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: Materials and Methods for the Treatment of Foreign Tissue BACKGROUND OF INVENTION The present invention relates to the problem of rejection of foreign tissue. In particular, it provides materials and methods for the amelioration of rejection of foreign tissue. Transplantation of organs and tissues between identical twins can be successfully achieved and does not lead to graft rejection. 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 recognises the graft or dressing as foreign, and mounts an immune response against the foreign tissue. This response depends upon genetically determined antigenic differences between the donor and the recipient and rejection can take place in at least two ways. The first involves antibody and complement and has been referred to as hyperacute graft rejection, while the second involves activated T-cells and is referred to as acute graft rejection. For this reason, a donor organ for transplantation is selected, which closely matches both the tissue and blood types of the recipient patient. Therefore, before the transplant operation, blood and tissue samples of both the patient and donor will be matched as closely as possible with respect to at least the ABO and Rh blood group systems and the MHC tissue type system. In some cases the rejection of the donor organ .will occur very quickly- (within minutes and up to 48 hours). This phenomenon is known as hyperacute graft rejection and it seems to occur when the recipient patient is pre- sensitized against the donors blood and/or tissue types. Such pre-sensitization of the recipient patient may occur spontaneously, or by virtue of previous grafts, blood transfusions or pregnancy. In such cases of pre- sensitization, the recipient patient will usually have pre-formed circulating antibodies specifically directed against the blood and/or tissue type of the donor. The pre-existing antibodies will then bind to the foreign cells transported into the recipient patient by the donor organ, and this cell-antibody binding will then stimulate complement mediated cytotoxici'ty or antibody-dependent cell-mediated cytotoxicity (ADCC). In complement mediated cytotoxicity, there will be fixation of the first protein component (Cl via Clq) of 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 activation of C3 causes platelet aggregation, accumulation of neutrophils, and the formation of an inflammatory exudate. As a result of this, bystander tissue cells can be damaged even though they are not the immediate tar-gets of the antibody recognition. In antibody-dependent cell-mediated cytotoxicity, the antibody coated foreign cells are recognized by cytotoxic cells (e.g., monocytes and polymorphonuclear cells) having Fc receptors. Binding will occur between the foreign cells and cytotoxic cells via the antibody- Fc receptor bridge and lysis of the foreign cells will follow. Thus, when the donor and patient recipient are matched for blood and tissue groups, tests will be carried out to check that serum from the patient recipient will not attack lymphocytes of the donor, in the presence of complement. Any such attack by the patients serum would be taken as a strong contra¬ indication against the success of the proposed graft. Even where the recipient patient has not been pre- sensitized against the donors blood and tissue types, acute rejection 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, affecting the whole immune system, making the patient vulnerable to bacterial and viral infection. Therefore, it is desirable to have a way of controlling the immune response to defined antigens only, in this case those antigens expressed by the donors cells and which are seen as foreign by the recipient patient. DESCRIPTION OF PRIOR ART Studies have been carried out in rodent models and in the human, to see if certain cells of the donor organ itself, or cells incidentally carried by the donor organ, are particularly effective in stimulating an immune response in the recipient receiving the donor organ (Hart D.N.J. , et al: Transplantation Vol 33, No.3:319, 1982 and Hart et al: Transplantation Vol 31, No.3:428, 1981). Dendritic cells (passenger leucocytes) in "the interstitium of both rat and human kidneys were identified as being intensely positive for the major histocompatibility complex (MHC) class II antigen. Studies have also shown that the survival of rat kidney allografts can be greatly 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, radiation and hydrocortisone therapy. The study showed that even small numbers of interstitial dendritic cells act as a potent stimulus to graft rejection (McKenzie J.L., et al. , Transplantation Proceedings, Vol. XVI No.4 (August), 1984). However, in these studies there were difficulties in the eradication of the dendritic cells 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 