Note: Descriptions are shown in the official language in which they were submitted.
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MODIFIED CELLS AND METHODS FOR INHIBITING XENOGRAFT
REJECTION
Ba~k~round of the Invention
A nurnber of ~ise~es are treated by the transplantation of tissue donated by other
hurnan (allografts) or obtained from ~nim~ls (xenografts). Examples of such diseases include
Parkinson's ~ e~e, which can be treated by transplantation of neural cells, and insulin-
dependent diabetes, which can be treated by transplantation of insulin-secreting pancreatic
islet cells. While the transplanted cells may have the capacity to perform the desired function
10 (e.g., secretion of insulin in response to the rising levels of giucose), the graft will soon fail as
a result of immllnnlogical rejection. Shortly after transplantation, cells of the immune system
of the recipient recognize the allogeneic or xenogeneic cells as foreign and proceed to attack
the graft through both humoral and cellular routes. Allogeneic or xenogeneic cells are
initially recognized by the recipient's immllne system through antigenic (let~rmin~ntS
15 expressed on the surface of the cells. The predominant antigens recognized as "non-self ' are
the major histoc.~.mp~tibility complex class I and class II antigens (MHC class I and class II).
MHC class I antigens are expressed on virtually all parenchymal cells (e.g., pancreatic islet
cells). In contrast, MHC class II antigens are expressed on a limited number of cell types,
primarily B cells, macrophages, dendritic cells, Langerhans cells and thymic epitheliurn. The
20 interaction of foreign MHC antigens with the T cell receptor on host T cells causes these host
cells to become activated. Following activation, these T cells proliferate and induce effector
functions which lead to cell lysis and destruction of the transplanted cells.
For transplantation to be a viable therapeutic option, approaches are needed to inhibit
rejection of transplanted cells by the immlme system of the recipient. One method for
25 inhibiting this rejection process is by ~rlministration of drugs that suppress the function of the
immllne system. Drugs such as cyclophosphamide and cyclosporin can inhibit the actions of
the immllne system and thus allow graft acceptance. However, these drugs generally need to
be ~-lministered to a graft recipient perm~nently (i.e., life-long) and their use results in
generalized immlln~suppression which leaves the recipient susceptible to infection and tumor
30 growth. Additionally, ~-lmini~tration of immunosuppressive drugs is often accompanied by
other serious side effects such as renal failure and hypertension. The requirement for life-
long ~lmini~stration of immunosuppressive drugs in transplant recipients illustrates the need
for better methods for transplanting cells such that rejection of the cells by the recipient's
immllne system is inhibited.
It has been shown that it is possible to alter an antigen on the surface of a cell to be
transplanted prior to transplantation to "mask" the antigen from normal recognition by cells
ofthe recipient's immune system (see Faustman & Coe (1991) Science 252:1700-1702 and
WO 92/04033). For example, MHC class I antigens on transplanted cells can be altered by
contacting the cells with a molecule which binds to the antigen. such as an antibody or
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fragment thereof (e.g., a F(ab')2 fragment) prior to transplantation. This alteration of MHC
class I antigens modifies the interaction between the antigens on the cells and immllne cells
in the recipient following transplantation, to thereby inhibit rejection of the transplanted cells.
Additional methods for inhibiting rejection of an allograft or xenograft following
5 transplantation in a host are needed.
Sllmm~ry of the Invention
This invention pertains to methods for transplanting cells into an allogeneic orxenogeneic recipient such that rejection of the cells by the recipient is inhibited. The
10 methods of the invention involve modification of donor cells prior to transplantation to
reduce the immunogenicity of the cells in a recipient. In the l~ler~lled embodiment, this
invention features treatment of donor cells to modify surface antigens prior to transplantation
such that upon transplantation into a recipient subject natural killer (NK) cell-mediated
rejection and/or lymphokine activated killer (LAK) cell-mediated rejection of the cell is
15 inhibited. As a result of this tre~tment, rejection of donor cells in the recipient is inhibited.
The present invention pertains to a cell (i.e., a donor cell) which has at least one
antigen on the cell surface which stim~ tes an immllne response against the cell when the
cell is transplanted into a recipient subject, for example, a xenogeneic subject. The antigen
on the surface of the cell is altered such that rejection of the cell is inhibited. Alteration of
the cell surface antigen can inhibit rejection of the cell by a variety of m~ch~nicm~ For
example, alteration of the antigen can modify an interaction between the antigen and a an
immune cell such as a lymphocyte, e.g., a T lymphocyte, a B lymphocyte, a natural killer
cell, or a lymphokine activated killer cell, in the recipient, thereby inhibiting an immllne
response against the cell in the recipient. The antigen(s) on the surface of the cells to be
altered is one which is capable of stimulating an immune response against the cell in the
recipient. An antigen on the surface of a cell can be altered prior to transplantation by
contacting the cell in vitro with a molecule which binds to the antigen. In one embodiment.
the molecule which binds to the antigen is an antibody, or fragment or derivative thereof,
which binds to the antigen but does not activate complement or induce lysis of the cell. A
30 ~lef~ d antibody fragment is an F(ab')2 fr~gment Alternatively, the molecule is a peptide
or derivative thereof (e.g., a peptide mimetic) which binds the antigen and interferes with an
interaction with an immune cell. In a pler~ d embodiment, the antigen on the cell surface
which is altered is an MHC class I antigen. Preferred antibodies which can be used to alter
MHC class I antigens on the surface of cells include the monoclonal antibodies W6/32 and
35 PT85, or fragments or derivatives thereof, or other antibodies which bind to the same
epitopes recognized by the W6/32 and PT85 antibodies. Other cell surface antigens which
can be altered include adhesion molecules, such as ICAM-l, ICAM-2 and LFA-3.
Preferred cells of the invention are porcine cells. The porcine cells can be endothelial
cells, hepatocytes. pancreatic islet cells, skeletal myocytes. skeletal myoblasts, cardiac
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myocytes, cardiac myoblasts, fibroblasts, epithelial cells, neural cells, e.g., mesencephalic
cells, striatal cells, cortical cells, bone maf~ow cells, hematopoietic cells, and lymphoid cells.
The cells can be within a tissue or an organ.
Another aspect of the present invention is a method for reducing the immunogenicit,v
of a cell for transplantation into a recipient subject. This method includes contacting a cell
which has at least one antigen on the cell surface which stim~ tes an immune response
against the cell in the recipient subject with at least one molecule which binds to the antigen
on the cell surface. When such a cell is transplanted into recipient subject, natural killer cell-
mediated rejection and/or lymphokine activated killer cell-mediated rejection of the cell is
10 inhibited. Preferred antigens and molecules which bind such antigens are described herein.
Preferred recipient subjects include hllm~n~
A further aspect of the present invention is a method for transplanting a cell into a
recipient subject such that rejection of the cell by the recipient subject, e.g., a xenogeneic
subject, is inhibited. This method includes ~lmini~t~rin~ to the subject a cell having at least
15 one antigen on the cell surface which stim~ tes an immlln~ response against the cell in the
recipient subject, wherein the at least one antigen on the cell surface is altered prior to
transplantation to inhibit natural killer cell-mediated rejection and/or lymphokine activated
killer cell-mediated rejection of the cell by the recipient subject. Preferred antigens,
molecules which bind such antigens, and donor cells are described herein.
In addition to inhibiting rejection of transplanted cells, the methods of the invention
induce donor cell-specific T cell tolerance or nonresponsiveness to the tr~n~pl~nt~d cells in
the transplant recipient. The invention thus provides methods for successful transplantation
of cells into an allogeneic or xenogeneic transplant recipient which avoids life-long
gener~li7~-d immlmo~ pl~ s~ion of the subject.
Brief Description of the D...~
Figure I shows a graph depicting the results of a cytolytic assay in which freshly
isolated human peripheral blood lymphocytes (PBL) were shown to lyse porcine PBLs.
Figure 2 shows a graph depicting the results of a cytolytic assay in which freshly
30 isolated human PBLs were shown to lyse porcine hepatocytes.
Figure 3 shows a graph depicting the results of a mixed Iymphocyte reaction (MLR)
in which untreated porcine kidney cells or porcine kidney cells treated with anti-MHC Class I
antibody PT85 F(ab')2 fragments were incubated with human PBLs. Human PBL
proliferation in response to the treated porcine kidney cells was reduced compared to the
3~ human PBL proliferation in response to the untreated porcine kidney cells.
Figure 4 shows the results of a FACS analysis of human cells isolated from an MLR
(the results of the MLR are described for Figure 3) and stained with monoclonal antibodies
for CD56. When human PBLs were stimulated with untreated porcine kidney cells, the
FACS staining revealed a remarkable increase in human cells expressing CD56 (NCAM,
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which is a marker for natural killer (NK) cells). However, when human PBLs were
stim~ ted with porcine kidney cells treated with anti-MHC Class I antibody PT85 F(ab')2
fragments, there was not a substantial increase in CD56 ~xplesshlg cells.
Figures 5A-S C show the results of a 5 I Cr release assay in which freshly isolated PBLs
from three individuals (A, B, and C) were used as effector cells against porcine PBLs in the
presence of pooled human serum (-); serum-free media (0); JY target cells (serum free) (o).
The 5ICr release assay revealed that freshly isolated human PBLs lysed porcine cells but not
human cells.
Figure 6 shows the results of a 5 I Cr release assay in which human PBLs cultured for
10 6 days with aa haplotype porcine PBLs were used as effectors against porcine PBLs. Target
cells were as follows: aa target cells (-); dd targetcells (0); JY target cells (o). The slCr
release assay revealed that human PBLs, after culture with porcine cells, lysed porcine cells
regardless of MHC restriction.
Figures 7A-7B show the results of a 5ICr release assay in which K562 or JY cells15 were used as cold target inhibitors against porcine PBLs. In Figure 7A, freshly isolated
human PBLs were used as effectors while in Figure 7B effector cells were harvested from a 6
day mixed culture of hurnan PBLs and porcine PBLs. Inhibitors were as follows: no
inhibitors (-); JY inhibitors (0); K562 inhibitors (o). The 5ICr release assay revealed that
K562 cells inhibit lysis of porcine cells when freshly isolated human PBLs are used as
20 ~rre-;Lol~ and JY cells do not inhibit lysis of porcine cells when freshly isolated human PBLs
are used as effectors.
Figures 8A-8C show the results of a slCr release assay in which unfractionated
human PBLs (Figure 8A), CD56 enriched PBLs (Figure 8B), and CD56 depleted cells
(Figure 8C) were used as human effector cells against porcine PBLs. Target cells were as
25 follows: K562 cells (-); porcine PBLs (0). The S I Cr release assay revealed that most of the
cytotoxic activity toward porcine cells and K562 cells is present in the CD56-enriched
population and not in the CD56-depleted population.
Figures 9A-9B show the results of a 5ICr release assay in which freshly isolatedhuman PBLs (Figure 9A) and human PBLs previously cultured with mitomycin C-treated
30 porcine PBLs for 6 days (Figure 9B) were used as effectors against porcine PBLs.
Inhibitors were as follows: no cold target inhibitors (-); K562 inhibitors (0); Daudi inhibitors
(o). The 5 I Cr release assay revealed that while K562 cells inhibited the unstimulated human
anti-porcine cytotoxicity as well as cytotoxicity after mixed culture, Daudi cells inhibited
cytotoxicity after mixed culture only.
Fig~re 10 shows the results of an ELISA in which the supen~t~nt of mixed cultures
of human PBLs and mitomycin-C-treated porcine PBLs was tested for IL-2 production.
Black bars, no antibody added; Hatched bars, anti-CD25 added. The ELISA showed that
human IL-2 is generated in these cultures.
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Figure 11 shows the results of a 5ICr release assay in which human PBLs, previously
cultured with mitomycin C-treated porcine PBLs for 6 days, were used as effectors against
porcine PBLs. No antibody added (-), control IgG added (o), anti-CD25 added (0). The slCr
release assay revealed that anti-CD25 antibody blocks generation of anti-porcine human
S cytotoxic cells.
Figure 12 shows the results of a 5 I Cr release assay in which a CD56-depleted
population of human PBLs were used as effectors against porcine PBLs. This population was
generated by depleting CDS6+ cells from human PBL pl~aldlions, cnltllring the cells for 6
days with llliLo",ychl C-treated porcine PBLs, and repeating the CDS6+ cell depletion. A: %
specific lysis by CDS6-depleted population, B: % specific lysis by CD56-depleted population
in the presence of anti-CD3; C: % specific lysis by CDS6-depleted population in the presence
of control IgG; D: % specific lysis by CDS6-depleted population in the presence of K562
cold target inhibitors; E: % specific lysis by CD56-depleted population in the presence of JY
cold target inhibitors. The 5lCr release assay demonstrated that, after depletion of CDS6+
cells, a T cell component of hurnan anti-porcine cytotoxicity was ~pa.
Detailed Description of the Invention
The invention re~Lu-~s cells and methods for transplanting cells into an allogeneic or
xenogeneic recipient such that rejection of tr~ncpl~nted cells by the recipient is inhibited.
