Removal of therapeutic anti-lymphocyte antibodies from human sera prior to anti-human leukocyte antibody testing.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.

Immuno-activation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy.

BACKGROUND: A stable reservoir of latently infected, resting CD4 T cells has been demonstrated in HIV-1-infected patients despite prolonged antiretroviral treatment. This is a major barrier for the eradication of HIV by antiretroviral agents alone. Activation of these cells in the presence of antiretroviral therapy might be a strategy to increase the turnover rate of this reservoir. METHODS: Three HIV-1-positive patients on potent antiretroviral therapy, in whom plasma viremia had been suppressed to below 5 copies/ml for at least 26 weeks, were treated with a combination of OKT3 (days 1-5) and recombinant human IL-2 (days 2 6). RESULTS: The side-effects were fever, headache, nausea, diarrhea, and in one of the patients transient renal failure and seizures. The regimen resulted in profound T cell activation. In one patient plasma HIV-1 RNA transiently increased with a peak at 1500 copies/ml. In the other two patients plasma HIV-1 RNA levels remained below the detection limit, but HIV-1 RNA levels in the lymph nodes increased two- to threefold. All patients developed antibodies against OKT3. CONCLUSION: OKT3/IL-2 resulted in T cell activation and proliferation, and could stimulate HIV replication in patients having achieved prolonged suppression of plasma viremia. OKT3/IL-2 therapy was toxic and rapidly induced antibodies against OKT3.

Anti-ICAM-1 monoclonal antibody R6.5 (Enlimomab) promotes activation of neutrophils in whole blood.

R6.5 (BIRR-1, Enlimomab), a murine IgG2a mAb to the human ICAM-1, inhibits leukocyte adhesion to the vascular endothelium, thereby decreasing leukocyte extravasation and inflammatory tissue injury. In initial clinical trials, R6.5 proved to be beneficial in reducing both disease activity in refractory rheumatoid arthritis and the incidence of acute rejection after kidney and liver allograft transplantations. However, adverse effects such as fever, leukopenia, or cutaneous reactions were not infrequent. We studied the effects of R6.5 on neutrophil function in whole blood samples ex vivo. Surprisingly, at the concentrations achieved in clinical trials, R6. 5 activated neutrophilic granulocytes, as indicated by a significant increase in expression of the adhesion molecule beta2-integrin CD11b, a concurrent decrease in L-selectin expression, and an enhancement of the oxidative burst activity. Neutrophil activation was not exerted by an anti-ICAM-1 mAb of the IgG1 isotype, by isotype-matched, irrelevant anti-2-phenyloxazolone mAb, or by F(ab')2 fragments of R6.5. Neutrophil activation was completely inhibited by soluble complement receptor type 1. We conclude that in whole blood, R6.5 activates resting neutrophils in a complement-dependent manner. This finding can explain, at least in part, the side effects associated with R6.5 therapy.

OKT3 monitoring in the treatment of steroid-resistant acute rejection of hepatotransplant recipients.

OKT3 is a monoclonal antibody used as T-specific immunosuppressor agent in the treatment of acute rejection of hepato- or renal-transplanted patients. The immunosuppressor effect is related to the elimination and modulation of T-cells after the binding between OKT3 and the specific antigen CD3+. This drug has been used in the treatment of acute rejection. The more frequent side effects is the immunogenic reaction Human Antibody Mouse Antibody (HAMA). The aim of this study is the evaluation of the dose and the administration route of the OKT3. The results of the antibody monitoring in the plasma of the treated patients and the analysis of the clinical data were evaluated to focus a valid therapeutic protocol as well as a more rational time sampling of the circulating drug to achieve a correct monitoring. The results show a gradual increase of the hematic concentration of the drug, positively correlating the clinical data of hepatic biopsy and lymphocytic screening. These results have permitted to modify the therapeutic protocol previously performed. It has been defined the administration route choosing i.v. infusion (5 mg/die/2 h), moreover it the therapy has been shortened to 6 days. The HAMA were also evaluated and the analysis of the data showed a negative results, suggesting the possibility of the OKT3 retreatment in the cases of rescue.

Increased risk of early rejection correlates with recovery of CD3 cell count after liver transplant in patients receiving OKT3 induction.

