Note: Descriptions are shown in the official language in which they were submitted.
CA 0223281~ 1998-03-23 '
W O 97/11607 PCTAUS9~/15577
M~-1~V OF I~nEIBITING IM~nnNE SYSl~ DE~l~llON OF
TRU~NSPLA~rrED VIABLE ~ELLS
This application claims the benefit o~ U.S. Provisional
Application No. 60/004,375, filed September 27, 1995, the
contents of which are hereby incorporated by reference.
The invention disclosed herein was made with Government
support under NIH ~rant No. RO1-DK39088. Accordingly, the
U.S. Government has certain rights in this invention.
Throughout this application, various references are
re~erred to within parentheses. Disclosures of these
publications in their entireties are hereby incorporated by
reference into this application to more fully describe the
state o~ the art to which this invention pertains. Full
bibliographic citation ~or these references may be found at
the end of this application, preceding the claims.
Backqround o~ the Invention
There is a critical need ~or better insulin replacement
therapy to circumvent the complications of insulin-
dependent diabetes mellitus (IDDM). Our goal is to develop
techniques for transplantation of microencapsulated,
xenogeneic islets to provide a durable, physiological
source of insulin to diabetic patients. It has previously
been shown that microcapsules are biocompatible and that
xenogeneic islet grafts contained in microcapsules
functioned indefinitely in the peritoneal cavity of mice
with streptozotocin-induced (SZN) diabetes. Thus,
microcapsules may be intact and stable in vivo and factors
that may be required for long-term survival and function of
the xenogeneic islets are accessible. The microcapsules
serve as a mechanical barrier that prevents cell-to-cell
contact between recipient lymphocytes and donor islets.
CA 0223281~ 1998-03-23
W O 97tll607 PCT~US96/15~77
The mechanical barrier primarily prevents host
sensitization rather than protecting the gra~t ~rom immune
destruction, because encapsulated islets are very rapidly
destroyed by recipients that are presensitized to the islet
donor cell antigens. Similarly, encapsulated xenogeneic
islets were rejected (in two weeks) by NOD mice, which i8
possibly due to presensitization of NODs to islet antigens.
Xenografts undergoing rejection in NOD mice were surrounded
by large numbers of activated macrophages and
immunoglobulins, with IL-l~, TNF~, both documented by
immunocytochemistry, and IL-4 messenger RNA detected by RT-
PCR. We postulate that NOD rejection is initiated by donor
antigens that are secreted by or shed from the encapsulated
islets and which are processed via the MHC (major
histocompatibility complex) class II pathway by host APC
(antigen presenting cells). These APC activate NOD CD4~ T
cells that develop into a Th2 response, with donor islet
destruction occurring via cytokine-mediated events.
We have also been able to improve the microencapsulation
process to permit long-term survival o~ concordant, rat
islet xenografts, even in NOD mice. Furthermore, we have
found that blockade of NOD co-stimulatory molecules with
CTLA4Ig significantly prolongs survival of discordant,
2~ rabbit islet xenogra~ts for up to 200 days. Thus, we have
been able to overcome problems associated with
transplanting encapsulated islet xenografts into autoimmune
diabetic recipients.
Insulin-Dependent Dia~etes Mellitus
The last several years have witnessed a remarkable increase
in or knowledge of the effects of therapies for insulin-
dependent diabetes mellitus (IDDM). The Diabetes Control
and Complications Trial (DCCT) ~ound that intensive insulin
therapy delayed the onset and slowed progression of
CA 0223281~ 1998-03-23 ~
W O 97/11607 PCT~US96/15577
retinopathy, nephropathy, and neuropathy in patients with
IDDM (1). Unfortunately, intensive insulin therapy is not
appropriate for many IDDM patients; and even with careful
monitoring, DCCT patients had increased episodes of severe
hypoglycemia (1). Ironically, results of the DCCT support
the rationale for pancreas and islet transplantation.
Since the inception o~ islet transplant experiments, it has
been the hope that such grafts might supply insulin more
homeostatically than exogenous insulin can, and that 'near-
normal' modulation of carbohydrate metabolism might preventthe secondary complications of IDDM (2). Clinical pancreas
allografts have improved outcomes with the advent o~
combination immunosuppression; and near normal o~ glucose
homeostasis follows most pancreatic allo- and auto-grafts
1~ ~3~. However, the first-year mortality o~ a human
pancreatic allograft remains high (10~), immunosuppression
is required, and only limited numbers of clinical whole-
organ pancreatic transplants are being done worldwide
(2,4,5).
The Rationale for Microencap~ulated Islet Xenograft~
Islet transplantation is an attractive therapy for patients
with IDDM, since problems related to the exocrine pancreas
may be avoided. However, allografts of donor human islets
have not been successful long-term (3~; and availability
and yield of human islets are limited. Therapeutic islet
transplants for large number of patients almost certainly
will require donor islets harvested from animals
~xenografts)(2,4).
The optimal source of xenogeneic islets for clinical use
remains controversial. Islets have been isolated ~rom
subhuman primates and xenografted into immunosuppressed,
diabetic rodents, with short-term reversal of diabetes (6).
However, there are significant ethical issues surrounding
~ CA 0223281~ 1998-03-23
W O 97/11607 PCTrUS96/15577
use of primates, Other promising sources are porcine,
bovine, canine, and rabbit islets, which function
remarkably well, (i.e., maintaining normoglycemia) in
diabetic rodents until transplant rejection occurs ~7-11).
Long-term human, bovine and porcine islet xenograft
sur~ival has been documented in nude mice and rats,
suggesting that sufficient islet-specific growth factors
are present in xenogeneic recipients (2,12-17). For
sociologic/ethical reasons, canine islets are not
clinically appropriate. Porcine islets are both dif~icult
to isolate (intact) and to maintain in vitro; nevertheless,
they are extremely promising for eventual clinical
application (18-21). Isolation o~ bovine islets is
technically easier (than porcine islets), and calf islets
are glucose-responsive (22). Recently, large scale rabbit
islets isolation has been developed (23)(see Preliminary
Studies). Rabbit pancreas is an attractive source of
islets. Rabbit, like porcine insulin, differs from human
insulin at only one amino acid, and rabbit islets are
glucose responsive (22,24). In addition, most humans do
not possess natural anti-rabbit antibodies, which might
improve the possibility of preventing xenogra~t rejection
(25). It is currently feasible to consider isolation of
1,000,000 donor islets/per human diabetic recipient ~rom
either calves, pigs or rabbits, utilizlng multiple donors.
The most significant obstacle to islet xenotransplantation
on human IDDM is the lack of an effective immunosuppressive
regiment to prevent cross-species graft rejection (2,26-
28) Recently, it has been reported that human islets will
survive long-term in SZN-diabetic mice treated either with
anti-CD4 antibody (16) or CTLA4Ig (a high affinity fusion
protein which blocks CD28-B7 interactions) (12), or by
exposure of donor islets to purified high affinity anti-HLA
(ab) 2 (29). However, with the exception of these studies,
CA 0223281~ 1998-03-23 ~
W O 97/11607 PCT~US96/15577
-- 5
indefinite survival of islet xenografts has rarely been
achieved, except with the aid o~ porous, mechanical
barriers. Both intra- and extra-vascular devices are under
development. However, potential clinical complications,
such as bleeding, coagulation, and bioincompatibility
mitigate against their current use in diabetic patients
(30,31). For example, acrylic-copolymer hollow ~ibers
placed subcutaneously maintained viabllity of human islet
allogra~ts for two weeks (50 islets per 1.5 cm
~iber)(65,000 M.W. permeability)(32).
However, to implant 500,000 islet would require ~150 meters
o~ these hollow ~ibers, which is not clinically feasible.
One of the most promising islet envelopment methods is the
polyamino acid-alginate microcapsule. A large number of
recent studies have shown that intraperitoneal xenografts
of encapsulated rat, dog, pig or human islets into
streptozotocin-diabetic mice or rats promptly normalized
blood glucose for 10-100~ days (7,19,33-39). Long-term
normalization of hyperglycemia by microencapsulated canine
islet allogra~ts, porcine islet xenografts, and one human
islet allograf~ has been reported (21,40-42). The
mechanisms hy which microcapsules protect islet xenogra~ts
~rom host destruction are not ~ully understood. However,
it has been suggested that prohibition of cell-cell contact
with host immunocytes is important (30,35). The marked
prolongation of widely unrelated encapsulated islet
xenografts in rodents with induced diabetes has prompted
studies in animals with spontaneous diabetes.
The Spontaneously Diabetic NOD Mouse As A Model O~ Human
IDDM
Nonobese diabetic (NOD) mice develop diabetes
spontaneously, beginning at approximately twelve weeks o~
CA oi23281~ l998-03-23
WO97/11607 PCT~S96/15~77
age. NOD mice are the most appropriate model for studying
the feasibility of islet xenotransplants because their
disease resembles human IDDM in several ways. Macrophage,
dendritic cell and lymphocytic infiltration of islets can
be detected as early as four weeks of age and precedes
overt hyperglycemia (43-46). NOD diabetes is T lymphocyte-
dependent (43-45); and it is associated with (MHC) Class II
genes (47-50). Cytotoxic T cells and antibodies specific
for beta cells or for insulin have been identified,
characterized and cloned from NOD mice (44,45,51-55). Loss
o~ tolerance to islet antigens in NODs correlates with
appearance of Thl immune responses to glutamic acid
decarboxylase, a factor which has been reported to be a
primary auto-antigen in human IDDM (5,657). The disease
can be induced in non-diabetic, ~yngeneic mice by transfer
of both CD8+ and CD4+ T cells or T-cell clones from diabetic
NODs (44,52,55,58); and inhibition of NOD macrophages or
CD4+ T lymphocytes or treatment with anti-Class II
monoclonal antibodies prevents or delays diabetes onset in
NOD mice (59,50). Defects in NOD macrophages, C5
complement and NK cell function have been reported (61).
It has been suggested that helper T-cells function to
activate CD8+ cells, which damage beta cells by direct
cytotoxic attack. However, some recent studies have
suggested that beta cell killing may be indirect, ~rom a
nonspecific inflammatory response which initially involves
CD4~ cells, but also includes infiltrating macrophages,
which release cytokines and oxygen free-radicals
(particularly nitric oxide), known beta cell toxins (62-
65). Because of similarities to IDDM, NOD mice are thebest model in which to study islet xenografts.
Recently, the Scid mutation has been back-crossed onto the
NOD background, resulting in immuno-deficient NOD-Scid mice
(66-69). These mice homologous for the Scid mutation,
CA 0223281~ 1998-03-23 -
W O 97/11607 PCTfUS96/15~77
which results in an inability to rearrange T-cell receptor
and immunoglobulin genes (66,67). The consequence is an
absence o~ T and B-lymphocytes. These mice do not develop
diabetes spontaneously; but they may be rendered diabetic
with multiple low-dose streptozotocin (MLD-SZN) regimens,
making them an optimal model for adoptive transfer
experiments (67-69). NOD-Scids express NOD MHC genes and
other genes that are relevant ~or development of the
disease. They mount robust macrophage and limited NK-cell
responses, but are ~unctionally T- and B-lymphocyte
deficient (69).
Islet Xenogra~ts into Diabetic NOD Mice
Unlike mice with SZN-induced diabetes, diabetic NOD mice
rapidly reject unencapsulated islet xenografts, allo~rafts
and isografts ~7,8,10,19,33,56,70,71). Conventional
immunosuppressive regimens have little ef~ect on this
reaction (10,71-73). Treatment of NOD recipients with
monoclonal antibodies directed against CD4+ helper T
lymphocytes or FK506 prolongs islet graft function (from 5
to 25 days)(7,8,10,73); but long-term islet graft survival
in NODs has not been reported.
Several laboratories have reported that intraperitoneal
microencapsulated islets (allo- and xeno-geneic) function
significantly longer than non-encapsulated controls, but
eventually are destroyed also by recipients with
spontaneous (autoimmune) diabetes (NOD mice or B~3
rats)(7,9,19,33,35,70,74-78). Rejection is accompanied by
an intense cellular reaction, composed primarily of
macrophages and lymphocytes, which entraps islet-containing
microcapsules and recurrence of hyperglycemia within 21
days, in both NOD and BB recipients (7,19,74,76,77). The
mechanism of encapsulated islet rejection by animals with
spontaneous diabetes remains incompletely understood, but
CA oi23281~ 1998-03-23
WO97/1l607 PCT~S96/1j577
the fact that it rarely occurs in mice with induced (SZN)
diabetes suggests that anti-islet autoimmunity may be
involved in islet graft destruction.
Me~h~n;sms o~ NOD De~truction of Encap~ulated Islet
Xenografts: MacrophageR, T-Cells, and Cytokines
It has been suggested by several investigators that
microcapsules, like other bioartificial membrane devices
promote survival of xenogeneic and allogeneic islets by:
(A) preventing or minimizing release of donor=antigen(s),
thereby reducing host sensitization, and/or (B) preventing
or reducing host effector mechanisms (i.e. T-cell contact,
anti-graft antibody binding, cytokine release).
Most studies of rejection of islets in microcapsules and
other membrane devices have focused on effector mechanisms.
For example, Halle (35) and Darquy and Reach (79) reported
that microcapsules protected donor islets from host
immunoglobulins, specifically human anti-islet antibodies
and complement effects, in vi tro. Although complement
components, are too large (>>150,000 Kd) to enter
conventional poly-l-lysine microcapsules, it is possible
that antibodies combine with shed donor antigens forming
complexes which bind to FcR of macrophages in vivo ( in the
peritoneal cavity) which could initiate cytokine release
causing encapsulated islet destruction (80). Complement
could facilitate binding of complexes to macrophages via
the C3b receptor or by the release of chemotactic peptides
that could increase the number of macrophages.
Involvement of NOD T-lymphocytes in rejection of
encapsulated islets has been proposed by Iwata, et al.
(81), who found significant prolongation of encapsulated
hamster-to-NOD mouse encapsulated islet xenografts when NOD
recipients were treated with deoxyspergualin (DSG), a T-
CA 0223281~ 1998-03-23 ~
W O 97/11607 P~T~US96/15577
cell inhibitory immunosuppressant (81). This data is
consistent with prior finding of several laboratories, that
treatment of NODs with monoclonal antibodies directed
against CD4+ helper T cells or FK-506 prolonged f~unction o:E
both encapsulated and nonencapsulated rat-to-NOD islet
xenograft (7,8,10,73) and these finding are similar to
observations o~ Auchincloss (27), Pierson (82) and Gill
(83), that CD4+ T cells play a dominate role in
xenoreactivity.
