Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION AND METHOD TO PREVENT GRAFT REJECTION AND
OTHER COUNTER-ADAPTIVE T LYMPHOCYTE MEDIATED IMMUNE
RESPONSES
FIELD OF THE INVENTION
This invention relates to the field of tissue
transplantation, and more particularly to the use of
monoclonal antibodies specific for T cell determinants
in blocking cell mediated immune responses resulting in
allograft or xeongraft rejection.
This invention further relates to the prevention
or reversal of graft organ rejection and other counter-
adaptive T lymphocyte mediated immune responses. The
invention provides compositions and an order and method
of treatment to reduce or prevent the rejection of
graft organs in primates or man, and to prevent disease
resulting from a poorly targeted T lymphocyte mediated
immune response.
BACKGROUND OF THE INVENTION
Organ transplantation between genetically non-
identical individuals invariably results in
immunological rejection of the organ through T cell
dependent mechanisms unless that rejection process is
bridled by administering drugs that suppress T cell
function. Both calcineurin phosphatase inhibitors and
glucocorticosteroids are used clinically, and both
prevent the T cell mediated release of activating
cytokines, particularly IL-2. Therapy with these
agents is imperfect however. Both act by impairing
signaling through the T cell antigen receptor (TCR?,
the sole mediator of T cell antigen specificity, and
act on all T cells indiscriminately. In addition, the
effect of these drugs is not lasting such that
cessation of immunosuppression has generally resulted
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in graft loss even after prolonged rejection free
survival. Thus, in order to avoid graft rejection,
transplant recipients must suffer the consequences of
non-specific immunosuppression. These consequences
include an increased risk of infection and malignancy
as well as significant drug related expense and
toxicity.
Data establishing that T cell activation requires
both TCR mediated signals and simultaneously delivered
costimulatory signals have accumulated over the past 20
years [1]. These important costimulatory signals are
provided at least in part by the T cell based CD28
molecule when bound to its counter receptors CD80(B7-1)
or CD86 (B7-2), hereafter referred to collectively as
B7, on antigen presenting cells (APCs) and perhaps
parenchymal cells [1,2,3]. The interaction of CD40 and
its T cell based ligand, CD40L (CD154), also plays an
important role in T cell activation at least in part by
up-regulating B7 [4,5]. In addition, CD40 and CD154
play a fundamental role in establishing T dependent B
cell activity [6,7]. Further studies have shown that
the T cell molecule CTLA4 (CD152), appears to down-
regulate costimulation and TCR mediated activation, at
least in part by competing with CD28 for B7 and by
delivering a unique negative signal to the TCR signal
transduction complex [8].
Several groups have shown in rodents that T cell
activation can be blocked and rodent allograft survival
prolonged by interfering with B7 interacting with its T
cell counter-receptors CD28 and CTLA4 utilizing the B7
specific fusion protein CTLA4-Ig [9-11]. Others have
demonstrated that B7 up-regulation can be prevented by
the CD154 specific monoclonal antibody MRI[4]. As both
agents appear to be dependent on TCR engagement for
their effectiveness, the specificity of the T cell
response can theoretically be exploited rather than
depending on pan T call suppression. In addition to in
vitro efficacy, these agents have shown dramatic in
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vivo effects in rodents, allowing for the acceptance of
fully mismatched skin grafts, a result not obtainable
with currently available immunosuppression [12]. It is
noteworthy however that all previously reported
techniques allowing long-term graft survival in rodents
have failed to work or have been associated with major
toxicity when tested in species higher on the
phylogenetic tree.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is a
combination of drugs to prevent rejection of
transplanted cells, tissues, or organs from either an
allogeneic or a xenogeneic source by administering
agents that interfere with T cell costimulatory
signaling via CD28 when given in conjuirction with
agents that interfere with the CD40:CD154 interaction.
Another object is a method of treatment to reverse
ongoing organ rejection by administering agents that
interfere with T cell costimulatory signaling via CD28
when given in conjunction with agents that interfere
with the CD40:CD154 interaction.
A third object recognizes that reversal of an
ongoing rejection process can be stopped by
administering agents that interfere with T cell
costimulatory signaling via CD28 when given in
conjunction with agents that interfere with the
CD40:CD154 interaction.
A fourth object is that for patients currently
being treated with standard immunosuppressive therapies
(e. g. glucocorticoids, calcineurin phosphatase
inhibitors, mycophenolate mofetil) to prevent the
rejection of a transplant or to prevent graft versus
. host disease, those toxic and expensive medications
could be discontinued and replaced with short course
therapy with agents that interfere with T cell
costimulatory signaling via CD28 when given in
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conjunction with agents that interfere with the
CD40:CD154 interaction.
