Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II double-blinded, randomized, dose-escalating trial

E. H. S. Choy, B. Hazleman1, M. Smith1, K. Moss2, L. Lisi, D. G. I. Scott3, J. Patel4, M. Sopwith4 and D. A. Isenberg2

GKT School of Medicine, King's College London, London
1 Addenbrooke's NHS Trust Hospital, Cambridge
2 University College London, London
3 Norfolk and Norwich Hospital, Norwich and
4 Celltech Research and Development, Slough, UK


    Abstract
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Objective. Biological products that neutralize tumour necrosis factor {alpha} (TNF-{alpha}) are beneficial in rheumatoid arthritis (RA). We studied the effects of CDP870, a novel anti-TNF-{alpha} antibody fragment modified to obtain a prolonged plasma half-life (~14 days).

Methods. Thirty-six patients were randomized in a double-blind, ascending-dose group study to a single intravenous infusion of placebo (n = 12) or 1, 5 or 20 mg/kg CDP870 (each n = 8). The patients were predominantly female (30/36), had a mean age of 56 yr and a mean duration of RA of 13 years. They had received a mean of five DMARDs or experimental therapies (with 1 month washout before the study started) and had active disease. Continuation of NSAIDs and up to 7.5 mg prednisolone daily was allowed. Following the blinded dosing period, 32 patients received a single open-label infusion of either 5 or 20 mg/kg CDP870.

Results. In the blinded dosing period, 6/12 placebo patients withdrew from the study (for deteriorating RA <=4 weeks after dosing). Two of 24 CDP870-treated patients withdrew, both in the 1 mg/kg group (for deteriorating RA or lost to follow up >4 weeks after dosing). The proportion of patients with ACR20 improvement for the per-protocol population with the last observation carried forward was 16.7, 50, 87.5 and 62.5% after 0, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P = 0.012, primary analysis) at 4 weeks and 16.7, 25, 75 and 75% (P = 0.032) at 8 weeks. The proportion of patients with ACR50 improvement for the per-protocol population with the last observation carried forward was 0, 12.5, 12.5 and 50% after 0, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P = 0.079) at 4 weeks and 0, 12.5, 12.5 and 50% (P = 0.079) at 8 weeks. Following the open-label dose of CDP870, similar beneficial effects were achieved.

Conclusion. CDP870 is effective, was very well tolerated in this small study, and has an extended duration of action following one or more intravenous doses.

KEY WORDS: Rheumatoid arthritis, Immunotherapy, Biologicals, Monoclonal antibodies, Tumour necrosis factor, Polyethylene glycol.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tumour necrosis factor {alpha} (TNF-{alpha}) is a proinflammatory cytokine that plays a major role in the pathogenesis of rheumatoid arthritis (RA) [1]. Mice transgenic for human TNF-{alpha} produce high levels of TNF-{alpha} constitutively and develop a spontaneous inflammatory, destructive polyarthritis resembling RA [2]. Furthermore, in collagen-induced arthritis, disease can be treated effectively with anti-TNF-{alpha} monoclonal antibody (mAb) [3]. In addition, increased levels of TNF-{alpha} are found both in the synovial joints and the peripheral blood of RA patients. When TNF-{alpha}-blocking agents are administered to RA patients, they reduce inflammation, improve symptoms and retard joint damage [4].

Two TNF-{alpha}-blocking agents, etanercept and infliximab, are currently licensed for the treatment of RA. Etanercept (Enbrel; Immunex Corporation, Seattle, WA, USA) is a recombinant human p75 soluble TNF receptor–human immunoglobulin G1 (IgG1) construct. It contains two TNF-{alpha}-binding domains linked to the Fc portion of human Ig. Infliximab (cA2; Centocor, Malvern, PA, USA) is a chimeric anti-TNF-{alpha} mAb that has a human Ig-{gamma}1 Fc. Both are recombinant proteins manufactured by mammalian cell culture techniques.

