Treatment of acute kidney allograft rejection with a non-mitogenic CD3 antibody

Abstract

T3/4.A is a non-mitogenic murine IgA mAb to human CD3 that was selected for clinical studies to provide an alternative for the mitogenic, T cell-activating, therapeutic mAb OKT3. Previously, we reported that T3/4.A is better tolerated in humans than the IgG2a-CD3 mAb T3/4.2a. Here we report the results of a phase II clinical trial to assess the immunosuppressive potential of T3/4.A. Eighteen first kidney transplant recipients with a first rejection episode were included. Baseline immunosuppression consisted of cyclosporin and prednisolone. Rejection treatment consisted of 5 mg mAb per day during 10 days. Fourteen patients responded, of whom four experienced a second rejection within 2 weeks, one experienced chronic rejection after 2·5 years, whereas the others remained rejection-free after treatment (median duration of follow-up 42 months). Four patients did not respond and eventually lost their graft. These results are similar to treatment results with OKT3, as reported in the literature. Following the first dose of T3/4.A, side effects were limited, and reduced compared to OKT3-treated controls. On the second day, 15 patients developed transient vomiting and/or diarrhoea, which coincided with elevated serum levels of proinflammatory cytokines. Minimal or even no side effects occurred during the remaining days, which is in sharp contrast to that seen generally during OKT3 treatment. Both T cell numbers and TCR expression were reduced during the therapy. We conclude that T3/4.A is a good alternative for OKT3 to treat rejection episodes in renal transplant recipients.

INTRODUCTION

OKT3 is a powerful immunosuppressive drug that is used almost exclusively in transplantation medicine. However, its use is hampered by severe side effects, including dyspnoea, fever, chills, headache, gastrointestinal symptoms, myalgia and arthralgia. Although these side effects are not restricted to the first day of treatment, they generally are maximal in the 12 h following the first administration and are therefore termed ‘first-dose reaction’. This first-dose reaction results from activation of T lymphocytes [1] and possibly of Fcγ-receptor I-bearing accessory cells, and from activation of the complement system [2]. All these processes combined induce cytokine release, neutrophil activation and activation of coagulation and fibrinolysis [2–4].

T lymphocyte activation in vivo is paralleled by mitogenicity in vitro [4,5]. Therefore, several groups have identified or designed non-mitogenic TCR/CD3 mAbs for clinical use [4,6–10]. CLB-T3/4.A (hereafter: IgA-CD3), a murine IgA antihuman CD3 monoclonal antibody (mAb), is non-mitogenic for human peripheral blood mononuclear cells (PBMC) in vitro. The mAb was developed as a switch variant antibody, derived from an originally IgG1-producing hybridoma [11]. Because of its lack of mitogenicity, the mAb was selected for clinical studies to provide a non-mitogenic alternative for the mitogenic CD3 mAb OKT3. Previously, we have reported that IgA-CD3 is better tolerated in humans than the switch variant antibody CLB-T3/4.2a, which is a murine IgG2a mAb, as is OKT3 [4]. Here, we report the results of a phase II clinical trial to assess the immunosuppressive efficacy of IgA-CD3 in adult patients who suffered from a first rejection episode after a first kidney transplantation.

PATIENTS AND METHODS

Inclusion of patients

Eligibility for this trial was based on the diagnosis of a first acute rejection episode in adult patients that had received a first kidney transplant. Exclusion was based on the following criteria: (1) presence of overhydration (>3% above dry weight), (2) serious infection, (3) earlier treatment with murine antibodies, (4) severe HLA sensitization (>80% panel reactive antibodies prior to transplantation), (5) ongoing primary non-function of the transplant, (6) hydronephrosis and/or (7) cyclosporin toxicity. Diagnosis of acute rejection was based on clinical and laboratory criteria (two of six: rise in plasma creatinine concentration, decreased urine production, rise in body temperature, eosinophilia, papillary oedema on ultrasound examination and/or decreased uptake in renal scintigraphy), and was followed by a core biopsy in all patients. Biopsies were scored according to the CCTT criteria, blindly and independently by two pathologists [12,13].

Written consent was obtained from all patients and the study was approved by the local ethical committee.

Preparation of therapeutic grade mAb

IgA-CD3 (CLB-T3/4.A) has been described previously [11]. For therapeutic use, the hybridoma was cultured in vitro. Harvest concentrates were purified on CM-sepharose, dialysed and filtered. Virus inactivation was performed by Solvent Detergent treatment. Thereafter, the product was purified further on Q-sepharose. Safety testing included in vivo testing in rabbits for pyrogenicity and unforeseen toxicity. Endotoxin was <0·23 EU per mg IgA. The product was formulated at 1·0 mg/ml in PBS with 1% human serum albumin added for stabilization.

Treatment protocol

All patients received prednisolone and cyclosporin as basic immunosuppression. Dosage of prednisolone was based on a fixed scheme (first and second postoperative day: 50 mg twice a day, from the third postoperative day 10 mg per day); dosage of cyclosporin was started i.v. (3 mg per kg per 24 h on the first and second postoperative day), then continued orally from the third postoperative day (Neoral, 10 mg per kg). Thereafter, the dose was adjusted according to whole blood trough levels (target levels between 100 and 200 ng/ml, as measured by radioimmunoassay).

First rejection episodes were treated with IgA-CD3, 5 mg per day for 10 days, given as an intravenous injection. Analysis of drug levels in the 15 patients who completed the course gave the following pharmacokinetic profile [14]: mean through levels of mAb increased during the first week from 41 ng/ml preceding the second dose to 316 ng/ml preceding the eighth dose, and decreased thereafter to 169 ng/ml at 24 h after the 10th dose. Except on the first day, monoexponential elimination kinetics were observed, with mean plasma half-lives ranging from 6 to 8·5 h. Mean peak levels at 30 min were about 1400 ng/ml above the preceding trough levels. On the first day, mAb levels showed a biphasic plasma disappearance curve, suggesting a distribution phase. The mean peak level at 30 min on the first day was 997 ng/ml. Within 1 h preceding the first mAb administration, 500 mg of methylprednisolone (i.v.) and 25 mg of promethazine (p.o.) were given, as is recommended for OKT3.

At the latest, after 7 days of experimental treatment, the clinical effect was evaluated. When refractory to the experimental treatment, the rejection episode was treated further with corticosteroids (500 mg methylprednisolone i.v. per day for 6 days).

Clinical assessment and control group

Response to therapy was evaluated based on the change in plasma creatinine concentration. Return of the plasma creatinine concentration to maximally 125% of the prerejection value, within 2 weeks after cessation of treatment, was considered to represent successful treatment [15]. Return of the plasma creatinine concentration to between 125% and 175% of the prerejection value was labelled as a partial success. All other cases, including all rerejections within 2 weeks after the last dose of IgA-CD3, were labelled as treatment failures.

Side effects were evaluated semiquantitatively, as described [16]. In short, patients were observed and questioned for the presence of (1) fever above 38·5°C, (2) chills, (3) dyspnoea with increased breathing frequency, (4) nausea or vomiting, (5) diarrhoea and (6) headache, in the time intervals ranging from 0 to 3 h, 3–6 h, 6–12 h, 12–24 h, 24–48 h and 48–72 h after the first administration. Each symptom was scored 1+. In this way, the side effects score ranged from 0 to 6 in a given time interval. We compared our patients with a historical group of patients who had been treated a few years previously with OKT3 for histologically proven acute kidney allograft rejection, and who had been evaluated prospectively according to this same scoring system in the frame of another study. For the control group, the same exclusion criteria were used that we apply here and the patients received the same basic immunosuppressive therapy. In addition, the control group received the same premedication (500 mg methylprednisolone and 25 mg promethazine) and had the same dosing regimen of the monoclonal antibody (5 mg per day for 10 days).

Immunological monitoring

Blood samples were drawn prior to IgA-CD3 treatment, during the therapy and in decreasing frequency after cessation of the therapy. EDTA-anticoagulated blood was used to determine white blood cell counts and differential counts. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood and used for flowcytometry. We used fresh unfixed cells. Flowcytometry was performed on a FACScan flowcytometer (Becton Dickinson, Erembodegem, Belgium). Analysis of flowcytometry data was performed with PC-LYSYS software (Becton Dickinson). The following mAbs were used: CD4-PECy5 and CD8-PECy5 from Dako; CD45-FITC + CD14-PE (Lymphogate), CD3-FITC (Leu-4) CD4-FITC, CD8-FITC, CD69 FITC, CD25 PE and irrelevant isotype control mAbs, as well as PE-labelled streptavidin, from Becton Dickinson; TEPC15 (mouse IgA) from Sigma (St Louis, MO, USA); antimouse kappa rat-IgG1-FITC from Immunotech (Marseille, France); irrelevant rat-IgG1-FITC from Pharmingen (San Diego, CA, USA); CD45RA FITC and biotinylated CD27 from CLB (Amsterdam, the Netherlands).

For analysis of lymphocyte subsets (CD4^pos: T helper cells; CD8^bright: cytotoxic T cells), a lymphocyte gate was drawn, based on forward scatter and side scatter. The total percentage of lymphocytes (CD45^bright, CD14^neg) within the lymphocyte gate was determined. Subset percentages were divided by the total lymphocyte percentage in the gate and multiplied with the absolute lymphocyte count to obtain absolute subset numbers. Naive, effector and memory subsets were defined as described [17–19]: naive cells are characterized by expression of both the CD45RA isoform and CD27, recently activated and effector type cells are CD27^neg, whereas memory cells have lost the CD45RA isoform, but do express CD27. For analysis of these subsets, CD4^pos and CD8^bright lymphocytes were gated, based on FSC, SSC and fluorescence, and of these the distribution over the four compartments as defined by the expression of CD45RA and CD27 was calculated.

For analysis of TCR/CD3 complex expression, two CD3 mAbs were used that bind in a mutually exclusive way to the CD3 e-chain: the mouse-IgG1 mAb Leu-4-FITC (control: irrelevant mouse-IgG1-FITC) and IgA-CD3 (control: TEPC15). Binding of IgA-CD3 and TEPC15 was detected with antimouse kappa rat-IgG1-FITC (control: rat-IgG1-FITC). CD3 antibodies and secondary antibodies were used each time in combination with either CD4-PECy5 or CD8-PECy5 antibodies, to identify T lymphocytes irrespective of TCR/CD3 expression. To permit double staining, the secondary antibody step (antimouse kappa-FITC) was followed by a blocking step with normal murine serum.

Free CD3-antigen was detected with Leu-4-FITC. IgA-CD3 bound in vivo (‘opsonization’) was detected with antimouse kappa-FITC. Total CD3-antigen was detected with antimouse kappa-FITC after prior in vitro saturation with IgA-CD3.

For analysis of activation markers, CD4^pos and CD8^bright lymphocytes were gated, based on FSC, SSC and fluorescence, and of these the positive proportions for CD25 and CD69 were calculated.

Serum was collected and stored at −80°C until further use in cytokine ELISAs. Interleukin (IL)-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α were determined with commercially available ELISA-kits (Duoset™ from Genzyme, Cambridge, MA, USA, Pelikine™ from the CLB and EASIA™ from Medgenix, Billerica, MA, USA, respectively). IL-6 [20] and IL-10 [21] were measured as described previously.

Data analysis

Where applicable, results are presented as a function of time. Time ‘zero’ was defined as the moment of the first injection of IgA-CD3. In addition, the first interval of 24 h was defined as ‘first day’, etc. Statistical analysis was performed with the Wilcoxon matched-pairs signed-rank sum test for paired observations and the Mann–Whitney test for independent samples. A probability (P) value <0·05 was considered to be significant.

RESULTS

Clinical results (1): IgA-CD3 is immunosuppressive

Eighteen patients with a first rejection episode were included. Basic characteristics are summarized in Table 1. The mean time elapsed from the day of transplantation until the first rejection episode was 16 days (range 5–47). Evaluation of the kidney biopsies revealed evidence of acute rejection according to the CCTT criteria in 17 cases (10 type I, six type II, one type III). In one patient, the biopsy did not contain representative material. However, a biopsy taken at the end of the therapy showed type III rejection, and this patient eventually lost his kidney.