suggested however, that even if human donor kidneys were depleted of dendritic cells, the presence of the HLA-DR antigen on human endothelial cells may still cause rejection. Subsequent experiments have also shown that repletion of these dendritic endothelial cells restores their immunogenicity (Lechler R.J., et al. J. Exp. Med. 1982; 155:31-40). In other laboratory animals, heart (Sone Y. , et al. Transplant. Proc. 1987; XIX: 599-604), pancreas (Lloyd D.M., et al. Transplant Proc. 1989; 21: 482-83) and kidneys (Otsubo et al. Transplant. Proc. 1983; 15: 797- 799) grafts can be made to survive longer by perfusion with antibodies against class II MHC antigens which inactivate or destroy these cells. The human kidney endothelium expresses MHC class II antigens, so that although the dendritic cells are strongly MHC II positive, monoclonal antibodies against these antigens cannot be used in the same way for human renal transplantation to destroy the interstitial dendritic cells, since the endothelial cells of the graft are likely to be damaged and as a consequence, the graft deprived of its blood supply and lost. Other investigations have looked at the functioning of heterologous antibody as an immunosuppressive agent. In an effect known as "passive enhancement", recipient rats pre-treated by injections of recipient anti-donor spleen serum, showed increased survival times of the kidney allograft. The effect is thought to be due to the opsonisation of passenger dendritic cells, which thereby removes part of the immunogenic stimulus for the recipients rejection response: (Hart, D.N.J., et al., Transplantation Vol.33 No.3, 1982). This 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 distinguished from kidney endothelial cells by their expression of leucocyte common antigen, (LC or LCA, also known as CD45 and T200) studies have been carried out in which human kidney allografts were perfused with anti-LC monoclonal antibodies prior to transplantation, in an effort to inactivate or destroy the dendritic cells. Two groups of patients received kidneys, perfused respectively with a mouse anti-LC of isotype IgG2a and a mouse anti-LC of isotype IgG3 (in the paper, the authors incorrectly stated the antibody as being a rat anti-LC of isotype IgG3). The former antibody is non-lytic via the route of complement fixation and lytic via the route of ADCC, whilst the latter is not lytic via either route. Lysis via ADCC depends on various host leucocytes moving into the kidney to exert 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 reducing the incidence of graft rejection and improving renal function as compared to non-perfused controls. The mouse IgG3 anti-LC appeared to have no such effect. (Taube D., et al. Transplantation Proceedings 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 more effectively inactivated or destroyed by use of an antibody which could also mediate complement-dependent lysis of the cells. However, there was widespread reluctance to use such an antibody in this technical field. The desirable effects of achieving comnplement- dependent lysis may be offset by the damaging effects triggered through the extensive activation of C3 and the induction of a local inflammatory response and bystander lysis. This in turn might lead to kidney damage and failure of the graft. Indeed this fear was confirmed when a pair of monoclonal antibodies, that in combination mediated lysis via both the complement and antibody-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 transplanted kidney failed and the response in this field of art was that use of perfusion antibodies which used the complement-mediated lytic pathway were contraindicated. The value of pairs of antibodies in achieving synergistic lysis has been investigated. In one study (Bindon C.I., et al. Transplantation Vol. 40, No. 5 1985) combinations of antibodies recognizing different epitopes of the LC antigen were investigated in an attempt to improve the lytic potency of the antibodies. The study showed that certain pairs of rat antibodies of isotype 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 practical disclosure of such use. Another report (Waldmann H. et al. Transplantation Proc. Vol XX No. 6 Suppl. 8, 1988: p.46-51) discusses the use of anti-CD4 rat monoclonal antibodies of isotype IgG2b, injected into mice to cause T cell depletion and immunosuppression. The effect could be improved by using synergistic pairs of antibodies to non-overlapping epitopes of the CD4 molecules. However, other antigens such as the CD45 as expressed on the interstitial dendritic cells were considered as poorly lytic with human complement whatever the 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. The importance of the antigen specificity was also considered in Bindon C.I., et al. Eur. J. Immunol. 