BACKGROUND: We evaluated the utility of CD3 cell counts for monitoring OKT3 induction immunosuppression and for predicting early rejection in liver recipients. METHODS: In 32 adults in whom OKT3 and steroids were used to induce immunosuppression, CD3 cell subsets were labeled with CD3 (IgG1)-fluorescein isothiocyanate monoclonal antibody and assayed by flow cytometry before orthotopic liver transplantation and within 2-4 days, 5-7 days, and 8-10 days after transplantation. Trough OKT3 levels were measured at the same points in 10 patients. Early rejection (before postoperative [POD] day 21) was proven by elevated liver function tests and biopsy. Six patients were excluded for death, retransplantation, or early cessation of OKT3. RESULTS: Eight of 26 patients (30.8%) had early rejection and 18 (69.2%) had no early rejection. All had depletion of CD3 cells to <10.2% of baseline at POD 2-4. On POD 8-10, the mean CD3 count in rejectors was 213.31+/-184.98/mm3 vs. 22.71+/-32.42/mm3 in nonrejectors (P<0.001). By POD 8-10, five of eight (62.5%) patients who rejected had CD3 count recovery to >75% of baseline. No nonrejecting patient recovered to >26% of baseline (P<0.001). OKT3 levels did not correlate with CD3 recovery or rejection. CONCLUSIONS: The incidence of early rejection correlates strongly with recovery of CD3 counts by POD 10. Higher baseline CD3 counts do not predict early rejection.

OKT3 escalating dose regimens provide effective therapy for renal allograft rejection.

Dose-response relationships for anti-CD3 monoclonal antibody (mAb) therapy remain undefined, particularly with respect to higher dose ranges. The clinical efficacy and safety of an OKT3 dosing regimen that incorporates higher doses (escalating dose regimens) was examined in a pilot trial. Patients undergoing acute rejection were treated with a 7-d course of OKT3 in which the daily OKT3 dose was escalated during treatment course (daily doses 5, 5, 5, 5, 10, 15, 25 mg). The total amount of OKT3 given was equal to a standard 14-d course (70 mg). A total of 10 primary cadaveric renal transplant recipients were treated, and data analyzed from a median follow up of 5 months (range 3-13 months). Pre-OKT3 immunosuppressive therapy consisted of ATGAM induction therapy (n = 8), and corticosteroid rejection therapy (n = 6, 18.6 +/- 11.4 mg/kg). Median time of first rejection was 32 d (12-48 d) and median time to OKT3 was 33 d (range 15-42 d). Pre-OKT3 histology (by Banff criteria) included: mild ACR (n = 6), moderate ACR (n = 2), AVR (n = 1), ACR and acute transplant glomerulopathy (n = 1). Rejection reversal rate with escalating dose OKT3 was 100%, and each patient experienced a rapid reversal of rejection (i.e. reversal within 14 d initiation of OKT3 therapy). Six recurrent rejection episodes were diagnosed in 5 patients with a median time to recurrent rejection of 30 d following cessation of OKT3 therapy. All recurrent rejection episodes were successfully treated (FK 506 n = 4, corticosteroids n = 1, and OKT3 n = 1). CMV disease was limited to a single episode of CMV viremia in one patient. PTLD was observed in one patient who had coexisting vascular rejection at the time of PTLD diagnosis. Short- and long-term graft function is excellent (pre-rejection baseline creatinine 1.8 +/- 0.4 mg/dl, current creatinine 1.75 +/- 0.4 mg/dl). Monitoring of OKT3 serum levels revealed that patients maintained therapeutic serum levels for an average of 4 d following the last OKT3 dose. Circulating CD3+ and CD5+ cells were maintained below baseline levels for at least 10 d following the last OKT3 dose. Anti-OKT3 antibody formation occurred in 22% of patients, however, anti-idiotypic responses were of low titer. Adverse reactions experienced during dose escalation were minimal compared to first dose reactions, and consisted primarily of mild headaches and arthralgias in a minority of patients. OKT3 EDR, by obviating monitoring and administration costs, are cost effective [OKT3 EDR $8088, OKT3 SDR (10 d) $9684, OKT3 SDR (14 d) $13,224]. In conclusion, escalating dose regimens of OKT3: 1) provide rejection reversal rates similar to standard dose regimens, 2) provide high OKT3 serum levels and reliable CD3+ cell depletion, 3) induce minimal adverse reactions during dose escalation, and 4) may decrease costs by obviating the need for monitoring peripheral blood T cells and by decreasing administration costs and outpatient visits.

The IgE humoral response in OKT3-treated patients. Incidence and fine specificity.