A fifth object is that for patients with a
transplanted organ undergoing chronic rejection, agents
that interfere with T cell costimulatory signaling via
CD28 when given in conjunction with agents that
interfere with the CD40:CD154 interaction can block
this undesired immune reaction.
A sixth and most general object is to prevent
and/or treat disease states resulting from a counter-
adaptive immune response such as the various T-
lymphocyte mediated autoimmune illnesses (e. g. insulin
dependent diabetes mellitus, multiple sclerosis, etc.)
and the various allergic disease states (e.g. hay
fever) .
A seventh object is to test the hypothesis that
CTLA4-Ig and the anti-human CD154 specific monoclonal
antibody are capable of inducing tolerance to
allografted or even xenografted tissues in humans, and
in a more general sense to ameliorate (prevent or
treat) all counter-adaptive T-lymphocyte mediated
disease states.
These and additional objects of the invention are
accomplished by:
1. Utilizing agents that interfere with the interaction
of the CD28 and/or CD152 (CTLA4) with their B7 family
ligands (CD80 and/or CD86) and with agents that
interfere with the interaction of CD40 and CD154
(CD40L). These agents will be administered
parenterally (intramuscularly, subcutaneously, or most
preferably intravenously) in a standard pharmaceutical
carrier (i.e. iv infusion with saline, water, or other
buffer) .
2. Agents will be administered after cells, tissue(s),
or organs) have been transplanted. Initial dosing
will be administered as soon as the graft is
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transplanted at a dose of between 5-20 mg/kg body
weight (each agent). Doses will then be administered
on days 2,4,6,8,12,15, and 28 post transplant.
Thereafter, should signs of immune rejection ensue,
dosing will be repeated to reverse the rejection
episode. During this retreatment, dosing will be
administered as per the initial induction therapy post
transplant.
3. This therapy employing agents that interfere with
the interaction of both CD28/CD152:B7 and CD40:CD154
will also be administered to individuals with signs
indicating that they are developing a disease
(including chronic rejection), or that are already
suffering with an illness, mediated completely or in
part by activated T cells (including patients with a
transplant currently receiving standard
immunosuppressive therapy). Such "counter-adaptive" T
cell responses also include diseases like the various
autoimmune illnesses (for example insulin dependent
diabetes mellitus, rheumatoid arthritis, multiple
sclerosis, inflammatory bowel disease, and systemic
lupus erythematosus) as well as in states resulting
from the sequela of an immune response like allergic
illnesses (hay fever). For these indications, the
therapy will be administered in doses ranging from 2-20
mg/kg body weight (each agent) as frequently as every
other day for up to 28 days.
4. The "treatment package" will be termed "immune re-
education" and will consist of the drugs to be
administered, the carrier solvent for those agents, and
the infusion system to be used to administer the agent.
This hypothesis is tested in a relevant pre-
clinical model. CTLA4-Ig and anti-CD154 were tested
alone and in combination on rhesus peripheral blood
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leukocytes in vitro, and in rhesus monkeys transplanted
with primarily vascularized renal allografts.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. The effect of CTLA4-Ig and humanized anti-human
CD154 alone and in combination on unidirectional rhesus
monkey mixed lymphocyte reactions. Increasing
concentrations of CTLA4-Ig result in progressive
suppression while the effects of humanized anti-human
CD154 are more modest. The combination is more
effective than either drug alone at 100 fold greater
concentrations. Results shown were reproduced in three
independent experiments. C.P.M. - counts per minute
from incorporated 3H-thymidine.
Fig. 2 (A) Survival and renal function as determined by
serum creatinine following unmodified allogeneic renal
transplantation (dashes) or transplantation following
induction with CTLA4-Ig alone (squares) or humanized
anti-human CD154 alone (diamonds). Open arrows
indicate retreatment during biopsy proven rejection.
Solid arrows continued survival. (B) Survival and
renal function as determined by serum creatinine
following unmodified allogenic renal transplantation
(dashes) or transplantation following induction with
both CTLA4-Ig and humanized anti-human CD154. Open
circles indicate treatment on days 0,2,4,6,8,10, and 12
post-transplant. Closed circles indicate treatment on
days 0,2,4,6,8,12,16, and 28 post-transplant. Open
arrows indicate retreatment during biopsy proven
rejection for the animal depicted in open circles.