The cells to be transplanted into a recipient are treated such that at least one antigen on the
surface of the cell is altered prior to transplantation to modify an interaction between the
antigen and an immune cell (e.g., a natural killer cell, a lymphokine activated killer cell) in
the recipient, thereby inhibiting rejection of the cells by the recipient. In addition to
~lmini~tration of the modified cell, the recipient can be treated with an agent which inhibits T
cell activity in the recipient to further inhibit rejection of the transplanted cells.
The cells and methods of the invention are described in further detail in the following
subsections.
I. Cells for Transplantation
One aspect of the invention relates to a modified or altered cell suitable for
transplantation. Preferred cells of the invention are porcine cells, such as embryonic porcine
cells. The porcine cells can be endothelial cells, hepatocytes, pancreatic islet cells, skeletal
~ myocytes, skeletal myoblasts, cardiac myocytes, cardiac myoblasts, fibroblasts, epithelial
cells, neural cells, e.g. mesencephalic cells, striatal cells, or cortical cells, bone marrow cells,
hematopoietic cells, and lymphoid cells. The cells can be isolated or within a tissue or an
organ.
In an unmodified or unaltered state, the antigen on the cell surface stimulates an
imrnune response against the cell (also referred to herein as the donor cell) when the cell is
~imini.~tered to a subject (also referred to herein as the recipient, host, or recipient subject).
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~y altering the antigen, the normal immunological recognition of the donor cell by the
immllne system cells of the recipient is disrupted and additionally, "abnormal"
immnnt~logical recognition of this altered form of the antigen can lead to donor cell-specific
long terrn unresponsiveness in the recipient. Thus, alteration of an antigen on the donor cell
S prior to ~tlmini~tering the cell to a recipient illL~ .rel~s with the initial phase of recognition of
the donor cell by the cells of the host's immune system subsequent to ~llmini~tration of the
cell. Furthermore, alteration of the antigen can induce immunological nonresponsiveness or
tolerance, thereby pl~v~lllillg the induction of the effector phases of an immlme response
(e.g., cytotoxic T cell generation, antibody production etc.) which are llltim~t~ly responsible
10 for rejection of foreign cells in a normal immune response. As used herein, the terms
"altered" and "modified" are used interchangeably and encomr~es changes that are made to
a donor cell antigen which reduce the immunogenicity of the antigen to thereby hll~lrt;l~ with
immlmological recognition of the antigen by the recipient's immlme system. Preferably
immnnological nonresponsiveness to the donor cells in the recipient subject is generated as a
15 result of alteration of the antigen. The terms "altered" and "modified" are not intended to
include complete elimin~tion of the antigen on the donor cell since delivery of an
in~ l;ate or insufficient signal to the host's immune cells may be neces.~ry to achieve
immunological nonresponsiveness.
Antigens to be altered according to the invention include antigens on a donor cell
20 which can interact with an immune cell (e.g., a hematopoietic cell, an NK cell, an LAK cell)
in an allogeneic or xenogeneic recipient and thereby stimlll~te a specific immlme response
against the donor cell in the recipient. The interaction between the antigen and the immlme
cell may be an indirect interaction (e.g., merli:~te~l by soluble factors which induce a response
in the hematopoietic cell, e.g., humoral mediated) or, preferably, is a direct interaction
25 between the antigen and a molecule present on the surface of the immune cell (i.e., cell-cell
mediated). As used herein, the phrase "imm~lne cell" is int~?ntle~l to include hematopoietic
cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, dendritic cells, and
other antigen presenting cells, NK cells, and LAK cells. In ~ r~ d embodiments, the
antigen is one which interacts with a T lymphocyte in the recipient (e.g., the antigen normally
30 binds to a receptor on the surface of a T lymphocyte), or with an NK cell or LAK cell in the
reclpient.
In a pler~ d embodiment, the antigen on the donor cell to be altered is an MHC
class I antigen. MHC class I antigens are present on almost all cell types. In a normal
immune response, self MHC molecules function to present antigenic peptides to a T cell
35 receptor (TCR) on the surface of self T Iymphocytes. In immune recognition of allogeneic or
xenogeneic cells, foreign MHC antigens (most likely together with a peptide bound thereto)
on donor cells are recognized by the T cell receptor on host T cells to elicit an immlme
response. In addition, foreign MHC class I antigens are known to be recognized by MHC
class I receptors on NK cells. MHC class I antigens on a donor cell are altered to interfere
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with their recognition by T cells, NK cells, or LAK cells in an allogeneic or xenogeneic host
(e.g., a portion of the MHC class I antigen which is normally recognized by the T cell
receptor, NK cells, or LAK cells is blocked or "masked" such that normal recognition of the
MHC class I antigen can no longer occur). Additionally, an altered form of an MHC class I
5 antigen which is exposed to host T cells, NK cells or LAK cells (i.e., available for
pres~nt~tion to the host cell receptor) may deliver an ina~ p,;ate or insufficient signal to
the host T cell such that, rather than stimulating an immllne response against the allogeneic or
xenogeneic cell, donor cell-specific T cell non-responsiveness, inhibition of NK-mediated
cell rejection, and/or inhibition of LAK-mediated cell rejection is in~ re~ For example, it is
10 known that T cells which receive an i~ op,iate or insufficient signal through their T cell
receptor (e.g., by binding to an MHC antigen in the absence of a costim~ tory signal, such as
that provided by B7) become anergic rather than activated and can remain refractory to
tion for long periods oftime (see, e.g, Damle et al. (1981) Proç. Natl. ~lcad. Sci.
USA 78:5096-SlQ0; Les~ r et al. (1986) Eur. J. Immunol. 16:1289-1295; Gimmi, et al.
(1991) Proc. Natl. Acad. Sci. USA 88: 6575-6579; Linsley et al. (1991) ~ Exp. Med. 173:721-
730; Koulova et al. (1991) J: Exp. Med. 173:759-762; Razi-Wolf, et al. (1992) Proc. Natl.
Acad. Sci. USA 89:4210-4214).
~ ltt~rn~tive to MHC class I antigens, the antigen to be altered on a donor cell can be
an MHC class II antigen. Similar to MHC class I antigens, MHC class II antigens function to
20 present antigenic peptides to a T cell receptor on T lymphocytes. However, MHC class II
antigens are present on a limited number of cell t,vpes (primarily B cells, macrophages,
~1en(1ritic cells, Langerhans cells and thymic epithelial cells). In addition to or alternative to
MHC antigens, other antigens on a donor cell which interact with molecules on host T cells
or NK cells and which are known to be involved in immunological rejection of allogeneic or
25 xenogeneic cells can be altered. Other donor cell antigens known to interact with host T cells
and contribute to rejection of a donor cell include molecules which function to increase the
avidity of the interaction between a donor cell and a host T cell. Due to this plop~l ~y, these
molecules are typically referred to as adhesion molecules (although they may serve other
functions in addition to increasing the adhesion between a donor cell and a host T cell).
30 Examples of pl~r~ d adhesion molecules which can be altered according to the invention
include LFA-3 and ICAM-l. These molecules are ligands for the CD2 and LFA-l receptors~
respectively, on T cells. By altering an adhesion molecule on the donor cell, (such as LFA-3.
ICAM-l or a similarly functioning molecule), the ability of the host's T cells to bind to and
interact with the donor cell is reduced. Both LFA-3 and ICAM-l are found on endothelial
35 cells found within blood vessels in transplanted organs such as kidney and heart. Altering
these antigens can facilitate transplantation of any vascularized implant, by altering
recognition of those antigens by CD2+ and LFA-l+ host T-lymphocytes.
The presence of MHC molecules or adhesion molecules such as LFA-3, ICAM- 1 etc.
on a particular donor cell can be ~e~se~l by standard procedures known in the art. For
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example, the donor cell can be reacted with a labeled antibody directed against the molecule
to be detected (e.g., MHC molecule, ICAM-l, LFA-l etc.) and the association ofthe labeled
antibody with the cell can be measured by a suitable technique (e.g., immunohistochemi~try,
flow cytometry etc.).
S A ~l~f~ d method for altering an antigen on a donor cell to inhibit an immune
response against the cell is to contact the cell with a molecule which binds to the antigen on
the cell surface. It is pl~rell~d that the cell be contacted with the molecule which binds to the
antigen prior to ~flmini~tering the cell to a recipient (i.e., the cell is contacted with the
molecule in vitro). For example, the cell can be incubated with the molecule which binds the
10 antigen under conditions which allow binding of the molecule to the antigen and then any
unbound molecule can be removed. Following ~-lmini~tration of the modified cell to a
recipient, the molecule remains bound to the antigen on the cell for a sufficient time to
illLelr~lc; with immllnnlogical recognition by host cells and induce non-responsiveness in the
recipient.
Preferably, the molecule for binding to an antigen on a donor cell is an antibody, or
fragment or derivative thereof which retains the ability to bind to the antigen. For use in
therapeutic applications, it is necess~ry that the antibody which binds the antigen to be altered
be unable to fix complement, thus ~l~;vellli~lg donor cell lysis. Antibody complement fix~tinn
can be prevented by deletion of an Fc portion of an antibody, by using an antibody isotype
which is not capable of fixing complement, or by using a complement fixing antibody in
conjunction with a drug which inhibits complement fixation. Alternatively, amino acid
residues within the Fc region which are necessaly for activating complement (see e.g., Tan et
al. (1990) Proc. Natl. Acad. Sci. USA 87:162-166; Duncan and Winter (1988) Nature 332:
738-740) can be mllt~tecl to reduce or elimin~te the complement-activating ability of an intact
antibody. Likewise, amino acids residues within the Fc region which are neces~y for
binding of the Fc region to Fc receptors (see e.g, Canfield, S.M. and S.L. Morrison (1991) J.
~xp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. Immunol. 147:2657-2662) can also be
mutated to reduce or elimin~te Fc receptor binding if an intact antibody is to be used.
A preferred antibody fragment for altering an antigen is an F(ab')2 fr~gment
Antibodies can be fr~gment~l using conventional techniques. For exarnple, the Fc portion of
an antibody can be removed by treating an intact antibody with pepsin, thereby generating an
F(ab')2 fragment. In a standard procedure for generating F(ab')2 fragments, intact antibodies
are incubated with immobilized pepsin and the digested antibody mixture is applied to an
immobilized protein A column. The free Fc portion binds to the column while the F(ab')2
fragments passes through the colurnn. The F(ab')2 fragments can be further purified by
HPLC or FPLC. F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab'
fr~gment~.
An antibody, or fragment or derivative thereof, to be used to alter an antigen can be
derived from polyclonal antisera cont~inin~ antibodies reactive with a number of epitopes on
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an antigen. Preferably, the antibody is a monoclonal antibody directed against the antigen.
Polyclonal and monoclonal antibodies can be prepared by standard techniques known in the
art. For example, a m~mmzll, (e.g., a mouse, hs~m~ter, or rabbit) can be immllni7~(1 with the
antigen or with a cell which expresses the antigen (e.g., on the cell surface) to elicit an
antibody response against the antigen in the m~mm~h Alternatively, tissue or a whole organ
which expresses the antigen can be used to elicit antibodies. The progress of i"""l~"iz~tion
can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or
other immnnn~ qy can be used with the antigen to assess the levels of antibodies. Following
i" " """i7~tion, antisera can be obtained and, if desired, polyclonal antibodies isolated from
10 the sera. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be
harvested from an immunized animal and fused with myeloma cells by standard somatic cell
fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art. For example, the hybridoma technique originally
developed by Kohler and Milstein ((1975) Nature 256:495-497) as well as other techniques
15 such as the human B-cell hybridoma technique (Kozbar et al., (1983) Immunol. Today 4:72),
and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al.
(1985) Monoclonal Antibodies in Cancer Therapy, Allen R. Bliss, Inc., pages 77-96) can be
used. Hybridoma cells can be screened immunochemically for production of antibodies
specifically reactive with the antigen and monoclonal antibodies isolated.
Another method of generating specific antibodies, or antibody fr~gm~onte, reactive
against the antigen is to screen expression libraries encoding immlmoglobulin genes, or
portions thereof, expressed in bacteria with the antigen (or a portion thereof). For exarnple,
complete Fab fr~gment~, VH regions, Fv regions and single chain antibodies can be
expressed in b~rtt~ri~ using phage e~rt:s~ion libraries. See e.g, Ward et al., (1989) Nature
25 341:544-546, Huse et al., (1989) Science 246:1275-1281; and McCafferty et al. (1990)
Nature 348:552-554. ~ltern~tively, a SCID-hu mouse can be used to produce antibodies, or
fr~ment~ thereof (available from Genpharm). Antibodies of the a~pl~pl;ate binding
specificity which are made by these techniques can be used to alter an antigen on a donor
cell.
