Pharmacokinetics, pharmacodynamics, short term efficacy and safety of RCT-18, a novel BLyS/APRIL fusion protein, in patients with rheumatoid arthritis

Authors

  • Xia Chen,

    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
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  • Qian Zhao,

    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
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  • Yong Hou,

    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
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  • Ji Jiang,

    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
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  • Wen Zhong,

    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
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  • Wenxiang Wang,

    1. RC Biotechnologies, Ltd., Yantai
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  • Xuejing Yao,

    1. RC Biotechnologies, Ltd., Yantai
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  • Lin Li,

    1. RC Biotechnologies, Ltd., Yantai
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  • Jianmin Fang,

    Corresponding author
    1. School of Life Science and Technology, Tongji University, Shanghai, China
    • Correspondence

      Professor Dr Pei Hu MD, Clinical Pharmacology Research Centre, Peking Union Medical College Hospital, 41 Damucang Alley, Xicheng District, Beijing 100032, China. Tel.: +86 10 6915 8366 Fax: +86 10 6915 8365 E-mail: hubei@pumch.cn; Professor Dr Fengchun Zhang MD, Department of Rheumatology, Peking Union Medical College Hospital, 1 Shuaifuyuan, Dongcheng District, Beijing 100730, China. E-mail: zhangfccra@aliyun.com; Professor Dr Jianmin Fang, School of Life Science, Tongji University, 1239 Siping Road, Shanghai, P.R. China. E-mail: jfang@tongji.edu.cn

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  • Fengchun Zhang,

    Corresponding author
    1. Department of Rheumatology, Peking Union Medical College Hospital, Beijing
    • Correspondence

      Professor Dr Pei Hu MD, Clinical Pharmacology Research Centre, Peking Union Medical College Hospital, 41 Damucang Alley, Xicheng District, Beijing 100032, China. Tel.: +86 10 6915 8366 Fax: +86 10 6915 8365 E-mail: hubei@pumch.cn; Professor Dr Fengchun Zhang MD, Department of Rheumatology, Peking Union Medical College Hospital, 1 Shuaifuyuan, Dongcheng District, Beijing 100730, China. E-mail: zhangfccra@aliyun.com; Professor Dr Jianmin Fang, School of Life Science, Tongji University, 1239 Siping Road, Shanghai, P.R. China. E-mail: jfang@tongji.edu.cn

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  • Pei Hu

    Corresponding author
    1. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing
    • Correspondence

      Professor Dr Pei Hu MD, Clinical Pharmacology Research Centre, Peking Union Medical College Hospital, 41 Damucang Alley, Xicheng District, Beijing 100032, China. Tel.: +86 10 6915 8366 Fax: +86 10 6915 8365 E-mail: hubei@pumch.cn; Professor Dr Fengchun Zhang MD, Department of Rheumatology, Peking Union Medical College Hospital, 1 Shuaifuyuan, Dongcheng District, Beijing 100730, China. E-mail: zhangfccra@aliyun.com; Professor Dr Jianmin Fang, School of Life Science, Tongji University, 1239 Siping Road, Shanghai, P.R. China. E-mail: jfang@tongji.edu.cn

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Abstract

Aim

RCT-18 is a recombinant fusion protein that interferes with the selection and survival of mature B-lymphocytes by inhibiting B-lymphocyte stimulator and a proliferation-inducing ligand.

Methods

This single blind, randomized, placebo controlled, clinical pharmacological study explored the short term efficacy and safety of RCT-18 in 21 rheumatoid arthritis (RA) patients with three different dosing regimens. The pharmacological behaviour of RCT-18 was also characterized through a six level biomarker cascade approach to identify potential predictors for clinical responses.

Results

Nine out of 10 patients (>80%) experienced moderate to good EULAR response at the end of 3 months with once or twice weekly doses of 180 mg RCT-18, whereas weekly administration of 360 mg RCT-18 or placebo, however, only resulted in moderate improvement in one patient in each group. Absence of IgM-type rheumatoid factor reduction, recovery of IgM 2 weeks after drug cessation, lack of decrease in the count of CD27(+) B-lymphocytes and a DAS28 change from baseline <6 in 4–6 weeks after the treatment initiation may indicate poor clinical response. No anti-drug antibody of RCT-18 was detected. The active treatments were well tolerated, although more mild to moderate infections were reported in patients receiving RCT-18.

Conclusion

The study results support further development of RCT-18 in RA patients and provide important information for future dose selection.

What is Already Known about this Subject

  • The B lymphocyte stimulator (BLyS) family ligands and receptors are critical players in the selection and survival of most mature B lymphocytes.
  • As a BLyS-targeting recombinant fusion protein, antacicept was found to be ineffective for active RA patients who responded inadequately to MTX or a TNF-α inhibitor, despite of its pharmacodynamic effects on IgM, IgG and IgA levels.
  • RCT-18 has been investigated in a single ascending dose study. The doses of 180 mg and 360 mg were shown to be pharmacodynamically active based on their effects on the ratio of IgM : IgG, while the dose of 540 mg was associated with an IgM-suppressing effect similar to those reported with atacicept.

What this Study Adds

  • RCT-18 statistically significantly reduced DAS28 score with a weekly injection of 180 mg for 3 successive weeks or twice weekly injection of 180 mg for eight times, but caused little clinical improvement with weekly injections of 360 mg for five times.
  • The biomarker cascade approach of this study may serve as the basis for establishing a quantitative relationship between the pharmacological effects of RCT-18 or other BLyS and/or APRIL targeting drugs and their clinical behaviours.

