Xenogeneic transplantation
Johan van den Bogaerde and David J G White
Imutran Ltd, Cambridge, UK
Hyperacute rejection, the previously insurmountable obstacle to pig-to-human
xenografts, has been overcome.There is reason to hope that concerted research will
overcome the remaining obstacles. Pigs will be produced expressing other
regulators of complement activation molecules in addition to decay accelerating
factor. Modification of antigenic structure of cells could also be achieved.There is
now some hope that large scale clinical xenotransplantation could become a
reality.
To avoid confusion, some fundamental definitions have been listed in
Table 1. Organ transplantation is currently the accepted therapy for end
stage renal, hepatic or cardiac failure. Other established transplantation
procedures include pancreas, small bowel, islet cells, and intracerebral
fetal tissue transplantation. Even optimal utilisation of cadaveric organs
in developed countries would not provide sufficient transplantable
organs to meet the ever increasing demand3'4. This organ shortage crisis
has stimulated research into the use of animal organs for clinical
transplantation.
Concordant xenograf ting
A large body of experimental work has examined the mechanisms of
rejection between closely related species2. Concordant xenografts are
rejected more gradually than discordant xenografts. There are, however,
differences between apparently concordant species combinations. For
example, mouse-to-rat xenografts are rejected by T cell dependent
Correspondence fo mechanisms 5 , while hamster-to-rat xenografts do not survive despite
Dr David JG white, complete T cell depletion6. Mouse-to-rat transplantation, therefore,
imutran Ltd (a Novartis represents an 'easy' concordant model, while hamster-to-rat is a
pf.orn.aAGcompany 'difficult' model. Hyperacute
rejection (HAR) of these 'difficult'
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©The Brrti»h Council 1997
Brituh Medical Bulletin 1997,53 (No 4)904-920
Xenogeneic transplantation
Table 1
Definitions
Alogeneic transplantation
The transplantation of tissue between members of the same species
Xenogeneic trantplantahon
The transplantation of tissue From one species to another
Concordant xenografhng' J
TransplantaKon of huue between closely related species, tuch as baboon-to-man, or
ham»ter-to-rat Organs are rejected over a period of days
Discordant xenograftmg'
J
Transplantation between spea'es which are phylogenehcdry distant, such as pig-topnmate Re|echon occurs within minutes or hours, and is similar to refection of
allograftsin a previously sensitised recipient The re|echon of discordant xenografts is
called hyperocute re|ection
Hyperocute re|edion (KAR)
The acute and severe rejection of discordant organ transplants, mediated by naturally
occurring pro-formed antibodies binding to carbohydrate epitopes on endotheGal
cells (EC) and platelets. Complement activation, thrombosis and neutrophil infiltration
follow antfeody binding
Heterotopic organ transplantation
The transplantation of a vascularised organ graft man anatomicaBy different position
from that in which it is normally found For example, a heart may be placed in the
abdomen of a recipient animal The transplanted heart does not maintain the cardiac
output of the recipient, but is fully vascularised, and can be monitored for signs of
refection
Orthotopic organ transplantation
The transplantation of organs in positions where they are normally found, for
example the donor heart replaces that of the recipient, and maintams cardiac output
'difficult' concordant xenografts is only achieved when complement is
depleted, or when anti-xenograft antibody production is blocked7'8.
Primate-to-human xenografts have been performed in the past9"11. In
1964, 9 month survival was achieved in a chimpanzee-to-human renal
transplant patient, using an immunosuppressive regimen of azathioprine, actinomycin C, and steroids12. In contrast to this, baboon-to-human
xenografts are rejected despite modern immunosuppressive regimens13.
Although formal proof is lacking, baboon-to-human xenografts appear
to belong to a 'difficult' concordant combination, while chimpanzee-tohuman xenografts are an 'easy' concordant combination. Chimpanzees
are an endangered species, and could never be considered as a donor
species for clinical xenotransplantation. Ethical and retroviral considerations, size constraints, and slow gestation periods have diminished
enthusiasm for baboon, or other primate xenografts14'15, and the recent
'Kennedy report' has barred the use of primates for organ donation in
the UK16.
Discordant xenotransplantation
The domestic pig has been identified as the suitable xenogeneic donor
species, since pigs are domesticated, have organs of sufficient size,
Bnhsh M e o W Bulletin 1997,53 (No 4)
905
Transplantation
multiply rapidly, and are sexually mature after 6 months17. However,
HAR occurs when pig organs are transplanted into primate recipients2.
