JPH05501256A - Materials and methods for processing foreign tissue - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] and methods for cleaning up Light plate ■Background The present invention relates to the problem of foreign tissue rejection. In particular, the present invention Materials and methods for mitigation of absolute reactions are provided.

Transplantation of organs and tissues between identical twins can be successfully accomplished and does not lead to graft rejection. allogeneic (aI iogenic) tissue and When performing organ transplants, or using wound dressings (drapers) on the skin of allogeneic cadavers. When used as a graft or dressing, the recipient's immune system recognize the tissue as foreign and mount an immune response against the foreign tissue. This response The answer depends on genetically determined antigenic differences between donor and recipient, and Rejection is minimal (can occur in two ways; the first is antibodies and complement). is involved and has been called hyperacute graft rejection; associated T cells and is called acute graft rejection.

For this reason, a donor that closely matches both the tissue type and blood type of the recipient patient. organs are selected for transplantation. Therefore, prior to the transplant operation, patient and Blood and tissue samples from both donors should be of at least ABO and Rh blood type. There will be as close a match as possible in terms of system and MHC tissue type system.

In some cases, rejection of donor organs will occur very rapidly (number within minutes and up to 48 hours). This phenomenon is known as hyperacute graft rejection. and the recipient patient has prior sensitization to the donor's blood and/or tissue type. This seems to happen when the machine is created. Such presensitization of receptor patients is spontaneous or may occur due to a previous graft, blood transfusion, or pregnancy. Ru. In such cases of sensitization, the recipient patient receives donor blood and/or usually have preformed circulating antibodies directed specifically against the tissue type. will. The pre-existing antibodies are then transferred to the recipient patient by the donor organ. binds to transported foreign cells, and this cell-antibody binding then results in complement-mediated cytotoxicity or or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC).

Leads to cleavage of C3 and activation of the lytic pathway of the complement system in complement-mediated cytotoxicity and the first protein component of the complement cascade (C1) that leads to cytolysis of foreign cells. There is a fixation of C1) via g. Furthermore, C3 activation is associated with platelet aggregation, neutrophils, and neutrophils. Causes the accumulation of bulbs and the formation of inflammatory exudates. As a result, the pie stander ( (bys tander) Synthetic cells are not direct targets of antibody recognition, but may be damaged. Sometimes it happens.

In antibody-dependent cell-mediated cytotoxicity, foreign cells coated with antibodies receive Tc receptors. recognized by cytotoxic cells (e.g. monocytes and polymorphonuclear cells) with Ru. Binding between foreign cells and cytotoxic cells via antibody-Fc receptor crosslinking will occur, followed by lysis of the foreign cells.

Thus, when the donor and patient recipient are matched for blood and tissue type , to ensure that serum from patient recipients does not attack donor lymphocytes in the presence of complement. Tests will be conducted to examine the Such an attack with the patient's serum would be understood as a strong contraindication to the success of the proposed transplant.

If the recipient patient has not been previously sensitized to the donor's blood and tissue type Even acute rejection episodes are common, and usually activated T Cell-mediated. After all, a patient with a donor organ, for example, a kidney, is immune Treated with inhibitors such as prednisolone and cyclosporine. However However, these immunosuppressive drugs are non-specific, affect the entire immune system, and may making them susceptible to bacterial and viral infections. Therefore, antigen, in this case expressed by the donor's cells and foreign and foreign by the recipient patient. It would be desirable to have a way to control the immune response only to the antigens that are seen. Delicious.

Sakioka Sumitora qk place In rodent models and humans, certain cells of the donor organ itself or cells that the donor's organ happens to have in the receptor that receives the donor's organ. Studies have been conducted to see if it is effective in stimulating the immune response (Hart D, N, J, et al.: Transuplation V ol, 33. No. 3:319, 1982 and Hart et al.: Transup lantation Vol, 31. NO, 3:428°1981). rat o Dendritic cells (parasenger leukocytes) in the interstitial tissue of the kidneys of both humans and humans. is strongly positive for major histocompatibility complex (MHC) class ■ antigens. It was identified as

Additionally, in a study, rat kidney allograft survival was significantly affected by MHCff prior to transplantation. can be greatly enhanced by depleting the dendrite cells of The MHC dendrite cells shown were treated with donor rats by cyclophosphamide, Depletion by treatment with a combination of radiation and hydrocortisone therapy It was done. In this study, even a small number of dendritic cells in the stromal tissue were found in the grafts. It has been shown to act as a potent stimulus against rejection (McKenz ie J, L.

