Department of Rheumatology, Guy's, King's College and St Thomas Hospitals School of Medicine, King's College London,
1 Glaxo Wellcome Research and Development, Stevenage,
2 Guy's and Lewisham Hospitals, London, UK and
3 Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina, USA
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Abstract |
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Method. Twenty-four patients in four cohorts (six patients in each cohort) were allocated to be treated with five consecutive daily doses of 4162W94 (10, 30, 100 or 300 mg i.v.). Disease activity was measured by the American College of Rheumatology (ACR) criteria and disease activity score (DAS). We also measured 4162W94 concentration, the percentage of 4162W94-coated CD4+ lymphocytes, percentage down-modulation of CD4, interleukin-6 (IL-6) and tumour necrosis factor (TNF
) levels in the PB and SF.
Results. A direct relationship between 4162W94 dose, biological response and clinical outcome was seen. Treatment with 10 and 30 mg of 4162W94 for 5 consecutive days resulted in transient coating and down-modulation of CD4+ lymphocytes, with little effect observed beyond the final dose. However, treatment with 100 and 300 mg resulted in sustained coating and/or down-modulation for 3 weeks and 4 weeks, respectively, in PB and >4 weeks in SF in one patient from the 300 mg cohort. There was a dose-related moderate but transient depression in the CD4+ lymphocyte count in most patients, with all but three returning to >0.40 x 109/l or >75% baseline by the end of the study period. Significant clinical improvement (ACR 20%) was seen in only 1/6 patients in each of the 10- and 30-mg cohorts; however, 3/6 and 5/5 patients in the 100 and 300-mg cohorts, respectively, were ACR 20% responders. In addition, there were significant reductions in PB acute phase reactants as well as SF IL-6 and TNF concentrations in parallel to clinical improvement.
Conclusion. Data from this pilot study suggest that 4162W94 is a clinically active novel immunotherapeutic agent that may suppress inflammation in RA.
KEY WORDS: Rheumatoid arthritis, Anti-CD4, Monoclonal antibodies, Pharmacokinetics, Pharmacodynamics, Clinical response.
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Introduction |
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A major goal for the treatment of autoimmune diseases is the selective and permanent induction of immunological tolerance among pathogenic T lymphocytes using a relatively short course of treatment. Tolerance to protein antigens can be readily induced in mice by administering anti-CD4 monoclonal antibodies (mAbs) at the same time as antigen [5, 6]. Treated animals mount normal responses to other antigens but fail to respond to subsequent challenge with the tolerizing antigen for as long as continued injections of the tolerizing antigen are given. Subsequently, anti-CD4 mAb-mediated immunological tolerance has been demonstrated in animal models of autoimmune disease [710]. Rats treated prophylactically with anti-CD4 mAb and then immunized with streptococcal cell walls not only failed to develop arthritis but were also resistant to further attempts to induce arthritis [8]. This tolerance to arthritis development did not require additional anti-CD4 mAb treatment and occurred at a time when antibodies could no longer be detected in the circulation. Furthermore, anti-CD4-mediated immunological tolerance can be induced even in models of established autoimmune disease, including diabetes [9] and established collagen-induced arthritis [10]. Clearly, if similar tolerance could be induced in established RA by anti-CD4 mAb, such a therapy would lead to prolonged disease remission, obviating the need for continuous, expensive and potentially toxic therapy.
Several murine and chimaeric depleting anti-CD4 mAbs have been studied in RA [1113]. When the chimaeric depleting anti-CD4 mAb cM-T412 was used clinically, it caused profound and extended dose-dependent CD4 lymphopenia [1416]. Pharmacokinetic (PK) and pharmacodynamic (PD) studies showed that the concentration of cM-T412 and the percentage of antibody-coated CD4 lymphocytes in the synovial fluid (SF) were much lower than in the corresponding peripheral blood (PB) [17]. Interestingly, the percentage of cM-T412-coated lymphocytes in the SF, but not the PB, correlated with clinical improvement. Unfortunately, the dose of cM-T412 required to achieve a consistently high concentration in the joint will inevitably lead to unacceptably profound CD4 lymphopenia. Hence, the strategy of using depleting anti-CD4 mAb has been abandoned. This problem may be circumvented by using non-depleting immunomodulatory anti-CD4 mAbs.