This acute and severe rejection process is largely mediated by naturally
occurring pre-formed antibodies which recognise glycoprotein antigens
on porcine endothelial cells (EC) and platelets18-19. After birth, all
humans develop these naturally-occurring antibodies following bacterial
colonisation of the gastrointestinal tract2-20. Antibody binding results in
EC, complement and platelet activation, as well as neutrophil infiltration21. Histologically, this is seen as EC disruption, interstitial
haemorrhage, platelet thrombosis, and infarction22.
Naturally occurring human anti-pig antibodies predominantly recognise the carbohydrate antigengalactose-alpha-(l,3)-galactose2329. Digestion of the galactose-alpha-(l,3) linkage abrogates binding of human
pre-formed IgM antibodies to pig endothelium30, confirming that this
antigen is the major epitope which is recognised by naturally occurring
xenoantibodies. Human serum contains both IgM and IgG antibodies to
these carbohydrate antigens, and up to 1 % of the total serum IgG may
consist of anti-alpha-galactosyl antibodies29. The enzyme which
catalyses the galactose-alpha-(l,3) linkage is expressed on all mammalian cells, except humans, old world monkeys, and the great apes.
Hyperacute rejection (HAR) is the first, and perhaps the most
formidable barrier which must be overcome, before attempting pig-tohuman transplantation. There are three possible methods of blocking
HAR. These are: (i) depletion of pre-formed antibodies; (ii) reduction of
target antigen expression; and (iii) inhibition of complement activation.
Depletion of pre-formed antibodies
Effective antibody depletion is difficult, since the binding of a single
pentavalent IgM molecule to an antigen on the cell surface may activate
the classical complement pathway, and result in cell lysis. Antibody
depletion also results in antibody rebound within 1-5 days, often
resulting in levels exceeding the original antibody titre. Specific
reduction of IgM xenoreactive antibodies has been achieved experimentally by using anti-heavy chain (anti-u) monoclonal antibodies31'32. These
studies demonstrated that IgM antibody levels could be reduced 100fold. The addition of plasmaphoresis, splenectomy, and even drugs
inhibiting antibody formation did not successfully block HAR31-33. The
median survival of guinea pig hearts in IgM depleted rats was only 62
min, and rejection was histologically identical to control animals32. In
the pig-to-primate model, antibody depletion by ex vivo perfusion
through pig organs resulted in 3-5 day survival of transplanted
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Bntii/i Medical BuH»hn 1997;53 (No 4)
Xenogeneic transplantation
vascularised pig organs22. The rejected organs demonstrated focal
vascular changes, similar to hyperacute rejection and, more importantly,
IgM deposition in the absence of complement deposition was observed
on EC. Serial transplantation of organs, as well as immunoabsorption of
antibody has also been attempted34.
Although HAR was delayed in many of these experiments, antibody
depletion was always temporary. A more serious problem with these
data is that most antibody depletion regimens probably also reduce or
alter complement levels. Other studies have suggested that the
alternative complement pathway can produce HAR in the absence of
antibodies35. Clinically, depletion of pre-formed xenoreactive antibodies
is probably not achievable, and does not appear sufficient to block
HAR36.
Reduction of target antigen expression
A novel approach to changing the antigens of the pig is the production of
pigs expressing human fucosyl transferases, which would convert the
alpha-galactose epitope into an antigen similar to that of human blood
group antigens37. This may reduce reactivity to the dominant carbohydrate antigen, but does not prevent binding of pre-formed xenoantibodies to other epitopes. The antigen modifying approach does not take
into account the induced antibody response to a diverse group of
xenoantigens, which probably produces high affinity, polyclonal
antibodies after xenotransplantation.
Inhibition of complement activation
The complement system by itself appears capable of discriminating self
from non-self in xenogeneic systems33-38. This realisation suggested that
blockade of complement activation was the only clinically viable
strategy in combating HAR. Increased organ survival could be achieved
in discordant species combinations by depleting complement, or by using
congenitally complement deficient recipients39"41. Complement depletion
is achievable in experimental systems, but is not a realistic clinical aim.