An antibody, or fragment thereof, produced in a non-human subject can be recognized
to varying degrees as foreign when the antibody is ~lmini~tered to a human subject (e.g.,
when a donor cell with an antibody bound thereto is ~lmini~t~red to a human subject) and an
immune response against the antibody may be generated in the subject. One approach for
minimi7ing or elimin~ting this problem is to produce chimeric or hl-m~ni7~d antibody
35 derivatives, i.e., antibody molecules comprising portions which are derived from non-human
antibodies and portions which are derived from human antibodies. Chimeric antibody
molecules can include, for example, an antigen binding domain from an antibody of a mouse.
rat, or other species, with human constant regions. A variety of approaches for making
chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci.
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U.S.A. 81, 6851 (1985), Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent ~o.
4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent
Publication EP171496, European Patent Publication 0173494, United Kingdom Patent GB
2177096B. For use in therapeutic applications, it is preferred that an antibody used to alter a
donor cell antigen not contain an Fc portion. Thus, a hllm~ni7~1 F(ab')2 fragment in which
parts of the variable region of the antibody, especially the conserved framework regions of
the antigen-binding domain, are of human origin and only the hypervariable regions are of
non-human origin is a ~l~fe-l~d antibody derivative. Such altered immllnoglobulin
molecules can be made by any of several techniques known in the art, (e.g., Teng et al., Proc.
0 Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279
(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and are preferably made according to
the te~chin~s of PCT Publication WO92/06193 or EP 0239400. Hllm~ni7~cl antibodies can
be commercially produced by, for example, Scotgen T imite-l, 2 Holly Road, Twickenham,
Middlesex, Great Britain.
Each of the cell surface antigens to be altered, e.g., MHC class I antigens, MHC class
II antigens, LFA-3 and ICAM-1 are well-characterized molecules and antibodies to these
antigens are commercially available. For example, an antibody directed against human MHC
class I antigens (i.e., an anti-HLA class I antibody),W6/32, is available from the American
Type Culture Collection (ATCC HB 95). This antibody was raised against human tonsillar
20 lymphocyte membranes and binds to HLA-A, HLA-B and HLA-C (R~ t~kle, C.J. et al.
(1978) Cell 14:9-20). Another anti-MHC class I antibody which can be used is PT85 (see
Davis, W.C. et al. (1984) Hybridoma Technology in Agricultural and Veterinary Research.
N.J. Stern and H.R. Gamble, eds., Rownman and Allenheld Publishers, Totowa, NJ, pl21;
commercially available from Veterinary Medicine Research Development, Pullman, WA).
25 This antibody was raised against swine leukocyte antigens (SLA) and binds to class I
antigens from several different species (e.g., pig, human, mouse, goat). An anti-ICAM-1
antibody can be obtained from AMAC, Inc., Maine. Hybridoma cells producing anti-LFA-3
can be obtained from the American Type Culture Collection, Rockville, Maryland.
A suitable antibody, or fragment or derivative thereof, for use in the invention can be
30 iclentified based upon its ability to inhibit the immunological rejection of allogeneic or
xenogeneic cells. Briefly, the antibody (or antibody fragment) is incubated for a short period
of time (e.g., 30 minlltec at room temperature) with cells or tissue to be transplanted and any
unbound antibody is washed away. The cells or tissue are then transplanted into a recipient
animal. The ability of the antibody pretre~tment to inhibit or prevent rejection of the
35 transplanted cells or tissue is then determined by monitoring for rejection of the cells or tissue
compared to untreated controls.
It is p.~f~ d that an antibody, or fragment or derivative thereof, which is used to
alter an antigen have an affinity for binding to the antigen of at least 10-7 M. The affinity of
an antibody or other molecule for binding to an antigen can be determined by conventional
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techniques (see Masan, D.W. and Williams, A.F. (1980) Biochem. J. 187:1-10). Briefly, the
antibody to be tested is labeled with l25I and incubated with cells ex~lc~ing the antigen at
increasing concentrations until equilibrium is reached. Data are plotted graphically as [bound
antibody]/~free antibody] versus [bound antibody] and the slope of the line is equal to the kD
(Scatchard analysis).
Other molecules which bind to an antigen on a donor cell and produce a functionally
similar result as antibodies, or fr~gmente or derivatives thereof, (e.g., other molecules which
illlc.r~ le with the interaction of the antigen with a hematopoietic cell and induce
immunological nol,.cs~o.lsiveness) can be used to alter the antigen on the donor cell. One
such molecule is a soluble form of a ligand for an antigen (e.g., a receptor) on the donor cell
which could be used to alter the antigen on the donor cell. For exarnple, a soluble form of
CD2 (i.e., comprising the extracellular domain of CD2 without the tr~nememkrane or
cytoplasmic domain) can be used to alter LFA-3 on the donor cell by binding to LFA-3 on
donor cells in a manner analogous to an antibody. Alternatively, a soluble form of LFA- 1
can be used to alter ICAM-l on the donor cell. A soluble form of a ligand can be made by
standard recombinant DNA procedures, using a recombinant cx~cssion vector co~
DNA encoding the ligand encompaeein~ an extracellular domain (i.e., lacking DNA encoding
the tr~nemembrane and cytoplasmic domains). The recombinant expression vector encoding
the extracellular domain of the ligand can be introduced into host cells to produce a soluble
ligand, which can then be isolated. Soluble ligands of use have a binding affinity for the
receptor on the donor cell sufficient to remain bound to the receptor to i-lL~-r~le with
immunological recognition and induce non-responsiveness when the cell is ~(1mini~tered to a
recipient (e.g., preferably, the affinity for binding of the soluble ligand to the receptor is at
least about 10-7 M). Additionally, the soluble ligand can be in the form of a fusion protein
comprising the receptor binding portion of the ligand fused to another protein or portion of a
protein. For example, an immunoglobulin fusion protein which includes an extracellular
domain, or functional portion of CD2 or LFA-l linked to an immunoglobulin heavy chain
constant region (e.g., the hinge, CH2 and CH3 regions of a human immunoglobulin such as
IgGl) can be used. Tmmlmoglobulin fusion proteins can be prepared~ for example, according
to the te~t~hinr~ of Capon, D.J. et al. (1989) Nature 337:525-531 and U.S. Patent No.
5,116,964 to Capon and Lasky.
Another type of molecule which can be used to alter an MHC antigen (e.g., and MHC
- class I antigen) is a peptide which binds to the MHC antigen and i~c~rt;~cs with the
interaction of the MHC antigen with a T lymphocyte, NK cell, or LAK cell. In one- 35 embodiment, the soluble peptide mimics a region of the T cell receptor which contacts the
MHC antigen. This peptide can be used to interfere with the interaction of the intact T cell
receptor (on a T lymphocyte) with the MHC antigen. Such a peptide binds to a region of the
MHC molecule which is specifically recognized by a portion of the T cell receptor (e.g., the
alpha-l or alpha-2 domain of an MHC class I antigen), thereby altering the MHC class I
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- 12-
antigen and inhibiting recognition of the antigen by the T cell receptor. In another
embodiment, the soluble peptide mimics a region of a T cell surface molecule which contacts
the MHC antigen, such as a region of the CD8 molecule which contacts an MHC class I
antigen or a region of a CD4 molecule which contacts an MHC class II antigen. For
5 example, a peptide which binds to a region of the alpha-3 loop of an MHC class I antigen can
be used to inhibit binding to CD8 to the antigen, thereby inhibiting recognition of the antigen
by T cells. T cell receptor-derived peptides have been used to inhibit MHC class I-restricted
immune responses (see e.g., Clayberger, C. et al. (1993) TransplantProc. 25:477-478) and
prolong allogeneic skin graft survival in vivo when injected subcutaneously into the recipient
(see e.g., Goss, J.A. et al. (1993) Proc. Natl. Acad. Sci. USA 90:9872-9876).
An antigen on a donor cell further can be altered by using two or more moleculeswhich bind to the same or different antigen. For example, two dirrt;~ lL antibodies with
specificity for two dirrelelll epitopes on the same antigen can be used (e.g., two different anti-
MHC class I antibodies can be used in combination). Alternatively, two dirr~,c;.ll types of
15 molecules which bind to the same antigen can be used (e.g., an anti-MHC class I antibody
and an MHC~ class I-binding peptide). A ~c;r~ ;d combination of anti-MHC class Iantibodies which can be used with human cells is the W6/32 antibody and the PT85 antibody
or F(ab')2 fr~gment~ thereof. When the donor cell to be ~ mini~t~red to a subject bears more
than one hematopoietic cell-interactive antigen, two or more tre~tment~ can be used together.
20 For exarnple, two antibodies, each directed against a different antigen (eg., an anti-MHC class
I antibody and an anti-ICAM-l antibody) can be used in combination or two different types
of molecules, each binding to a different antigen, can be used (e.g., an anti-ICAM-l antibody
and an MHC class I-binding peptide). ~ltern~tively, polyclonal antisera generated against
the entire donor cell or tissue co~ donor cells can be used, following removal of the Fc
25 region, to alter multiple cell surface antigens of the donor cells.
The ability of two dirre,cnl monoclonal antibodies which bind to the same antigen tO
bind to dirrelell~ epitopes on the antigen can be ~ietermined using a competition binding
assay. Briefly, one monoclonal antibody is labeled and used to stain cells which express the
antigen. The ability of the unlabeled second monoclonal antibody to inhibit the binding of
30 the first labeled monoclonal antibody to the antigen on the cells is then assessed. If the
second monoclonal antibody binds to a different epitope on the antigen than does the first
antibody7 the second antibody will be unable to competitively inhibit the binding of the first
antibody to the antigen.
A preferred method for altering at least two different epitopes on an antigen on a
35 donor cell to inhibit an immune response a~ainst the cell is to contact the cell with at least
two different molecules which bind to the epitopes. It is preferred that the cell be contacted
with at least two different molecules which bind to the dirrt:l~llL epitopes prior to
~flmini~tcring the cell to a recipient (i.e., the cell is contacted with the molecule in vitro). For
example, the cell can be incubated with the molecules which bind to the epitopes under
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-13-
conditions which allow binding of the molecules to the epitopes and then any unbound
molecules can be removed. Following zlt1mini~tration of the donor cell to a recipient, the
molecules remain bound to the epitopes on the surface antigen for a sufficient time to
hll~lrer~ with immlmnlogical recognition by host cells and induce non-responsiveness in the
recipient.
.~ltern~tive to binding a molecule (e.g., an antibody) to an antigen on a donor cell to
inhibit immunological rejection of the cell, the antigen on the donor cell can be altered by
other means. For example, the antigen can be directly altered (e.g., mllt~t~l) such that it can
no longer interact normally with an immune cell, e.g., a T lymphocyte), an NK cell, or an
LAK cell, in an allogeneic or xenogeneic recipient and intll1cçs immlmological non-
responsiveness to the donor cell in the recipient. For example, a mutated form of a class I
MHC antigen or adhesion molecule (e.g., LFA-3 or ICAM-l) which does not contribute to T
cell activation but rather delivers an ina~,u~liate or insufficient signal to a T cell upon
binding to a receptor on the T cell can be created by mutagenesis and selection. A nucleic
acid encoding the m~lt~tecl form of the antigen can then be inserted into the genome of a non-
human animal, either as a transgene or by homologous recombination (to replace the
endogenous gene encoding the wild-type antigen). Cells from the non-human animal which
express the mut~tecl form of the antigen can then be used as donor cells for tr~n~pl~nt~tion
into an allogeneic or xenogeneic recipient.
.Alt~rn~tively, an antigen on the donor cell can be altered by downmotl~ tin~ or:~lt~.rin~ its level of ~:x~-es~ion on the surface of the donor cell such that the interaction
between the antigen and a recipient immlme cell is modified. By decreasing the level of
surface expression of one or more antigens on the donor cell, the avidity of the interaction
between the donor cell and the immllne cell e.g., T lymphocyte, NK cell, LAK cell, is
reclllce~l The level of surface expression of an antigen on the donor cell can be down-
modulated by inhibiting the transcription, translation or transport of the antigen to the cell
surface. Agents which decrease surface ex~les~,ion of the antigen can be contacted with the
donor cell. For example, a number of oncogenic viruses have been demonstrated to decrease
MHC class I e~ ssion in infected cells (see e.g, Travers et al. (1980) Int'l. Symp. on Aging
in Cancer, 175180; Rees et al. (1988) Br. .~: Cancer, 57:374-377). In addition, it has been
found that this effect on MHC class I expression can be achieved using fr~gment~ of viral
genomes, in addition to intact virus. For example, transfection of cultured kidney cells with
fragments of adenovirus causes eliminsltion of surface MHC class I antigenic expression
(Whoshi et al. (1988) J. Exp. Med. 168:2153-2164). For purposes of decreasing MHC class I
- 35 ~ es~,ion on the surfaces of donor cells, viral fragments which are non-infectious are
preferable to whole viruses.
Alternatively, the level of an antigen on the donor cell surface can be altered by
capping the antigen. Capping is a term referring to the use of antibodies to cause aggregation
and inactivation of surface antigens. To induce capping, a tissue is contacted with a first
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antibody specific for an antigen to be altered, to allow forrnation of antigen-antibody immllne
complexes. Subsequently, the tissue is contacted with a second antibody which forms
immllnP complexes with the first antibody. As a result of tre~tment with the second antibody,
the first antibody is aggregated to form a cap at a single location on the cell surface. The
technique of capping is well known and has been described, e.g., in Taylor et al. (1971), Nat.