We recently described a case of anaphylaxis occurring at the time of retreatment with OKT3 of a renal allograft recipient in whom, for the first time, high anti-OKT3 IgE levels were documented. This led us to examine a large series of sera from 181 OKT3-treated patients to better define the frequency of IgE sensitization, its fine specificity (anti-isotypic and/or anti-idiotypic) and its relation to the appearance of IgG anti-OKT3 antibodies (Abs). Six patients out of the 181 assayed have developed anti-OKT3 IgE Abs as detected by ELISA. The earliest time of appearance of IgE anti-OKT3 Abs was 10 days after starting OKT3 (range, 10-25). The IgE response peaked by day 18 (range, 11-35) and had usually disappeared at 3 months after treatment. A more careful dissection of the fine specificity of the IgE response revealed that three of the four patients tested had developed an exclusive anti-idiotypic response. In the last patient, an anti-isotypic component was present since anti-OKT3 IgE Abs also reacted with control IgG2a, IgG2b, and IgG3 monoclonal antibodies. Importantly, anti-OKT3 IgE Abs were only detected in heavily sensitized patients also showing high titers of IgG specific Abs by ELISA (> or = 1/1000) as well as "blocking" anti-OKT3 antibodies, as assessed by immunofluorescence. We conclude that (1) exposure to OKT3 may lead to specific IgE sensitization that, however, only appears in about 38% of the patients; (2) IgE Abs mostly appear in patients also showing high levels of conventional IgG anti-OKT3 Abs including the presence of "blocking" anti-idiotypic Abs, and (3) IgE Abs may be directed to both idiotypic and isotypic determinants of the monoclonal antibody.

Monitoring of allograft recipients for the development of HLA-specific antibodies: elimination of OKT3 as a complicating factor.

In this report we demonstrate that the use of immunoabsorbent beads to remove the OKT3 monoclonal antibody (MoAb) from the sera of transplant recipients is necessary in order to avoid the false positive reactivity in panel reactive antibody (PRA) assay. We have shown that the presence of OKT3 MoAb in patient's sera can give a positive reactivity in PRA, which may be interpreted as antibody development. Rabbit antimouse immunoglobulin covalently linked to sepharose can effectively remove the OKT3 MoAb from patients sera, but has no effect on anti-HLA antibodies. The absorbance of OKT3 MoAb, therefore, is necessary to obtain accurate results in respect to humoral rejection, which may lead to mismanagement of patients.

The OKT3 Antibody Response Study: a multicentre study of human anti-mouse antibody (HAMA) production following OKT3 use in solid organ transplantation.

Human anti-murine antibody titres following patient exposure to the monoclonal antibody Orthoclone OKT3 (muromonab-CD3) are determined by laboratories using diverse analytical methods which are not standardized and whose concordance is not established. A multicentre study group therefore compared testing for IgG anti-OKT3 antibody among seven laboratories. A set of 270 sera was obtained from 30 heart, 30 kidney and 30 liver transplant recipients with no previous exposure to OKT3 who were receiving OKT3 for induction immunosuppression. Sera were collected from each patient prior to and at 24 +/- 2 days and 31 +/- 2 days following initial OKT3 exposure. Identical aliquots of all 270 sera were tested for IgG anti-OKT3 antibody by each laboratory. In addition, the limit of detection of each laboratory's method was estimated by titration of an affinity-purified IgG anti-OKT3 reference material of known concentration. Anti-OKT3 antibody formation differed greatly among the three organ groups. Cardiac patients demonstrated the least sensitization and almost exclusively lower titres, while kidney recipients had more frequent and higher titre antibody formation. Liver recipients yielded the highest sensitization rate and the most frequent high titre sera. Importantly, the seven laboratories differed widely in the number of pretreatment sera reported as positive (ranging from 0% to 41% among laboratories), the number of post-OKT3 sera reported as positive (17-63%), the number of post-OKT3 samples with titre > or = 1000 (2-31%), and the number of patients sensitized 19-69%). Concordance among laboratories was highly variable, with interlaboratory agreement ranging from 38% to 83% on the sample titres assigned to 180 post-OKT3 sera. Many of the discordant results were consistent with differences in the limit of detection of the analytical methods, which ranged from 0.19 microgram/ml to > or = 15 micrograms/ml, a nearly 100-fold difference among laboratories. This study demonstrated the presence of both good concordance and significant discordance among laboratories in determining human anti-mouse antibody titres, and demonstrated that common titre categories (100, 1000, 10,000) were not equivalent among laboratories. The level of concordance among methods should be considered when comparing anti-OKT3 antibody results from different centres and their correlation with clinical events. Universal comparative testing, patterned after proficiency testing programmes, is needed to assess differences among laboratories and to bring uniformity and a sound interpretative basis to this field of testing.

