CA2069342A1 - Materials and methods for the treatment of foreign tissue - Google Patents

Abstract

Materials and Methods for the Treatment of Foreign Tissue Abstract The specification discloses materials and methods for the treatment of foreign tissue which utilise a preparation which comprises an anti-CD45 specific antibody system which mediates complement-dependent cell lysis in the presence of human complement.

Description

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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 :
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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 .: ;, ::

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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 .,;: .
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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 , ~ ~

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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 ,:
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` - 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 , ;, :. .

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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.

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.

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3 ~ 2 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 , ". '~
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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.

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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~

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 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 . ~ . .

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"; 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.

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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.

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' "' ~' ~ "'' ' -~ 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
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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 ~ ~

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.

Claims (14)

1. A preparation for the treatment of foreign tissue which comprises an anti-CD45 specific antibody system which mediate complement-dependent cell lysis in the presence of human complement.

20 A preparation according to claim 1 wherein the antibody system comprises polyclonal antiserum.

3. A preparation according to claim 1 wherein the antibody system comprises two monoclonal antibodies which act synergistically in mediating complement-dependent cell lysis.

4. A preparation according to claim 3 wherein the synergistic monoclonal antibodies are against different epitopes selected from the P, Q, R, S and T epitopes of CD45.

5. A preparation according to claim 4 wherein the synergistic pair of monoclonal antibodies is selected from the group consisting of:
anti-P: anti-Q
anti-P: anti-R
anti-P: anti-S

anti-Q: anti-R
anti-Q: anti-S
anti-Q: anti-T
anti-R: anti-S

6. A preparation according to claim 5 wherein the synergistic pair of monoclonal antibodies is anti-P and anti-Q.

7. A preparation according to claim 1 wherein the antibodies are rat monoclonal antibodies.

8. A preparation according to claim 7 wherein the rat monoclonal antibodies are of isotype IgG2b.

9. A preparation according to claim 8 wherein the antibodies are YTH 54.12 and YTH 24.5.

10. A method of preparing foreign tissue ex-vivo prior to transplantation or use as a dressing material, which comprises treating the tissue with a preparation of any one of claims 1 to 9.

11. A method according to claim 10 wherein the foreign tissue is skin and said skin is soaked in said preparation.

12. A method according to claim 10 wherein the foreign tissue is an organ and said organ is perfused by said preparation.

13. A method of treating a patient which comprises transplanting or dressing said patient with foreign tissue which has been treated with a preparation of any one of claims 1 to 9.

14. In a method of performing an allograft or xenograft organs transplantation to a human subject or providing said human subject with an allogeneic or xenogeneic dressing, the improvement which comprises treating the organ or dressing with a preparation of any one of claims 1 to 9 prior to transplantation.