Objectives Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease associated with diffuse immune cell dysfunction. CD40–CD40 ligand (CD40L) interaction activates B cells, antigen-presenting cells and platelets. CD40L blockade might provide an innovative treatment for systemic autoimmune disorders. We investigated the safety and clinical activity of dapirolizumab pegol, a polyethylene glycol conjugated anti-CD40L Fab' fragment, in patients with SLE.
Methods This 32-week randomised, double-blind, multicentre study (NCT01764594) evaluated repeated intravenous administration of dapirolizumab pegol in patients with SLE who were positive for/had history of antidouble stranded DNA/antinuclear antibodies and were on stable doses of immunomodulatory therapies (if applicable). Sixteen patients were randomised to 30 mg/kg dapirolizumab pegol followed by 15 mg/kg every 2 weeks for 10 weeks; eight patients received a matched placebo regimen. Randomisation was stratified by evidence of antiphospholipid antibodies. Patients were followed for 18 weeks after the final dose.
Results No serious treatment-emergent adverse events, thromboembolic events or deaths occurred. Adverse events were mild or moderate, transient and resolved without intervention. One patient withdrew due to infection.
Efficacy assessments were conducted only in patients with high disease activity at baseline. Five of 11 (46%) dapirolizumab pegol-treated patients achieved British Isles Lupus Assessment Group-based Composite Lupus Assessment response (vs 1/7; 14% placebo) and 5/12 (42%) evaluable for SLE Responder Index-4 responded by week 12 (vs 1/7; 14% placebo). Mechanism-related gene expression changes were observed in blood RNA samples.
Conclusions Dapirolizumab pegol could be an effective biological treatment for SLE. Further studies are required to address efficacy and safety.
Trial registration number NCT01764594.
- autoimmune disease
- systemic lupus erythematosus
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Systemic lupus erythematosus (SLE) is a multisystem and complex autoimmune disease that results in morbidity, an increased mortality rate and a poor quality of life.1–3 The pathogenesis involves several mechanisms, including proinflammatory presentation of potential autoantigens by the innate immune system, hyperactivation of the adaptive immune system, formation of pathogenic autoantibodies and the deposition of immune complexes capable of affecting diverse organ systems.4 5
CD40 ligand (CD40L, also known as CD154) has been shown to be an important immune-inflammatory modulator and as such it is a credible candidate for pharmacological intervention.6 7 It is widely expressed on naïve and activated CD4+ T cells and platelets; the receptor, CD40, is expressed constitutively on a wide range of cells including antigen presenting cells and B cells.8–10 CD40–CD40L interaction has been shown to be essential for normal T cell/B cell functional interactions, including the T cell-dependent humoral immune response, T cell activation of antigen presenting cells, augmentation of CD8+ T cell responses, immunoglobulin (Ig) class-switching and induction of dendritic cell maturation.10–12 Blockade of CD40–CD40L may decrease the immune activation seen in autoimmune disorders, and this approach has been shown to be efficacious in diverse models of experimental autoimmunity.13
In previous studies, the monoclonal anti-CD40L IgG1 antibody, hu5c8 (ruplizumab; BG9588), showed evidence of potential efficacy in patients with lupus nephritis and idiopathic thrombocytopenic purpura;14 15 however, clinical trials were halted because of a higher than expected occurrence of thromboembolic events.15 16 It is proposed that the observed treatment-related (TR) thromboembolic events occurred as a result of platelet activation and aggregation, due to the formation of anti-CD40L antibody and soluble CD40L immune complexes that tether to platelets via binding of sCD40L to surface-expressed CD40 and activate platelets through interactions of the Fc with Fc gamma receptor IIA on the platelet surface.17 18 Dapirolizumab pegol (CDP7657), an anti-CD40L Fab’ antibody fragment conjugated to polyethylene glycol (PEG),19 was designed to address the potential safety problems caused by the Fc moiety, while retaining favourable pharmacokinetics (PK). In vitro assays have demonstrated that dapirolizumab pegol is a potent antagonist of CD40L binding to CD40, and dose-dependent inhibition of antibody responses with dapirolizumab pegol have been demonstrated in both humanised severe combined immune deficient mice and cynomolgus macaques.19 20 No histopathological evidence of increased thrombovasculopathy or in vitro platelet activation with dapirolizumab pegol compared with placebo was observed in these models.