A prominence of macrophages/monocytes in peri-microcapsular
infiltrates o~ encapsulated islet allogra~ts and xenografts
in NOD mice and BB rats has been reported (7,33,36,74,76-
78,84). Cytokines known to be products of macrophages,
including IL-1 and TNF (62,77,85,86), may be involved
destruction o~ encapsulated islets. Both I~-1 and TNF have
been reported to reduce insulin secretion and cause
progressive damage of islet cells in vitro (58,62-64,85-
87). Cytokine-mediated injury might occur directly or
indirectly, by activation o~ an intraperitoneal
in~lammatory response (30,77). Recently, it has been
reported by Dr. J. Corbett (IPITA con~. 6/95), that there
are as many as ten macrophages within each islet. IL-1
induces nitric oxide synthase (NOS)(63-65), with resultant
generation o~ nitric oxide (NO), which causes injury to
mitochondria and to DNA in beta cells (63-65).
Furthermore, this pathway o~ islet damage is worsened by
TNF (88,89). Theoretically, macrophages from within donor
islets and host peritoneal cavity or within the down islets
could be involved in cytokine-mediated damage to
encapsulated islets.
Studies of cytokine messenger RNA pro~iles in hamster-to-
rat liver and pig-to-mouse islet xenografts have ~ound
selective increases in Th2 cytokines (IL-4, IL-5, IL-10)
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
- 10 -
and no change from normal in IL-2 (11,90). These are
distinctly different from those of O'Connell, et al.
(91,92), who reported IL-2 messenger RNA in biopsies of
allograft rejections of nonencapsulated islets. Increased
Th2 activity relative to Thl (93-95) activity is distinct
from the known NOD 'Thl' anti-islet immune response
~56,57,96). The Th2 response is characteristic of evoked
antibody responses to foreign antigens and suggests that
humoral reactions to encapsulated xenografts may be of
critical importance. Furthermore, strategies designed to
abrogate 'Th2' responses may significantly prolong
encapsulated islet xenograft survival. The 'Th2' helper T-
cell cytokine mRNA profile i5 characteristic of antibody
responses to foreign antigens.
Co~timulatory Molecules, APC'~ and Islet Xenogra~t
Destruction by NOD Mice
Involvement of APCs in immune responses to islet xenografts
is suggested by recent studies of Lenschow, et al. (12),
who found that blockade of the co-stimulatory molecule, B7
with the soluble fusion protein, CTLA4Ig, prolonged human-
to-mouse islet xenografts in SZN-diabetic mice. Several
studies, in vitro and in ~ivo, have shown that foreign
molecules which interact with the T cell receptor
(peptides, specific antibodies, mitogens) fail on their own
to stimulate naive T cells to proliferate (95,97), and may
induce antigen-specific anergy. At least one additional
(costimulatory) signal is required, and it is delivered by
APCs. In mice, one such costimulatory pathway involves the
interaction of the T-cell surface antigen, CD28 with either
one of two ligand, B7-1 and B7-2, on the APCs (95,97-102).
Once this full interaction of T-cells and APCs occurs,
however, subsequent re-exposure of T-cells to peptide,
mitogen, etc. will result in proliferation in the absence
of costimulation. (95).
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
CTLA4 is a cell surface protein that is closely related to
CD28; however, unlike C328, CTLA4 is expressed only on
activated T-cells. B7-1 has a high affinity for CLTA4 than
CD28; and it has been suggested that CT~A4 may modulate
functions of CD28 (97,103,104). CTLA4Ig is a recombinant
soluble fusion protein, combining the extracellular binding
domain of the CTLA4 molecule with constant region of the
IgGl gene. Both human and murine CTLA4Ig have been shown
to inhibit T-lymphocyte responses in mice (141,142).
Administration of CTLA4Ig to mice has been shown to induce
antigen-specific unresponsiveness (in a murine lupus
model)(97,99,105) and long-term acceptance of murine
cardiac allografts (106,107). In addition, Lenschow, et
al., found that it induced tolerance to human islets in
SZN-diabetic mice (12). CTLA4Ig has also been reported to
reduce the incidence o~ diabetes in NODs (108). There are
no reports of effects of CTLA4Ig on islet graft survival in
spontaneously-diabetic recipients, such as NOD mice.
However, our studies show that CTLA4Ig significantly
prolongs survival of encapsulated rabbit isle~s in NOD
recipients.
Recent studies have further illuminated helper T-cell-APC
interactions, with recognition of the importance of binding
of the APC-CD40 antigen to its ligand, GP39, on helper T-
cells (109,110). A monoclonal hamster anti-murine GP39
anti~ody (MRl) blocks helper T-cell interactions with APCs,
macrophages, effector T-cells and B-lymphocytes (109,110).
Dr. A. Rossini has reported recently (IPITA conf. 6/95)
that MR1 plus B7 negative donor spleen cells day 7 allows
long-term survival of both allo- and xeno-geneic islets in
SZN-diabetic mice.
The T~llnQgenicity of Encapsulated Islets and ~ec~n;sm~ of
Graft of Destruction
CA 0223281~ 1998-03-23
W O 97/11607 PCTrUS96/15577
- 12 -
Empty microcapsules have been reported to elicit no
cellular responses (33,35,36). On the other hand, others
have found reactions to empty capsules, (30,76,77,111,112).
Impurities in reagents ~uch as contamination with endotoxin
or high concentrations of mannuronate most likely
contribute to bioincompatibility (113). It is apparent
that some formulations of poly-l-lysine microcapsules are
biocompatible and some are not. Until standardized
reagents are available, immunologic studies are
microencapsulated islets can only be interpreted when
investigators include empty microcapsule controls which
document their biocompatibility.
Recently, de Vos, et al. (114) reported incomplete
encapsulation or actual protrusion of islets through
microcapsule membranes in some microcapsules, and suggested
this biomechanical imperfection is one factor in
microcapsule destruction. Similar observations have been
made by Chang (115), who found incorporation of islets and
hepatocytes within the walls of poly-l-lysine alginate
microcapsules. Several other investigators have published
photomicrographs of encapsulated islets showing obvious
entrapment of islets in capsules, walls, but did not
comment on this problem (35,116,117). Inco~plete
encapsulation would be anticipated to result in premature
capsule fracture and exposure of donor islets to host
cells; but there are no reports analyzing this as a source
of donor antigen exposure, sensitization and host.
Relatively few studies have focused on the role of donor
islet antigen(s) released from microcapsules in initiating
host immune responses. Ricker, et al. (33~ reported
similar, intense cellular reactions by NOD mice to rat
insulinoma, hepatoma and pheochromocytoma cell lines in
microcapsules and concluded that the NOD immune reaction
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/15577
- 13 -
was not islet-specific. Horcher, et al. (36) reported 15-
week survival of 6/7 encapsulated Lewis rat islet
isogra~ts, compared to failure of 8/lo encapsulated Wistar-
to-Lewis islet allogra~ts within 56 days. Isograft
biopsies showed viable islets, intact capsules and no
' pericapsular immune reaction (36), while biopsies of ~ailed
allografts revealed pericapsular cellular responses and
nonviable islets. This is the only report ln the
literature with encapsulated islet isogra~t controls.
Although the Lewis rat model is not one with autolmmune
diabetes, the results are significant, and suggest that
donor antigen~s) are the stimulus for subsequent host
responses.
CA 022328l~ l998-03-23
W O 97/11607 PCTAUS96/15577
- 14 -
S~ y of the Invention
This invention provides a method of inhibiting viable cells
transplanted into a subject from being destroyed by the
subject's immune system which comprises: a) containing the
viable cells, or tissue comprising the viable cells, prior
to transplantation within a device comprising a
semipermeable membranei and b) treating the subject with a
substance which inhibits an immune-system costimulation
event in an amount effective to inhibit the subject~s
immune system from responding to said contained cells or
tissue.
In one embodiment, the substance which inhibits an immune-
system costimulation event is CTLA4. Accordingly, this
invention further provides a method of inhibiting viable
cells transplanted into a subject from being destroyed by
the sub]ect~s immune system which comprises: a) containing
the viable cells, or tissue comprising the viable cells,
prior to transplantation within a device comprising a
semipermeable membrane; and b) treating the subject with
CTLA4 in an amount effective to inhibit the subject~s
immune system from responding to said contained cells or
tissue.
This invention also provides a method of treating diabetes
in a subject which comprises: a) containing viable insulin-
producing cells, or tissue comprising viable insulin-
producing cells, within a device comprising a semipermeable
membrane so as to obtain contained viable insulin-producing
cells; b) transplanting contained viable insulin-producing
cells obtained in step (a) into the subject in an amount
effective to treat diabetes in the subject; and c) treating
the subject with a substance which inhibits an immune-
system costimulation event in an amount effective to
inhibit the subject's immune system from responding to an
CA 02232815 1998-03-23
W O 97/11607 PCT/US96/15577
- 15 -
amount of contained viable insulin-producing cells
according to step (b).
CA 0223281~ 1998-03-23
W O 97/11607 PCT~U~96/15577
Brie~ Description of the Drawinq~
Fiqure 1: Encapsulated Lewis rat islet, day ~150
after xenografting to unmodified diabetic
NOD H&E. (x250). The microcapsule is a
"double-wall" microcapsule.
Fiqure 2: Survival of islet xenograft, "double-wall"
microcapsule.
10 Fiqure 3: Comparison of survival of rabbit islets
encapsulated in microcapsules with a
permeability of up to 70,000 Kd to survival
of rabbit islets in microcapsules having a
permeability of 100,000 Kd.
Fiqure 4: Effect of Lewis rat splenocyte priming on
Lewis rat-to-NOD microencapsulated islet
transplantation.
20 Fiqure 5: Effect of Lewis rat islet priming on Lewis
rat-to-NOD encapsulated islet
transplantation.
Fiqure 6: Microencapsulated dog islet, day #80, from
peritoneum of NOD mouse treated with Gkl.5.
H&E (x250).
Fiaure 7: Functioning, encapsulated rabbit islets,
biopsied day #86, from peritoneum of NOD
mouse, treated with CTLA4Ig. Note absence
of NOD cell response and the presence of
viable islets within capsule. H&E (x400).
Fiqure 8: Effects of microencapsulation of islets
combined with CTLA4Ig treatment on islet
CA 0223281~ 1998-03-23
W O 97/11607 PCTAJS96/15577
xenografts.
Fiqure 9: Survival of microencapsulated mouse INS-
CTLA4 islets transplanted into NODs. These
islets express CTLA4.
Fiqure 10: Effects of transplanting rat islets into
streptozotocin SZN - diabetic NOD-Scid
mice.
Figure 11: E~fects of transplanting rabbit islets into
streptozotocin (SZN)-diabetic NOD-Scid
mice.
.
15 Fiqure 12: Effects of transplanting microencapsulated
rabbit islets into streptozotocin (SZN)-
diabetic NOD-Scid mice.
Fiaure 13: Functioning, encapsulated rabbit islets,
biopsied day #86, from peritoneum of NOD
mouse, treated with ~TLA4Ig. Note absence
of NOD cell response and viable islets
within capsule. H&E. (x400). Arrows point
to outside of capsule wall.
Fiqure 14: Yield of Islets from Neonatal Porcine
Pancreas (Total Islet #).
Fiqure 15: In Vitro Insulin Release form
Nonencapsulated (N) and Encapsulated (E)
Neonatal Porcine Islets (uU/1000
islets/24hr.)
Fiqure 16: Dispersed neonatal porcine ~'islets", in
tissue culture, day #5. Anti-insulin
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15~77
- 18 -
immunocytochemistry demonstrates 5-10~ beta
cells. Approx. 400X.
Fiqure 17: Neonatal islet in microcapsule, biopsied
day # 103 from SZN-diabetic NOD-Scid mouse.
anti-insulin immunohistochemistry, showing
intensely insulin-positive beta cells,
occupying approximately 80~ of islet.
Approx. 400X. Arrow points to outer
-10 surface of microcapsule membrane.
Fiqure 18: Non-encapsulated intrasplenic/portal
neonatal procine islet xenograft in
streptozotocin diabetic NOD-Scid mouse.
Biopsies (not shown) revealed viable
porcine islets in both liver and splenic
parenchyma.
N=1
T=Transplant
S=Sacrificed for biopsies of spleen and
liver
Fiqure 19: Intraperitoneal microencapsulated neonatal
porcine islet xenograft into
streptozotocin-diabetic NOD-Scid mouse.
Biopsied day #103 (see Fig. 20).
N=1
T=Transplant
S=Sacrificed - -
Fiqure 20: Neonatal porcine islet in mirocapsule,
biopsied day ~103 after xenotransplantation
to SZN-diabetic NOD-Scid mouse. H & E, X
400. Arrow points to inner surface o~
microcapsule membrane.
~ =~ ~ =
CA 02232815 1998-03-23
W O 97/~1607 PCT~US9611~577
-- 19
Fiqure 21: Encapsuled Neonatal Porcine Islet
Xenografts (N-5) in NODs, treated with
CTLA4Ig, 200 ~g i.p. Q.O.D., x 20 days.
NOD 880 was biopsied at day #101 (see Fig.
22).
S=Sacri~iced for biopsy
(---)=Gra~t ~ailure
Fiqure 22: Microencapsulated neonatal porcine islet,
lo biopsied 101 days after xenotransplantation
i.p. to spontaneously diabetic NOD mouse.
CTLA4Ig, 200 ,ug i.p. Q.O.D., days ~ 0-21.
Arrow points to inside o~ intact
microcapsule wall. No pericapsular NOD
cellular response. H. ~ E. x200.
Fiqure 23: Adjacent section of same biopsy Anti-
insulin immunocytochemistry demonstrates
that most cells are insulin-positive beta
cells. x400.
Fiqure 24: Intraperitoneal microencapsulated neonatal
porcine islet xenografts in NOD mice
treated with CTLA4Ig , which does not Eix
complement.
Fiqure 25: Spleen cells were cultured at 2X106 cells/ml
in 96-well plates with no antigen, 10 empty
capsules, 10 capsules containing neonatal
pig islets, 4 x 103 neonatal pig islets that
were unirradiated or irradiated with 2000R.
Spleen cells were obtained ~rom normal NOD
mice (panel A); diabetic NOD mice (panel
B); diabetice NOD mice that were
transplanted with encapsulated, neonatal
CA 0223281~ 1998-03-23
W O 97/11607 PCTrUS96/15577
- 20 -
pig islets and injected with CTLA4Ig (panel
D) as described in Fig.24. After 4~ hrs
incubation, 3H-TdR was added and the cells
harvested 18 hrs later. Results represent
the average +SD o~ triplicate cultures.
