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Efficacy and safety of tregalizumab in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a phase IIb, randomised, placebo-controlled trial
  1. Ronald F van Vollenhoven1,
  2. Edward Clark Keystone2,
  3. Vibeke Strand3,
  4. Cesar Pacheco-Tena4,
  5. Jiří Vencovský5,
  6. Frank Behrens6,
  7. Arthur Racewicz7,
  8. Daniela Zipp8,
  9. Faiza Rharbaoui8,
  10. Ralf Wolter8,
  11. Luise Knierim8,
  12. Rainer Schmeidl8,
  13. Xuefei Zhou8,
  14. Silke Aigner8,
  15. Benjamin Dälken8,
  16. Andrea Wartenberg-Demand8
  17. on behalf of the TREAT2b study team
    1. 1 Department of Rheumatology, Amsterdam Rheumatology and Immunology Center ARC, Amsterdam, The Netherlands
    2. 2 The Rebecca MacDonald Centre For Arthritis, Mount Sinai Hospital, Toronto, Ontario, Canada
    3. 3 Division of Immunology/Rheumatology, Stanford University School of Medicine, Palo Alto, California, USA
    4. 4 Investigacion y Biomedicina de Chihuahua, Chihuahua, Mexico
    5. 5 Institute of Rheumatology, Prague, Czech Republic
    6. 6 Center for Innovative Diagnostics and Therapy in Rheumatology/Immunology (CIRI), Goethe University Frankfurt, Frankfurt, Germany
    7. 7 ZDROWIE Osteo-Medic, Bialystok, Poland
    8. 8 Biotest AG, Dreieich, Germany
    1. Correspondence to Dr Ronald F van Vollenhoven, Department of Rheumatology, Amsterdam Rheumatology and immunology Center ARC, Amsterdam 1018 HV, The Netherlands; r.f.vanvollenhoven{at}


    Objective To evaluate the efficacy, biological activity and safety of tregalizumab in patients with active rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX).

    Methods 321 patients were randomised (1:1:1:1) to placebo or tregalizumab 25, 100 or 200 mg once-weekly subcutaneously in addition to MTX treatment. Responders at week 12 continued the same treatment, and non-responders at week 12 were escalated to the next higher tregalizumab dose level or re-randomised from placebo to active treatment. After 24 weeks, patients could continue treatment with tregalizumab for 24 weeks (extension phase). The primary endpoint was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. Safety and biological activity were monitored through week 48.

    Results At week 12, ACR20 response rates were not statistically significantly different between placebo and any of the tregalizumab doses. Tregalizumab injections were well tolerated; most adverse events were mild to moderate and comparable among treatment and placebo groups. Biological activity was shown by dose-dependent CD4 downmodulation.

    Conclusion Treatment with tregalizumab did not show significant clinical efficacy in patients with active RA compared with placebo but resulted in the expected biological effect on CD4 modulation. Tregalizumab was generally well tolerated, and no new safety findings were identified.

    Trial registration number NCT01999192; Results.

    • rheumatoid arthritis
    • t cells
    • autoimmune diseases
    • Dmards (biologic)

    Statistics from


    Chronic inflammation of synovial tissues in peripheral joints1 2 and immune response dysregulation are key characteristics of rheumatoid arthritis (RA). Regulatory T cells (Tregs) play key roles in immune responses, and their function may be altered in RA.1 Improvement of Tregs function in RA has been reported with tumour necrosis factor inhibitors (TNFis),3 methotrexate (MTX)4 and abatacept5–7 and in patients with RA who responded to TNFis, increased Tregs function caused inhibition of interleukin-6 and inactivation of Th17 cells.8 Furthermore, treatment with tocilizumab enhanced CD39+ Tregs activity.9 It was therefore proposed that therapeutic modulation of Tregs may attenuate autoimmune diseases including RA.10

    Tregalizumab is a humanised, monoclonal, anti-CD4 IgG antibody that binds a specific epitope on the CD4 molecule, leading to the selective activation of Tregs (online supplementary figure 1).11–13 Tregalizumab binds to CD4 on all cells, inducing an agonistic signal in CD4 T cells and Tregs, and this signal leads to activation of Tregs but not normal T cells. Downmodulation of CD4 on T cells and Tregs also occurs. Tregalizumab-induced signals can be blocked by inhibition of the CD4-associated kinase Lck.13 Lck inhibition blocks activation of Tregs and downmodulation of CD4. Thus, downmodulation of CD4 can be used to monitor the agonistic activity of tregalizumab.

