Note: Descriptions are shown in the official language in which they were submitted.
- ~ -
ZZ ~C~ o,O 2~ 34~
Ma~erials and Methods for the_Trea~ment
of Foreign Tissue
BACKGROUND OF INVENTION
The present invention relates to the problem of
re~ection of foreign tissue. In particular, it provides
materials and methods for the amelioration of rejection
o foreign tissue.
Transplantation vf organs and tissues between
identlca:L twins can be successfully achieved and does not
lead to grat re~ectlon. Where allogenic tissue and
organ transplants are carried out, or where allogeneic
cadaver skin is used as a wound dressing, the immune
system of the recipient recogn.lses the graft or dressing
as foreign, and mounts an lmmune response agains~ the
foreign tissue. This response depends upon genetically
determined anti~enic di~ferences between the donor and
the recipient and rejection can take placP in at least
two ways. The first involves antibody and complement and
has been referred to as hyperacute graft reJection, wh~l~
the second involves activated T-cells and is referred to
as acute graft re~ection.
For this reason, a donor organ for transplantation
ls selected, which closely matches both the tissue and
blood types of the recipient patient. Therefore, before
the transplant opPration, blood and tissue samples ~f
both the patient and donor will be matched as closely as
:
:: .
.
2 ~
pos~ible with respect to at least the ABO and Rh blood
group systems and the MHC tissue type system.
In some cases the re~ectlon o~ the donor organ will
occur very quickly (within mtnutes and up to 48 hours).
This phenomenon is known as hyperacut~ graft rejection
and it seems to occur when the rec:ipient patient is pre-
sensitized ayainst the donors blood andtor tissue types.
Such pre sensitization of the recipient patient may occur
spontaneously, or by virtue of previous grafts, blood
transuslons or pre~nancy. In such cases of pra-
sensitization~ the reciptent patient will usually have
pre-formed circulating antibodies specifis::ally directed
against the blood and/or tissue type of the donor. The
pre-existing antibodies will then bind to the oreign
cells transported into the reciE~ient patient by the donor
organ, and this cell-antlbody binding will then stimulate
complement mediated cytotoxicity or antibody-dependent
ce~l-mediated cytotoxicity (ADCC).
In complement mediated cytotoxicity, there will be
fixation of the first protein component (Cl via Clq) o
the complement cascade leading to cleavage of C3 and
activation of the lytic pathway of the complement system
and to cytolysis of the foreign cells. In addition the
~ 9~Q9Q~'o~
activation of C3 causes pla~elet ~,
accumulation of neutrophils, and the formation o an
inflammatory exudate. As a result of thi~, bystander
.: ;,
::
.. ~ ~ .;:
3 ~ 2
tissue cells can be damaged even though they are not the
immediate targets of the antibody recognition.
In antibody-depende~t cell-mediated cytoto~icity,
the antibody coated foreign cells are recognized by
cytotoxic cells (e.~., monocytes and pol~morphonuolear
cells) havlng Fc receptors. Binding will occur between
the foreign cells and cytotoxic c~115 vla the antibody-
Fc receptor bridge and lysis of the foreign cells will
follow.
Thus, when the donor and patie~t reciplent are
matched for blood and tissue groups, tests will be
carried out to check that serum from the patient
recipient will not attack l~mphocyte~ of th~ donor, ~n
the presence of complement. Any such attack by the
patients serum would be taken as a strong contra-
indication a~ainst the success of the proposed graft.
Even where the recipient ~)atient has not been pre-
sensitized against the donors blood and tissue types,
acute re~ection episodes are common and are usually
mediated by activated T cells~ Consequently patients
having donor organs e.g, kidneys are treated with
immunosuppressive agents such as prednisolone and
cyclosporin. However, such immunosuppressive agents are
non-specific, af*ecting the whole immune system, making
the patient vulnerable to bacterial and viral infection.
Therefore, it is desirable to have a way of controlling
.,;: .
'
,;
2 0 ~ 2
the immune response to defined antigens only, in this
case those antigens expressed by the donors cells and
whlch are seen as forelgn by the recipient patient.
DESCRIPTION gF PRIOR ART
Studies have been carried out in rodent models and in the
human, to see if certain cells o the donor organ itself,
or cells incidentally carried by the donor organ, are
particularly efect~ve in stimulating an immune response
i~ the recipient receiving the donor organ (Hart D.N.J.,
et al: Transplantation Vol 33, No.3:319, 1982 and Hart et
alo Transplantation Vol 31, No.3:428, 1981). Dendritlc
oells (passenger leucocyte~) in the interstitium of both
rat and human kidneys were identified as beiny intensely
positive for the ma~or histocompatibility complex (MHC)
class II antigen.