Table 1.

Baseline characteristics

Sex (male/female)	11/7
Recipient age (mean, range)	42 years (18–61)
Recipient race (negroid/other)	2/16
Donation (living/postmortem)	3/15
Cold ischaemia time (mean, range)	23 h (2–46)
HLA mismatches (mean)
A	0·72
B	0·89
DR	0·83
Underlying kidney disease
Chronic glomerulonephritis	5
Hypertension	5
Polycystic renal disease	2
Unknown cause	6

Fifteen patients received a full treatment course of IgA-CD3; three patients were switched to corticosteroids after 5, 6 and 7 days, respectively, as they showed deterioration of renal function during the experimental therapy. A fourth patient was switched to corticosteroids after 10 days, because the experimental treatment course did not result in improvement of renal function.

In all four non-responding patients, corticosteroid therapy was not effective and graft loss occurred. Biopsy findings at the start of IgA-CD3 therapy were not related to failure: two of the four had a type I rejection, one had type II, and the fourth patient's biopsy yielded no representative material. As mentioned above, a second biopsy in this last patient, taken after 10 days of IgA-CD3 treatment, showed a type III rejection. At nephrectomy, performed after unsuccessful corticosteroid therapy, three of the four had type III rejection. The fourth kidney showed type II rejection, together with a massive thrombosis of the renal artery, and infarction.

Fourteen patients showed improvement of renal function in response to the treatment. Of them, four experienced a second rejection within 2 weeks of cessation of the experimental treatment. These rejections were treated successfully with corticosteroids, after which a third maintenance immunosuppressive drug was added. All survived with a functioning transplant.

Of the remaining 10 responding patients, one died with functioning graft, one had recurrence of IgA nephropathy 10 months after successful rejection treatment and died after return to dialysis, and one patient developed chronic rejection and returned to dialysis 30 months after the treatment. Median follow-up time was 42 months.

In summary, 14 of 18 patients initially responded. However, according to the strict criteria for response to therapy, as mentioned above, the treatment was successful in eight of 18 patients, partially successful in two of 18 patients and failed in eight of 18 patients. At 1 year, patient survival was 100%, graft survival was 78% (14/18) of whom 11 had a creatinine clearance of more than 50 ml/min (two of the three surviving with a creatinine clearance less than 50 ml/min had a clearance less than 50 ml/min from the start).

Clinical results (2): IgA-CD3 induces less side effects than OKT3

In the 3 h following the first administration of IgA-CD3, fever or chills were observed in 15 of 18 patients. Headache and vomiting were infrequent and dyspnoea, which is observed frequently with the use of OKT3, was observed only once. The median side effects score in the first 3 h was two, and thereafter zero during the remainder of the day. This represents a remarkable reduction in toxicity compared with the control group of OKT3 treated patients (Fig. 1), and confirms the results of our previous study with IgA-CD3, where the mAb was compared to the isotype switch variant T3/4·2a (murine IgG2a CD3 mAb) [4].

Fig. 1.

Side effects scores: mean of the 18 patients described in this paper compared with 10 patients treated with OKT3. Statistically significant differences (Mann–Whitney) are indicated by *. 0 refers to side effects score zero in the IgA-CD3 group and ? refers to missing data on day 3 in the OKT3 group.

Following the second administration, no side effects were observed until 5 to more than 12 h after. By that time, 15 of 18 patients developed vomiting or diarrhoea, accompanied by fever or chills in five patients. In two of these five patients hypovolaemia, hypotension and anuria occurred. These symptoms also rapidly subsided. In the control group, side effects beyond the second day had not been scored prospectively (Fig. 1). However, as is generally known, most patients remain bedridden during the whole OKT3 treatment course. Specifically, some patients suffer most from gastrointestinal side effects, others most from headache and myalgia. In contrast, all IgA-CD3 treated patients were mobilized and without side effects in the second part of the treatment course.