Fiqure 26: Lymphokine production in cultures o~ spleen
cells ~rom the mice described in Fig. 24
were determined by ELISA. Spleen cells
~rom normal or diabetic NOD mice were
cultured with unirradiated neonatal, pig
islets as described in Fig. 24.
Supernatent fluids were harvested after 24
hrs of incubation and assayed for IL-4, IL-
10 and IFN~ using a sandwich ELISA and the
appropriate recombinant cytokines as
standards.
~iqure 27: Model o~ immune response to micro
encapsulated, xenogeneic islets by
autoimmune, NOD mice. Secreted insulin
clearly crosses the membrane o~ double
walled microcapsules and regulated glucose
levels in engrafted mice. 1): Potentially,
other donor proteins or protein fragments
of less than 100,000mw (AgX) that are shed
or secreted b~ islets diffuse out of
microcapsules and are endocytosed by
dendritic cells. 2): Dendritic cells
process proteins via the MHC class II
pathway and present peptide X complexed
with class II and co-stimulatory molecules
to CD4+ T cells. In the presence of the
appropriate cytokines, CD4' T cells are
activated and develop into Th2 cells that
,
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
express CD40L (GP39). B cells with sur~ace
IgM that bind AgX endocytose and process it
into peptides that bind MHC class II which
are expressed on the surface of B cells.
Th2 specific peptide X complexed with class
II binds B cells and the interaction of
CD40 with CD40L ~GP39) causes the
activation of B cells. 3): Activated B
cells mature into plasma cells under the
lo direction o~ Th2 lymphokines. 4~: Plasma
cells secrete speci~ic antibody that ~orms
complexed with AgX. 5): Binding o~
complexes to FCR activated macrophages to
secrete a variety of mediators including
IL-1, TNF~ and nitric oxide (NO), all of
which have toxic e~ects on islets and all
of which are small enough to cross the
double-walled microcapsules.
Detailed DescriPtion o~ the Invention
This invention provides a method of inhibiting viable cells
transplanted into a subject from being destroyed by the
subject's immune system which comprises: a) containing the
viable cells, or tissue comprising the viable cells, prior
to transplantation within a device comprising a
semipermeable membrane; and b) treating the subject with a
substance which inhibits an immune-system costimulation
event in an amount effective to inhibit the subject's
immune system from responding to said contained cells or
tissue.
As used herein, an "immune-system costimulation event" is
an interaction between an APC and a T-cell required in
conjunction with the binding o~ an MHC-bound antigen on the
surface of the APC to the T cell receptor. Immune-system
CA oi23281~ 1998-03-23
WO97~11607 PCT~S96/15577
costimulation events include any specific binding of an APC
cell-surface molecule (other than an MHC-bound antigen) to
a specific ligand on a T cell. Such speci~ic bindings
include, but are not limited to, binding of a B7 molecule
(present on the surface of an APC) to a CTLA4 receptor or
a CD28 receptor on the surface of a T cell, and binding of
a CD40 molecule (present on the surface o~ an APC) to GP39
~on the surface of a T cell).
Substances which inhibit immune-system costimulation events
are known in the art and include, but are not limited to,
T cell or APC cell-surface-molecule analogs, such as MRl
(which blocks the binding of CD40 expressed on the surface
o~ an APC to GP39 expressed on the sur~ace of a T cell), or
CTLA4 (which blocks the binding o~ a B7 molecule to a CD28
receptor or a CT~A4 receptor).
In one embodiment of the method for inhibiting destruction
of viable transplanted cells described herein, the
substance which inhibits an immune-system costimulation
event is CTLA4. The term CTLA4, for purposes of this
invention, is meant to indicate any proteinaceous construct
which comprises an amino acid sequence which is the same as
or sufficiently the same as the amino acid sequence of the
CTLA4 receptor such that the proteinaceous construct is
capable of binding to a B7 molecule, thereby blocking the
B7 molecule from binding to a CTLA4. receptor on a T cell.
Proteinaceous constructs are well known in the art and
indicate any molecule which comprises amino acid moieties
linked to one another by peptide bonds; including peptides,
polypeptides, and molecules comprising peptide and/or
peptide subunits. Thus, the term CTLA4 includes, but is
not limited to, molecules expressed by the gene encoding
the B7-binding site o~ the CTLA4 receptor in genetically
engineered cells, molecules expressed by mutants o~ the
CA 0223281~ 1998-03-23 ~
WO 97/11607 P ~ nJS96/15577
gene encoding the B7-binding site of the CTLA4 receptor
which molecules are capable of binding to a B7 molecule,
and synthetic amino acid ch~; n-S having an amino acid
sequence which is the same as or suf~iciently the same as
the amino acid sequence of the CTLA4 receptor such that
they are able to bind to s7. CTI.A4 also includes soluble
CTLA4 comprising the extracellular binding domain of the
CTLA4 receptor, such as CTLA4 Ig. Accordingly, the term
CTLA4 ~or purpo~es of this invention also includes CTLA4Ig,
i.e. a recombinant soluble ~usion protein which combines
the extracellular binding domain of the CTLA4 receptor with
the constant region of IgGl.
In an embodiment of this invention, the substance which
inhibits an immune-system costimulation event also alters
the cytokine profile of the subject so as to protect the
contained cells or tissue from the subject's immune system.
The term "cytokine profile" means the type and quantity of
each type of cytokine produced in a subject at a given
time. Cytokines are proteins which have an immune effect
and which are released by white blood cells. Examples of
cytokines include, but are not limited to interferon (such
as gamma-interferon), tumor necrosis factor, interleukin
~IL) 1, IL-2, IL-4, IL-6, and IL-10. For example, the
substance may be a substance which increases the production
of gamma-interferon in the subject. An example of a
substance which alters the cytokine profile of a subject so
as to protect contained cells or tissue grafted into the
subject is CTLA4Ig.
In another embodiment, the substance which inhibits an
immune-system costimulation event binds complement.
Substances which bind complement favor prolonged survival
of contained cells or tissue grafted into the subject. An
example of a substance which binds complement is CTLA4Ig.
CA 0223281~ 1998-03-23
W O 97111607 PCT~US96/15577
- 24 -
This invention also provides a method of inhibiting viable
cells transplanted into a subject from being destroyed by
the subject's immune system which comprises: a) containing
the viable cells, or tissue comprising the viable cells,
prior to transplantation within a device comprising a
semipermeable membrane; and b) treating the subject with
CTI.A4 in an amount effective to inhibit the subject's
immune system from responding to said contained cells or
tissue.
Devices comprising a semipermeable membrane useful for
transplantation of viable cells or tissue are well-known to
those of ordinary skill in the art, and any such device may
be used in the subject invention. Devices useful for the
subject invention may be comprised of various materials and
may be formed into various shapes, such materials and
shapes being well known in the art. Any particular device
for an application o~ this invention is selectable based on
factors including, but not limited to, the biocompatibility
of the material with the subject, the site o~
transplantation, whether the transplantation is
intravascular or extravascular, the method of
transplantation, availability, and economy. Examples o~
suitable shapes for devices include, but are not limited
to, hollow fibers, discs, and spheres. Suitable materials
include, but are not limited to, agarose hydrogel,
plastics, polymers, and polyamino acids. A device may be
comprised of more than one material.
In a preferred embodiment of the subject invention, the
device is a microcapsule. As used herein, the term
"microcapsule" means any polyamino acid spherical capsule.
Microcapsules as defined herein and their methods of
manufacture are well known in the art and include, but are
not limited, single layered, double layered, or
CA 0223281~ 1998-03-23
WO 97/11607 PCTAUS96/15577
- 25 -
multilayered polyamino acid spheres, as well as polyamino
acid spheres comprising a layer or more than one layer of
alginate.
.,
The viable cells or the tissue comprising the viable cells
in the a~orementioned method o~ this invention may be
derived from any source for viable cells. In one
embodiment, the viable cells or the tissue are derived from
a xenogeneic donor, i.e. a subject which is a di~erent
species from the subject into which the viable cells or
tissue are transplanted. In another embodiment, the viable
cells or the tissue comprising the viable cells are derived
from an allogeneic donor, i.e. a subject which is of the
same species as the subject into which the viable cells or
tissue are transplanted. In a ~urther embodiment, the
viable cells or the tissue comprising the viable cells are
derived ~rom the subject into which they are transplanted,
i.e. they are, inter alia, obtained from the subject,
contained within the device, and transplanted back into the
subject. Viable cells obtained from the sub~ect may, for
example, be genetically engineered after they are obtained
and before they are transplanted back into the subject.
The viable cells or tissue comprising viable cells may be
obtained from any donor. In one embodiment, the donor is
a mammal. Such a mammaliàn donor may, for example, be a
calve, a pig, a rabbit, a rat, a mouse, or a human. The
viable cells or tissue comprising viable cells may be
obtained ~rom a mammalian neonate, such as a neonatal pig.
The subject o~ the invented method described herein may be
any subject into which transplantation of viable cells is
desired. In one embodiment, the subject is a human. If
the subject is a human, the viable cells, or tissue
containing them, are in one embodiment derived from a
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/1~577
- 26 -
mammal, ~or example a human.
In another embodiment, the subject is a domesticated
animal. As used herein, a domesticated animal is any
animal subjected to human intervention. Domesticated
animals include, ~or example, farm animals which are raised
by humans and which are used as a resource ~or products for
human consumption. Such products include, but are not
limited to, meat, milk, and leather. Examples of
domesticated animals include, but are not limited to, cows,
pigs, sheep, horses, and chickens. Domesticated animals
use~ul in applications o~ the sub~ect invention may be
adults, infants, or domesticated animals at any other
developmental stage.
In one embodiment wherein the subject is a domesticated
animal, the viable cells comprise cells which secrete a
hormone which promotes growth in the domesticated animal.
Such hormones are well known to those of ordinary skill,
including hormones such as growth hormone and insulin. The
viable cells secreting such a hormone are in one embodiment
genetically enyineered to secrete the hormone. That is
they have been genetically engineered to contain the gene
encoding the hormone and are capable of expressing the
gene.
In the aforementioned method of this invention, the viable
cells in one embodiment comprise cells which secrete a
biologically active substance. The term "biologically
active substance" as used herein means any substance which
is capable of eliciting a physiological response in a
subject. The biologically active substance may illicit a
response in the subject into which the cells producing it
are transplanted. Cells which secrete biologically active
substances are well known in the art, and any such cells
CA 0223281~ 1998-03-23
W O 97/11607 PCTAJS96/15577
- 27 -
may be used in the subject invention
In one embodiment, the cells which secrete a biologically
active substance are endocrine cells. Endocrine cells are
well known to those of ordinary skill in the art and
include, but are not limited to, insulin-producing cells,
hepatocytes, parathyroid cells, and pituitary cells. In
another embodiment, the cells which secrete a biologically
active substance are neuroectodermal cells.
Neuroectodermal cells are also well known in the art, and
include, but are not limited to, adrenal cells and
lymphocytes.
In another embodiment, the cells are genetically engineered
to secrete a biologically active substance. For example,
the cells may be genetically engineered to secrete a
biologically active substance useful for treating the
subject into which they are transplanted. Thus, the
subject method provides a novel, useful, and advantageous
drug delivery system for treatment of subjects afflicted
with conditions including, but not limited to, cancer and
HIV infection. If the subject is afflicted with cancer,
the transplanted viable cells may, for example, be
genetically engineered to secrete Interleukin-2, a
cytokine, or a lymphokine. If the subject is infected with
HIV, the transplanted viable cells may, for example, be
genetically engineered to secrete a substance which
stimulate~ lymphocyte production in the subject, such as a
T cell growth factor or the HIV T cell receptor.
In the method of the subject invention, the permeability of
the semipermeable membrane of the device is determined
based on factors well known in the art, for example, the
size of the cells or tissue being contained, the size of
any substances needed to permeate the membrane in order to
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
- 28 -
sustain the cells or tissue, and the size of any
biologically active substances secreted by the cells which
are desired to permeate from the device. In one
embodiment, the semipermeable membrane is impermeable to
lymphocytes. In another embodiment, the semipermeable
membrane is impermeable to lymphocytes and immunoglobulins.
Using a semipermeable membrane which is impermeable to
immunoglobulins and/or lymphocytes prevents contact between
the immunoglobulins and/or lymphocytes of the subject and
the contained viable cells, and thereby prevents
destruction of the contained cells which would result ~rom
such contact.
Any suitable method o~ treatment may be used in the subject
invention to treat the subject with the substance which
inhibits an immune-system costimulation event, and such
methods are well-known in the art. For example, the
substance may be administered by injection to the subject
in the ~orm o~ a pharmaceutically acceptable composition.
I~ the substance is CTLA4, CTLA4Ig may be directly
administered to the subject, or in another embodiment,
cells genetically engineered to secrete CTLA4, that is
cells which have been genetically engineered to contain a
gene encoding a molecule capable o~ binding to a B7
molecule and to express that molecule, may be transplanted
into the subject.
In another embodiment o~ the invention, treatment o~ the
subject with the substance comprises transplanting into the
subject cells genetically engineered to secrete the
substance. If cells genetically engineered to secrete the
substance are transplanted into the subject, such cells may
themselves be contained within a device comprising a
semipermeable membrane prior to transplantation. In
different embodiments, the semipermeable membrane o~ the
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96~1~577
- 29 -
device containing the cells secreting the substance is
impermeable to immunoglobulins and/or lymphocytes, thereby
preventing destruction of these cells which would otherwise
result from such contact.
In the aforementioned embodiments, treatment with the
substance may occur before, after, of contemporaneously
with transplantation of the viable cells or tissue.
In another embodiment of the subject invention, treating
the subject with the substance comprises containing cells
genetically engineered to secrete the substance within the
device containing the viable cells or tissue prior to
transplantation.
In a ~urther embodiment of the invention, treating the
subject with the substance comprises genetically
engineering the viable cells transplanted into the subject
to secrete the substance prior to transplantation.
The amount of the substance effective to inhibit the
subject's immune system from responding to said contained
cells or tissue is determined by factors well-known to
those of skill in the art, including, but not limited to,
the amount of viable cells or tissue transplanted into the
subject, and the size and weight of the subject.