CDP870 (Celltech Research and Development, Slough, Berkshire, UK) is a novel compound that neutralizes TNF-{alpha} in vitro. It comprises an engineered human anti-TNF-{alpha} antibody Fab' fragment that is linked chemically to polyethylene glycol (PEG). The Fab' fragment is made by microbial fermentation rather than in mammalian cell culture. The attachment of PEG increases the circulating half-life of Fab to approximately 14 days. We evaluated the safety and efficacy of intravenous CDP870 in a randomized double-blind, placebo-controlled, dose-escalating trial in patients with RA.


    Methods
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 Methods
 Results
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Patients
Patients aged 18–75 yr who satisfied the 1987 revised American College of Rheumatology (ACR) diagnostic criteria for RA [5] were recruited from out-patient rheumatology clinics in London, Cambridge, Norfolk and Norwich (UK). Patients were required to have clinically active disease, defined by having at least three of the following criteria: at least three swollen joints (28-joint count), at least six painful or tender joints (28-joint count), >=45 min of early morning stiffness and erythrocyte sedimentation rate (ESR) >=28 mm/h. They must have failed treatment with at least one disease-modifying anti-rheumatic drug (DMARD) and have been off treatment for at least 4 weeks. Corticosteroids were permitted if the dose was <=7.5 mg/day of prednisolone. Pregnant women, nursing women and women of childbearing potential not using an effective method of contraception were excluded. Patients were also excluded if they had a history of malignancy, concomitant severe uncontrolled medical conditions, previous failure of TNF-{alpha}-neutralizing therapy or allergy to PEG. Written informed consent was obtained from each patient before enrolment. The study was approved by the research ethics committees of King's College Hospital, Addenbrooke's Hospital, Norfolk and Norwich Hospital as well as Middlesex Hospital and University College Hospitals London.

Treatment protocol
Thirty-six RA patients were divided into three groups, each of which were to receive an increasing dose of the trial drug (1, 5 or 20 mg/kg). Each group of 12 patients was randomly divided so that eight received CDP870 and four received placebo. CDP870 was given as a single intravenous infusion (100 ml in total) over 60 min. Placebo (sodium acetate buffer) was given similarly as a single intravenous infusion of 100 ml over 60 min. Treatment was given on an out-patient basis. After 8 weeks, patients had the opportunity to receive an open-label infusion of 1, 5 or 20 mg/kg of CDP870 within 2 weeks of the blinded dosing period if they tolerated the initial infusion.

Clinical assessment
RA disease activity was assessed based on the World Health Organization and International League of Associations for Rheumatology [6] and European League Against Rheumatism (EULAR) [7] core data sets. In brief, these include pain score, assessor and patient global assessment of disease activity measured by visual analogue scales (0–10 cm), tender and swollen joint count (max. 28), health assessment questionnaire (0–3), ESR (mm/h) and C-reactive protein (CRP, mg/dl). Changes in disease activity were assessed with the Disease Activity Score (DAS) [8] and the ACR response criteria [9]. Assessments were carried out before treatment and 1, 2, 4, 6 and 8 weeks after both blinded and open-label therapy. Patients were also assessed for safety and tolerance of the study drug. Haematology, biochemistry, anti-CDP870 antibodies and adverse events were assessed at each visit.

CDP870 plasma concentration and anti-CDP870 antibodies
CDP870 was measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of patients' plasma were incubated in microtitre plates (Nunc, Rochester, NY, USA) coated with recombinant human TNF-{alpha} (Strathmann Biotech, Hanover, Germany). Captured CDP870 was revealed with horseradish peroxidase (HRP)-conjugated goat anti-human {kappa} light chain (Cappel, ICN, Costa Mesa, CA, USA) followed by tetramethyl benzidine (TMB) substrate.

Antibodies to CDP870 were screened (at 1/10 plasma dilution) using a double-antigen sandwich ELISA with biotinylated CDP870 as the second layer. Bound antibodies were revealed using HRP–streptavidin and TMB substrate. The assay was calibrated using a hyperimmune rabbit IgG standard. A unit of activity is equivalent to 1 µg of the rabbit standard. Patients were considered positive for antibodies if there was a greater than two-fold increase in anti-CDP870 levels over a baseline cut-off and pre-infusion level.