We report a phase I study designed to evaluate the safety, tolerability and PK of repeat intravenous dosing of dapirolizumab pegol in patients with active SLE. The study also aimed to assess the effect of dapirolizumab pegol on disease activity and CD40L pathway modulation.
Trial design and interventions
A randomised, double-blind, multicentre, placebo-controlled, exploratory phase I study was performed at multiple sites across Europe (Belgium, Bulgaria, Germany, Poland, Romania, Russia and Spain). Patients were randomised (2:1) to receive intravenous-administered dapirolizumab pegol 30 mg/kg followed by dapirolizumab pegol 15 mg/kg every 2 weeks for 10 weeks (total of 5 doses) or to receive a matching placebo regimen (see online supplementary figure S1). Participants were randomised according to evidence of antiphospholipid (aPL) antibodies; these could include historic reports of all known antiphospholipid antibodies. The expected duration of study participation was 32 weeks, comprising an initial 4-week screening period, a 10-week dosing period and an 18-week follow-up period (see online supplementary figure S1). Dosing in the study was intended to achieve a probable therapeutic concentration of dapirolizumab pegol. Data from human studies completed with another anti-CD40L antibody (ruplizumab; BG9588) indicated quantifiable effects on antidouble stranded (ds) DNA antibody levels in patients with SLE15 when a concentration of >100 µg/mL was achieved (UCB data on file). This was further supported by preclinical data showing a dose-dependent inhibition of antibody response with dapirolizumab pegol in cynomolgus macaques.19
Supplementary file 1
Patients were aged 18–75 years with a diagnosis of SLE satisfying the American College of Rheumatology classification and a Safety of Estrogen in Lupus Erythematosus National Assessment Modification to the Systemic Lupus Erythematosus Disease Activity Index - 2000 (SELENA SLEDAI) score ≥4 at screening.21 22 All patients had positive anti-dsDNA antibodies (defined as >10 IU/L using an enzyme-labelled anti-isotope assay (ELiA) (Thermo Fisher Scientific: Phadia)) or were antinuclear antibody (ANA) positive (>1:80 using an ELiA assay (Thermo Fisher Scientific: Phadia)) at screening or had a previously documented positive anti-dsDNA antibody or ANA assay.
Patients taking corticosteroids, antimalarial drugs or immunosuppressants were required to be on a stable dose (no greater than 20 mg/day for oral prednisolone or equivalent) for at least 28 days before the first dose of dapirolizumab pegol and were required to maintain this dose for at least 28 days following the start of dapirolizumab pegol administration. There were no restrictions on prior biological therapy use to treat SLE.
Patients with active, neuropsychiatric SLE, active severe glomerulonephritis, an existing or documented acute renal flare in the previous 6 months and/or decreased renal function (estimated glomerular filtration rate <30 mL/min/1.73 m2; urinary protein >2 g/24 hours or urinary protein:creatinine ratio of >200 mg/mmol) were excluded. Also excluded were patients with a significantly increased risk of thromboembolic events (history of thromboembolism within 1 year prior to screening, vascular graft, valvular heart disease, atrial fibrillation, presence of at least one Factor V Leiden mutant allele, a positive test for aPL antibodies with no stable anticoagulation treatment for at least 28 days prior to the first dose of dapirolizumab pegol).
The primary objective of this study was to evaluate the safety and tolerability of repeated dapirolizumab pegol administration in patients with active SLE. The secondary objective was to assess the PK of dapirolizumab pegol; exploratory objectives monitored disease activity and/or CD40L pathway modulation (via messenger RNA (mRNA) changes and laboratory measures including platelet counts, autoantibodies and complement components) following repeated intravenous administration of dapirolizumab pegol.
Disease activity measures included the British Isles Lupus Assessment Group (BILAG) 2004 index assessment using centralised grading, which reflects disease activity within the last 4 weeks22 23 and SELENA SLEDAI composite scoring to reflect disease activity within the last 10 days. Responder indices included the BILAG-based Composite Lupus Assessment (BICLA) and the Systemic Lupus Erythematosus Responder Index-4 (SRI-4).24 25 Physician global assessment was collected via a visual analogue scale ranging from 1–100, and the assessor was also blind to the Subject Global Assessment.