Inhibiting the subject's immune system from responding to
the contained viable cells or tissue by the method of the
subject invention involves an inhibition of immunoglobulin
production in the subject and an inhibition of macrophage
activation in the subject. Such immunoglobulins and
activated macrophages would otherwise be capable of
reacting with, and destroying, the contained viable cells
or tissue
CA 0223281~ 1998-03-23
W O97111607 PCTAJS96/15577
- 30 -
This invention also provides a method of treating diabetes
in a sub~ect which comprises: a) containing viable insulin-
producing cells, or tissue comprislng viable insulin-
producing cells, within a device comprising a semipermeablemembrane so as to obtain contained viable insulin-producing
cells; b) transplanting contained viable lnsulin-producing
cells obtained in step (a) into the subject in an amount
effective to treat diabetes in the subject; and c) treating
the subject with a substance which inhibits an immune-
system costimulation event in an amount effective to
inhibit the subject's immune system from responding to an
amount of contained viable insulin-producing cells
according to step (b).
Substances which inhibit an immune-system costimulation
event are known in the art, and any such substance may be
used in the method for treating diabetes described herein.
Substances which inhibit an immune-system costimulation
event which may be used in the subject method for treating
diabetes are described above. In one embodiment, the
~3ubstance is CTLA4.
The viable insulin-producing cells, or tissue comprising
viable insulin-producing cells, may be obtained from any
known source for insulin-producing cells or tissue
comprising insulin-producing cells.
In one embodiment of the subject invention, viable insulin-
producing cells are derived from pancreatic islet tissue.In another embodiment, the viable insulin-producing cells
comprise cells which have been genetically engineered prior
to transplantation to secrete insulin. The viable cells or
tissue may be derived from a xenogeneic donor, an
allogeneic donor, or they may be derived from the subject
CA 0223281~ 1998-03-23 -
W O 97/11607 PCT~US96/15577
- 31 -
prior to transplantation. If the cells are derived from
the subject, in one embodiment, they are genetically
engineered to produce insulin after they have been removed
~rom the subject, prior to being transplanted back into the
subject.
The viable insulin-producing cells or tissue comprising
viable insulin-producing cells, such a pancreatic islet
tissue, may be obtained ~rom any donor. In one embodiment,
the donor is a mammal. Such a m~mm~lian donor may, for
example, be a calve, a pig, a rabbit, a rat, a mouse, or a
human. The viable insulin-producing cells or tissue
comprising viable insulin-producing cells, such as
pancreatic islet tissue, may be obtained from a mammalian
neonate, such as a neonatal pig. In one embodiment, the
viable insulin-producing cells or tissue comprising viable
insulin-producing cells used in the subject invention
comprises neonatal porcine (pig) pancreatic cells.
The sub~ect of the invented method described herein may be
any subject into which transplantation of viable cells is
desired. In one embodiment, the subject is a human. If
the subject is a human, the viable cells, or tissue
containing them, are in one embodiment derived ~rom a
m~mm~l, for example a human.
Devices comprising a semipermeable membrane are well-known
to those of ordinary skill as described above, and any such
device may be used in the subject method of treating
diabetes. In different embodiments of the method, the
device is a hollow fiber, a disk, and a sphere. In another
embodiment of the method, the device is a microcapsule as
described above.
The method of treating diabetes described herein may be
CA oi23281~ 1998-03-23
WO97/llC07 PCT~S96/15577
- 32 -
applied to any subject for whom diabetes treatment is
desired. In one embodiment of the invented method for
treating diabetes in a subject, the subject is afflicted
with insulin-dependent diabetes mellitus (IDDM). In
another embodiment of the method, the subject is a mammal,
for example a human.
The amount of contained viable insulin-producing cells
transplanted into the subject ef~ective to treat diabetes
in the subject depends on factors known to those of
ordinary skill, including, but not limited to, factors such
as the weight of the subject, and the severity of the
diabetes.
The permeability of the semipermeable membrane of the
device in the subject method of treating diabetes is
determined by factors known to those of ordinary skill,
including those factors for determining permeability
described above. In different embodiments of the method,
the semipermeable membrane is impermeable to
immunoglobulins and/or lymphocytes.
Treatment of the subject with the substance which inhibits
an immune-system costimulation event in the subject method
of treating diabetes includes those methods of treatment
described above. If the substance is CTLA4, treatment may
comprise administering CTLA4Ig to the subject, for example
by injecting CTLA4Ig into the subject. Treatment with the
substance may, as described above, comprise transplanting
into the subject cells genetically engineered to secrete
the substance. Such genetically engineered cells may
themselves be contained within a device comprising a
semipermeable membrane prior to transplantation. If
treatment with the substance comprises transplanting into
the subject cells genetically engineered to secrete the
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
substance contained within a device comprising a
semipermeable membrane, the device is in di~ferent
embodiments impermeable to lmmunoglobulins and/or
lymphocytes.
In the aforementioned methods of treating the subject with
a substance, such as CTLA4, capable of inhibiting an
immune-system costimulation event, treatment may occur
before, after, or contemporaneously with transplantation of
the contained viable insulin-producing cells into the
subject.
In another embodiment of the subject method o~ treating
diabetes, treating the subject with the substance capable
of inhibiting an immune-system costimulation event
comprises containing cells genetically engineered to
secrete the substance within the device containing the
viable insulin-producing cells or tissue prior to
transplantation.
In another embodiment of the subject method of treating
diabetes, treating the subject with the substance comprises
genetically engineering the viable insulin-producing cells
to secrete the substance prior to transplantation.
Inhibiting the subject's immune system from responding to
contained viable insulin-producing cells or tissue by the
subject method of treating diabetes involves an inhibition
of immunoglobulin production and of macrophage activation
in the subject which would otherwise react with and lead to
the destruction of the viable insulin-producing cells or
tissue.
This invention will be better understood from the
t'Experimental Details~ section which follows. However, one
CA oi23281~ 1998-03-23
WO97/11~7 PCT~S96/15577
- 34 -
skilled in the art will readily appreciate that the
specific methods and results discussed therein are not
intended to limit, and rather merely illustrate, the
invention as described more fully in the claims which
follow thereafter.
Experimental Details
Improvements in Microcapsule Design
An improved formulation of poly-l-lysine-alginate
microencapsulation which allows nearly indefinite survival
of rat islets in spontaneously diabetic NOD mice is the
"double-wall~ microcapsule (Figures l and 2). This double-
wall microcapsule is more durable than conventional
microcapsules, with fewer capsule wall defects, has a
measured membrane permeability of approximately l00,000 Kd,
and excludes IgG (unlike conventional design capsules,
which allowed passage of IgG and 148,000 Kd fluoresceinated
dextran)(9,l9,20,ll8). These data support the relevance of
encapsulated islet xenografts for eventual application in
humans with IDDM.
Poly-h-Lysine (PLL) Concentration Alters P~ ~-hility o~
PLL-Alginate Microcapsules
It was postulated that microencapsulated islet xenogra~t
survival would be influenced by microcapsule permeability.
We found that microcapsule permeability may be altered by
increasing or decreasing the concentration of PLL (poly-l-
lysine~ in the microcapsule formula. Red blood cells were
encapsulated in alginate via an air jet system and then
incubated with various polyamino acids including PLL. The
RBCs were then lysed and hemoglobin (MW 64,500) ef~lux was
measured spectophotometrically at 480nm as a ~unction of
time alongside a concurrent control. Permeability
coefficient was calculated according to the following
formula: (2.303 ~ Cf~ Vt ~ S) / (Ci ~ At), where Cl and C~
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/15577
are the initial and final he~oglobin concentrations, Vt and
At are the total volumes and areas of capsules
respectively, and S = 810pe of ln (Ct-Cf) / (Ci-Ct)(119).
PhL substitutions (poly-l-ornithine, alanine, aspartate and
histidine) did not result in viable capsules. PLL
molecular weight alterations did not effect permeability.
PLL concentration was the most critical factor in altering
capsule di~~usion. These observations are supported by
the recent findings of other investigators (119). There
was a thirteen ~old decrease in hemoglobin efflux occurring
in capsules that had a fourfold increase in PLL (see Table
1~. In experiments, encapsulated rabbit islet survival in
NODs is prolonged using microcapsules with permeability
<70,000 Kd vs. 100,000 Kd (see Figure 3).
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/15577
- 36 -
o
o
~ o
oo
o ",
~ ~
~ --' I
3 o ~
g
1_ ~ o
.~ ~ ~
o
.~ o
C V~
V ._ ~
~,, ~o
~
c
_I
Q~
E ~ ~~ E E
CA 022328l5 l998-03-23
W O 97/11607 PCT~US96tl5577
- 37 -
Microcapsule~ Prevent or Delay Host Sensitization
To clarify the mechanism of long-term microcapsule
protection o~ xenogeneic rat islets, experiments were
performed in which paired diabetic NODs were pre-treated
with saline or Lewis rat islets (2QO intra-peritoneally) or
o6 Lewis rat splenocytes intra-peritoneally. ~ncapsulated
~ewis islets were xenografted into presensitized and
control NODs 14 days later. As shown in Figures 4 and 5,
lo both islet- and splenocyte pretreatment resulted in rapid
graft rejection while non-presensitized NODs accepted
encapsulated islet xenografts long-term. These data
suggest that a major function o~ microcapsules is to
prevent host sensitization, rather than to protect grafts
~5 from the e~fector arm of the response. Thus, maneuvers
which reduce islet Immunogenicity may be synergistic with
islet encapsulation.
Comparison~ o~ Encap~ulated I let Iso-, Allo- and Xenograft
Sur~i~al in NODs
We have found that microencapsulation allowed islet
xenograft survival in NODs of 79 ~t 15 days (N=8) (X + SE)
for Lewis rat islets, vs. 20 :: 2 days (N=7) for rabbit
islets and 14 i 4 (N=3) for dog islets (Table 2), with
2s similar peri-microcapsule NOD cell accumulations at
rejection. NODs also rejected encapsulated, allogenic
Balb/c islets in 73 + 31 days (N=4) and encapsulated
isologous NOD islets in 44 + 7 days (N=4)(Table 2).
However/ biopsies of these allo- and isologous grafts, at
rejection, have shown ~ew host macrophages adherent to
microcapsules, while free peritoneal cells (thus far not
characterized) were present. Thus encapsulated islet
xenograft rejection is distinct from iso- and allo-gra~t
rejection in this model.
CA Oi2328l5 l998-03-23
W097tll607 PCT~S96/15577
- 38 -
Table 2.
I~let Iso-, Allo- and Xenoqra~t~ in NOD Mice
Group Donor-Recip Technique Rx. (N) Surv
(days)@
1 NOD-NOD CAP/I.P. (-) 4 44 + 7
2 Balb-NOD CAP/I.P. (-) 4 6,7,7
3 LeRat-NOD CAP/I.P. (-) 8 5,5
4 Dog-NOD CAP/I.P. (-) 3 73 + 31
5 Rabbit-NOD CAP/I.P. (-) 7 79 + 15
6 Rabbit-NOD CAP/I.P. CyA 4 14 + 4
7 Rabbit-NOD CAP/I.P. CTLA4Ig 7 20 + 2
8 Rabbit-NOD Splenic CTLA4Ig 2 22 + 3
9 Rabbit-NOD-Scid Splenic - 1 22 + 6
10 Rabbit-NOD-Scid CAP/I.P. - 1 98 +
25#
11 LeRat-NOD-Scid Splenic - 2 6
12 Rabbit-NOD-Scid Splenic - 1 1198
13 Rabbit-NOD-Scid CAP/I.P. - 4 56 + 11
14 LeRat-NOD-Scid Splenic - 2 1248
15 Cal~-NOD CAP/I.P.(-) 1 24
16 Pig-NOD CAP/I.P.(-) 2 6,8
17 Human-NOD CAP/I.P.(-) 1 6
CA 02232815 1998-03-23
W O 97fll607 PCT~US96/15~77
=Pc.002 vs. Group 7; @= Mean ~ SEM; #=P<.05 vs. Group 7;
*#=P~ . 003 vs. Group 7
CAP/I.P.= microencapsulated islet gra~t to peritoneal
cavity; Splenic - Nonencapsulated islets grafted beneath
splenic capsule.
We have also ~ound that microencapsulation prolongs the
~unctional survival of islet xenografts in NODs, when
compared to survival of unencapsulated islets injected into
the spleen. The same is true ~or islet allografts and for
islet isografts into NODs (Table 3).
Table 3.
Bene~icial E~ect o~ "Double-Wall" Microencapsulation of
Survival of Islet Iso-~ Allo-and Xenografts in NOD Mice
Donor-Recip Technique (N)Surv(days)@
NOD-NOD CAP/I.P. 4 44~7
NOD-NOD Splenic 3 6,7,7
Balb-NOD CAP/I.P. 4 73+31
Balb-NOD Splenic 2 5,5
Lewis Rat-NOD CAP/I.P. 8 79~15
Lewis Rat-NOD Splenic 9 l9i3
Dog-NOD CAP/I.P. 3 14+4
Dog-NOD Splenic 2 o,o
Rabbit-NOD CAP/I.P. 7 20~2
Rabbit-NOD Splenic 2 5,6
Neonatal Pig-NOD CAP/I.P. 8 27~13
Neonatal Pig-NOD Splenic 3 6il
'pc.01 vs. spl(nic: ~=Mean + SEM.CAP/I.P.
microencapsulated islet gra~t to peritoneal cavity;
Splenic = Nonencapsulated islets grafted beneath splenic
capsule.
CA b223281~ l998-03-23
WO97/11607 PCT~S96/15577
-- ~0
Functioning and rejected encapsulated xenografts were
biopsied ~rom the peritoneal gra~t sites of spontaneously
diabetic NOD mice, on days #4-~50 post-transplantation.
Controls included normal mouse peritoneal fluid and
peritoneal fluid ~rom NOD mice bearing empty capsules or
capsules with functioning (recipient normoglycemic) rat
islets ~20,74). However, cell number increased
dramatically at rejection on days #14 and ~50. Pipetting
of biopsied capsules freed adherent cells. Flow cytometric
analyses revealed that 20-50~ of non-adherent peritoneal
cells were B220+ (B cells), and that the majority of free
peritoneal cells and cells adherent to microcapsules were
Macl+ (20,74). The percentages of CD4+ and ~D8~ peritoneal
cells were low (4-9~). By FACS analysis, the phenotype of
peritoneal Macl cells shifted from predominantly Granl- to
Gran 1+ during rejection of xenogeneic islets in
microcapsules (vs. empty capsules)(20,74,120). These
~indings were confirmed by immunocytochemistry (20,74). In
additionJ immunocytochemistry documented IgG and IgM around
microcapsules, and IL-1 and TNF alpha both around and
within microcapsules (20,74).