Introduction

In the last decade, the treatments for rheumatoid arthritis (RA) have moved from broad spectrum immunomodulatory agents to molecules targeting specific cytokines or cells that are involved in the pathogenesis of RA [1]. The B lymphocyte stimulator (BLyS) family ligands and receptors are critical players in the selection and survival of most mature B lymphocytes. Recently, development of frank humoral autoimmunity in BLyS transgenic mice and the observation of elevated BLyS levels in systemic lupus erythematosus (SLE), RA and Sjogren's syndrome have connected BLyS to a potential therapeutic target for rheumatic diseases [2, 3].

RCT-18 is a novel recombinant fusion protein constructed with the soluble extracellular portion of the trans-membrane activator and calcium-modulating cyclophylin ligand (CAML) interactor (TACI) molecule and the Fc portion of human IgG. It binds and neutralizes the activity of BLyS and a proliferation-inducing ligand (APRIL) [4]. Preclinical studies have demonstrated dose-dependent efficacy of this BLyS/APRIL-targeting agent in animal models with collagen-induced arthritis [5] and adjuvant-induced arthritis [6, 7]. These effects were accompanied with drug-induced reduction on circulating immunoglobulin (IgM, IgG1, IgG2a) concentrations and cytokines [6]. Nevertheless, at doses higher than 3.3 mg kg–1, no apparent dose–response relationship was observed with RCT-18 in a mouse model with collagen-induced arthritis [5], implying effect saturation with the high doses.

RCT-18 is currently under clinical development. A first-in-human single ascending dose (SAD) study has been completed in 28 Chinese RA patients [8]. The results suggest good safety and tolerability of RCT-18 at subcutaneous doses up to 540 mg. However, the development of RCT-18 was overshadowed by the lack of efficacy with atacicept, another BLyS/APRIL-targeting TACI-Ig fusion protein, in RA patients who inadequately responded to methotrexate [9] or a tumour necrosis factor α inhibitor [10]. Even worse, atacicept was found intolerable in SLE patients due to excessive immunoglobulin suppression and consequently severe infections [11]. On the contrary, the results of early clinical studies with belimumab or tabalumab (both are monoclonal antibodies against BLyS) were encouraging in SLE patients [12] and RA patients [13-15]. Reasons for the differences in clinical behaviour among these BLyS-targeting biologics are unclear, but the differences in pharmacological activities, study design, patients selecting criteria and even clinical end points [16] may provide some explanations. In the phase I study with single and repeated doses of atacicept, immediate recovery of IgM level was observed after the last administration of atacicept with 2 weekly doses of 70 mg to 630 mg atacicept. However, with single doses of atacicept, the trough IgM concentration was reached only 2 weeks after dosing and was followed by a gradual recovery for 4–6 weeks [17]. In a 52 week open label study with tabalumab, maintenance of IgM reduction was observed for at least 30 weeks after the last drug administration, although the maximum effect size on IgM was smaller than that of atacicept [14]. As robust IgM reduction does not seem to be linked to clinical efficacy of these BLyS-targeting agents and excessive IgM depression was found accompanied with severe infections [11], the highest dose of RCT-18 (540 mg) investigated in the SAD study [8] was not selected for this multiple dose study. Instead, 180 mg RCT-18 and 360 mg RCT-18 were recognized previously as the pharmacodynamically effective doses based on their effects on the ratio of IgM : IgG [8] and were employed in the present study.

In this study, we explored the short term efficacy and safety of three RCT-18 dosing regimens in 21 RA patients. The pharmacological behaviour, including pharmacokinetics, pharmacodynamics and immunogenicity, of RCT-18 was also characterized through a six-level biomarker cascade approach to identify potential predictors for clinical responses. The purpose was to find an optimal dosing regimen(s) for future clinical development of the drug.

Methods

Study design

This single blind, randomized, placebo controlled study was performed in a single study centre. The trial was conducted in compliance with the Declaration of Helsinki, as well as with Good Clinical Practice and applicable regulatory requirements. Approval was obtained from the local Ethics Committee before study initiation and written informed consent was obtained by a delegated rheumatologist from all study patients before performing any study procedure. The trial was registered in the Centre for Drug Evaluation of CFDA under the code of ‘CXSL1000017’.

Patients came to the research unit for a comprehensive medical screening within 28 days prior to their first study dose. During this visit, subjects received a screening number and were asked for demographic characteristics, present and past medical history, and current treatments for RA and/or other on-going medical disorders. Physical examination, including counting of swollen and tender joints was done, together with blood and urine sampling for haematology, urinalysis, blood chemistry and coagulation function tests. The eligibility of the patients was automatically verified using the electronic data management system (Promasys version 6.0, Leiden, The Netherlands) based on pre-defined protocol -specific validation rules and the data entered into the study database.

Eligible subjects were allocated with a subject number by the study nurse and they registered at the study unit on the day before dosing (day −1). Based on the sequence of their enrolment (i.e. the screening number), patients were randomized 2 : 1 within each cohort to receive RCT-18 or placebo with one of the following three subcutaneous (s.c.) dosing regimens in chronological order (Table 1): 1) 180 mg RCT-18 or placebo s.c. weekly for three times, 2) 180 mg RCT-18 or placebo s.c. twice weekly for seven times, followed by a last injection in the 5th week and 3) 360 mg RCT-18 or placebo s.c. weekly for five times.