The correlation of cM-T412 SF PD effect and clinical outcome, as well as the finding that tolerance induction may require the persistence of antibody in the circulation for a prolonged period [6], emphasizes the importance of PK and PD studies in the early clinical development of anti-CD4 mAbs. Likewise, data on anti-tumour necrosis factor (TNF
) mAbs in RA patients have shown that the plasma antibody concentration correlates with the duration of clinical response [18]. However, as the PK of mAbs is affected by the intrinsic properties of both the antibody, such as the Fc, and those of the target antigen, generalization to the behaviour of new mAbs is not possible. In this open-label, dose-escalating study, we examined the PK and PD of a humanized IgG1 anti-CD4 mAb, 4162W94 (Glaxo Wellcome), in patients with RA. We measured the biological and immunological effects in the PB and SF to assess whether it was possible to deliver significant antibody into the synovial joint and inhibit local inflammation, with the ultimate goal of inducing SF T-cell tolerance.
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Methods |
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Antibody
4162W94 was engineered by grafting the complementarity-determining regions from the variable region of a rat anti-CD4 mAb into the human IgG1 constant region framework [20]. It was produced from a non-secreting murine myeloma (NSO) cell line [21] grown in a closed system of controlled fermenters using protein-free media. After harvesting, mAb was concentrated by ultrafiltration and purified by sequential protein A affinity chromatography, ion-exchange chromatography on an S-Sepharose column and size exclusion chromatography through Superdex 200. Therapeutic mAb was formulated at 10 mg/ml in 50 mM citrate buffer containing 0.05 mM disodium edetate and 0.01% polysorbate 80, sterilized by filtration, filled into 1 ml glass ampoules and stored at 28°C prior to use. 4162W94 mAb does not mediate complement lysis and has only weak activity in an antibody-dependent cellular cytotoxicity assay [21].
Treatment protocol
Patients observed a 4-week DMARD washout period prior to the first day of mAb dosing. Patients were allowed to continue non-steroidal anti-inflammatory drugs and 10 mg/day or less of oral prednisolone, provided the dose had remained stable during the 4 weeks prior to the first day of mAb dosing and continued to be stable throughout the study. Parenteral and intra-articular steroids were not allowed from the beginning of the washout period to the end of the study. Patients were sequentially enrolled into 4 cohorts each of six patients, to receive daily doses of 10, 30, 100 and 300 mg respectively, for 5 consecutive days. 4162W94 was administered i.v., in the out-patient clinic, over 2 h as a freshly prepared solution in 200 ml of normal saline. Vital signs were monitored closely during and after infusion until stable.
Clinical assessment
Arthritis activity was assessed using the ACR composite scoring criteria [22] at screening and on days 0, 7, 14, 28, 42, 56 and 84. A disability index for physical function in RA was measured using a modified Health Assessment Questionnaire (HAQ) [23]. Either ESR or C-reactive protein (CRP) was acceptable to assess the acute-phase response.
Pharmacokinetic studies
4162W94 was measured in plasma and SF by a non-competitive time-resolved fluorescence immunoassay. Microtitre plates were coated with recombinant soluble human CD4, washed and then blocked with bovine serum albumin. Test plasma or spiked normal control plasma was diluted 1/10 in blocking buffer prior to addition to the plates. After incubation at room temperature for 2 h, the plates were washed and biotinylated anti-4162W94 anti-idiotypic mAb (FC210) was added and the plates were incubated for 1 h at room temperature. FC210 was selected because it recognizes an idiotypic epitope exposed on 4162W94 bound to the CD4 antigen. After washing, europium-conjugated streptavidin was added and, following incubation at 37°C for 30 min, the plates were washed prior to addition of enhancement solution. This solution releases lanthanide ions, which are detected by time-resolved fluorescence. 4162W94 concentrations were determined against a standard curve plotted as log concentration vs log fluorescence. The limit of quantification in both plasma and SF for the assay was 0.5 µg/ml. Plasma 4162W94 concentrations were determined before and after infusion on days 04, 2 h after infusion on day 4, and on days 7, 14, 28, 42, 56 and 84. SF levels were measured, when samples were available, before infusion on day 0 and at intervals after (2 h after infusion on day 4 and on days 7, 14 and 28) the treatment course of 4162W94 in 100- and 300-mg cohort patients who had a significant knee effusion.