Studies have also shown that the survival of rat
kldney allografts can be great:ly enhanced by depleting
the MHC II dendritic cells before transplantation. The
MHC II dendritic cells were depleted by treating the
donor rats with combinations of cyclophosphamide,
radiat1on and hydrocortisone therapy. The study showed
that ev~n small numbers of interstitial dendritic cells
aat as a potent stimulus to graft rejection (McKenzie
J.L., et al., Transplantation Proceedings, VolO XVI No.4
(August), 1984). However, in these studies there were
difficulties in the eradication of the dendritic cells
, ~ ~
- - \
2 ~
and therefore effective donor pre-treatment by the
techniques may be difficult to apply clinically. The
authors suggested that prior to transplantation, donor
organs may be perfused with monoclonal antibodies against
MHC antigens or dendritic cells. The authors also
su~gested however, that even if human donor kidneys were
depleted of dendritic cells, the presence of the HLA-DR
antigen on human endothellal cells may still cause
re~ctlon.
Subsequent experiments have also shown that
repletion of these dendritic endothallal cells restores
their lmmunogenicity (Lechler R.J., e~ al. JO Exp. Med.
lg~2; 155:31-40).
In other laboratory animals, heart (Sone Y., et al.
Transplant. Proc. 1987; XIX: 599-604), pancxeas (Lloyd
D.M., et al. Transplant Proc. 1989; 21: 482-83) and
kidneys ttsubo et al. Transplant. Proc. 1983; 15: 797-
799) grafts can be made to suxvive longer by perfusion
with antibodies against class II MHC antigens which
lnactivate or destroy these cells.
The human kidney endothel1um expresses MHC class II
a~tigens, so that although the dendritic cells are
strongly MHC II positive, monoclonal antibodies against
these antigens cannot be used in ~he same way for human
renal transplantation to destroy the interstitial
dendritic cells, since the endothelial cells of the graft
.
3 ~ 2
are li~ely to be damaged and as a consequence, the graft
deprived of its blood supply and 105t.
Other investigatlons have looked at the functioning
of he~erologous antibody as an immunosuppressive agent.
In ~n effect known as "passive enhancement", recipient
rats pre-treated by in~ections of recipient anti-donor
spleen serum, showed increased survival times of the
kidney allograft. The effect ls thought to be due to the
opsonisation of passenger dendritlc cells, whirh thereby
removes part of the immunogenic stimulu~ for the
recipients re~ection response (Har~, D.N.3., et al.,
Transplantation Vol.33 No.3, 1982).
Thts work led to the hypothesis that depletion of
donor dendritic cells prior to transplantation may have
the same effect as passive enhancement.
Since interstitial dendritic cells can be
dlstinguished from kidney endothelial cells by thelr
expression of leucocyte common antigen, (LC or LCA, also
known as CD45 and T200) st~die~ have been carried out in
which human kidney allografts were perfused with anti-LC
monoclonal antihodies pr~or to transplantation, in an
effort to inactivate or destroy the dendritic cells. Two
groups of patients received kidneys, perfused
respectively with a mcuse anti-LC of isotype IgG2a and a
mouse anti-LC o isotype IgG3 (in the paper, the authors
incorrectly stated the antibody as being a rat anti-LC of
,:
. . , ' . , ' . ,~..,:
,:
` - 2 ~ 4 2
isotype IgG33. The former antibody is non-lytic via the
route of complement fiYation and lyt$c via the route of
ADCC, whilst the latter is not lytlc via either route.
Lysis via ADCC depends on various host leu~ocytes moving
into the kidney to e~ert their cytotoxic effects. The
results suggested (test numbers were small and the study
was neither randomized nor controlled) that the mouse
IgG2a anti-LC had favourable effects in reducin~ the
incidence of graft re~ection and improving renal function
as compared to non-perfused controls. The mouse IgG3
an~i-LC appeared to have no such effect. ~Taube D., et
al. rr n~plantation Proceeding~ Vol.XIX, No.l pl961-1963
1987).
However, for maximum effect in the inactivation or
destruction of undesired cells, an antibody should be
used which mediates as many mechanisms as possible of the
immune response.