Table 1. Randomized allocation of the study medication in each cohort
CohortAdministrationTreatment allocation
RCT-18nPlacebon
  1. image represents a subcutaneous injection of 180 mg RCT-18 or matching placebo.
1image180mgQW*34Placebo2
2image180mgBIW*86Placebo3
3image360mgQW*54Placebo2
Total  14 7

Both RCT-18 and the placebo preparation were supplied as lyophilized powder. The investigational drugs were dissolved into colourless solutions with matching volume of aquae pro injectione within 2 h prior to dosing. Thus the appearance of the administered injections was visually discernable. The study medication was prepared and labelled with subject number by two nurses in a specific room prior to administration. In order to avoid information bias, all patients were kept unknown about the identity of their administered treatment or the content of the treatment allocation form during the entire study.

Patients

Patients of either gender, aged between 18 and 65 years, being diagnosed as having rheumatoid arthritis according to the American College of Rheumatology (ACR) criteria [18] for at least 1 year and maintaining on current anti-RA treatments for at least 3 months, without significant concomitant illness, recent severe infections or organ dysfunction, were enrolled into the study. Active RA patients were defined as subjects who had a pre-dose DAS28 [19] score ≥ 3.2.

Use of rituximab (anti-CD20 antibody) or anti-BLyS antibody within 6 months before the first study dose or injection of any vaccine within 1 month before the study was not allowed. Subjects who were positive in HIV-Ab, HCV-Ab or HBsAg test, or had an induration larger than 15 mm in diameter 72 h after injection of the tuberculin PPD were excluded from the study. All patients were instructed to use medically acceptable contraceptive methods to avoid pregnancy during the study and for 3 months after their last study administration.

As a clinical pharmacological study, this trial was designed to address the biomarker cascade associated with the pharmacological behaviour of RCT-18 at six escalating levels [20]: level 1, concentration of drug: free RCT-18 and total RCT-18 (i.e. the total systemic level of RCT-18, either in free form or in binding form]), level 2, molecular target occupancy: BLyS-RCT-18 complex, anti-drug antibody, level 3, molecular/\cellular target activation: counts of CD19(+) lymphocytes, CD27(+) B cells, and IgD(+) B cells, level 4, physiological measures: immunoglobulins, such as IgM, IgG, IgA, level 5, pathophysiological measures: IgM type rheumatoid factor and level 6, clinical responses: DAS28 score. Figure 1 provides an overview of the biomarker cascade and illustrates our hypothesis about the between level interactions.

Figure 1.

An overview of the six level biomarker cascade and the hypothesized between level interactions

Clinical and pharmacodynamic assessments

The disease status of all subjects was evaluated by the same qualified physician throughout the study with the Disease Activity Score 28-joint assessment (DAS28) [19], as well as the counts of tender and swollen joints (out of 68 joints and 66 joints, respectively), patients' self-rating of pain and disease activity using a 0–100 mm visual analogue scale (VAS), erythrocyte sedimentation rate (ESR), physician's global assessment of disease activity using a 0–100 mm VAS and patients' assessment of physical function using the Health Assessment Questionnaire (HAQ). The clinical response of each patient was categorized based on the DAS28 score change from baseline according to the European League Against Rheumatism (EULAR) criteria [21].

Circulating concentrations of three immunoglobulin subtypes (IgM, IgA and IgG), IgM-type rheumatoid factor (IgM-RF) and the counts of lymphocyte subtypes, i.e. CD19(+) lymphocytes (total B cells), CD27(+) B cells and IgD(+) B cells, were repeatedly examined in cohort 1 on days 0 (pre-dose), 1, 7, 14, 19, 21, 28, 35, 42, 56, 70 and 84 and in cohort 2 and 3 on days 0 (pre-dose), 1, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84, 98 and 105.

Pharmacokinetic (PK) assessments

Serial blood samples were collected to determine the serum concentrations of total RCT-18, free RCT-18 and BLyS-RCT-18 complex, respectively. The level of anti-drug antibody was also measured at pre-dose and up to 84 (for cohort 1) or 105 days (for cohorts 2 and 3) after the first dose.

PK samples were analyzed using a validated ELISA method with a lower limit of quantification (LLOQ) of 15 ng ml-1 for free and total RCT-18 and a LLOQ of 20 ng ml-1 for RCT-18-BLyS complex [8].

Pharmacokinetic assessments were performed through non-compartmental analysis using WinNonlin (version 6.3; Pharsight Corp., Mountain View, CA, USA).

Immunogenicity assessments

An immunogenicity ELISA assay was developed to quantify the total Ig antibody response to RCT-18 in human serum [8]. RCT-18 was immobilized on 96-well plates, and the blocked and diluted serum samples (dilution fold between 5 and 1215, with an escalation step of 3) were incubated for 1 h, during which possible anti-RCT-18 antibodies (ADAs) would bind to the RCT-18 on the plate. Biotinylated RCT-18 in combination with horseradish peroxidase (HRP)-anti human IgG-Fc was used to generate a signal. Equivalently diluted pre-dose serum samples were used in this assay as a negative control. The cut-off signal-to-noise ratio is set ≥2.1 for a positive result.

Safety assessments

Adverse events, haematology, blood chemistry, urinalysis, 12-lead electrocardiograms, vital signs and physical examinations were frequently monitored during the study.

Statistical analysis

All statistical analyses were done with SAS® v9.2 (SAS Institute, Cary, North Carolina, USA).