Pharmacodynamic studies
Coating of CD4+ lymphocytes with 4162W94 was assessed by immunofluorescence and flow cytometry with a biotinylated anti-4162W94 anti-idiotypic antibody (FC210) detected with streptavidin-phycoerythrin. Down-modulation of the CD4 molecule was calculated using the ratio of the mean fluorescent intensity (MFI) of CD4 to CD45 (which does not co-modulate with CD4) on the cell surface; the MFI ratio was expressed as the percentage of the pretreatment value and the percentage of down-modulation was defined as 100% minus this derived percentage. In addition, interleukin-6 (IL-6) and TNF were measured in the SF by ELISA (Medgenix, Ratingen, Germany) kits. The schedule for PD measurements was the same as for PK sampling.
Laboratory monitoring
The following laboratory parameters were measured prior to therapy on day 0: complete blood count, ESR, CRP, biochemistry and urinalysis. Mononuclear cell phenotypes for CD3, CD4, CD8, CD14, CD16 and CD20 were measured using dual-colour immunofluorescence staining and analysed by flow cytometry as previously described [17]. The laboratory measurements and mononuclear cell phenotype studies were repeated on days 1, 2, 3, 4, 7, 14, 28, 42, 56 and 84.
Safety monitoring
Adverse events were recorded during each assessment visit. PB CD4 lymphocyte number was measured as described above.
Plasma anti-4162W94 antibodies were detected using the method described by Cobbold et al. [24] before treatment and on days 7, 14, 28, 42, 56 and 84. Briefly, microtitre plates were coated with 4162W94 and blocked with bovine serum albumin. Patient plasma and a positive control, anti-4162W94 anti-idiotype mAb (FC210) in normal human plasma, were diluted to 1/10 in blocking buffer and added to the plates. After incubation at room temperature for 2 h, the presence of antibodies against 4162W94 was detected by addition of europium-labelled 4162W94 for 1 h at 37°C followed by washing and addition of enhancement solution for the measurement of bound europium by time-resolved fluorimetry.
Statistical analysis
Only patients who received the full protocolled dose of 4162W94 were included in the analysis of plasma PK and PB coating/down-modulation data (all SF data were analysed). Only patients who took at least 80% of their protocolled dose and attended at least one post-dosing assessment were considered evaluable for the analysis of clinical response. Data were analysed on an intention-to-treat basis. Patients requiring additional DMARDs or steroid therapy were classified as treatment failures from that time point. A clinical response was defined as at least a 20% improvement in joint pain and swelling, coupled with a similar improvement in three of the other five measures of disease activity, including ESR or CRP, as defined by the ACR [22]. A modified DAS was also calculated from the above data [25]. Changes in the individual measures of disease activity within a cohort were compared at each time point against baseline by two-tailed paired Student's t-test. Spearman's rank correlation was used to assess the associations between (i) plasma 4162W94 concentration and both coating and down-modulation, and (ii) modified DAS and both plasma concentration and coating.
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Results |
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The median age at enrolment was 53 yr (range 3774). The median number of discontinued DMARDs prior to enrolment was 3 (range 17). Twenty-two patients were positive for rheumatoid factor and eight had rheumatoid nodules.
Adverse events
Seventeen of the 25 patients experienced at least one predefined infusion-related adverse experience (i.e. fever, chills/rigors, headache, nausea, vomiting, diarrhoea, dyspnoea or hypotension). Most of these events were mild or moderate in intensity and occurred on the first day of dosing and resolved within 8 h. Neither the frequency nor the severity of these infusion-related events appeared to be dose-related. In some patients, the appearance of these symptoms was associated with the appearance of TNF in the plasma during the 3-h period following the start of antibody infusion (data not shown), suggesting that these reactions were a result of cytokine release, as has been noted with infusion of other antibodies [26, 27]. Systolic hypotension, defined as <90 mmHg and a fall of >20 mmHg below the pre-dose value, occurred in patient 1 in the 10-mg cohort on the first day of dosing. Non-specific skin rash was reported in one patient in each of the 10-, 30- and 100-mg cohorts and in three patients in the 300-mg cohort. All cases of rash were graded as mild in intensity. Most patients were given topical hydrocortisone cream and oral chlorpheniramine to relieve symptoms of rash with resolution in 231 days. There were three serious adverse experiences reported in the study: a fatal rupture of an aortic aneurysm (patient 1), which was felt to be unrelated to the study treatment, an episode of severe reversible airway constriction in an asthmatic patient during dosing, and an unexplained episode of collapse, presumed vasovagal in nature, followed by full recovery several hours after the end of the first infusion. No opportunistic or other serious infections were reported.