Therefore in theory, a foreign cell would be mor~
efectively inactivated or destroyed by use of an
antibody which could also mediate complement-depandent
lysis of the cells. However, there was widespread
reluctance to usa such an antlbody in this technical
field. The desirable effects of achieving comnplement-
dependent lysis may be offset by the damaging effects
triggered through the extensive ac~ivation of C3 and the
a~d ~ ~n~a~ ~ I`S.
induction of a loc~l inflammatory response~ Thi~ in turn
2 ~
migh~ lead to kidney damage and failure of the grat.
Indeed this fear was confirmed when a pair of monoclonal
antibodies, that in combination mediated lysis via both
the complement and an~lbody-dependent cell mediated
pathways, were used to perfuse a kidney before
transplantation. (Cambridge, England, 1986, unpublished
results). To the bitter disappointment of all concerned,
the transplant~d kidney failed and the response ~n this
ield of art was that use of perfusion an~ibodles which
used the complement-medl2ted lytic pathway were
contrainclicated.
The value of pairs of antibodies in achieving
synergistic lys~s has been investlgated. In one study
(Bindon C.I., et al. Transp].antation Vol. 40, No. 5
1985) combinations of antibodi.es recognizing different
epitopes of tha LC antigen ~were investigated in an
attempt to improve the lytic potency of the antibodies.
The study showed that certain pairs of rat antibodie~ of
iso~ype IgG2b although not lytic separately in the
presence of human complement, became lytic when used
together. The study also showed that the presence of a
rat IgG2a antibody interfered with the lytic ability of
the synergistic pair. The authors suggested that such
pairs of synergistic antibodies may be useful for purging
organ grafts of interstitial passenger leucocytes to
reduce their immunogenicity, although there was no
2~3~2
practical disclosure of such use.
Another repor~ (Waldma~n H. et al. Transplantation
Proc. Vol XX No. 6 Suppl. 8, 1988: p.46-51) discusses the
use o~ anti-CD4 rat monoclonal antibodie~ of isotyps
IyG2b, injected into mice to cause T cell d~pletion and
immunosuppression. The effect could be improv~d ~y using
~ynergistic pairs of antibodies to non-overlapping
epitope~ of the CD4 molecules. However, other antigens
such as the CD45 as e~pressed on ~he interstitial
dendritlc cells were considered as poorly lytic with
human complemen~ whatever ~he isotype of the monoclonal
antibodies, and therefore that the antigen is a critical
variable for achieving complement lysis. The authors
suggested that they were examining the effects of
perfusing a donor kidney prior to transplantation with a
synergistic pair of anti-CD45 antibodies. ,;
~ he importance of the antigen specificlty was also
consldered in Bindon C~ et al. Eur. J. Immunol. 1988
18: 1507-1514O This study showed that of many hundreds
o rat monoclonal antibodies, only a few (<5~) were lytic
with human complement and that some antigens ar~ better
tar~et~ for complement mediated lysis than others
irrespective of antibody isotype. Generally the LC
antigen CD45 was a fairly poor antigen for human
complement mediated lysis.
Five distinct epitopes have been distinguished on
.
2~ 3~2
CD45: P, Q, ~, S and T (Hale, G., et al. in Leucocyte
Typ~ng III. Edited by AoJ~ McMichael. Ox~ord University
Press). Synergist$o lysis was found between pairs of
antibodies against two different epitopes selected from
P, Q, R OT S~ However, where the T epitope is involved~
there is only synergistic lysis between an anti-Q: anti-T
antibody pair and furthermore an anti-T antlbody would
interfere with the lytic activity of anti-P: anti~R and
anti-Q: anti-R synergistic pairs.
In a study related to ~he present lnventlon (Brewer
Y., et al. ~ransplantation Proceedings. Vol. 21. No. 1
(February~ 1989: pp. 1772-1773), pattents received kidney
allograts perfused with: (i) a pa~r of rat IgG2b anti-LC
monoclonal antibodies which when mixed together are
complement fixing and lytic, or ~ a mouse IgG~a
complement-fixing, non-lytic antl-LC monoclonal antibody.
The r~-sults reported that allograft function was
significantly better in patients with allografts having a
>50~ uptake of antibody. However, there was no
significant difference in antibody uptake between groups
(i) and (ii), and the results in term~ of allogr~f~
rejection and function were not distinguished according
to th~ type o perfusion antibody.
~ he present inventors have now produced as an
~mbodiment of the present invention, a monoclonal
antibody system which media~es cell lysis by both the
3 ~ ~
comp~ement and antibody-dependent cell-mediated pathways,
but wh~ch has no effect in the stimulation o rejection
of the grafted donor organ. The monoclonal antibody
system is anti-LC (also called anti-CD45).
