maintained for a sufficient time period to give birth to a
transgenic
mammal having at least 1 cell, e.g. a bone marrow cell, e.g. a hematopoietic
cell, which
expresses the recombinant nucleic acid molecule of the present invention that
has
developed from the genetically altered mammalian egg.
At two-four weeks of age (post-natal), tail sections are taken from the
piglets and
digested with Proteinase K. DNA from the samples is phenol-chloroform
extracted,
then digested with various restriction enzymes. The DNA digests are
electrophoresed on
a Tris-borate gel, blotted on nitrocellulose, and hybridized with a probe
consisting of the
at least a portion of the coding region of the recombinant cDNA of interest
which had
been labeled by extension of random hexamers. Under conditions of high
stringency,
this probe should not hybridize with the endogenous pig gene. and will allow
the
identification of transgenic pigs.
The methods of the invention can also include inducing tolerance to the
galactosyl a(1, 3) galactose moiety by administering to the recipient blood
group B
antigen. This can be done prior to exposure to the galactosyl a(1, 3)
galactose moiety
tolerogen. so as to induce a first level of tolerance to the galactosyl a(1,
3) galactose
moiety .
Equivalents
Those skilled in the art will recognize. or be able to ascertain using no more
than
routine experimentation, many equivalents of the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
Other embodiments are within the following claims.
What is claimed is:
