Tumor necrosis factor-alpha (TNFa)* is a cytokine released by macrophages and activated T cells that has been shown to be the primary cytokine responsible for the OKT3-induced cytokine release syndrome or acute clinical syndrome (OKT3-ACS)(1-5). This syndrome is manifested clinically by fever, chills, diarrhea, arthralgias, and other symptoms, as well as initial worsening of renal dysfunction in renal allograft recipients receiving OKT3 for rejection (6, 7). TNFa is known to be an important cytokine mediator of acute rejection as well(8-13) TNFR:Fc is a dimer of the p80 TNF receptor linked by the Fc portion of immunoglobulin that binds both TNFa and LT, thereby neutralizing their effects in vitro(14-17). We have previously demonstrated the efficacy of TNFR:Fc as an immunosuppressive agent in cynomolgus renal allograft recipients(15-17). There is evidence in animal and human studies that anti-TNF monoclonal antibody modulates OKT3-induced cytokine release and may be effective in preventing the OKT3-ACS(18, 19). TNFR:Fc has many features that make it more attractive for clinical use than anti-TNF monoclonal antibody. These include a higher affinity of TNFR:Fc for TNF, minimal immunogenicity, and the more physiologic nature of the human receptor versus a xenogeneic monoclonal antibody (14, 15) and unpublished data, Immunex). The purpose of this study was to investigate the therapeutic potential of TNFR:Fc in decreasing the severity of the OKT3-ACS and in restoring renal function in renal allograft recipients being treated with OKT3 for steroid-resistant rejection.
MATERIALS AND METHODS
Twelve renal allograft recipients diagnosed with steroid-resistant rejection were prospectively randomized to receive OKT3 alone or in conjunction with TNFR:Fc. Each patient received two boluses of 500 mg of methylprednisolone without improvement in renal function prior to initiation of OKT3. All rejection episodes were histopathologically confirmed. TNFR:Fc was provided by Immunex Research and Development Corp. Six patients were randomized to TNFR:Fc and six patients to OKT3 (controls). OKT3 was given as a single 5-10 mg i.v. injection daily for 10 to 14 days. All patients were given a bolus of 500 mg of methylprednisolone IV, as well as 350 mg of acetominophen and 25 mg of diphenhydramine orally prior to the first dose of OKT3. Five of the six patients in the TNFR:Fc received.05 mg/kg i.v. and one received.15 mg/kg i.v. TNFR:Fc was given as a single infusion 30 min prior to each of the first two doses of OKT3. Patients were evaluated clinically every 4 hr for manifestations of OKT3-ACS, and serum samples were obtained daily to monitor changes in BUN and creatinine. Specific clinical signs and symptoms monitored included fever, chills, diarrhea, vomiting, headache, arthralgias, and respiratory distress. In addition, serial serum samples were obtained preinjection and at 30 min, 1 hr, 2 hr, 4 hr, 12-16 hr, and 24 hr after the first two OKT3 doses and daily thereafter for the determination of TNFa antigenic levels (protein content) and TNF receptor protein levels and TNFa bioactivity. TNFa antigenic levels were assessed by ELISA (Endogen, Boston, MA) and TNFa bioactivity was determined by L929 assay(20).6 TNF receptor levels were measured by ELISA (Biosource International, Camarillo, CA). Methods of production and purification of TNFR:Fc have been previously published(14).
The total number of symptoms for the first two days of treatment was compared between groups using the one-sided Wilcoxon test. The occurrence of each symptom during the first two days was compared between groups using a one-sided Fisher's exact test. The serum creatinine expressed as a percentage of the initial value prior to initiation of OKT3 was averaged over the first six days of treatment. These values were compared between the two groups using the one-sided Wilcoxon test.
There were no adverse side effects seen in patients who received TNFR:Fc, other than those expected with OKT3-ACS. There was a significant difference in the frequency of chills between the two groups of patients. Three TNFR:Fc-treated patients had chills, while all control patients had chills(P<.05). There was also a significant difference in the duration of OKT3-ACS symptoms in the TNFR:Fc-treated group. TNFR:Fc patients had significantly fewer symptoms by day 2 of OKT3 therapy than controls(P=0.032). A summary of symptomatology monitored is shown inTable 1.
