TRANSPLANT International

Review article

9 Springer-Verlag1992

Transplant Int (1992) 5:234-246

Monoclonal antibodies in renal transplantation: a review K. J. Parlevliet and P. T. A. Schellekens

Renal Transplant Unit, Department of Internal Medicine,AcademicMedicalCenter, Universityof Amsterdam,Meibergdreef9, NL-1105 AZ Amsterdam,The Netherlands Received May 22,1991/Receivedafter revision October 29, 1991/AcceptedJanuary 13,1992

Abstract. OKT3 is the first anti-CD3 monoclonal antibody available for treatment in humans. Over the last few years it has proven to be a very powerful immunosuppressive agent in renal transplantation. Clinical studies have shown that OKT3 is superior to high-dose steroids as first-line treatment for acute renal allograft rejection. Furthermore, it is comparable to antithymocyte globulin (ATG) in treating steroid-resistant rejection and is also effective as rescue treatment in ATG- and antilymphocyte globulin- (ALG-) resistant rejection. Despite its excellent rejection-reversal rate, OKT3 treatment is followed by a substantital percentage of re-rejections, most of which respond well to steroids. In the early post-transplantation period, a prophylactic course of OKT3 is very effective in preventing acute rejections, and in this respect it is probably equivalent to ATG. Indirect evidence exists that a prophylactic course of OKT3 may be beneficial in immunologically high-risk patients and in patients with delayed graft function. However, more clinical studies are required to answer the question whether OKT3 should be given as induction treatment, as first-line treatment, or as rescue treatment. To answer this question, the side effects of OKT3 should also be taken into account. First-dose-related side effects, although frequent and disturbing, are usually transient and seldom life-threatening, provided overhydration has been corrected and steroids have been given before the first administration. These side effects are attributed to the release of cytokines as a result of T-cell activation or lysis. After exposure of patients to OKT3 an increased incidence of infections and malignanies has been reported. However, it is not yet clear whether this is due to OKT3 as such, or whether it merely reflects the total burden of immunosuppression. Xenosensitization represents an important limitation to OKT3 treatment, although a second or third course can still be effective in patients with low antibody titers. The precise immunosuppressive mechanism of anti-CD3 monoclonal antibodies is yet unknown. Monitoring of patients treated

Correspondence to: K. J. Parlevliet

with OKT3 revealed CD3 and/or T-cell antigen receptor depletion and immunological incompetence of remaining T cells. More clinical data are required to establish the correct dose and duration of OKT3 treatment. In conclusion, OKT3 is a powerful immunosuppressive agent but its real value in renal transplantation remains to be determined. A practical approach may be to reserve it for the treatment of steroid-resistant rejections. Research strategies for the development of new monoclonal antibodies for future use in renal transplant patients should be aimed at evading the major hazards of OKT3 treatment, namely side effects and xenoimmunization. Many promising monoclonal antibodies are currently under study: theoretically, anti-CD3/TCR monoclonal antibodies that are not mitogenic (such as CLB-T3/4.A, BMA031, T10.B9.1A-31A, and OKT3 F[ab']2 fragments) may be expected to cause less immediate toxicity than OKT3. However, their immunosuppressive properties still have to be established. Monoclonal antibodies that are reactive with more restricted T-cell markers such as the interleukin-2 receptor (33B3.1 and anti-Tac), CD4 (BL4), and CD8 (Leu 2a) are generally better tolerated than OKT3. On theoretical grounds they may also be expected to cause fewer long-term complications. However, their immunosuppressive properties also remain to be established. In patients with high titers of anti-idiotypic antibodies to OKT3, WT32 and perhaps RIV9 and T10B9.1A-31A may be good alternatives. Key words: Monoclonal antibodies, in kidney transplantation - Kidney transplantation, monoclonal antibodies Antibodies, monoclonal, in kidney transplantation

Since the introduction of the hybridoma technique for the production of monoclonal antibodies (mAbs) by K6hler and Milstein in 1975 [113], mAbs have been increasingly used not only in the laboratory but also in hospitals, in particular for the treatment of renal allograft rejection. Briefly, hybridoma cells result from the fusion of a murine myeloma cell line with spleen cells of a mouse that has

235 been immunized with an antigen. Each hybridoma cell has the potential to produce one type of antibody. By means of a limiting dilution technique it is possible to produce clones of hybridomas, each of which can provide a nearly limitless amount of one specific mAb, directed against one epitope on the immunizing antigen. Before the introduction of mAbs, polyclonal antibodies such as antithymocyte globulin (ATG) or antilymphocyte globulin (ALG), produced by immunizing rabbits or horses with thymocytes or lymphocytes, were considered most effective in treating renal allograft rejection [44, 73, 93, 120, 135, 162, 176, 178]. However, their clinical use is hampered by a few important drawbacks. Since these polyclonal antibodies are derived from individual animals, there is considerable batch-to-batch variability with respect to specificity and affinity,which affects their immunosuppressive properties and toxicity in an unpredictable way [176]. MAbs lack this batch-to-batch variability as they are characterized by absolute identity and monospecificity. Furthermore, a course of ATG or ALG often has to be terminated within 2 or 3 weeks because of clinical intolerance due to acute serum sickness. As we shall see, mAbs do not cause acute serum sickness. Lastly, but not least, the administration of polyclonal antibodies in peripheral veins is complicated by a high rate of thrombotic complications and therefore requires the presence of a central venous catheter. At Ortho Pharmaceutical Corporation Kung et al. developed a panel of mAbs to human T-cell surface antigens - the OKT series, the third of which was Orthoclone OKT3 [116]. Over the last 5 years it has proved to be a powerful immunosuppressive agent and at this moment OKT3 is still the only mAb that is commercially available for administration in transplant patients. After a short introduction to the immunobiological backgrounds of OKT3 and other anti-CD3 mAbs, we shall focus on the clinical experience with OKT3; finally we shall briefly discuss possible future developments and mention some other mAbs currently under study for treatment of renal transplant patients.

Immunobiological backgrounds of OKT3 and other anti-CD3 monoclonal antibodies OKT3 is a murine mAb of the IgG2a class directed against the CD3 molecule, which is present on the surface of thymocytes and mature human T cells [116]. The CD3 molecule is closely associated with the T-cell antigen receptor (TCR) in the so-called CD3/TCR complex. This complex plays a vital role in T-cell function: antigen recognition by the TCR results in signal transduction via the CD3 molecule and subsequent T-cell proliferation and activation of cytotoxic cells [41, 77, 97, 98]. In vitro at high concentrations, OKT3 blocks antigen-stimulated T-cell proliferation and the activation of cytotoxic T cells [5, 29, 30, 122, 155, 160, 161]. In doing so, it blocks both the T-cell induction phase and the effector functions involved in atlograft rejection. In vivo, administration of OKT3 initially results in coating of all circulating T cells. Within minutes, these OKT3-coated cells then disappear almost completely from the circulation, as evidenced by depletion of CD2-,

CD3-, CD4-, and CD8-expressing cells [32-34, 47, 48, 69, 86, 168, 175, 207]. Whether this is due to cell lysis or sequestration of opsonized cells by the mononuclear phagocytic system (MPS) remains unclear. Because murine immunoglobulins only poorly activate human complement, it appears that direct, complement-mediated cell lysis is not involved. During ianti-CD3 treatment T-cell depletion in lymph nodes and spleen was found to be incomplete; also anti-CD3 treatment did not prevent the recurrence of CD3 + cells in thymectomized mice [89], suggesting that at least some CD3 § cells escape lysis. After a few (2-5) days' treatment with OKT3- T cells reappear in the circulation. However, these T cells are functionally inactive [11, 29, 32, 122, 155,161,215]. It is generally assumed that the disappearance of the CD3 molecule from the surface of the T lymphocyte - a process called "antigenic modulation" contributes to the immunosuppressive effect of anti-CD3 mAbs [5, 11, 34]. This is substantiated by the observation that in cases where OKT3 § cells reappear in peripheral blood during OKT3 treatment, OKT3 is ineffective [175]. Remarkable and as yet unexplained is the finding that the above-mentioned OKT3- T cells react weakly with anti-Leu 4, a mAb that recognizes a CD3 epitope distinct from OKT3 [71, 72,218,224]. However, these weakly antiLeu 4 + cells do not express the TCR, they are functionally inactive, and their presence does not correlate with ineffectiveness of OKT3 treatment. The cause of this discordant CD3 expression is not understood. The detection of large numbers of OKT3-coated cells in the circulation during OKT3 treatment suggests that masking of the CD3 molecule may also play a role [26]. However, which of the abovementioned mechanisms - blocking and/or modulation of the CD3/TCR complex or disappearance of T cells from the circulation - is responsible for the immunosuppressive effect of OKT3 in vivo remains undefined. During OKT3 treatment CD3 modulation has also been observed in the transplanted kidney, although data on this subject are conflicting [17, 18, 42, 107, 108]. Upon discontinuation of OKT3 treatment the number of CD3 + cells in peripheral blood rapidly returns to normal values, but in a murine model it was found that normal immunocompetence was not recovered until 2 months after treatment [89]. In vitro, apart from its immunosuppressive action at high concentrations, OKT3 at low concentrations is known to be a potent T-cell mitogen [30, 214]. Monocytes are probably required for this T-cell activation in two ways: first, by binding of the antibodies of their Fc receptors (FcR), which causes cross-linking of the CD3/TCR complex, and, second, by producing interleukin-1 (IL-1) [31, 150, 164, 213]. However, FcR polymorphism on human monocytes for murine immunoglobulins results in a donor-dependent variation of anti-CD3 mAbs of different (sub)classes to induce T-cell proliferation: whereas all human monocytes carry the appropriate FcR for murine IgG2a and IgG3, only 70 To of the donors express FcR for murine IgG1, only 10 % express FcR for IgG2b, and all donors completely lack the proper FcR for murine IgA [27, 28,172,192-194, 213]. Hence, murine anti-CD3 mAbs of the IgG2a and IgG3 isotypes can cause T-cell activation in 100 To of human donors, whereas an IgA anti-CD3 mAb and anti-CD3 F(ab')2 fragments never can. Further-

236 more, F(ab')2 fragments are immunosuppressive in mice without causing T-cell activation [91, 92], suggesting that mitogenicity of anti-CD3 mAbs is not a prerequisite for immunosuppressive capacity. The possible implications for toxicity of the different mitogenic properties of antiCD3 mAbs of different isotypes will be discussed below. The question whether OKT3-mediated T-cell activation may even enhance the process of acute allograft rejection remains to be resolved.

Present state of knowledge regarding OKT3

Efficacy in treatment of acute renal allograft rejection In the early 1980s the first clinical studies on OKT3 for treatment of acute allograft rejection were published [15, 47, 48, 139, 196]. They were uncontrolled studies, describing the experiences with a course of OKT3 in renal transplant patients with acute allograft rejection. The most important observation was the reversal of rejection in almost all patients within 2-7 days after the start of OKT3 treatment. A course of OKT3 usually consisted of one intravenous bolus injection of 5 mg daily for 10 or 14 days. As far as we know this dosage was not based on a phase-I trial. Based on in vitro studies, trough levels in the range of 1 gg/ml were considered to be effective and were usually reached within a few days [78, 81]. These trials were followed by a controlled study from the Ortho Multicenter Transplant Study Group in which 123 patients with acute rejection of a cadaveric renal transplant were treated with either OKT3 or with high-dose steroids [148]. The rejection-reversal rate after OKT3 (94%) was significantly better than after high-dose steroids (75 %). The therapeutic superiority of OKT3 was also reflected in an improved 1-year graft survival of 62 % in the OKT3 group, as compared with 45 % in the steroid-treated group. These excellent results with OKT3 have been confirmed by many reports [6, 7, 9,13, 45, 49, 50, 55, 69, 77, 79, 80, 82, 88, 94, 95,104, 114, 125, 126, 132, 138, 140, 141,144, 146, 149, 156, 168,186-188, 197,198,200,204, 219,220,223]. The general conclusion is that OKT3 is very effective as a first-line treatment of acute renal allograft rejection. In this respect it is superior to high-dose steroids. Moreover, OKT3 was as effective as ATG in treating steroid-resistant acute rejection episodes [9], and it proved to be effective as rescue treatment in ATG- and ALG-resistant rejection [49, 144, 146,156, 219]. OKT3 appeared to be most effective in treating acute cellular rejection [223]: only a few patients were reported with predominantly vascular acute renal allograft rejection that responded well to OKT3 [55,171]. Furthermore, it was equally effective in patients with azathioprine and cyclosporine maintenance immunosuppression. There has been a reluctance to continue cyclosporine during OKT3 treatment, based on fear of overimmunosuppression and nephrotoxicity [75, 179]. Indeed, some authors report a diminished rejection-reversal rate when cyclosporine is continued during OKT3 treatment [88], but this was not confirmed by others [94-96]. Despite the excellent rejection-reversal rate, the number of recurrent rejections after OKT3 was substantial, amounting to 67 % within the 1st month after cesssation of OKT3

[50, 77,140,141,146,148,223]. Fortunately, the majority of these re-rej ections responded well to high-dose steroids. In an attempt to answer the question whether OKT3 is more effective when used as first-line treatment or whether it should only be given in steroid-resistant acute rejections, Kovarik et al. and Deierhoi et al. reported comparative results of OKT3 and steroids for primary treatment of acute rejections and rescue treatment. Kovariak et al. [1 t4] found that the rejection-reversal rate was significantly better when OKT3 was given as first-line, while D eierhoi et al. [54] found no difference in rejection-reversal rate whether OKT3 was given as first-line or as rescue treatment. However, irrespective of the fact that patients were not randomized, the interpretation of these results is hampered by the short interval between first-line and rescue treatment, which makes it impossible to differentiate between a late response to first-line treatment and an early response to rescue treatment. Furthermore, the question whether OKT3 is superior to steroids cannot be answered without considering the side effects (see below).

Efficacy for prophylaxis of acute renal allograft rejection The hypothesis that blocking of T-cell function at the very moment of transplantation could induce immunologic tolerance to the graft led to the prophylactic use of OKT3. The first experiences with prophylactic treatment began to be published from 1986 onwards [23, 24, 42, 53, 77, 105, 115, 128, 142, 168, 169, 201, 202, 207, 217]. The average duration of a full prophylactic course of OKT3 was 15 days (varying from 6 to 30 days) and the average daily OKT3 dose amounted to 5 mg. However, recent reports indicate that lower doses may perhaps be equally effective [145]. Basic immunosuppression during OKT3 prophylaxis usually consisted of prednisone and azathioprine and was extended with cyclosporine towards the end of the course, provided graft function was satisfactory. The majority of these reports were of controlled studies. Basic immunosuppression in the control group consisted of prednisone and azathioprine with or without cyclosporine. It was noted that rejections hardly ever occurred during OKT3 prophylaxis and that OKT3 significantly delayed the mean onset of rejection. Between these reports there is a large variation in rejection rate in both the OKT3 and the control group that can be explained by variations in the period of follow-up and the amount of basic immunosuppression. Overall there were a significantly lower number of acute rejections in the OKT3 prophylaxis group (varying from 6 % to 29 %) than in the control group (varying from 50 % to 94 %), a difference that was still present after i year [51, 53, 76, 77, 105, 106, 142, 202]. Therefore, OKT3 prophylaxis was considered very effective in preventing acute rejections compared to conventional basic immunosuppression, although the mere occurrence of acute rejection after OKT3 induction did not favor the notion of immunologic tolerance. In a nonrandomized prospective study of 110 renal transplant patients with triple therapy (prednisone, azathioprine, and cyclosporine) as basic immunosuppression, OKT3 prophylaxis was superior to A L G in preventing acute rejections [128]. However, in three randomized

237 prospective studies induction therapy with OKT3 and ATG gave more or less similar survival of patients and grafts [67, 85, 124]. In immunologically high-risk patients OKT3 prophylaxis resulted in improved graft survival compared to a historical control group [23,169]. Since cyclosporine can prolong the duration of acute tubular necrosis (ATN) after transplantation, and a severe rejection can remain unnoticed during a period of initial graft dysfunction, while on the other hand withholding cyclosporine may increase the risk of rejection, special attention was given to prophylactic use of OKT3 in patients with delayed graft function. This led to somewhat conflicting data. Thislethwaite et al. [201] found an excellent graft survival rate (97 %) in patients with delayed graft function who were prophylactically treated with OKT3. However, as there was no control group and the follow-up was only 2-8 months, this observation does not permit any definite conclusions. Similar results were reported by Cohen et al. [42] and Benvenisty et al. [4], who compared graft survival in patients with delayed graft function receiving OKT3 prophylaxis with a historical control group: they found a much better 1-year graft survival rate in the OKT3 prophylaxis group (83 % and 80 % respectively vs. 55 %). In a prospective study Kahana et al. [106] found a decrease in the duration of ATN in the OKT3 induction group compared to a control group treated with triple therapy, while Toussaint et al. [51,202] reported an increased incidence of delayed graft function following OKT3 prophylaxis compared to cyclosporine, and suggested that OKT3 might be nephrotoxic. This issue certainly needs further investigation. In an attempt to answer the strategical question of whether OKT3 is preferable as induction treatment or whether it should be reserved for the treatment of acute rejection episodes, Kreis et al. [115] prospectively compared the results of OKT3 prophylaxis to the results of OKT3 treatment for acute rejection in patients with the same basal immunosuppression (prednisone and azathioprine). They found a difference in graft survival - although not statistically significant - between the two groups: 18-month graft survival was 89.3 % in the OKT3 prophylaxis group and 66 % in the OKT3 antirejectiontreated group. Obviously, these results cannot be extrapolated to cyclosporine-treated patients. Also, this question can only be answered when the side effects of OKT3 are taken into account (see below).

Side effects The first administration of OKT3 is almost invariably followed by a spectrum of symptoms, including pyrexia, dyspnea, headache, nausea, vomiting, and diarrhea [45, 148, 199]. These symptoms usually start within 1 h after administration of the first dose of OKT3, reverse spontaneously after several hours, and tend to diminish after the subsequent OKT3 administrations. In patients who were overhydrated at the start of OKT3 treatment, acute life-threatening pulmonary edema has been described [148, 199]. For this reason the beginning of OKT3 treatment, either therapeutic or prophylactic, is nowadays always preceded by correction of volume status, if necessary

by means of dialysis or ultrafiltration. With this regimen the occurrence of pulmonary edema after the initial administration of OKT3 has become rare [167], although significant hypoxia is not uncommon [16]. According to the instructions from the manufacturer the first dose of OKT3 should also be preceded by an intravenous bolus of steroids, acetaminophen, and antihistamines in an attempt to mitigate these first dose-related symptoms. The benefit of steroids has only been demonstrated in mice [2, 61], while the giving of acetaminophen and antihistamines lacks any scientific base. Between the 2nd and the 5th day of OKT3 treatment pruritus, rash, aseptic meningitis, and altered mental status have been reported [199]. In our experience severe complaints of general malaise may persist throughout OKT3 treatment. Most patients are therefore hospitalized during OKT3 treatment, although some authors treat their patients partly on an out-patient basis [149,201]. After cessation of OKT3 treatment there is usually fast recovery. The complex of symptoms that arises after the first dose of OKT3 bears a marked similarity to the syndrome of endotoxic shock mediated by tumor necrosis factor-~ (TNF-cz) or interleukin-2 (IL-2) [131, 163, 166, 203]. Indeed, very soon after the first administration of OKT3 in renal transplant patients a systemic rise in TNF-a and interferon- 7 (IFN-?) was detected, in some patients accompanied by an increase in IL-2 [1, 36, 37, 39, 70, 74]. The highest levels of cytokines were observed in patients who had not received steroids prior to the first dose of OKT3. This inhibitory effect of corticosteroids on cytokine production and toxicity was also observed in mice [2, 61]. Recently, a systemic rise in interleukin-6 (IL-6) levels was also reported during OKT3 treatment [8]. However, TNFcz, IFN-7, and IL-2 reached their maximum levels during the first 4 h after the first dose of OKT3, whereas IL-6 reached its maximum at 48 h. In general, this anti-CD3 induced cytokine release parallels the clinical symptoms described above and therefore is held responsible for them. This view is supported by the observation in mice that administration of anti-TNF antibodies prior to the injection of anti-CD3 mAbs reduces toxicity [63]. Apart from causing systemic illness, these cytokines are also held responsible for the increased incidence of ATN reported after OKT3 prophylaxis in some studies [51, 74, 75,202]. The release of cytokines has been attributed to T-cell lysis, T-cell activation, or both [60, 62, 185, 189, 190, 196]. The observation in a murine model that an anti-CD3 mAb causes significant toxicity without at the same time causing T-cell lysis may suggest that these symptoms are at least partly caused by T-cell activation [89]. This is in line with observations in vitro that FcR cross-linking on monocytes induces secretion of TNF-c~ [52], and that T-cell activation by mAbs that are reactive with the CD3/TCR complex results in production of TNF-cz, IFN-g, and IL-2 [31, 130, 151,164, 185, 189, 221,222]. Recently, T-cell activation was also demonstrated in vivo after administration of anti-CD3 mAbs [58, 59, 91]. Apart from the symptom complex described above, which is directly related to the administration of OKT3, OKT3 treatment is, like all other immunosuppressive

238 regimens, followed by an increased susceptibility to infection. Bacterial as well as viral, predominantly cytomegalovirus (CMV), infections have been reported after OKT3 treatment. In a few controlled studies on this subject the infection rates after OKT3 treatment, high-dose steroid treatment, and A L G treatment were not essentially different and varied with the period of follow-up, from about 25 % after 3 months to about 50 % after a follow-up period of i year [106, 128, 148]. Two other controlled studies report an increased infection rate after ALG prophylaxis, compared to OKT3, that did not influence long-term patient survival [67, 85]. The same was reported when OKT3 prophylaxis was compared to triple therapy [76]. In retrospect, multiple exposures to OKT3 increased the risk of infection, especially by CMV [129, 199]. Most studies on this subject are uncontrolled or use historical control groups [12, 14, 49, 50, 54,134,144, 147,223]. Oh et al. [147] report that OKT3, when used to treat steroid-resistant rejection (i. e., given after a full course of high-dose steroids) increases the risk for infection compared to that in a historical control group in which steroid-resistant rejections were treated with ALG. Munda et al. [134[ found in retrospect that there was no difference between the total amount of OKT3, pulse steroids, or polyclonal antilymphocyte antibodies given as induction therapy or treatment for rejection to infected and noninfected graft recipients. Deierhoi et al. [54] found no difference in infection rate whether OKT3 was used as primary treatment for allograft rejection (and pulse steroids as rescue) or whether OKT3 was used as rescue (and pulse steroids as primary) therapy for allograft rejections. Pancytopenia was reported in two patients after administration of OKT3 [14]. Both occurred in the setting of multidrug immunosuppression including polyclonal ALG, and both patients recovered with supportive therapy. Recently, retrospective studies reported an increased incidence of lymphoproliferative disorders after OKT3 treatment [153, 191]. In our opinion the prophylactic use of OKT3 at the currently used dosage in uncomplicated transplant cases is not justified by clinical data. An exception can be made for immunologically high-risk patients and patients with delayed graft function, although the supposed benefit of OKT3 prophylaxis in those patients is largely based upon studies using historical control groups.