1988 18: 1507-1514. This study showed that of many hundreds of rat monoclonal antibodies, only a few (<5%) were lytic with human complement and that some antigens are better targets 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 CD45: P, Q, R, S and T (Hale, G., et al. in Leucocyte Typing III. Edited by A.J. McMichael. Oxford University Press). Synergistic lysis was found between pairs of antibodies against two different epitopes selected from P, Q, R or 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 antibody would interfere with the lytic activity of anti-P: anti-R and anti-Q: anti-R synergistic pairs. In a study related to the present invention (Brewer Y. , et al. Transplantation Proceedings. Vol. 21. No. 1 (February) 1989: pp. 1772-1773), patients received kidney allografts perfused with: (i) a pair of rat IgG2b anti-LC monoclonal antibodies which when mixed together are complement fixing and lytic, or (ii) a mouse IgG2a complement-fixing, non-lytic anti-LC monoclonal antibody. The results 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 terms of allograft rejection and function were not distinguished according to the type of perfusion antibody. The present inventors have now produced as an embodiment of the present invention, a monoclonal antibody system which mediates cell lysis by both the complement and antibody-dependent cell-mediated pathways, but which has no effect in the stimulation of 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 foreign tissue which comprises an anti- CD45 specific antibody system which mediates complement- dependent cell lysis in the presence of human complement. 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. Typically, the foreign tissue may be xenogeneic or allogeneic. The antibody ' system may comprise a single antibody having the necessary specificity or polyclonal antiserum which provides antibodies having the required specificity. Alternatively, the antibody system may comprise two or more monoclonal antibodies, which most preferably act synergistically in effecting complement- mediated cell lysis, but may not mediate complement- dependent cell lysis separately. The synergistic monoclonal antibodies may be against different epitopes selected from the P, Q, R, S or T epitopes of CD45. Thus synergistic pairs of monoclonal antibodies may be of any of the following combinations: 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, Particularly the synergistic pair may be anti-P and anti-Q. The antibodies may be rat monoclonal antibodies, particularly of isotype IgG2b. The present invention also provides 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 as described. The foreign tissue may be xenogeneic or allogeneic. The foreign tissue may be skin, in which case the skin is soaked in said preparation. Alternatively, the foreign tissue may be an organ such as the kidney, in which case the organ may be perfused with said preparation. The method and preparations as provided by the invention are applicable to any foreign tissue where there is a need to reduce the immunogenic effect 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 treated with a preparation as described. The patient may be suffering from conditions related to the defective or inefficient operation of an organ such as the kidney. Alternatively, the patient may be suffering from a skin condition such as burns, leg ulcers or the like. DESCRIPTION OF EMBODIMENTS In order that the present invention is more clearly understood embodiments will be described in more detail. Preparation of Monoclonal Antibodies Sources of cells used. Human tonsils removed in routine tonsillectomies were used. Humans peripheral blood was obtained from laboratory volunteers and defibrinated by shaking with glass beads. Cell-handling medium Unless otherwi.se stated, cells were suspended and washed in Iscove's modified Dulbecco's medium buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) and containing 1% bovine serum albumin (BSA). This will be referred to simply as medi m. 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 connective tissue. The cells were washed twice in lOmM tris HC1 pH 7.3 containing 0.15 M NaCl, and resuspended in the same buffer at 5xl0°/ml_. Cell membranes were prepared by lysis with Tween 40 (Standring R., et al. Biochim. Biophys Acta 1978; 50-85) and stored at -70°C 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, Kidlington, Oxford, England, 0X5 1JE) is coupled to CNBr- activated Sepharose (Pharmacia) at a ratio of 4mg antibody to 1ml of gel (packed volume) according to the manufacture's instructions. Cell membranes (approximately lO 1 '- * cell equivalents) were resuspended in lysis buffer PBS containing 0.5% Nonidet P40, 0.1% SDS, and ImM phenyl methyl sulphonyl fluoride) and mixed with 2ml of antibody coupled Sepharose. The mixture was rotated for 2hr at 4 C C and then placed in a column. Unbound 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 suitable elution buffers. Two column volumes were collected and dialyzed overnight against PBS containing 0.02% sodium azide. The purified antigen was stored at -20°C (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 10° human peripheral mononuclear cells enriched for T cells. One month and . 