IN VITRO ANTIBODY PERFUSION OF KIDNEY GRAFTS

Abstract: In vitro treatment of a kidney prior to transplantation into a human recipient to reduce the risk of rejection, comprises perfusing the kidney with a solution of antibody specific to passenger leukocytes (e.g. anti-CD45 antibody) under cold ischaemia (CI) conditions with the renal vein occluded, and incubating the perfused kidney under CI conditions for at least 20 minutes. The volume of perfusion liquid is preferably at least 50 ml and the concentration of mAb about 40 mu g/ml or more. This has been found to increase mAb uptake by the target cells and the extent of mAb saturation of the target cells, and seems to be associated with a reduced incidence of graft rejection.

Description: IN VITRO ANTIBODY PERFUSION OF KIDNEY GRAFTS This invention relates to the treatment of kidney grafts to reduce the risk of rejection. More particularly it relates to the perfusion of kidneys in vitro with a liquid containing antibodies directed against passenger leukocytes in the kidney. The broad concept is disclosed in our International Patent Application PCT/GB90/01621, with respect to the in vitro perfusion of kidneys with liquid containing a lytic pair of anti-CD45 monoclonal antibodies (mAbs). That was based on the work reported in Brewer et al 1989 1 who reported on a blind trial in which patients received first cadaver kidney allografts that had been perfused with a pair of complement-fixing rat anti-CD45 mAbs, in an attempt to reduce the immunogenicity of passenger leukocytes. The technique reported involved the preparation of vials containing 2 mg of each antibody in 2 ml phosphate buffered saline. The allograft was prepared for transplantation by standard method. The renal veins were clamped and the contents of the vial in 50 ml 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 transplants prior to wound closure and used to measure allograft uptake of anti-CD45 mAbs. The authors reported that the perfusion technique needs to be improved, as at least 25% of the grafts were poorly perfused with an antibody uptake of less than 50%. In a similar report by Brewer et al 1989 2 only 39% of the grafts had an antibody uptake of more than 75%. 62% of the grafts had an uptake of more than 50% and 38% had an uptake of less than 50%. This reference 2 purportedly used an antibody perfusion technique as described in Taube et al 1987 5 . This earlier Taube et al reference, did not document antibody uptake, although it referred to the injection of 2mg of a single mouse monoclonal antibody (F10-89-4) against leukocyte common antigen in 100 ml preservation medium. Following injection of the antibody, the kidney was allowed to incubate for 20 minutes. However, the later Brewer et al reference 2 also concerned the injection of a pair of rat lgG2b anti-leukocyte common antigen antibodies YTH 24.5 and YTH 54.12. Therefore, it is not clear whether in Brewer et al each antibody separately or the antibodies in total were injected in accordance with the teaching of Taube et al 1987. Nevertheless these literature reports suggest that there is a need to establish ways of achieving maximum binding to one or more sets of cells in an organ, by ex- vivo perfusion of an antibody with the appropriate specificity. We therefore set out to establish the conditions required to maximise uptake of perfused anti-CD45 mAbs. In view of the shortage of human organs, which largely precludes their experimental use, we employed a pig kidney model. The parameters examined were (1) mAb concentration, (2) mAb incubation time, (3) volume of perfusate, (4) homogeneity of uptake, (5) different cold ischaemia times (CITs) and (6) the effect of clamping of the renal vein. In addition, we report here some in vitro studies on human kidneys and data from clinical studies in human patients. The results of all these studies (pig and human) indicate the following. 1. The renal vein should be clamped (occluded) during perfusion, so that a hydrostatic pressure is built up, presumably causing the perfusate and antibody to move into the interstitium of the kidney. 2. The volume of liquid is suitably about 50 ml. Significantly lower volumes seem to produce a deterioration in antibody uptake and saturation by the target cells. A small improvement may be obtained by using higher volumes, eg 100 ml, but the risk of rupture of some part of the organ places a practical limit on the volume in the interests of safety, suggesting that volumes significantly higher than 100 ml are best avoided. 