SUMMARY OF INVENTION
Thus, the present invention provides a preparation for
the treatment of foraign tissue which compr1ses an anti-
CD45 specific antibody system which medlates complement-
dependent cell lysis in the presence of human complemen~.
The treatment may be ex-vivo. The foreign tissue may be
any tissue which would be seen by a recipient as not
originating from said recipient. ~ypically, the foreign
tissue may be xenogeneic or allogeneic.
The an~ibody system may comprise a single antibody
having the necessary specificity or polyclonal antiserum
whtch provldes antibodies having the required
speclficity. Alternatively, the antibody system may
comprise two or more monoclonal antibodies r which most
preferably act synerglstically in effecting complement-
media~ed cell lysis, but may not mediate complement-
dependent cell lysis separately. The synergis~ic
monoclonal antibodies may be against different epitOpeQ
selected from the P, Q, R, S or T epi~opes of CD45. Thus
synergistic pairs of monoclonal antibodies ~ay be of any
o~ ~he following combinations:
antl-P: anti-Q
anti-P: antl-R
v~ 3 ~ 2
12 ;
anti-P: anti-S
anti-Q: anti R
anti-Q: anti-S
anti-Q: anti-T -~
anti-R: anti-S~
Particularly the synergistic palr may be anti-P and
anti-Q. The antibodies may be rat monoclonal antibodles,
particularly of isotype IgG2bO .
The present invention also provides a method oP
preparing foreign tissue ex-vivo prior to transplantation
or use as a dressing material, wh~ch comprises treating
the tissue with a preparat~on as described. The forelgn
t~ssue may be xenogeneic or allogeneic. The foreign
tissue may be skin, in which case the skin is soaked in
said preparation. Alternatlvely, the foreign tissue may
be an organ such as the kidney, in whlch case the organ
may ~e perfused with said preparation. The method and
preparations as provided by the invention are applicable
to any foreign tissue where there ls a need to reduce the
lmmunogenic effec* of cells (such as passenger
leucocytes) which express leucocyte common antigen
(CD45).
The present invention also provides a method of
treating a patient which comprises transplanting or
dressing said patient with foreign tissue which has been
, ;, :. .
~. .:. ',.,, : ' '' ''" ~
2~6~3~?J
13
treated with a preparation as described. The patient may
be suffering from conditions related to the defective or
inefficient operatlon of an organ such as the. kidney.
Alternatively, the patient may b~ ~uffering from a skin
conditlon such as burns, leg ulcers or the like.
DESCRIPTION OF EME3ODIMENTS
In order tha~ the presen~ inventlon .ts more clearly
understood embodiments will be described in more detail.
Preparation of Monoclonal Antibodles
Sources of cells used.
Human tonsils removed in routine tonsillectomies
were used. Humans peripheral blood was obtained from
laboratory volunteers and deibrinated by shaking with
gl~ss beads.
Cell-handling medium
Unless otherw~se stated, cells were suspended and
washed in Iscove's modified Dlllbecco's medium buffered
with N-2-hydroxyethylpiperazlne-N'-2-ethanesulphonic acid
(HEPES) and containing 1~ bovine serum albumin (BSA~.
Thi~ will be referred to simply as medium.
Purification of LC antigen from human tonsil cells
-- Cell suspensions was prepared from human tonsil by
chopping with a scalpel blade, followed by passage
through a sieve to remove connecti~e tissue. The cells
were washed twice in lOmM tris HCl p~ 7.3 containing 0015
M NaCl, and resuspended in the same buffer at 5xlO3/ml.
, .
3 ~ 2
14
Cell membranes were prepared by lysis with Tween 40
(Standring R., et al. ~iochim. Biophys Acta 1978; 50-85)
and stored at -70C until required.
For the affinity purification, an anti-LC monoclonal
antibody (e.g. YTH 54.12, YTH 24.5 and F10 89-4 all
available from Serotec, Bankside, Station Approach,
Kldlington, Oxford, England, OX5 lJE) is coupled to CNBr-
activated Sepharose (Pharmacia) at a ratio of 4mg
antibody to lml of gel (packed volume) according to the
manufacture's instructions. Cell membrane
(approximately 101 cell equivalents) were resuspended in
lysiR buffer PBS containing 0.5~ Nonidet P40, 0.1% SDS,
and lmM phenyl methyl sulphonyl fluoride) and mixed with
2ml of antibody coupled Sepharose. The mixture w~s
rotated for 2hr at 4C and t:hen placed in a column.