Variations in serum OKT3 concentration based upon age, sex, transplanted organ, treatment regimen, and anti-OKT3 antibody status.

An essential parameter of the efficacy of OKT3 therapy is serial determinations of serum OKT3 levels. We hypothesized that precise monitoring of these levels would optimize treatment protocols. Therefore, enzyme-linked immunosorbent assay (ELISA) technology was utilized to measure OKT3 serum concentrations daily during 263 OKT3 treatment courses in recipients of solid organ grafts. Patient characteristics were: mean age 33 years (0.1-71), 147 male/116 female, 134 kidney/82 liver/47 heart, 122 prophylaxis/141 rejection, and 213 conventional dosing/50 increased dosing. Mean OKT3 levels were higher in women than in men at all time points from day 1 to day 14, reaching the greatest difference between groups on day 7 (849 versus 598 ng/ml, p = 0.004). Patients receiving OKT3 as a component of a prophylactic protocol had higher levels than those receiving the drug for treatment of rejection from day 1 to day 6, with the greatest difference between groups occurring on day 1 (678 versus 333 ng/ml, p < 0.00001). However, from day 7 to day 14 patients receiving OKT3 prophylactically had lower mean OKT3 levels than did those receiving OKT3 for rejection, with the greatest difference between groups occurring on day 11 (555 versus 784 ng/ml, p < 0.05). Liver transplant recipients had significantly higher OKT3 levels than did kidney or heart transplants at all time points. However, more liver patients required increased OKT3 doses to modulate peripheral blood CD3+ cells to < 25/mm3. Kidney recipients had higher levels than did heart recipients. Children < 10 years of age had higher OKT3 levels than did older patients at all time points.(ABSTRACT TRUNCATED AT 250 WORDS)

OKT3 serum levels as a guide for prophylactic therapy: a pilot study in kidney transplant recipients.

The use of OKT3 as prophylaxis in renal transplantation results in a reduced incidence of graft rejection and appears to have beneficial effects on long-term kidney graft survival. However, we and others have observed that patients still experience rejection during the period of OKT3 prophylaxis given at the regular 5 mg/day dose. Many of these patients had no circulating CD3+ cells at the time of rejection, but their OKT3 serum levels were distinctly low (< 500 ng/ml). This led us to adjust OKT3 doses (5 or 10 mg) daily, according to the patients' OKT3 levels, in order to maintain an OKT3 concentration of around 1000 ng/ml. In addition, patients were randomized to receive either 5 mg (group 1, n = 15) or 10 ng (group 2, n = 14) OKT3 as the initial three doses. Concomitant immunosuppression consisted of azathioprine and steroids, with the introduction of cyclosporin A on day 11. Patient survival was 100% after 3 months of follow-up. The intensity of OKT3 first-dose reactions was similar in both groups. Intragraft thrombosis, initially observed in a previous group of patients who received a fixed 10 mg/day OKT3 prophylaxis, occurred in three patients in group 1 and resulted in two graft losses. The cumulative OKT3 dose was similar in both groups (mean +/- SEM 98 +/- 2 mg in group 1 vs 102 +/- 3 mg in group 2) and higher than the 70 mg usually administered. Group 2 patients had higher OKT3 serum levels during the first 4 days of therapy. No correlation could be found between patient weight and cumulative OKT3 dose (r = 0.29).(ABSTRACT TRUNCATED AT 250 WORDS)

[The importance of the host nutritional and immune status on the prognosis of urinary tract infection in the elderly].

This study was performed to evaluate the role of the nutritional and immune status on the prognosis of urinary tract infections (UTI) in the elderly. 192 patients among the 790 inpatients were diagnosed as UTI. Age-related increase in the prevalence of UTI was accompanied with poor prognosis in the patients with hypoalbuminemia (< 2.6 g/dl) and lymphocytopenia (< 700/mm3). To study the immunologic basis for susceptibility to UTI in the aged further, we compared the T-cell functions between outpatients without serious disease and inpatients with chronic UTI. The absolute numbers of lymphocyte, OKT3 (pan T-cell marker), OKT4 (Helper/Inducer marker) and Interleukin (IL)-2 as well as serum albumin concentration were decreased in the patients with UTI. A prospective study was done to confirm that these nutritional and immunological changes become risk factors in the prognosis of UTI with long-term and low-dose chemotherapy. In the patients continued with bacteriuria (Non-responders, n = 6), serum albumin concentration and IL-2 production were significantly lower than the patients who became free from bacteriuria (Responders, n = 7). These results suggest that poor nutrition-related immune dysfunction contributes to the vulnerability of elderly patients to UTI and becomes risks for he prognosis of UTI.