3. The concentration of monoclonal antibody would seem to be suitably about 40μg/ml. This value would apply to each antibody species if there is more than one to be used. Thus, for example, if the objective is to target a lytic antibody to the passenger leukocytes, then a single lytic mAb could be applied at 40μg/ml, or a lytic pair (such as YTH24.5 YTH54.12) applied at 40μg/ml each. Much lower concentrations, such as lOμg/ml, or even 20μg/ml, seemed to produce significantly less saturation, although high uptake levels could apparently be obtained with prolonged incubation (see below). 40μg/ml is therefore seen as effective in that case both for good saturation and for a good uptake level achieved in a reasonable time. Significantly higher mAb concentrations are therefore unlikely to produce markedly better results. 4. The data also indicate that following perfusion a period of incubation under cold ischaemia (CI) conditions is effective at achieving good uptake and saturation. In this connection, the distinction between cold ischaemia and warm ischaemia (WI) should be understood. Following removal of the organ from the donor there is a period of warm ischaemia before the organ can be chilled to the desired low temperature. This low (CI) temperature is not a specific value, but rather is generally that which is achieved by keeping the organ on (melting) ice. It will therefore normally be expected to be in the range 0-4°C, but may rise somewhat, say to 8°C. This latter could particularly be the case when the organ is perfused, since the perfusion liquid will normally have been kept in a refrigerator, where the temperature is not strictly regulated, but may be as high as 8-10°C, even though ideally it should be rather lower, eg around 4-6°C. During the surgical procedure to introduce the organ into the recipient, the organ will warm up from its CI condition to a WI condition. Ultimately, of course, its temperature will be that of the recipient's body when the blood flow is established, but during the operation the organ will warm up prior to opening the blood vessels, possibly reaching a temperature as high as 20°C or so, which would still be regarded as WI, since biological functions, and consequent deterioration of the organ in vitro, are generally active at such temperatures. Thus, whereas in the surgical procedures described by Brewer et al, 1989 (supra) the total time from injection of the perfusate to opening of the blood vessels was described as about 20 minutes, a significant proportion of that time would be regarded as WI, since it included the surgical procedure. In contrast, the present disclosure contemplates incubation of the organ with the mAb in vitro under CI conditions (say, less than 12°C, and preferably less than 8°C) for a period prior to allowing WI conditions to occur during the surgical procedure. This CI incubation time with the mAb is preferably at least 20 minutes, and may be longer, as the data show. This produces a very significant improvement in the % antibody uptake over that reported in Brewer 1989 (supra) and also good antibody saturation levels. The clinical data presented herewith also demonstrate an apparent correlation of this improvement with reduced incidence of graft rejection. PIG STUDIES Methods Harvesting, preservation and preparation of kidneys Kidneys were obtained from outbred pigs at an abattoir, typically after 20-30 minutes of warm ischaemia. The kidneys were perfused on site via the renal artery, with cold organ preservation solution (OPS * , Royal Free Hospital, London). Kidneys were cold-perfused until the venous effluent, appeared colourless (400-500ml). They were then sealed in polythene bags containing cold OPS, stored on ice, and returned to the laboratory. After the appropriate cold ischaemia time (CIT, see later), redundant non-renal tissue was dissected off, and the kidneys were weighed. The renal veins were next isolated and clamped. * The composition of this is given in Appendix 1 of this description. Monoclonal antibody perfusion The mAb employed was a mouse IgG2a anti-pig CD45 (common epitope), Pgl/45.9 (cell line provided by Dr Richard Binns, AFRC, Babraham, Cambridge, England and antibody purified by Cantab Pharmaceuticals Research Limited). Immediately prior to kidney mAb perfusion, the mAb was diluted in various volumes of cold OPS, in 50ml plastic syringes (Sherwood Medical, Ballymoney, Northern Ireland). A 2.6mm diameter Medicut cannula (Sherwood Medical) was cut short, and placed on the end of the syringe. This was introduced into the renal artery, which was then sealed by finger pressure alone. The mAb solution was slowly injected at a constant rate of 20ml/minute. The syringe was then removed, and the renal artery left patent (only minimal mAb reflux was observed). The kidneys were returned to the polythene bags employed previously, and again stored on ice. After varying periods of incubation, wedge biopsies were taken, layed on 1.5cm 2 cork tiles coated with Cryo-M-Bed embedding