OKT3 is a murine anti-human CD3 monoclonal antibody (mAb*) that was first used to treat renal allograft rejection in humans in the early 1980s (1). Food and Drug Administration approval was granted for OKT3 for treatment of acute renal allograft rejection on the basis of the ability to reverse a majority of acute renal allograft rejection episodes. Ten to twenty-five percent of patients in the initial clinical experiences with OKT3, however, experienced treatment failure and required additional anti-rejection immunosuppressive therapy. The significant toxicity of OKT3 was obvious in the initial experience in the first several patients (1). A systemic first dose reaction was consistently observed with murine OKT3 therapy, consisting of fever, chills, rigors, tachycardia, tachypnea, diarrhea, nausea, vomiting, and, in severe cases, pulmonary edema, and even death. Despite the fact that OKT3 provided rejection reversal in a large majority of patients, more than half the patients developed idiotypic and/or isotypic antibodies to the murine OKT3 antibody (2).
In vitro studies performed in the early 1980s demonstrated the potent T-cell activation properties of OKT3 (2-4). Knowledge of the potent ability of OKT3 to induce cytokine production by T cells in vitro led to clinical studies that demonstrated high levels of IL-2, TNF-alpha, and gamma-interferon in the serum of murine OKT3-treated patients within hours of the first dose (5, 6). Development of a murine model of anti-CD3 mAb therapy advanced the understanding of the immunomodulatory effects of anti-CD3 mAb (7-10). These murine studies provided the seminal observations that immune activating effects of anti-CD3 mAbs could be avoided by eliminating the Fc receptor (FcR) binding activities of the mAb and that immunosuppressive effects could be retained (7-11). As a result, studies were initiated to evaluate and develop FcR nonbinding anti-human CD3 mAbs that could provide potent immunosuppression while avoiding in vivo T cell activation, cytokine production, and the resultant morbidity of murine OKT3 (11-14).
Several approaches were undertaken to identify FcR non-binding anti-CD3 mAbs for clinical therapy (11-14). These approaches included digest fragment preparations of OKT3 (11), anti-CD3 mAbs of defined epitope specificity and mAb isotype (12), and, finally, genetically engineered antibodies that lacked FcR-binding properties (14). Of these approaches, the genetically engineered antibodies offered the advantages of mAb humanization, and, therefore, the potential for reduced immunogenicity. Cloning of the antibody (Ab) genes also provided the potential for inducing specific mutations in Ab structure, and, thus, function. The huOKT3γ1(Ala-Ala) antibody contains such mutations in the CH2 region, in which amino acids at positions 234 and 235 have been mutated to alanine residues (14). This mutation resulted in a marked diminution in FcR binding affinity, thereby reducing the in vitro T cell activation properties (14).
The present study is a phase I pilot study of huOKT3γ1(Ala-Ala) therapy in the setting of acute renal allograft rejection. This experience represents the initial clinical experience of humanized anti-CD3 therapy and demonstrates that it may provide effective therapy of acute renal allograft rejection with minimal first dose effects.
Definitions. Rejection was defined as an increase in serum creatinine of at least 15% above baseline value (baseline serum creatinine defined as the median of five consecutive serum creatinine determinations immediately before the rejection diagnosis), with a renal allograft biopsy specimen demonstrating acute rejection as defined by strict Banff criteria (15). HuOKT3γ1(Ala-Ala) treatment success was defined as the return of serum creatinine to baseline with a subsequent biopsy showing no rejection, and treatment failure defined as failure of serum creatinine to return to baseline with biopsy specimen evidence of ongoing rejection. Time to rejection reversal was defined as the number of days of huOKT3γ1(Ala-Ala) therapy required for the serum creatinine to return to prerejection baseline value.
Trial design. Acute renal allograft rejection in kidney and kidney-pancreas transplant recipients was treated with huOKT3γ1(Ala-Ala). Solumedrol (500 mg) was given i.v. as a bolus 2 hr before the first huOKT3v1(Ala-Ala) dose only. huOKT3γ1(Ala-Ala) therapy was initiated at a dose of 5 mg/day, and dose adjustments consisted of a doubling of the daily dose to 10 mg/day to reach target serum trough levels of 1000 ng/ml. The target level of 1000 ng/ml was chosen because this concentration of huOKT3γ1(Ala-Ala) provided complete CD3 coating and saturation in vitro (Xu D, Zivin ZA, unpublished observations).