T cell numbers and TCR/CD3 expression

Total lymphocyte numbers in peripheral blood decreased dramatically within 5 min after the first administration of IgA-CD3 (data not shown). At 30 min, FACS analysis showed that this decrease was due almost exclusively to a decrease in T cell numbers (Fig. 2a). T cells were enumerated on the basis of CD4 and CD8 expression, as the expression of TCR/CD3 on the remaining T cells decreased to such an extent that they could not be identified reliably with TCR or CD3 monoclonal antibodies (see below). During treatment, T cells gradually reappeared with reduced expression of TCR/CD3, and from the eighth day onwards T cell numbers before dosing were about 80% of pretreatment values. On the second, fourth and tenth days, when sequential FACS analysis was performed, we again observed T cell depletion from the peripheral blood which was, in contrast to the first day, only partial.

Fig. 2.

T cell numbers (a), CD4/CD8 ratio (b) and CD4^pos T cell numbers (c) in 15 patients who completed the course, and naive/memory ratio in CD4^pos T cells in 14 patients (d) as one patient did not lose the CD45RA isoform upon activation. In panels (a–c), cell numbers or ratios are significantly different from pretreatment values until t = 5, and in panel (d) until t = 4 (Wilcoxon). Post-treatment values were not significantly different from pretreatment values (Wilcoxon).

Subset analysis of T cells showed that CD4^pos T cells were somewhat more susceptible to depletion than CD8^pos T cells (Fig. 2b). In addition, naive T cells were more susceptible to depletion than memory T cells (Fig. 2c,d and data not shown). By the end of treatment, the pretreatment balance between the subsets was restored. CD27^neg cells (both CD45RA^pos and CD45RA^neg) comprised only about 10% of cells throughout the treatment course (data not shown). No relation was found between the extent of depletion of T cells or of T cell subsets and the response to therapy (data not shown).

During the treatment, TCR/CD3 expression (both ‘total’ and ‘free’) was decreased (Fig. 3a–d). We observed little binding of murine IgA to T cells, as measured by direct labelling with antimurine kappa antibodies (Fig. 3e,f). Apparently, TCR/CD3 complex internalization upon binding of the IgA-CD3 mAb is so swift that only on the first day of treatment, at 30 min, could we observe significant ‘opsonization’ of T cells. Alternatively, ‘opsonization’ could be invisible because ‘opsonized’ cells disappear immediately from the peripheral blood. Within 1–2 weeks after cessation of the treatment, TCR/CD3 expression returned to normal (data not shown).

Fig. 3.

TCR/CD3 expression: phenotypic changes in CD4^pos T cells at 72 h (preceding the fourth mAb dose, b,d,f) compared to t = 0 (a,c,e). (a,b) free CD3; (c,d) total CD3; (e,f) opsonization. Histograms of one representative individual, showing fluorescence of relevant antibodies (black line) and of control antibodies (grey line) on the x-axis, and cell number on the y-axis.

Inflammatory response: humoral and cellular markers

The side effects that were described above coincided with waves of cytokine release: a first peak of cytokine release was observed around 1 h after the first dose, a second peak around the evening of the second day. In addition, the side effects corresponded with cellular activation.

Based on what is known of the pathogenesis of OKT3-related side effects, we measured TNF (TNF-α), IFN-γ, IL-2 and IL-6 (Fig. 4a–d). In addition we measured IL-10, which acts as an antagonist for TNF and is a prototype anti-inflammatory cytokine (Fig. 4e). TNF and IL-10 were the first cytokines to appear in the bloodstream (within 30 min after the first dose of IgA-CD3); immediately thereafter, IL-2 could be detected. IFN-γ and IL-6 followed slower kinetics. On the second day, all cytokines were released again, peak levels being reached 6–12 h after the dose. In all patients, second-day levels of TNF and IL-10 were much lower than first-day levels. In contrast, IL-2, IFN-γ and IL-6 peak levels on the second day were higher than on the first day. The highest IL-2 concentration of 14000 pg/ml was measured in the evening of the second day in one of the two patients who developed hypovolaemia and anuria. However, in general no relation was found between individual side effects on either day and individual cytokine levels. Also, TNF/IL-10 ratios did not correlate with individual side effects on both days.