2 months later, it received similar cells intravenously. Three days after the final dose, the rat spleen cells were fused with the rat myeloma line Y3 Ag 1.2.3. using polyethylenglycol. (Galfre G. , et al. Nature 277:131, 1979). The fusion was labelled YTH. Hybrid cells were grown in selective medium and the hybrid myelomas were cloned and recloned on soft agar. To obtain antibody, cells were grown to stationary phase in Iscove's modified Dulbecco's medium containing 1% fetal cell serum, or as ascitic tumours in (DA X LOU) Fl rats. Immunoglobin fractions were isolated from the ascitic fluids by precipitation with (NH)2Sθ and were redissolved in phosphate-buffered saline (PBS) containing 0.02% sodium azide for storage at 4°C or -30°C. Binding to fixed cells or to purified LC antigen was carried out using a solid-phase enzyme-linked binding assay (Cobbold S.P. et al 1981 J. Immunol. Methods 44: 125). The test monoclonal antibody was incubated in microtiter plates containing adsorbed purified antigen (lO^-lO^ cell equivalents per well) or fixed cells, followed by a detection reagent of monoclonal mouse anti(rat lg) coupled to β-galactosidase. Finally the enzyme substrate was added and a colour change measured in a spectrophotometer. Isotyping of Monoclonal Antibodies Hybrid myeloma cells were grown in (U. 14 C) lysine, and samples of the culture supernatant were electrophoresed on dodecylsulfate-polyacrylamide gels to determine the sizes of heavy and light chains. (Galfre et al. 1981 Methods Enzymol 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, Oxford U.K., p.122). The central well contained 10 μl of subclass-specific antiserum, and each outer well contained 10 μl of -'-'^C-labelled culture supernatant plus sufficient carrier rat lg previously titrated to give optimal precipitation. When the precipitin 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 subclass of that antibody. PERFUSION OF ALLOGENEIC DONOR KIDNEYS PRIOR TO TRANSPLANTATION Patients and Controls 77 patients were entered into the trial. 39 patients received allografts perfused with monoclonal antibodies and 38 patients received allografts perfused with a similar volume of human albumin solution. All patients were recipients of first cadaver allografts. The kidneys were maintained in Marshalls solution supplied by Baxter Healthcare Ltd, Thorpe Lea Road, Egham, Surrey, TW20 8HY. Another suitable organ preservation solutions is UW solutions of DuPont (UK) Ltd, Pharmaceuticals Division, Wedgwood Way, Stevenage, Herts. SGI 4QN. Others are known in the art. Monoclonal Antibodies and Perfusion Technique The renal allografts were perfused with a mixture of the anti-CD45 monoclonal antibodies YTH 54.12 and YTH 24.5 which had been purified from ascites fluid using HPLC chromatography according to standard techniques. They are complement-fixing rat monoclonal antibodies of isotype lgG2b which together are lymphocytotoxic in the presence of human complement (Bindon, et al. Transplantation 1985; 40: 538-546). Vials containing 2mg of each antibody in 2ml phosphate buffered saline were prepared. A solution of human albumin was used as a control because it looks superficially the same as the antibody solution and was stored in identical vials. The vials were marked with a code. The relation of the code to the contents was not known to the clinical team until the end of the trial. Thus pretreatment of the graft with the monoclonal antibody pair or albumin was random. A modification of a previously described technique was used (Taube D., et al. Transplant Proc. 1987; XIX: 1961-1963). The allograft was prepared for transplantation by the standard method. The renal veins were clamped. The contents of the vial (in 50ml Marshall's solution at 4°C) were injected into the renal arteries. About 20 minutes later, when the arteries and veins had been anastomosed, the clamps were removed and the transplant operation was completed in the usual way. Biopsy specimens were taken from the majority 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 of CD45 the monoclonal antibodies. 