Therapeutic drug monitoring of tumour necrosis factor alpha inhibitors in inflammatory joint diseases
Thesis by Johanna Elin Gehin
Department of Medical Biochemistry Oslo University Hospital
Institute of Clinical Medicine Faculty of Medicine
University of Oslo
Division of Rheumatology and Research Diakonhjemmet Hospital
© Johanna Elin Gehin, 2022
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-348-0045-0
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Graphics Center, University of Oslo.
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Acknowledgements
The work in this thesis would not have been possible without the contribution of numerous persons to whom I am very grateful. This thesis builds on the pivotal work of my colleagues in establishing the analyses for measurement of biopharmaceutical blood concentrations in our department.
I would especially like to acknowledge my impressive team of supervisors; Nils Bolstad, Guro Løvik Goll, Silje Watterdal Syversen and Prof. Trine Bjøro. I sincerely thank my principal supervisor, Nils Bolstad, for introducing me to the field of therapeutic drug monitoring (TDM) of biopharmaceuticals. Thank you for sharing your extensive knowledge and the numerous hours you have patiently spent by the laboratory bench to teach me practical assay development, which I have come to greatly appreciate. I admire your endless enthusiasm to solve mysteries and problems, which has led us into numerous projects, experiments and scientific conversations. I also appreciate our not so scientific
conversations.
Guro, you are one of the pioneers within TDM of biopharmaceuticals in Norway. I truly appreciate our collaboration and thank you for sharing your extensive knowledge and writing skills. Your endless enthusiasm and warm and including personality inspires everyone.
Silje, thank you for always taking the time to help and support me, even at times when I know you are extremely busy with other obligations. I admire your scientific competence and it has been truly inspiring to follow you as a leader of the NOR-DRUM trial. Thank you for involving me in the NOR-DRUM trial and encouraging me to join the EULAR TDM task force as a fellow.
Trine, I appreciate your skilled and warm leadership and I am grateful that you have let research and development become an integrated part of my work. Your vast experience within research and supervision of PhD-candidates has been essential for conducting this PhD project.
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I thank David Warren for his support and for sharing his extensive knowledge with me. This thesis would not have been possible without your expertise in developing assays and reagents. I am grateful to work with all the friendly and highly competent colleagues in the tumour marker laboratory and research group and especially thank Rolf Klaasen for
contributing to this project. I am also grateful to our former colleagues Kjell Nustad and Elisabeth Paus for sharing their vast laboratory experience. I thank Ragnhild Nome for our everyday conversations and for listening and encouraging me whenever needed.
Further, I thank the former and present leaders of the department and section, Jens-Petter Berg, Olav Klingenberg and Karin Toska, for allowing me to do a PhD-project while employed at the department. I am grateful to the Faculty of Medicine at the University of Oslo for providing valuable education and for accepting me in the PhD-program.
I sincerely thank the Division of Rheumatology and Research at Diakonhjemmet Hospital for providing the research biobanks and datasets included in this thesis. I particularly thank Prof.
Emeritus Tore K. Kvien, Elisabeth Lie and Prof. Hilde Berner Hammer for their tremendous efforts in establishing and managing the cohort studies which this thesis builds on. I am grateful to all patients and study personnel. I also thank Joseph Sexton for patiently answering statistical queries, Maria Dahl Mjaavatten and Ellen Sauar Norli for their contributions and head of Research Prof. Espen Haavardsholm for support.
Further, I thank co-authors Eldri Kveine Strand, Liz Paucar Loli and Ada Wierød for their contributions and I thank Lillehammer Hospital for Rheumatic Diseases and the Dept. of Rheumatology, Vestre Viken Hospital Trust, for contributing to the NOR-DMARD and NOR- VEAC studies.
Last, but not least, I thank my family and friends. Special thanks to my parents and brother Niclas for your care and encouragement. I also thank my family-in-law and particularly Hilde Sofie for your support. The final thanks go to my husband Carl Fredrik and our wonderful children, Victor and Julie, for your endless love and support. You mean everything to me.