Transcriptional analysis (mRNA signature profiling)
Peripheral blood samples used for gene expression analyses were collected using PAXgene Blood RNA tubes (PreAnalytix GmbH). RNA was isolated using the Agencourt RNAdvance Blood kit (Beckman). cDNA was prepared with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For quantitative real-time PCR, custom primers and probe sequences were designed using the NCBI Nucleotide website and Primer Express software and ordered from Applied Biosystems (see online supplementary table S1). A panel of genes expressed by B cells and plasma cells was selected for analysis of CD40 pathway modulation. In addition, gene transcripts associated with SLE disease activity, including type I interferon (IFN)-response genes, and those associated with various cellular subsets, were evaluated. All transcripts were normalised to the geometric mean of four housekeeping genes. For each patient, transcript expression for each gene at each timepoint was expressed relative to day 0 (fold change from baseline).
For the analysis, genes were grouped into functional domains associated with either their cellular expression (such as B cells, T cells and natural killer cells) or based on their coordinated expression pattern and association with SLE disease activity (such as IFN-response genes) as described by Chaussabel et al.26 Internal decision criteria to indicate an effect on gene expression were designed to identify consistent changes, recognising the inherent architecture of the data and controlling the false positive rate (see online supplementary material). The prespecified significance level was chosen to achieve an overall false positive rate of 5% when comparing two treatment groups of size 8 and 16 and were finalised prior to assay of any transcriptomic samples.
Randomisation and blinding
A randomisation list was generated by the contract research organisation (CRO) using the Interactive Voice Response/Interactive Web Response Systems. Participants were randomised according to the presence of aPL antibodies. Patients received a five-digit subject number at screening, which was used along with the generated randomisation code to allocate phial numbers to the subject at each treatment. Throughout the study, investigators, study site and CRO staff, with the exception of pharmacy monitors, remained blinded to treatment allocations, as did patients and sponsor staff.
The study was conducted in accordance with International Conference on Harmonisation Good Clinical Practice requirements. The study was run under the aegis of a Data Monitoring Committee formed to monitor the ongoing safety of the study; the study could have been stopped for any significant safety concern, especially the observation of more than one thromboembolic event.
The full analysis set (FAS) consisted of all randomised patients who received at least one dose of study treatment (dapirolizumab pegol or placebo). The pharmacodynamic per protocol analysis set was a subset of the FAS including those patients who had no important protocol deviations affecting pharmacodynamic variables. The evaluable population for the PK analysis consisted of those patients in the FAS who had no important protocol deviations affecting PK variables and had data from at least one PK sample available.
All statistical analyses were considered exploratory. A statistical analysis plan was completed in advance of database lock and study unblinding. BILAG-BICLA and SRI-4, were implemented to define responders and were analysed separately (see online supplementary material). No escape treatment rule was implemented; no patients received escape treatment. The efficacy analysis was performed in a subset of patients with a baseline SELENA SLEDAI score ≥6 and at least two BILAG grade Bs. The number of responders in each treatment group was calculated for both BICLA and SRI-4, and the responder rates were compared between treatments using Fisher’s exact test.
Statistical analysis was performed for measurements of anti-dsDNA, complement C3, complement C4 and for IgG, IgA and the ratio of IgA:IgG. Each variable was subject to a repeated-measures analysis of covariancewith visit as the repeated measure and visit, treatment and visit-by-treatment interaction as fixed effects. Baseline was included as covariate. In the mRNA expression analysis, each transcript type was subject to analysis of variance with subject as random effect and visit, treatment and visit-by-treatment interaction as fixed effects. All original and derived parameters were listed and described using summary statistics. The outputs from these analyses informed the internal decision criteria described above.
In total 68 patients were screened, 44 were screen failures (majority due to inclusion and exclusion criteria linked to laboratory parameters) and 24 patients were randomised to treatment (dapirolizumab pegol n=16, placebo n=8; figure 1). Baseline characteristics are displayed in table 1 and were broadly similar for each treatment group.
Based on the primary safety variables, multiple doses of dapirolizumab pegol were well tolerated in patients with SLE. The overall incidence of treatment-emergent adverse events (TEAEs) in the dapirolizumab pegol group was 87.5% (14/16 patients) compared with 62.5% in the placebo group (5/8 patients; see online supplementary table S2). All TEAEs in the dapirolizumab pegol group were considered mild or moderate in intensity; none were severe. No serious AEs were reported, and no patients died during the study. No TEAEs related to thromboembolic events or laboratory findings suggestive of thromboembolic events were reported in either the dapirolizumab pegol or placebo group.
