Guidelines for the optimal use of muromonab CD3 in transplantation.

Muromonab CD3 (OKT3), the murine anti-CD3 monoclonal antibody (mAb) of the IgG-2a class, directed against the CD3 molecule on the surface of human T cells, has proven to be a powerful immunosuppressive agent in solid organ transplantation and has been shown to be superior to high-dose steroids as first-line treatment of acute allograft rejection. It is comparable to antithymocyte globulin (ATG) in treating steroid-resistant rejection and it is also effective as rescue treatment in ATG-resistant rejection. However, OKT3 treatment is followed by a substantial percentage of re-rejections, most of which respond well to steroids. In the early post-transplant period, a prophylactic course of OKT3 can delay the onset of acute rejection, in particular in immunologically high-risk patients. First-dose-related adverse effects (pyrexia, dyspnoea, headache, nausea, vomiting and diarrhoea) of OKT3 are severe, but usually transient and seldom life-threatening, provided overhydration has been corrected and steroids have been given before the first administration. These adverse effects are partly attributed to the release of cytokines as a result of mononuclear cell activation. In addition, complement activation and subsequent activation of neutrophils may play a role in their pathogenesis. After exposure of patients to OKT3 an increased incidence of infections and malignancies has been reported. However, most likely this merely reflects the total burden of immunosuppression. Xenosensitisation represents an important limitation to OKT3 treatment, although a second or third course can still be effective in patients with low antibody titres. In practice, the initiation of OKT3 treatment should be preceded by correction of overhydration, if necessary by means of dialysis or ultrafiltration. According to the instructions from the manufacturer the first dose of OKT3 is preceded by paracetamol (acetaminophen), an antihistamine and intravenous (IV) corticosteroids, in an attempt to mitigate the first dose-related symptoms. A regimen consisting of administration of 2 IV doses of 250mg methylprednisolone, given 6 hours and 1 hour before the first OKT3 injection, followed by a 2-hour infusion of OKT3, is most effective in diminishing OKT3-induced adverse effects.

Different CD3/T cell receptor monoclonal antibodies have distinct capacities to induce adhesion of T lymphocytes to endothelium.

Murine CD3/T cell receptor (TCR) monoclonal antibodies (mAbs) induce immediate peripheral lymphocytopenias of different degree and duration. Lymphocytopenia is of short duration after the administration of immunoglobulin A CD3 mAb, but it persists much longer after the administration of immunoglobulin G2a CD3 mAb. Peripheral lymphocytopenia after the administration of WT31, a murine immunoglobulin G1 TCR mAb, appears to be dependent on the polymorphism of Fc(gamma)RIIa. In high responders, lymphocytopenia is comparable to that observed after immunoglobulin G2a CD3 mAb; in low responders, no lymphocytopenia occurs. In vitro, both immunoglobulin A and immunoglobulin G2a CD3 mAbs induce immediate activation of CD11a/CD18, with concomitant up-regulation of CD11b/CD18 on T cells, each of which is shown to be involved in the concurrent adhesion of T cells to endothelium. WT31 induces an immediate activation of CD11a/CD18 as well as T cell adhesion to endothelium in Fc(gamma)RIIa high responders only, interestingly without changes in the level of expression of CD11b/CD18. We conclude that the immediate occurrence of peripheral lymphocytopenia after the administration of CD3/TCR mAb is mediated by changes in the level of expression or avidity (or both) of adhesion molecules on T cells, whereas the persistence of this lymphocytopenia depends on the isotype of the CD3/TCR mAb and on the presence of suitable Fc receptors.

Administration of OKT3 as a two-hour infusion attenuates first-dose side effects.

BACKGROUND: Use of the murine CD3 monoclonal antibody OKT3 is limited by first-dose side effects, which are thought to be caused by the release of inflammatory mediators. Because these processes might be influenced by the speed of administration, we compared a 2-hr OKT3 infusion with the bolus infusion usually applied nowadays. METHODS: Eighteen renal allograft recipients were prophylactically treated with OKT3 and randomized to receive the first dose either as a 2-hr infusion or as an intravenous bolus infusion. Clinical side effects score and the occurrence of complement activation, cytokine release, and activation of neutrophils were determined. RESULTS: Two-hour infusion of OKT3 completely prevented the occurrence of dyspnea, reduced the incidence of other side effects, and attenuated complement activation. Cytokine release and depletion of peripheral blood lymphocytes were similar in both groups. CONCLUSIONS: Thus, complement activation seems to play an additional role in the development of side effects after the first OKT3 dose.

Increased dermal expression of ICAM-1 and VCAM-1 after administration of OKT3 in man.