A more feasible approach would be the expression of human
complement inhibiting molecules on xenografts. Xenogeneic research
has become focused on the human regulators of complement activation42^*4, and the utilisation of these molecules to block hyperacute
rejection.
Bnfich M»dical Bulletin 1997^3 (No 4)
907
Transplantation
Classical
Pathway
C4+C2
\
C3 Convertase
C4b2a
C3
C3 Convertase
\
C3b
C5-C9 (MAC)
C3bBb
C3+B
Alternative
Pathway
Classical
Pathway
C4+C2
IC4b
C3 Convertase
C4b2a
[DAF
| DAF
C3 Convertaae
Fig. 1 (a) The normal
complement cascade, (b)
Inhibitors of complement
activation, and their Jite
of action.
C3bBE
"*"
C3+B
Altsmatlve
P ^
The complement cascade/ and regulators of
complement activation
The complement system is an amplification cascade consisting of 30 or
more proteins, which make up 10% of total serum proteins45. Both the
classical and the alternative pathway result in the production of C3b.
The classical pathway is activated by immune complex binding to Cl,
which in turn generates C4b2a, a C3 convertase. The alternative
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Bnhsh MedicalBu».tm 1997,53 (No 4)
Xenogeneic transplantation
pathway C3 convertase is C3bBb, which is stabilised by properdin. C3b
is spontaneously converted to C3Bb. In the absence of regulatory
elements, a positive amplification loop is formed, resulting in the
production of C3b (see Fig. la). The alternative pathway is activated
non-specifically, and is usually maintained in a state of low level
activation by complement inhibitory molecules.
The C3b molecule binds to cell surfaces, and becomes a C5
convertase. The formation of C5b results in the assembly of the C5b—
C9 molecule. This complex is also called the membrane attack complex
(MAC), and causes cell lysis.
There are 5 membrane bound regulators of complement activation
(RCA), which are broadly divided into two groups: those that downregulate the C3/C5 convertases, and those that inhibit the formation of
the MAC46. Decay accelerating factor (DAF), membrane cofactor
protein (MCP), and complement receptor 1 (CR1) increase destruction
of the C3/C5 convertases47'48. Homologous restriction factor (FiRF) and
CD59 block the formation of the MAC.
The genes encoding the three major membrane bound RCA molecules,
DAF, MCP, and CR1 are on chromosome 142>43. The DAF (CD55)
molecule is found on the q32 area of chromosome I42. The gene consists
of 11 exons42, and has 4 short consensus repeats (SCR) of approximately
60 ammo acids. Four short consensus repeats are also found on MCP,
while CR1 has 30. DAF is attached to the cell membrane by a glycosyl
phosphatidylinositol (GPI) anchor46. DAF usually has a molecular
weight of 70 kD, but a 140 kD form has been documented49.
Human RCA molecules and xenotransplantation
Pig complement inhibiting molecules have not been as well characterised
as their human counterparts, but it is clear that human complement is
not efficiently inactivated by pig RCA molecules38. The hypothesis, that
the expression of human RCA molecules on pig cells could inhibit FLAR,
was advanced50. The membrane bound RCA molecules would need to be
utilised, since soluble complement inhibitors might cause systemic
complement inhibition, without protecting xenografts.
DAF splits C2a from the C4b2a molecule, thus inactivating the C3
convertase of the classical complement pathway (see Fig. lb). DAF also
splits Bb from the alternative pathway C3 convertase C3bBb. DAF,
therefore, blocks activation of both the classical and the alternative
complement pathway. Apart from CR1, the other membrane bound
RCA molecules block complement activation at a later stage. CR1 has
the activity of both DAF and MCP, but is not a candidate molecule for
Bnht/i Medico/ Bullthn 1997^3 (No 4)
909
Transplantation
complement down-regulation bound to cell surfaces, since its primary
function is the binding of immune complexes. Chromium release assays
demonstrated that transfection of DAF and MCP protected pig EC from
human complement induced lysis51, but DAF was more effective than
MCP, and DAF/MCP transfectants were no more effective than DAF
transfectants52. DAF was, therefore, selected as a molecule which could
potentially inhibit human complement activation on pig cells. The
production of transgenic pigs, expressing the human DAF molecule on
transplantable organs was undertaken to determine whether this could
combat FLAR of pig organs in primate recipients53"56.