While there was some difference in symptomatology between the two groups, the rate of improvement in renal function was more dramatic. Reversal of renal dysfunction began in the first 24 hr in patients treated with TNFR:Fc, while the control patients had a mean delay of 48 hr with an initial worsening of renal dysfunction. The serum creatinine expressed as a percentage of initial creatinine was significantly lower in the TNFR:Fc-treated group during the first six days of therapy as compared with controls (P=0.032). Five of six patients who received TNFR:Fc had a decline in serum creatinine within 24 hr after receiving OKT3 while one remained the same. In the control group, four of six patients had worsening of renal dysfunction with an initial increase in serum creatinine within 24 hr after treatment.Figure 1 shows the rate of decline in serum creatinine as a percentage of the creatinine at initiation of therapy; the more prompt return of renal function in the TNFR:Fc-treated group is demonstrated by the steeper slope.
Antigenic TNFa levels were higher in the TNFR:Fc patients at day 1 and day 2 than in the controls. TNFa levels increased in the control patients from less than 10 pg/ml before OKT3 to a mean peak of 30±13 pg/ml on day 1 and decreased to pretreatment levels by day 2. TNFa levels in the TNFR:Fc-treated patients rose from less than 10 pg/ml prior to OKT3 therapy to a mean peak level of 235±135 pg/ml (range 130-320) on day 1.6 In contrast, the L929 bioassay detected little TNFa bioactivity (<50 pg/ml) in day 1 sera from TNFR:Fc-treated patients, whereas significant TNFa bioactivity with mean peak level of 60±35 was detected in day 1 sera of control patients. Figure 2 demonstrates typical TNFa antigenic levels and bioactivity in a control patient and TNFR:Fc-treated patient respectively. These findings are consistent with the known carrier ability of TNFR:Fc. TNF receptor level concentrations increased from 32±3 ng/ml pretreatment to greater than 600 ng/ml on day 1 of TNFR:Fc treatment and remained higher than 100 ng/ml for longer than 13 days. The calculated half-life of TNFR:Fc based on these values was approximately 4.5 days. TNF receptor levels in control patients were 25±2 ng/ml following OKT3 therapy reflecting intrinsic soluble TNF receptors and 0.43±0.1 in untreated normal volunteers.Figure 3 shows an example of the difference in TNF receptor levels in a control patient and TNFR:Fc-treated patient and the long half-life of TNFR:Fc.6
OKT3 has proven to be a potent immunosuppressive drug that is very effective as an induction agent and in reversing steroid-resistant acute rejection (7, 21). However, OKT3 therapy is associated with a systemic adverse reaction referred to as OKT3-ACS(6, 7). This syndrome is caused by massive cytokine release triggered by T cell activation and subsequent lympholysis(3, 4). TNFa has been implicated as the primary cytokine responsible for the OKT3-ACS, with contributing effects from gamma interferon and possibly IL-2 and IL-6(3, 4, 18, 19). This cytokine release responsible for the OKT3-ACS has been the major limitation to the use of OKT3, and therefore provides the stimulus to prevent its occurrence. Previous studies have shown that anti-TNF therapy may effectively decrease the severity of the OKT3-ACS (19). Our study is the first report of the use of TNF receptors for this purpose.
TNFR:Fc is a dimer of the p80 TNF receptor linked by the Fc portion of IgG1, which binds both TNFa and LT and neutralizes their effects(14). As TNF and LT exist predominantly in trimeric forms, the dimeric construct of TNFR:Fc gives this molecule higher affinity for TNF than monomeric forms of the receptor (14, 22). The Fc peptide imparts a longer half-life to the molecule. As expected, the TNFR:Fc fusion peptide has a 3000-fold greater affinity for TNF than monomeric TNF receptor and 160-fold greater affinity than anti-TNF monoclonal antibody (14, 15). These qualities suggest that TNFR:Fc would be even more effective than anti-TNF in limiting the OKT3-ACS.