Unbou~d material was removed by washing with several
column volumes of lysis buffer, and bound antigen was
eluted using 50mM diethylamine pH 11.5 containing 0.5 M
NaCl or other suitabla elution buffers. Two column
volumes were collected and dialyzed overnight agalnst PBS
containlng 0.02% sodium azide. The purified antigen was
stored at -20C (Bindon C.I., et al. Transplantation
Vol.40 No.5 1985).
Immunization, Cell Fusion and Production of Monoclonal
Antibodies
A DA rat was immunized subcutanously with 5-10 x lQ6
, ". '~
,
` '.~ '
', :: . ,,; ,
2 ~ 3 ~ ~
human peripheral mononuclear cells enriched for T cells.
One mo~th and 2 months later, lt recelved similar cells
intravenously. Three days after the final dose, the rat
spleen cells were fused wikh the rat myeloma line Y3 Ag
1.2.3. using polyethylenglycol. (Galfr~ G., et al.
Na~ure 277:131, 1979). The fusion was labelled YTH.
Hybrid cells were grown in selective m~dium and the
hybrid myelomas were cloned and recloned on ~oft agar.
To o~tain antibody, cells were grown to stationary
phase in Iscove's modi~ied Dulbecco's medium containlng
1% ~etal cell sarum, or as ascitic tumours in (DA X LOU)
Fl rats. Immunoglobln fractions wer~ isolated from the
ascitic fluids by precipita-tion with (NH)2S04 and were
redissolved in phosphate-buffered sallne ~PBS~ containing
0.02% sodium azide for storage at 4C or -30~C.
Binding to fixed cells or to purified LC antigen was
carried out using a solid phalse enzyme-linked binding
assay (Cobbold S.P. et al 1981 J. Immunol. Methods 44:
125). The test monoclonal antibody was ~ncubated in
microtlter plates containing adsorbed purified antigen
(106-107 cell equivalents per well) or fixed cells,
followed by a detection reagent of monoclonal mouse
anti(rat Ig) coupled to ~-galactosidase. Finally the
enz~me substrate was added and a colour change measured
in a spectrophotometerO
Isotyping o Monoclonal Antibodies
.
Hybrld myeloma cells were grown in (U.1~C) lysine,
and samples of the culture supernatant were
electrophoresed on dodecylsulfate-polya~rylamide gels to
determine the si~es of heavy and light chains. (Galfré
et al. 1981 Methods Enz~mol 73:3). The subclass of the
IgG antibodies was established by Ouchterlony double
diffusion in 2% agar (Johnstone A., and R. Thorpe 1982.
Immunochemistry in Practice, Blackwell Scientific
Publications, O~ford U.K., p.1223. The cen~ral well
contained 10 ~1 of subclass-specific antiserum, and each
outer well contained 10 ,ul of 14G-labelled culture
supernatant plus suficient carrie~ rat lg previously
titrated to give optlmal precip~tation. When the
precipitln lines had developed, the plate was dried and
exposed to X-ray film for about 5 days. For each
monoclonal antibody, only one antiserum gave a
radioactive precipitin line corresponding with the
sul3class of that antibody.
PERFUSION OF ALLOGENEIC DONOR KIDNEYS PRIOR TO
$RANSPLANTATION
Patients and Controls
77 patients were entered into the trial. 39
patients received allografts perfused with monoclonal
antibodies and 3B patients received allografts perfused
wlth a similar volume of human albumin solution. All
patients were recipients of firs~ cadaver allografts.
~ `: . ' ':: ': .' :
2~3~2
The kidneys were maintained tn Marshalls solutlon
supplied by Baxter Healthcare Ltd, Thorpe Lea Road,
Egham, Surrey, TW20 8HY. Ano~her suitable organ
preservation solution is UW solutlons cf DuPont (UK)
~td, Pharmaceuticals Dlvision, Wedgwood Way, Stevenage,
Herts. SG1 4QM. Others are known in the art.
Monoclonal Antibodies and Perfusion Techni~ue
The renal allografts were perfused with a mi~ture of
the anti-CD45 monoclonal antibodies YTH 54.12 and YTH
24.5 whlch had been purified from ascites fluid using
HPLC chromatography according to standard techniques.
They are complement-fixing rat monoclonal antibodies of
iso~ype lgG2b which together are l~mphocytotoxic in the
pressnce of human complement (Bindon, et al.