Transuplation Proceedings, Vol, XV I No. 4 (August), 1984). However, in these studies , eradication of dendritic cells is difficult, and therefore, effective proposals by these techniques are limited. Donor preparation may be difficult to apply clinically.

As the authors suggest, prior to transplantation, the donor organ is isolated from dendritic cells or MM. It can be perfused with antibodies against the C antigen. However, the authors further show As suggested, even when human donor kidneys lack dendritic cells, human The presence of HLA-DR antigens on endothelial cells may still cause rejection .

Subsequent studies further demonstrated that the replication of these dendritic endothelial cells was linked to their immune system. (Lecher RJ, et al. J. Exp. Med. 1982;155:3l-40).

In other laboratory animals, the heart (Sorts Y, et al. Transupplant).

Proc, 1987; XIX:599-604), pancreas (Lloyd D, M , et al. Transuprant.

Proc, 1989; 21:482-83) and kidney (Otsubo et al. nsuplant.

Proc, 1983: 15: 797-799> grafts Perfusion with antibodies against class II MHC antigens that inactivate or destroy cells. This allows them to survive longer.

Since the human kidney endothelium expresses MHC class ■ antigens, dendritic cells are strongly HCII positive, but the monoclonal antibodies against these antibodies are from the same person. can be used to destroy dendritic cells in interstitial tissue for human kidney transplants. I can't do it. Because the endothelial cells of the graft appear to be damaged and eventually , because the graft is deprived and loses its blood supply.

Other research has been directed at functionalizing xenoantibodies on immunosuppressants. "Passive Augmentation" In a known effect, pre-treatment by injection of receptor anti-donor spleen serum Recipient rats showed increased renal allograft survival time. This effect is This is thought to be due to the opsonization of Panosenger dendritic cells1. This eliminates part of the immunogenic stimulus for the receptor's rejection response. : (Hart D, Nj, et al.: Transuplation Vol. 33. No. 3.1982).

This study shows that depletion of donor dendrite cells prior to transplantation has the same effect as passive augmentation. This led to the hypothesis that it is possible to do this yourself.

Dendritic cells in the interstitial tissue are similar to endothelial cells in the kidney due to the expression of common antigens on leukocytes. Because they can be distinguished (as LC or LCA, CD45 and T2O0) (known) further studies were carried out. In other words, it inactivates dendritic cells. Human kidney allografts are treated with anti-LC monochromes prior to transplantation in an effort to destroy or destroy human kidney allografts. perfused with local antibodies. Two groups of patients were treated with isotype I Perfusion with mouse anti-LC of gG2a and mouse anti-LC of isotype I gG3. (In this paper, the authors show that the antibody is of the isotype IgG3 (incorrectly stated as rat anti-CL). The former antibody is responsible for complement fixation. non-cytolytic via the ADCC route and cytolytic via the ADCC route. However, the latter is non-cytolytic no matter which route it is taken. Cell lysis via ADCC The solution is that various host leukocytes migrate into the kidney and exert their cytotoxic effects. Depends on. As these results suggest (the number of trials is small and studies are Mouse IgG2a anti-LC (unrandomized and uncontrolled) Reduced incidence of graft rejection and improved renal function compared to non-perfused controls It had a favorable effect. Mouse IgG3 anti-LC has this effect. (Taube D, et al. Transuplation Proceedings.

Vol, XIX No. 1 p1961-19631987).

However, to maximize the effect of undesirable cell inactivation or destruction Antibodies that mediate as many immune response mechanisms as possible should be used.