Our data demonstrate that TNFR:Fc can be safely administered to renal allograft recipients with steroid-resistant rejeciton. Furthermore, we found that patients treated with TNFR:Fc had shorter duration of symptoms related to OKT3 and a decreased incidence of chills as compared with controls. Further investigation of TNFR:Fc at higher doses is necessary to determine the extent to which this molecule may ameliorate the symptoms of OKT3-ACS. In addition, some symptoms of OKT3-ACS may be related to the release of other cytokines such as gamma interferon, IL-2, or IL-6. Interestingly, TNFR:Fc was efficacious in improving the rate of return of renal function in these patients and preventing the initial worsening of serum creatinine usually seen following initiation of OKT3 therapy for acute rejection. This effect of TNFR:Fc may be related to neutralization of TNF bioactivity, since the release of TNF, as well as IL-2 and gamma interferon, has been reported to impair renal function associated with OKT3 (6, 23-25). We have also demonstrated for the first time in humans that TNFR:Fc is an antagonist of TNFa, while at the same time exhibiting carrier activity. This property of TNFR:Fc was demonstrated by the high TNFa antigenic levels detected in TNFR:Fc-treated patients with concomitant low or undetectable TNFa bioactivity. The long half-life of TNFR:Fc was shown by the high levels of TNF receptors seen more than 13 days after TNFR:Fc treatment, which was administered only during the first two days of OKT3 therapy. In contrast, levels of naturally occurring soluble TNF receptors in the control group dropped to pre-OKT3 levels by day 2.
TNFa and LT also appear to be integrally involved in the acute rejection process through direct cytotoxic effects, as well as stimulation of T cell development (9-11, 26-29). Soluble TNF receptors have been shown to stabilize TNFa bioactivity and protect against damaging effects of TNFa in vitro and in vivo(30, 31). We have previously shown that TNFR:Fc given at a higher dosage of 1 mg/kg/day effectively delays the onset of acute rejection when used as an induction immunosuppressive agent either alone or in conjunction with cyclosporine (15, 16). There are also preliminary data in cynomolgus renal allograft recipients suggesting that TNFR:Fc may be effective in reversing acute rejection (unpublished observation). The mechanism by which TNFR:Fc exerts its immunosuppressive effects appears to be related to inhibition of direct cytolytic effects of TNFa, as well as prevention of TNFa-mediated T cell activation and effector cell function (15-17). The immunosuppressive properties of TNFR:Fc may also be partially responsible for the earlier return of renal function seen in the present study. Thus, it appears that TNFR:Fc may be synergystic with OKT3 in reversing renal allograft rejection.
In conclusion, TNFR:Fc can be safely administered to renal allograft recipients being treated with OKT3 for steroid-resistant rejection, and it may be effective in decreasing the severity of OKT3-ACS. TNFR:Fc appears to eliminate the initial worsening of serum creatinine usually seen following OKT3 therapy for acute rejection, resulting in a more rapid reversal of renal dysfunction. This effect may be related to synergy with OKT3 in reversing rejection along with direct antagonistic action of TNFR:Fc toward TNFa. TNFR:Fc appears to show potential both in immunosuppressive protocols and in limiting OKT3-ACS-however, further investigation at higher doses is necessary to fully evaluate its efficacy.
Acknowledgments. We thank Dr. David Schoenfeld for his assistance with statistical analysis.
Presented at the 21st Annual Meeting of the American Society of Transplant Surgeons, May 17-19, 1995, Chicago, IL.
The views expressed herein are those of the authors and do not necessarily represent the views of the U.S. Air Force.
Abbreviations: LT, lymphotoxin or TNF beta; OKT3-ACS, OKT3-induced acute clinical syndrome; TNFa, tumor necrosis factor-alpha; TNF, tumor necrosis factors alpha and beta; TNFR:Fc, recombinant human soluble dimeric tumor necrosis factor receptor.
Wee SL, Eason JD, et al. Biological effect and fate of a soluble dimeric 80 Kd TNF receptor in renal transplant recipients on OKT3 therapy, manuscript in preparation.
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