Transplantation 1985; 40: 538-546).
V~als containing 2mg o each antibody in 2ml
phosphate buffered saline were prepared. A solution of
human albumin was used as a control because it looks
superficlally the same as the antibody sDlution and was
stored in identical vialsO The vlals were marked wlth a
code~ The relation of the code to the contents was not
kno~ to the clinical team until ~he end of the trial.
Thus pretreatmerlt of the graft wl~h the monoclonal
antibody pair or albumin was random. A modification of a
previously described technique was used ( Taube ~., et al.
Transplant Proc. 1987; XIX: 1961-1963). The allograft
`- 2~3~
18
was prepared for transplantation by the standard method.
~he renal veins were clampedO The contents of the vial
(in 50ml Marshall's solution at 4C) were injected into
the renal arteries. About 20 m~nutes later, when the
arteries and veins had been anastomosed, the clamps were
removed and the transplant opPratlon wa~ completed in the
u~ual way. Biopsy specimens were taken from the ma~ori~y
of the transplants before wound closure. One core was
used for routine histopathology and the other, snap
frozen in liquid nitrogen was used to measure allograft
uptake o:E CD45 th~ monoclonal antibodi~s. 5 ~m frozen
sections were pretreated wi$h avidin/biotln (Vector Labs
Ltd, 16 Wulfric Square, Br~tton, Peterborough, Cambs.)
before incubation with biotlnylated anti-rat lgG
(Vector), peroxidase labelled strep-avidin biotin complex
(strep-ABC, Dako Ltd., 16 Manor Courtyard, ~ughenden Ave,
High Wycombe, BUG~S . HP13 5RE ), and diaminobenzidine.
Then they were counterstained with haematoxylin. To
count the total number o~ dendritic cells, sequential
sections were similarly processed having been
preincubated with YTH 54.12 and YTH 24.5. Sections
treated with strep~AB~ alone, were used as controls.
After labelling with CD45, dendritic cells were
identified by their characteristic morphology. A minimum
of 15 high-power fields were examined. The number of
dendritic cells which were positive after staining with
. , :,
!, . .
-` ~0~342
19
anti-rat Ig alone, was e~pressed as a percentage of ~he
number sta~ned with added CD45 antibodies. This
percentage is a measure of the effectiveness of
perfusion.
Immunosupress$on and Post-operat$ve Mana~ement
All patients were immunosupressed with cyclosporin (Cs)
and prednisnlone. 8mg/kg of Cs was g~ven orally before
transplantation, followed by 16mg/kg per day, in 2
dividad doses, for three days following the operation.
The dose was reduced to lOmg/kg per day for the next four
days and then adJusted to maintain whole blood trough
levels of 200-300 ng/ml measured by the standard
radioimmunoasæay (S~ndoz). Patiants received 20mg/day of
prednisolone or the first month, 15mg/day for the second
month and 10 mg/day subsequently. Patients with peak
panel reactive ant~bodies >50% were treated with anti~
thymocyte globulin (2mg/kg per day) for 10 days as
previously described (~almer A., et al. $ransplant Proc.
1988; 20: 198-200). Allogra~t rejection was diagnosed by
biopsy and managed as previou.~ly described, l~aube D.H.,
et al. Lancet 1985; ii: 171 17A). Cyclosporin
nephrotoxicity was diagnosed when the biopsy specimen
showed no eYidence of rejection, Cs levels were high, and
allo~raft function improved with reduction in Cs dose or
change to azathioprine.
-
Statistical Analysis
Student ' s test and the x2 test were used .
Results
The two groups were essentially similar ~ ~able I ) .
No side-effects attributable to monoclonal antibody
p~rfusion were observed.
Actuarial patient survival in the two groups was
similar. Patent survival was ~7~ and 92~ at 1 year and
18 months pos~-transplant, respect~vely, in the group of
patlents receiviny monoclonal ant~ body perused
allografts and 94~ at 1 year and 18 months post-
transplant in the controls. 2 patients receiving
monoclonal antibody perfused allografts died (1 after an
intracerebral haemorrhage, 3 ~onths post-transplant and 1
from carcinomatosis lS months post-transplant). Both
patients had functioning allografts. 2 control patients
died, both a~ 1 month poct-~ransplant (1 from
cytomegalovirus pneumonitis and 1 from systemic
candidi2sis). Neither of their allografts was
functioning.