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Preface/Background
This PhD-project was undertaken at the Department of Medical Biochemistry, Oslo University Hospital, Radiumhospitalet, in collaboration with the Division of Rheumatology and Research, Diakonhjemmet Hospital (from 2018 to 2021). The Department of Medical Biochemistry, where I am employed, has a long tradition of producing monoclonal
antibodies for analytical purposes and developing in-house immunometric assays, particularly for measurement of tumour markers. Since 2013, we have offered
measurements of serum concentration of biopharmaceuticals for routine clinical care and research.
Since the establishment of the analytical service in our laboratory, we have had a close collaboration with rheumatologists at Diakonhjemmet Hospital, both in routine practice and research. The biobank samples and clinical data used in this PhD-project were obtained in longitudinal observational studies that were initiated and conducted at the Division of Rheumatology and Research at Diakonhjemmet Hospital.
Therapeutic drug monitoring (TDM) has been proposed as a strategy to optimise efficacy, safety and cost-effectiveness of treatment with tumour necrosis factor alpha inhibitors (TNFi) and has been widely implemented in routine clinical care in treatment with TNFi in Norway. Knowledge on therapeutic ranges and immunogenicity is essential in TDM. The present PhD-project was initiated with the primary aim of identifying therapeutic ranges, as well as immunogenicity, of TNFi where evidence was lacking for patients with inflammatory joint diseases. In addition to the main papers in this thesis, I had the privilege of participating as a fellow in the European Alliance of Associations for Rheumatology (EULAR) task force TDM of biopharmaceuticals in inflammatory rheumatic and musculoskeletal disease during the course of this PhD-project. The work led to the first evidence-based points to consider for the employment of TDM of biopharmaceuticals in rheumatology. Furthermore, my supervisors and I had important roles in the steering committee of the Norwegian Drug Monitoring (NOR-DRUM) trial, which investigated the effectiveness of tailoring infliximab treatment with TDM. The results and impact of these highly relevant projects will be discussed at the end of this thesis.
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List of contents
Acknowledgements ... 3
Preface/Background ... 5
Thesis summary ... 8
Norsk sammendrag ... 10
Abbreviations ... 12
List of papers ... 14
1 Introduction ... 15
1.1 Inflammatory joint diseases ... 15
1.2 Tumour necrosis factor alpha and its role in pathogenesis of chronic inflammatory diseases ... 17
1.3 Pharmacological treatment of IJDs ... 18
1.4 Biopharmaceuticals ... 19
1.5 Tumour necrosis factor alpha inhibitors ... 20
1.6 Immunogenicity of TNFi ... 30
1.7 Therapeutic drug monitoring of TNFi in rheumatology ... 38
1.8 Technologies for measurement biopharmaceutical blood concentrations and ADAb 43 1.9 Disease activity scores and criteria for treatment response in IJDs ... 47
1.10 Heterophilic antibodies and rheumatoid factor interference in immunoassays ... 49
2 Aims ... 52
3 Results / summary of papers ... 54
4 Methodological considerations ... 61
4.1 General considerations ... 61
4.2 Study design and study populations... 62
4.3 Clinical disease activity- and response measures used in this thesis ... 65
4.4 Other assessments of disease activity ... 69
4.5 Adverse events and drug survival ... 70
4.6 Measurement of TNFi concentrations and ADAb ... 70
4.7 Characterisation of RF reactivity to antibodies from other species used in immunoassays ... 85
4.8 Testing of interference in commercial immunoassays ... 87
4.9 Statistical analyses ... 88
4.10 Validity ... 93
5 Ethical considerations ... 94
5.1 Study design ... 94
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5.2 Marketing of biopharmaceuticals ... 94
5.3 Laboratory animal science ... 95
5.4 Insufficient antibody interference protection in commercial immunoassays ... 95
6 Discussion of main results ... 97
6.1 Between-individual variation, identification of therapeutic ranges and clinical relevance of ADAb ... 97
6.2 RF reactivity to animal antibodies and immunoassay interference (paper IV) ... 105
6.3 Main conclusions ... 106
6.4 Implications for clinical practice and research ... 107
6.5 Ongoing projects and clinical utility of TDM ... 109
6.6 Cost-effectiveness of TDM ... 114
6.7 Future aspects ... 114
7 References ... 115
8 Papers I-IV ... 133
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Thesis summary
The aim of this PhD-project was to explore therapeutic drug monitoring (TDM) as a potential tool to optimise treatment with tumour necrosis factor alpha (TNF) inhibitors in patients with inflammatory joint diseases. The project was a collaboration between the Department of Medical Biochemisty, Oslo University Hospital, and the Division of Rheumatology and Research, Diakonhjemmet Hospital.