Analysi~ o~ Cytokine MeQ~enger RNA (mRNA) in Encapsulated
I~let Xenograft3 Biopsie~ from NODs
To elucidate the pathogenesis of NOD destruction of
encapsulated islets, ~RNA was extracted from recipient NO~
peritoneal cells and expression of mRNA for IL-2, IL 4, and
IL-10 was studied by RT-PCR, as previously described (121) .
Integrity of RNA samples was assessed by inspection of
northern transfer and hybridization with the probe for the
3' untranslated region of beta actin (121). IL-4 was
detected in the majority of xenografts undergoing
rejection. IL-10 expression was variable (Table 4 ) . IL-2
CA 02232815 1998-03-23
W ~ g7ill607 PCTfJS96/lS577
- 41 -
was detected during autoimmune destruction of NOD
isografts, (and in one allograft) but only rarely in
rejecting xenografts (Table 4). These data suggest that
the primary T cell response in rejecting encapsulated islet
xenogra~ts is "Th2-like". This interpretation is
consistent with the observation that large numbers of
activated macrophages and immunoglobulins are associated
with rejecting encapsulated islet xenografts in NODs.
Thus, it is possible that rejection of encapsulated islet
xenogra~ts is initiated by soluble, or shed, xenoantigens
that are processed via the Class II pathway by host APC.
These APC then activate Th2 cells via B7/CD28 dependent
mechanisms. We postulate that formation of antigen-
antibody complexes in the peritoneal cavity activates
macrophage~ to release cytokines that are directly toxic to
encapsulated islets.
CA 022328l5 l998-03-23
WO 97/11607 PCT~US96/1~77
- 42 -
Table ~.
CYTOKINE mRNA IN BIOPSIES OF ENCAPSULATED XENO- ISLETS IN
NOD MICE
~s
lslet Donor NOD# SamPle Day Rejected Day BioPsied IL2 IL4 IL10
NOD 194 FC- 39 40 + + -
291 FC 14 21 +
Balb~c 487 Ca~ 12 14 + - -
Rat 154 Cap 18 20 - +
154 FC 18 20 - +
58 Cap 34 38 - + +
165 Cap 21 28 - +
54 Cap 136 143 + + +
54 FC 136 143 +
107 FC 41 45
453 CaP 132 134 +
Canine141 Cap 17 24 - + +
268 Cap 13 14 - - -
268 FC 13 14
69 FC 18 24 - + +
Rabbit 91 Cap 35 49
91 FC 35 49 - +
151 Cap 28 32 + +
46 FC 12 15 - +
FC 18 21 - +
152 FC Funct.15 - +
157 FC Funct.15 +
Human 136 CaP 6 8 - + +
Cap = Cells adherent to capsules
'F C = Free peritoneal cells
4 0 ~ = RT- PCR ~ - ) is undetachable and (+) is detachable
CA 02232815 1998-03-23
W O 97/11607 PCT~US96115~77
- 43 -
The NOD-MHC i8 Nece~ary :Eor Rejection o:E Encap~ulated
Islet Xenogra~ts
Both NOD and (SZN-diabetic) B10 H-297 ~expresses the NOD-
MHC-linked disease allele) rejected encapsulated rat
islets, while NOD.H-2b mice, which express all of the non-
MHC-linked diabetes susceptibility genes, accepted
encapsulated rat islets for >100 days (similar to B10
controls)(75). This suggests that the NOD-MHC may
contribute to destructive responses against encapsulated
islets which are distinct from diabetes susceptibility,
since neither B10.H-2 g7 nor NOD.4-2b mice develop diabetes
spontaneously (20,75). The possibility that SZN treatment
of BIO.H-2g7 mice may have initiated an autoimmune response
was considered; however, 2/2 non-diabetic (no SZN
treatment) B10.H-2 g7 mice rejected encapsulated rat
islets ~by biopsy histology, day X603~75).
~8' Depletion Does Not Protect Encapsulated I~let
Xenografts in NODs
It was found that treatment of NOD recipients of
encapsulated rabbit islets with either monoclonal antibody
53.6 .7J (100~g i.p. day -5 and then twice weekly)(anti-CD8)
or cyclosporine (CyA), 30. Mg/kg, s.c., daily had no effect
on graft survival (Table 2). CD8+ cell depletion was
conf}rmed by flow cytometry of NOD spleen and peritoneal
cells. Biopsies of failed grafts revealed intense host
cellular responses and non-viable islets within intact
microcapsules. These data are consistent with prior
observations, that CD4+ (but not CD8+) T-cells play a
dominate role in non-encapsulated islet xenograft rejection
(83j. They also are consistent with a predominantly Th2
NOD rejection mechanism of encapsulated islet xenografts.
CA b223281~ l998-03-23
wos7/ll6o7 PCT~S96/15577
- 44 -
Co-stimulatory Blockade Prolongs Encapsulated Islets
Xenogra~ts in Diabetic NOD~
It was shown previously that inhibition o~ CD4+ helper T-
cells by administration of monoclonal antibody GK 1.5 to
diabetic NOD recipients resulted in significantly increased
survival (~100 days) of both encapsulated rat and dog
islets (7,84)(Figure 6). The experiments herein show that
treatment of NOD mice with CTLA4Ig (200~g i.p. day #0, and
QOD until day #90) significantly prolonged encapsulated
rabbit islet survival, from 20 ~ 2 days to 98 ~ 25 days
(p<.05)(see Table 2 and Figures 7 and 8).
This suggests that an "indirect" pathway of antigen
presentation is dominant in NOD responses to encapsulated
islet xenografts. Unlike findings with human islet
transplanted to SZN-diabetic mice (12), CTLA4Ig alone did
not increase nonencapsulated rabbit or rat islet survival
in NODs (intrasplenic or renal subcapsule)(Table 2),
suggesting that encapsulation and CTLA4Ig both were
required to prolong graft survival.
Furthermore, the experiments herein show that encapsulated
female islets from INSCTLA4 mice, which express CTLA4 on
the beta cell insulin promoter, function long-term in NODs
~see Figure 9). Unencapsulated INSCTLA4 islets were
rejected by NODS in 6 -7 days. These data suggest that
indefinite survival of discordant islet xenografts may be
achieved by combinations of donor islet encapsulation and
limited host immunomodulation. These data also support the
working hypothesis that donor antigen(s) are shed from
microcapsules and processed by APCB which activate CD4~ T
cells via B7/CD2 8-dependent mechanisms. In this model,
CTLA4-transgenic mice secrete CTLA4, along with insulin,
and CTLA4 inhibits antigen presentation. Interestingly,
3~ female mice secrete more CTLA4 than do male mice in this
CA 0223281~ 1998-03-23, ~
W O 97/11607 PCT~US96/15577
- 45 -
transgenic model (pers. Comm.).
NOD-Scid Mice Accept Rat and Rabbit I~let Xenogra~t~ Long-
Term
These experiments demonstrate that NOD-scid mice are
susceptible to M~D-SZN diabetes (30mg/kg daily x5); and
reversal of NOD-scid diabetes with xenografts of
nonencapsulated and encapsulated rat and rabbit islets for
greater than 50 days is documented (see Figures 10,11, and
12 and Table 2). Thus, the NOD-scid mice will serve as a
good recipient model ~or the trans~er of antibodies and/or
T cells for studies of the mechanisms by which encapsulated
islets are rejected. We noted recurrent hyperglycemia in
3/4 NOD-scids receiving ~icroencapsulated ra~bit islets, on
days #51, #68, and #70. Biopsies revealed disrupted
capsules and minimal cellular failure for technical
reasons, since empty microcapsule controls done
concurrently, showed broken microcapsules (in 1/3) and
intact microcapsules (in 2/3) at day #50.
Co~timulation Blockade with CTLA4Ig
Method:
Adult New Zealand rabbit islets were isolated by duct-
injection, collagenase digestion. Rabbit islets (approx.
2000) were encapsulated in double-wall, poly-1-lysine-
alginate microcapsules and xenografted intraperitoneally in
NODs, as previously reported (7,20~. Controls received
approximately 2000 unencapsulated rabbit islets xenografted
beneath the splenic or renal capsule, as previously
described (7,20).
Murine CLTA4Ig, provided by Bristol-Myers-Squibb, Seattle,
WA, was administered at 200ug intraperitoneally (i.p.),
day-1 and then Q.O.D. for 14 or 92 days, or until graft
rejection.
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15S77
- 46 -
Controls inc~uded NODs receiving identically encapsulated
rabbit islets (i.p), and given no additional treatments,
cyclosporine 30mg/kg s.c., day-I, and then daily, or
monoclonal anti-CD8 antibody ~53.6.7.7 (A.T.C.C.), 10~g
i.p. day-5, +2, and then weekly.
Biopsies of long-term functioning peritoneal microcapsules
were done periodically, using metafane anesthesia and
sterile technique. Removal of 100-200 microcapsules
allowed histologic light microscopic studies without
altering gra~t-related normogycemia.
~t 180 days after successful encapsulated rabbit islet
xenogra~ting, splenectomy was per~ormed on one long-term
functioning, biopsy-proven, CTLA4Ig-treated NOD. These
splenocytes (107) were passively trans~erred,
intraperitoneally, to two naive diabetic NODs, which
subsequently received identically encapsulated fresh rabbit
islets - (donor-type New Zealand, not inbred),
intraperitoneally, on day 10-14 after splenocyte trans~er.
Statistical difference between groups were assessed by use
Student's "t"-tested and by ANOVA.
Results:
Treatment of NODs with CTLA4Ig prolonged survival of
intraperitoneal poly-l-lysine-alginate microencapsulated
donor rabbit islet xenogra~ts (CAP/I.P.) In spontaneously
diabetic NODs, when compared to either islet
microencapsulation or host CTLA4Ig treatment alone. The
longest functioning grafts were in NODs treated for 92 days
with CTLA4IgK, but mean gra~t survival was not
statistically different from that of NODs which received
CTLA4Ig for only 14 days (See Table 5). By contrast,
recipient NOD treatment with cyclosporine A ~CyA),
monoclonal antibodies speci~ic ~or CD8 (53.6.7.7) or CTL4Ig
CA 0223281~ 1998-03-23
W O 97/11607 PCTAUS96/15~77
- 47 -
alone were ineffective (See Table 5). Biopsies o~ long-
term surviving encapsulated rabbit islets from NODs
documented intact microcapsules, viable donor islets, and
absence of per-capsular NOD cellular response (See Figure
13).
Biopsies o~ ~ailed CTLA4Ig-treated, encapsulated rabbit
islet xenografts showed primarily disrupted (broken)
microcapsules, few viable islets, and minimal pericapsular
cellular reaction. Biopsies of intrasplenic rabbit islets
at rejection showed nuclear and cytoplasmic damage and
nonviable islets. Biopsies of controls receiving
intraperitoneal encapsulated rabbit islets, plus ~yA or
53.6.7.7 recipient treatments or no treatment, performed at
rejection on days 12-52 post-grafting, uniformly showed
marked pericapsular accumulations of macrophages,
neutrophils, and lymphocytes, as previously described
(1~3,3,144).
Both NODS receiving encapsulated rabbit islets 10-14 days
following passive transfer or 1O7 splenocytes from a long-
term normoglycemic NOD, (with functioning encapsulated
rabbit islets, off CTLA4Ig treatment for 90 days) rejected
their grafts in 10-12 days, with graft biopsies which were
indistinguishable from untreated control NODs. Biopsies of
pancreas from NODs in all experimental groups showed
uniform absence of islets, and occasional accumulation of
lymphocytes in perivascular areas.
CA 022328l5 l998-03-23
W O 97/11607 PCT~US96/15577
- 48 -
Table 5:
EFFECTS OF CTLA4I~, CvA AND ANTI-CD8
MONOCLONAL ANTIBODY ON ENCAPSULATED RABBIT
ISLET XENOGRAFT ::iU~VlVAr~ IN DIABETIC NOD MICE
Graft Survival
Grou~ Donor-Reci~Techniaue Rx. (N) X+SE Davs
#1 Rabbit-NODCAP/I.P. None 720i2 12,16,18,18,
20,28,28
#2 Rabbit-NODCAP/I.P. CyA 422_3 13,24,26,26
#3 Rabbit-NODCAP/I.P. 53.6.7.7 4 5_9 14,15,18,52
7.7
#4 Rabbit-NODCAP/I.P. CTLA4Ig 8 108_24~ 37d,43,47,58
(x92 days) 148,1515,173,
205d
#5 Rabbit-NODCAP/I.P. CTLA4Ig 470+8 ~ 48,66,81,83
(x14 days)
#6 Rabbit-NODRenal/ CTLA4Ig 36i1' 5~8~,6~r~,6
Splenic
#7 Rabbit-NODRenal/ None 2 _ 5(a),6
S~lenic
3 = sacrificed, functioning graft.
d s died, functioning graft.
'p~.005 vs. Group 1, ~"t~-test).
p~.0001 vs. Group 1, (~'t~'-test).
CTLA4Ig, 200~g day -1, then Q.O.D., i.p.
CyA - 30mg/ky day -1, then Q.D., 8 . C .
63.6.7.7 -100~g, day -5,+2, then weekly, i.p.
(r) = renal subcapsule, not encapsulated
(s) = splenic subcapsule
= P = .31 vs. Group #4, ANOVA
~arge-Scale Neonatal Porcine Islet I~olation
We believe the neonatal pig is the most promising xenogeneic
CA 02232815 1998-03-23
W O 97~11607 PCT~US96/15577
- 49 -
source of donor islets. A reproducible method for
isolation o~ large numbers of functionally viable islets
~rom neonatal procine donors has been developed (146,147).
With this technique, 30,000-100,000 islets may be obtained
from each donor pig (Figure 14). Neonatal pig islet cells
continue to secrete insulin in vitro after
microencapsulation. (Figure 15). These neonatal pig islets
are actually dispersed neonatal porcine pancreatic cells
which reaggregate to ~orm 1'islet"-like spheroids with
approximately 5-10~ beta cells (Figure 16), which is
~ignificantly higher than the 1-2~ beta cell concentration
in the adult procine pancreas. Furthermore, biopsies of
these "isletsl' 100 days following xenotransplantatlon
reveal increa~ed numbers o~ intensely insulin-positive
islet cells (Figure 17). These neonatal pig islets have an
added advantage over adult islets, in that they appear to
differentiate and proliferate within microcapsules after
transplantation.
Both Encapsulated and Non-encapsulated Neonatal Porcine
Iqlets Reverse SZN-Diabetes in NOD-Scid Mice.