Adverse events. Adverse events were prospectively defined and monitored. The severity of each adverse event was recorded on a daily basis during huOKT3γ1(Ala-Ala) therapy based on World Health Organization (WHO) criteria. For analysis, seven adverse events were chosen (fever, tachycardia, tachypnea, headache, vomiting, diarrhea, arthralgias). A single point was recorded for each adverse event of WHO Class I severity, two points for each WHO Class II adverse event, three for each class III event, and four for each class IV event for each patient each day. The total number of total WHO points per patient per day for the seven selected adverse events was calculated and compared with data previously published for murine OKT3 therapy (16).
Peripheral blood T cell monitoring. Circulating peripheral blood T cells were monitored by flow cytometric analysis. MAbs used for immunophenotyping included the following (all purchased from Becton Dickinson): CD45-PerCP, CD2-PE, CD19-FITC, mouse immunoglobulin (Ig)-fluorescein isothiocyanate (FITC), CD4-PE, CD8-PE, and CD3-FITC. OKT3 was obtained from R. W. Johnson PRI (Raritan, New Jersey). OKT3 was FITC coupled by dissolving FITC (Sigma St. Louis, MO) in N,N-dimethyl formamide (Fisher Scientific) to give a 10 mg/ml solution. FITC/dimethylformamide was added to purified mAb at 1:10 weight/weight and incubated at 25°C for 4 hr, followed by dialysis into phosphate-buffered saline containing an anion exchange resin (AG1-X8, 200-400 mesh, chloride form [Bio-Rad, New York, NY]). Aggregates were removed by centrifugation.
Staining of peripheral blood lymphocytes (PBL) with fluoro-chrome-labeled mAbs was performed in whole blood and analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), using forward and side light scatter to gate on lymphocyte populations defined by CD45 expression. Individual lymphocyte subsets were read as a percentage of the lymphocyte gate and converted to actual numbers using total lymphocyte counts obtained from analysis of heparinized whole blood using a Roche Cobus Minos SSTEL hemocytometer.
CD3 coating/modulation. CD3 coating/modulation of PBL from huOKT3γ1(Ala-Ala)-treated patients was quantitated using OKT3-FITC and measuring absorbance through flow cytometric method. The formulae used for calculating CD3 coating was: (Equation 1)
huOKT3γ1(Ala-Ala) serum level assay. HuOKT3γ1(Ala-Ala) serum levels were determined using a flow cytometry-based assay. Normal human PBL were incubated with serum samples from huOKT3γ1(Ala-Ala)-treated patients, or with known concentrations of huOKT3γ1(Ala-Ala) in normal human serum for 30 min at room temperature. Without washing, OKT3-FITC was added and the PBL were incubated for 30 min on ice. Cells were then washed, fixed with paraformaldehyde, and analyzed by flow cytometric method. A standard curve was generated using fluorescence intensity data from control samples with known huOKT3γ1(Ala-Ala) concentrations. HuOKT3γ1(Ala-Ala) concentrations in serum from huOKT3γ1(Ala-Ala)-treated patients were determined by extrapolating from the standard curve.
Cytokine assay. IL-2 and IL-10 levels in serum samples from huOKT3γ1(Ala-Ala)-treated patients were quantitated using commercial ELISA-based kits (Endogen, Cambridge, MA).
Anti-huOKT3v1(Ala-Ala) antibody ELISA. An ELISA was developed to detect antibodies that reacted with the murine OKT3 idiotype. Using patient sera that possessed idiotypic antibodies to murine OKT3, we have previously shown idiotypic cross-reactivity between humanized and murine OKT3 (13). The ELISA was performed by first coating standard 96-well, flat-bottom ELISA plates (CoStar) with OKT3 (1 μg/100 μl in bicarbonate buffer) overnight at 4°C. Plates were washed and blocked with 1% bovine serum albumin and serial dilutions of control sera (i.e., sera containing idiotypic anti-murine OKT3 antibodies) were added as well as 1:100 dilutions of sera from huOKT3γ1(Ala-Ala)-treated patients. Plates were incubated for 1 hr and washed and peroxidase-coupled anti-human Ig polyclonal Ab or peroxidase-coupled anti-human IgM antibody was added. Plates were washed, substrate was added, and optical density was measured by spectrophotometry at 405 nm. Renal allograft biopsies were performed on an outpatient basis using real time, and the diagnosis of rejection made using Banff criteria (15).