Inflammatory joint diseases, including rheumatoid arthritis, axial spondyloarthritis and psoriatic arthritis, are chronic immune-mediated inflammatory diseases that constitute a significant burden for the individual patient and society. Antibody-based biopharmaceuticals have revolutionised treatment of these diseases in the last two decades. TNF inhibitors constitute a group of effective and frequently used biopharmaceuticals in treatment of inflammatory joint diseases. However, a considerable proportion of patients fail to achieve, or lose response, over time. Subtherapeutic biopharmaceutical serum concentrations and immunogenicity are major reasons for lack of clinical effect. Individualised dosing based on serum concentration measurements, TDM, has been proposed as a strategy to optimise treatment with TNF inhibitors. Knowledge on therapeutic ranges and immunogenicity is necessary for validation of TDM as a tool in research and clinical practice. The aim of this project was to study the variation in serum concentrations, identify therapeutic ranges and the incidence and clinical consequences of neutralising anti-drug antibodies (ADAb), for TNF inhibitors where minimal data existed. We employed biobank serum samples and clinical data from patients with rheumatoid arthritis, psoriatic arthritis and axial spondyloarthritis included in prospective observational studies initiated at the Division of Rheumatology and Research, Diakonhjemmet Hospital. Serum TNF inhibitor concentrations and ADAb were measured using in-house automated fluorescence assays.
The results of paper I demonstrated large variation in serum concentrations of certolizumab pegol (a PEGylated humanised anti-TNFα antibody Fab’ fragment), and identified the
therapeutic range of 20-40 mg/L. The study revealed anti-certolizumab antibodies in 6.1% of patients, and these were associated with low drug concentration and reduced clinical
response. Our study was the first to identify the therapeutic range for certolizumab pegol across inflammatory joint diseases and indicated a rationale for TDM to optimise treatment with certolizumab pegol.
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In paper II, the association between serum concentration of golimumab (a human monoclonal anti-TNFα antibody) and treatment response was investigated. The results indicated better treatment responses among patients with serum concentrations ≥1.0 mg/L, compared to <1.0 mg/L. Furthermore, the proportion of responders was highest among patients with serum concentrations ≥4.0 mg/L, but the difference was not statistically significant. Anti-golimumab antibodies were detected in 6% and were associated with low drug concentration and lack of response.
The aim of paper III was to identify the therapeutic range for etanercept (a TNF receptor fusion protein) in patients with rheumatoid arthritis. To this end, sensitive and objective markers of inflammation, including ultrasound examination of joints and plasma levels of calprotectin, were used, in addition to clinical disease activity measures.
Despite an association between etanercept serum conconcentration and reduction in plasma calprotectin, C-reactive protein and DAS28 in the longitudinal analyses, a clinically relevant therapeutic range could not be identified due to the lack of an association between etanercept serum concentration and any of the disease activity measures at 3, 6 or 12 months. No anti-etanercept antibodies were detected. The results suggest that for etanercept, TDM is unlikely to benefit rheumatoid arthritis patients in general.
Animal antibodies are frequently used in immunometric assays to quantify analytes in human blood samples. In paper IV, we revealed high frequencies of human antibodies binding animal antibodies used in immunometric assays, particularly mouse IgG1 and rabbit IgG, among rheumatoid arthritis patients with rheumatoid factor. Furthermore, falsely elevated results were revealed in commercial immunoassays with insufficient protection against rheumatoid factor interference. The study highlighted the importance of being aware of the risk of false test results caused by interference in rheumatoid factor positive rheumatoid arthritis patients.