Production of transgenic pigs expressing hDAF
Transgenic pigs can be produced by injection of DNA constructs into the
nuclei of a fertilized egg. The expression of the gene depends on both the
sequence of the relevant gene, and regulatory elements, which are
usually, but not invariably upstream of the transgene. The genomic DAF
gene, with all its exons, introns, and promoter regions spans
approximately 80-90 kb, which is too large for transfer in plasmids or
cosmids. DNA which is produced by RNA transcription contains only
exons and not the intervening introns. This type of DNA is called cDNA
(c = copy). Although cDNA is easier to work with than genomic DNA, it
lacks the correct promoters, resulting in low gene expression after
injection57. The addition of promoter sequences improves gene
expression58.
Minigene constructs can consist of both genomic and cDNA. They are
easily produced, injected, and incorporate efficiently into the host
genome. The disadvantage of minigenes is that their expression is
determined by where they are inserted into the host genome59. If the gene
is incorporated in an inactive area, protein expression does not occur,
whereas incorporation into an area where promoter and enhancer
sequences are active allows successful expression. Alternative RNA
splicing has been documented for MCP60, producing up to 14 different
MCP isoforms, which may have functional implications. Since minigene
constructs lack the control elements which regulate this process,
transgenic proteins may be less efficient than naturally occurring
protems.
The first step in the production of transgenic pigs was the screening of
yeast artificial chromosome libraries (YAC) from ICRF and ICI human
YAC libraries61. YACs contain up to 1000 kb fragments of human DNA,
and genomic DNA for DAF, MCP and CD59 was obtained. A minigene
construct was produce which contained both genomic and cDNA. A
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Medical Bvllehn 1997,53 (No. 4)
Xenogeneic transplantation
4 kb section of genomic DNA consisted of the signal peptide, the first
exon, and a section of the first intron, while a 2 kb cDNA segment
contained to the last 10 exons of the DAF gene. These minigenes were
then injected into pronuclei of more than 2000 fertilised ova, and placed
into 85 surrogate mothers. The majority of these animals (65%) became
pregnant, and 15% of the offspring demonstrated human DNA by slot
blot analysis50-53.
Structural and functional characterisation of hDAF in
transgenic pigs
The successful incorporation of human DAF (hDAF) in pigs represented
an important first step in testing the hypothesis that recipient RCA
molecules could inhibit hyperacute rejection. The presence of the gene
did not mean that a functionally normal hDAF molecule was expressed
in relevant tissues, at an appropriate concentration. Reverse transcnptase polymerase chain reaction (RT-PCR), followed by Northern blot
analysis confirmed the presence of mRNA encoding hDAF in transgenic
animals. The correlation between mRNA and hDAF expression on the
cell surface was good62. Immunoprecipitation of the protein demonstrated that the molecular weight of the transgenic protein was the same
as the naturally occurring human protein63'64. Detailed analysis using a
panel of monoclonal antibodies further confirmed that the 4 SCR,
necessary for hDAF function, were present on the transgenic protein.
These data proved that the incorporation of hDAF into pig cells, resulted
in the formation of mRNA, which was correctly transcribed, producing
intact hDAF molecules on the cell surface.
Immunohistochemical staining demonstrated the presence of hDAF on
all the organs routinely used in allogeneic transplantation (heart, kidney,
liver, lung and pancreas). Detailed, semi quantitative analysis of hDAF
expression in 30 different lines of transgenic pigs using double
determinant immunoassays and Scatchard analysis was undertaken
(Cozzi E et al. manuscript submitted). This analysis included comparative studies of human cadaveric tissue. There was considerable
variability in expression of hDAF in transgenic animals, not only
between different animals, but also between organs from the same
ammal. For example, muscle expression of hDAF was 6 times higher
than heart expression in one animal.
The liver was the organ in which hDAF expression was most
frequently observed, and hDAF protein was measurable in more than
90% of liver tissues from transgenic pigs. The heart was the organ in
which hDAF was least frequently detected, and the transgene was
Bnhsfi Mtdical Buffehn 1997^3 (No. 4)
911
Transplantation
expressed in only 18 of 30 samples. 26 organs from 12 different lines of
pigs were found to express amounts of hDAF comparable to, or greater
than, those found in equivalent human tissue.
High expression of hDAF in transplantable organs did not guarantee
EC expression. Some animals did express high levels of hDAF on both
EC and parenchyma of transplantable organs.