Immunological effects
No anti-4162W94 antibody response was found in any patient in the study at any time point.
Although CD4 lymphocyte counts fell by up to 80% from baseline values in the higher dose cohorts during the 5-day dosing period, there was a dose-related recovery toward baseline following 4162W94 therapy, the median CD4 cell count returning to within 75% of baseline in the 300-mg cohort. Therefore, in spite of a transient fall in CD4 lymphocyte counts during the dosing period, 4162W94 appeared to be relatively non-depleting (Fig. 1). At the end of study, only three patients (one in the 100-mg cohort and two in the 300-mg cohort) had a CD4 lymphocyte count of <0.40 x 109/l and <75% of baseline; none had <0.20 x 109/l. This differs from a previous study using depleting anti-CD4 mAbs, in which all patients developed prolonged CD4 lymphopenia [28]. The effect on other mononuclear cell phenotypes was less marked (data not shown). The CD3 lymphocyte count changes reflected the changes noted for CD4 lymphocytes, and there was no apparent effect on CD8 lymphocytes. 4162W94 appeared to have a similar, although more modest, effect on CD14 monocytes compared with CD4 lymphocytes, with a transient fall noted during the dosing period. There was no effect noted on CD20 B-lymphocyte or CD16 natural killer cell numbers during dosing with 4162W94.
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Pharmacokinetics
An increase in the plasma 4162W94 concentration was observed with increasing dose and was linear at higher doses, most likely reflecting the saturation of CD4 receptors and the accumulation of free mAb. Following 5 successive days of dosing, the peak plasma concentration of 4162W94 in the 100- and 300-mg cohorts was 90 and 257 µg/ml, respectively, falling to approximately 4 µg/ml after 14 and 28 days. Although collection of serial SF samples was planned for all patients enrolled in the 100- and 300-mg cohorts, these samples were obtained from only six patients (four in the 100-mg cohort and two in the 300-mg cohort). Individual patients results are shown in Fig. 2. In contrast to the PB 4162W94 concentrations, the peak SF concentration in these cohorts was 16 and 73 µg/ml, falling to approximately 1 µg/ml after 14 and 28 days. The derived plasma PK parameters of this mAb were typical of a large molecule: small volume of distribution (
5 l), low clearance (0.030.04 l/h at 300 mg) and long half-life (46 days at 300 mg).
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Pharmacodynamics
A dose-related response in both magnitude and duration of CD4 molecule coating and down-modulation on PB and SF lymphocytes was observed (Figs 2 and 3
). The biological effect was transient in the 10- and 30-mg cohorts, such that median PB levels (coating and down-modulation) in the latter group peaked at approximately 80% and fell sharply following the last dose. Median PB levels of coating plateaued at approximately 85% and 100% in the 100- and 300-mg cohorts, respectively, and were sustained above 7080% for 3 weeks and 4 weeks, falling to 10% and 6% at week 4 and 6, respectively, in these groups (Fig. 3
). 4162W94 coating of CD4-positive lymphocytes in the SF plateaued at >95% in both cohorts, falling to 20% at 4 weeks in the 100-mg group but remaining near 100% in the 300-mg group up to 4 weeks. Median PB down-modulation plateaued rapidly at >95% in both the 100- and 300-mg cohorts, remaining above 7080% for 2 weeks and 3 weeks, respectively, and fell to 12% and 14% at week 3 and 4 (Fig. 4
). Down-modulation of CD4-positive SF lymphocytes peaked at 72% and 96%, falling to undetectable levels in the 100-mg cohort after 2 weeks but remaining above 80% in the 300-mg cohort (one patient) at 4 weeks. In contrast to coating, median CD4 down-modulation values were lower in SF compared to PB in paired samples. Using Spearman's rank correlation, statistically significant correlations were found between plasma 4162W94 level and PB CD4 down-modulation (r = 0.81, P = 0.0001) as well as PB CD4 lymphocytes coated with 4162W94 (r = 0.77, P = 0.0001).