New Biol. 233:225-227; and Santiso et al. (i986), Blood, 67:343-349. To alter MHC class I
antigens, donor cells are incubated with a first antibody (e.g., W6/32 antibody, PT85
antibody) reactive with MHC class I molecules, followed by incubation with a second
antibody reactive with the donor species, e.g., goat anti-mouse antibody, to result in
1 0 aggregation.
In yet another embodiment, cells which are ~lmini~tered to a subject according to the
methods of the invention are genetically modified to express a gene product. The genetically
modified cells can be transplanted into a recipient subject to deliver the gene product to the
subject. Cells can be genetically modified to express a gene product by introducing nucleic
acid encoding the gene product into the cell. For example, a cell can be infected with a
recombinant virus (e.g., rekovirus, adenovirus) which contains the nucleic acid of interest. A
non-human cell which is genetically modified to express a human gene product can be used
to deliver the human gene product to a human subject by altering at least one antigen on the
surface of the non-human cell and transplanting the cell into the recipient subject.
A cell can modified to express a gene product by introducing genetic m~t~ri~l, such as
a nucleic acid molecule (e.g., RNA or, more preferably, DNA) into the cell. The nucleic acid
molecule introduced into the cell encodes a gene product to be expressed by the cell. The
term "gene product" as used herein is inten~ cl to include proteins, peptides and functional
RNA molecules. Generally, the gene product encoded by the nucleic acid molecule is the
desired gene product to be supplied to a subject. Alternatively, the encoded gene product is
one which induces the e~ ;ssion of the desired gene product by the cell (e.g., the introduced
genetic material encodes a transcription factor which induces the transcription of the gene
product to be supplied to the subject).
A nucleic acid molecule introduced into a cell is in a forrn suitable for expression in
the cell of the gene product encoded by the nucleic acid. Accordingly, the nucleic acid
molecule includes coding and regulatory sequences required for transcription of a gene (or
portion thereof) and, when the gene product is a protein or peptide, translation of the gene
product encoded by the gene. Regulatory sequences which can be included in the nucleic -
acid molecule include promoters, enhancers and polyadenylation signals, as well as
sequences necessary for transport of an encoded protein or peptide. for exarnple N-terminal
signal sequences for transport of proteins or peptides to the surface of the cell or for secretion.
Nucleotide sequences which regulate expression of a gene product (e.g., prornoter and
enhancer sequences) are selected based upon the type of cell in which the gene product is to
be expressed and the desired level of expression of the gene product. For example~ a
-
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- 15-
promoter known to confer cell-type specific ex~res~ion of a gene linked to the promoter can
be used. A promoter specific for myoblast gene ~x~l~s~ion can be linked to a gene of interest
to confer muscle-specific ~x~les~ion of that gene product. Muscle-specific regulatory
elements which are known in the art include upstream regions from the dystrophin gene
(Klamut et al. (1989) Mol. Cell. Biol. 9:2396), the creatine kinase gene (Buskin and
~ çhk~ (1989) Mol. Cell Biol. 9:2627) and the troponin gene (Mar and Ordahl (1988)
Proc. Natl. Acad. Sci. USA. 85:6404). Regulatory elements specific for other cell types are
known in the art (e.g., the albumin çnh~nc er for liver-specific ~xl)lc;ssion; insulin regulatory
elemçnt~ for pancreatic islet cell-specific ~ ession; various neural cell-specific re~ulatory
10 element~, incl~ ing neural dystrophin, neural enolase and A4 amyloid promoters).
.Alt~ tively, a regulatory element which can direct constitutive ~x~lession of a gene in a
variety of dir~elell~ cell types, such as a viral regulatory element, can be used. Examples of
viral promoters commonly used to drive gene expression include those derived from polyoma
virus, Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
15 ~ ltern~tively, a regulatory element which provides inducible ~ Lession of a gene linked
thereto can be used. The use of an inducible regulatory element (e.g., an inducible promoter)
allows for modulation of the production of the gene product in the cell. Examples of
potentially useful inducible regulatory systems for use in eukaryotic cells include hormone-
regulated elements (e.g., see Mader, S. and White, J.H. (1993) Proc. Natl. Acad. Sci. US~l
90:5603-5607), synthetic ligand-regulated elements (see, e.g. Spencer, D.M. et al. (1993)
Science 262:1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et
al. (1993) Biochemistry 32 ro607-l06l3; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA
89:10149-10153). Additional tissue-specific or inducible regulatory systems which may be
developed can also be used in accordance with the invention.
There are a number of techniques known in the art for introducing genetic material
into a cell that can be applied to modify a cell of the invention. In one embodiment, the
nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid
molecule introduced into a cell to be modified consists only of the nucleic acid encoding the
gene product and the nPcçc~ry regulatory elements. Alternatively, the nucleic acid encoding
the gene product (including the necessary regulatory elements) is contained within a plasmid
vector. Examples of plasmid expression vectors include CDM8 (Seed, B., Nature 329:840
(1987)) and pMT2PC (~nfrn~n, et al., EMBO J. 6:187-195 (1987)). In another
~ embodiment, the nucleic acid molecule to be introduced into a cell is contained within a viral
vector. In this situation, the nucleic acid encoding the gene product is inserted into the viral
genome (or a partial viral genome). The regulatory elements directing the expression of the
gene product can be included with the nucleic acid inserted into the viral genome (i.e.. linked
to the gene inserted into the viral genome) or can be provided by the viral genome itself.
Examples of methods which can be used to introduce naked nucleic acid into cells and viral-
mediated transfer of nucleic acid into cells are described separately in the subsections below.
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-16-
A. Tntroduction of Naked Nucleic Acid into Cells
1. Transfection m~ 7ted by CaP04: Naked DNA can be introduced into cells by
5 forming a precipitate co~,t~ g the DNA and calcium phosphate. For example, a HEPES-
buffered saline solution can be mixed with a solution col~ calcium chloride and DNA
to form a precipitate and the precipitate is then incubated with cells. A glycerol or dimethyl
sulfoxide shock step can be added to increase the amount of DNA taken up by certain cells.
CaPO4-mediated transfection can be used to stably (or transiently) transfect cells and is only
10 applicable to in vitro modification of cells. Protocols for CaPO4- mccli~te~l transfection can
be found in Current Protocols in Molecular Biolo~y~ Ausubel, F.M. et al. (eds.) Greene
Publishing Associates, (1989), Section 9.1 and in Molecular Clonin~: A Laboratorv Manual.
2nd Fdition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.32-
16.40 or other standard laboratory m~n
2. Transfection m~inted by DE~E-dextran: Naked DNA can be introduced into
cells by forming a lllixlul~ of the DNA and DEAE-dextran and inrllb~ting the mixLulc with
the cells. A dimethylsulfoxide or chloroquine shock step can be added to increase the amount
of DNA uptake. DEAE-dextran transfection is only applicable to in vitro modification of
20 cells and can be used to introduce DNA transiently into cells but is not ~.c~.lcd for creating
stably transfected cells. Thus, this method can be used for short term production of a gene
product but is not a method of choice for long-term production of a gene product. Protocols
for DEAE-dextran-mediated transfection can be found in Cu~rent Protocols in Molecular
Biolo~y, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.2 and in
25 Molecular C~onin~: A Laboratory Manll~l 2nd Fdition. Sambrook et al. Cold Spring Harbor
Laboratory Press, (1989), Sections 16.41-16.46 or other standard laboratory mzlnll~l~
3. Electroporation: Naked DNA can also be introduced into cells by incubating the
cells and the DNA together in an al~l,lop~iate buffer and subjecting the cells to a high-voltage
30 electric pulse. The efficiency with which DNA is introduced into cells by electroporation is
influenced by the strength of the applied field, the length of the electric pulse, the
temperature, the conformation and concentration of the DN~ and the ionic composition of
the media. Electroporation can be used to stably (or transiently) transfect a wide variety of
cell types and is only applicable to in vitro modification of cells. Protocols for
35 electroporating cells can be found in Current Protocols ;n Molecular Biolo~v, Ausubel, F.M.
et al. (eds.) Greene Publishing Associates, (1989), Section 9.3 and in Molecular Clonin~: A
Laboratory Manual. 2nd Edition. Sambrook et al. Cold Spring Harbor Laboratory Press,
(1989), Sections 16.54-16.55 or other standard laboratory manuals.
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4. Liposome-me~inte~ transfection ("lipofection"): Naked DNA can be introduced
into cells by mixing the DNA with a liposome suspension co~ cationic lipids. The
DNA/liposome complex is then incubated with cells. Liposome mediated transfection can be
used to stably (or 1T~n~iently) transfect cells in culture in vitro. Protocols can be found in
Current Protocols ;n Molecular Biolo~y, Ausubel, F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.4 and other standard laboratory m~nl~le Additionally, gene
delivery in vivo has been accomplished using liposomes, See for example Nicolau et al.
(1987) Meth. Enz. 149:157-176; Wang and Huang (1987) Proc. NatL Acad Sci. USA
84:7851-7855; Brigharn et al. (1989) Am. J: Med. Sci. 298:278; and Gould-Fogerite et al.
(1989) Gene 84:429-438.
5. Direct Injection: Naked DNA can be introduced into cells by directly injecting the
DNA into the cells. For an in vitro culture of cells, DNA can be inkoduced by
microinjection. Since each cell is microinjected individually, this approach is very labor
hlLtll~ive when modifying large numbers of cells. However, a situation wherein
microinjection is a method of choice is in the production of kansgenic ~nim~l~ (discussed in
greater detail below). In this situation, the DNA is stably introduced into a fertilized oocyte
which is then allowed to develop into an animal. The resultant animal contains cells carrying
the DNA introduced into the oocyte. Direct injection has also been used to inkoduce naked
DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332: 815-818; Wolffet al. (1990)
Science 247:1465-1468). A delivery al~pal~u~ (e.g., a "gene gun") for injecting DNA into
cells in vivo can be used. Such an a~ us is cornmercially available (e.g., from BioRad).
6. Receptor-Mediated DNA Uptake: Naked DNA can also be inkoduced into cells by
complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-
surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263:14621;
Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of
the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated
endocytosis. Receptors to which a DNA-ligand complex have targeted include the transferrin
receptor and the asialoglyco~ Lehl receptor. A DNA-ligand complex linked to adenovirus
capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can
be used to avoid degradation of the complex by intracellular lysosomes (see for example
Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90:2122-2126). Receptor-mediated DNA uptake can be used to introduce
DNA into cells either in vitro or in vivo and, additionally, has the added feature that DNA can
be selectively targeted to a particular cell type by use of a ligand which binds to a receptor
selectively expressed on a target cell of interest.
Generally, when naked DNA is introduced into cells in culture (e.g., by one of the
transfection techniques described above) only a small fraction of cells (about 1 out of 105)
CA 02217131 1997-10-20
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- 18 -
typically integrate the transfected DNA into their genomes (i.e., the DNA is mslint~ined in the
cell episomally). Thus, in order to identify cells which have taken up exogenous DNA, it is
advantageous to transfect nucleic acid encoding a selectable marker into the cell along with
the nucleic acid(s) of interest. Preferred selectable markers include those which confer
5 resistance to drugs such as G418, hygromycin and methotrexate. Selectable markers may be
introduced on the same plasmid as the gene(s) of interest or may be introduced on a separate
plasmid.
An ~ltt-rn~tive method for generating a cell that is modified to express a gene product
involving introducing naked DNA into cells is to create a transgenic animal which contains
10 cells modified to express the gene product of interest. A transgenic animal is an animal
having cells that contain a transgene, wherein the transgene was introduced into the animal or
an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA
molecule which is integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal, thereby directing the
15 ~ ,ession of an encoded gene product in one or more cell types or tissues of the transgenic
animal. Thus, a transgenic animal e~ e~hlg a gene product of interest in one or more cell
types within the animal can be created, for example, by introducing a nucleic acid encoding
the gene product (typically linked to a~lop.iate regulatory elements, such as a tissue-specific
~nh~n~er) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing
20 the oocyte to develop in a pseudopregnant female foster animal. Methods for generating
transgenic ~nim~l~, particularly ~nim~l~ such as mice, have become conventional in the art
and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and Hogan, B.
et al., (1986) A Labo,~Lo,.~ Manual, Cold Spring Harbor, New York, Cold Spring Harbor
Laboratory. A transgenic founder animal can be used to breed more zlnim~l~ carrying the
25 transgene. Cells of the transgenic animal which express a gene product of interest can then
be used to deliver the gene product to a subject in accordance with the invention.