    Supplementary file 1

    Phase I/II studies of tregalizumab in healthy volunteers and patients with RA or psoriasis indicated good tolerability up to 200 mg. This placebo-controlled phase IIb clinical trial (TREAT2b) aimed to assess the efficacy, biological activity and safety of tregalizumab in patients with active RA and an inadequate response to MTX.



    Eligible patients (n=321) had active RA according to the 1987 American College of Rheumatology (ACR) or 2010 ACR/European League Against Rheumatism criteria, with functional classes I‒III for ≥6 months, and an inadequate response to MTX. Detailed criteria are included in the online supplementary material.

    Study design

    Phase IIb, double-blind, randomised, placebo-controlled, multinational (14 countries throughout Europe and North America), multicentre (84 sites), parallel-group, clinical trial conducted in accordance with the International Conference Harmonisation for Good Clinical Practice guidelines, the Declaration of Helsinki and local regulatory requirements. Enrolling patients from 28 October 2013, it comprised a screening period (days -28 to -7), Main Phase (24 weeks placebo controlled), Extension Phase (24-week active treatment only) and 4-week follow-up (online supplementary figure 2). The extension phase was terminated early on 31 July 2015. An interactive web response system was used for randomisation and resupply, using the stratification criteria: prior exposure to a TNFi (yes or no), duration of MTX treatment (3‒6 months or >6 months) and C reactive protein (CRP) levels (≤ULN or >ULN). Patients were randomised (1:1:1:1) to placebo or tregalizumab (25, 100 or 200 mg), administered as two subcutaneous injections once weekly for up to 24 weeks. Patients received study drug administered at the study site during visits with supplies provided for self-administration until the next visit.

    Assessments at week 12 classified patients as responders or non-responders based on improvement from baseline in tender joint count (TJC) and swollen joint count (SJC) ≥20%. Week 12 responders continued on the same treatment for 12 weeks, non-responders on placebo were switched to active treatment and those on tregalizumab were escalated to the next highest dose; patients who received 200 mg continued receiving the same dose, all in a blinded manner.


    The primary efficacy endpoint was the proportion of patients achieving an ACR2014 at week 12. Secondary endpoints included ACR20 response at week 24 and also ACR50/ACR70 response(s); Disease Activity Score (DAS) remission (DAS in 28 joints (DAS28) <2.6)15; low disease activity by DAS28 (DAS28 <3.2);15 Simplified Disease Activity Index (SDAI ≤11)16; and Clinical Disease Activity Index (CDAI ≤10)16 at weeks 12 and 24.


    Safety and tolerability were evaluated by adverse events, physical examination and vital signs, ECG, chest X-ray, laboratory parameters and infection status. CD4 lymphocyte count, autoantibodies and antidrug antibodies (ADAs) were assessed. Treatment-emergent adverse events (TEAEs) were reported for all patients who received at least one tregalizumab dose.

    Pharmacokinetics and pharmacodynamics

    Tregalizumab plasma concentrations were determined prior to study drug administration, at baseline, regularly throughout the study and at follow-up. ADA tests were conducted at follow-up visits. The expression of CD4 receptors was measured at different visits (prior to drug administration) and normalised to baseline levels to derive a percentage of CD4 modulation.



    Patients were randomised to receive MTX combined with either placebo or one of the tregalizumab doses. Online supplementary table 1 and figures 2 and 3 show the treatment compositions for each arm and an overview of patient disposition.

    Baseline characteristics of patients and previous MTX dose were comparable across treatment groups (online supplementary tables 2 and 3).


    A numerically higher proportion of responders was observed in the tregalizumab groups; however, these were not significantly different from placebo (figure 1A) (primary efficacy endpoint not achieved). Similarly, no differences were detected in the ACR20/50/70 responses among the treatment groups at either 12 or 24 weeks (figure 1A,B). Results after week 12 are presented according to the treatment that each patient was assigned at week 0. Placebo non-responders at week 12 were switched to tregalizumab (28/72 patients), and non-responders who had received tregalizumab were escalated to the next higher dose, all in a blinded manner (18 patients increased dose from 25 mg to 100 mg; 19 increased from 100 mg to 200 mg) (online supplementary figure 3). Thus, due to the study design, comparisons with placebo are only meaningful for time points up to week 12.