Solid arrows indicate continued survival free of
rejection since transplantation.
Fig. 3. (A) Renal allograft histology showing acute
cellular rejection following unmodified renal
allotransplantation in rhesus monkeys. (B) Renal
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allograft histology showing acute cellular rejection
prior to reversal with humanized anti-human CD154. (C)
Normal renal allograft histology from an animal with
normal renal function 163 days following
transplantation and induction with CTLA4-Ig and
humanized anti-human CD154. (D) A perivascular
lymphoid aggregate with the allograft shown in C.
These nests of lymphocytes exist in the allograft
despite normal function and the absence of
immunosuppression. All micrographs are 250x.
Fig. 4. Mixed lymphocyte responses against donor
lymphocytes and third party lymphocytes for two rhesus
monkeys 150 days after allotransplantation with
rejection free survival and normal renal function and
without any chronic therapy. Both donor and third
party responsiveness is maintained. On the other hand,
in data NOT shown, skin grafts placed on a rhesus
monkey 6 months following successful
allotransplantation revealed donor specific tolerance.
Three skin grafts were placed: one from the host (an
autograft to control for surgical technique), one from
the allogeneic kidney donor, and one from a third party
donor. Only the third party donor skin was rejected at
day 14 (and counting) since the grafting. This data
indicates that functional donor specific tolerance has
been achieved despite failure of the allo-MLR to
reflect it. -
A more complete appreciation of the invention will
be readily obtained by reference to the following
Description of the Preferred Embodiments and the
accompanying drawings in which like numerals in
different figures represent the same structures or
elements. The representations in each of the figures
is diagrammatic and no attempt is made to indicate
actual scales or precise ratios. Proportional
relationships are shown as approximations.
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_g_
DESCRIPTION OF PREFERRED EMBODIMENTS
This invention is applicable to both xeno- and
alto- transplants, and for more general application to
disease states resulting from counter-adaptive T-
lymphocyte responses. The invention comprises a
composition involving the parenteral administration for
an agent interfering with the T cell costimulatory
receptors' (CD28/CD152? ability to bind with B7 in
close time sequence to administration of an agent
preventing signaling through CD152.
The best mode now known initial experience in
primates with a new class of reagents directed at
modifying T cell costimulation, rather than focused on
T cell suppression or elimination. Herein strategies
designed to interfere with the interaction of B7 and
its counter-receptors CD28 and/or CD152, or with the up
regulation of B7 expression are shown to have dramatic
effects on T cell responsiveness in vitro, and on
allograft survival in vivo- including prevention of
rejection and the reversal of established, biopsy
proven rejection. In addition, these data demonstrate
that anti-rejection activity can persist long after
drug administration has stopped. Finally, data is
presented to indicate that donor-specific tolerance can
be achieved.
It is encouraging that this regimen was remarkably
simple, involving two agents administered through a
standard peripheral intravenous catheter and that it
was tolerated so well by the recipients. This is in
stark contrast to other regimens used to achieve
lasting graft acceptance in primates requiring ionizing
radiation, administration of donor derived bone marrow
and significant perioperative immunosuppression
[15,16]. The animals treated in this study displayed
no evidence to T cell activation or the cytokine
release typically observed following treatment with
antibodies directed at CD3, and prolonged survival has
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not carried with it a demonstrable cost in terms of
opportunistic infection. In addition, no alterations
in peripheral blood hematological parameters were noted
during these studies. Long-term survival was achieved
without apparent clearing or global reductions in any
lymphocyte subset and without loss of in vitro T cell
responsiveness. It is therefore unlikely that the
observed effect is attributable to T cell destruction
following antibody or fusion protein opsonization. The
results are striking. Such success is outbred rhesus
monkeys suggests that allograft tolerance is an
achievable goal in humans using this or a similar
therapeutic approach.
The mechanism and relative contribution of each
agent remains a matter of speculation at this juncture.