5 μm frozen sections were pretreated with avidin/biotin (Vector Labs Ltd, 16 Wulfric Square, Bretton, Peterborough, Cambs. ) before incubation with biotinylated anti-rat IgG (Vector), peroxidase labelled strep-avidin biotin complex (strep-ABC, Dako Ltd., 16 Manor Courtyard, Hughenden Ave, High Wycombe, Bucks. HP13 5RE), and diaminobenzidine. Then they were counterstained with haematoxylin. To count the total number of dendritic cells, sequential sections were similarly processed having been preincubated with YTH 54.12 and YTH 24.5. Sections treated with strep-ABC 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 anti-rat lg alone, was expressed as a percentage of the number stained with added CD45 antibodies. This percentage is a measure of the effectiveness of perfusion. Immunosupression and Post-operative Management All patients were immunosupressed with cyclosporin (Cs) and prednisolone. 8mg/kg of Cs was given orally before transplantation, followed by 16mg/kg per day, in 2 divided 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 radioimmunoassay (Sandoz). Patients received 20mg/day of prednisolone for the first month, 15mg/day for the second month and 10 mg/day subsequently. Patients with peak panel reactive antibodies >50% were treated with anti- thymocyte globulin (2mg/kg per day) for 10 days as previously described (Palmer A., et al. Transplant Proc. 1988; 20: 198-200). Allograft rejection was diagnosed by biopsy and managed as previously described, (Taube D.H., et al. Lancet 1985; ii: 171-174). Cyclosporin nephrotoxicity was diagnosed when the biopsy specimen showed no evidence of rejection, Cs levels were high, and allograft function improved with reduction in Cs dose or change to azathioprine. Statistical Analysis Student's test and the X- *** " test were used. Results The two groups were essentially similar (Table I). No side-effects attributable to monoclonal antibody perfusion were observed. Actuarial patient survival in the two groups was similar. Patent survival was 97% and 92% at 1 year and 18 months post-transplant, respectively, in the group of patients receiving monoclonal antibody perfused 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 months post-transplant and 1 from carcinomatosis 18 months post-transplant). Both patients had functioning allografts. 2 control patients died, both at 1 month post-transplant (1 from cytomegalovirus pneumonitis and 1 from systemic candidiasis) . Neither of their allografts was functioning. Allograft survival was the same in both groups (Table II). 5 patients (3 with monoclonal antibody perfused allografts and 2 controls) lost their grafts within 48 hours of transplantation for technical reasons; 1 patient had uncontrollable bleeding at the time of surgery; 3 patients lost allografts (2 monoclonal antibody perfused and 1 control) as a result of primary renal arterial or venous thromboses; 1 patient had an ischaemic leg after surgery, the graft was abandoned and later removed. The 3 monoclonal antibody perfused graft were lost beyond 48 h, none as a result of rejection, one from irreversible renal artery stenosis, a second graft was abandoned because the patient developed Pneumocystis carinii pneumonia and immunosuppression stopped, a third graft was lost due to probable chronic Cs nephrotoxicity. Four control patients lost grafts, two as a result of irreversible rejection, and two from probable chronic Cs nephrotoxicity. Allograft function, measured as plasma creatinine, was slightly better in the group of patients with monoclonal antibody perfused allografts (Table III), but of statistical significance only at 3 months and 1 year post 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 12 had more than one rejection crisis. Biopsy specimens were analysed for CD45 monoclonal antibody uptake. The results suggest that rejection may be associated with a low uptake of the antibody. However the numbers are too small for statistical analysis. In this trial, pretreatment of human renal allografts with rat CD45 monoclonal antibodies reduced the incidence of rejection and possibly improved allograft function. Pre-treatment of human kidney allografts with small amounts of CD45 monoclonal antibodies does not appear to be harmful to either the allograft or the patient. Although 5 allografts (3 perfused with monoclonal antibodies and 2 controls) were lost as a result of complications at the time of operation, there is no evidence to implicate monoclonal antibody perfusion as a cause of graft loss. Allograft function (Table II) may be improved. The pre-treatment of human renal allografts with CD45 monoclonal antibodies will not make them non- immunogenic, even if all the passenger leucocytes are inactivated or destroyed. As previously mentioned, the human renal vascular endothelium and renal tubular epithelium express MHC antigens. (Hart, D.N.J., et al. Transplantation 1981; 31 428-33). This perfusion technique is simple, safe and cheap and may be applicable to other organs such as the heart, liver and pancreas. TABLE I - PATIENT, CONTROL, AND ALLOGRAFT DETAILS Standard deviation in parentheses TABLE II - ALLOGRAFT SURVIVAL TABLE III-ALLOGRAFT FUNCTION Mean plasma creat: nine μmni/1 (SD) 3mo 6mo 12mo 18mo Monoclonal antibody perfused grafts 174(64) 171(64) 155(42) 