Alternatively, an animal collf;li"i"g a gene which has been modified by homologous
recombination can be constructed to express a gene product of interest. For example, an
endogenous gene carried in the genome of the animal can be altered by homologousrecombination (for instance, all or a portion of a gene could be replaced by the human
homologue of the gene to "hl-m~ni7.?" the gene product encoded by the gene) or an
endogenous gene can be "knocked out" (i.e., inactivated by mutation). For example, an
endogenous gene in a cell can be knocked out to prevent production of that gene product and
then nucleic acid encoding a dirrt;l~nt (preferred) gene product is introduced into the cell. To
35 create an animal with homologously recombined nucleic acid, a vector is prepared which
contains the DNA which is to replace or interrupt the endogenous DNA flanked by DNA
homologous to the endogenous DNA (see for example Thomas, K.R. and Capecchi. M. R.
(1987) Cell 51:503). The vector is introduced into an embryonal stem cell line (e.g.~ by
electroporation) and cells which have homologously recombined the DNA are selected (see
-
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- 19-
for example Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see for example
Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J.
Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
S into a suitable pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ cells can be used to
breed ~nim~l~ in which all cells of the animal contain the homologously recombined DNA.
Cells of the animal co~ the homologously recombined DNA which express a gene
product of interest can then be used to deliver the gene product to a subject in accordance
with the invention.
13. Viral-Mediated Gene Transfer
A ~lc;r~;lled approach for introducing nucleic acid encoding a gene product into a cell
is by use of a viral vector co~ g nucleic acid, e.g. a cDNA, encoding the gene product.
Infection of cells with a viral vector has the advantage that a large ~lOpOl lion of cells receive
the nucleic acid, which can obviate the need for selection of cells which have received the
nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA
c(-nt~int?~ in the viral vector, are expressed efficiently in cells which have taken up viral
vector nucleic acid and viral vector systems can be used either in vitro or in vivo.
1. Retroviruses: Defective retroviruses are well characterized for use in gene transfer
for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Arecombinant retrovirus can be constructed having a nucleic acid encoding a gene product of
interest inserted into the l~,LloVildl genome. Additionally, portions of the retroviral genome
can be removed to render the retrovirus replication defective. The replication defective
retrovirus is then packaged into virions which can be used to infect a target cell through the
use of a helper virus by standard techniques. Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in
Current Protocols in Molecular Biolo~y~ Ausubel, F.M. et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard laboratory m~nt~ . Examples of
suitable rekoviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled
in the art. Examples of suitable p~ ing virus lines include ~Crip, ~Cre, y/2 and ~Am.
Retroviruses have been used to introduce a variety of ~enes into many dirr~;lGll~ cell types,
including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone
- 35 marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-
1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.
(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad.
Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry
et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science
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254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644; Kay
et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al. (1993) J: ImmunoZ. 150:4104-4115; U.S. PatentNo. 4,868,116;
U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468;
PCT Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors
require target cell division in order for the retroviral genome (and foreign nucleic acid
inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the
cell. Thus, it may be n~cç~ry to stim~ tç replication of the target cell.
2. Adenoviruses: The genome of an adenovirus can be manipulated such that it
encodes and expresses a gene product of interest but is inactivated in terms of its ability to
replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al.
(1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad
15 type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those
skilled in the art. Recombinant adenoviruses are advantageous in that they do not require
dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety
of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial
cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz
20 and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al.
(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the genome of a host cell but
remains episomal, thereby avoiding potential problems that can occur as a result of
insertional mutagenesis in situations where introduced DNA becomes integrated into the host
25 genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for
foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al.
cited supra; Haj-Ahmand and Graham (1986) J: Virol. 57:267). Most replication-defective
adenoviral vectors ~;w,~ in use are deleted for all or parts of the viral E1 and E3 genes but
retain as much as 80% of the adenoviral genetic material.
3. Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturally occurring
defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper
virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al.
Curr. TopicsinMicro. andImmunol. (1992) 158:97-129). Itisalsooneofthefewviruses
35 that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable
integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;
,S~mlllski et al. (1989) J: Virol. 63:3822-3828; and McT allghlin et al. (1989) J. Virol.
62: 1963-1973). Vectors cont~ining as little as 300 base pairs of AAV can be packaged and
can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as
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that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce
DNA into cells. A variety of nucleic acids have been introduced into diLL~"~;,.t cell types
using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. 'Sci. USA
81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081, Wondisford et al. (1988)
Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611 -619; and Flotte et al.
(1993) ~ Biol. Chem. 268:3781-3790).
The efficacy of a particular expression vector system and method of introducing
nucleic acid into a cell can be ~cses~e~l by standard approaches routinely used in the art. For
example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g.,
10 Southern blotting) and RNA produced by transcription of introduced DNA can be detected,
for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase
chain reaction (RT-PCR). The gene product can be detected by an a~pro~liate assay, for
example by immunological detection of a produced protein, such as with a specific antibody,
or by a functional assay to detect a functional activity of the gene product, such as an
15 enzymatic assay. If the gene product of interest to be expressed by a cell is not readily
assayable, an ~x~lession system can first be optimized using a reporter gene linked to the
regulatory elements and vector to be used. The reporter gene encodes a gene product which
is easily detectable and, thus, can be used to evaluate the efficacy of the system. Standard
reporter genes used in the art include genes encoding ,~-galactosidase, chloramphenicol acetyl
20 transferase, luciferase and human growth hormone.
When the method used to introduce nucleic acid into a population of cells results in
modification of a large proportion of the cells and efficient expression of the gene product by
the cells (e.g., as is often the case when using a viral e~ylession vector), the modified
population of cells may be used without further isolation or subcloning of individual cells
25 within the population. That is, there may be sufficient production of the gene product by the
population of cells such that no further cell isolation is needed. Alternatively, it may be
desirable to grow a homogenous population of identically modified cells from a single
modified cell to isolate cells which efficiently express the gene product. Such a population of
uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning
30 followed by exp~n~lin~ the single cell in culture into a clonal population of cells by standard
techniques.
C. OtherMethods for Modifyin~ a Cell to Fxpress a Gene Product
Alternative to introducing a nucleic acid molecule into a cell to modify the cell to
35 express a gene product, a cell can be modified by inducing or increasing the level of
expression of the gene product by a cell. For example, a cell may be capable of expressing a
particular gene product but fails to do so without additional treatment of the cell. Similarly~
the cell may express insufficient amounts of the gene product for the desired purpose. Thus.
an agent which stim~ te~ expression of a gene product can be used to induce or increase
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expression of a gene product by the cell. For example, cells can be contacted with an agent in
vitro in a culture medium. The agent which stimulates expression of a gene product may
function, for instance, by increasing transcription of the gene encoding the product, by
increasing the rate of translation or stability (e.g., a post transcriptional modification such as a
poly A tail) of an mRNA encoding the product or by increasing stability, transport or
localization of the gene product. Examples of agents which can be used to induce expression
of a gene product include cytokines and growth factors.
Another type of agent which can be used to induce or increase expression of a gene
product by a cell is a transcription factor which upregulates transcription of the gene
10 encoding the product. A transcription factor which upregulates the ~ es~,iOn of a gene
encoding a gene product of interest can be provided to a cell, for example, by introducing into
the cell a nucleic acid molecule encoding the transcription factor. Thus, this approach
represents an alternative type of nucleic acid molecule which can be introduced into the cell
(for example by one of the previously ~ cucsed methods). In this case, the introduced
15 nucleic acid does not directly encode the gene product of interest but rather causes production
of the gene product by the cell indirectly by in~ ing ~lcssion of the gene product.
In yet another method, a cell is modified to express a gene product by coupling the
gene product to the cell, preferably to the surface of the cell. For example, a protein can be
obtained by purifying the cell from a biological source or expressing the protein
20 recombinantly using standard recombinant DNA technology. The isolated protein can then
be coupled to the cell. The terms "coupled" or "coupling" refer to a chemical, enzymatic or
other means (e.g., by binding to an antibody on the surface of the cell or genetic engineering
of linkages) by which a gene product can be linked to a cell such that the gene product is in a
form suitable for delivering the gene product to a subject. For example, a protein can be
~5 chemically crosslinked to a cell surface using commercially available crosslinkin~ reagents
(Pierce, Rockford IL). Other approaches to coupling a gene product to a cell include the use
of a bispecific antibody which binds both the gene product and a cell-surface molecule on the
cell or modification of the gene product to include a lipophilic tail (e.g., by inositol phosphate
linkage) which can insert into a cell membrane.
In yet another embodiment, a recipient subject into which altered cells of the
invention are transplanted is also treated with a T cell inhibitory agent to further inhibit
rejection of the transplanted cells. The T cell inhibitory agent inhibits T cell activity. For
example, the T cell inhibitory agent can be an immunosuppressive drug. A preferred
immunosuppressive drug is cyclosporin A. Other immunosuppressive drugs which can be
35 used include FK506 and RS-61443. Such immunosuppressive drugs can be used in
conjunction with a steroid (e.g., glucocorticoids such as prednisone, methylprednisolone and
dexamethasone) or chemotherapeutic agents (e.g., azathioprine and cyclophosphamide), or
both. Alternatively, the T cell inhibitory agent can be one or more antibodies which deplete
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T cell activity, such as antibodies directed against T cell surface molecules (e.g., anti-CD2,
anti-CD3, anti-CD4 and/or anti-CD8 antibodies).
II. Methods of the Invention
Another aspect of the invention pertains to methods for re~ ing the immunogenicity
of a cell for transplantation wherein the cell has at least one antigen on the cell surface which
stimulates an immlme response against the cell in the recipient subject. These methods
include contacting the cell with at least one molecule which binds to the antigen on the cell
surface such that, when the cell is transplanted into a recipient subject, rejection of the cell is
inhibited. The terrn "cont~rtin~" is intenc1etl to encompass either incubating the cell with the
molecule which binds to the cell surface antigen in vitro or ~imini.~ttqrin~ the molecule which
binds to the cell surface antigen to a subject (e.g., a transplant recipient). In a ~ler~.led
embodiment, it is the NK cell-mediated rejection or the LAK cell-mediated rejection of the
cell which is inhibited. As used herein, the phrase "NK cell-mediated rejection" refers to an
l S imrnune response which can lead to or does lead to rejection of a cell in vivo, or lysis of a cell
in vitro and in which natural killer cells play either a direct or an indirect role. For example,
NK cells can kill target cells by at least two me-~h~ni~ms: antibody dependent cellular
cytotoxicity (ADCC) or antibody independent cellular cytotoxicity. NK cells are
characterized by the t;~ ion of the low affinity receptor for IgG, Fc-gamma-RIII (CD16)
and neural cell adhesion molecule (NCAM, CDS6). Binding of the NK cell CD16 to the Fc
region of an IgG-coated cell results in lysis of the target (ADCC). Cytotoxicity by the
antibody independent mech~nicm does not appear to require CD16 (Trinchieri, G. (1994) J.
Exp. Med. 180:417-421). As used herein, the phrase "LAK cell-me~ ted rejection" refers to
an immlme response which can lead to or does lead to rejection of a cell in vivo, or lysis of a
cell in vitro and in which lymphokine activated killer cells play either a direct or an indirect
role. LAK cells and NK cells share many of the same cell surface markers. LAK cells are
positive for the cell surface markers CD56, CD16, and CD25. LAK cells can be
distinguished from NK cells in that they lyse certain cells types, e.g., Daudi cells (see Figure
9) while NK cells do not. An inhibition of rejection of cells of the present invention refers to
prolongation of the cells' survival or prevention of rejection of the cells. Cells which can be
used in these methods and methods of altering the cells are described in Section I above.
After a cell is modified or altered as described above, the cell can be ~clminicTered to a
recipient. Accordingly, another aspect of the invention pertains to methods for transplanting
a cell into a recipient subject such that rejection of the cell by the recipient subject is
inhibited. As used herein, the term "subject" is intenclerl to include living org~nicmc in which
an immune response is elicited against allogeneic or xenogeneic cells, e.g., mzlmm~lc
preferably hllm~nc Other examples of subjects include monkeys, pigs, dogs, cats, mice, rats,
and transgenic species thereof. A "recipient subject" is a subject into which cells have been
kansplanted or are to be transplanted. A recipient subject can be allogeneic to the
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kansplanted cells (i.e., of the same species) or can be xenogeneic to the transplanted cells
(i.e., of a different species). The methods involve ~timinietering to the subject a cell having at
least one antigen on the cell surface which stimulates an immune response against the cell in
the recipient subject. Prior to transplantation, the cell is modified or altered as described
5 above such that rejection of the cell is inhibited. In pler~lled embodiments, the mech~nieme
of rejection which are inhibited are T cell-me~ te-l, NK cell-mediated, and/or LAK cell-
mediated rejection of the cell.
The cell is ~-iminietered to the subject in an amount and by a route which suitable for
the desired therapeutic result. The cell used in these methods can be within a tissue or organ.