The successes of CD154 blockade alone suggest that any
basal costimulation signaling is less important in
maintaining the rejection response than B7 up-
regulation. Indeed, anti-CD154 resulted in impressive
rejection free survival when used alone, while CTLA4-
Ig's effects were more transient. Given the recent
discovery that CD154 is expressed on non-myeloid cells
such as vascular endothelium and smooth muscle [17],
and that B7-1 can be induced on fibroblasts [3] and
hepatocytes [18], non-T cell events may be critical in
establishing reactivity against the allograft. By
denying the immune system access to significant
parenchyma) adhesion and costimulatory signals at the
time of transplantation, graft recognition and
destruction may be prevented. The differences in
activation induced by donor parenchyma and activation
induced by lymphoid cells could explain the
preservation of in vitro reactivity to donor
lymphocytes despite normal graft function, and the
general poor correlation between MLR reactivity and
clinical graft outcome. Nonetheless, the effects of
CTLA4-Ig and humanized anti-human CD154 were shown to
be synergistic both in vitro and in vivo. Perhaps,
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CTLA4-Ig provides insurance against B7 expression that
escapes the effects of humanized anti-human CD154. In
that instance, considerable time seems to be required
to mount an effective acute rejection with the few
cells that escape initial blockade.
As this strategy was successful in reversing
established, biopsy proven acute rejection, it would
appear that the rejection process must be maintained by
continuous costimulation, rather than a process that,
once set into motion, proceeds unless the effector
cells are eliminated or rendered incapable of TCR
signaling. Teleologically, the body is best served by
inflammation that is easily controlled. Thus, in the
absence of direction to attack, retreat may be the
tacit order. This suggests that exploitation of the
immune system's natural propensity to down-regulate may
be more advantageous than pan-suppression.
The rhesus monkey model used in this study has
been shown repeatedly to be a rigorous test of immune
manipulation - one that is exquisitely sensitive to
even minor changes in allograft function or adverse
effects on recipient wound healing and immune function
[13,15,19]. In addition, it has obvious biological
similarity to human renal transplantation.
Specifically, genes that encode MFiC proteins are well
conversed between rhesus monkeys and humans [20-22?,
and their rejection of vascularized organs closely
parallel that seen clinically [13,15,19.
Nevertheless, issues of optimal dosing and treatment
time course remain to be resolved. While rodent models
have been successful with a single dose of CTLA4-Ig
given on post-operative day 2 in combination with donor
specific transfusion [91, it is clear that a more
aggressive approach is required in primates.
Nonetheless, a transient well tolerated treatment that
exploits the specificity of the immune system and gives
lasting rejection free survival would appear to be
nearing clinical applicability.
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Having described the invention, the following
examples are given to illustrate specific applications
of the invention including the best mode now known to
perform the invention. These specific examples are not
intended to limit the scope of the invention described
in this application.
MATERIALS AND METHODS
Reagents
Human CTLA4-Ig and a control fusion protein-IgGl
were prepared as previously described [2] and shipped
in solution by Genetics Institute, Cambridge, MA. The
anti-CD40 ligand antibody humanized anti-human CD154
was prepared as previously described [6,7] and shipped
in solution by Biogen Corporation, Cambridge, MA. The
hamster anti-mouse CD28 monoclonal antibody PV-1 (IgGl,
clone G62) was purified from hybridoma culture
supernatants and used as in isotype control monoclonal
antibody.
MHC Typing and Donor-recipient Selection
Donor-recipient combinations and animals chosen
for third party cells were selected based on genetic
non-identity at both MHC class I and class II. Class I
disparity was established by one-dimensional
isoelectric focusing as previously described [13].
Class II disparity was established based on the results
of unidirectional mixed lymphocyte reactions (MLRs).
In addition, the animal's DRB loci were verified to be
disparate by denaturing gradient gel electrophoresis w
and direct sequencing of the second exon of DRB as
previously described [14). Vigorous in vitro T cell
responsiveness of the recipient towards the donor was
confirmed in vitro for all donor-recipient pairs. The
experiments described in this study were conducted
according to the principles set forth in the "Guide for
the Care and Use of Laboratory Animals" Institute of
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Laboratory Animals Resources, National Research
Council, DHHS, Pub. No. (NIH) 86-23 (19850).
In Vitro Cellular Analysis
Unidirectional MLRs were performed on all animals
prior to transplantation and on rejection free
survivors after 100 days. Each animal was tested
against all potential donors to establish the highest
responder pairs for transplantation. Responder cells
(3 x 105) were incubated with irradiated stimulator
cells (1 x 105) at 37°C for 5 days. Cells were pulse-
labeled with 3H-thymidine and proliferation was
monitored by 3H-thymidine incorporation. Polyclonal
stimulation with Concanavilin A (25 mcg/ml) served as a
positive control. A stimulation index was calculated
by normalization to self reactivity, which in alI cases
was near background incorporation. For in vitro dose
response studies, CTLA4-Ig or humanized anti-human
CD154 was added to the MLR on day 1 at concentrations
ranging from 100 mcg/ml to 0.01 mcg/ml. Combined
treatments were performed by varying the CTLA4-Ig
concentration and holding the humanized anti-human
CD154 concentration steady at 50 mcg/ml.