This PhD-project has contributed to novel and clinically useful knowledge with respect to individualised dosing of TNF inhibitors based on serum concentration measurements in patients with inflammatory joint diseases. In addition, awareness of the risk of rheumatoid factor interference in immunometric assays was raised. This has implications for design of assays for measurement of biopharmaceutical concentration and ADAb.
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Norsk sammendrag
Formålet med dette doktorgradsarbeidet var å studere terapeutisk legemiddelmonitorering (TDM) som verktøy for å optimalisere behandling med tumor nekrose faktor (TNF)-hemmere hos pasienter med inflammatoriske leddsykdommer. Prosjektet er et samarbeidsprosjekt mellom Avdeling for Medisinsk Biokjemi, Oslo Universitetssykehus, og Klinikk for
Revmatologi, forskning og poliklinikk, Diakonhjemmet sykehus.
Inflammatoriske leddsykdommer, inkludert revmatoid artritt, aksial spondyloartritt og psoriasisartritt, er nokså utbredte kroniske inflammatoriske sykdommer som fører til
belastninger og kostnader for individet og samfunnet. Biologiske antistoffbaserte legemidler har revolusjonert behandlingen av disse sykdommene de siste tyve årene. TNF-hemmere er en gruppe biologiske legemidler som er effektive og mye brukt i behandlingen av
inflammatoriske leddsykdommer. En del pasienter responderer imidlertid ikke tilstrekkelig på behandlingen eller taper effekt over tid. Manglende effekt av TNF-hemmere kan blant annet knyttes til lav medikamentkonsentrasjon i blodet og utvikling av immunrespons mot medikamentet. Individuell tilpasning av legemiddeldose ut fra konsentrasjonsmålinger i blod, TDM, har blitt foreslått som en strategi for å optimalisere behandlingen med TNF- hemmere. Kunnskap om terapeutisk område og immunogenisitet er nødvendig for å kunne validere bruk av TDM i forskning og klinisk praksis. I dette prosjektet ønsket vi å undersøke variasjon i serumkonsentrasjon, identifisere terapeutiske områder og studere forekomst og konsekvenser av dannelse av nøytraliserende antistoff for TNF-hemmere hvor kunnskap manglet. Vi benyttet biobankprøver og innsamlede kliniske data fra pasienter med revmatoid artritt, psoriasisartritt og aksial spondyloartritt inkludert i prospektive observasjonelle studier initiert ved Klinikk for Revmatologi, forskning og poliklinikk, Diakonhjemmet sykehus. Serumkonsentrasjon av TNF-hemmere og antistoff mot medikament ble målt ved hjelp av egenutviklede (in-house) automatiserte fluorescens analyser.
I artikkel I viste vi at det var stor spredning i serumkonsentrasjon for certolizumab pegol (et PEGylert humanisert anti-TNFα antistoff Fab’ fragment), og identifiserte et terapeutisk område mellom 20-40 mg/L. Videre fant vi antistoff mot certolizumab pegol hos 6.1% av pasientene, og disse var assosiert med lav medikamentkonsentrasjon og nedsatt
behandlingseffekt. Studien vår er den første som har identifisert et terapeutisk område for certolizumab pegol på tvers av inflammatoriske leddsykdommer og indikerer at TDM kan være et nyttig verktøy for å optimalisere behandling med certolizumab pegol.
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I artikkel II undersøkte vi sammenhengen mellom serumkonsentrasjon og effekt for golimumab, et humant monoklonalt anti-TNFα antistoff. Resultatene viste bedret
behandlingsrespons hos pasienter med serumkonsentrasjon ≥1.0 mg/L, sammenlignet med
<1.0 mg/L. Videre var andelen som responderte på behandlingen høyest blant de som hadde serumkonsentrasjon ≥4.0 mg/L, men forskjellen var ikke statistisk signifikant. Antistoff mot golimumab ble funnet hos 6 % og var assosiert med lav medikamentkonsentrasjon og manglende respons.