OKT3 induces a systemic release of cytokines and a profound peripheral lymphocytopenia. In vitro, tumor necrosis factor-alpha, interleukin-1, and interferon-gamma increase adhesion molecule expression on vascular endothelium. To investigate the effects of OKT3 induced cytokine release on endothelial- and lymphocyte adhesion molecule expression in vivo, we studied sequential skin biopsies of six renal allograft recipients treated for acute rejection with 5 mg OKT3. An additional group of six patients treated for acute rejection with 50 mg methylprednisolone served as a control group. Compared to pre-treatment biopsies, biopsies taken 4.5- and 24 hours after the first OKT3 dose showed a maximal increase in VCAM-1 and ICAM-1 expression, respectively. In parallel, an increased number of CD2+, CD11a+, and CD49d+ mononuclear cells in the skin was observed in all OKT3 treated patients. No changes were observed after methylprednisolone treatment. We conclude that the OKT3 induced cytokine release induces increased ICAM-1- and VCAM-1 expression on vascular endothelium, leading to increased influx of CD2+ lymphocytes which may contribute to the peripheral lymphocytopenia after OKT3.

CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes.

The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.

Activation and increased expression of adhesion molecules on peripheral blood lymphocytes is a mechanism for the immediate lymphocytopenia after administration of OKT3.

We investigated the mechanism by which antihuman CD3 monoclonal antibodies of the isotypes IgG2a (eg, OKT3) and IgA (eg, IXA) can induce the rapid disappearance of virtually all circulating T lymphocytes. We hypothesize that upregulation of adhesion molecules on the lymphocyte membrane contributes to this effect. However, this hypothesis is difficult to test, because of the inherent lymphocytopenia and/or shifts in lymphocyte populations between intra and extra-vascular compartments. Therefore, studies in vitro were performed, as well. Analysis of peripheral blood lymphocytes isolated at several times after addition of OKT3 or IXA to whole blood of healthy individuals showed an immediate increase in the proportion of T cells expressing NKI-L16, an activation epitope on CD11a/CD18. Likewise, an increase in CD11b/CD18 expression occurred. In parallel experiments, a transiently increased adhesion of T cells to endothelial cell monolayers was observed. This adhesion could be completely blocked by anti-CD18 or anti-CD11a monoclonal antibodies and only partly by an anti-CD11b antibody. Our data indicate that upregulation of activation epitopes of CD11a/CD18, as well as increased expression of CD11b/CD18 on T lymphocytes, may result in increased adhesion of these cells to intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on vascular endothelium. This phenomenon may, at least, partly explain the rapidly occurring peripheral lymphocytopenia observed in vivo.

Biphasic granulocytopenia after administration of the first dose of OKT3.

The effects of the administration of OKT3, a second immunoglobulin G2a (IgG2a) anti-CD3 monoclonal antibody (mAb), and its isotype switch variant IgA on granulocyte kinetics were compared for 5 hours after the first administration of the mAb. In addition, in vivo and in vitro studies were performed on alterations in expression of CD11b and CD62L induced by these mAbs. Within 15 minutes after administration OKT3 and IgG2a anti-CD3 mAbs induced a significant decrease in circulating granulocytes, whereas IgA anti-CD3 mAb did not. Apparently the initial decrease in circulating granulocytes depends on the heavy chain of the administered anti-CD3 mAb, resulting in immunocytoadherence and sequestration in the lungs. Increased adherence to pulmonary endothelium by altered expression of CD11b and CD62L plays a minor role in this first granulocytopenia, because each mAb exerted the same effects on these adhesion molecules in vitro. The second decrease in granulocyte counts occurred 60 minutes after administration of each mAb and correlated with a significant increase in expression of CD11b and CD62L in vivo and with upregulation of CD11b and down-regulation of CD62L in vitro. These alterations could be related to the presence of tumor necrosis factor-alpha both in vivo and in vitro. Thus granulocyte kinetics from 30 minutes after administration of each anti-CD3 mAb resemble neutrophil kinetics induced by TNF-alpha.

Pretreatment with divided doses of steroids strongly decreases side effects of OKT3.

The aim of this study was to attenuate side effects of OKT3 by variation of the time interval between administration of corticosteroids and OKT3 in renal allograft recipients. In view of a maximal lymphocytopenia at six hours following MPNS, we postulated a greater preventive action on side effects from administration of methylprednisolone (MPNS) at six hours preceding the first dose of OKT3 compared to administration immediately before. Two groups of renal transplant patients treated for acute rejection with 5 mg OKT3 were studied. Ten patients received 500 mg MPNS six hours and ten patients one hour before administration of OKT3. We measured clinical side effects, body temperature, TNF and IL-6. There were no differences between the two groups regarding clinical side effects and peak body temperatures. However, MPNS administered six hours before administration of OKT3 diminished TNF release; MPNS one hour before decreased IL-6 release. We studied an additional group of six patients receiving 250 mg MPNS six hours before, followed by 250 mg one hour before OKT3. This group experienced significantly less side effects and lower body temperature. In addition, IL-6 levels were significantly decreased. We conclude that two times 250 mg MPNS administered six hours and one hour before the first administration of OKT3 effectively attenuates adverse reactions following administration of OKT3.