Fig. 4.

Cytokines in serum after administration of IgA-CD3 in 18 patients. On the evening of the second day some samples were missing, as indicated. (a) TNF (TNF-α); (b) IFN-γ; (c) IL-2; (d) IL-6; (e) IL-10. In all panels, peak levels on both days were siginificantly increased as compared to predose levels (Wilcoxon).

Besides cytokine release, induction of activation markers on peripheral blood T cells also demonstrated enhanced activity of the T cell system. Figure 5 shows the induction of early (CD69) and late (CD25) activation markers on peripheral blood T cells. By the second half of the treatment course, when a return of T lymphocytes was observed, expression of the activation markers decreased again, although in the majority of patients it returned to baseline only after cessation of therapy.

Fig. 5.

Activation markers: phenotypic changes in CD4^pos T cells at 48 h (preceding the third mAb dose, b,d) compared to t = 0 (a,c). (a,b) CD69; (c,d) CD25. Histograms of one representative individual, showing fluorescence of relevant antibodies (black line) and of control antibodies (grey line) on the x-axis, and cell number on the y-axis.

DISCUSSION

In this paper we show that IgA-CD3, which was previously shown to have remarkably fewer side effects than an IgG2a CD3 mAb, is immunosuppressive and can reverse acute rejection episodes after renal transplantation. Our findings are in agreement with those of others who worked with non-mitogenic CD3 antibodies [8–10]. It is difficult to compare our treatment results to those obtained with OKT3 in the literature because of differences in basic immunosuppression. In the earlier literature, including the Ortho-trial [22], OKT3 was given as rejection treatment to patients on azathioprine/prednisolone maintenance therapy. In the subsequent literature OKT3 was given to patients on triple therapy, sometimes after quadruple induction, and seldomly as a first-line rejection treatment. Only Hirsch et al. [23] described 24 patients on cyclosporin maintenance therapy in whom OKT3 was used as first-line treatment of acute renal allograft rejection: 17 responded (71%, compare 14/18 or 78% in our group) and actuarial 6-month graft survival was 77% (78% in our group). Note also that most studies, in contrast to our study, record initial response rather than eventual success of treatment.

Which factors are responsible for the treatment failures is not immediately clear. Recently we described the pharmacokinetics and pharmacodynamics of IgA-CD3 in the same 18 patients [14]. The appearance of human antimouse antibodies might influence the pharmacokinetics and thus the clinical efficacy of IgA-CD3. Two weeks after the treatment, human antimouse antibodies were detectable in 14 of the 18 patients. However, we were not able to show their presence during the treatment [14]. We found no relation between antibody formation and pharmacokinetics or treatment failure and, in addition, responders and non-responders had similar pharmacokinetics. We studied the mechanistically important effect parameters of TCR/CD3 down-modulation and T cell depletion. We could describe accurately the relation between mAb serum concentrations and these effect parameters, but responders and non-responders were not different with regard to these parameters [14].

The cause of treatment failure may therefore be a predominance of either NK cells or monocytes/macrophages in the pathogenesis of these rejection episodes [24]. Also, non-immunological kidney damage may have obscured the issue.

The data on the immunological monitoring of the patients suggest once more that CD3 mAbs do not have a major lasting effect on the number of T cells in peripheral blood. As cell numbers rapidly returned to normal, with restoration of baseline subset distribution, large-scale cell death is improbable. The disappearance and reappearance of T cells is therefore probably caused by an effect of the mAb on T cell recirculation. We hypothesize that T cells, upon administration of IgA-CD3, become partially activated with expression of adhesion molecules [25] and migrate to the secondary lymphoid organs (naive and memory cells) or to peripheral tissues (memory cells only). Cells migrated to the secondary lymphoid organs probably encounter a more optimal co-stimulatory environment, which might contribute to further activation and cause them to remain in the secondary lymphoid organs for a while. The observation that naive CD4^pos T cells have a delay of a few days in returning to the peripheral blood, as compared to other subsets (Fig. 2c,d), supports this hypothesis [26].