Statistical analysis
The study was exploratory in nature and sample size was based on previous experience with similar agents. The efficacy of CDP870 was analysed by calculating the DAS28 score and ACR20/50 responses for intention to treat and per-protocol using a closed testing procedure. The DAS28 score was calculated as follows: DAS28 = 0.555 x square root of (28 tender joint count) + 0.284 x square root of (28 swollen joint count) + 0.7 x ln(ESR)+0.0142x(patient's global assessment). First, the pooled active groups were compared with patients treated with placebo. If this comparison was significant at the 5% level, each dosage group was compared with the placebo group. All comparisons were two-tailed with a significance level of 5%. All P-values were derived from exploratory analysis and should not be used for inferential interpretation.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient characteristics
Thirty-six patients with RA were recruited. Their demographic details are given in Table 1Go. The mean age was 56 yr and 30 patients were female. The mean duration of RA was 13 yr and 21 patients were positive for rheumatoid factor. Patients in the different groups had similar demographic characteristics. In the blinded dosing period, 6/12 placebo patients withdrew from the study for deteriorating RA <=4 weeks after dosing. Two of 24 CDP870-treated patients withdrew, both in the 1 mg/kg group, for deteriorating RA or loss to follow-up >4 weeks after dosing. The difference was statistically significant (P=0.009, Fisher's exact test).


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TABLE 1. Demographic details of patients (mean±S.D.)

 

Clinical efficacy
The proportion of patients with ACR20 improvement for the per-protocol population with the last observation carried forward was 16.7, 50, 87.5 and 62.5% after placebo and 1, 5 and 20 mg/kg after CDP870 respectively (combined treatment effect, P=0.012) at 4 weeks and 16.7, 25, 75 and 75% (P=0.032) at 8 weeks. The proportion of patients with ACR50 improvement for the per-protocol population with the last observation carried forward was 0, 12.5, 12.5 and 50% after 0, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P=0.079) at 4 weeks and 0, 12.5, 12.5 and 50% (P=0.079) at 8 weeks. Reduction in DAS28 scores (median) for the per-protocol population with the last observation carried forward was 0.15, 1.14, 1.91 and 1.95 after placebo, 1, 5 and 20 mg/kg CDP870 respectively (combined treatment effect, P=0.001) at 4 weeks and 0.31, 0.09, 2.09 and 1.76 (P=0.008) at 8 weeks (Fig. 1Go). Changes in individual components of the World Health Organization and International League of Associations for Rheumatology core data set are shown in Fig. 2Go.



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FIG. 1. DAS in patients treated with different doses of CDP870 and placebo. Median and interquartile range are presented for the per-protocol population with the last observation carried forward.

 


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FIG. 2. Tender joint count, swollen joint count, pain score (cm), patient and assessor's global assessment of disease activity (cm), modified Health Assessment Questionnaire (HAQ) score, C-reactive protein (CRP) concentration and ESR in patients treated with different doses of CDP870 and placebo. Median and interquartile range are presented for the per-protocol population with the last observation carried forward.

 
Following the open-label dose of CDP870, similar beneficial effects were achieved. Of the 36 patients recruited into the study, 32 received a second infusion of CDP870. The proportion of patients with ACR20 improvement since before the first infusion was 72.2 and 55.6% after 5 and 20 mg/kg CDP870 respectively at 4 weeks and 55.6 and 66.7% at 8 weeks.

Adverse events
Treatment was well tolerated, with no infusion-related reaction. No allergic reaction or skin rash was reported. In the double-blind phase, there were 19, 38, 8 and 14 adverse events in the placebo, 1, 5 and 20 mg/kg groups respectively. The commonest was headache, with nine episodes in five patients (one placebo, three at 1 mg/kg, one at 20 mg/kg). One patient who received placebo and three patients who received CDP870 (one at 5 mg/kg and two at 20 mg/kg) developed lower respiratory tract infections. These were reported as mild or moderate. They were treated with oral antibiotics and resolved over a 1–2 week period. Three patients each in the 1 and 5 mg/kg groups and one in the 20 mg/kg group developed a urinary tract infection 1–2 months after CDP870 treatment. One adverse event was described as severe, which was an episode of neck pain occurring 3 days after infusion with 1 mg/kg. An increase in antinuclear antibody was seen in four patients: one in the placebo group (negative to 1/40), two in the 1 mg/kg group (negative to 1/40, negative to 1/80) and one in the 20 mg/kg group (negative to 1/40). No change was found in anti-DNA or anti-cardiolipin antibodies.