    Figure 1

    (A) Proportion of patients with an ACR20, ACR50 or ACR70 response at week 12 (full analysis set, observed cases (OC)); (B) proportion of patients with an ACR20, ACR50 or ACR70 response at week 24 (full analysis set, OC); (C) mean tender joint count (TJC: 68) by treatment group and visit (OC) for main and extension phases (full analysis set); (D) mean swollen joint count (SJC: 66) by treatment group and visit (OC) for main and extension phases (full analysis set). Patients are presented according to the treatment they were randomised at week 0: for OCs, patients who switched from placebo to active medication or to a higher active dose at week 12 or 24 are presented with their subsequent responses against the treatment they were randomised to at week 0. The dashed placebo lines indicate that from week 12 onwards, the placebo group contains patients receiving active treatment. From week 24 onwards, all placebo patients received active treatment. Moreover, the 25 mg and 100 mg dose groups contain patients that were switched to higher dose levels from week 12 onwards. The actual treatments for each treatment group from week 12 to week 24 are listed in online supplementary table 1. Data shown in figure parts A and B are described in online supplementary table 4, and data shown in figure parts C and D are described in online supplementary table 5. ACR, American College of Rheumatology; TGZ, tregalizumab.

    The number of patients with DAS remission (DAS28 <2.6) and with low disease activity (DAS28 <3.2, SDAI ≤11 and CDAI ≤10) was low, and there were no significant differences between the tregalizumab groups and placebo (online supplementary table 4). Improvements were detected throughout the study in several parameters but with no statistically significant differences among the treatment groups. Decreases from baseline to weeks 12, 24 and 48 were observed for SDAI, CDAI, TJC (figure 1C), SJC (figure 1D), patient’s global assessment of pain and patient’s and physician’s global assessment of disease activity (online supplementary table 4). In general, improvements in all parameters seen at week 24 were maintained until the end of the extension phase in each of the treatment groups with some (ACR50, ACR70) increasing considerably in the tregalizumab 200 mg group at week 48 (online supplementary table 4 and figure 1).


    Tregalizumab injections were generally well tolerated, and the incidence of treatment-related TEAEs was similar across all groups (table 1). Most TEAEs reported with tregalizumab were mild to moderate in intensity with only a few patients (3.9%) experiencing severe TEAEs. TEAEs during the extension phase were similar with no indication that increasing the dose increased the incidence of TEAEs.

    Table 1

    Overall summary of treatment emergent adverse events and most frequent adverse events (≥2% in any treatment group) for the safety set of main phase 1 and for the modified safety set (main and extension phases combined)

    Overall, the incidence of serious AEs was low (6.3 events per 100 patient-years) during the main and extension phases combined. There were three deaths during the main phase 1; none was considered related to treatment.

    No clinically significant changes in laboratory parameters or reduction in mean CD4 T cells counts (online supplementary table 6) after tregalizumab treatment were observed. Tregalizumab did not induce cytokine release syndrome and did not induce secretion of proinflammatory cytokines (data not shown).


    Pre-dose tregalizumab plasma levels were variable between patients within dose groups, with levels remaining the same range over multiple dosing, suggesting no accumulation of tregalizumab (online supplementary table 7). Mean tregalizumab levels in responders increased marginally from week 12 to week 24, and also week 48 (online supplementary table 7).

    ADA tests were conducted at follow-up visits; only 14 patients were ADA positive (14/321=4.3%), and analysis of tregalizumab plasma levels by ADA status showed no detectable differences between ADA-positive and ADA-negative subgroups.

    CD4 modulation

    In all tregalizumab treatment groups, there was a clear dose-dependent decrease of CD4 expression from baseline. The largest mean decrease was observed in the 200 mg group. Nonetheless, between-patient variability was large within each dose group. Expression levels remained unchanged in the placebo group (figure 2).

    Figure 2

    Mean reduction (down-modulation) of baseline-normalised CD4 expression level (%) for Main Phase 1 (0–12 weeks). The cell surface expression of CD4 was measured pre-dose by flow cytometry using an anti-CD4 antibody that does not compete with tregalizumab for binding. The measured fluorescence intensity was directly proportional to the number of CD4 molecules still present on the cell surface. As the expression level can be relatively different between patients, it was normalised on the expression level measured at baseline.