compound (Bright Instrument Co. Ltd., Huntingdon, England), covered with further Cryo-M-Bed, and snap-frozen in liquid nitrogen. Biopsies were stored at -70°C. Study Design (i) Determination of optimal concentration and incubation times of perfused antibody. Three groups of five kidneys were perfused with 4, 2 or 1 mg mAb diluted in 100ml OPS (effective concentrations of 40, 20 and 10 μg/ml respectively). A volume of 100ml was chosen as this was found to be the largest volume that would not visibly over-distend the kidney or renal vein. Cortical wedge biopsies were then taken at 20, 40 and 60 minutes, 2, 4, 8 and 14 hours post-mAb perfusion. One kidney was subsequently perfused with 6 mg, and one with 8 mg mAb, in 100 ml OPS. (ii) Determination of minimum effective perfusate volume, and homogeneity of uptake. To minimise any possible deleterious effects of injecting solutions into the kidney against a clamped renal vein, studies were performed to determine the minimum volume of perfusate that would effect maximal mAb uptake. Each of three groups of five kidneys was perfused with 25ml, 50ml or 100ml of mAb solution, all of the same mAb concentration (40μg/ml, as determined by study (i)). mAb incubation time was 1 hour (again determined by study (i) ), after which six cortical and six medullary wedge biopsies were taken from each kidney, from the anterior and posterior sides of the upper, middle and lower thirds, and one proximal ureteric biopsy was also taken, (iii) The effect of prolonged cold ischaemia. Five kidneys were perfused with 50ml OPS containing 40μg/ml mAb, following a CIT of approximately 45 hours. After a one hour mAb incubation, six cortical and six medullary biopsies were taken as in (ii). (iv) Effect of leaving renal vein undamped during mAb perfusion. Five kidneys were perfused with 40μg/ml mAb in 50ml OPS, without clamping of the renal vein. After one hour, six cortical and six medullary biopsies were taken as in (ii). Immunohistology 5μm cryosections were cut (using a Bright cryotome) onto multi-well glass slides (CA Hendley Ltd, Essex, UK), and orientated so that the most superficial part of the biopsy was always facing the left of the slide. Sections were double immuno-labelled, in duplicate, to determine (a) the percentage of CD45+ cells which had taken up perfused antibody ("uptake"), and (b) the degree of saturation of CD45 binding sites by the perfused antibody, as follows. After acetone fixation for 10 mins at room temperature, washing in Tris buffered saline, pH 7.6 (TBS) and blocking with 20% normal rabbit serum (Sigma, UK) in TBS, sections were incubated with alkaline phosphatase conjugated rabbit anti-mouse Igs (RAM-AP) (Dako Ltd, UK) for one hour, diluted 1:25 in TBS/1% bovine serum albumin, which had been pre-incubated with 5% normal swine serum (Dako), and developed with Fast Red TR (Sigma) and naphthol AS-MX phosphate sodium salt in Tris 0.1M pH8.2, with ImM levamisole to block endogenous alkaline phosphatase, for 20 minutes at room temperature. Thus CD45+ cells labelled by perfused antibody would stain red. After further blocking with 50% normal goat serum (Sigma)/5% dried skimmed milk powder in TBS for 10 mins, further Pgl/45.9 (50μg/ml in TBS/BSA) was incubated on the sections for one hour at room temperature, which would bind to any CD45 antigens not bound by the perfused antibody. Detection was by the subsequent application of 5nm colloidal gold-conjugated goat anti-mouse (GAM) Igs (Biocell Research Laboratories, Cardiff, UK) for one hour, diluted 1:25 in 5% dried milk powder in TBS. Cross-reactivity with the rabbit anti-mouse reagent and background swine Igs in the sections was prevented by pre-incubation of the GAM antibodies with both 5% normal rabbit and 5% swine sera. After post-fixation with formol/acetone for 2 minutes at 4°C, gold labelling was visualised as a black stain by incubation of the slides with a silver enhancer solution (Biocell) for 15 minutes at room temperature. Sections were finally counterstained in Meyer's haematoxylin, and mounted using Loctite 357 engineering adhesive (Loctite UK, Hertfordshire, UK). Thus, cells labelled by perfused mAb stained red, whilst those labelled by slide-applied mAb stained black. Cells labelled by both perfused and slide-applied mAb were double-stained. The use of dried milk powder eliminated background silver staining 3 . Post- ixation with formol/acetone preserved cellular morphology from the deleterious effect of the silver enhancing solution 3 . Controls A negative control perfusion was performed, where one kidney of a pair was perfused with lmg of the mouse IgG2a monoclonal myeloma protein UPC10 (Sigma M7769), diluted in 100 mis OPS, whilst the contra-lateral kidney was perfused with lmg of PG1/45.9 diluted in