Side effects on the first day of therapy were limited. Side effects on the second day of treatment, especially the fluid loss associated with fever and gastrointestinal symptoms were, in some patients, of greater clinical impact than the first dose reactions. Subjective evaluation by the patients themselves was in agreement with this observation.

Altogether, the side effects were not fully absent, as would be expected for a non-mitogenic CD3 mAb. For the first-dose effects our findings are in agreement with those of others, who also found a reduction but not absence of side effects [6–8]. The side effects on day 2, however, are largely unexplained and are without precedent. Why did we not observe these in our preceding study? First, a dosage difference exists: we gave here five times as much of the mAb (5 mg once a day instead of 0·5 mg twice a day), as no detectable serum concentration of the mAb was reached in the previous study [4]. Secondly, the patients in the previous study were treated prophylactically with the mAb, whereas now we treated acute rejection episodes, so that the mAb was entered into an already activated immune system.

The observed side effects were shown to coincide with release of TNF, IFN-γ, IL-6 and IL-10, as was shown previously for OKT3. To determine the source of the cytokine release, we also measured the T cell specific cytokine IL-2. In addition, we measured the expression of activation markers on T cells. The results indicate that T cells were activated by IgA-CD3. Previously, we showed that IgA-CD3 acts as a partial agonist in vitro and induces proliferation and cytokine production when in an appropriate co-stimulatory environment [27]. Above we indicated that secondary lymphoid organs could be such an environment. Naive cells that after partial activation upon the first administration migrated to the secondary lymphoid organs and that were activated further there might be the source of the second release of cytokines.

Compared to two other non-mitogenic mAbs used for treatment of renal allograft rejection [9,10], IgA-CD3 appears to have a more T cell activating profile. Although such an indirect comparison is always difficult because of differences between the studies, we can speculate about the causes. The antibody described by Woodle et al. [9] acts as a partial agonist in vitro, similar to IgA-CD3 [27,28]. Therefore, the difference in T cell activation in vivo is probably only a quantitative difference. In this respect it should be noted that IgA-CD3 has a high affinity for its target antigen, being about 10 times the affinity of OKT3 [29]. Initially, we thought of this as a benefit, as it would allow us to use lower dosages. However, this high affinity might contribute to the severity of the first dose reaction, as T cell signalling is concentrated within a few minutes.

IgA-CD3-treated patients experienced fewer side effects on the first day than OKT3 treated controls, but still had high cytokine concentrations. An explanation could be that with IgA-CD3 the complement system was hardly activated (unpublished data), whereas OKT3 induces significant complement activation, as was shown earlier by our group [2]. In addition, we hypothesize that IgA-CD3 and OKT3 differ in their capacity to release anti-inflammatory cytokines such as IL-10.

In conclusion, we report here that IgA-CD3 is a good alternative for OKT3 in the treatment of acute rejection episodes after renal transplantation, both by its similar efficacy and by its minor toxicity as compared to IgG2a CD3 mAbs. In order to diminish the toxicity further, we consider changing the dosing scheme in the future. Administration of OKT3 as a 2-h infusion has been shown to result in fewer side effects [30]. In the same way, continuous administration of IgA-CD3 might result in spreading the cytokine release and the concomitant side effects over a longer time interval, thereby reducing the side effects further. Continuous administration might also improve the efficacy [14]. To prevent or reduce antimAb antibody formation a human IgM construct, derived from the same hybridoma as IgA-CD3, might be worth studying [31].

In recent years, the position of mAbs in renal transplantation has changed. Whereas new humanized CD25 antibodies have become available for induction treatment, the use of OKT3 as a therapy for acute rejection has been minimized by the advent of new immunosuppressive agents, such as mycophenolate mofetil, tacrolimus and sirolimus. However, CD3 mAbs may be of use in autoimmune diseases, as has been shown in animal models [32] and recently in humans [33]. Therefore, the search for CD3 mAbs with minimal side effects remains of importance.