Given the explorative nature of this study, no calculation was performed for sample size estimation. The number of subjects was determined according to the requirement of the China Food and Drug Administration (CFDA) for PK studies [22].

The safety analysis set was defined as all patients who received at least one dose of the investigational treatment. The intent-to-treat analysis set was defined as all patients who were randomized. The per protocol analysis set included patients who received all study administrations and had complete and valid data in all study evaluations. Since no patient discontinued their study prematurely and there was no major protocol deviation, the three above mentioned data sets were eventually identical in this study. In addition, there was a subset of patients with baseline DAS28 score ≥ 3.2 for the analysis of efficacy.

Pharmacodynamic and efficacy parameters were summarized and graphically presented as ratios over the baseline values or changes from baseline over time. Continuous efficacy parameters were summarized by time point, using changes from baseline. Categorical parameters were summarized upon the last study assessment using only frequencies and incidences. The overall response of the pharmacodynamic and efficacy parameters (i.e. IgM, IgG, IgA, IgM-type rheumatoid factor and DAS28 score) were expressed as least square means and analyzed by mixed model analyses of covariance (ancova), with treatment as fixed effects, with subject as random effect, and with the baseline value as covariate, where baseline was defined as the average of the available values obtained prior to dosing. Treatment effects of the three active treatment regimens (‘180mgQW*3’, ‘180mgBIW*8’, and ‘360mgQW*5’) were compared with placebo within the ancova model. All variables were analyzed untransformed. Treatment effects were reported as contrasts, where the average of the measurements up to the last time point and the difference between each active treatment and placebo was calculated within the statistical model. Contrasts were reported along with 95% confidence intervals (CIs) and analyses were two-sided with a significance level of 0.05. The average response was believed to reflect the general effect of the study treatment during the observation period and be superior to the maximum value since the latter may be more prone to a potential placebo effect.

The PK parameters were summarized with descriptive statistics by treatment. Individual plasma concentration measurements were averaged by time points for graphical presentation of the mean concentration–time profile by treatment.

All adverse events were described with preferred terms and classified into system organ classifications (SOCs) based on MedDRA version 15.0.

Results

Study population

The study lasted about 8 months, with the first patient visit occurring on 28 February 2012, and the last patient follow-up taking place on 11 October 2012. Twenty-one RA patients, four males (19.0%) and 17 females (81.0%), aged between 19 and 60 years, were randomized into the study. Overall, 14 patients received RCT-18 in three different groups (four in ‘180mgQW*3’, four in ‘360mgQW*5’ and six in ‘180mgBIW*8’) and seven patients received placebo (a combined group from the three cohorts). All patients completed their study per protocol. Table 2 summarized the demographic characteristics and baseline measurements of patients by treatment. Body weight was generally balanced among the three RCT-18 groups. In the subgroup of patients defined as ‘active RA’, DAS28 score, count of swollen joints and count of tender joints were comparable among the four treatment groups.

Table 2. Demographic characteristics and baseline measurements
Group 180mgQW*3180mgBIW*8360mgQW*5Placebo
All patients n = 4n = 6n = 4n = 7
  • TJC tender joint count; SJC swelling joint count;
  • ESR erythrocyte sedimentation rate;
  • HAQ Health Assessment Questionnaire.
  • * Patients whose baseline DAS28 score was ≥3.2.
Age (years)Mean (SD)38.3 (14.8)47.7 (12.6)49.0 (7.6)51.0 (6.4)
Weight (kg)Mean (SD)60.6 (13.3)59.4 (11.3)63.1 (12.7)69.7 (9.5)
GenderFemale (%)3 (75.0%)5 (83.3%)4 (100%)5 (71.4%)
SJCMedian (range)3 (0–9)5 (1–18)5 (2–7)3 (0–5)
TJCMedian (range)13.5 (1–23)12.5 (2–34)12 (5–27)9 (3–29)
HAQMean (SD)1.20 (0.85)0.57 (0.24)1.18 (0.69)0.66 (0.48)
DAS28Mean (SD)3.97 (2.08)4.92 (2.64)4.71 (2.22)3.70 (1.33)
ESR (mm h–1)Mean (SD)10 (5)36 (23)19 (11)15 (10)
IgM-RF (g l–1)Mean (SD)1193 (1279)2109 (1568)5743 (1594)2916 (2885)
IgA (g l–1)Mean (SD)2.03 (0.61)2.89 (0.98)3.16 (0.49)2.47 (0.68)
IgG (g l–1)Mean (SD)10.8 (3.7)13.3 (4.0)12.5 (3.7)11.7 (3.0)
IgM (g l–1)Mean (SD)1.03 (0.29)1.17 (0.70)1.94 (0.55)1.00 (0.37)
RAactive* (n = 3)(n = 6)(n = 4)(n = 3)
DAS28activeMean (SD)4.61(0.69)4.92(1.29)4.71(0.74)5.03(0.75)
SJCactiveMedian (range)4(2–9)5(2–18)5(2–7)3(3–4)
TJCactiveMedian (range)14(13–23)7.5(2–34)12(5–27)20(9–29)

Upon entry into the study, each subject had been under stable anti-RA treatment for at least 3 months, which included synthetic disease-modified anti-rheumatic drugs (synthetic DMARDs), non-steroid anti-inflammatory drugs (NSAIDs), steroid hormones, traditional Chinese medications, folk remedies or combined therapies. Methotrexate (MTX) (n = 11, 52.4%), leflunomide (n = 9, 42.9%) and prednisone (n = 8, 38.1%) were the most common concomitant treatments in the selected RA patients.