Allograft survival was the same in both groups
(Table II)o 5 patients (3 with monoclonal antibody
perfused allografts and 2 controls) lost their grafts
wlthin 48 hours of ~ransplantation for technical reasons;
1 patient had uncontrollable bleeding at the time of
surgery; 3 patients lost allografts (2 monoclonal
. ~ . .
~ - .
"; 2 ~ 2
antlbody perfused and 1 control~ as a res~lt of primary
renal arterial or venous thromboses; 1 pati~nt had an
ischaem~c leg after surgery~ the graft was abandoned and
later removed. The 3 monoclonal antibody perfused ~raft
were lost beyond 48 h, none as a result of re~ec~ion, one
from irreversible renal artery stenosis, a second graft
was abandoned because the patient developed Pneumocystls
carinii pneumonia and immunosuppression stopped, a thlrd
graft was lost due to probable chronic Cs nephrotoxici~y.
Four con-trol patients lost grats, two as a result of
irreversible reJection, and two from probabls chron~c Cs
nephroto~icity.
Allograft function, measuxed as plasma creatinine,
was slightly better ln the group of patients wi~h
monoclonal antibody perfused allografts (Table III), but
of statistical signiicance only at 3 months and 1 year
po t transplant (p=0.04).
Rejection episodes occurred in 7 of the patients who
received monoclonal antibody perfused grafts. 2 of these
patients had more than 1 episode. 24 controls had
reJections episodes, of which 1~ had more than one
rejection crisis.
Biopsy specimens were analysed for CD45 monoclonal
antibody uptake. The results suggest ~hat rejection may
be associated with a low uptake of the antibody. How~ver
the numbers are too small for statistical analysis.
:``
22
In this trial, pretreatment of human renal
allografts with rat CD45 monoclonal antibodies reduced
the incidence of re~ection and posslhly improved
allograft function.
Pre-treatment o human kidney allografts with small
amounts o CD45 monoclonal anti~odies does not appear to
be harmful to either the allograft or the patient.
Although 5 allo~rafts (3 p~rfused with monoclonal
antibodies and 2 controls) were lost as a result of
complications a~ the time of op~ration, there is no
evidence to implicate monoclonal antibody perfu~ion as a
cause of graft loss. Allograft function (Table II~ may
be impr~ved.
The pre-treatmen~ of human renal allografts with
CD45 monoclonal antibodies will not make them non-
immunogenic, even i all tha passen~er leucocytes are
inactivated or destroyed. As ]previously mentloned, the
human renal vascular endo~helium and renal ~ubular
epithelium express MHC antigen~. (Hart, D.N.J., et al~
Transplantation 1981; 31 428-33). Thi~ perfusion
technigue is simple, safe and cheap and may be applicable
to other organs such as the heart, liver and pancreas.
.
`'~
: I , . . ~ ' .
" ' ' ' '' ~
' "' ~' ~ "'' '
-~ 2 ~ 2
TABLE I - PATIENT, CONTROL, AND ALLOGRAFT DETAILS
Monoclonal Albumin
Antlbody Perfused
_Perfused
No of patients analysed 39 38
Maan age, 52(12) yrs 47(14) yrs
Mean (~) peak panel
reactivity 12(22) 8~13)
Mean no of I ILA class
mismatches 2~5(1.0) 2.8(0.7)
Mean donor age, 36(18j yrs 38 (10) yrs
Mean cold ischaemia 16(7) hrs 17 ~10) hrs
tlme
Mean follow-up after
transplant, 22~9) mo 20(8) mo
Standard deviation in parentheses
TABLE II - ALLOGRAFT SURVIVAL
_ Allo~raft survival (~)
6mo 12mo 18mo
Monoclonal antibody
perfused grafts34t87) 33(87) 25(79)
Albumin perfused grafts 34(~37) 32(87) 26(77)
TABLE III-ALLOGRAFI' FUNCTION
_, . _ .
Mean pla~ ;ma creat: nlne ~mn L/l (SD3
3mo 6mo 12mo 18mo
Monoclonal antibody _ _
pQrfused grafts 174(643 171(64) 155(42) 151(37)
Albumin perfused
grafts 221(115-~ 196(95) 188(783 185(81)
: .
2 ~ 2
~4
WOUND DRESSINGS
In burn patien~s, the burn needs to be dressed while the
patient's epithellal cells are being expanded in in vitro
culture. Allogeneic skin taken from a dead individual,
usually a donor of kidneys for transplantation, is a ve~y
good dressing, but it wlll be rejected by the patient's
own immune system a~ it will be seen as foreign.