the same volume. Non¬ specific binding of slide-applied Pgl/45.9 was checked by comparing the labelling produced on unperfused pig kidney sections with that produced by substitution of the primary with TBS/BSA or UPC10. Cross-reactivity of the GAM-gold reagent with the RAM-AP reagent was checked each time a new supply or either of these reagents was obtained, by double- labelling perfused kidney sections as described above, but substituting TBS/BSA for Pgl/45.9. Cell counting Biopsies were examined under high power (x400) using a Leitz Laborlux microscope. Field area was calculated as 0.15mm 2 using a stage micrometer (Graticules Ltd.). The field was divided into four quadrants with a cross-lines graticule (Graticules Ltd. ) to aid cell counting. Any cell stained red or black within the interstitium was considered a CD45+ cell. The majority of these, especially in the renal cortex, were dendritic in shape, but cell morphology was not used as a criterion. Biopsies were coded and randomised with those from at least one other kidney perfused under different conditions, and cells counted by a single investigator. Ten contiguous high power fields (hpf), starting from the bottom left of the section, were counted from each biopsy. If a field included a glomerulus, the microscope stage was moved along until none was visible. For each kidney, the uptake of perfused mAb by CD45+ cells was calculated as follows: No. of cells stained red X 100 No. of cells stained red + no. of cells stained black only In addition, a score was given to each biopsy, again blind to perfusion conditions, to represent the saturation of CD45 binding sites on individual cells as follows: 1 = all labelled cells black only; 2 = double-labelled cells present but predominantly black stained; 3 = double- labelled cells present, red and black intensities equal; 4 = double-labelled cells present but predominantly red- stained; 5 = all cells labelled red only. Statistics Results are expressed as the median (and range) of the five kidneys within each group. Comparisons of percentage uptake and saturation scores between biopsies and kidneys were made by Wilcoxon rank sum analysis. Results It became clear during the course of the study that the anti-CD45 monoclonal antibody Pgl/45.9 recognises a polymorphic determinant of the CD45 molecule's common region, as a proportion of the kidneys (approximately 25%) did not stain at all using the double-labelling protocol of this study. These kidneys were therefore excluded from the study. Preliminary immuno-histological studies on un-perfused kidneys revealed CD45+ cells to be present in the cortical interstitium, where they were dendritic in shape (IDCs) and randomly distributed throughout the cortex; in the glomerulus, where they were more rounded, and most probably resident macrophages 4 ; and in the medulla, where a variety of different cell sizes and shapes were positively stained. Negative control perfusion and immuno-histology Whilst therewas clearly demonstrated cellular labelling of interstitial cells by the kidney perfused with lmg PG1/45.9, no cell binding could be demonstrated in sections of the contra-lateral kidney perfused with UPC10, excluding significant Fc receptor binding. Similarly, there was no positive labelling of un-perfused pig kidney sections treated with TBS/BSA or UPC10 as the primary reagent. On no occasion was any positive immuno-gold silver staining produced on mAb-perfused kidney sections which were double- stained with the omission of the slide-applied Pgl/45.9. This excluded significant cross-reactivity of the GAM-gold reagent with the perfused mAb-RAM-AP complex. Determination of optimal concentration and incubation time of perfused antibody The median cold ischaemia time prior to mAb perfusion for these 15 kidneys was 3 hours (range 3 - 4 hours). The variation in mAb uptake and antigen saturation with time is shown in Tables la and lb. mAb uptake can be seen to have been rapid, and was maximal within one hour of mAb perfusion. mAb % uptake was similarly good in kidneys perfused with any of the three volumes, whilst CD45 antigen saturation by perfused mAb was consistently better with 40μg/ml than with 20 or lOμg/ml, reaching statistical significance at several time points. No improvement in uptake was seen in two kidneys perfused with 60 or 80μg/ml mAb (% uptake 100% and 100%, saturation score 3.0 and 4.0 respectively). On the basis of these results, a concentration of mAb of 40μg/ml, and a mAb incubation time of one hour, was used in subsequent studies. Determination of minimum effective perfusate volume, and homogeneity of uptake mAb perfusion was performed after a median CIT of 19 hours (range 19 - 21 hours). The results are shown in Table 2. Uptake by cortical CD45+ cells was