In the open phase, one patient who received 20 mg/kg CDP870 died from complications following rapid drainage of a large, chronic rheumatoid pericardial effusion. No infective agent was isolated from either the pericardial fluid or peripheral blood. In the opinion of the investigator, this event was unrelated to treatment with CDP870.

CDP870 plasma concentration and anti-CDP870 levels
As expected, for all dose levels of CDP870, the peak plasma concentration occurred at the end of infusion and was proportional to dose; the plasma concentration declined slowly thereafter. The plasma concentration profile of CDP870 appeared very similar to that previously observed in volunteers in whom the half-life was calculated to be ~14 days. On redosing, a profile similar to that obtained with single-dose infusion was observed.

Following a single intravenous infusion, anti-CDP870 levels were low or undetectable. Following a second cycle of treatment, antibodies were detected in all treatment groups. The incidence varied with the dose, but was lower in the higher dose groups.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Neutralizing TNF-{alpha} is an effective treatment strategy in RA. Currently, this requires the use of biological agents, such as chimeric mAbs and soluble receptor–human Fc fusion protein, which are expensive to manufacture. An optimal TNF-{alpha}-neutralizing agent needs to bind TNF-{alpha} with high affinity and have a long plasma half-life, low antigenicity and high tolerability and safety. It also needs to be accessible to all patients with RA who would benefit from TNF-{alpha} blockade. One technology to achieve these objectives is the conjugation with PEG of a TNF-{alpha}-binding antibody fragment made in E. coli. In this preliminary study, we found that the PEGylated anti-TNF-{alpha} Fab' CDP870 was effective and well tolerated by patients with RA.

In vitro studies have shown that CDP870 has similar TNF-{alpha}-neutralizing activity to the murine anti-TNF-{alpha} parent antibody. This study confirms that CDP870 reduced inflammation and improved symptoms in RA. Clinical improvement, as measured by the ACR20 response criteria, in the 5 and 20 mg/kg groups (75%, 75%) was comparable to that of etanercept (60%) [10] and infliximab (50%) [11]. At the middle and highest dose levels tested, the therapeutic effect lasted 8 weeks, which is comparable to previous mAbs [12, 13]. A previous study has shown that the therapeutic effect of anti-TNF-{alpha} antibody is related to its plasma half-life and the generation of circulating antibodies [14]. Our study showed that CDP870 has a plasma half-life of 14 days, which is that of a whole antibody [13] and much longer than the half-life of unconjugated Fab' fragments. At higher doses, CDP870 generated only very low levels of antibody response. A longer study with repeat dosing is currently being undertaken to investigate this more thoroughly.

One of the important objectives of this study was to examine the tolerability and safety of administering this PEGylated Fab'. In our study CDP870 appeared well tolerated, but further study will be needed to assess long-term safety, especially the risks of demyelinating disease, infection and skin rashes, which have been reported with etanercept and infliximab.

In summary, CDP870 is a novel PEGylated anti-TNF-{alpha} Fab' fragment that is therapeutically effective in RA and was well tolerated in this short-term study. A larger study with repeated dosing is currently under way to assess its long-term safety and therapeutic effect in RA.


    Acknowledgments
 
We would like to thank Ms Maxine Troop, who helped to monitor and coordinate the study, and Ms Catherine Granier, who performed the statistical analysis.


    Notes
 
Correspondence to: E. Choy, Academic Department of Rheumatology, GKT School of Medicine, King's College Hospital (Dulwich), East Dulwich Grove, London SE22 8PT, UK. Back


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Brennan FM, Maini RN, Feldmann M. TNF alpha—a pivotal role in rheumatoid arthritis? Br J Rheumatol 1992;31:293–8.[ISI][Medline]
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  8. Prevoo ML, van't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis [see comments]. Arthritis Rheum 1995;38:44–8.[ISI][Medline]
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Submitted 5 February 2002; Accepted 11 April 2002





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