Peripheral blood lymphocyte phenotype analysis was
performed prior to transplantation and periodically
during and after drug therapy. Assays evaluated 0.2 ml
of heparinized whole blood diluted with phosphate
buffered saline and 1% fetal calf serum. FITC labeled
T11, B1 (Coulter), and FN18 (the generous gift of Dr.
David M. Neville, Jr.) monoclonal antibodies were used
to assess the percentage of CD2 (T cell/NK cell), CD20
(B cell), and CD3 (T cell) positive cells respectively.
Red blood cells were removed from the preparation by
ACK lysis buffer (0.15 M NH4C1, 1.0 mM KHC03, 0.1 mM Na2
EDTA, pH 7.3) treatment following staining. Cells were
subjected to flow cytometry immediately, or following
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fixation in 1% paraformaldehyde. Flow cytometry was
performed using a Becton Dickinson FACSCAN.
Renal Allografts
Renal allotransplantation was performed as
previously described [13]. Briefly, outbred juvenile
(1-3 years of age) rhesus monkeys, seronegative for
simian immunodeficiency virus, simian retrovirus, and
l0 herpes B virus, were obtained from the Primate Center
(University of Wisconsin) or LABS (Yemassee, SC).
Procedures were performed under general anesthesia
using ketamine (1 mg/kg, i.m.), xylazine (1 mg/kg,
i.m.) and halothane (1%, inhaled). Transplantation was
performed between genetically distinct donor-recipient
pairs as determined by the MHC analysis described
above. The animals were heparinized during organ
harvest and implantation (100 units/kg). The allograft
was implanted using standard microvascular techniques
to create an end to side anastamosis between the donor
renal artery and recipient distal aorta as well as the
donor renal vein and recipient vena cava. A primary
ureteroneocystostomy was then created. Bilateral
native nephrectomy was completed prior to closure.
Animals were treated with intravenous fluid for
approximately 36 hours until oral intake was adequate.
Trimethaprim-sulfa was administered for 3 days for
surgical antibiotic prophylaxis. Each animal received
81 mg of aspirin on the day of surgery. The need for
analgesia was assessed frequently and analgesia was
maintained with intramuscular butorphanol. Animals
were weighed weekly. Skin sutures were removed after 7
to 10 days. CTLA4-Ig and/or humanized anti-human
CDI54, was given intravenously at doses and dosing
schedules varying based on accumulating experience with
the agents. No other immunopharmaceuticals were
administered. Serum creatinine, and whole blood
electrolytes (Na', K', Ca2') and hemoglobin were
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determined every other day until stable and then
weekly.
Pathological Analysis
Biopsies were performed on animals suspected of
having rejection using a 20-gauge needle core device
(Biopty-Cut, Bard). Standard staining with hematoxylin
and eosin was performed on frozen or formalin fixed
tissue to confirm the diagnosis of rejection. Animals
were euthanized at the time of anuria or if a weight
loss of 15% of pre-transplant body weight occurred in
accordance with AAALAC standards. All animals
underwent complete gross and histopathological
evaluation at the time of death.
RESULTS
CTLA4-Ig and humanized anti-human CD154 synergistically
prevent T cell proliferation in vitro.
Both CTLA4-Ig and humanized anti-human CD154
inhibited rhesus MLRs in a dose dependent fashion (Fig.
1). CTLA4-Ig was, however, more effective than
humanized anti-human CD154 as a single agent in
preventing T cell proliferation. Substantial reduction
in.thymidine incorporation was seen at a CTLA4-Ig
concentration of 0.1 mcg/ml, and further inhibition was
achieved at higher concentrations. Modest reduction in
proliferation was achieved with humanized anti-human
CD154 concentrations of 0.01 mcg/ml but inhibition was
not substantially improved by increasing
concentrations. Both agents acted synergistically, the
combination inhibiting proliferation approximately 100
times more effectively than either agent alone did.
Dose response studies were repeated for 3 separate
naive animals with identical results. CTLA4-Ig and
humanized anti-human CD154 synergistically prevent
allograft rejection in vivo.
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Twelve renal allotranspiants were performed (Fig.
2). Four animals received transplants without any
immunological intervention. These animals rejected in
5,7,7 and 8 days. Histological examination of their
kidneys showed acute cellular rejection characterized
by diffuse interstitial and tubular lymphocytic
infiltration with edema and cellular necrosis (Fig.