Therefore, it is theoretically possible to use antibodies that can also mediate complement-dependent cytolysis of foreign cells. With its use, foreign cells can be more effectively inactivated or destroyed. death However, in this technical field, there are no recommendations for using such antibodies. There was widespread resistance. The desired effect of achieving complement-dependent cell lysis is that of C3. through widespread activation and induction of local inflammatory responses and high-stander cell lysis. This will be offset by the damaging effects initiated by This, in turn, causes kidney damage and will lead to graft dysfunction. In fact, this fear is a pair of monochrome null antibodies (which in combination inhibit complement-dependent cell-mediated pathways and antibody-dependent Perfuse the kidney before transplantation with a cell-mediated pathway (which mediates cell lysis through both the cell-mediated pathway) (Cambridge, UK, 1986, results not yet published). all To my bitter disappointment at the problem, the transplanted kidney was destroyed and this technique Responses in the field contraindicate the use of perfused antibodies using the complement-mediated cytolytic pathway. It was said that the

The value of antibody pairs in achieving synergistic cell lysis has been investigated. , one lab Research (Bindon C, 1, et al. Transplantation Vol.

40, No. 51985), in an attempt to improve the cytolytic efficacy of antibodies. Therefore, combinations of antibodies that recognize different epitopes of the LC antigen were investigated. child In a study, certain pairs of rat antibodies of isotype IgG2b Although not cytolytic separately in the presence of human complement, when used together - It was shown that it is decomposable. In addition, in this study, rat IgG2a The presence of antibodies was shown to interfere with the cytolytic ability of the synergistic pair. The authors suggest According to the authors, such synergistic antibody pairs purge organ grafts and remove interstitial tissue. Decreases immunogenicity of tissue pansenger leukocytes by removing them There is no actual disclosure of such use.

Other reports (Waldllann H, et al. Transuprant, TranS upplant, Proc.

Vol, XχNo, 65upp1.8. p, 46-51) is isotype Tg G2b anti-CD4 rat monoclonal antibody was used to inject into mice. Discusses T-cell exhaustion and immunosuppression. This effect is caused by the CD4 molecule By using synergistic pairs of antibodies directed against non-overlapping epitopes of However, other antigens, e.g. CD45 expressed on dendritic cells depends on the isotype of the monoclonal antibody. However, it is less cytolytic than human complement, and therefore antigens are less lytic than human complement. It was considered to be an important variable for achieving the solution. Donor kidneys are treated with antibiotics before transplantation. The authors suggest that they are testing the effect of perfusing with a synergistic pair of CD45 antibodies. Ta.

The importance of antigen specificity is also discussed in Bindon C, 1, et al. Eur, J.

Discussed in In+muno1.198818:1507-1514. child In a study, out of hundreds of rat monoclonal antibodies, only a few ( 5%) are cell lytic together with human complement, and some antigens are soluble in antibodies. better for complement-mediated cell lysis than others independent of isotype shown to be a target. Generally, the LC antigen CD45 is a human complement-mediated cytolytic It was a much poorer antigen for the solution.

Five distinct epitopes are distinguished on CD45: P, Q, R, S and and T (Hale G, et Leucocyte Typing meA, J, McMichael, [, University of Oxford). Synergistic dissolution is P, Q , between a pair of antibodies against two different epitopes selected from R or S. It's been found. However, if the T epitope is involved, the anti-Q:anti-T antibody There is synergistic cell lysis only between the pair, and in addition anti-T antibodies and anti-Q:anti-R synergistic pair.

Research related to the present invention (Brewer Y, et al. n　Pr.

ceed i ngs, Vo 1.21 + No, 1 (Februar y) 1989: p, 1772-1773), (i) - mixed together A pair of rat IgG2b anti-LCs that are complement-fixing and cytolytic when Monoclonal antibody, or (11) mouse IgG2a complement-fixing, non-cytolytic The patient received a kidney allograft perfused with anti-LC monoclonal antibody. result As suggested by Significantly better in two patients. However, groups (i) and (ii) There was no significant difference in antibody uptake between and in allograft rejection and function. The results presented were not differentiated according to the type of perfused antibody.

The inventors herein provide that, in one embodiment of the invention, complement-dependent cell-mediated mediate cell lysis by both the antibody- and antibody-dependent cell-mediated pathways; monoclonal antibody systems that are ineffective in stimulating rejection of donor organs. Manufactured. This monoclonal antibody system is anti-LC (also called anti-CD45). ).