I artikkel III ønsket vi å finne et terapeutisk målområde for etanercept, et TNF-reseptor fusjons protein, hos pasienter med revmatoid artritt. Til dette formålet benyttet vi flere sensitive og objektive mål på sykdomsaktivitet, inkludert ultralyd av ledd og plasmanivå av betennelsesmarkøren kalprotektin. Selv om etanerceptkonsentrasjon var assosiert med reduksjon i plasma kalprotektin, C-reaktivt protein og DAS28 i longitudinelle analyser, kunne et klinisk relevant terapeutisk område ikke identifiseres da det ikke var sammenheng mellom etanercept serumkonsentrasjon og noen av sykdomsaktivitetsmålene ved verken 3, 6 eller 12 måneder. Ingen av pasientene utviklet antistoff mot etanercept. Funnene våre tyder på at serumkonsentrasjonsmåling av etanercept neppe er klinisk nyttig som en generell strategi hos revmatoid artritt pasienter.
Antistoffer fra dyr benyttes i immunologiske analyser for å måle konsentrasjon av analytter i blodprøver fra mennesker. I artikkel IV fant vi høy forekomst av antistoffer som binder dyreantistoffer, særlig mus IgG1 og kanin IgG, hos revmatoid artritt pasienter med revmatoid faktor. I tillegg ga en betydelig andel av testede revmatoid faktor positive prøver falskt forhøyede svar i kommersielle immunometriske analyser som ikke er godt nok beskyttet mot interferens fra antistoffer. Funnene viser at alle som er involvert i behandling av revmatoid artritt pasienter med revmatoid faktor, må være oppmerksomme på muligheten for feil svar grunnet risiko for interferens fra revmatoid faktor.
Dette prosjektet har bidratt med ny og klinisk nyttig kunnskap for individuell tilpasning av legemiddeldose for TNF-hemmer ut fra konsentrasjonsmålinger i serum hos pasienter med inflammatoriske leddsykdommer. Videre har vi vist at man bør være oppmerksom på risiko for interferens fra revmatoid faktor i immunologiske analyser.
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Abbreviations
ADAb Anti-drug antibody
ASDAS Ankylosing Spondylitis Disease Activity Score AU/L Arbitrary units per litre
AxSpA Axial spondyloarthritis
BASDAI Bath Ankylosing Spondylitis Disease Activity Index bDMARD Biologic disease modifying anti-rheumatic drug
CI Confidence interval
CRP C-reactive protein
csDMARD Conventional synthetic disease modifying anti-rheumatic drug DAPSA Disease Activity index for Psoriatic Arthritis
DAS28 Disease Activity Score 28-joint count DMARD Disease modifying anti-rheumatic drug ELISA Enzyme-linked immunosorbent assay ESR Erythrocyte sedimentation rate
EULAR European Alliance of Associations for Rheumatology
Fab Antigen-binding fragment
Fc Fragment crystallisable
FcRn Neonatal Fc receptor
FcγR Fc gamma receptor
HLA Human leukocyte antigen
Ig Immunoglobulin
IJD Inflammatory joint disease
IL Interleukin
LC-MS/MS Liquid chromatography-mass spectrometry
mAb Monoclonal antibody
NOR-DMARD Norwegian Disease Modifying Anti-Rheumatic Drug study NOR-DRUM Norwegian Drug Monitoring study
NOR-VEAC Norwegian Very Early Arthritis Clinic PsA Psoriatic arthritis
RA Rheumatoid arthritis
RCT Randomised clinical trial
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RF Rheumatoid factor
RIA Radio immunoassay
SDAI Simplified Disease Activity Index SJC28 Swollen joint count 28
SLR Systematic literature review SpA Spondyloarthritis
TDM Therapeutic drug monitoring TJC28 Tender joint count 28
TNFα Tumour necrosis factor alpha
TNFi Tumour necrosis factor alpha inhibitor
ULRABIT Ultrasonography of Rheumatoid Arthritis patients starting Biological Treatment
VAS Visual analogue score
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List of papers
Paper I: Gehin JE, Goll GL, Warren DJ, Syversen SW, Sexton J, Strand EK, Kvien TK, Bolstad N, Lie E. Associations between certolizumab pegol serum levels, anti- drug antibodies and treatment response in patients with inflammatory joint diseases: data from the NOR-DMARD study. Arthritis Res Ther. 2019 Nov 29;21(1):256.