Therapeutic response. Patient demographic and immunologic data are presented in Table 1. Most rejection episodes treated in this study were vigorous rejection episodes; five were Banff grade IIB or grade III. Two patients were kidney/pancreas transplant recipients, and five patients were kidney transplant recipients. HuOKT3γ1(Ala-Ala) therapy provided successful treatment of rejection in five of seven patients (Table 2). HuOKT3γ1(Ala-Ala) therapy was given for 10.1±2.5 days, with a mean total dose of 76±27 mg given. Median time to rejection reversal was 4 days, whereas the median time for serum creatinine to begin decreasing was 2 days (Fig. 1). Patient and graft survival were both 100%, with a median follow-up of 12 months (range 10-17 months). Recurrent rejection was observed in only a single patient, and responded well to tacrolimus therapy.
Two patients were classified under treatment failure (Table 2). The first patient (patient 3) experienced a Banff grade IIB rejection on post-treatment day (PTD) 22 and had huOKT3γ1(Ala-Ala) therapy initiated the following day. The serum creatinine rose progressively during huOKT3γ1(Ala-Ala) therapy, increasing to 3.0 mg/dl on treatment day (TD) 6. By protocol, failure to achieve a decrease in serum creatinine by TD 6 required a repeat renal allograft biopsy. The biopsy specimen revealed an ongoing, unimproved Banff grade IIB rejection despite a serum huOKT3γ1(Ala-Ala) level of 2800 ng/ml. On TD 7, huOKT3γ1(Ala-Ala) therapy was stopped, and murine OKT3 therapy initiated. After 7 days of murine OKT3 therapy, significant improvement in renal function was not observed, and a repeat biopsy revealed grade IIB rejection. Murine OKT3 therapy was discontinued, and high dose corticosteroid, tacrolimus, and mycophenolate therapy were initiated. After 5 days of corticosteroid/tacrolimus/mycophenolate therapy, the serum creatinine had decreased to 2.5 mg/dl and a repeat biopsy specimen revealed borderline acute rejection with 15-20% tubular loss. Eight weeks later, a renal allograft biopsy revealed no acute rejection. One year after huOKT3γ1(Ala-Ala) and additional anti-rejection therapy, the serum creatinine is stable at 2.1 mg/dl.
The second patient classified under treatment failure (patient 4) was a kidney/pancreas transplant recipient who experienced a Banff grade III rejection on PTD 18. HuOKT3γ1(Ala-Ala) therapy was initiated on PTD 19. After an early fall in serum creatinine, a slow, progressive increase in serum creatinine was observed and on TD 12, and after 12 days of huOKT3γ1(Ala-Ala) therapy, a renal allograft biopsy was performed and the specimen revealed some histologic improvement with a Banff grade IIB rejection. The specimen from repeat renal allograft biopsies performed 4 and 7 days after cessation of huOKT3γ1(Ala-Ala) therapy showed progressive histologic improvement with Banff grade IIA and grade I rejections, respectively. Another renal allograft biopsy performed 22 days after cessation of huOKT3γ1(Ala-Ala) therapy showed a worsening histologic picture, with a Banff grade IIB rejection, requiring high dose corticosteroid and tacrolimus therapy. Seven weeks after cessation of huOKT3γ1(Ala-Ala) therapy, a renal allograft biopsy specimen revealed no acute rejection. Nine months after huOKT3γ1(Ala-Ala) therapy, serum creatinine is 1.9 mg/dl.
Histologic responses to huOKT3γ1(Ala-Ala) therapy were determined by performing renal allograft biopsies during and after therapy (Table 3). Renal allograft biopsies were performed during huOKT3γ1(Ala-Ala) therapy in five patients. Both patients that experienced treatment failure showed grade IIB rejection during huOKT3γ1(Ala-Ala) therapy (one on TD 6 and another on TD 12). In contrast, biopsies performed in patients experiencing successful huOKT3γ1(Ala-Ala) therapy showed histologic reversal of rejection during therapy. Two patients who had successful huOKT3γ1(Ala-Ala) therapy did not have biopsies during therapy; however, biopsies specimens taken within 4 weeks after stopping huOKT3γ1(Ala-Ala) therapy both revealed no rejection.