excellent and almost uniform with all three volumes, with no significant differences between the three groups. Medullary CD45+ cell uptake, however, was better with 50 and 100 mis than with 25 mis (though only significantly so for the 100 ml volume). Saturation scores were similar in all three groups. Double-staining of the ureteric biopsies revealed that CD45+ cells in the urothelium were well labelled by perfused antibody (median % uptake 100% in all three groups), but that CD45+ cells within the lamina propria were variably labelled, with no clear relation to perfusate volume. The effect of prolonged cold ischaemia Five kidneys were perfused with 2mg mAb in 50mls cold OPS after a median CIT of 45 hours (range 45-46 hours). Results are shown in Table 2. There was no statistically significant difference between these two groups. Effect of leaving renal vein undamped during mAb perfusion Results are presented in Table 2. Although median cortical uptake was 100%, many sections showed patchy uptake, a finding only seen very rarely in the cortices of kidneys perfused with 50 mis with the renal vein clamped. This finding was more pronounced in the medulla. The clear difference between the two groups did not reach statistical significance however. In none of the perfused kidneys in this study was there any evidence of capsular, renal artery or renal vein damage after antibody injection, whichever volume was employed. Discussion The effectiveness of anti-dendritic cell treatment of kidney grafts will depend on the specific elimination or inactivation of most or all of these cells, without causing graft damage. The perfusion of monoclonal antibodies into kidneys prior to transplantation offers a means of targeting such cells. We have shown that the simple injection of a small volume of mAb solution into the renal artery, whilst the renal vein was clamped, enabled the vast majority of these cells to be labelled, once the appropriate mAb concentration, incubation time and perfusate volume had been determined. Antibody uptake was essentially homogeneous in both the cortex and medulla, using the larger perfusate volumes, and this effect was not diminished by longer cold ischaemia times. Occlusion of the renal vein during mAb perfusion and incubation has here been shown to improve mAb distribution throughout the kidney. The likely mechanism is that it creates a higher hydrostatic pressure within the post- glomerular capillaries (and arterioles and venules). This would encourage the flow of intra-vascular solutes into the interstitium. No renal artery, vein or capsular rupture has been observed, either in this study using pig kidneys or in clinical trials 1 - 5 with human kidneys, in which perfusion volumes of up to lOOmls were employed. Utilisation of a 50ml volume for perfusion should allow a wide margin of safety. The pig kidney model may be used to determine a protocol for human kidney pre-treatment. Since all IgG molecules are of a similar size, their access to the interstitium, assuming similar porosity of pig and human post-glomerular capillary endothelium, will be comparable. Also, the availability of CD45 antigens for antibody binding after prolonged cold ischaemia in pig kidneys is likely to be mirrored in human kidneys. HUMAN STUDIES In vitro studies To date, three human kidneys have become available for in vitro study. The first, "HK1", was obtained as a nephrectomy from a patient with severe renal artery stenosis. Consequently the organ was diseased, shrunken and scarred. The cold ischaemia time for this kidney was 7 hours (i.e. prior to antibody perfusion). After renal vein ligation, the kidney was perfused (by injection into renal artery) with 4 mg of the rat anti-human IgG2b mAb pair YTH 24.5 and YTH 54.12 [2mg of each antibody] . The total 4 mg mAb was given in a total volume of only 50 ml preservation fluid (RFS) owing to the very small size of this particular kidney. Thus each individual antibody was injected into the kidney at a concentration of 40 μg/ml to provide a total dose of 2 mg of each antibody. The 50 ml volume containing the 2 mg of each antibody was injected over 2.5 minutes. The kidney was left to incubate for 12 hours, and then washed out with 300 mis of ice-cold (approximately 4°C) organ preservation fluid. The washing with preservation fluid was carried out by use of a 1 m gravity controlled drip and took approximately 5 minutes. Measurement of the amount of mAbs which leaked out of the kidney during perfusion, 12 hour incubation, and which were washed out of the kidney, were made by collecting of the fluid, and analysis of mAb concentration by a rat IgG2b EIA. Of the total 4 mg of antibody injected into HK1, 1.8 mg leaked out during antibody perfusion, 103μg leaked out during the 12 