Analysis of the number of human DAF genes incorporated into
transgenic pig cells was undertaken, and an attempt was made to
correlate this with hDAF expression in pig tissues. No correlation
between gene number and hDAF expression could be demonstrated. For
example, the animal with the highest gene copy number (13 copies of the
transgene), expressed very low levels of hDAF in all the transplantable
tissues analysed. The two most promising pig lines incorporated between
6 and 8 copies of the gene, and expressed hDAF on parenchyma and
endothehum of all the transplantable organs analysed. In 75% of these,
organs expressed levels of hDAF greater than in the equivalent human
tissues.
The transgenic hDAF molecules were then shown to inhibit human
complement activation. EC are the first targets of HAR, and were thus
used in these experiments. Expression of hDAF on pig cells prevented
iC3b deposition on the cell surface, after exposure to human serum
(Cozzi E. manuscript submitted). The transgenic animals, not expressing
high levels of hDAF on EC, demonstrated substantial iC3b deposition.
This confirmed that transgenic hDAF, expressed on EC, inhibited human
complement activation.
Neutrophil adhesion to EC occurs when complement is activated, and
is mediated by binding of neutrophil P2 integrin (CDllb/CD18) to
iC3b65. During HAR, complement activation leads to neutrophil binding
to EC57, and contributes to inflammation, thrombosis and rejection.
Human neutrophils did not bind to pig EC expressing hDAF, but did
bind to control pig endothelium (Richards et al. manuscript submitted).
Both human complement deposition and neutrophil binding was
blocked by hDAF on transgenic cells. The hDAF molecule protected
pig endothehal cells from complement and neutrophil activation.
These data confirmed that structurally and functionally normal hDAF
was expressed at high levels on both parenchyma and EC of selected
lines of transgenic pigs. The endothelium is the first target of antibody/
complement mediated HAR, so hDAF expression on vascular endothelium is obviously necessary. Although there is no a priori reason to
believe that parenchymal expression of hDAF is necessary for successful
organ transplantation in the pig-to-primate model, vascular injury,
following complement activation on EC could result in the exposure of
underlying parenchymal tissue to xenoreactive antibodies. Human RCA
molecules in parenchymal tissue should, therefore, confer protection in
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Snhsh Medical Bulletm 1997^3 (No 4)
Xenogeneic transplantation
the event of such vascular injury. Thus, high expression of hDAF should
ideally be found on both EC and parenchymal tissues. In vivo
experiments were only performed using organs with both endothelial
and parenchymal expression of hDAF. Since no transplants have been
performed using organs where hDAF is only expressed on the
endothelium, no in vivo data exist which would substantiate these
suppositions.
Ex vivo organ studies, and xenogeneic transplantation
studies
Pig hearts were connected to an ex vivo perfusion circuit, and cardiac
output, heart weight, and coronary blood flow measured. The variables
of this model can be well controlled, but pig hearts perfused with pig
blood stop beating after approximately 128 min due to physiological
factors associated with the ex vivo nature of the circuit66. Perfusion of
pig hearts with human blood resulted in HAR, with a 26 min heart
survival (Fig. 2). The ex vivo perfusion circuit demonstrated that
transgenic pig hearts expressing hDAF were protected from HAR.
Histological examination demonstrated that C3 deposition did not occur
when hDAF was expressed on the endothelium. The expression of high
levels of hDAF was as successful as total complement depletion with
cobra venom factor in prolonging heart function. Myocardial parenchymal expression of the transgene did prolong heart survival, although
this was substantially less than those hearts in which endothelial cells
expressed hDAF. Deposition of human anti-pig antibodies on endothelium was demonstrated, but the presence of functioning hDAF blocked
HAR. In addition, antibody depletion of perfusing serum did not block
deposition of C3, presumably via the alternative complement pathway,
in hearts from non-transgenic animals. The presence of hDAF also
reduced activation of neutrophils and platelets.
This ex vivo model proved that hDAF expression could abolish HAR
of pig hearts by fresh human blood. Transplantation of transgenic pig
organs into non-human primates could now be performed. During all
these operations particular care was taken not to cause haemodilution,
or infuse hepann which could reduce complement activity. Before
operation, the recipient monkeys were tested for the presence of anti-pig
endothelial and red blood cell antibodies. The anti-pig antibody titres
were at least as high as those found in pooled human serum.