10 Accordingly, in these embo-liment.e, the tissue or organ is transplanted into the recipient by
conventional techniques for transplantation. Acceptance of transplanted cells, tissues or
organs can be ~letermined morphologically (e.g., with skin grafts by ex~mining the
transplanted tissue or by biopsy) or by ~qee~eement of the functional activity of the graft. For
example, acceptance of pancreatic islet cells can be determined by measuring insulin
15 production, acceptance of liver cells can be determin~-l by assessing albumin production and
acceptance of neural cells can be ~letermined by ~eeeeein~ neural cell function. To ~letermine
whether, for example, the mech~niem of rejection that is inhibited is NK cell-mediated
rejection, NK cells can be isolated from the recipient subject's circulation or from a site in or
near the graft (e.g., from a lymph node draining the graft area), or from a tissue section of the
20 graft. The NK cells can then be cultured and their response to cells of the sarne type as those
that were transplanted into the recipient subject can be measured. If the NK cells appear
nonresponsive to the kansplant cells relative to control NK cells or NK cells cultured under
the same conditions, then NK cell-me~ te~l rejection is most likely inhibited. To deterrnine
whether, for example, the merh~niem of rejection that is inhibited is LAK cell-mediated
25 rejection, the above t;x~lhllents can be repeated wherein LAK cells are substituted for NK
cells.
The methods of present invention can include additional in vitro tre~tment of the cells
prior to transplantation and/or additional in vivo tre~tment of the recipient following
transplantation to further inhibit immunological rejection of transplanted cells. For example,
30 an antigen on a donor cell can be altered by using two or more molecules which bind to the
same or different antigen as described in Section I above. In addition, a recipient subject can
be treated prior to, during and/or following transplantation with an agent which inhibits T cell
activity in the subject. The temporal relationship between ~fimini~tration of the cell and
~lminietration of the agent depends in part upon the nature of the agent used to inhibit T cell
35 activity. Typically, the two compositions are ~-lminietered contemporaneously, e.g. within
several days of each other. Preferably, the cell and the agent are ~-lminietered to the subject
simultaneously or the agent is ~timinietered to the subject prior to ~iminietration of the cell.
As used herein, an agent which inhibits T cell activity is defined as an agent which
results in removal (e.g., sequestration) or destruction of T cells within a subject or inhibits T
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cell functions within the subject (i.e., T cells may still be present in the subject but are in a
non-functional state, such that they are unable to proliferate or elicit or ~lrOllll effector
functions, e.g., cytokine production, cytotoxicity etc.). The term "T cell" encompasses
mature peripheral blood T cells lymphocytes. The agent which inhibits T cell activity may
5 also inhibit the activity or maturation of imm~tllre T cells (e.g., thymocytes).
A ~lef~ d agent for use in inhibiting T cell activity in a recipient subject is an
immllnosuppressive drug. The term '~immlml~suppressive drug" is int~ntl~l to include
ph~rm~relltical agents which inhibit or illL~;lr~lc with normal immllne function. A ~ler~lled
immlln~ u~ s~ive drug is cyclosporin A. Other immlmc,suppressive drugs which can be
used include FK506 and RS-61443. In one embodiment, the immnno~u~les~ive drug is5~1minietered in conjunction with at least one other therapeutic agent. Additional therapeutic
agents which can be ~-1minietçred include steroids (e.g., glucocorticoids such as prednisone,
methyl prednisolone and ~e~methasone) and chemoth~ uLic agents (e.g., azathioprine
and cyclosphosphamide). In another embodiment, an immlmQsuppressive drug is
15 ~-1minietered in conjunction with both a steroid and a chemotherapeutic agent. Suitable
immuno~u~piessive drugs are commercially available (e.g., cyclosporin A is available from
Sandoz, Corp., East Hanover, NJ).
An immllno~u~ e drug is ~imini~etered in a formulation which is compatible
with the route of ~tlminietration. Suitable routes of ~rlminietration include intravenous
20 injection (either as a single infusion, multiple infusions or as an intravenous drip over time),
intraperitoneal injection, intr~mllecnl~r injection and oral ~iminietration. For intravenous
injection, the drug can be dissolved in a physiologically acceptable carrier or diluent (e.g., a
buffered saline solution) which is sterile and allows for syringability. Dispersions of drugs
can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
25 Convenient routes of ~-iminietration and carriers for immunosuppressive drugs are known in
the art. For example, cyclosporin A can be ~-lminietered intravenously in a saline solution, or
orally, hlLl~liloneally or illll~lluscularly in olive oil or other suitable carrier or diluent.
An immlmosuppressive drug is ~1minietered to a recipient subject at a dosage
sufficient to achieve the desired therapeutic effect (e.g., inhibition of rejection of transplanted
30 cells). Dosage ranges for immunosuppressive drugs, and other agents which can be
co~tlminietered therewith (e.g., steroids and chemotherapeutic agents), are known in the art
(see e.g., Freed et al. New Engl. J. Med. (1992) 327:1549: Spencer et al. (1992) New Engl. J.
Med. 327:1541; Widner et al. (1992) New Engl. J. Med. 327:1556; Lindvall et al. (1992) Ann.
Neurol. 31: 155; and Lindvall et al. (1992) Arch. Neurol. 46:615). A preferred dosage range
- 35 for immunosuppressive drugs, suitable for treatment of hl-m~ne, is about 1-30 mg/kg of body
weight per day. A preferred dosage range for cyclosporin A is about 1-10 mg/kg of body
weight per day, more preferably about 1-5 mg/kg of body weight per day. Dosages can be
adjusted to m~int~in an optimal level of the immunosuppressive drug in the serum of the
recipient subject. For example. dosages can be adjusted to mzlint~in a preferred serum level
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for cyclosporin A in a human subject of about 100-200 ng/ml. It is to be noted that dosage
values may vary according to factors such as the disease state, age, sex, and weight of the
individual. Dosage regimens may be adjusted over time to provide the o~Lhllulll therapeutic
response according to the individual need and the professional j~ gment of the person
~lmini.ctering or supervising the ~1mini~tration of the compositions, and that the dosage
ranges set forth herein are exemplary only and are not intçncle~l to limit the scope or practice
of the cl~ime-1 composition.
In one embodiment of the invention, an immuno~ les~ive drug is ~lmini.~tered to a
subject transiently for a sufficient time to induce tolerance to the transplanted cells in the
10 subject. Transient ~tlmini~tration of an immuno~u~les~ e drug has been found to induce
long-term graft-specific tolerance in a graft recipient (see Brunson et al. (1991)
Transplantation 52:545; ~-ltchin~on et al. (1981) Transplantation 32:210; Green et al. (1979)
Lancet 2:123; Hall et al. (1985) J. Exp. Med. 162:1683). Administration ofthe drug to the
subject can begin prior to transplantation of the cells into the subject. For example, initiation
15 of drug ~flmini.~tration can be a few days (e.g., one to three days) before transplantation.
Alternatively, drug ~-imini.~tration can begin the day of transplantation or a few days
(generally not more than three days) after transplantation. Administration of the drug is
continl~?cl for sufficient time to induce donor cell-specific tolerance in the recipient such that
donor cells will continue to be accepted by the recipient when drug ~11mini.~tration ceases.
20 For example, the drug can be ~lmini~tered for as short as three days or as long as three
months following transplantation. Typically, the drug is ~lmini~tered for at least one week
but not more than one month following transplantation. Induction of tolerance to the
transplanted cells in a subject is indicated by the continued acceptance of the transplanted
cells after ~tlmini~tration of the immlln~u~ ;s~i~e drug has ceased. Acceptance of
25 transplanted tissue can be determine~l morphologically (e.g., with skin grafts by ex~mining
the transplanted tissue or by biopsy) or by ~e~e~Sment of the functional activity of the graft.
For example, acceptance of pancreatic islet cells can be deterrnined by measuring insulin
production, acceptance of liver cells can be cl~L~. " ,i"ed by ~.ses~ing liver function or
acceptance of neural cells can be det~rmined by assessing neural cell function.
Another type of agent which can be used to inhibit T cell activity in a subject is an
antibody, or fragment or derivative thereof, which depletes or sequesters T cells in a
recipient. Antibodies which are capable of depleting or sequestering T cells in vivo when
~rlministered to a subject are known in the art. Typically, these antibodies bind to an antigen
on the surface of a T cell. Polyclonal antisera can be used, for example anti-lymphocyte
35 serum. Alternatively, one or more monoclonal antibodies can be used. Preferred T cell-
depleting antibodies include monoclonal antibodies which bind to CD2, CD3, CD4 or CD8
on the surface of T cells. Antibodies which bind to these antigens are known in the art and
are available (e.g., from American Type Culture Collection). A preferred monoclonal
antibody for binding to CD3 on human T cells is OKT3 (ATCC CRL 8001). The binding of
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an antibody to surface antigens on a T cell can facilitate sequestration of T cells in a subject
and/or destruction of T cells in a subject by endogenous mech~nieme. Alternatively, a T cell-
depleting antibody which binds to an antigen on a T cell surface can be conjugated to a toxin
(e.g., ricin) or other cytotoxic molecule (e.g., a radioactive isotope) to facilitate destruction of
T cells upon binding of the antibody to the T cells.
Another type of antibody which can be used to inhibit T cell activity in a recipient
subject is an antibody which inhibits T cell proliferation. For example, an antibody directed
against a T cell growth factor, such as IL-2, or a T cell growth factor receptor, such as the IL-
2 receptor, can inhibit proliferation of T cells (see e.g, DeSilva, D.R. et al. (1991) J:
0 Immunol. 147:3261-3267). Accordingly, an anti-IL-2 or an anti-IL-2 receptor antibody can
be ~lminietered to a recipient to inhibit rejection of a transplanted cell (see e.g Wood et al.
(1992) Neuroscience 49:410). Additionally, both an anti-IL-2 and an anti-IL-2 receptor
antibody can be cc.~-1minietered to inhibit T cell activity or can be ~lminiet~red with another
antibody (e.g., which binds to a surface antigen on T cells).
An antibody which depletes, sequesters or inhibits T cells within a recipient can be
slrlminietered at a dose and for an a~~ l;ate time to inhibit rejection of cells upon
transplantation. Antibodies are preferably ~timinietered intravenously in a ph~rm~ee~ltically
acceptable carrier or diluent (e.g., a sterile saline solution). Antibody ~riminietration can
begin prior to transplantation (e.g., one to five days prior to transplantation) and can continue
on a daily basis after transplantation to achieve the desired effect (e.g., up to fourteen days
after transplantation). A plt;r~ d dosage range for ~-1minietration of an antibody to a human
subject is about 0.1-0.3 mg/kg of body weight per day. Alternatively, a single high dose of
antibody (e.g., a bolus at a dosage of about 10 mg/kg of body weight) can be ~lminietered to
a human subject on the day of transplantation. The effectiveness of antibody trç~tment in
depleting T cells from the peripheral blood can be determined by col l lp~ ,g T cell counts in
blood samples taken from the subject before and after antibody trc~tment. Dosage regima
can be adjusted over time to provide the optimum therapeutic response according to the
individual need and the professional judgment of the person ~lminietering or supervising the
~lminietration of the compositions. Dosage ranges set forth herein are exemplary only and
are not inten(1c-1 to limit the scope or practice of the claimed composition.
III. Uses of the Method of the Invention
~ Cells having at least one surface antigen altered according to the invention can be
~iminietered to a subject (i.e., transplanted into the subject) for therapeutic purposes. A cell
- 35 can be z~Aminietered to a subject by any applop,iate route which results in delivery of cell to a
desired location in the subject. For example, cells can be ~q~lminietered intravenously,
subcutaneously, intramuscularly, intracerebrally, subcapsularly (e.g., under the kidney
capsule) or intraperitoneally. Cells can be ~lmini~tered in a physiologically compatible
carrier, such as a buffered saline solution. When cells are within a tissue or organ, the tissue
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or organ can be transplanted into a suitable location in the subject by conventional techniques
to ~llmini~ter the cells to the subject.
The methods of the invention can be applied to any type of cell which is suitable for
transplantation (i.e., any type of cell which can be isolated or obtained in a form that can be
S transplanted to another subject). The cells can be human cells or non-human cells. Preferred
non-human cells are porcine cells. Preferred cell types for use in the method of the invention
are cells which can provide a therapeutic function in a disease or disorder. F.x~mples of such
cells include muscle cells (e.g., myoblasts, myocytes, myotubes), liver cells, pancreatic islet
cells, neural cells, e.g., mesencephalic cells, striatal cells, and cortical cells, and
10 hematopoietic cells. For example, muscle cells can be transplanted into subjects suffering
from a muscular dy~LIophy (e.g., Duchenne muscular dystrophy), pancreatic islet cells can be
transplanted into a subject suffering from diabetes, neural cells can be transplanted into a
subject suffering from Parkinson's ~lice~Ç, Huntington's ~liee~ç, Alzheimer's t1i~e~e, or
epilepsy, liver cells can be transplanted into a subject with hepatic cell dysfunction (e.g. in
15 hypercholesterolemia, hemophilia B or inherited emphysema), and hematopoietic cells can be
transplanted into patients with hematopoietic or imm~ln- logical dysfunction. Liver tissue can
be obtained, for example, from brain dead donors or from non-human ~nim~l~ such as pigs.