Figure 2 demonstrates the pharmacological, pharmacodynamical and clinical response profiles to RCT-18 under the three dosing regimens, which were compared with those of placebo wherever applicable.

Figure 2.

Pharmacological profiles of three dosing regimens of RCT-18 vs. placebo at six escalating levels. image 180mgBIW, image 180mgQW, image 360mgQW, image placebo

Efficacy

DAS28 assessments

Clinical efficacy was explored in subgroups of patients with moderate to severe RA activity at baseline (i.e. baseline DAS28 score ≥ 3.2). A total of 16 patients met this criterion. Mean pre-dose DAS28 scores were generally balanced among the four groups (Table 2). Compared with the placebo group, the DAS28 response was statistically improved with ‘180mgQW*3’ and ‘180mgBIW*8’ (both P < 0.05), but showed no difference with ‘360mgQW*5’ (P = 0.7498) (Table 3). Overall, the DAS28 score steadily decreased during the 3 month observation in the ‘180mgQW*3’ group and the ‘180mgBIW*8’ group, but underwent minimal changes from baseline scores with ‘360mgQW*5’ RCT-18 and placebo (Figure 2). In the groups of ‘180mgQW*3’ and ‘180mgBIW*8’, although the last dose of RCT-18 was administered on day14 and day 28, respectively, the beneficial effect on DAS28 proceeded in a similar rate of improvement up to the last assessment (on day 84 or day105, respectively).

Table 3. Summary of pharmacokinetic parameters for total RCT-18, free RCT-18 and the BLyS-RCT-18 complex with the study treatment
AnalytePK ParameterUnit180mgBIW (n = 6)180mgQW (n = 4)360mgQW (n = 4)
gMeangCVgMeangCVgMeangCV
  • * tmax and tmax1 are presented as median and minimum-maximuam.
  • tmax1 time to maximal serum concentration during the first interval between drug administrations;
  • AUC(0,τ,ss) area under the concentration–time curve during the interval between drug administrations at steady-state;
  • CLss/F apparent clearance at steady-state;
  • Cmax,ss maximal serum concentration at steady-state;
  • R[AUC] accumulation ratio based on AUC(0,τ);
  • R[Cmax] accumulation ratio based on Cmax;
  • t1/2,ss elimination half-life at steady-state;
  • Vz,ss/F volume of distribution at steady-state;
  • AUC(0,∞) area under the concentration–time curve from time zero to infinity;
  • AUC(0,tlast) area under the concentration–time curve from time zero to the last sampling time point;
  • Cmax maximal serum concentration;
  • tmax time to maximal serum concentration.
Free RCT-18tmax1*days11––1.510.3––11.51.5––1.5
 AUC(0,τss)ng ml–1 day16 16044.1%16 93127.0%50 59840.1%
 CLss/Fml day–111 13944.1%10 63227.0%711540.1%
 Cmax,ssng ml–1746740.1%550417.5%16 84336.4%
 R[AUC]1.523.9%2.217.9%1.612.8%
 R[Cmax]1.521.8%2.121.3%1.721.9%
 t1/2,ssdays15.240.5%6.050.0%14.512.9%
 Vz,ss/Fml244 63828.6%92 36733.7%148 47938.7%
 AUC(0,∞)ng ml-1 day135 23241.4%34 67230.9%169 53042.7%
Total RCT-18tmax1*days1.31–-1.511–-1.51.51.5–-1.5
 AUC(0,τss)ng ml-1 day19 25639.9%21 12723.9%64 59340.4%
 CLss/Fml day–1934839.9%852023.9%557340.4%
 Cmax,ssng ml-1814843.4%537616.8%16 51441.4%
 R[AUC]1.618.7%1.819.3%1.717.2%
 R[Cmax]1.517.8%1.628.1%1.413.8%
 t1/2,ssdays15.811.3%15.75.0%12.86.1%
 Vz,ss/Fml213 55532.8%193 46821.4%103 30936.3%
 AUC(0,∞)ng ml-1 day194 76839.2%63 17924.3%264 40239.2%
BLyS-RCT-18 complextmax*days45.530–-565656–-564031–-56
 CmaxIU ml-128 98743.7%29 32914.2%65 91920.8%
 AUC(0,tlast)IU ml-1 day2 041 18941.0%1 800 54214.9%4 623 15210.8%

With regard to clinically significant improvement, 100% (3/3) ‘180mgQW*3’-treated patients and 83.3% (5/6) ‘180mgBIW*8’-treated patients exhibited moderate to good EULAR responses [21] at the end of the 3 month follow-up period. In contrast, only 25% (1/4) ‘360mgQW*5’-treated patients and 33.3% (1/3) placebo-treated patients experienced moderate response. All of the remaining patients had ‘no EULAR response’ during the study.