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Clinical responses
Overall, 43% of the evaluable patients fulfilled the ACR 20% response criteria following treatment with 4162W94 (Table 1). For responders across all dose cohorts, the mean time to response was 13 days and the mean duration of response was 29 days. Two patients were still responding at the end of the 3-month study. A doseresponse relationship was noted, one of six patients achieving an ACR 20% response in each of the 10- and 30-mg cohorts, three of six patients in the 100-mg cohort and five of five in the 300-mg cohort. The clinical response was also evaluated using the modified DAS (Table 2
). There was a statistically significant reduction in the modified DAS of patients in the 100-mg cohort (P < 0.05) from day 7 to 56 and in the 300-mg cohort from day 14 (P < 0.01) to 42. Acute-phase responses were also reduced after treatment. There was a significant correlation between the median modified DAS with median plasma 4162W94 concentration (r = - 0.46, P < 0.05) and median 4162W94-coated CD4 lymphocytes (r = - 0.5, P < 0.001).
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Discussion |
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The main objective of this study was to assess the PK and PD effects of 4162W94 in both PB and SF. The results confirm our previous finding that anti-CD4 mAb can penetrate into the SF only after saturating PB targets by administering high doses of mAb [17]. The SF 4162W94 concentration after treatment with 100 mg daily for 5 days was very low; nevertheless, it was sufficient to coat all CD4-positive lymphocytes. Increasing the dose to 300 mg increased the SF mAb concentration (although the SF concentration was less than 30% of the corresponding plasma concentration), resulting in a longer duration of down-modulation and the coating of SF CD4+ lymphocytes. In contrast to coating, down-modulation was lower in the SF than PB, indicating a difference in biological effects between the two compartments. In vitro studies have shown that down-modulation of CD4 on resting T cells by 4162W94 depends on cross-linking of the bound antibody by FcR-positive accessory cells [30]. The reduction of down-modulation in the SF may have been due to immune complexes, known to be present in SF at high concentration, competing with 4162W94-coated cells for available Fc receptors.
Treatment with 100 or 300 mg of 4162W94 daily for 5 days was associated with significant improvement in the disease measured by the ACR 20% response criteria [22] or moderate improvement by the modified DAS [25]. Clearly, a randomized double-blind placebo-controlled trial of 4162W94 therapy in RA is required to formally assess the clinical efficacy of this biological agent. A primatized non-depleting anti-CD4 mAb has demonstrated efficacy in such a placebo-controlled trial [31]. Nevertheless, we have shown that there was reduction in acute-phase responses associated with disease improvement. Since ESR and CRP are unlikely to be affected by a placebo response, these data suggest that the disease improvement was genuine. The reduction of the acute-phase response may have been a secondary result of disease improvement and damping of inflammation through the effect of 4162W94 on T lymphocytes; however, given the dramatic reduction in SF TNF and IL-6 observed following administration of 4162W94, it is also possible that this mAb acts directly on human macrophages to reduce the release of proinflammatory cytokines. TNF
can drive the release of IL-6 from macrophages and IL-6 stimulates the release of acute-phase reactants from the liver. Since both macrophages and T cells can produce TNF
and IL-6 and both express membrane CD4, albeit at very low levels on macrophages, the relevant cellular target and mechanism for reduction of ESR and CRP by 4162W94 is not clear.
The primary goal of non-depleting anti-CD4 mAb treatment of RA is to induce long-term immunological tolerance. Therefore, one of the aims of future studies will be to see whether treatment with 4162W94 can produce prolonged clinical improvement. In this study, we have seen prolonged improvement in only two patients, both in the 100-mg cohort. However, a single course of treatment may be insufficient since in animal models more prolonged blockage of CD4 was necessary to achieve tolerance [6]. As no anti-4162W94 antibody response was detected in any patient at any time point, repeat treatment with 4162W94 is feasible and may produce more sustained disease improvement.
In summary, 4162W94 is a relatively non-depleting anti-CD4 monoclonal antibody with a tolerable side-effect profile, which has measurable dose-related PD, PK and clinical response.
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Acknowledgments |
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Notes |
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References |
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