Adverse events and cytokine release. In general, few new-onset adverse events were noted during huOKT3γ1(Ala-Ala) therapy. New-onset adverse events were considered adverse events that did not exist immediately before huOKT3γ1(Ala-Ala) therapy or, if present before therapy, were observe to increase in severity during therapy. Specifically, new-onset hypertension, chest pain, headache, diarrhea, and arthralgias were not observed. Low-grade fever was noted in two patients on TD 1. Mild tachycardia was noted in two patients on TD 2. Mild dyspnea was noted in one patient on TD 3, and vomiting was observed in two patients (TD 2 and TD 3). The timing and nature of these symptoms suggest that they may not be related to huOKT3 therapy. First dose reactions to huOKT3γ1(Ala-Ala) were monitored daily using a WHO severity scale-based scoring system previously reported by our group (Fig. 2)(16). This analysis of adverse event data showed no increase in adverse event severity during the first 3 days of huOKT3γ1(Ala-Ala) therapy compared with pre-treatment. This observation is in marked contrast to the increases in symptom severity observed previously in control patients treated with murine OKT3 (16). The only infection observed within 6 months of huOKT3γ1(Ala-Ala) therapy was a single episode of herpes zoster cutaneous eruption that responded promptly to acyclovir therapy.
IL-2 and IL-10 levels were determined before and at 2 and 4 hr after the first huOKT3γ1(Ala-Ala) dose (Fig. 3). Mean serum IL-10 levels before and 2 hr after huOKT3γ1(Ala-Ala) administration were 70±81 and 424±402 pg/ml (P<0.05). In contrast, serum IL-2 levels remained unchanged after the first huOKT3γ1(Ala-Ala) dose (before, 12.5±6.6 ng/ml and 2 hr after, 12.5±5.5 ng/ml [P=0.70]).
HuOKT3γ1(Ala-Ala) dosing and therapeutic monitoring. HuOKT3γ1(Ala-Ala) dosing was initiated at 5 mg/day in the first two patients. However, the time to reach the target level of 1000 ng/ml at the 5 mg/day dosing level was not rapidly achieved, and the dose was increased according to the protocol in individual patients (Fig. 4). Therefore, huOKT3γ1(Ala-Ala) dosing was initiated at 10 mg/day in the next two patients (Patients 3 and 4). Because the time to achieve the target level was not greatly shortened, an initial dose of 5 mg/day was used in the next three patients. Although the time to achieve serum levels of 1000 ng/ml required 2-4 days of therapy, both the 5 and 10 mg/day doses provided trough serum levels that demonstrated progressive elevation during the remaining treatment period. Pharmacokinetic analysis was performed after the last huOKT3γ1(Ala-Ala) doses (Fig. 5). These data revealed a half-life (t1/2) of 142±32 hr after the final huOKT3γ1(Ala-Ala) dose.
Measurement of circulating CD2+, CD4+, and CD8+ cells revealed a marked clearance from the peripheral circulation of T cells after the initial dose, with a progressive increase occurring over the ensuing days of therapy (Fig. 6). Quantitation of CD3 coating/modulation was performed in each patient, and extensive coating/modulation was observed during and after therapy for several days (Fig. 7). However, this assay did not distinguish, between coating and modulation.
Humoral responses to the huOKT3γ1(Ala-Ala) Ab were serially determined using an ELISA-based assay at weeks 4, 8, 12, and 16. Using this assay, no anti-huOKT3γ1(Ala-Ala) Abs were detected.
This initial clinical experience with huOKT3γ1(Ala-Ala) provides evidence of its in vivo immunosuppressive properties in humans and indicates that huOKT3γ1(Ala-Ala) can provide effective therapy for vigorous acute renal allograft rejection episodes; five of seven patients experienced rejection reversal with huOKT3γ1(Ala-Ala) therapy. HuOKT3γ1(Ala-Ala) also provided durable immunosuppressive effects after therapy; only one recurrent rejection episode was observed, and no renal allografts were lost. The rejection episodes treated in this study were vigorous; most of the rejections were Banff grade IIB or grade III, with the two treatment failures occurring in patients experiencing grade IIb and grade III rejections. In one of these patients, conversion to murine OKT3 also failed to control the rejection process. It is important to note that the rejection episodes treated in this study were first rejection episodes, therefore the ability of huOKT3γ1(Ala-Ala) to reverse steroid-resistant rejection episodes remains to be established.