The first series of experiments examined heterotopic heart transplants
from transgenic and control pigs into cynomolgus monkeys {Macaca
fasctcularis)67. In this experiment, 8 hearts from transgenic pigs were
Bntish Medical Bulletin 1997;53 (No 4)
913
Transplantation
Cardiac output of Pig hearts perfused with fresh Human blood
Normal pJgN-16
DAF on •ndothelliim N*5
DAF on Myocardium only N=3
Transcjenlc no OAF detected N=11
f
1
E
3
O
u
CO
1
1CO
u
2-
60
90
120
150
160
210
240
Time in minutes
Fig. 2
Survival of pig hearts in an ex vivo perfusion circuit Hearts from control and trantgenic pigs are shown.
transplanted into non-immunosuppressed cynomolgus monkeys. Hyperacute rejection of transgenic, hDAF expressing hearts did not occur67.
The median survival of transgenic hearts was 5.1 days (range 97-126 h).
During these experiments, some of the control hearts survived longer
than was expected. Previous studies by others had documented a survival
of less than 1 h in pig-to-cynomolgus xenotransplants68, but 5 of the 10
controls survived for a mean of 86.4 h. The 5 remaining control hearts
survived for only 2.6 (+3)h. Histology demonstrated prominent
thrombosis and necrosis in these control hearts, suggesting antibody
mediated rejection. The explanation for this surprisingly long survival of
some control hearts, despite the presence of naturally occurring
antibodies, was not readily apparent. One explanation is variability of
carbohydrate antigens on the donor pig endothelium. Lower levels of
galactose-alpha-(l,3)-galactose was demonstrated in longer surviving
control hearts. Down-regulation of these antigens could, however, have
occurred during the longer period of survival in the donor monkeys.
Another possibility is that some pigs may express molecules which
function as human CD59 molecules, blocking formation of the MAC69.
914
Brihth M*dKal Bullmhn 1997;33 (No 4)
Xenogeneic transplantation
Further experiments are being performed to explain the prolonged
survival of the pig control organs.
Having demonstrated that HAR could be controlled, the achievement
of prolonged xenograft survival was the next goal. Cyclosponne,
cyclophosphamide and steroid regimens had been shown to produce
long term survival in the 'difficult' hamster-to-rat concordant combination. This regimen had also been shown to inhibit the production of
induced anti-xenograft antibodies7, and was, therefore, used as the first
immunosuppressive regimen in transgenic pig-to-primate xenotransplants.
Cynomolgus monkeys were given 80-180 mg/kg/day of cyclosporine,
which produced a clinically acceptable blood level of approximately 400
ng/ml in recipients. Cyclophosphamide is more toxic, and was
administered at doses of 10-20 mg/kg on alternate days67. Methylprednisolone was also administered at 1 mg/kg as a tapering dose. The white
cell count was maintained at approximately 2000 cells/ul, which is
compatible with human climcal immunosuppressive regimens. The doses
of cyclosporine and cyclophosphamide needed to be much higher than
equivalent human dosage schedules, since absorption in 3—4 kg
cynomolgus monkeys is poor. This regimen produced median heterotopic transgenic heart survival of 40 days in 10 treated monkeys (range
6-62 days). The 5 non transgenic control hearts were rejected
hyperacutely (median 55 min). Five animals from the transgenic heart
group had to be euthanased due to gastrointestinal toxicity, resulting in
severe diarrhoea. The hearts from all 5 of these animals were
histologically completely normal, with no evidence of complement or
immunoglobuhn deposition. All these hearts demonstrated C4 and
immunoglobulin deposition; however, only the two hearts that were
rejected showed evidence of C3 and C9.
This immunusuppressive regimen was used for both orthotopic heart
transplantation and life sustaining kidney transplants (manuscripts in
preparation). These experiments demonstrated that transgenic pig
organs could maintam fluid balance and sustain cardiac output in
primate recipients.