Lord's victory and seal Thus, the present invention provides anti-CD cells that mediate complement-dependent cell lysis in the presence of human complement. A preparation for treating foreign tissue is provided comprising a 45-specific antibody system.

This treatment can be performed ex vivo. Exogenous MWli is not derived from said receptor. It can be any tissue that would be seen by a receptor as a tissue. Noriyoshi Typically, the foreign tissue can be a xenograft or an allograft.

Antibody systems can consist of a single antibody with the required specificity or an antibody with the required specificity. The antiserum may comprise a polyclonal antiserum. Alternatively, antibodies The systems most preferably act synergistically in performing complement-mediated cell lysis, but Two or more models that are unable to mediate complement-dependent cell lysis in each It can comprise a noclonal antibody. Synergistic monoclonal antibodies are CD 45 P, Q, R.

be directed against different epitopes selected from S or T epitopes. I can do it. Thus, a synergistic pair of monoclonal antibodies can be used for any combination of It can be anything you want: 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 In particular, the synergistic pair can be anti-P and anti-Q.

The antibody is a rat monoclonal antibody, especially isotype Ig (1, 2b). can be done.

The present invention also relates to transplantation or dressing comprising treatment with the aforementioned preparations. A method is provided for preparing foreign tissue in vitro prior to use as a modeling material. outside The tissue can be heterogeneous or homogeneous. The foreign tissue may be skin. In this case the skin is immersed in the preparation.

Alternatively, the foreign tissue can be an organ, for example a kidney, in which case perfuses the organ with the preparation. The methods and preparations provided by the invention are , cells expressing leukocyte common antigen (CD45) (e.g., parasenger leukocytes) ) applied to any foreign tissue if necessary to reduce the immunogenic effects of be able to.

The present invention also provides for transplanting foreign tissue treated with the aforementioned preparation into a patient or A method of treating a patient is provided, the method comprising dressing a patient. the patient, Suffering from conditions related to defective or inefficient surgery of organs, e.g. kidneys It may happen. Alternatively, the patient may have a skin condition, such as a burn or leg ulcer. Sometimes it bothers me.

Jitoshi l Maki ■ Escape In order that the present invention may be more clearly understood, the embodiments will be described in more detail. Ru.

Human tonsils removed during routine tonsillectomy were used. Human peripheral blood in the laboratory defibrillation by shaking with glass beads. It was rinsing.

Week 1 "Q" Unless otherwise specified, N-2-hydroxyethylpiperazine-N'-2-ethanesulfate buffered with HEPES and 1% bovine serum albumin (BSA). Contains Iscove modified Dulbecco (Du 1 becco) Cells were suspended in medium and washed therein. This is simply called a medium.

+1 of LC from humans cutting out human tonsils with a scalpel blade and then passing them through a sieve to remove connective tissue Cell suspensions were prepared from tonsils by. Cells containing 0.15M NaCl washed twice in 10o+M Tris-HCI (pH 7,3) and Resuspend in buffer at 5×10”/ml. Cell lysis with Tween 40. To prepare cell membranes by: Standring R, et al. Biochim .

Biophys Acta 1978; 8O-85) and until needed - Stored at 70°C.

For affinity purification, anti-LC monoclosal antibodies (e.g. 1, YTH5 4,12,’! 'TH24,5 and Flo-89-4, all of which are Bankside, Station Approach (Serotec, Bankside, 5tat, ion Approach), Kittlington, Onoxford, UK , available from OX5 1JE) and CNBr-activated Sephas parose) (Phamr+*acia) according to the manufacturer's instructions. Coupling at a ratio of body/1 ml gel (packed volume). Cell membrane (Habo 10"I II cells) and 0.5% Nonidet P 40.0 ．． A solution containing 1% SDS and 11 phenylmethylsulfonyl fluoride. Resuspend in lysis buffer PBSO and add 2 ml of antibody cambred Sepha Mixed with loin. This mixture was rotated for 2 hours at 4”C and then I put it in the room. Unbound material washed with several column volumes of cell lysis buffer and bound antigen in 50 mM diet containing 0.5 M NaCl. Elution was carried out with thylamine (pH 11,5) or other suitable elution buffer. 2 power ram Collect the volume and add to PBS containing 0.02% 1~lium azide. Dialyzed. The purified antigen was stored at -20''C (Bindon C).

r, et al. Transplantation Vol. 40. No, 51985) .