Humoral immune response

An antibody response against murine immunoglobulin poses an important limitation to OKT3 treatment. In early clinical studies the administration of OKT3, with or without basic immunosuppression, led to the appearance of antibodies to OKT3 together with a high rate of recurrent rejection [7, 33, 34, 47, 48, 99,105, 135,148,177, 196, 223]. The percentage of patients that became sensitized to OKT3 after one complete course varied between 50 and 100. Usually, these antibodies appeared between 9 days and 4 weeks after the initiation of OKT3 treatment. Further characterization of these antibodies reveals a restriction to two categories: anti-idiotypic antibodies, which are reactive with the antigen binding site of OKT3, and anti-

isotypic antibodies, which are reactive with the heavy chain [3, 33-36, 56, 99, 100, 129, 196, 199]. The majority of the antibodies to OKT3 are anti-idiotypic antibodies [34, 100, 129, 196, 199]. Anti-idiotypic antibodies may abrogate the therapeutic effect of OKT3 in two ways: they can inhibit the binding of OKT3 to T lymphocytes and they can accelerate the elimination of OKT3 [3, 34]. Both result in a reappearance of CD3 § cells and subsequent allograft rejection [34]. Anti-isotypic antibodies generally do not interfere with the immunosuppressive activity of OKT3 [3, 34]. Surprisingly, despite the occurrence of antibodies, no signs of immune complex disease and only one case of IgE-mediated anaphylaxis have been reported during OKT3 treatment [143]. From several clinical studies it has become clear that a second or third course of OKT3 can still be effective in reversing acute renal allograft rejection, provided no high-titer antibodies to OKT3 are present [7, 64, 65,174]. Generally, the success rate of retreatment with OKT3 varies inversely with the total antibody titer: in patients with low titers ( < 1:100) the rejection-reversal rate averages 90 %, whereas in patients with higher antibody titers the percentage of rejectionreversal varies between 0 % and 25 %. Some authors report an increased depletion of CD3 by doubling or tripling the dose of OKT3 in patients with low-titer antibodies [43, 64], although this was not confirmed by others [143]. Overall, there is a strong inverse correlation between titer of antibodies to OKT3, OKT3 levels, the extent of CD3 depletion, and the rate of rejection reversal. However, some patients have been reported in whom acute cellular renal allograft rejection occurred during OKT3 treatment, while at the same time no antibodies to OKT3 could be demonstrated, OKT3 trough levels were in the therapeutic range, and there was effective CD3 modulation [83,123]. It also became clear that the occurrence and titer of antibodies to OKT3 varies inversely with basic immunosuppression during OKT3 treatment [53, 77, 78, 90, 9496, 140, 141,169, 170]. In general, it is advisable to determine whether a patient has mounted an antibody response after the first course of OKT3. In patients with low antibody titers or a purely isotypic antibody response treatment with OKT3 can be repeated, while in patients with high titers of antibodies alternative antirejection therapy should be used. During retreatment with OKT3 the amount of CD3 + cells should be carefully monitored; if CD3 depletion is insufficient, an increase in OKT3 dosage should be considered. Development of other monocional antibodies for future use in renal transplantation

The rationale for the development of new mAbs to be used as immunosuppressive agents in renal transplantation is to evade the main hazards of OKT3 treatment, namely side effects - both first-dose related and long-term - and xenoimmunization. As we have already mentioned, the complex of symptoms after the first administration of OKT3 is at least partly caused by T-cell activation, probably as a result of interaction with FcR on monocytes [1, 31, 36, 37, 39, 52, 150,