Recently, the Scid mutation has been back-crossed onto the
NOD background, resulting in immuno-deficient NOD-Scid mice
~66,6~,68,69). These mice are homozygous for the Scid
mutation, which results in an inability to rearrange T-cell
receptor and immunoglobulin genes (48,79). Consequently,
these mice lack T and B-lymphocytes. NOD-Scid mice do not
develop diabetes spontaneously; but they may be rendered
diabetic with multiple low-dose streptozotocin (MLD-SZN),
~67,68,69) NOD-Scids express NOD MHC genes and other genes
that are required for development of diabetes, upon
transfer of lymphocytes from diabetic NODs.
To document functional viability of neonatal procine
islets, we xenografted them into SZN-diabetic normalized
-
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/15577
- 50 -
hyperglycemia in streptozotocin-diabetic NOD-Scid mice for
~100 days (Figures 18, 19, 20~. This data demonstrates
that neonatal procine islets survive and function
physiologically in xenogeneic recipients for prolonged
periods, in the absence of an immunological attack.
We have found that CTLA4Ig significantly prolonged survival
of encapsulated rabbit and porcine islets in NOD
recipients/ whereas CTLA4Ig alone did not protect non-
encapsulated islet xenografts in NOD mice (Table 6 andFigure 21).
_
:
CA 02232815 1998-03-23
W O g7/11607 - 51 - P ~ AJS96/15577
U
u
a
.,
~4
r~
J-
o
X X X
~ ' N , rd
'' ~DN
.~ ~ ' U) ,I t'
o
~1 In ~ ~ N ~1 ~1
o O
~ N
ta a~
V~N ~ +1 5-
XN H r-- ~D I N ~1 Lr) ~ a
V
~ 3
o
O r ~ ~ ~ H t ~ ~ ~ O
J Z V V V Z Z; ~ ~ Z . ~
,~ ~ ~ a ~ ~
h .. ~ rl . ~ ~ C
~ q t~ ~ t~ o
U~ U 41 C,) H H H a ~ ~ a ~ O
U ~ U ~ ~ ~
r~ ~ lV t-
E-l -rl ~ O ~
O I ~ , ~ r
r L ' . ~ '
t~ t
CA 0223281~ 1998-03-23
W O g7/11607 PCT~US96/1~577
- 52 -
Biopsies o~ long-term functioning encapsulated neonatal
porcine islet xenografts showed viable porcine islets
within intact microcapsules and absence of host NOD
pericapsular reactivity was observed in biopsies of long-
term normoglycemic NODs (Figure 22 and 23).
To analyze the potential mechanisms of action of CTLA4Ig in
this model, we substituted a recently devised mutant of
CTLA4Ig, which does not fix complement (CTLA4Ig ) (145).
0 As shown in Figure 24, our studies have revealed that
CTLA4Ig~ does not prolong graft survival above that of
capsules alone. The data are distinct from findings with
murine allografts, which are prolonged significantly by
either conventional CT~A4Ig or mutant CTLA4Ig . These
results suggest that mechanisms of prolongation of graft
survival by CTLA4Ig may be dif~erent for allogeneic and
xenogeneic islet grafts. The results suggest that the
cytokine pro~ile in a subject can be altered in favor of
graft protection. In the sytem studied in this experiment,
conventional CTLA4Ig altered the cytokine production so as
to protect the graft by increasing gamma-interferon
production in the host. Conversely, in the studied system,
an increase in IL-10 production induced by CTLA4Ig~
treatment favored graft rejection.
We also measured proliferative responses by spleen cells
from a matched pair of diabetic NOD mice that were
transplanted with the same batch of encapsulated, neonatal
pig islets but were treated with either CTLA4Ig or the non-
complement fixing CTLA4Ig~ (Figure 25). In this
experiment, normal or diabetic NOD mice did not proliferate
when stimulated by neonatal pig islets (panel A and B).
The reason for the inconsistent response of nontransplanted
NOD mice is not yet known but is under investigation.
Empty capsules did not induce proliferation in any of the
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
- 53 -
spleen cells but islets and encapsulated islets recognized
by T-cells are small enough to exit from microcapsules.
However, more experiments may verify this interpretation.
As usual, background responses of spleen cells ~rom mice
rejecting grafts (panel D) were higher than those from mice
that were not rejecting grafts ~panel C).
These results suggest that spleen cells from both mice
engra~ted with encapsulated islets were primed in vivo, and
lo are somewhat surprising given the fact that the mouse that
received CTLA4Ig showed no signs of rejection. These
results did not address the possibility that there might be
different fluids from cultures stimulated with neonatal pig
islets for lymphokines by ELISA (Figure 26). These results
indicate that lymphokines were produced only by mice that
were engrafted with neonatal, pig islets. More
importantly, spleen cells from the mouse that had accepted
its graft long term ~treated with CThA4Ig) produced a
preponderance of INF~ and low levels of IL-10. These
results suggest that CT~A4Ig induced long term tolerance to
neonatal pig islets that is associated with T cells that
produce INF~. Rejection of xenogeneic islet graft occurred
when lymphokines shifted to IL-10. Thus, graft rejection
is associated with a Th2-like response, whereas graft
survival is associated with Thl-like responses. These
findings are consistent with our working model (Figure 27).
These results differ somewhat from the picture obtained by
analyzing mRNA level at the site of rejection where IL-4
predominated in mice that rejected the encapsulated,
xenogeneic islets.
Discussion:
On the basis of our data, we develop a model to describe
t~e mechanisms that we think are involved in rejection of
microencapsulated xenogeneic islets by autoimmune, NOD mice
CA 0223281~ 1998-03-23
W O 97/11607 PCTAUS96/15577
- 54 -
(Figure 27). Secreted insulin clearly crosses the membrane
of double walled microcapsu~es and regulates glucose levels
in engrafted mice. Potentially, other donor proteins or
protein fragments of less than 100,000 mw (AgX) that are
s shed or secreted by islets diffuse out of the microcapsule
and are endocytosed by dendritic cells. Dendritic cells
process proteins via the MHC class II pathway and present
peptide X complexed with class II and co-stimulatory
molecules to CD4~T cells. In the presence of the
appropriate cytokines, CD4~T cells are activated and
develop into Th2 cells that express CD4OL. B cells with
surface IgM that binds AgX endocytose and process it into
peptides that bind MHC class II which are expressed on the
surface of B cells. Th2 specific peptide X complexed with
class II binds B cells and the interaction of CD40 with
C~40L causes the activation of B cells. Activated B cells
mature into plasma cells under the direction of Th2
lymphokines. Plasma cells secrete specific antibody that
forms complexes with AgX.
Antibodies are not able to directly damage the encapsulated
islets because they are too large to enter the capsules.
However, antibodies could be involved in the recruitment
and activation of macrophages which are the predominant
population in the peritoneal cavity of NODs rejecting
encapsulated islet xenografts. Specific antibodies in the
peritoneal cavity could form complexes with antigens shed
or secreted from the capsules. Such antigen-antibody
comp~exes efficiently bind to FcR expressed on the surface
of peritoneal macrophages. Binding of complexes to FcR
activates macrophages to secrete a variety of mediators
including IL-1, TNF~ and nitric oxide (NO) (122,123), all
of which have toxic effects on islets and all of which are
small enough to cross a double walled microcapsule. The
effector arm could be further augmented by the activation
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
of complement (c) by antigen complexes. C3b bound to the
complexes enhances the activation of macrophages by
increasing the binding of the complexes via the C3b
receptor (124) and small peptides such as C3b released
during complement activation induce local in~lammatory
responses thereby attracting more macrophages into the
peritoneal cavity (125).
We demonstrated synergy of donor islet microencapsulation
0 and NOD CTLA4Ig treatment in prolonging islet xenograft
survival. Our data represent the longest biopsy-proven
survival of discordant islet xenografts in NODs reported to
date. Neither CTLA4Ig nor encapsulation alone were
e~ective. Furthermore, splenocytes from a long-term
successful graft recipient did not transfer donor-specific
unresponsiveness. Failure o~ anti-CD8 and CyA therapies is
consistent with our hypothesis of a primarily Th2 type
response in this model.
23 There is considerable evidence that xeno-recognition
~unlike allorecognition) occurs primarily via the so-called
"indirect" antigen presentation pathway, by which host APC
present peptides scavenged from extracellular (donor)
proteins to host helper T-cells (27,137,29,138). Our
recent report, that the host MHC is critical to NOD
rejection of encapsulated islet xenografts (75), and our
prior observations, that helper T-cells are essential for
this response (7), both are consistent with an "indirect"
pathway. Our prior ~indings of more rapid destruction of
3~ encapsulated "discordant" (widely unrelated) islets
~canine, rabbit, bovine, porcine) than "concordant"
~closely related)(rat) islets (20), also support this
hypothesis, since the "directn pathway would favor an
accelerated reaction to "concordant" donor tissue.
Furthermore, our current data suggest that "indirect"
-
CA 02232815 1998-03-23
W O 97J11607 PCT~US96115577
- 56 -
antigen presentation may be blocked by CTLA4Ig in this
model of encapsulated islet xenotransplantation. In
conclusion, we have found that neither microencapsulation
nor CTLA4Ig alone prevent NOD destruction o~ rabbit islets.
However, we have observed synergy between CTLA4Ig treatment
of NOD recipients plus encapsulation with significantly
prolonged discordant islet xenograft survival.
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
- 57 -
References
1. The Diabetes Control and ~omplications Trials Research
Group. The ef~ect of intensive treatment o~ diabetes
on the development and progressions of long-term
complications in insulin-dependent diabetes mellitus.
NEJM 1993j329:977-986.
2. Lacy P. Status of islet cell transplantation.
Diabetes Review 1993jl:76-92.
3. Parker C. Naji A. Perspectives and islet
transplantation for diabetes-cures or curiosities?
NEJM 1992;327:1861-1868
4. Warnock G., Rajotte R. Human pancreatic islet
transplantation. Transplantation Reviews 1992j6:195-
208.
5. Remuzzi F., Ruggenenti P., Mauer S. Pancreas and
kidney/pancreas transplants; experimental medicine or
real improvement? Lancet 1994;343:27-31.
6. Weber C., Hardy M., Riveria S., Bailey-Braxton D.,
Michler R., Thomas W., Chabot J., Pi-Sunyer F., Wood
M., Reemtsma K. Diabetic mouse bioassay for functional
and immunologic human and primate islet xenograft
survival. Transplant proceedings 1986;18:823-828.
7. Weber C., Zabinski S., Koschintzky T., Rajotte R.,
Wicker L., D'Agati V., Peterson L., Norton J.,
Reemtsma K. The role of CD4+ helper T cells in
destruction of microencapsulated islet xenografts in
NOD mice. Transplantation 1990j49:396-404.
CA 0223281~ 1998-03-23
W O 97/11607 PCTAJS96115577
- 58 -
8. Mandel T., Koulmanda M., Loudovaris R., Bacelj A.
Islet gra~ts in NOD mice: A comparison of iso_, allo,
and pig xenografts. Transplant Proceedings
1989;21:3813-3814.
9. Weber C., D'Agati V., Ward L., Costanzo M., Rajotte
R., Reemtsma K. Humoral reaction to microencapsulated
rat, canine, porcine islet xenografts in spontaneously
diabetic NOD mice. Transplantation Proceedings
1993;25:462-463.
10. Akita K., Ogawa M., Mandel T. Effect of FK506 and
anti-CD4 therapy on fetal pig pancreas xenografts and
host lymphoid cells in NOD/Lt, CBA, and BALB/c mice.
Cell Transplantation 1994;3:61-73.
11. Morris C., Fung M., Simeonovic S., Wilson D., Hapel
A. Cyokine expression in CDA.H mice following
xenotransplantation of fetal pig proislets.
Transplantation Proceedings 1994j26:1304-1305.
12. Lenschow D., Zeng Y., Thistlewaite J., Montag A.,
Brady W., Gibon M., Linsley P., Bluestone J. Long-
term survival of xenogeneic pancreatic islet grafts
induced by CTLAlg. Science 1992;257:789-795.
13. Falqui L., Finke E., Carel J., Scharp D., Lacy P.
Marked prolongation of human islet xenograft survival
~human-to-mouse) by low temperature culture and
temporary immunosuppression with human and mouse
antilymphocyte sera. Transplantation lg91;51:1322-
1325.
14. Ricordi C., Scharp D., Lacy P. Reversal of diabetes
in nude mice after transplantation o~ fresh and seven
CA 02232815 1998-03-23
W O 97/11607 PCTAUS96/15577
- 59 -
day cultured (24~C) human pancreatic islets.
Transplantation 1988j45:994-996.
15. Ricordi C., Kneteman N., Scharp D., Lacy P.
Transplantation o~ cryopreserved human pancreatic
islets into diabetic nude mice. World J Surgery
1988j12:861-865.
16. Ricordi C., Lacy P., Sterbenz K., Davie J. Low-
temperature culture of human islets or in vivo
treatment with L3T4 antibody produces a marked
prolongation is islet (human-to mouse).
Transplantation 1987j44:465-468.
17. Ricordi C., Lacy P., Sterbenz K., Davie M. Low-
temperature culture of human islets plus in vivo
treatment of L3T4 antibody produces a marked
prongation of islet human-to-mouse xenograft
survival. Proc Natl Acad Sci 1987j84:8080-8084.
18. Ricordi C., Finke E., Lacy P. A method for the mass
isolation of islets from the adult pig pancreas.
Diabetes 1986j35:64g.
19. Weber C., Costanzo M., Zabinski S., Krekun S.,
Koschitzky T., D'Agati V., Wicker L., Ra}otte R.,
Reemtsma K. Xenografts of microencapsulated rat,
canine, porcine, and human islets into streptozotocin
~SZN)-and spontaneously diabetic NOD mice. In:
3~ Ricordi C. (Eds.) Pancreatic Islet Transplantation,
R.G. Landes, Austin, 1992:177-190.
20. Weber C., Reemtsma K. Microcapsulation in small
animals-II: Xenografts. In: Lanza R., Chick W.
~Eds.). PANCREATIC ISLET TRANSPLANTATION SERIES: VOL
CA 0223281~ 1998-03-23
W O 97/11607 PCTAUS96/15577
- 60 -
III: IMMUNOISOLATION OF PANCREATIC ISLETS. R.
Landes, Austin, 1994:59-79.
21. Zhou D., Sun Y., Vacek I., Ma P., Sun A.
Normalization of diabetes in cynomolgus monkeys by
xenotransplantation of microencapsulated porcine
islets. Transp Proc 1994j26:1091-1092.
22. Giannarelli R., Marchetti P., Villani G., DiCarlo A.,
Cosimi S., Andreozzi M., Cruschelli L., Masieco P.,
Coppelli A., Navalesi R. Preparation of pure, viable
porcine and bovine islets by a simple method.