Men - Change - 111 Melting Rei 1s 4Σ Immunization by subcutaneous injection with human peripheral mononuclear cells enriched for T cells of It became. After 1 month and 2M months, similar cells were administered intravenously to the 71-year-olds.

Three days after the last administration, spleen cells of rat I were transferred to rat myeloma line Y3 Ag1. ．． 2.3 and fused using polyethylene glycol (Galfre G, Nature 277:13], 1979). The fusion was labeled with Y''VH.

The hybrid cells are grown in selective media and the hybrid myeloma is removed. I corned and recloned on soft agar.

To obtain antibodies, cells were incubated with Iscoub containing 1% fetal bovine serum. ve) grown to stationary phase in modified Dulbecco medium. or (DAXI, 0U) grown as an ascites tumor in Fl. Ta. By precipitating the immunoglobulin fraction from ascites with (NH)2SO, isolated and in phosphate buffer (PB) containing 0.02% sodium azide. S) and stored at 4°C or -30°C.

Binding to fixed cells or purified LC antigen was determined using a solid-phase enzyme-linked assay. (Cobbold S, P. et al. 1981 J.

fa+n+uno1. Methods 44:125) o Adsorbed and purified microorganisms containing antigen (106-107 cell equivalents/well) or fixed cells. incubating the test monoclonal antibody in a crotiter plate; A monoclonal mouse anti(random) coupled to β-galactosidase was then used. The cells were incubated with a detection reagent for (Ig). Finally, add the enzyme substrate, The color change was then measured using a spectrophotometer.

and culture supernatant samples were electrophoresed on dodecyl sulfate-polyacrylamide gels. The size of the heavy and light chains was determined by electrophoresis (Galfre et al. 1981 Methods Enzymol, 73:3). 2% cold IgG subclass Established in the sky by Ouchterlony's double diffusion method (Johnsotone A. and R. Thorpe 1982. Imm. unochemistry in Practice, BlackwellSc ientific Publication, Oxford, UK, p. 12 2).

The center well contains 10 d of subclass-specific antiserum, and each outer well contains 10 d of subclass-specific antiserum. To determine the optimal precipitate, perform titer assay using 1OIII I4C-labeled culture supernatant sample. Contained 1 g of carrier rats sufficient for feeding. When a line of precipitation occurs The plates were dried and exposed to X-ray film for approximately 5 days.

For each monoclonal antibody, only one antiserum is compatible with that antibody subclass. gave a corresponding radioactive precipitate.

μ order for stick planting [MiU1 publication lΔ(flow) μ person ministry hawk de batan Seventy-seven patients participated in the study. Thirty-nine patients were perfused with monoclonal antibodies. received allografts and 38 patients received a similar volume of human albumin solution. Allografts were perfused with All patients received their first cadaveric allograft It is the body. Kidneys are provided by Baxter Healthcare Limited (Baxter He althcare Ltd,), Thorpe Lea Road, Nigeham, Surrey, of Marshalls solution supplied by TW20 8HY kept inside. Other suitable organ preservation solutions are available from DuPont Pharmacy Ikal Division (Pharmaceuticals Division) , Uejiusodo Way, Stephanage, Half, SGI 4QN UW solution Met. Others are known in the art.

Monoclonal and 2'1' - HPLC chromatography according to standard techniques Anti-CD45 monoclonal antibody YTH54 purified from ascites using , 12 and YT H24,5, the renal allografts were perfused. So These are isotype IgG2b complement fixing monoclonal antibodies. - together with lymphocytotoxicity in the presence of human complement (Bindon et al. transplantation 1985;40:538-546).

Two vials of each antibody in 2ra1 phosphate buffer were prepared. human albumin The sample solution is superficially identical to the antibody solution, so it is used as a control1. and stored in the same vial. Vials were marked with a code. to the contents The clinical team was not informed of the relationship between the codes until the end of the study. . Thus, pretreatment of the graft with monoclonal antibody pairs or albumin It was random. A previously described technique was modified and used (Taube D, TransplantProc, 1987:XIX:1961-1963), Allografts were prepared for transplantation by standard methods. clamp the renal veins Ta. The contents of the vial (in Marshall's solution, 4°C) were injected into the renal artery. Ta. Approximately 20 minutes later, when the artery and vein were anastomosed, the clamp was removed and the transfer Replanting was completed in the usual manner.