Obviously the more frequently that the patient requires
surgery to redress the burnt areas, ths more stress and
tra~ma the pa~lent suffers. The present applica~ion
provid s for the use of anti-CD45 antlbodies to reduce
the ant~genicity of the allogeneic dressing or graft and
to provide a novel dres~ing material which can be
tolerated by the patient for a longer period of time.
The technique can be applied to other skin
transplant/skin dressing situations. For e~ample, in
preparing leg ulcers for final grafting with expanded
syngeneic skin culturesO ::
In relation to the treatment of burns and leg ulcers
there are two main uses for skin. The first is to
provide a wound dressing whlch remains in place, prevents
water-loss, and reduces the risks of infection. The
second appllcation i~ to replace the ne~d for the
patient's skin to grow back over the wound.
Currently, there are several different types of skin
available as a dressing: autologous, ~llogenic (also
known as homograft in this technical field) and ~enogenic
(usually pig). The skin can be prepared in different
ways: either used directly, s~ored at 4 for up to 7
days, or frozen in glycerol; cultured to produce a shset
of growing epithelial cells bu~ probably lacking dermal
cells; and finally as a free~e-dried strip prepara~ion
which is used exclusively with plg skin.
.
There are also a number of artificial skin preparations
whlch are non-viable and really represent a biological
dres-~ing rather than skin graft.
Allo and xenografts are simply dressings. In contrast,
a~ autograft acts hoth as a dressing and as potentially
new skin for the recipient if the graft takes. With
severely burnt patients and wlth the elderlyg lt is often
difficult to obtain sufficient material as a split skin
graf~ from the patient themsalf to cover both of these
requirements. This is particularly true with extensive
burns and with ulcers which require dressing in
preparation for grating. It is quite common for
patlents to unders~o repeated dressing~ with allc~grafts
from different donors while the appropriate heallng
process takes place and the autograft ~s prepar~d. With
leg ulcers it may be useful to dress the ulcer wi~h
allograft skin prior to an attempted autograft
treatment.In these circumstance~; to prolong the survlval
of an allograft would have considerable advantage.
Previous attempts to achieve this have involved topical
application of s-terolds and UV irradiation of the
allograft.
~ he present applicants teach that the problem of
re~ection of non-patient derived skin as a wound dressing
or as a ~raft, can be overcome by pretreating the non-
patient derived skin with the antibody systems as
previously described in the specification.
Skin grafts may be taken from ~onors according to
techniques well known in the art and less than 24 hours
after death. Strict aseptic procedure must be observed.
Generally, the grafts are wrapped in a saline soaked swab
and placed in a contai~er for transport to eg. a skin
culture laboratory. There the skin may be treated with
15~ (v/v) glycerol in an isotonic solu~ion eg. 0.9% NaCl
3 ~ ~
26
or a tissue culture medium such as DMEM (Dulbecco's
Modified Eagles Medium) , sealed in polythene bags and
frozen under controlled conditions. The aim of the
freezing process is to control the rate of cooling to one
degree per mlnute, down to below 30c~ The frozen skin
packs are then stored until use in a -70a freezer.
When required for use, the skin iQ removed from the
freezer and plunged into a water bath at 37c, where it
thaws rapidly. ~he bag can then be opened and the skin
used in the desired way. Once thawed th~ skin cannot bs
refrozen.
The~e techniques are well known in the art and
reported throughout the literature (Cochrane, T. (1968)
The 1DW temperature storage of skln: a preliminary
report. ~rit. J. Plast. ~urg. 21, 118-125; May, 5.12~
(1980) Human skln cryopreservation m~thodoloyies.
Cryobiology. 17, 616: Kearney, J~N: Wheldon, L.A and
Gowland G., (1990) Effect of cryobiological variables on
the survival of skin using a defined murine model.
Cryobiology 27, 164-170; May S.R and Declement F.A.
(1980) Skln banking methodology:An evaluation of package
format, cooling and warming rates and storage efficiency.
CrYGb1010g~ 17, 33-45.)
The present applicants provide that at some stage in
the preparation of the skin for use as a dressing or
graft, it i~ soaked in an antibody system as provided by
the present invention. The soaking may be carried out
either before freezing the skin for storage or after
defrosting the skin.. The soaking may be carried out for
a period o time which allows the binding of the anti-
CD45 antibodies to cells ~eg. passenger leucocytes)
expressins the CD45 antigen. Suitably tha soaking may be
for 1 to 24 hours.