Pharmacokinetics

The serum concentrations of total RCT-18 and free RCT-18 peaked within 1–1.5 days after each RCT-18 dose. Once or twice weekly repeat administrations resulted in mild accumulations of total RCT-18 and free RCT-18, as well as saturated PK of BLyS-RCT-18 (Table 4, Figure 3). With the three investigated dosing regimens, the overall exposure of free RCT-18 was approximately 80% of that of total RCT-18, while the peak level was generally comparable between free RCT-18 and total RCT-18. Such findings suggested that free RCT-18 accounted for nearly 100% of the total RCT-18 peak concentration (Cmax,ss), while the difference of overall exposure (AUC(0,τss)) between the two analytes might be explained by the relatively slow elimination of the BLyS-RCT-18 complex and/or other binding forms of RCT-18. Due to the difference of administered RCT-18 doses, the total exposure of total and free RCT-18 was smaller following ‘180mgQW*3’ than that after ‘180mgBIW*8’ or ‘360mgQW*5’, respectively (Table 4). However, the total exposure of the BlyS-RCT-18 complex was rather comparable between the treatment of ‘180mgQW*3’ and ‘180mgBIW*5’, but approximately 2–3-fold higher with the treatment of ‘360mgQW*5’ (Table 4).

Table 4. Results of the ancova analyses comparing the pharmacodynamic effects and clinical efficacy of ‘180mgQW*3’, ‘180mgBIW*8’, ‘360mgQW*5’ and placebo
ItemUnitTreatmentLSMDifference vs. placebo95% CI
  • NA not applicable; 95% CI 95% confidence interval; DAS28 DAS28 score.
  • * The ancova analysis was performed in patients with a baseline DAS28 score ≥ 3.2.
IgMg l–1180mgBIW*80.929–0.27(–0.40, –0.15)
  180mgQW*30.962–0.24(–0.38, –0.10)
  360mgQW*50.976–0.23(–0.40, –0.05)
  Placebo1.203NANA
DAS28*-180mgBIW*83.389–0.87(–1.69, –0.05)
  180mgQW*33.262–0.99(–1.85, –0.14)
  360mgQW*54.3920.14(–0.75, 1.03)
  Placebo4.255NANA
Figure 3.

Mean serum concentration–time profile of A) free RCT-18, B) composite RCT-18 and C) BlyS-RCT-18 complex in semi-log scale with the treatment of ‘180mgQW*3’, ‘180mgBIW*8’ and ‘360mgQW*5’ RCT-18. image 180mgQW (n = 4), image 180mgBIW (n = 6), image 360mgQW (n = 4)

Immunogenicity

No anti-RCT-18 antibody was detected by the immunogenicity assay during the observation period of each study cohort.

Pharmacodynamics

Immunoglobulins

Substantial reductions of IgM were observed following administration of RCT-18 (Figure 2). Regardless of the dosing regimen, the maximal IgM decrement generally appeared on day 28, which was 2 weeks after the last dose of the ‘180mgQW*3’ treatment (on day 14), but was close to the last dose of the ‘180mgBIW*8’ and ‘360mgQW*5’ treatment (both on day 28). Thereafter, IgM levels stayed at the nadir (i.e. approximately 30–40% reduction from baseline) and remained at an over 20% reduction until day 84 with the ‘180mgQW*3’ treatment and until day 105 with the ‘180mgBIW*8’ treatment. Under the treatment of ‘360mgQW*5’, however, circulating IgM concentration gradually rose and returned to baseline level on day 84. Such a profile was observed in each of the four subjects who were randomized to this treatment. Hence the mean profile of this dose group virtually reflected the IgM changes in individual patients.

The response profiles of IgG and IgA (Figure 2) were similar to those of IgM with the three dosing regimens, but the relative effect size was smaller on IgG. The maximal IgG decrement was about 20% from baseline with the treatment of ‘180mgBIW*8’ and ‘360mgQW*5’, but was approximately 10% in the ‘180mgQW*3’ group. For IgA, the treatment of ‘180mgQW*3’, ‘180mgBIW*8’ and ‘360mgQW*8’ resulted in around 30% suppression from baseline and a similar recovery curve was also evident with the doses of ‘360mgQW’ after the trough point occurring on around day 28.

According to the ancova analysis, the average decreases of IgM (Table 4), IgG and IgA were all statistically larger with the three RCT-18 treatment regimens compared with the change after placebo (all P < 0.05).

Lymphocyte subpopulation

An initial elevation in the subpopulations of CD19(+) lymphocytes (i.e. total B cells) and IgD(+) B cells was seen during the first 1–3 weeks, followed by a decline that led to maximally 50% reduction of the cell count on days 63–105 with the three RCT-18 dosing regimens (Figure 2). With the treatment of ‘360mgQW*5’, the peak positive effect was much larger than those with the other two active treatments. Such effect difference was rather obvious on the count of IgD(+) B cells. The mea7surements of CD27(+) B cell count were quite variable but changed in a similar pattern (Figure 2). Its response profile with the treatment of ‘180mgBIW*8’ was not distinguishable from that with placebo. However, the biphase response profile with ‘180mgQW*3’ was similar to the abovementioned responses of other lymphocyte subpopulations. The treatment of ‘360mgQW*5’ resulted in a sudden and huge increase in the count of CD27(+) B cells, which subsequently declined to and fluctuated around its baseline level.

Rheumatoid factors

As is shown in Figure 2, the serum concentration of IgM-type rheumatoid factor (IgM-RF) gradually decreased with 180mgQW*3 and 180mgBIW*8, but fluctuated around baseline and then elevated with placebo or 360mgQW*5. The decrement of IgM-RF continued up to day 84 with 180 mg QW RCT-18 but partially reversed since day 49 with ‘180mgBIW*8’. ancova analysis suggested that there was no statistically significant difference in IgM-RF between each RCT-18 group and the placebo group (all P > 0.05).