239 151, 164]. Murine anti-CD3 mAbs of different (sub)classes differ in their mitogenic properties in the following way, according to the presence or absence of proper FcR on human monocytes: IgG1 mAbs cause Tcell activation in 70 % of human donors, IgG2a in 100 %, IgG2b in 10 %, IgA and F(ab')2 fragments not at all [221]. Therefore, mAbs that do not interact with FcR, such as for instance IgA mAbs or F(ab')2 fragments, may result in a better tolerance. This hypothesis is supported by the observation that anti-CD3 mAbs injected into mice caused T-cell activation and serious toxicity, while their F(ab')2 fragments did not [89, 91, 92]. To our knowledge no antiCD3 F(ab')2 fragments have been tested in humans yet. Theoretically, a mAb that specifically targets those T cells involved in allograft rejection may leave physiological host immune surveillance at least partly intact and thus decrease the risks of immunosuppression. Antigenic stimulation induces T cells to synthesize IL-2 and at the samemoment to express high-affinitymembrane receptors for IL-2. The binding of IL-2 with its specific membrane receptor is a potent stimulus for T-cell activation [165, 211]. Thus, IL-2 production and the induction of IL-2 receptor (IL-2R) positive cells are supposed to play a major role in acute allograft rejection [158, 173,206]. In animal studies mAbs that are reactive with the IL-2R have been shown to be effective in preventing acute allograft rejection [109,117, 159, 173]. Similar results have been reported on anti-CD4 and anti-CDSmAbs [46, 101-103,216]. The problem of xenoimmunization can theoretically be circumvented by using humanized mAbs, although this may not prevent an anti-idiotypic response. In highly sensitized patients the subsequent use of mAbs directed to different epitopes on the CD3 molecule and which do not cross-react with anti-idiotypic antibodies to OKT3 may again result in improved CD3 depletion. We shall briefly discuss here some newly developed mAbs that are currently being tested in renal transplant patients or are considered very promising in this respect. First, we shall mention some newly developed mAbs that are, like OKT3, reactive with the CD3/TCR complex and thus with all T cells. Next, Campath-lM will be discussed. Finally, we shall mention some mAbs that are reactive with more restricted T-cell markers such as IL-2R, CD4, and CD8.

MAbs that are reactive with the CD3/TCR complex

WT 32 is a murine mAb of the IgG2a subclass that is reactive with the CD3 molecule. In a small clinical study rejection reversal was achieved in seven out of eight renal transplant patients with acute rejection [195]. Recently prophylactic administration of WT 32 was also demonstrated to be effective after renal transplantation [66]. Side effects and xenoimmunization were more or less comparable to those induced by OKT3. Blocking assays in vitro suggest that previous immunization to WT 32 does not interfere with the binding of OKT3 to the CD3 molecule [136, 137]. Therefore, WT 32 may be a good alternative, especially in patients with high titers of anti-idiotypic antibodies to OKT3.

CLB-T3/4.A is a murine mAb of the IgA class that is reactive with the CD3 molecule [10, 127]. Its avidity for the CD3 molecule is approximately ten times as high as that of OKT3. A preliminary study in chimpanzees has demonstrated that the administration of CLB-T3/4.A causes a depletion of CD3 + cells without side effects and without release of IL-6 [152]. This is consistent with the presumed relation between FcR interaction, T-cell activation, and toxicity of anti-CD3 mAbs. Therefore, CLB-T3/4.A may possess the immunosuppressive properties of OKT3, while it will probably have fewer side effects. No clinical data are available as yet. RIV 9 is a murine mAb of the IgG3 isotype that is reactive with the CD3 molecule. Experiments in vitro revealed that RIV 9 binds via its Fc moiety to the same monocyte FcR as OKT3 (which is an IgG2a) [27]. Recently five renal transplant patients were prophylactically treated with RIV 9 [205]. Side effects and T-cell depletion were more or less comparable to those of OKT3. Since antibodies provoked by RIV 9 did not cross-react with OKT3, sequential therapy seems possible. BMA 031 is a murine mAb of the IgG2b class that is reactive with the TCR [119]. In vitro an IgG2b mAb activates T lymphocytesin only 10 % of all humans. This is consistent with the observation that administration of BMA 031 to renal transplant patients was not accompanied by major side effects [38, 87, 121, 180]. However, a prophylactic course of BMA 031 over a period of 7 days after transplantation in conjunction with cyclosporine, azathioprine, and 9 steroids resultedin a rejection frequency of 78 % (of which 44 % occurred during or immediately after treatment). Immunologic monitoring revealed only a transient CD3 depletion during BMA 031 treatment. A modified approach, by which BMA 031 was given in a tenfold dose intraoperatively and 48 h later, yielded more promising results: a nearly complete T-cell depletion was observed that lasted more than 5 days [180]. During a follow-up period of between 2 weeks and 4.5 months no rejection episodes occurred and the tenfold dose was also tolerated excellently. In immunologically high-risk patients the high-dose approach resulted in a 1-year graft survival rate of 67 %, compared to 79 % in a historical control group that was treated with ALG/ATG [57]. Blocking assays in vitro suggest that previous immunization to BMA031 does not interfere with the binding of OKT3 to the CD3 molecule [136], which would allow sequential therapy. T10B9.1A-31A is a murine IgMk mAb that is reactive with the TCR [208, 209]. Unlike OKT3, it is not mitogenic in vitro and in the presence of complement it is lytic for T lymphocytes. In a small clinical study, T10B 9.1A-31A was comparable to OKT3 in reversing acute rejection [210]. Immunologic monitoring revealed comodulation of the TCR and the CD3 molecule. Side effects were similar though milder than those of OKT3 and could be suppressed by pretreatment with steroids. In two patients re-rejections occurred that were successfully treated with OKT3. Indeed, the fact that T10B 9.1A-31A and OKT3 are of different isotype and idiotype opens perspectives for sequentialtherapy.