Not only did huOKT3γ1(Ala-Ala) provide effective rejection reversal, but rejection reversal was prompt, as evidenced by a rapid return of serum creatinine values to the prerejection baseline values. This prompt decline in serum creatinine is more rapid than what is traditionally observed with murine OKT3 and is similar to that observed with another anti-T cell receptor for antigen (TCR) mAb, T10B9.A1-31, in humans (17). Previous demonstration of elevations in serum cytokine levels (IL-2, γ-interferon, TNF-α) after the first OKT3 dose led to the proposal that cytokine release may contribute to the transient increase in serum creatinine levels often observed after initiation of OKT3 therapy. A plausible explanation for the rapid decline in serum creatinine observed with both huOKT3γ1(Ala-Ala) and T10B9.A1-31 is the lack of associated cytokine release (17). The promptness of the biochemical response to huOKT3γ1(Ala-Ala) therapy is supported by documentation of a prompt histologic response seen on renal allograft biopsies.
One of the primary objectives in developing the huOKT3γ1(Ala-Ala) antibody was to abolish first dose reactions. Prospective assessment of adverse events with grading by WHO severity scoring revealed minimal evidence of adverse reactions. The lack of adverse events observed clinically is also supported by the lack of IL-2 release observed after the first huOKT3γ1(Ala-Ala) dose. Thus, the lack of first dose reactions reduces the morbidity associated with OKT3 therapy and also may allow significant reductions in corticosteroid use by avoiding the need for high-dose corticosteroid premedication before the first OKT3 dose. Although the patients in this phase I trial received only 500 mg of methylprednisone before the first huOKT3γ1(Ala-Ala) dose (about 1/3 of the dose used in the control murine OKT3-treated patients), grading of first dose-related adverse events revealed minimal response in comparison with murine OKT3-treated patients. This observation strongly supports elimination of corticosteroid premedication in future huOKT3γ1(Ala-Ala) trials.
Another primary objective in developing the huOKT3γ1(Ala-Ala) antibody was to reduce or eliminate the humoral response. Lack of detection of anti-huOKT3γ1(Ala-Ala) antibodies in the present study suggests that the humoral response to huOKT3γ1(Ala-Ala) may be substantially less than the response to murine OKT3. The lack of a humoral response to huOKT3γ1(Ala-Ala) provides an additional advantage of allowing retreatment should recurrent rejection occur.
The t1/2 of huOKT3γ1(Ala-Ala) seems to be substantially longer than that of murine OKT3. The longer t1/2 and the lack of side effects could allow larger doses to be administered at longer intervals in future trials, permitting a significant reduction in the number of doses, and, thus, possibly eliminating the need for monitoring serum levels or circulating T cells (because the Ab could be delivered in great excess).
Peripheral blood T cell monitoring revealed a significant clearance of T cells from the peripheral circulation after induction of huOKT3γ1(Ala-Ala) therapy, with a subsequent progressive return of peripheral blood T cell counts toward pretreatment levels. This observation indicates that the effects of huOKT3γ1(Ala-Ala) on peripheral blood T cells are similar to those previously reported with murine OKT3 (18).
Quantitation of CD3 coating by the huOKT3γ1(Ala-Ala) Ab revealed near-complete (i.e., >90%) CD3 saturation by TD 5. The requirement of 4-5 days to reach extensive CD3 coating is consistent with the time required to reach serum levels in excess of 1000 ng/ml. The observation of prolonged, extensive CD3 coating for several days after cessation of huOKT3γ1(Ala-Ala) administration is consistent with the persistence of high huOKT3γ1(Ala-Ala) levels resulting from the prolonged t1/2 of the Ab.