Safety

All 21 patients reported 146 treatment emergent adverse events (TEAEs) during the study (Table 5). There were no serious AEs or premature study discontinuations due to AE(s). All AEs were mild to moderate in severity and were recovered with or without medical interventions.

Table 5. AEs experienced by at least two RCT-18 treated patients and had higher incidence in the combined RCT-18 groups
Preferred termPlacebo (n = 7)180mgQW (n = 4)180mgBIW (n = 6)360mgQW (n = 4)RCT-18 (n = 14)
Upper respiratory tract infection2(28.6%)2(50%)5(83.3%)4(100%)11(78.6%)
Hepatic enzyme increased1(14.3%)1(25%)1(16.7%)1(25%)3(21.4%)
Hypoesthesia1(14.3%)0(0%)0(0%)3(75%)3(21.4%)
Dizziness1(14.3%)0(0%)2(33.3%)2(50%)4(28.6%)
Rash1(14.3%)0(0%)3(50%)1(25%)4(28.6%)
Fatigue1(14.3%)0(0%)1(16.7%)2(50%)3(21.4%)
Chest discomfort0(0%)0(0%)1(16.7%)1(25%)2(14.3%)
Administration site reaction0(0%)1(25%)6(100%)3(75%)10(71.43%)
Gingivitis0(0%)1(25%)1(16.7%)0(0%)2(14.3%)
Abdominal pain0(0%)0(0%)1(16.7%)1(25%)2(14.3%)
Abdominal pain upper1(14.3%)1(25%)1(16.7%)1(25%)3(21.4%)
Subject with any AEs7(100%)4(100%)4(100%)6(100%)14(100%)

The AEs were most frequently categorized as musculoskeletal and connective tissue disorders (18, 85.71%), skin and subcutaneous tissue disorders (14, 66.67%), general disorders and administration site conditions (13,61.9%) and infections and infestations (13,61.9%) according to the SOCs of MedDRA v15.0. Multiple-dose RCT-18 treatments were associated with approximately 3-fold more general disorders and administration site conditions and infections and infestations in all patients who were treated with RCT-18 compared with those with placebo (n [incidence%]), RCT-18 vs. placebo: 11 [78.57%] vs. 2[28.57%] for both SOCs. Table 3 summarizes AEs experienced by at least two patients and the incidence was higher in the general RCT-18 group.

Discussion

Once or twice weekly doses of 180 mg or 360 mg RCT-18 were generally safe and well tolerated in the study population. Similar to the findings in a previous single ascending dose study [8] and the study in patients with SLE [23], there were more infectious events reported by patients treated with RCT-18, indicating a propensity for infections. Nevertheless, in our study, all infectious events were mild to moderate in severity and short term use of symptomatic treatments and/or anti-infection drugs led to good outcome. These findings indicated the necessity of avoiding cases with ongoing infections and monitoring infectious events shortly before and during the treatment with RCT-18.

Our study showed a divergence between drug levels, biologic activity and clinical activity. The treatments of 180 mg QW*3 and 180 mg BIW*8 caused clinically and statistically significant DAS28 improvement in most RA patients, whereas the treatment of 360 mg QW*5 was ineffective. Such efficacy outcomes were unexpected, as the total administered dose of the ‘360mgQW*5’ treatment was only 20% higher than the ‘180mgBIW*8’ treatment. Noteworthy, the plateau level of the BlyS-RCT-18 complex was approximately 2-fold higher with 360 mg QW RCT-18 compared with that following 180 mg QW or 180 mg BIW RCT-18, and the latter two treatments resulted in a fairly comparable plateau level of the complex. The formation of the BlyS-RCT-18 complex was assumed to be determined by the exposure of RCT-18 and the circulating level of the antigen, BLyS. Because we did not measure the concentration of BLyS in this study, an important point of information was missing for quantitative modelling of the observed profile of the BlyS-RCT-18 complex.

On the other hand, we had intensive data to delineate the biomarker cascade from the concentration of drug to the clinical responses (Figure 1). The dynamic profiles of these biomarkers and their pharmacological/(patho-)physiological relationship provide us with an opportunity to streamline the in vivo behaviour of RCT-18. In this study, sustained IgM and IgM-RF reduction, as well as clinical improvement were observed after the last dose of ‘180mgQW*3’ and ‘180mgBIW*8’ RCT-18. In contrast, prompt recovery of immunoglobulins and lack of IgM-RF and DAS28 improvement were evident following the last dose of ‘360mgQW*5’ RCT-18. The effect profiles of the former two dosing regimens were distinct from those after multiple doses of atacicept [16]. In patients dosed with 360 mg QW RCT-18, a substantially high level of the BLyS-RCT-18 complex was related to excessive stimulation and no subsequent suppression on the count of CD27(+) B cells, to immediate recovery of IgM after drug withdrawal, and to the lack of improvement in IgM-RF and DAS28. Similar associations have been revealed in two previous studies with RCT-18. In the SAD study [8], a numerically similar exposure of the BLyS-RCT-18 complex was seen after single injection of 360 mg RCT-18, which was even higher than that after 540 mg RCT-18. A single dose of 540 mg RCT-18 resulted in an exposure of the BLyS-RCT-18 complex comparable with that obtained with ‘180 mg QW*3’ or ‘180 mg BIW*8 RCT-18’ in the current study. Although measurements of the lymphocyte subpopulation were not available in the SAD study, we did find similar ‘recovery’ in IgM response, leading to smaller IgM change with 360 mg RCT-18 compared with that induced by 540 mg RCT-18 [8]. In the study performed in SLE patients, the dosage of RCT-18 and the resultant level of the BLyS-RCT-18 complex were similar to those obtained in the current study [23]. However, a similar over-stimulation of CD27(+) B cells was seen in the SLE patients, which probably contributed to the partial or full recovery of IgM, IgG and IgA after the last dose [23]. These results confirmed the U-shaped relationship between exposure of the BLyS-RCT-18 complex and the IgM/IgM-RF responses. CD27(+)B cells are functionally responsible for the generation of IgM, IgA and IgG [24, 25]. Hence decrease in the count of CD27(+) B cells might mediate the sustained reduction of immunoglobulins and IgM-RF after dosing cessation, while the excessive stimulation and lack of subsequent suppression in count of CD27(+) B cells are thought to explain the recovery of immunoglobulins and the minimal change in IgM-RF (Figure 1). Despite of the similarity in the complex exposure, the extent of IgM responses to single doses of 360 mg RCT-18 and 540 mg RCT-18 were both smaller than those with weekly 360 mg RCT-18 and those with 180 mg weekly or biweekly RCT-18, respectively. Such inconsistency inferred the possibility that free RCT-18 itself may have a direct inhibitive effect on IgM production, but not on IgM-RF (Figure 1).

