The effect of certolizumab drug concentration and anti-drug antibodies on TNF neutralisation L.C. Berkhout1, E.H. Vogelzang2, M.H. Hart1, F.C. Loeff1, L. Dijk1, N.I.L. Derksen1, R. Wieringa3, W.A van Leeuwen3, C.L.M. Krieckaert2, A. de Vries3, M.T. Nurmohamed2,4, G.J. Wolbink1,2, T. Rispens1 1Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, Academic Medical Centre, University of Amsterdam, The Netherlands; 2Amsterdam Rheumatology and immunology Center | Reade, Amsterdam, The Netherlands; 3Biologics Lab, Sanquin Diagnostic Services, Amsterdam, The Netherlands; 4Amsterdam Rheumatology and immunology Center | Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands. Abstract Objective Tumour necrosis factor (TNF) inhibitors like certolizumab, elicit an immunogenic response leading to the formation of anti-drug antibodies (ADAs). We sought to mechanistically investigate the relationship between certolizumab concentrations, ADAs, and the effective TNF neutralising capacity in sera of rheumatoid arthritis (RA) patients. TNF neutralising capacity of certolizumab was compared to the neutralising capacity of adalimumab. Methods Serum samples were collected from 40 consecutive certolizumab-treated RA patients at baseline and 4, 16, 28 and 52 weeks after treatment initiation [Dutch Trial Register NTR (Nederlands Trial Register) Trial NL2824 no. 2965]. Certolizumab concentration and ADA titre were measured with a certolizumab bridging enzyme-linked immunosorbent assay (ELISA) and a drug-tolerant radioimmunoassay (RIA), respectively. TNF neutralisation by certolizumab and adalimumab, in presence or absence of ADAs, was analysed with the TNF-sensitive WEHI bioassay. Results Despite a high incidence of ADAs during one year of follow-up (65%; 26/40 patients), certolizumab levels of >10 μg/ml were measured in most patients. The capacity for TNF neutralisation highly correlated with certolizumab serum concentration, whereas no association with ADAs was observed. Similar results were obtained for adalimumab. The relative in vitro neutralising potency was higher for certolizumab compared to adalimumab. Conclusion Anti-certolizumab antibodies were detected in a large proportion of patients, but in most cases where ADAs were detected, certolizumab was also present in high concentrations, directly correlating with in vitro neutralising capacity. These results indicate that measurement of certolizumab drug levels, rather than ADAs, have direct clinical signifcance. Key words certolizumab, anti-drug antibodies, TNF, rheumatoid arthritis, adalimumab Clinical and Experimental Rheumatology 2020; 38:Clinical 306-313. and Experimental Rheumatology 2020 TNF neutralisation by certolizumab / L.C. Berkhout et al. Lea C. Berkhout, Msc Introduction viously, we have shown that the anti- Erik H. Vogelzang, MD, LLM Biological disease-modifying anti- body response to a range of therapeutic Margreet H. Hart, Bsc rheumatic drugs (bDMARDs) have antibodies, including certolizumab and Floris C. Loeff, Msc been developed to inhibit the activity of adalimumab, is highly restricted to Lisanne Dijk, Msc Ninotska I.L. Derksen, Msc infammatory cytokines such as tumour the antigen binding site, thereby pre- Roeland Wieringa, PhD necrosis factor (TNF). These TNF in- dominantly neutralising (13, 14). In a W. Astrid van Leeuwen hibitors have proven to be a successful number of studies, increasing serum Charlotte L.M. Krieckaert, MD, PhD treatment option for patients with rheu- concentrations of TNF inhibitors, in- Annick de Vries, PhD matoid arthritis (RA) and other infam- cluding certolizumab, were associated Michael T. Nurmohamed, MD, Prof. matory disorders (1, 2). Therapeutic with better clinical outcome (4, 7, 15- Gerrit J. Wolbink, MD, PhD antibodies such as certolizumab have 18). Furthermore, the amount of TNF Theo Rispens, PhD been shown to elicit an immunogenic inhibition will depend on the strength Please address correspondence to: response leading to the formation of of binding between TNF inhibitor and Theo Rispens, Department of Immunopathology, anti-drug antibodies (ADAs). How- ADAs on the one hand and TNF inhibi- Sanquin Research, ever, the reported incidence, and levels tor and TNF on the other hand. In other Plesmanlaan 125, of anti-certolizumab antibodies varies words, the balance between TNF inhib- 1066 CX, Amsterdam, The Netherlands. highly between different studies (~5- itor concentration, ADA titre and TNF E-mail: [email protected] 37% of the patients) (2-4). In a recent concentration plays a role in determin- Received on February 19, 2019; accepted study by Jani et al. detection of ADAs ing the TNF neutralising effcacy of the in revised form on May 29, 2019. in certolizumab-treated RA patients TNF