Transplantation Proceedings 1994j26:630-631.
23. Jos C., Connolly J., Deardon D., Pearson R., Parrot
N., 3Ohnson R. A simple method for isolation from
the rabbit pancreas. Transplantation 1994;58:390-
392.
24. Reemtsma K., Weber C., Kazin M., Pi-Sunyer F.,
Nilaver G., Fenoglio C. Xenogeneic islets o~
Langerhans for human transplantation: Functional and
morphologic studies of primate, bovine and rabbit
islets. In: Friedman E., L'Esperance L. (Eds).
~iabetic-Renal-Retinal Syndrome Vol. 3, Grune and
Stratton, New York, 1986:521-546.
25. Forty J., Cary N., White D., Wallwork J. Hyperacure
rejection of rabbit hearts by human blood is mediated
by the alternative pathway o~ complement.
Transplantation Proceedings 1992jl24:488-489.
26. Platt J., Back F. The barrier to
xenotransplantation. Transplantation 1991j52:937-
947
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/~5577
- 61 -
27. Auchincloss H. Xenogeneic transplantation.
Transplantation 1988; 46:1-20
28. Lanza R.P., Beyer A.M., Chick W.L. Xenogeneic
humoral responses to islets transplanted in biohybrid
di~usion chambers. Transplantation 1994j57:1371-
1375.
29. Faustman F., Coe C. Prevention o~ xenograft
rejection by masking donor HLA class I antigens.
Science 1991;252:1700-1702.
30. Colton C., Avgoustiniatos E. Bioengineering in
development of the hybrid arti~icial pancreas. J.
Biochem En~. 1991jll3:152-170.
31. Lanza R., Sullivan S., Chick W. Islet
transplantation with immunoisolation. Diabetes
1992j41:1503-1510.
32. Scharp D., Swanson C., Olack B., Latta P., Hegre O.,
~oherty E., Gentile F., Flavin K., Ansara M., Lacy P.
Protection o~ encapsulated human islets implanted
without immunosuppression in patients with Type I or
Type II diabetes and in nondiabetic control subjects.
~iabetes 1994;43:1167-1170.
33. Ricker A., Stockberger S., Halban P., Eisenbarth F.,
Bonner-Weir S. Hyperimmune response to
microencapsulated xenogeneic tissue in non obese
diabetic mice. In: Jaworski M. (Eds). The Immunoloqy
of Diabetes Mellitus. Elseview, Amsterdam, 1986:193-
200.
34. Weber C., Zabinski S., Norton J., Koschitzky T.,
CA 0223281~ 1998-03-23
W O 97/11607 PCTAUS96/15577
D'Agati V., Reemtsma K. The ~uture role o~
microencapsulation in xenotransplantation. In: Hardy
M. (Eds). Xenoqraft 25, Elseview, Amsterdam,
1989:297-308.
s
35. ~alle J., Bourassa S., Leblond F., Chevalier S.,
Beaudry M., Chapdelaine A., Cousineau S., Saintonge
J., Yale J. Protection of islets of langerhans from
antibodies by microencapsulation with alginate-poly-
l-lysine membranes. Transplantation 1993;44:350-354.
36. Horcher A., Zekorn T., Siebers U., Klock G., Fra~k
H., Houben R., Bretzel R.G., Zimmerman U., Federlin
K. Transplantation of microencapsulated islets in
rates: Evidence for induction of fibrotic overgrowth
by islets alloantigens released from microcapsules.
Transpl Proc 1994j26:784-786.
37. Sibers R., Zekorn T., Horcher A., Klock G., Houben
R., Frank H., Bretzel R.G. , Zimmerman U., Federlin
K. Microencapsulated transplantation of allogeneic
islets into specifically presensitized recipients.
Transpl Proc 1994j26:787-788.
38. Lanza R., Kuhtreiber W., Ecker D., Staruk J., Chich
W., Xenotransplantation of porcine and bovine islets
without immunosuppression using uncreated alginate
microspheres. Transplantation 1995j59:1377-1384.
39. Lanza R., Ecker D., Kuhtrieber W., Staruk J., Marsh
J r Chick W. A slmple method for transplanting
~iscordant islets into rats using alginate gel
spheres. Transplantation 1995;59:1485-1487.
4~. Soon-Shiong P., Feldman E., Nelson R., Komtebedde J.,
CA 02232815 1998-03-23
WO 97111607 PCT~US96/15577
- 63 -
Smidsrod o., Skauk-Braek G., Espevik T., Heintz R.,
~ee M. Successful reversal of spontaneous diabetes
in dogs by using intraperitoneal microencapsulated
islets. Transplantation 1992;54:769-774.
41. Soon-Shiong P., Heintz R.E., Merideth N., Yao Q.X.,
Yao Z., Zheng T., Murphy M., Moloney M.K., Schmehl
M., Harris M., Mendez R., Sandford P.A. Insulin
independence in a type I diabetic patient after
encapsulated islet transplantation. Lancet
1994;343:950-951.
42. Heald K., Jay T., Downing R. Assessment of the
reproducibility of alginate encapsulation of
pancreatic islets using the MTT colorimetric assay.
Cell transplantation 1994j3:333-337.
43. Jarpe A., Hickman M., Anderson J. Flow cytometric
enumeration of mononuclear cell populations
infiltrating the islets of Langerhans in prediabetic
NOD mice: Development o~ model of autoimmune
insulitis for Type I diabetes. Regional Immunology
l9g0j3:305-317.
2S 44. Miller B., Appal M., O'Neil J., Wicker L. Both the
Lyt-2+ and L3T4+T call subsets are required for the
trans~er o~ diabetes in nonobese diabetic mice. J.
Immunol 1988;140:52-58.
45. Haskins K., Portas M., Bradley B. T-lymphocyte clone
specific for pancreatic islet antigen. Diabetes
1988j37:1444-1448.
46. Jansen A., Homo-Delarche F., Hooijkaas J., Leenen P.,
Dardenne M., Drexhage H. Immunohistochemical
CA 022328l5 l998-03-23
W O 97111607 PCT~US96/15577
- 64 -
characterlzation of monocytes-macrophages and
dendritic cells involved in the initiation of the
insulitis and B-cell destruction in NOD mice.
Diabetes 1994;43:667-674.
47. Prins J., Todd J., Rodrigues N., Ghosh S., Hogarth
M., Wicker L. Ga~ney E., Fishcer P., Sirotina A.,
Peterson L. Linkage on Chromosome 3 of autoimmune
diabetes and defective Fc receptor ~or IgG in NOD
mice. Science 1993j260:695-695.
48. Lipes M., Rosenzweig A., Tan K., Tanigawa G., Lass
D., Seidman J., Eisenbarth G. Progression to
diabetes in nonobese diabetic (NOD) mice with
transgenic T cell response. Science 1993;259:1165-
1169.
49. Gelber C., Pabrosky L., Singer S., McAteer D., Tisch
R., Jolicoeur C., Buelow R., McDevitt H., ~athman G.
Isolation of nonobese diabetic mouse T-cells that
recognize novel autoantigens involved in the early
events of diabetes. Diabetes 1994;43:33-39.
50. Podolin P., Pressey A., DeLarato N., Fischer P.,
Peterson L., Wicker L. I-E~ nonobese diabetic mice
develop insulitis and diabetes. J Exp Med
lg93;178:793-803.
51. Haskins K., Portas M., Bergman B., La~erty K.,
Bradley B. Pancreatic islet-speci~ic T-cell clones
from nonobese diabetic mice. PNAS 1989;86:8000-8004.
52. Haskins K., McDuffie M. Acceleration of diabetes in
young NOD mice with a CD4f islet-specific T cell
clone. Science 1990j249:1433-1436.
CA 02232815 1998-03-23
W O 97/11607 PCT~US96/15577
53. Supon P., Stecha P., Haskins K. Anti-islet cell
antibodies ~rom NOD mice. Diabetes 1990j39:1366-
1392.
54. Bergman B., Haskins K. Islet-speci~ic T-cell clones
from the NOD mouse respond to B-granule antigen.
Diabetes 1994;43:197-203.
55. Peterson J., Pike B., McDuffie M., Haskins K. Islet-
specific T-cell clones transfer diabetes to nonobese
diabetic (NOD) Fl mice. J. Imminol 1994jl53:2800-
2806
56. Kau~man D., Clare-Salzier M., Tian J. Spo~taneous
loss of T-cell tolerance to flutamic acid
decarboxylase in murine insulin-dependent diabetes.
Nature 1993j365:69-72.
57. Tisch R., Yang X., Singer S., Liblau R., Fugger L.,
McDevitt H. Immune response to glutamic acid
decarboxylase correlates with insulitis in non-obese
diabetic mice. Nature 1993j366:72-75.
58. Dylan D., Gill R., Schloot N., Wegmann D. Epitope
speci~icity, cytokine production profile and
diabetogenic activity so insulin-speci~ic T cell
clones isolated from H=NOD mice. Eur J. Immunology
1995;25:1062.
59. Wang Y., Hao L., Gill R., Lafferty K. Autoimmune
diabetes in NOD mouse is L3T4 T-lymphocyte dependent.
Diabetes 1987;36:535-538.
60. Boitard D., Bendelac A., Richard M., Carnaud C., Bach
J. Prevention o~ diabetes in nonobese diabetic mice
CA 022328l~ l998-03-23
W O 97/11607 PCT~US96/1~577
- 66 -
by anti-l-A monoclonal antibodies: Transfer of
protection by splenic T cells. PNAS 1988j85:9719-
9723.
61. Serrese D., Gaskins H., Leiter E. Defects in the
differentiation and function of antigen presenting
cells in NOD/lt. Mice. J Imunol 1993j150:2534-2543.
62. Rabinovitch A., Sumoski W., Rajotte R., Warnock G.
Cytotoxic effects of cytokines on human pancreatic
islet cells in monolayer culture. J. Of Clinical
Endocrinology and Metabolism 1990j71:152-156.
63. Bergmann L., Kroncke K., Suschek D., Kolb H., Kolb-
Bachofen C. Cytotoxic action of IL-lB against
pancreatic islets in mediated via nitric oxide
formation and is inhibited by NG -monomethyl-L-
arginine. FEBS letter 1992;299:103-106.
64. Xenos D., Stevens R., Gores P., Casanova D., Farney
A., Sutherland D., Platt J. IL-1 induced inhibition
of B-cell function is mediated through nitric oxide.
Transpl Proc 1993j25:994-994.
65. Anderson H., Jorgensen K., Egeberg J. Mandrup-Poulsen
R., Berup J. Nicotinamide prevents Interleukin-I
effects on accumulated insulin release and nitric
oxide production in rat islets of Langerhans.
Diabetes 1994j43:770-777.
66. Christianson S., Shultz L., Leiter D. Adoptive
transfer of diabetes into immunodeficient NOD-
Scid/Scid mice. Diabetes 1993j42:44-45.
67. Gerling I., Friedman H., Greiner D., Schultz L.,
CA 02232815 1998-03-23
W O 97/11607 PCT~US9611~577
- 67 -
Leither E. Multiple low-dose streptozotocin-induced
diabetes in NOD-Scid/Scid mice in the absence of
functional lymphocytes. Diabetes 1994;43:433-440.
68. Rohane P., Shimada A., Ki m D., Edwards C., Charlton
B., Shultz L., Fathman C. Islet infiltrating
lymphocytes from prediabetic NOD mice rapidly transfer
diabetes to NOD-scid/scid mice. Diabetes 1995j44:550-
554.
69. Shultz L., Schweitzer P., Christian S., Gott B.,
Schweitzer I., Tennent B., McKenna S., Mobraaten L.,
Rajan R., Greiner D., Leiter E. Multlple defects in
innate and adaptive immunologic function in NOD/LtSz-
scid mice. J of Immunology 1995j154:180-191.
7Q. Weber C., Krekun S., Loschitzky S., Zabinski S.,
D'Agati C., Hardy M., Reemtsma K. Prolonged
functional survival of rat-to-NOD mouse islet
xenografts by ultraviolet-B (W-B) irradiation plus
microencapsulation of donor islets. Transplantation
Proceedings 1991j23:764-766.
71. Lafferty K. Circumventing rejection of islet grafts;
An overview. In: Van Schifgaarde R., Hardy M. (Eds).
Elsevier , Amsterdam, 1988:279-291.
72. Ricker A., Bhatia V., Bonner-Weir S., Eisenbarth G.
Microencapsulated xenogeneic islet grafts in NOD
mouse: Dexamethasone and inflammatory response.
Diabetologia 1989;32:53.
73. Lafferty K.J., Hao L. Approaches to the prevention of
immune destruction of transplanted pancreatic islets.
Transpl Proc 1994j26:399-400.
CA 022328l~ l998-03-23
W O 97/11607 PCT~US96/15577
- 68 -
74. Weber C., Price J., Costanzo M., Becker A., Stall A.
NOD mouse peritoneal cellular response to poly-l-
lysine-alginate microencapsulated rat islets.
Transplantation Proceedings 1994;26::L116-1119.
75. Weber C., Tanna A., Costanzo M., Price J., Peterson
L., Wicker L. Effects o~ host genetic background on
survival or rat -, mouse islet xenografts.
Transplantation Proceedings 1994j26:1186-1188.
76. Wijsman J., Atkinson P., Mazheri R. Histological and
immunopathological analysis of recovered encapsulated
allogeneic islets from transplanted diabetic BB/W
rates. Transplantation 1992j54:588-592.
77. Cole D., Waterfall M., McIntyre M., Baird J.
Microencapsulated islet grafts in the BB.E rat a
possible role for cytokines in graft failure.
Diabetologia 1992j35:231-237.
78. Mazaheri R., Atkinson P., Stiller C., Dupre J., Vose
J., O;Shea F. Transplantation of encapsulated
allogeneic islets into diabetic BB/W rates: Effects o~
immunosuppression. Transplantation 1991;51:750-754.
79. Darquy S., Reach G. Immunoisolation of pancreatic B
cells by microencapsulation. Diabetolgia
18=985j28:776-780-.
80. Baldwin W., Pruitt S., Brauer R., Daha M., Sanfillippo
F. Complement in organ transplantation.
Transplantation 1995j59:797-808.
81. Iwata H., Takagi R., Amemiya H. Marked prolongation
of islet xenograft survival (hampster-to-mouse) by
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/15577
- 69 -
microencapsulation and administration of 15-
deoxyspergualin. Transplantation Proceedings
1992j24:1516-1518.