    There are several possible explanations for not achieving clinical improvements with tregalizumab in patients with RA (and an inadequate response to MTX treatment) in this trial. It is possible that Tregs were not effectively activated in vivo, even though binding of the target was demonstrated. Alternatively, the absence of significant results by stimulating Tregs may be due to a less important role for Tregs in RA than previously thought.17 Moreover, some evidence suggests that B cells from patients with RA are more resistant to the effect of Tregs.18

    The continuous increase in ACR20/50/70 responses through week 48, particularly in the 200 mg tregalizumab group, suggests the possibility of higher efficacy after long-term use. Thus, it is possible that a convincing therapeutic effect would have emerged after a longer treatment period. A delayed time to clinical response might be related to the unique mechanism of activating Tregs.

    Trial limitations must also be considered when interpreting the results of this study. The efficacy of tregalizumab may be limited to specific subgroups of patients, but no such profiling was undertaken in this study. As in all trials with a negative outcome, a true difference in the primary outcome could be missed due to chance (type 2 error). However, this study was 90% powered, so risk of such an error was low.

    The lack of clinical efficacy by activating Tregs in RA may be disease specific. Patients with RA seem to exhibit high thioredoxin-1 levels, possibly diminishing tregalizumab binding and signalling.19 Thus, tregalizumab may be effective in other immune conditions such as allergic asthma or transplantation. Smaller clinical studies in psoriasis20 showed that subcutaneous administration of tregalizumab was well tolerated with a potential dose-related efficacy.

    This observation would support the use of tregalizumab in other autoimmune or immunological diseases where efficacy could be demonstrated in future studies.

    Although the primary efficacy endpoint was not met in this large phase IIb trial in RA, the safety profile demonstrated and the observed biological activity of tregalizumab are consistent with the proposed mechanism of action and supports further investigation of tregalizumab in other immunological or autoimmune diseases.


    The coordinating investigator was RFvV. Quintiles was responsible for study management, pharmacovigilance/drug safety, monitoring, data management, statistics/biometrics, medical writing, central laboratory and study medication vendor management. An independent Data and Safety Monitoring Board (DSMB) was established to evaluate safety and efficacy data from the study to ensure the continuation of the study was appropriate and to make recommendations to the sponsor. On behalf of the TREAT2b study team, the authors would like to thank all patients, site staff and investigators, and coordinators at each of the clinical sites, and the Independent Review Committee, who contributed to the study. Editorial assistance was provided by 4C Consultants International.


    Supplementary materials

    Related Data


    • Handling editor Josef S Smolen

    • Contributors RFvV, ECK, VS, FB, RS, SA, BD and AW-D were involved in the design of the study; CP-T, JV, FB, AR, DZ, FR, RW, LK and XZ performed research; CP-T, JV, FB and AR collected data; RFvV, ECK, VS, FB, DZ, FR, RW, LK, RS, XZ, SA, BD and AW-D analysed and interpreted data; RFvV wrote the manuscript; all authors reviewed and approved the manuscript’s content before submission.

    • Funding This study was sponsored by Biotest AG. AbbVie provided financial support to Biotest for scientific and clinical evaluation of tregalizumab (BT-061) including this work from June 2011 to June 2015.

    • Competing interests RFvV has received research support and grants from AbbVie, BMS, GSK, Pfizer and UCB and has been a consultant and received honoraria from AbbVie, AstraZeneca, Biotest, BMS, Celgene, Crescendo, GSK, Janssen, Lilly, Novartis, Pfizer, Roche and UCB. ECK has received research funding from Abbott Laboratories, Amgen, AstraZeneca, BMS, Roche, Janssen, Lilly, Novartis, Pfizer, Sanofi-Aventis and UCB; has served as a consultant/advisory board member for Abbott Laboratories, AstraZeneca, Biotest, BMS, Crescendo, Roche, Genentech, Janssen, Lilly, Merck, Pfizer, and UCB; and has received speaker honoraria from Amgen, Abbott Laboratories, Astrazeneca, BMS Canada, Roche, Janssen, Pfizer, Sanofi Genzyme, and UCB. VS has been a consultant for Biotest. CP-T has received support from Abbvie, BMS, Roche, UCB, Janssen, Amgen, AstraZeneca, Pfizer, GSK, Ely Lilly, Sanofi, Celltrion, Vertex and Novo-Nordisk. FB has received research grants/support from Roche, Pfizer and Chugai and has been a consultant for AbbVie, Biotest, BMS, Janssen, Lilly, Pfizer, UCB, Novartis, Genzyme, AstraZeneca, MSD/Merck. DZ, FR, RW, LK, RS, XZ, SA, BD and AW-D are or were employees of Biotest.