Biopsy specimens were taken from the majority of the grafts before the wound closed. one One core is used for routine histopathology, and the other is rinsed in liquid nitrogen. CD45 monoclonal antibodies were applied to allografts using deep frozen samples. Uptake was measured. 5! Pre-treat frozen sections of M. with avidin/biotin. νecter Labs Ltd, 16 Ulfrinok Square, Brelat Petersburg, Cambridgeshire) followed by a biotinylated anti-Rat. IgG (νector), peroxidase-labeled streptavidin Ochin complex (strep-ABC, Dako Ltd, 16 Manor Cote Yard, Hugenden Ahe, High Wycombe, Packinghamshire, HP135RE) and diaminohengin. then , they were counterstained with hematoxylin. To count the total number of dendritic cells pre-incubated with YTH54,l2 and YTH24,5 for Sequential sections were processed similarly. Sections treated with 5trep-ABC alone were used as controls. and used it. After labeling with CD45, dendritic cells adopt their characteristic morphology. Identified it more. A minimum of 15 high force fields were tested. Stained with anti-depressant I-Ig alone The number of dendritic cells that are positive after Expressed as a fraction. This percentage is a measure of the effectiveness of perfusion.

and Tobi' All patients were immunosuppressed with cyclosporine (Cs) and prednisolone.

8■/kg of Cs was administered orally before transplantation, then 16■/kg/day was divided into two. The dose was given for 3 days after surgery. Reduce dose to 10 kg/day for next 4 days a little, then 20 as determined by standard radioimmunoassay (Sandoz). Adjustments were made to maintain whole blood trough levels between 0 and 300 ng/n+1. patient Prednisolone 20 days/day during the first month, 10 days during the second month and subsequently / gave a day. Patients with >50% of the peak panel of reactive antibodies were treated with antithymocyte Globulin (2 kg/day) was treated for 10 days as previously described (Pa lmer A, et al, Transplant Proc, 1988; 20 :198-200).

diagnosing allograft rejection by biopsy as previously described; and managed (Taube D, H, et al. Lancet 1985; ii: 1 71-174). When the biopsy specimen shows no evidence of rejection, Diagnose cyclosporine nephrotoxicity, Cs levels are high, and allograft function improved with decreasing Cs dosage or switching to azathioprine.

Haibunimo content analysis Student's test and X2 test were used.

quince The two groups were essentially similar (Table I). Attributable to monoclonal antibody perfusion No possible side effects were observed.

Patient survival rates in the two groups were similar. patient survival rate in a group given allografts perfused with monoclonal antibodies. At 1 year and 18 months after transplantation, the rates were 97% and 92%, respectively. In controls, the rate was 94% at 1 year and 18 months post-transplant. monoku Two patients who received allografts perfused with local antibodies died (one Intracerebral hemorrhage, 3 months post-transplant and 1 patient, carcinomatosis, 18 months post-transplant). Both patients Had a functioning allograft. Two control patients both died one month after transplantation. (one due to cytomegalovirus pneumonia and one due to candidiasis) ). None of their allografts were functioning.

Allograft survival was the same in both groups (Table ■). 5 patients subjects (3 with monoclonal antibody-perfused allografts and 2 controls) Lost their graft within 48 hours for operative reasons; one patient lost his graft at the time of surgery. Three patients had uncontrollable bleeding; three patients had temporary renal arterial or venous bleeding. Allograft loss as a result of thrombosis (two patients perfused with monoclonal antibodies and a One patient had an ischemic leg after surgery and the graft was rejected and later removed. did. Grafts perfused with three monoclonal antibodies were lost over 48 hours. The second transplant, not as a result of rejection, but one from irreversible narrowing of the renal arteries. The patient had Pneu+nocystis carinii pneumonia and The third graft was rejected due to cessation of immunosuppression, probably due to chronic Cs renal cells. was lost due to cytotoxicity. Four control patients had graft loss and two had irreversible graft loss. as a result of clinical rejection, and two were probably lost due to chronic Cs renal cytotoxicity. Ta.