hour incubation and 49μg was washed out by the 300 ml of preservation fluid. The results are illustrated in Fig. 1. Immuno-staining of CD45+ cells by the perfused mAbs on sections taken after this washout appeared excellent, though the results have not been quantified to date. A pair of human kidneys ( "HK2 and 3") from a single organ donor became available after they were rejected for transplantation purposes in view of mild renal artery stenosis, though the kidneys were normal macroscopically. After renal vein ligation, HK2 was perfused with 4mg of anti-human CD45 mAb pair YTH 24.5 and YTH 54.12 (2 mg of each antibody). The total 4 mg mAb was given in a total volume of 100 mis. Thus each individual antibody was injected into the kidney at a concentration of 20 μg/ml to provide a total dose of 2 mg of each antibody. The 100 ml volume at 20 μg/ml was divided between two 50 mis syringes. The injection time was 5 minutes (2.5 minutes/syringe). HK3 was perfused with 100 mis of diluent only. Biopsies were taken 1, 2, 4 and 12 hours later. The double-staining technique as described for the pig kidneys had not been developed when this kidney was first stained. Thus, percentage uptake was determined by calculating the perfusion-labelled and total CD45+ cell counts from sequential sections. Paired sections were stained to detect either: (A) CD45+ cells labelled by perfused antibody alone; or (B) Total CD45+ cell count. Both groups of slides are first treated with 20% normal rabbit serum (Sigma, Poole, Dorset, UK) in Tris buffered saline (TBS) for 10 minutes to block non-specific binding of the mAb. This was then gently rinsed off. Group B slides then had 50 μls of 10 μg/ml of the pair of perfusion antibodies, diluted in TBS/1% BSA (Sigma), added onto the sections, whilst Group A slides had only TBS/1% BSA buffer. Incubation was for one hour at room temperature. Following three two-minute washes in TBS, all sections were then layered with affinity-purified, rabbit anti-rat immunglobulins, (Dako Ltd.), 1:25 with TBS/1% BSA, having been pre-incubated with 5% normal human serum (Dako Ltd. ) for one hour at room temperature to remove cross-reacting anti-human immunoglobulin activity. Incubation is again for one hour at room temperature. After a further three two-minute washes in TBS, the slides were incubated with rat APAAP (alkaline phosphatase anti-alkaline phosphatase), dilution 1/25 in TBS/BSA for 1 temperature. The slides were then given three further two-minute TBS washes followed by 10 minute reincubations with rabbit anti-rat and rat APAAP reagents respectively, with three two minute TBS washes between and after them. Alkaline phosphatase substrate was then filtered onto the slides and left for 20 minutes. The substrate was composed of naphthol AS-MX di- sodium phosphate salt (Sigma) and Fast Red salt TR (Sigma), with levamisole added to block endogenous alkaline phosphatase activity. Slides were then washed in running tap water for 5 minutes, counterstained with Mayer's Haematoxylin, washed for a further 5 minutes in running water, air dried, then mounted with Loctite UV-activated engineering adhesive (Loctite UK). The results are shown in Fig. 2. In the figure, U=upper third of kidney, M=middle third of kidney and L=lower third of kidney, denoting where biopsies were taken from. The results show good uptake at 1 hour with the antibody perfusion volume and concentration used. Uptake did not appear to be significantly improved by increasing the antibody incubation time. There were no apparent significant differences in uptake between the upper, middle and lower region of the kidney. The applicants intend using double staining to validate these findings. The data disclosed herein for perfusion of kidneys with anti-CD45 mAbs may apply also to the perfusion of kidneys with other antibodies, monoclonal, polyclonal or mixed, and indeed to the perfusion of other organs with antibodies. CLINICAL DATA Table 3 shows the uptake and saturation of antibody onto human kidneys using the perfusion protocol developed in the pig. The double staining protocol used is shown in Appendix 2. 