The most commonly reported TEAEs in the dapirolizumab pegol group were nasopharyngitis (6/16; 37.5%), headache (4/16; 25%), upper respiratory tract infection (3/16; 18.8%), anaemia and diarrhoea (2/16; 12.5% for each) (table 2). One subject (12.5%) in the placebo group reported severe TEAEs of musculoskeletal pain and neck pain. One subject (6.3%) in the dapirolizumab pegol group discontinued the study because of an upper respiratory tract infection; the event was deemed mild and not considered by the investigator to be related to treatment. There was a numerically greater incidence in TEAEs related to infection in the dapirolizumab pegol group (11/16; 68.8%) compared with the placebo group (3/8; 37.5%). However, none of the infections were serious or considered opportunistic. There were no noteworthy TEAEs related to infusion reactions.
Four (25.0%) dapirolizumab pegol-treated patients and three (37.5%) placebo-treated patients were considered to have treatment-related (TR)-TEAEs, as determined by the investigator. In the dapirolizumab pegol group, these included nasopharyngitis (2/16; 12.5%), diarrhoea, herpes simplex, paronychia, increased lipase, hypernatraemia, headache and dysuria (1/16; 6.3%, each). In the placebo group, TR-TEAEs included feeling hot, musculoskeletal pain, neck pain and hot flush (1/8; 12.5% each). There were no clinically significant abnormalities and no substantive differences between the dapirolizumab pegol and placebo groups in laboratory values, including platelet, lymphocyte and neutrophil counts, vital signs or ECGs. There were no increases in the doses of concomitant corticosteroids, antimalarial drugs or immunosuppressants during the study.
Overall, there was greater improvement in the clinical measures of disease activity in the dapirolizumab pegol group than in the placebo group. Improvements from baseline were observed for the SELENA SLEDAI (figure 2), Subject’s Global Assessment of Disease, and BILAG total score. There was a greater proportion of BICLA (5/11; 45.5%) and SRI-4 (5/12; 41.7%) responders in the dapirolizumab pegol group at week 12, compared with the placebo group (1/7; 14.3% for both BICLA and SRI-4).
At the mRNA transcript level, expression changes in genes within the plasma cell and B cell domains were noted, consistent with modulation of the CD40 pathway. Among the plasma cell genes, the dapirolizumab pegol group exhibited more rapid and greater decreases in the expression of several Ig-associated genes (secretory IgA, IgG, Igκ, Igλ and J chain) starting at week 2 and maintained over the treatment period, compared with placebo (figure 3A). Among the B cell genes, the dapirolizumab pegol group showed a transient increase in CD19 and CD20 RNA transcripts at week 2 (figure 3B). Several of the patients in the dapirolizumab pegol group exhibited a >2-fold reduction in the expression of type I IFN-response genes (MX1 (n=6), OAS1 (n=4), IFITM3 (n=9), G1P2 (n=5)) for at least two time points (figure 4). These changes were more dramatic in the responder group. No other consistent changes in other functional group expression patterns were observed (data not shown).
There was a treatment difference in anti-dsDNA antibody concentrations (p=0.0168) at week 12; however, there were only seven patients in the dapirolizumab pegol group and four patients in the placebo group with elevated titres (>15 IU/mL) at baseline. No signals were detected in other laboratory measures, including anti-C1q, aPL and complements C3 and C4. In the dapirolizumab pegol group, a reduction in IgG concentrations (p=0.0436) and small reductions in total Ig and IgA concentrations were observed at week 12 compared with the placebo group at the same time point.
PK assessments showed that the exposure of dapirolizumab pegol was maintained with a trough concentration >100 µg/mL (online supplementary figure 2); the doses selected for the study were based on the trough level at which anti-dsDNA antibody decrease was observed in the hu5c8 study.15 The estimated half-life of dapirolizumab pegol was in the range of 7.8–14 days, geometric mean AUCw0–10 was 22 326 day*µg/mL and geometric mean Cmax at week 10 was 582.2 µg/mL. The PK of dapirolizumab pegol was not affected by the presence of antidrug or anti-PEG antibodies.
Studies in animal models have shown that blockade of CD40L is efficacious in inflammatory and autoimmune conditions.19 20 While caution is needed when extrapolating such data to humans, CD40L blockade could be an innovative approach for the treatment of SLE.27 Dapirolizumab pegol is a purified recombinant, humanised Fab’ antibody fragment covalently bound to PEG that targets CD40L. The primary objective of this study was to evaluate the safety and tolerability of repeated intravenous doses of dapirolizumab pegol in patients with mild to moderate SLE. The secondary objective was to assess PK; exploratory objectives included evaluation of the effects of dapirolizumab pegol on disease activity, biomarkers of disease activity and CD40L pathway modulation.