We have previously shown that, although FcR nonbinding anti-CD3 mAbs (either OKT3 F(ab=)2 fragments (11), IgG2b anti-human CD3 mAbs (12), and humanized OKT3 mAbs (13) demonstrate extensive CD3 coating of normal human PBL in vitro, the degree of CD3 modulation is substantially reduced in comparison with FcR-binding mAbs, such as murine OKT3. Quantitation of CD3 coating and modulation in the present study however, indicates that extensive CD3 modulation may be observed with anti-human CD3 mAbs with reduced FcR binding activity, such as the huOKT3γ1(Ala-Ala) Ab. This observation suggests that the in vitro observations indicating that both T-cell activation and CD3 modulating properties are similarly proportional to the FcR binding activity of an individual anti-CD3 mAb (11-13) do not necessarily hold in in vivo studies.
The murine model of anti-CD3 mAb therapy has provided a means for studying the effects of alterations in FcR-binding properties of anti-CD3 mAbs on their immunosuppressive and immune activating effects (7-10). These studies have shown that both FcR-binding and FcR-nonbinding anti-murine CD3 mAbs induce depletion of T cells in secondary lymphoid organs (7, 8). Data regarding induction of anergy or hyporesponsiveness, however, are less clear. FcR-binding anti-CD3 mAbs clearly induce a CD8+ T cell anergy (8), whereas FcR-nonbinding anti-CD3 mAbs seem to induce a defect in CD4+ T cell function (7), and more recent in vitro studies with an FcR-nonbinding-murine CD3 mAb indicate that they induce a partial TCR signal that is thought to be analogous to TCR signals induced by altered peptide ligands and is biochemically distinct from the TCR signal delivered by FcR-binding anti-CD3 mAbs (10, 19). FcR-binding anti-CD3 mAb induce a strong TCR signal characterized by marked TCR ζ phosphorylation, ZAP-70 association, ZAP-70 phosphorylation, PLCγ1 activation, phosphoinositidyl turnover, and calcium release (10, 19). In contrast, recent in vitro studies with FcR-nonbinding anti-murine CD3 mAb have shown a marked reduction in TCR ζ phosphorylation, ZAP 70 association, ZAP 70 phosphorylation, and calcium release. Furthermore, additional in vitro studies with FcR-nonbinding anti-murine CD3 mAbs have indicated that a selective TH1 defect may be induced (10). If true, this observation argues strongly for continued clinical evaluation of FcR-nonbinding anti-CD3 mAbs, because such selective immunosuppression delivered with a nontoxic mAb may provide immunosuppression through mechanisms that hold significant theoretical advantage.
Achievement of very low rates of acute rejection using modern immunosuppressive agents (e.g., tacrolimus, mycophenolate, and rapamycin) have lessened the apparent need for biologic agents in the minds of some clinicians. In response to this view, proponents of biologic agents have proposed that significant advantages still exist for their use and development. These advantages include the following: 1) avoidance of the need for patient compliance, 2) use in tolerance-inducing regimens, and 3) ability to allow reduction in the doses of nephrotoxic immunosuppressive agents in the early period after the transplant.
With respect to rejection therapy, non-FcR binding anti-CD3 mAbs provide advantages over other agents currently used in the clinical setting. First, therapy with huOKT3γ1(Ala-Ala) provides an attractive, potentially effective, and minimally toxic therapy as first line treatment of acute rejection. Furthermore, huOKT3γ1(Ala-Ala) may provide lower rates of recurrent rejection than corticosteroids. HuOKT3γ1(Ala-Ala) also provides a means for avoiding nephrotoxicity (e.g., with tacrolimus), bone marrow, and gastrointestinal toxicity (e.g., with mycophenolate).
This initial clinical experience with a humanized version of the murine OKT3 mAb, huOKT3γ1(Ala-Ala), indicates that it can provide effective reversal of vigorous acute renal allograft rejection episodes. In contrast to murine OKT3, huOKT3γ1(Ala-Ala) therapy does not induce first dose reactions or anti-OKT3 antibody formation. The results of this phase I pilot study thus support conducting additional trials with huOKT3γ1(Ala-Ala) as an induction agent and in rejection treatment.
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*Abbreviations used: Ab, antibody; CDR, complementarity-determining region; FcR, Fc receptor; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; Ig, immunoglobulin; PBL, peripheral blood lymphocyte; PTD, posttransplant day; TCR, T cell receptor for antigen; TD, treatment day; t1/2, half-life; WHO, World Health Organization.