Allograft function, measured as plasma taleatinin, was determined by monoclonal antibody slightly better in the group of patients with allografts perfused with (Table U) was statistically significant only at 3 months and 1 year after transplantation (p=0.04 ).

Rejection episodes occur in patients who receive grafts perfused with monoclonal antibodies. It occurred in 7 of the patients. Two of these patients had more than one epi It had a sword.

24 controls had an episode of rejection, of which 12 had more than one rejection. There was a danger of an absolute reaction.

Biopsy specimens were analyzed for absorption of CD45 monoclonal antibody.

The results suggest that rejection can be linked to low absorption of antibodies.

However, the numbers are too small for statistical analysis.

In this study, human kidney allografts using the CD45 monoclonal antibody Pretreatment of explants reduces the incidence of rejection1 and possibly modifies allograft function. Good.

Pretreatment of human kidney allografts with a small amount of CD45 monoclonal antibody It does not appear to be harmful to either the graft or the patient. 5 allografts The sections (three perfused with monoclonal antibodies and two controls) were combined at the time of surgery. monoclonal antibody perfusion as a cause of graft loss. There is no evidence to explain it. Can improve allograft function (Table ■) .

Pretreatment of human kidney allografts with CD45 monoclonal antibodies Even when Lasenger lymphocytes are inactivated or destroyed, they can be immunogenized. It will not be considered sexual. As previously described, human renal vascular endothelium and renal tubular The endothelium expresses MHC antigens (Hart, D, N, J, et al. Transplant ation 1981; 31428-33). This perfusion is simple, safe and inexpensive and can be applied to other organs such as heart, liver and pancreas. Ru.

Table - Detailed Table for Patients, Controls and Allografts - Allograft Survival Table ■ - Allograft function In burn patients, while the patient's epithelial cells are expanding in in vitro culture. It is necessary to dress the burn. Dead individuals, usually due to transplantation Allogeneic skin from kidney donors is an excellent dressing; Since it is seen as foreign, it will be rejected by the patient's own immune system. Akira Requires the patient to redress the burnt area to ensure The more frequently the patient suffers from stress and trauma. This t@ is anti- reducing the antigenicity of allogeneic dressings or grafts using CD45 antibodies; and offer new dressing materials that patients can tolerate for longer periods of time. provide

This technique can be applied in other skin graft/skin dressing cases . For example, to remove leg ulcers for final grafting using expanded native skin cultures. 1! When preparing.

There are two main uses for the skin regarding the treatment of burns and leg ulcers.

The first is a wound that stays in place, prevents water loss, and reduces the risk of staining. It is to provide dressing. The second application is to grow back on top of the wound. It is to replace the patient's skin need.

There are currently several different types of skin available as dressings: autologous, allogeneic (also known in the art as homograft) and heterogeneous (usually porcine). Skin can be prepared in different ways: direct use Store at 4°C for up to 7 days, or freeze in glycerol. Conclusion 2: Producing a sheet of skin cells that grow in culture (but probably skin cells U7 was finally prepared as a lyophilized strip preparation, and this Use this with pig skin. Also, non-livable and cutaneous A number of artificial skins that actually represent biological dressings rather than grafts There are several preparations.

Allografts and xenografts are simply dressings.

In contrast, autografts do not contain 7 receptors when transplanted. and new skin. Severely burned patients and middle-aged people If too many people split enough material to cover both of these requirements As a skin graft, it is often difficult to obtain from the fool himself. ,-reha5 , extensive trauma and ulcers requiring dressings in the preparation of graft reservoirs. This is especially true. from different providers while the proper healing process occurs. Perform the dressing in duplicate (-) on the allograft, and prepare the autograft (-). It is very normal for fools to have a leg ulcer. Dressing with allograft skin followed by the intended autograft procedure is that it is useful.

In these cases, prolonging allograft survival has considerable benefits. Ru. Previous attempts to accomplish this have included topical steroids and congeners. Included was UV irradiation of the graft.