Paper II: Gehin JE, Warren DJ, Syversen SW, Lie E, Sexton J, Loli L, Wierød A, Bjøro T, Kvien TK, Bolstad N, Goll GL. Serum golimumab concentration and anti-drug antibodies are associated with treatment response and drug survival in patients with inflammatory joint diseases: data from the NOR-DMARD study.
Scand J Rheumatol. 2021 Mar 2:1-10.
Paper III: Gehin JE, Syversen SW, Warren DJ, Goll GL, Sexton J, Bolstad N, Hammer HB.
Serum etanercept concentrations in relation to disease activity and treatment response assessed by ultrasound, biomarkers and clinical disease activity scores: Results from a prospective observational study of patients with
rheumatoid arthritis. Accepted for publication RMD Open 16 November 2021, doi: 10.1136/ rmdopen-2021-001985.
Paper IV: Gehin JE, Klaasen RA, Norli ES, Warren DJ, Syversen SW, Goll GL, Bjøro T, Kvien TK, Mjaavatten MD, Bolstad N. Rheumatoid factor and falsely elevated results in commercial immunoassays: data from an early arthritis cohort. Rheumatol Int. 2021 Sep;41(9):1657-1665.
The published papers are reprinted under the terms of the Creative Commons Attribution 4.0 International License.
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1 Introduction
Inflammatory joint diseases (IJDs) constitute a group of chronic immune-mediated inflammatory diseases of unknown origin. IJDs include rheumatoid arthritis, axial
spondyloarthritis and psoriatic arthritis, and are all characterised by inflammatory changes in synovial tissues of joints, cartilage and bone, as well as various extra-articular
manifestations. IJDs constitute a significant burden for the individual patient and inflict substantial direct and indirect costs on society (1, 2). The treatment of IJDs has evolved considerably from the empirical use of salicylates and gold salts in the first part of the 20th century, to the use of targeted inhibitors of the inflammatory process, such as the tumour necrosis factor alpha inhibitors (TNFi) introduced in the late 1990s (3). TNFi are the most frequently used class of biopharmaceuticals, commonly referred to as therapeutic
antibodies, which are monoclonal antibodies or molecules derived thereof. TNFi specifically target tumour necrosis factor alpha, a pro-inflammatory cytokine that plays a key role in the pathogenesis of IJDs. TNFi have revolutionised the treatment of chronic inflammatory diseases, including IJDs. However, TNFi are costly treatments and not all patients respond adequately (4-6). Therapeutic drug monitoring (TDM), refers to measurements of blood concentrations to guide individial dose adjustments, and has been proposed as a strategy to optimise efficacy and cost-effectiveness of treatment with TNFi (7, 8). TDM has been widely implemented in routine clinical care in treatment with TNFi and other biopharmaceuticals in Norway.
The following introduction will provide an overview of the role of TNFi in treatment of IJD, methods for assessment of disease activity and treatment response in IJDs, as well as the rationale for TDM of TNFi in treatment of IJD, and available technologies for measurement of serum biopharmaceutical concentrations and immunogenicity.
1.1 Inflammatory joint diseases
1.1.1 Rheumatoid arthritis
Rheumatoid arthritis (RA) is a chronic progressive inflammatory disease of multifactorial etiology (9). The prevalence of RA is about 0.5-1.1% in Europe and North America (10). The incidence of RA increases with age (11) and women are affected more frequently than men
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(9). Synovial joints and cartilage are mainly affected, and symmetric polyarthritis of small and medium size joints is the hallmark of RA. Early and effective treatment is essential to reduce symptoms and to prevent irreversible structural damage. RA is also associated with extraarticular manifestations, such as an increased risk of cardiovascular disease, anemia, amyloidosis and osteoporosis. Another feature of RA is the presence of autoantibodies.
Rheumatoid factor (RF), a group of autoantibodies with reactivity to the Fc of human
immunoglobulin (Ig) G, and anti-citrullinated peptide antibodies are present in 50-85% of RA, with variation between cohorts (12, 13).