50 ml of Baxter's perfusion solution nominally at 4°C and containing two antibodies (YTH24.5 and YTH54.12), each at 40 μg/ml, were injected into the renal artery of the excised kidney over a period of 2.5 minutes while the renal vein was clamped. The clamp was left on the renal vein for a period of at least 40 minutes. In this trial the time on ice before starting the operation to connect the kidney to the patients vasculature was not specified precisely and the 40 minute period before the clamp was removed would have covered some time on ice (nominally 4°C) and part of the time necessary to make the connections. As the latter typically takes 15 to 20 minutes it is reasonable to suppose that the kidneys were on ice for at least 20 minutes and probably nearer 30 minutes. For the remainder of the time they will have been warming up. The data show an improvement in uptake compared to the published data (Brewer 1989, supra). The relationship between uptake and whether the patient suffered a rejection episode is striking. Relative binding of the lvtic pair of mAbs YTH24.5 and YTH54.12 Fig. 3 is a graph showing the relative binding of the two antibodies to a CD45-positive human B cell line called Raji. Relative binding is measured as the mean fluorescence intensity (MFI) in a FACS apparatus. The graph demonstrates that good uptake is achieved in solution at mAb concentrations of about 2μg/ml, and also that the maximum relative binding is achieved at about the same concentration for the combined (lytic) pair as for the mAbs individually. The fact that much greater mAb concentrations (preferably ca 40 μg/ml) are required to achieve the desired effect when actually applied to a kidney illustrates the difference between a simple in vitro experiment in solution and the practical application of the findings to a kidney graft. The graph in Fig. 4 shows the percentage of maximal activity of the antibody pair binding to Raji cells compared to the percentage maximal activity of lysis of Raji cells coated with anti CD45 antibodies in the presence of human complement. The antibody concentration in μg/ml is the total immunoglobulin concentration while the antibodies are reacting with the cells. The concentration of each antibody separately is one half of the amount shown. The lysis measured by 51 Cr release was carried out essentially as described in Bindon et al 3 replacing Raji cells for human lymphocytes. The antibody binding experiment was carried out as follows. 250,000 Raji cells in 25 microlitres of phosphate buffered saline containing 1% BSA (FACS buffer) were incubated with 25 microlitres of the different concentration of immunoglobulin. After one hour at 4°C the cells were washed three times and the antibody bound to the surface of the cells detected by using a saturating amount of an FITC conjugated affinity purified anti-rat immunoglobulin. The cells were incubated for a further 30 minutes at 4°C washed twice in FACS buffer and once in PBS 1% BSA. The cells were finally resuspended and analyzed on a Becton Dickinson FACS scan using standard procedures. REFERENCES 1. Brewer Y, Taube D, Bewick M et al. Effect on graft perfusion with two CD45 monoclonal antibodies on incidence of kidney allograft rejection. Lancet 1989; 2:935. 2. Brewer Y, Bewick M, Palmer A, Severn A, Welsh K, Taube D. Prevent of renal allograft rejection by perfusion with antileucocyte common monoclonal antibodies is dependent on good uptake of antibody by interstitial dendritic cells. Transplant Proc 1989;21:1772. 3. De Waele M, Renmans W, Segers E, De Valck V, Jochmans K, Van Camp B. An immunogold-silver staining method for detection of cell surface antigens in cell smears. J Histochem cytochem 1989;37:1855. 4. Schreiner GF, Unanue ER. Origin of the rat mesangial phagocyte and its expression of the leucocyte common antigen. Lab Invest 1984; 51:515. 5. Taube D, Welsh KI, Bewick M et al. Pretreatment of human renal allografts with monoclonal antibodies to induce long-term tolerence. Transplant Proc 1987; Vol 19 no. 1:1961. 6. Bindon et al, Transplantation, 40, 538-544 (1985) Appendix 1 Composition of Royal Free Hospital organ preservation fluid: Raff nose pentahydrate 1.86% Lactobionate 3.861% Potassium acid phosphate 0.355% Mg sulphate 0.262% Reduced glutathione 0.962% Allopurinol 0.014% Sodium hydroxide 0.154% Potassium hydroxide 0.585% Water for injection up to 100% pH 6.8 Appendix 2 PHASE II TRIAL OF CD45 PERFUSION OF HUMAN RENAL ALLOGRAFTS DOUBLE STAINING PROTOCOL FOR BIOPSIES POST-PERFUSION 1. 5Mm cryosections cut in duplicate onto 2-well glass slides (PH-128, CA Hendley, Essex) 2. Air-dried overnight 3. Fixation in 100% acetone, 10 mins RT 4. Wash in TBS, 5 minutes 5. Normal rabbit serum 20% (Sigma) in TBS, 10 mins RT 6. Alkaline phosphatase-conjugated rabbit anti-rat Igs (Sigma A9529), 1/25 in TBS/1%BSA, 30 mins RT 7. Wash in TBS, 2 mins x 3 8. Rat APAAP (Dako D488) 1/25 in TBS/1%BSA, 30 mins RT 9. Wash in TBS, 2 mins x 3 10. Fast Red 20mg (Sigma F2768) in 10 mis Tris 0.1M pH 8.2, with 4mg naphthol AS-MX phosphate (di-sodium salt, Sigma N5000) and 4mM levamisole: 20 mins RT 11. Wash in TBS, 2 mins x 3 12. Hydrogen peroxide 0.3% in distilled water, 20 mins RT 13. Wash in TBS 5 mins x 1 14. Avidin 0.01% (Vector UK) in TBS + 0.01% sodium azide, 20 mins RT 15. Wash in TBS, 2 mins x 3 16. Biotin 0.001% (Sigma B4501) in TBS + 0.01% sodium azide, 20 mins RT 17. Wash in TBS, 2 mins x 3 18. Normal