These experiments confirm that FLAR does not occur if donor organs
have recipient DAF on endothelial cells. The human DAF molecules do
block cynomolgus complement, but less efficiently than human
complement. This makes the abolition of HAR in transgenic organs
expressing hDAF, but transplanted into cynomolgus monkeys, even
more remarkable. In immunosuppressed animals rejection was not the
primary cause of graft failure (2/10). Drug toxicity resulted in 50% (5/
10) having to be euthanased with functioning xenografts. Rejection was
associated with the presence of anti-graft antibodies, but there was no
correlation between the titre of antibodies or the antibody isotype, and
Bntuh M,d,cal Bulletin 1997J3 (No 4)
915
Transplantation
rejection. Furthermore, anti-pig antibodies were routinely observed in
animals where no rejection occurred. It is possible that high affinity antigraft antibodies bind to the xenograft, and are thus not detected in the
serum. Measurement of serum antibodies may not accurately reflect the
antibody profile within the xenograft. In those grafts which were
rejected, C9 deposition was documented. This could be due to high
affinity antibodies overwhelming the complement inhibiting properties
of hDAF. The addition of CD59 might control the activation of the
terminal components of the complement cascade. In this regard, a recent
publication has described heterotopic cardiac xenotransplants from pigs
transgenic for both hDAF and human CD59 into baboons70. Control
hearts survived for 60-90 min, whereas two transgenic pig hearts were
rejected at 6 and 69 h, respectively. These disappointing results, in
comparison with the results reported above, might be due to the
expression of the two transgenic RCA molecules, which was lower than
equivalent human expression.
Ongoing experiments are being performed, using baboons and
cynomolgus monkeys as recipients. Immunosuppressive regimens, which
are potentially more effective at reducing anti-graft antibody formation
with less toxicity are being studied. Newer compounds such as
rapamycin, brequinar sodium, deoxyspergualine, and leflunamide are
all likely candidates71"74. These drugs may effectively inhibit xenoantibody production.
The ideal regimen would combine low toxicity with profound
suppression of inducible antibody formation. If a specific regimen could
successfully combat inducible antibody formation, cellular rejection of
discordant xenografts could be addressed. Cross species binding between
donor class II MHC molecules and costimulatory molecules such as CD4
on recipient T cells might be less avid than corresponding allogeneic
binding, thus reducing T cell responses75. It could, therefore, be
relatively simple to combat cellular rejection in discordant xenografts,
once induced antibody formation has been overcome.
The road forward
The production of an ideal hDAF expressing pig is not complete. All the
organs used in this study were derived from heterozygous pigs. The ideal
pig would express high levels of hDAF on organs and endothelium, and
would be bred to homozygosity. Of all the pigs produced to date, only
one line fulfils these stringent criteria. This illustrates the difficulties
inherent in the production of transgenic animals for possible clinical use.
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Bntnh Medical Bulletin 1997,53 (No 4)
Xenogeneic transplantation
In addition, breeding to homozygosity might cause undesirable effects on
the stability and health of the pigs.
The other human RCA molecules could be incorporated into a pig,
already expressing hDAF. Mouse and pig lines expressing MCP, CD59
or DAF have been produced56'61-76, and transgenic animals expressing
high levels of two or more human RCA molecules is a possibility.
Producing pigs which do not express the carbohydrate antigens which
are recognised by pre-formed antibodies has already been suggested26'77.
Gene knockout of a multitude of xenoantigens is clearly not practical,
but gene knockout of certain specific antigens, or other immunologically
important molecules, in combination with transgenic expression of RCA
may improve results still further. It is to be hoped that immunosuppression after transplantation of the 'custom made' transgenic animals of the
future would be easier than in allogeneic transplants.
There are three further issues which need to be addressed before large
scale clinical xenotransplantation becomes a reality. These are: (i) ethical
considerations; (li) infectious and retroviral constraints; and (lii)
physiological barriers. Ethical considerations are important, and have
effectively caused a prohibition on clinical primate to human xenotransplants. The use of pig organs appears to be ethically acceptable.
Retroviral considerations are important, and the transplantation of pig
organs into immunosuppressed recipients, may allow the spread of
hitherto unknown pig viruses in humans. Pig tissues have been
transplanted into human recipients for many years, without any viral
complications. The possibility of pig retroviral infection in xenograft
recipients still requires further research.
Xenotransplants may not function in a way compatible with human
physiology. Preliminary data have demonstrated that both heart and
kidney transplants perform physiologically in primate recipients. In the
absence of long term survival of xenografts in humans, these three issues
will remain theoretical considerations. Before clinical xenografting
becomes a reality, immunologists and transplant surgeons must devise
clinically acceptable methods of ensuring long term survival of
xenografts in humans.
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