inhibitor. © Copyright CLINICAL AND was not associated with the 12 months In the present study we describe the in- EXPERIMENTAL RHEUMATOLOGY 2020. European League Against Rheumatism cidence of anti-certolizumab antibod- (EULAR) response (4). In contrast, in ies, as well as the relationship between certolizumab-treated Crohn’s disease serum certolizumab concentrations and patients persistent ADAs were corre- the TNF neutralising capacity in pres- lated with reduced effcacy, while tran- ence and absence of ADAs. We com- sient ADAs were not (5). These mixed pared the certolizumab neutralising results are seemingly in contrast with capacity with the neutralising capacity many studies demonstrating clear cor- of adalimumab, since these drugs are relations between ADA formation to structurally different and have a differ- adalimumab or infiximab and a lower ent binding strength for TNF (19). likelihood of minimal disease activity or clinical remission (6-10). Materials and methods Reprints will not be available from the The detection of ADAs in patients var- Details about the methodology can be author. ies widely between studies, depending found in the Supplementary fle. Brief- Funding: this work was supported by on duration of follow-up, concomitant ly, certolizumab concentration and ZonMw, the Netherlands Organisation for medication, the type of TNF inhibi- anti-certolizumab antibody titre were Health Research and Development, in the tor and the type of assay that is used measured with a rabbit anti-certolizum- program 2Treat (grant no. 436001001). The certolizumab and adalimumab cohort to detect ADAs; a golden standard for ab bridging ELISA and a one-tiered or studies were partially supported by UCB the quantifcation of ADAs is missing two-tiered certolizumab RIA, respec- (IIS-2014-101540) and AbbVie (11). In particular, drug concentration tively, in 40 consecutive RA patients (ACA-NETH-05-012), respectively. may profoundly affect the detection of starting certolizumab treatment [Dutch UCB and AbbVie had no involvement in ADAs, depending on how drug-tolerant Trial Register NTR (Nederlands Trial the study design; in the collection, analysis, the ADA assay is. Importantly, ADAs Register) Trial NL2824 no. 2965]. The and interpretation of data. and drug levels will affect each other study was approved by the medical eth- Competing interests: mutually. ics committee of the Slotervaart Hospi- M.T. Nurmohamed has received consultancy ADAs are generally expected to only tal and Reade Medical Research Ethics fees from Abbott,Roche, Pfzer, MSD, UCB, SOBI and BMS, and payment for lectures affect treatment effcacy by lower- Committee, Amsterdam, the Nether- from Abbott, Roche and Pfzer. ing exposure to free active drug, via lands (CCMO NL35209.048.11). All G.J. Wolbink has received a research neutralisation and/or enhanced clear- patients gave written informed consent. grant from Pfzer (paid to the institution) ance. Hence, ADAs might only infu- To determine the TNF neutralising ac- and honoraria for lectures from UCB, ence clinical response when they affect tivity of certolizumab in patient sera, Pfzer, AbbVie, Biogen and BMS. pharmacokinetics (PK) to a noticeable in presence or absence of ADAs, the T. Rispens has received honoraria for degree (12). When enough free active TNF-sensitive WEHI bioassay was lectures from Pfzer, AbbVie and Regeneron, and a research grant from Genmab. drug is left to bind to its target, despite used. The TNF neutralising capacity of The other co-authors have declared no the presence of ADAs, ADAs are un- certolizumab was compared with the competing interests. likely to impair clinical response. Pre- neutralising capacity of adalimumab. A Clinical and Experimental Rheumatology 2020 307 TNF neutralisation by certolizumab / L.C. Berkhout et al. Fig. 1. Development of bridging ELISA for certolizumab. A cross-linking assay in which for both capture and detection polyclonal rabbit anti-idiotype antibodies were used, resulted in a sensitive assay to quantify certolizumab serum concentrations. A: Schematic overview of the certolizumab concentration assay. Certolizumab in serum is captured by polyclonal rabbit-anti certolizumab antibodies. Subsequently, biotinylated F(ab’)2 fragments of the same polyclonal rabbit anti-certolizumab antibodies are added for detection of certolizumab. B: Representative calibration curve of the certolizumab concentration assay. C: Inhibition of certolizumab specifc signal by increasing concentration of TNF. The concentration assay does not detect certolizumab bound to its target, assuring that only functional certolizumab is measured in this assay. Table I. Demographics, previous and con- tres (20–830 AU/ml anti-certolizumab antibodies