rat serum 20% (Sigma) in TBS, 20 mins RT 19. Biotinylated rat anti-human CD45 (YTH 24.5/54.12 1:1, Cantab Pharmaceuticals Research Ltd), 5μg each Mab/ml in TBS/1%BSA 1 hour RT 20. Wash in TBS, 2 mins x 3 21. StreptABC Complex-HRP (Dako K377) in Tris buffer 0.05M, pH 7.6, 30 mins RT 22. Wash in TBS, 2 mins x 3 23. DAB-nickel HRP substrate (Vector kit SK-4100) 15 mins RT 24 Wash in tap water 5 mins 25. Haematoxylin counterstain 26. Wash in tap water 5 mins, then distilled water rinse 27. Air dry 28. Mount slides in Loctite 357 UV activated engineering adhesive (Loctite UK) Abbreviations: TBS Tris buffered saline, 0.15M, pH 7.6 BSA Bove serum albumin Tables la and lb Effect of mAb incubation time and perfusate concentration on CD45+ cell uptake and saturation (lOOmls perfusate volume). Table la % Uptake [mAb] Time post-Mab perfusion 20mins 40mins 60mins 2hrs 4hrs 8hrs 14hrs (No significant differences between groups at any time point) Table lb Saturation Scores [mAb] Time post-Mab perfusion 20mins 40mins 60mins 2hrs 4hrs 8hrs 14hrs *p<0.05 vs 20μg/ml +p<0.05 vs lOμg/ml Table 2 Effect of mAb perfusate volume, cold ischaemia time and renal vein clamping on mAb uptake. Perfusate Renal Vien CIT CORTEX MEDULLA volume status (hrs) % uptake+ range Sat core- % uptake+ range Sat Sc 25 mis Clamped 20 82.6 34.3-96.1 2.0 50 mis Clamped 20 98.5 30.0-100 3.0 100 mis Clamped 20 98.7 93. 5-99.9 4.0 50 is Clamped 45 99.0 90.3-99.0 3.0 50 mis Un amped 20 64.8 48.4-92.1 3.0 + median median saturation score * p0.05 vs 100 mis Table 3 Anti-CD45 Pre-treatment of human renal allografts Results of double-staining biopsies Patient %uptake Sat score Reject no. (median) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. (not clamped) 11. + 12. + 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Claims: Claims 1. A method of in vitro treatment of a kidney prior to transplantation into a human recipient, which comprises perfusing the kidney with a liquid containing antibody specific to passenger leukocytes, wherein the liquid is injected into the vasculature of the kidney with the outlet veins occluded whereby a hydrostatic pressure is produced in the kidney, both the kidney and the injecting liquid being at a temperature appropriate to cold ischaemia, the perfused kidney being incubated under cold ischaemia conditions for at least 20 minutes. 2. A method according to claim 1 wherein said antibody is monoclonal antibody. 3. A method according to claim 1 or claim 2 wherein the CI incubation time of the perfused kidney is at least 40 minutes. 4. A method according to claim 3 wherein the CI incubation time of the perfused kidney is at least one hour. 5. A method according to claim 1 wherein the incubation time of the perfused kidney is at least 40 minutes prior to anastomosis. 6. A method according to any one of the preceding claims wherein the volume of liquid and the concentration of antibody are selected for achieving antibody uptake by at least 80% of the passenger leukocytes. 7. A method according to any one of the preceding claims wherein the volume of liquid is greater than 25 ml. 8. A method according to claim 7 wherein the volume of liquid is at least 50 ml. 9. A method according to claim 8 wherein the volume of liquid is about 100 ml. 10. A method according to any one of the preceding claims wherein the concentration of leukocyte specific antibody is greater than 20 μg/ml. 11. A method according to claim 10 wherein the concentration of leukocyte antibody is at least about 40 μg/ml. 12. A method according to any one of the preceding claims wherein the volume of liquid, the concentration of antibody and the incubation time are selected for achieving a median saturation level of the target cells by the antibody of at least about 4 on a scale of 1-5 as visually assessed after double immunolabelling by EIA, wherein value 1 represents no observable binding of the cell by the antibody, and value 5 represents apparently saturated binding of the cell by the antibody. 13. A method according to any one of the preceding claims wherein the antibody is specific to CD45. 14. A method according to claim 12 wherein the antibody is capable of complement mediated cell lysis. 15. A method according to claim 13 wherein the antibody comprises a pair of anti-CD45 monoclonals which together are capable of complement mediated cell lysis, but individually are not lytic. 16. A method according to claim 14 wherein the antibodies are monoclonal antibodies YTH 24.5 and YTH 54.12.

Inventors: GOLDBERG LAWRENCE CHARLES, TAUBE HIRSCH DAVID, GOLDBERG, LAWRENCE, CHARLES, TAUBE, HIRSCH, DAVID
Applicants: CANTAB PHARMA RES, CANTAB PHARMACEUTICALS RESEARCH LIMITED
Application year: 1992
Main classification: A61K39
IPC codes: A01N1, A61K38, A61K39, A61P37, C07K16