240 Campath-l M

Campath-lM is a rat mAb of the IgM class that is able to fix human complement and that recognize more than 95 % of human peripheral mononuclear cells (T cells, B cells, and monocytes) [68, 84]. It has been evaluated as a prophylactic agent following renal allografting and has been shown to be a powerful immunosuppressant: patients who received a 10-day course of C a m p a t h - l M immediately postoperatively, in addition to cyclosporine, showed a significantly lower incidence of early acute cellular rejection than control patients who received cyclosporine alone. Immunologic monitoring revealed a very rapid but transient lymphocyte depletion that was ascribed to cell lysis. Side effects were usually mild and consisted of pyrexia and pruritus. However, a disturbingly high incidence of serious infections was noted in the Campath-lM treatment group compared to the cyclosporine group, probably as a result of the mAb's broad specificity. Thus, Campath-lM may be very effective as a prophylactic and antirejection agent after renal transplantation, but the high rate of infection will probably limit its extensive use in clinical practice.

MAbs that are reactive with more restricted T-cell markers such as IL-2R, CD4, and CD8

33B3.1 is a rat mAb of the IgG2a isotype reactive with the 55 kD o~ chain of the IL-2R. Since 1987 several clinical studies have been performed with 33B3.1 [19-22, 118, 181-184]. It appeared that a prophylactic 2-week course of 33B3.1, started immediately following transplantation, was more effective in preventing early rejection than prednisone and azathioprine given in a historical control group of renal transplant patients, and equally effective compared to A T G induction [19, 154]. A randomized controlled trial of 33B3.1 versus A T G induction showed that both were equally effective in preventing acute renal allograft rejection [22, 184]. Furthermore, the incidence of infections was lower in the 33B3.1 than in the ATG group. Sensitization to 33B3.1 occurs in about 80 % of patients after one full prophylactic course [184]. Results of a therapeutic course of 33B3.1 are less convincing: when given as a treatment for acute rejection 33B3.1 only reversed six of ten rejection episodes [20, 21]. Therefore, 33B3.1 is considered less efficient for treatment than for prevention of acute kidney rejection. Anti-Tac is a murine mAb of the IgG2a isotype, reactive with the 55 kD c~ chain of the IL-2R. Since 1989 several studies have been conducted with anti-Tac [25, 110, 157, 212]. It was shown that a prophylactic 10-day course of anti-Tac, started on the 1st post-transplant day, was more effective in preventing acute renal allograft rejection than conventional immunosuppression alone, consisting of prednisone and cyclosporine with or without azathioprine [25,111,112]. No major side effects were reported. However, another study using anti-Tac, azathioprine, and prednisone for the 1st post-transplant week versus standard triple therapy was terminated when four of the first

five patients treated with anti-Tac developed acute rejection episodes [110, 118]. Thus, without concomitant cyclosporine anti-Tac does not seem to prevent rejection. Fortunately, the fact that each of these rejection episodes was reversed with OKT3 suggests that the use of anti-Tac does not preclude subsequent treatment with OKT3. Immunologic monitoring during anti-Tac prophylaxis revealed that despite adequate levels of anti-Tac, no removal or cytodestruction of IL-2R positive cells flom the circulation occurred [25,110,157]. Before and 4 days after anti-Tac treatment in vitro alloreactivity of T lymphocytes was unchanged [25]. It has therefore been suggested that the effect of anti-Tac is limited to the period of administration, with recovery after a few days' lag period. BL4 is a murine m A b of the IgG2a isotype that is reactive with the human CD4 molecule. Twelve renal transplant patients received 10-15 mg BIA prophylactically for 3-14 days together with prednisolone and azathioprine [133]. No side effects were reported. Acute rejection occurred in four patients and was suspected in four other patients. Following BL4 infusion a partial and transient CD4 depletion was observed. The inability to achieve immunosuppression in this study was ascribed to an insufficient dosage of BL4. Leu 2a is a murine mAb of the IgG1 subclass that is reactive with the CD8 molecule. In a small clinical study rejection reversal was achieved in four out of six renal transplant patients with acute steroid resistant rejection [216]. Only minor side effects were reported, such as fever and diarrhea. No more clinical data are available as yet. Acknowledgements. We are grateful to Dr. J.M.Witmink, Dr. R. J. M. ten Berge, and Dr. W. Zeylemaker for critically reading this manuscript, and to Mrs. J. M. Cloos and Mrs. Y. Robberse for secretarial assistance.

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