151(37) Albumin perfused grafts 221(115) 196(95) 188(78) 185(81) WOUND DRESSINGS In burn patients, the burn needs to be dressed while the patient's epithelial cells are being expanded in in vitro culture. Allogeneic skin taken from a dead individual, usually a donor of kidneys for transplantation, is a very good dressing, but it will be rejected by the patient's own immune system as it will be seen as foreign. Obviously the more frequently that the patient requires surgery to redress the burnt areas, the more stress and trauma the patient suffers. The present application provides for the use of anti-CD45 antibodies to reduce the antigenicity of the allogeneic dressing or graft and to provide a novel dressing 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 example, in preparing leg ulcers for final grafting with expanded syngeneic skin cultures. 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 which remains in place, prevents water-loss, and reduces the risks of infection. The second application is to replace the need for the patient's skin to grow back over the wound. Currently, there are several different types of skin available as a dressing: autologoμs, allogenic (also known as homograft in this technical field) and xenogenic (usually pig). The skin can be prepared in different ways: either used directly, stored at 4° for up to 7 days, or frozen in glycerol; cultured to produce a sheet of growing epithelial cells but probably lacking dermal cells; and finally as a freeze-dried strip preparation which is used exclusively with pig skin. There are also a number of artificial skin preparations which are non-viable and really represent a biological dressing rather than skin graft. Allo and xenografts are simply dressings. In contrast, an autograft acts both as a dressing and as potentially new skin for the recipient if the graft takes. With severely burnt patients and with the elderly, it is often difficult to obtain sufficient material as a split skin graft from the patient themself to cover both of these requirements. This is particularly true with extensive burns and with ulcers which require dressing in preparation for grafting. It is quite common for patients to undergo repeated dressings with allografts from different donors while the appropriate healing process takes place and the autograft is prepared. With leg ulcers it may be useful to dress the ulcer with allograft skin prior to an attempted autograft treatment.In these circumstances to prolong the survival of an allograft would have considerable advantage. Previous attempts to achieve this have involved topical application of steroids and UV irradiation of the allograft. The present applicants teach that the problem of rejection of non-patient derived skin as a wound dressing or as a graft, 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 donors 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 container for transport to eg. a skin culture laboratory. There the skin may be treated with 15% (v/v) glycerol in an isotonic solution eg. 0.9% NaCl 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 minute, down to below -30°c. The frozen skin packs are then stored until use in a -70°c freezer. When required for use, the skin is removed from the freezer and plunged into a water bath at 37°c, where it thaws rapidly. The bag can then be opened and. the skin used in the desired way. Once thawed the skin cannot be refrozen. These techniques are well known in the art and reported throughout the literature (Cochrane, T. (1968) The low temperature storage of skin: a preliminary report. Brit. J. Plast. Surg. 21 . , 118-125; May, 5.12. (1980) Human skin cryopreservation methodologies. 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 2^7, 164-170; May S.R and Declement F.A. (1980) Skin banking methodology:An evaluation of package format, cooling and warming rates and storage efficiency. Cryobiology V7_, 33-45.) The present applicants provide that at some stage in the preparation of the skin for use as a dressing or graft, it is 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 of time which allows the binding of the anti- CD45 antibodies to cells (eg. passenger leucocytes) expressing the CD45 antigen. Suitably the soaking may be for 1 to 24 hours.

Claims: CLAIMS 1. A preparation for the treatment of foreign tissue which comprises an anti-CD45 specific antibody system which mediates complement-dependent cell lysis in the presence of human complement. 2. 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.

Inventors: HALE GEOFFREY, TAUBE DAVID HIRSCH, THICK MICHAEL GORDON, WALDMANN HERMAN, WELSH KENNETH IAN, HALE, GEOFFREY, TAUBE, DAVID, HIRSCH, THICK, MICHAEL, GORDON, WALDMANN, HERMAN, WELSH, KENNETH, IAN
Applicants: LYNXVALE LTD, LYNXVALE LTD.
Application year: 1990
Main classification: A61K39
IPC codes: A01N1, A61K38, A61K39, C07K16