The cells can be dissociated by digestion with collagenase. Viable cells can be obtained and
washed by centrifugation (at 700 x g), elution, and resuspension. At least one antigen on the
20 surface of the liver cells (e.g., MHC class I antigen) is altered as described herein. Following
alteration of the antigen(s), cells are ~lmini~t-pred through the portal vein to the liver of the
recipient patient. In another embodiment, nerve cells obtained from a source (such as an
abortus) are treated to alter a surface antigen and stereotaxically localized into the desired
area of the brain, such as the corpus striatum or hippocampus. Dopaminergic or GABA-ergic
25 neurons are used for the tre~fment of Parkinson's or Huntington's disease, respectively. In
another embodiment, muscle cells can be obtained from a donor (e.g., by biopsy of a living
related donor or from a brain dead donor) using a 14-16 gauge cutting trochar into a 1-2 inch
skin incision. The fresh muscle plug can be lightly digested to a single cell suspension using
collagenase, trypsin and dispase at 37~C. Floating debris is removed with a pipette and media
30 washes and the viable cell pellet is counted after centrifugation at 1000 rpm for 10 mimltPc
The cell count is then used to calculate the amount of antibody fr~ment~ (or other suitable
molecule, e.g. peptide) to be used to alter a surface antigen on the muscle cells. Muscle cells
are injected intramuscularly into a recipient patient in need of an increased store of muscle.
e.g., an elderly patient with severe muscle wasting, or injected into a muscle group of a
35 patient afflicted with Becker's or Duchenne muscular dy~llophy.
Recipient subjects are further treated with a T cell inhibitory agent according to the
invention. Treatment can begin prior to, concurrent with or following transplantation of cells.
The combination therapy taught by the invention provides a therapeutic regimen for
transplantation of allogeneic or xenogeneic cells into a recipient subject which is more
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effective than either alteration of donor cell surface antigens or tre~tment of the recipient with
a T cell inhibitory agent alone.
This invention is further illustrated by the following Lxamples which should not be
construed as limiting. The contents of all references and published patents and patent
5 applications cited throughout the application are hereby incorporated by reference.
THE FOLLOWING MATERIALS AND METHODS WERE USED IN
EXAMPLES I AND II:
10 Cell~ culture media and rea~ents
Culture media consisted of RPMI supplemented with 10% human AB negative (or
fetal calf) heat inactivated serum, 2mM L-gl~ 7 Penicillin (lOOU/ml), SLl~;~Lollly~;hl
(100 mg/ml), and 30mM HEPES. Human and porcine PBLs were isolated from whole blood
fractionated on Ficoll/Hypaque. PKl 5 cells are a transformed pig kidney cell line purchased
15 from AInerican Type Culture Collection (Accession No.: CCL 33).
P~ lion of F(ab')2 fr~ment~
F(ab')2 fr~gment~ of antibodies W6/32 and PT85 were generated using immobilized
pepsin, as follows. Purified antibody was added, at 20 mg/ml in pH 4.7 digestion buffer and
20 digested for 4.0 hours. The crude digest was removed from the pepsin and immediately
neutralized with pH 7.0 binding buffer. The antibody lllixLule was applied to an immobilized
Protein A column and the elute was collected for the F(ab')2 fr~,o;ment~ Dialysis against
phosphate buffered saline for 24 h using 50,000 molecular weight cut-off tubing was then
performed to rid the digest of cont~min~ting Fc fragments. CHAPS buffer was added to the
25 dialysis bag at a concentration of 1 OmM. The completeness of the digest and purification of
the F(ab')2 were monitored by silver staining of 15% SDS polyacrylamide gels. Final
purification of the fragments was achieved by using a Superose 12 HPLC column. The
completeness of Fc removal was demonstrated in an in vitro assay in which binding of the
m~teri~l to a target cell was followed with the addition of complement, and cytolysis of the
30 pre-loaded target cells was measured by chromium release.
F(ab')2 fr~gment~ were incubated with porcine cells described herein at a
concentration of 1 ,ug of antibody per approximately 1 million cells for 30 min. at room
- temperature. After incubation, porcine cells were washed once with Hanks balanced salt
solution cu~ t~g 2% heat-inactivated fetal calf serum.~ 35
Cytolytic assays.
The cytolytic activity of freshly isolated human PBLs was assessed in a 4 hour 5 l Cr
release assay in which effector cells were tested against porcine PBLs. Targets were treated
for 3 days with PHA (lmg/ml) in order to blast the cells and labeled with 5 1 Cr for 1 hour at
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37~C. Targets were used at 5 x 103/well. Percent specific lysis was determined as the
(Experimental cpm - spontaneous cpm/Maximum cpm-spontaneous cpm) x 100 = %
cytotoxicity.
Mixed Iyn~hocyte reaction
PK15 cells were grown on tissue culture flasks, trypsinized and replated at 1 x 104
cells/ml in DMEM with 10% FCS and added to 24 well flat bottom plates. Cells were
allowed to adhere overnight at 37~C. The cells were then treated with mitomycin C (100
mg/ml) in serum free DMEM for 1 hour at 37~C. The cells were washed 3 times and
10 ~ J~ed for m~skin~. PT85 IgG2F(ab')2 was added to the a~ opl;ate wells at 10 ,ug/ml.
After 2 hours at 4~C or 16 hours at 37~C all wells were washed 3 times in PBS. Human
PBLs were added at 2 x 1 o6 cells/well in a final volume of 2 ml. Plates were incubated for
six days at 37~C, % C~2 and then harvested for FACS analysis. Cell samples from each
well were pulsed with 3H-thymidine for 16 hours and then triplicate samples of 200 ,ul were
15 harvested an a Packard Filt~rm~te 196 cell harvester onto 96 well Unifilter plates.
Incorporated radioactivity was measured on a Packard Top Count microplate scintillation
counter.
Flow cytofluorimetric analysis
One x 105 cells were stained with the ~plupliate antibody followed by
fluoresceinated goat anti-mouse IgG. Control samples were stained with the fluoresceinated
reagent alone. All samples were then analyzed on a flow cytometer (FACScan, Becton
Dickinson and Co), gated to remove nonviable cells.
EXAMPLE I: LYSIS OF PORCINE CELLS BY HUMAN CELLS
IN THE ABSE~CE OF PRIMING
NK cells are known to Iyse target cells nonspecifically by a non-MHC restricted
30 mec.h~ni~m in the absence of priming whereas resting T or B lymphocytes should not mediate
spontaneous killing (Moretta, L. et al. (1994) Adv. Immunol. 55:341-380). To determine
whether freshly isolated human PBLs lyse porcine cells, a cytolytic assay was conducted as
described in the Materials and Methods Section above. Freshly isolated human PBLs were
able to Iyse 5 l Cr labeled porcine PBLs and hepatocytes. See Figures 1 and 2. In a control
35 incubation, freshly isolated human PBLs were not able to lyse 5ICr labeled allogeneic cells.
The same cytolytic assays were performed in the presence of human serum and in the
presence of fetal calf serum. The results of these assays (see Figures 1 and 2) indicated that
Iysis of porcine targets is not due to ADCC as a result of human antibodies that recognize pig
cells since a similar level of lysis were seen when the assay was done in the presence of
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human serum (presumably co~ g natural antibodies) and in the presence of fetal calf
serum (cont~ining no natural antibodies).
EXAMPLE II: EXPANSION OF NATURAL KT~ T.F~ CELLS
IN MIXED LYMPHOCYTE REACTION
Unprimed lysis of porcine cells by human PBLs shows a role for NK cells in killing
of porcine cells. In order to address what role NK cells may play in m~skin~, a test was
performed to determine if NK cells could be detected in an in vitro assay wherein both
10 antibody-treated OEnd ullLIe~l~d porcine cells were cultured with hurnan PBLs. In a mixed
lymphocyte reaction (MLR, see Abbas, A.K. et al. (1994) Cellular and Molecular
Tmmllnology, 2nd ed. (W.B. Saunders Company, Philadelphia) pp. 341-343, for a general
description of OEn MLR), porcine cell stim~ tion of human PBL proliferation was measured
by inco,~old~ion of 3H-thymidine (Figure 3). Treatment of the porcine cells with anti-class I
15 monoclonal antibody, PT85, F(ab')2 fr~gment~ resulted in reduced stimulation of human
PBLs as measured in CPM (Figure 3).
Cells from the MLR were stained with monoclonal antibodies for cell surface m~rkers
and analyzed by FACS. Figure 4 shows the results of FACS st~ining of humOEn cells isolated
from the MLR. CD56 (NCAM) is a marker for NK cells. When human PBLs were
20 stim~ ted with u~ d porcine cells in the MLR, a rem~rk~le increase in cells expressing
CD56 was observed. However, when human PBLs were stim~ ted by treated porcine cells,
a lOEge increase in CD56 staining cells was not observed. See Figure 4. NK cells, like T
cells, express the IL-2 receptor (CD25).
The results in Examples I and II above show a role for NK cells in the human anti-
porcine response in vitro. Unprimed lysis of porcine cells can be detected in a S ICr release
assay OEnd allogeneic cells OEe not lysed. The human anti-porcine MLR shows an outgrowth
of CD56 positive cells when the stimulator cells OEe untreated. This population of cells does
not appeOE to grow out when the stim~ tQrs have been plell~Led with the m~kin~ antibody.
THE FOLLOWING MATERIALS AND METHODS WERE USED IN EXAMPLES
III-VII:
.
P~ lion of periphelal blood Iymphocytes
PBLs were isolated from human or pig whole blood by Ficoll-Hypaque gradient
centrifugation. Coligan, J.E. et al. (1991) Current Protocols in Immunology Vol. 2, Coico. R.
ed. (John Wiley and Sons, New York) ch. 7. Human blood was donated by healthy
volunteers. Porcine blood was from Yorkshire and Hanford strains (Tufts School of
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Vt;l~ uy Medicine) and from inbred minipigs (M~c~chll~etts General Hospital) of the aa
or dd haplotypes. Sachs, D.H. et al. (1976) Transplantation 22:559.
Cell lilles. cu~ture conditions and nledia
K562, JY and Daudi cells were obtained from the American Type Culture Collection.
Cultured cells were grown in DMEM supplementecl with 10% heat inactivated fetal bovine
serum (FBS) (Hyclone) and 100 U/ml penicillin/100 ,ug/ml streptomycin (BioWhittaker) or
50 ~lg/ml g~lll~llycin (Gibco) in a 37~C humidified incubator with 5% C02. For cytotoxicity
assays and mixed culture conditions, AIM-V (Gibco) media, which is serum free, was used.
10 When media co.l~ g human serum was needed, RPMI-1640 (BioWhittaker) was
supplemente~l with 10% AB pooled heat-inactivated human serum (PelFreeze), 2 mM
glul~...i-.e (BioWhittaker), 100 U/ml penicillin/100 ~lg/ml ~ll~lolllycin (BioWhittaker) and
10 mM HEPES (BioWhittaker). The media used in ~l~ pald~ion oftarget cells was RPMI-
1640 supplementecl with 10% fetal bovine serum, 2 mM glllt~min~, 100 U/ml penicillin/100
~lg/ml streptomycin or 50 ,ug/ml genl~llycin and 10 mM HEPES.
Cytotoxicity assays
Chromium release assays were performed basically as described (Coligan, J.E. et al.
(1991) Current Protocols in Immunology Vol. 2, Coico, R. ed. (John Wiley and Sons, New
York) ch. 7). Briefly, porcine PBLs used as targets were treated for 3 days with 5 ~lg/ml
concanavalin A (Sigma). JY cells used as targets were harvested and resuspended in media.
Target cells were labeled with 51 Cr. The cells were then washed 3 times before addition to
the assay mix. Effector PBLs were added to a 96-well round-bottom microtiter plate ranging
from 2.5 x 104/well to 5 x 105/ well. Targets were added at 2 to 5 x 103/well in a total of 200
~Ll. The plate was centrifuged for 4 minutes at 750 rpm to allow cell-cell contact. The plate
was then incubated for 3 to 4 hours in a 37~C humidified incubator with 5% CO2. One
hundred microliters of supern~t~nt was placed in a Luma plate (Packard). The plate was left
to dry for 18 to 20 hours and then read in a TopCount scintillation counter (Packard). Percent
specific lysis was deferrnine-1 by the following formula:
% Specific Lysis = 100 x (experimental - spontaneous release~
(maximum - spontaneous release)
For cold target inhibition assays (Colonna, M. et al. (1993) P~oc. Natl. Acad. Sci. USA
90:1200), the effector to target ratio was 100 to 1. K562, Daudi or JY cells were added
directly to the cytotoxicity assay at the indicated cold to hot ratio one-half hour before targets
were added to the plates. Anti-CD3 (OKT3, Ortho Diagnostics) or control IgG was added to
the cytotoxicity assay at a final concentration of 40 ~g/ml.
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Mixed human/porcine culture conditions
Two x 106 porcine stim~ tor cells were treated with 50~g/ml mitomycin-C (Sigma)
in 2 ml PBS for 30 ...;..~l~es in a 37~C humidified incubator with 5% CO2. The stimul~tors
were washed 3 times in PBS. Two x 106 human effector cells were cocultured with the
5 porcine stimlll~tr~r cells in a 24 well flat-bottom plate in Aim-V media for 6 days in a 37~C
humidified incubator with 5% CO2. Effectors were harvested, washed and used in acytotoxicity assay. Anti-CD25 (Ph~rmin~en, azide-free) was added to the mixed culture at a
final concentration of 15 ,ug/ml.