In previous clinical studies of CD40L inhibition using monoclonal full-length IgG1 antibodies, the incidence of thromboembolic events has been higher than expected.15 16 28 The Fc portion of the full-length antibody has a critical role in the mechanism leading to thromboembolic events.17 18 Since dapirolizumab pegol lacks the Fc portion, it would not be expected to be associated with increased thromboembolic risk, a hypothesis supported by the absence of thromboembolic events in the present study and a previous non-human primate study in Rhesus monkeys.19 Multiple doses of dapirolizumab pegol were well tolerated. There was a higher incidence of non-serious infection in the dapirolizumab pegol group. No TEAEs related to thromboembolic events or laboratory findings suggestive of thromboembolic events were reported during the study, and there was no evidence of enhanced procoagulatory effects. A trend towards an increased infection rate was noted in the dapirolizumab pegol arm, but this was not accompanied by changes in total lymphocyte or neutrophil counts. These safety findings are comparable with those from a previous single-dose, double-blind, first-in-human, phase I study of dapirolizumab pegol (NCT01093911).29 None of the study subjects experienced any dose limiting toxicities and no thromboembolic events were reported.29
In terms of clinical measures of disease activity, there was greater improvement in the dapirolizumab pegol group compared with the placebo group. Improvements from baseline were observed in SELENA SLEDAI, Subject’s Global Assessment of Disease, BILAG total score, BICLA and SRI-4 responders. In addition, statistically significant changes were observed in expression of genes associated with B cell and plasma cell function, as were reductions in the expression of IFN-response genes, consistent with known functions of CD40L.
The promising safety and preliminary efficacy findings reported here must be interpreted with caution as this small, exploratory study was not powered to demonstrate statistical significance on all outcomes reported. Only one dose level was evaluated, so no dose effects were studied; however, a phase II dose-ranging study to better define the optimal therapeutic dose and regimen is underway (NCT02804763).
This study has shown that dapirolizumab pegol, administered in multiple doses over 12 weeks, appears well tolerated in patients with mild to moderate SLE with no major safety concerns. We observed gene transcription changes associated with inhibition of the CD40–CD40L interaction and improvement in clinical measures of disease activity in the dapirolizumab pegol group compared with the placebo group. These results support further investigation of dapirolizumab pegol as a novel treatment for SLE.
The study was funded by UCB Pharma and Biogen. The authors would like to acknowledge Norm Allaire, Julie Czerkowicz and other Biogen colleagues for their contributions to the gene expression analysis; Huo Li and Carrie Wager of Biogen for normalisation of the qPCR data. The authors would like to acknowledge Laura Griffin, PhD, of iMed Comms, an Ashfield Company, part of UDG Healthcare plc for medical writing support that was funded by UCB Pharma and Biogen. The authors would like to thank the investigators, patients and their families who participated in this study.
Handling editor Tore K Kvien
Contributors CC, AMR, LCB and MZ contributed to the conception and design of the study; CC, TD, FH, GIJ, CO and MU were involved in the acquisition of data; CC, AMR, PJC, TD, FH, GIJ, CO, CS, MU, LCB and MZ contributed to the analysis and interpretation of data. All authors contributed to drafting and/or revising the manuscript.
Funding UCB Pharma and Biogen.
Competing interests CC, PJC, GIJ, CO and MZ are full-time employees of UCB Pharma and hold stock awards and/or options. CS is a full-time employee of UCB Biosciences GmbH and holds stock awards and/or options. AMR was a full-time employee and stock holder of Biogen at the time the study was conducted. LCB is a full time employee and stockholder of Biogen. FH has received consultancy fees from UCB, Sanofi, Eli Lilly, Baxter, BMS and research grants from Deutsche Forschungsgemeinschaft, IMI (PRECISESADS) and attended speakers’ bureau for GSK, Roche Pharma and Pfizer. TD has received consultancy fees and research grants from UCB, Biogen, Roche, Sanofi, Eli Lilly, Jansen and research grants from Deutsche Forschungsgemeinschaft and EU Horizon 2020 (Harmonics). MU has served as Chair of the Data and Safety Monitoring Committee for the study.
Patient consent A patient consent form was completed.
Ethics approval This was a national, regional, Independent Ethics Committee or Institutional Review Board.
Provenance and peer review Not commissioned; externally peer reviewed.
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