In the present invention, as a wound dressing or as a graft, The problem of induced skin rejection is a problem, as mentioned above in this specification. , to be overcome by pre-treating skin derived from non-patient sources with an antibody system. Teach them what they can do.

Skin grafts were harvested according to techniques well known in the field and post-mortem for 24 hours. Can be taken from the provider within hours. Strict sterile procedures are not observed Must not be. Generally, the graft is enclosed in a saline-soaked swab and and placed in a container for transport, for example, to a skin culture laboratory. Therefore, 4 skin in an isotonic solution, e.g. 15% (V/V) glycol in 7 volumes of 0.9% NaCl. Serol or tissue culture medium, e.g. DMEM (Dulbenko's modified Eagle's medium) ), sealed in a polythene hang and under controlled conditions. Can be frozen. The purpose of the freezing method is to increase the cooling rate to 1°C/min from -30°C. It is to adjust to. The batch of frozen skin was then heated to 70°C at -70°C. Store in a dryer until use.

When needed for use, remove the skin from the freezer and store at 37°C. Submerge it in water, where it will rapidly melt. Then open the bag and peel The skin can be used in any desired manner. Once thawed, the skin cannot be refrozen. I can't do it.

These techniques are well known in the field and have been reported throughout the literature. (Cochrane, T. (1986) Low temperature storage of the skin Wa: Preliminary report. Brlt, J, PIas, Surg, 21, 118-125; May .

5.12. (1980) Method for cryogenic preservation of human skin. Cryobiolog y, 17+616; Kearney, J.N., and Gohland G. (1980) Introducing cryobiology to skin survival using a defined murine model. Effect of variables.

Cryobiology 27, 164-170; May S, R, and D eclea+ent F, A.

(1980) Methods of skin banking: package format, cooling and Evaluation of heating rate and storage efficiency. Cryobiology 17°33-45) .

According to the invention, a step in the preparation of skin as a dressing or graft , it is provided that the skin is immersed in the antibody system provided by the present invention. Ru. This soaking is done before freezing the skin for storage or before thawing the skin. It can be carried out after Soaking is performed on cells expressing the CD45 antigen (e.g. carried out for a period of time to allow binding of the anti-CD45 antibody to parasenger lymphocytes). can do.

Suitably, soaking can be for between 1 and 24 hours.

Submission of translation of written amendment (Article 184-8 of the Patent Law) April 20, 1992

Claims (14)

[Claims] 1. Anti-CD45-specific antibody system that mediates complement-dependent cell lysis in the presence of human complement A preparation for treating foreign tissue comprising: 2. A preparation according to claim 1, wherein the antibody system comprises a polyclonal antiserum. . 3. The antibody system has two monochromes that act synergistically in complement-dependent cell lysis. 2. A preparation according to claim 1, comprising a null antibody. 4. The synergistic monoclonal antibody targets the P, Q, R, S and T genes of CD45. according to claim 3, which are directed to different epitopes selected from pitopes. Preparation of. 5. The synergistic pair of monoclonal antibodies is 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 5. A preparation according to claim 4, selected from the group consisting of: 6. 6. The synergistic pair of monoclonal antibodies is anti-P and anti-Q. preparations. 7. 2. A preparation according to claim 1, wherein the antibody is a rat monoclonal antibody. 8. Claims that the rat monoclonal antibody is of the isotype IgG2b. Preparation according to item 7. 9. 9. The antibodies are YTH54.12 and YTH24.5. Preparation of. 10. treating foreign tissue with a preparation according to any of paragraphs 1 to 9 above. The foreign tissue is prepared in vitro before being used as a graft or dressing material. How to prepare. 11. the foreign tissue is skin, and the skin is immersed in the preparation; The method according to claim 10. 12. Claim: said foreign tissue is an organ, and said organ is perfused with said preparation. The method according to item 10. 13. Transferring foreign tissue treated with a preparation according to any of paragraphs 1 to 9 above to a patient. A method of treating a patient comprising implanting or dressing. 14. Allograft or xenograft organs are transplanted into human subjects or In a method of providing an allograft or xenograft dressing to a human patient, the method comprises: treating the organ or dressing with a preparation according to any of paragraphs 1 to 9 above; An improvement consisting of.