10 Ma~netic bead cell separatio~s and FA~S analysis
Human PBLs were prepared from Ficoll/Hypaque gradients. In order to isolate a
CD56-enriched population, 2 x 107 PBLs were incubated with 10 ~lg/ml anti-CD3 in 1 ml
PBS at 4~C on a rotating platform. After 30 .-,i..~"ç~, the cells were washed 3 times with PBS
and 4 x 108 goat anti-mouse coated m~gn~tic beads (Dynal) in 1 ml PBS with 1% heat
inactivated FBS were added. The llliX.~UlC was incubated as before for an additional hour.
For a CD56-depleted population, the same procedure was performed but instead of anti-CD3,
anti-CD56 antibody (ph~rrningen~ B159.5) was added. Purified populations were analyzed
by flow cytometry (Coligan, J.E. et al. (1991) Current Protocols in Immunology Vol. 2,
Coico, R. ed. (John Wiley and Sons, New York) ch. 7) with primary antibodies, anti-CD3 and
anti-CD56. FITC-conjugated goat anti-mouse antibody was used as the secondary antibody.
Control incubations were carried out without primary antibody.
IL-2 detection
Supern~t~nt~ (100 ~1) from mixed human/porcine cultures were collected each day
after the start of the cultures. IL-2 was detected in these supçrn~t~nt~ by ELISA (Endogen).
Anti-CD25 was added at a final concentration of 15 ~lg/ml to prevent the utilization of IL-2.
EXAMPLE III: PBLS FROM NORMAL HUMAN DONORS HAVE
CYTOLYTIC ACTIVITY TOWARD PORCINE PBLS
Hurnan serum contains natural antibodies which have been shown to be toxic to
porcine cells and are thought to be responsible for hyperacute rejection of porcine organs
(Satake, M. et al. (19g3) Clin. Transplant. 7:281, Kirk, A.D. et al. (1993) Transplantation
56:785, Satake, M. et al. (1994) Xenotransplantation l :24). To dirrert;llliate between ADCC
and that which is antibody independent killing, lysis of porcine cells by human cells in the
presence or absence of human serum was tested. The results of this experiment are shown in
Figures 5A-5C. PBLs from normal blood donors were isolated and used as effector cells for
cytotoxic activity against slCr-labeled porcine PBLs. Figures 5A-5C show that normal
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human subjects have anti-porcine cytotoxic activity; human cells were-not lysed in this assay
(Figures 5A-5B, JY cells). In particular, Figures 5A-5C show that while non-serum
dependent cytotoxicity against porcine cells is present in human PBL pl~aldlions, an
increase in lysis in the presence of serum was not always apparent. The m~gnitl1de of non-
serum dependent lysis varied among individuals. Allogeneic target cells were not lysed in
these assays.
The ability of human cells to lyse porcine cells does not appear to be limited to some
individuals as all human subjects tested (n=20) showed cytotoxicity toward porcine cells. In
addition, susceptibility of target cell donor was not restricted to a particular type of pig, as
10 outbred stocks and inbred minipigs were all sensitive targets. To test whether this
phenomenon was specific to the human/pig combination, or whether hurnan PBLs had lytic
activity against other xenogeneic target cells, mouse (Balb/c) and rat (Sprague/Dawley)
spleen cells were labeled with 5 ICr and used as targets for hDan PBLs. While porcine cells
were lysed at 100:1 effector to target ratio (25% specific release), rat and mouse cells were
15 not lysed under these conditions. The results from these studies demonstrated that peripheral
blood lymphocytes from normal donors have cytolytic activity toward porcine PBLs.
EXAMPLE IV: CYTOTOXIC LYMPHOCYTE RESPONSE BY MIXED
CULTURE OF HUMAN LYMPHOCYTES WITH PORCINE
CELLS IS NOT MHC-RESTRICTED
The antibody-independent cytolytic activity discussed above could be due to
cytotoxic T cells (CTLs), NK cells or other cytolytic cells. To test whether there was an
25 MHC-restricted CTL response by mixed culture of human lymphocytes with porcine cells~
mitomycin C-treated PBLs were isolated from an NIH inbred minipig of the aa haplotype
and cultured with human PBLs. After 6 days, effector function was assessed on 5 ICr-labeled
target cells from aa or dd pigs. As shown in Figure 6, human effector cells from the mixed
culture lysed porcine target cells equally, regardless of the stimulator haplotype. The lysis of
30 porcine targets was increased after coculture relative to unprimed lysis (Figures SA-5C).
Repeated stimulation of human PBLs with porcine stimulators did not yield MHC-restricted
lysis of porcine cells.
The lack of MHC restriction in this assay indicated that an effector cell that did not
require priming by MHC-peptide complexes could account for the unprimed lysis observed
35 above. The lysis observed after mixed human-porcine culture is due to cells, e.g., NK cells
that do not recognize antigen in the context of MHC.
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EXAMPLE V: HUMAN NATURAL KTT.T,l~R CELLS LYSE PORCINE CELLS
To test whether NK cells were responsible for the lysis of porcine cells, cold target
inhibition was used with the human NK cell target, K562. As shown in Figure 7A unlabeled
S K562 cells inhibited the lysis of porcine cells when freshly isolated human PBLs were used
as effectors. The lysis of porcine target cells was not inhibited by the NK-resistant cell line,
JY. The ability of K562 cells to inhibit the lysis mediated by the human anti-porcine
cytotoxic cells generated in mixed culture was also tested. Figure 7B shows that the
cytotoxic cells in the mixed culture are inhibited by K562 cells. These results indicate that
10 NK cells are responsible for the lysis of porcine cells by freshly isolated human cells or
human cells cocultured with porcine stimnl~tors.
To directly test whether human NK cells are responsible for lysis of porcine cells,
cells expressing CD56, a marker for NK cells, were partially purified from PBL ~ lions.
CD56+ cells were enriched by negative selection with anti-CD3 monoclonal antibody and
15 goat anti-mouse antibody coated magnetic beads as described above. These cells were then
used as effectors in the S l Cr release assay with porcine cells as the targets. Table I shows the
results of FACS analysis of the enriched NK cell population which indicates a 3-fold
purification of CD56+ cells. For comp~n~on, CD56+ cells were depleted by a negative
selection scheme with anti-CD56 monoclonal antibody and goat anti-mouse coated m~gnetic
20 beads. For control purposes, tests were also performed to ~letermine whether the CD56-
enriched and depleted populations would be active against K562 cells. Figures 8A-8C
illustrate that most of the cytotoxic activity toward porcine cells and K562 cells is present in
the CD56-enriched population and not in the CD56-depleted population.
TABLE I: Percent Stainingl of CD56-enriched and CD56-depleted population
Control anti-CD3 anti-CD56
Unfractionated <1 36 10
CD56+-enriched2 <1 15 38
CD56+-depleted <1 84 <1
1. Percent staining as determined by FACS analysis.
2- Enrichrnent and depletion of CD56+ cells was performed by negative selection with
antibody and magnetic beads.
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EXAMPLE VI: CYTOTOXIC HUMAN EFFECTORS RAISED AGAINST
PORCINE STIMULATORS ARE PHENOTYPICALLY
DISTINCT FROM FRESH HUMAN CYTOTOXIC CELLS
S Experiments were con-lllrte~l to dett?rmine whether mixed culture of human PBLs and
porcine cells led to changes in the human anti-porcine lytic activity. After mixed culture of
human PBLs and porcine stimulators, specific lysis ranged from 70 to 90% at an effector to
target ratio of 100 to 1, whereas lysis due to fresh human effectors under these conditions
ranged from 8 to 30%. While this increase in activity may be due to the outgrowth of cells
during mixed culture, analysis of the mixed culture after 6 days revealed an increase in CD56
st~ining. Gross changes in the various cell populations (CD4+, CD8+ and CD56+) with and
without porcine stimulator cells were also detected.
The increase in lytic activity produced during human/porcine mixed culture is due to
an increase in the number of NK cells or their differentiated coull~el~L~ lymphokine
activated killer cells (LAK). The results shown in Figures 7A-7B demonstrate that the NK
cell target, K562, acts as a cold target inhibitor to human anti-porcine cytotoxicity. Tests
were also performed to det~rmine whether the LAK target, Daudi (Biron, C.A. et al. (1989)
New Engl. J. Med. 320:1731), would act as a cold target inhibitor of this response. Figures
9A-9B show that while K562 cells inhibited the unstim~ te~1 human anti-porcine
cytotoxicity as well as cytotoxicity after mixed culture, Daudi cells inhibited the cytotoxicity
after mixed culture only. This indicates that during the mixed culture, LAK cells develop due
to the production of cytokines by the culture.
Since the generation of LAK cells from NK cells is dependent on IL-2, an assessment
was made as to whether IL-2 is generated by mixed human/porcine culture. Figure 10 shows
that human IL-2 is generated in these cultures. Significant IL-2 was measured only in the
presence of anti-IL-2 r~~ antibody, anti-CD25, suggesting that in the absence of the
antibody, IL-2 is difficult to measure due to consumption by the cells in the culture.
To test whether IL-2 in the mixed cultures is required for the increase in specific lysis,
anti-CD25 antibody was added to the cultures to block the utilization of IL~2. Figure 11
shows that in the presence of anti-CD25 the specific lysis of porcine targets is reduced,
indicating that the generation of LAK cells in these cultures is at least partially dependent on
the utilization of IL-2 by the human lymphocytes.
EXAMPLE VII: PRESENCE OF HUMAN CYTOTOXIC T CELL COMPONENT
AMONG HUMAN ANTI-PORCINE CYTOTOXIC CELLS
To measure a CTL response in the human/porcine mixed culture which might be
obscured by a strong NK component, a test was performed to determine whether removal of
CD56+ cells would reveal a T cell response. Human PBLs were depleted of CD56~ cells and
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put into culture with mitomycin C-treated porcine PBLs. Cells were harvested on day 6 and
the depletion of CD56+ cells was repeated. The cells recovered were then used as erre-;~ol, in
the 5 ICr release assay against porcine PBL targets. Figure 12 shows that ~;ylOt~iC activity is
~1Ptecte(l in these cultures after NK cell depletion. This activity is partially blocked by anti-
S CD3 antibodies (B). The cytotoxicity rem~inin~ is not inhibitable with cold K562 cells (D)which suggests that it is not due to NK cells. Thus, the T cell component of the human anti-
porcine response is measurable but appears to be overwhelmed by NK me~ te~l lysis.
As shown in Fx~mI~les III-VII, freshly isolated human PBLs have the ability to lyse
10 porcine cells. In addition, while antibody-mediated mech~ni~m~ account for variable levels
of ~;y ~u~oxicity, reproducible killing, in the absence of human serum is attributable to natural
killer cells. As described above, the cell line, K562, an established target for human NK
cells, acted as a cold target inhibitor in the human anti-porcine cytotoxicity assay. In
ition, when the human PBL population was enriched for CD56+ cells, anti-porcine
lS cytotoxicity increased, as did NK cell activity. Depleting the CD56+ cells reduced
significantly the anti-porcine lytic activity in these pop~ tion~ Mixed culture of human and
porcine cells prior to the cytotoxicity assay increased the level of anti-porcine cytotoxicity.
This activity did not appear to be due to T cells as it was not MHC restricted. IL-2 was
g~ner~tecl in these cultures and ~pe~,d to be required for the increase in anti-porcine
20 cytotoxicity. IL-2 generated in the human/porcine mixed culture leads to the differentiation
of NK cells to LAK cells. Cold target inhibition studies collrl-med that LAK cells were
generated in the mixed culture. When NK cells were removed from human/porcine mixed
cultures, a T cell component to the human anti-porcine cytotoxicity was detecte~l Therefore,
the human anti-porcine cellular cytotoxic response is due to multiple cell types that include T
25 cells in addition to NK and LAK cells.
Other~mbodiments
Other embotiiment~ are within the scope of the invention and the following claims.
For example, in another embodiment, cells which are ~flmini~tered to a subject according to
30 the methods of the invention are present within a tissue or organ. When cells are within a
tissue, antigens on the surface of the cells (e.g., MHC class I antigens) can be altered by
contacting the entire tissue with a molecule (e.g., antibody) which binds to the antigen (e.g..
~ incubating the tissue in a solution cont~ining the molecule which binds the antigen).
Alternatively, when a cell is within an organ, antigens on the surface of the cells (e.g., MHC
35 class I antigens) can be altered by perfusing the organ with a solution cont~ining a molecule
(e.g. antibody) which binds to the antigen. An organ can be perfused with a solution
c~ i"il~g the molecule using conventional techniques for organ perfusion.
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Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific embo-liment.~ of the invention
described herein. Such equivalents are intended to be encompassed by the following clairns.
s