82. Pierson R., Winn H., Russell P., Auchincloss H. CD-4
positive lymphocytes play a dominant role in murine
xenogeneic responses. Transplantation Proceedings
1989j21:519-521.
10 83. Gill R., Wolf L., Daniel D., Coulombe M. CD4+ T cells
are both necessary and sufficient for islet xenograft
rejections. Transp Proc 1994;26:1203-1204.
84. Weber C., Zabinski S., Koschitzky T., Wicker L.,
Ra~otte R., Peterson L., D'Agati V., Reemtsma K.
Microencapsulated dog and rat islet xenogra~ts into
streptozotocin-diabetic and NOD mice. Hoem Metab Res
1990;35:219-226.
85. Cirulli V., Halban P., Rouiller D. Tumor necrosis
factor a modifices adhesion properties of rat islet B
cells. J Clim Invest 1993j92:1868-1876.
86. Campbell I., Iscara A., Harrison L. Interferon gamma
and tumor necrosis factor alpha: cytotoxicity to
murine islets of Langethans. J Immunol 1988;141:1325-
1329.
87. Rabionovitch A. Immunoregulatory and cytokine
imbalances in the pathogenesis of IDDM. Diabetes
1994;43:613-621.
88. Ablamunits V., Baranove F., Mandrup-Poulsen R., Nerup
J. In vitro inhibition of insulin release by blood
mononuclear cells from insulin-dependent diabetic and
3~ healthy subjects: synergistic action of IL-1 and TNF.
..
CA 022328l~ l998-03-23
W O 97/11607 PCTAJS96/15577
- 70 -
Cel~: Transplantation 1994j3:55-60.
89. Mandrup-Poulsen T., Bendtzen D., Dinarello C., Nerup
J. ~uman tumor necrosis factor potentiates
interleukinmediated rate of pancreatic B-cell
cytotoxity. J Immunol 1987jl39:4077-4082.
90. Thai N., Wang S., Valdivia L., Celli S., Reilly M.
Demetris A., Simmons R., Stazi T., Fung J. Cytokine
messenger RNA profiles in hamster-to-rat liver
xenografts. Transplantation Proceedings 1993j25:444-
445.
91. O'Connell P., Pacheoco-Silca A., Nickerson P., Muggia
R., Bastos M., Kelly V., Strom R. Unmodified
pancreatic islet allograft re~ection results in
preferential expression of certain T cell activation
transcripts. J Immunol 1993; 150 :1093 -1104.
20 92. Nickerson P., Pacheco-Silca A., O'Connell P., Steurer
W., Kelly V., Strom R. Analysis of cytokine
transcripts in pancreatic islet cell allografts during
rejection and tolerance induction. Transplantation
Proceedings 1993; 25:984-985.
93. Lowry R., Takeuchi T. The Thl, Th2 paradigm and
transplantation tolerance R. Landes , Austin. 1994;
In press.
94. Lederer J., Liou J., Todd M., Glicher l~ ichtman A.
Regulation of cytokine gene expression in T helper
cell subsets. J Immunol 1994jl:78-86.
95. Janeway C., Bottomly K. Signals and signs for
35 lymphocyte responses. Cell 1994j76:275-285.
CA 0223281~ 1998-03-23
W O 97/11607 PCTAJS96/15577
96. Aguilar-Diosdada M., Parkinson D., Corbett J., Kwon
G., Marshall C., Gingerich R., Santiafo J., McDaniel
M. Potential autoantigens in IDDM: expression of
carboxypeptidase-H and insulin but not glutamate
decarboxylase on the B-cell surface. Diabetes
1994j43:418-425.
97. Jenkins M. The ups and downs of T cell costimulation.
Immunity 1994;~:443-446.
98. Liu Y., Jones B., Brady W., Janeway C., Linsley P.
Co-stimulation of murine CD4 T cell growth:
cooperation between B7 and heat-stable antigen. Eur
J Immunol 1992j22:2855-2859.
99. Harding F. McArthur J., Fross J., Raulet D., Alliston
J. CD28-mediated signaling co-stimulates murine T
cells and prevents induction of anergy in T-cell
clones . Nature 1992;356:607-609.
100. Galvin F., Greeman G., Razi-Wolf Z., Hall W.,
Benacerraf B., Nadler L., Reiser H. Murine B7 antigen
provides a sufficient costimulatory signal for
antigen-specific and MHC-restricted T cell activation.
25 J Immunol 1992j149:3802-3808.
101. Hathcock K., Laszio G., Dickler H., Bradshaw J.,
Linsley P., Hodes R. Identification of an alternative
CTLA-4 ligand costimulatory for T cell activation.
30 Science 1993;262:905-907.
1~2. Perrin P., Scott D., Quigley L., Albert P., Feder O.,
Gray G., Abe R., June C., Racke M. Role of B7:
CD28/CTLA-4 in the induction of chronic relapsing
35 experimental allergic encephalomyelitis. J of
CA 0223281~ 1998-03-23
W O 97/11607 PCTAUS96tlS577
- 72 -
Immunology 1995;154:1481-1490.
103. Gimmi C., Freeman G., Gribben J., Gray G., Nadler h.
Human T-cell anergy is induced by antigen presentation
in the absence o:E B7 costimulation. Immunology
1993;90:6586-6~90.
104. Guerder S., Meyerhoff J., Flavell R., The role o~ the
T-cell costimulatory B7-1 in autoimmunity and the
induction and maintenance of tolerance of peripheral
antigen. Immunity 1994;1: 155-166.
105. Finck B., Linsley P., Wofsy D. Treatment of murine
lupus with GTLA4Ig. Science 1994;265: 1225-1227.
106. Bolling S., Turka L., Wei R., Linsely P., Thompson C.,
Lin H. Inhibition of B7-induced CD28 T-cell activation
with CThA4Ig prevents cardiac allograft rejection;
evidence ~or costimulation. Transplantation of
tolerance induced by CTLA-4lg. Transplantation
1994;57: 1701-1706.
108. henschow D., Ho S., Sattar H., Rhee L., Gray G.,
Nabavi N., Herold K, Bluestone J. Dif~erential e~~ects
of anti-B7-1 and anti-B7-2 monoclonal antibody
treatment on the development of diabetes in the
nonobese diabetic mouse. J of Exp Medicine 1995j181:
1145-1155.
109. Durie F., Fava R., Foy T., Aruffo A., Ledbetter J.,
Noelle R, Prevention o~ collagen-induced arthritis
with an antibody to gp39, the Ligand ~or CD40.
Science 1993j261: 1328-1330.
110. Mohan C., Shi Y., haman J., Datta S. Interaction
-
CA 0223281~ 1998-03-23
W O 97/11607 PCr~US96~15577
between CD40 and its ligand gp39 in the development of
murine lupus nephritis. The American Association of
Immunologists 1995jl54: 1470-1480.
-
111. Gin H., Cadic C., Baquey C., Dupuy B. Peritoneal
exudates from microencapsulated rat islets of
Langerhans xenograftedmicepresentingcharacteristics
o~ potentially cytotoxic non-speci~ic in~lammation.
J of Microencapsulation 1992j9: 489-494.
112. Clayton H., London N., Colloby P., Bell P., James R.
The effect of capsule composition on the
biocompatibility of alginate-poly-l-lysine capsules.
J. Microencapsulation 1991j8: 221-233.
113. Soon-Shiohg P., Oterlie M., Skjak-Braek G., Smidsrod
O., Heintz R., Lanza R.P., Espevik T. An immunologic
basis for the fibrotic reaction to implanted
microcapsules. Transpl Proc 1991j23: 758-759.
114. DeVos P., Wolters G., VanSchil~gaarde R. Possible
relationship between fibrotic overgrowth of alginate-
polysine-alginate microencapsulated pancreatic islets
and the microcapsule integrity. Transp Proc 1994j26:
782-783.
115. Chang T. Artificial cells in immobilization
biotechnology. Art Cells & Immob Biotech 1992j20:
1121-1143.
116. Lum Z., Tai I., Krestow M., Norton J., Vacek I., Sun
A. Prolonged reversal of diabetic state in NOD mice
by xenografts of microencapsulated rat islets.
Diabetes 1991;40: 1511-1516.
CA 022328l~ l998-03-23
W O 97/11607 PCT~US96/15S77
- 74 -
117. Chicheportiche D., Reach G. In vitro kinetics of
insulin release by microencapsulated rat islets:
effects of the size of the microcapsules.
Diabetologia 1988;31: 54-57.
118. Weber C., Constanzo M., Kredun S., D'Agati V. Causes
of destruction o~ microencapsulated islet grafts:
Characteristics of a 'double-wall' poly-1-lysine-
alginate microcapsule. Diabetes, Nutrition and
Metabolism 1993;1: 167-171.
119. Vanenbossche G., Van Oostveldt P., Demeester ~., Remon
J. The molecular weight cut-off of microcapsules is
determined by the reaction between alginate and
polylysine. Biotechnology and Bioengineering 1993;42:
381-386.
12Q. Linderman G., Adams J., Cory S., Harris A. B-lymphoid
to granulocytic with during hematopoiesis in a
transgenic mouse strain. Immunity 1994jl: 517-527.
121. Takeuchi T., Lowry R., Konoieczny B. Heart allografts
in murine systems. Transplantation 1992;53: 1281-
1294.
122. Ravetch J., Kinet J. J Ann Rev Immunol 1993;9: 457-
492.
123. Takai T., Li M., Sylvestre D., Clynes R., Ravetch J.
FcR y chain deletion results inpleiotrophic effector
cell defects. Cell 1994j76: 519-529.
124. Krych M., Atkinson J., Holers v. Complement receptors.
Curr Opin Immunol 1992;4: 8-13.
CA 022328l~ l998-03-23
W O g7/11607 PCTnUS96/15577
- 7~ -
125. Frank M., Fries: The role of complement in
inflammation and phagocytosis. Immunol Today 1991;12:
322-326.
.~
126. Whiteley P.J., Jensen P.E. Pierce C.W., A~ruzzini
A.F., Kapp J.A. Helper T-cell clones that recognize
autologous insulin are stimulated in nonresponder mice
by pork insulin. Proc Natl Acad Sci USA 1998;85:
2723-2727.
127. Jensen P.E., Kapp J.A. Stimulation of helper T cells
and dominant suppressor T cells that recognize
autologous insulin. J Mol Cell Immunol 1985;2: 133-
133.
128. Poindexter N.J., Landon C., Whiteley P.J., Kapp J.A.
Comparison of the T cells receptors on insulin-
speci~ic hybridomas from insulin transgenic and
nontransgenic mice. Loss of a subpopulation of self-
reactive clones. J Immunol 1992j149: 38-44.
129. Ke Y., Li Y., Kapp J.A. Ovalbumin injected with
complete Freund's adjuvant stimulates cytolytic
responses. Eur J Immunol 1995j25: 549-553.
130. Seder R., Paul W. Acquisition of lymphokine-producing
phenotype by CD 4+ T cells. Annu Rev Immunology
1994;12: 635-635.
3~ 131. Beckerman K., Rogers H., Corbett J., Schreiber R.,
McDaniel M., Unanue E. Release of nitric oxide during
the T-cell independent pathway of macrophage
activation ~ Immunol 1993;150: 888-895.
132. Weinberg B., Granger D., Pisetsky D., Seldin M.,
CA 0223281~ 1998-03-23
W O 97/11607 PCT~US96/1~577
Misukonis M., Mason N., Pippen A., Ruiz P., Wood E.,
Gilkeson G. The role of nitric oxide in the
pathogenesis of spontaneous murine autoimmune disease:
Increased nitric oxide production and nitric oxide
synthase expression in MRL-lpr/lpr mice, and reduction
of spontaneous glomerulonephritis and arthritis by
orally administered Ng -monomethyl-l-arginine. J. Exp
Med 1994j179: 651-660.
133. Ravetch~J. Fc Receptors: Rubor Redux. Cell 1994;78:
553-560.
134. Unreels J., Scigliano E., Freedman V. Structure and
function of human an murine receptors for IgG. Annual
Review of Immunology 1988j6: 251-281.
135. Platt J., Lindman B., Geller R., Noreen H., Swanson
J., Dalmasso A., Bach F. The role of natural
antibodies in the activation of xenogenic endothelial
cells. Transplantation 1991j52: 1027-1043.
135. Baxter G., Cooke A. Complement lytic activity has no
role in the pathogenesis of autoimmune diabetes in NOD
mice. Diabetes 199~;42: 1574-1578.
137. Moses R., Winn H., Auchincloss H. Evidence that
multiple defects in cell-surface molecule interactions
across species differences are responsible for
diminished xenogenic T cell responses. Transplatation
1992j53: 203-209.
138. Moses R., Pierson R., Winn H., Auchincloss H.
Xenogeneic proliferation and lymphokine production are
dependent on CD4+ helper T cells and self antigen-
presenting cells in the mouse. J Exp Med 1990;172:
. CA 022328l5 l998-03-23
W O 97/11607 PCT~US96/15577
567-575.
139. Hardy M., Lau H., Weber C., Reemtsma K. Pancreatic
islet Transplantation; Induction of graft acceptance
by ultraviolet irradiation of donor tissue. Ann Surg
- 1984j200: 441-450.
140. Sullivan F., Ricordi C., Hauptfeld V., Lacy P. Effect
of low-temperature culture and site of transplantation
on hamster islet xenografts survival (hamster-to-
mouse). Transplatation 1987j44: 465-468.
141. Hering B., Bretzel R., Federlin K., Horm Metabol Res
1988j20:537-545.
142. Lacy P., Lacy E., Finke E., Yasunami Y. Diabetes
1982j31:109-111.
143. Nathan D. NEJM 1993;328:176-1685.
144. Robertson R. NEJM 1992j327: 1861-1868.
145. Linsley P.S., Brady W., Umes M., Grosmaire L.S., Damie
N.K., Ledbetter J.A. CTLA-4 is a second receptor for
the B cell activation antigen B7. J EXp Med 1991; 74:
561-569.
146. Korbutt G.S., Ao Z., Warnock G.L., Flashner M.,
Rajotte R.V. Successful reversal of diabetes in nude
3~ mice by transplantation of microencapsulated procine
neonatal islet cell aggregats. Transplantation
Proceedings 1995j27: 3212.
:L47. Korbutt G.S., Ao Z., Warnock G.L., Rajotte R.V. Large-
scale isolation of viable porcine neonatal islet cell
CA 02232815 1998-03-23
W O 97111607 PCT~US96/15577
- 78 -
(NIC) aggregates. Transplantation Proceedings
1995;27:3267.