Prevention of adverse clinical outcome by monitoring of cardiac transplant patients for murine monoclonal CD3 antibody (OKT3) sensitization.

We have previously reported that patients sensitized to murine monoclonal CD3 antibody (OKT3) and maintained on such therapy for induction of immunosuppression have a high mortality and/or allograft loss. In this follow-up study, we retrospectively reviewed all patients routinely and serially monitored by flow cytometry for plasma levels of OKT3 during a 21-month period beginning 1/90. A total of 112 patients were monitored during this period. We retrospectively tabulated the incidence of OKT3 sensitization, rejection pattern and impact on survival of withdrawal of OKT3 at the time of sensitization as compared with the previous study in which withdrawal was not done. Nine patients were excluded from analysis because of withdrawal for reasons other than sensitization: cytokine encephalopathy, infection, postoperative complications, or severe rejection. Twelve patients had OKT3 therapy aborted because of failure to achieve steady-state OKT3 levels or because of decline in levels while on therapy. These patients were thus defined as being sensitized to OKT3. No patient was aborted because of return of CD3 cells in the blood. Only one of the 12 patients sensitized to OKT3 died. Of 91 patients with steady-state OKT3 levels, 6 had high plasma levels (> 1000 ng/ml) and 6 had low plasma levels (< 500 ng/ml). None of these patients had OKT3 therapy aborted and all are alive. Twelve of these 91 patients had successful retreatment with OKT3 for refractory rejection, indicating that absence of sensitization on induction predicts safety of retreatment with OKT3. We also examined the frequency of associated human antimouse antibody (HAMA) production using the blocking assay modified from Jaffers and Mayes. Only the sensitized patients exhibited a significant association with HAMA production (6/7 tested, P = 0.05) Classification of the rejection pattern of the sensitized patients confirmed our previous results: eight of 12 had vascular rejection and 4/12 had mixed rejection. These patterns were prospectively determined. We conclude that serial monitoring of patients for plasma levels of OKT3 is an effective strategy to prevent adverse outcomes of induction with this agent.

OKT3 induction in pediatric renal transplantation.

Twenty cadaveric renal transplant patients (mean age 12.5 +/- 4.8 years) received 14 days (mean 13.0 +/- 2.8 days) of OKT3 (mean dose 0.16 +/- 0.09 mg/kg) along with prednisone 0.5 mg/kg per day, azathioprine 2 mg/kg per day and cyclosporine 5 mg/kg per day which was increased to obtain therapeutic levels before the discontinuation of OKT3. Actuarial patient and graft survival were 100% and 50%, respectively, at both 1 and 5 years. Four children lost their grafts within the first 48 h. One loss was technical in origin, the remaining 3 had pathological evidence of vascular thrombosis. Of the remaining 16 children, 12 (75%) experienced rejection episodes within the first 2 months post transplant (mean 27 +/- 15 days). Successful reversal of early rejection episodes was achieved in 11 of 12 patients. Clinically significant cytomegalovirus infection occurred in 4 patients and resulted in graft loss in 2 patients. Circulating OKT3 levels ranging from 1,000 to 32,000 ng/ml were seen in all patients within the first 48 h. There was a rapid and total depletion of circulating CD3-positive lymphocytes in all patients. Anti-isotypic and anti-idiotypic OKT3 antibodies were assessed by enzyme-linked immunosorbent assay (ELISA), and blocking anti-idiotypic antibodies were detected by immunofluorescence inhibition assay. Positive OKT3 antibody titers were detected in 11 children by ELISA and 10 children by immunofluorescence inhibition assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Laboratory monitoring of therapy with OKT3 and other murine monoclonal antibodies.

The first monoclonal antibody (MAb) approved by the Food and Drug Administration (FDA) for therapeutic use, OKT3, is now a standard treatment for organ allograft rejection. This article reviews the interpretation of laboratory tests used to manage patients treated with MAb. Emphasis is placed on OKT3, with which the experience is also the greatest, although comparisons with other MAbs in clinical trials is also discussed.