In the present study, the treatment of ‘360mgQW*5’ did not affect disease status of the RA patients. However, both ‘180mgQW*3’ and ‘180mgBIW*8’ provided significant improvements in patients with moderate to severe activity of RA. Such results indicated that weekly administration of 360 mg RCT-18 was less likely to be clinically beneficial for active RA patients, while weekly or twice weekly doses of 180 mg RCT-18 were probably attributed to clinical improvement. Since the effect of 180 mg QW was similar to that of 180 mg BIW, we would recommend the former dosing regimen for future clinical development. Moreover, the study findings established causal links from count of CD27(+) B cells to immunoglobulins, to IgM-RF and to DAS28 score. As the bioassay method of the RCT-18 analytes is relatively difficult and expensive to perform, it is more practical to monitor those distal biomarkers for the prediction of clinical efficacy [26]. Considering the potential influence of RCT-18 on immunoglobulins, we would recommend monitoring count of CD27(+) B cells, IgM-RF and DAS28 in clinical follow-up.

The main limitation of this study was that we were unable to measure directly the systemic and/or local (synovial) concentrations of BLyS, APRIL and/or other inflammatory cytokines. Such information would help us understand the in vivo process with different dosing regimens of RCT-18. Small sample size was another drawback of this trial. However, as a data intensive clinical pharmacological study, the PK, pharmacodynamics and short term efficacy and safety of RCT-18 was explored in a simultaneous manner, which enabled us to build up a bridge between the RCT-18 dosage and its clinical effects on RA patients via the PK and pharmacodynamic biomarkers.

In conclusion, our study demonstrated good safety and tolerability of multiple dose RCT-18 in Chinese RA patients. The treatments of ‘180mgQW*3’ and ‘180mgBIW*8’ exhibited clinical improvements in DAS28 score but ‘360mgQW*5’ had a lack of effect. The significantly higher exposure of the BLyS-RCT-18 complex with ‘360mgQW*5’ vs. those with ‘180mgQW*3’ and ‘180mgBIW*8’ may account for such divergence. In addition, the study results suggested monitoring the count of CD27(+) B cells, the level of IgM-RF and DAS28 may be useful for the prediction of clinical response. These findings help us define the dose range and dosing regimen for future studies.

Competing Interests

All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare CX, JJ, ZQ, ZW, HP, HY and ZF had no support from any organization for the submitted work, WW and LL were full-time employees of RC Biotechnologies, Ltd. Yantai, China and FJ and YX were full-time employees of School of Life Science in Tongji University and received support from RC Biotechnologies, Ltd for the submitted work in the previous 3 years. There were no other relationships or activities that could appear to have influenced the submitted work.

Authors' contributions

The authors of this work are Xia Chen, Yong Hou, Ji Jiang, Qian Zhao, Wen Zhong, Wenxiang Wang, Xuejing Yao, Lin Li, Jianming Fang, Fengchun Zhang, Pei Hu. X. Chen was the primary medical writer of this paper. W. Zhong performed the statistical analysis of the work. Y. Hou and F.C. Zhang were responsible for the recruitment, informed consent and medical care of the RA patients during the study. Q. Zhao, J. Jiang and X.J. Yao were responsible for the bioassay of pharmacokinetic and immunogenicity samples. L. Li, W.X. Wang and J.M. Fang were in charge of study coordination and study management. W.X. Wang and J.M. Fang provided scientific advice and support regarding preclinical information of the investigational product. P. Hu was the principle investigator of this study. All authors participated in the study design, and reviewed and approved the submission of this manuscript. All authors met ICMJE authorship criteria and no person not named as an author qualified for authorship on this manuscript.

We acknowledge all subjects for their contributions to the study. This study was funded by RC Biotechnologies, Ltd, Yantai, China. The work of this study was supported by the National Programme on Key Research Project of New Drug Innovation (No. 2012ZX09303006-002) to Pei Hu, the National High Technology Research and Development Programme (863 Programme) of China, grant #2012AA02A302 to Jianmin Fang; National Science and Technology Projects for ‘Major Drug Innovation and Development’, grants #2013ZX09401002 to Jianmin Fang and #2013ZX09101019 to Wenxiang Wang.

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