    • Ethics approval Ethics Committee for Multicenter Trials, Veritas IRB, Multicentricka Eticka komise, Eticka komise Revmatologicky ustav, Tallinn Medical Research Ethics Committee, Ethik-Kommission der Medizinischen, Fakultaet der LMU Muenchen, Egeszsegugyi Tudomanyos Tanacs Klinikai Farmakologiai Etikai Bizottsaga, Lithuanian Bioethics Committee, CEI Hospital Dalinde, Comite Bioetico para la Investigacion Clinica, Comite de Etica de la Fac de Med de la UANL y Hospital Universitario Dr. Jose Eleuterio Gonzalez, Comite de Etica e Investigacion Christus Muguerza del Parque SA de CV, Comite de Etica en Investigacion de Comite Mexicano para la Prevencion de la Osteoporosis, A.C., Comité Independiente de Ética e Investigación del Centro de Estudios de Inv. Básica y Clínica S.C., Comite Institucional de Revision (Comite de Etica e Investigacion) Hospital Aranda de la Parra, Comite para la Asesoria en Etica Medica COMET, Komisja Bioetyczna przy Okregowej Izbie Lekarskiej w Krakowie.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Collaborators Jacob Aelion, Jorge Aguilar Arreola, Maria Araujo Arias, Johan Back, Asta Baranauskaite, Frank Behrens, Ralph Bennett, Arthur Bookman, Jan Brzezicki, Irena Butrimiene, Melvin Churchill, Svetlana Djacenko, Eva Dokoupilova, Edit Drescher, Anna Dudek, William Edwards, Larisa Eliseeva, Olga Ershova, Isabelle Fortin, Dagmar Galatikova, Ignacio Garcia de la Torre, Olena Garmish, Yuriy Gasanov, Andriy Gnylorybov, Marc Goldberg, Oleksandr Golovchenko, Ivan Gordeev, Falk Hiepe, Gabriela Huerta Sil, Viola Husarova, Oleg Iaremenko, Slawomir Jeka, Daniela Kamburova, Arthur Kavanaugh, Peter Keszthelyi, Edward Keystone, Larisa Knyazeva, Diana Krechikova, Stefka Kuzmanova, Angelika Lapcikova, Dennis Levinson, Jesus Alberto Lopez Garcia, Iurii Lymar, Natalia Marinova, Sergey Moiseev, Lucie Musilova, Magdolna Nagy, Sabeen Najam, Jovan Nedovic, Eleonora Nemeth, Libor Novosad, Luis Ochoa-Ortega, Boycho Oparanov, Cesar Pacheco Tena, Dimitar Penev, Yves Pesant, Lucie Podrazilova, Grazyna Pulka, Artur Racewicz, Tatiana Raskina, Dmytro Rekalov, Olga Reshetko, Janett Carmen Riega Torres, Andrea Rubbert-Roth, Anna Rychlewska-Hanczewska, Ludmila Savina, Hendrik Schulze-Koops, Mirjana Sefik Bukilica, Sergii Shevchuk, Eszter Simoncsics, Lubomira Simova, Karina Sitek-Ziolkowska, Andrea Skublova, Beata Sliwowska, Svetlana Smakotina, Wolfgang Spieler, Mykola Stanislavchuk, Dusan Stefanovic, Zuzana Stejfova, Rumen Stoilov, Maria Strapkova, Raisa Stryuk, Gabriella Sulyok, Anna Sylwestrzak, Istvan Szombati, Jan Theander, Sona Tomkova, Robert Trapp, Vira Tseluyko, Jaak Tälli, Jiri Vencovsky, Petr Vitek, Stoyanka Vladeva, Jacqueline Vo, Ronald van Vollenhoven, Nada Vujasinovic-Stupar, Siegfried Wassenberg, Juergen Wollenhaupt, Agnieszka Zielinska.

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