3A). One animal was given a 5-day course of CTLA4-Ig
(10 mg/kg/d) beginning at the time of transplantation
and had graft survival prolonged to 20 days (Fig. 2A).
Graft loss was due to cellular rejection
indistinguishable from that seen in the control
animals. One animal was treated with CTLA4-Ig 20 mg/kg
on the day of transplantation followed by a 12 day
course of 10 mg/kg every other day and had graft
survival prolonged to 30 days-lFig. 2A~-. Again,-graft
loss was due to acute cellular rejection.
Extrapolating from previously published work in the rat
heterotopic cardiac allograft model of Turka, et al [9]
a donor specific transfusion of lymph node derived
lymphocytes (108) was given at the time of
transplantation to this 2 animals.
Two animals were treated with humanized anti-human
CD154 alone (Fig. 2A). Both animals received 20 mg/kg
every other day beginning on the day of surgery and
continuing for 14 post-operative days (8 doses total).
Both animals experienced extended rejection free
survival, although transient creatinine elevations were
recorded during the second and fourth post-operative
weeks. Both animals rejected between 95 and 100 days
post-transplant. Biopsy was performed on each animal
to confirm the diagnosis (Fig. 3B). Both animals were
then retreated with 7 doses of humanized anti-human
CD154 (20 mg/kg; one animal every other day and one
animal daily) and both returned to normal graft
function with no demonstrable adverse effects. They
remain alive and well greater than 150 days_af er
transplantation at the time of this writing. __
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Two animals were given 20 mg/kg each of CTLA4-Ig
and humanized anti-human CD154 following
transplantation (Fig. 2B). Again, each drug was given
every other day beginning on the day of surgery and
continuing for 14 post-operative days. One animal
rejected 32 days after surgery. The other remained
free of rejection for 100 days, but like those animals
treated with humanized anti-human CD154 alone, rejected
at that time. Similarly, a biopsy showed acute
cellular rejection. The initial regimen of CTLA4-Ig
and humanized anti-human CD154 was repeated and the
creatinine returned to baseline (1.0). MLR analysis
following this treatment showed a donor specific loss
of reactivity. Third party responsiveness was
maintained. At 165 days post transplant, the animal
was sacrificed as required by protocol due to weight
loss. Graft function at that time was normal. At
autopsy, the animal was found to have shigella and
camphylobacter enterocolitis, a common infection in
rhesus monkeys. This illness had infected multiple
animals in the original primate colony, including
several untreated animals. No other pathological
abnormality was found; specifically, there was no
evidence of lymphoproliferative disease or
opportunistic infection. Histologically, the graft had
isolated nests of lymphocytes in the interstitium, but
no evidence of tubular infiltration, glomerular damage,
or parenchymal necrosis (Fig. 3C).
Like the animals treated with humanized anti-human
CD154 alone, both of these animals had transient
increases in their creatinine combined with an increase
in graft size during the fourth post-operative week.
It was hypothesized that this graft swelling reflected
a second wave of infiltrating lymphocytes and therefore
led to a modified dosage schedule such that both
reagents were given on the day of surgery and on post-
operative days 2,4,6,8, 12, 16, and 28.
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Two animals were treated with this modified
regimen (Fig. 2B). Both have experienced rejection
free survival, free of illness or alterations in renal
function for greater than 150 days. Both remain alive
and well at the time of this writing. After 100 days
of rejection free survival, MLRs were repeated against
donor cells and third party cells. No changes in in
vitro reactivity were observed (data not shown). These
studies were repeated after 150 days of rejection free
survival with identical results (Fig. 4). Both animals
maintain vigorous in vitro responses toward donor and
third party cells but fail to reject their allografts.
No animal has demonstrated toxicity from any of
the therapies employed. Specifically, there has been
no fever, anorexia, or hemodynamic abnormalities, and
no opportunistic infections have occurred. Animals
have been housed in standard conditions and have been
allowed contact with the other -animals in the colony.
They have maintained normal Weight gain. Laboratory
chemistries and hematological parameters such as
hemoglobin and white blood cell counts have remained
normal. The percentages of cells expressing CD2, CD3
and CD20 were unaffected by any treatment regimen (data
not shown). Specifically, no reductions in T cell
counts were observed during or after treatment in any
animal.
Obviously, many modifications and variations of
the present invention are possible in light of the
above teachings. It is therefore to be understood
that, within the scope of the appended claims, the
invention may be practiced otherwise than as
specifically described.