1.1.2 Psoriatic arthritis
Psoriatic arthritis (PsA) is an IJD associated with psoriasis. The estimated prevalence of PsA in Europe is between 0.1-0.5% (14) and the estimated prevalence of PsA among patients with psoriasis varies between 6 to 42% (15). The large variation in the reported prevalence can be explained both by different populations and the lack of widely accepted classification
criteria. Women and men are affected more or less equally (15). PsA is usually preceded by psoriatic skin disease, but arthritis might be the first manifestation. The pattern of joint involvement is variable and both peripheral and axial joints may be affected (15). Affection of the distal interphalangeal joints, dactylitis, enthesitis, and nail changes such as pitting and onycholysis are commonly seen in PsA.
1.1.3 Axial spondyloarthritis
Axial spondyloarthritis (axSpA), also known as Bechterew’s disease, is a chronic
inflammatory disease affecting the axial skeleton. AxSpA includes both non-radiographic and radiographic disease. The estimated prevalence of axSpA range from 0.2-1.6% in different populations (16). Men are affected approximately twice as often as women and about 80%
are younger than 30 years when diagnosed (17, 18). Inflammation in the sacroiliac joint is the hallmark of axSpA, and inflammation elsewhere in the axial skeleton, enthesitis, peripheral arthritis and anterior uveitis are other features of axSpA (18). Chronic
inflammation of the axial skeleton causes inflammatory back pain and structural changes, such as growth of new bone, which can lead to spinal stiffness and loss of spinal mobility over time (19). There is a strong genetic association between human leukocyte antigen
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(HLA)-B27 and axSpA, as over 80% of patients with advanced disease are HLA-B27 positive (19).
1.2 Tumour necrosis factor alpha and its role in pathogenesis of chronic inflammatory diseases
Tumour necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of IJDs and other chronic inflammatory diseases. TNFα is mainly produced by monocytes and macrophages, but also by B cells, T cells and fibroblasts (20). Its structure is a homotrimer, composed of three identical subunits, each with a molecular weight of 17 kilodalton. TNFα exerts its effects by binding two transmembrane receptors, TNF receptor type-1 (p55) and TNF receptor type-2 (p75). Soluble forms of the TNF receptor are made by shredding of the receptors from the cell surface and the soluble receptors may act as natural TNFα-inhibitors (21). TNF receptor type-1 is expressed on cells of all tissues whilst TNF receptor type-2is mainly expressed on leukocytes, endothelial cells and neurons (22). The ligand, TNFα, also exists both in transmembrane and soluble forms. Transmembrane TNFα is the precursor form and is cleaved by metalloproteinase TNFα converting enzyme to soluble TNFα (23). Both the soluble and transmembrane forms of TNFα are biologically active, but have some distinct effects, such as the remote effects of soluble TNFα and it has been suggested that transmembrane TNFα is involved in cell-to-cell signaling (reversed signaling) by acting as a cell surface receptor (22).
TNFα exerts its pro-inflammatory effects by both autocrine stimulation as well as paracrine induction of other inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8 and
granulocyte-macrophage colony stimulating factor. Experiments have shown that application of blocking anti-TNFα antibodies reduced the production of IL-1, IL-6, IL-8 and granulocyte- monocyte colony stimulating factor in cultures of synovial cells from RA patients (24). TNFα also stimulates expression of fibroblast adhesion molecules which leads to recruitment of leukocytes into inflammatory sites, by interacting with ligands on leukocyte surfaces (20).
Importantly, TNFα is present in high concentrations in tissue and synovial fluids in inflamed joints in RA and PsA (25) and high amounts of TNFα messenger ribonucleic acid has been found in sacroiliac joint biopsy specimens in patients with ankylosing spondylitis (26).
18 1.3 Pharmacological treatment of IJDs
In the management of patients with IJDs, early and effective treatment is essential to prevent irreversible alterations and destruction of joint and cartilage. Treatment strategies based on tight monitoring and treat-to-target are highlighted in international/European recommendations for management of IJD (27-29). Moreover, multidisciplinary treatment strategies including both non-pharmacological and pharmacological therapies are needed.
The following section will focus of pharmacological therapies.
1.3.1 RA and PsA
According to the European Alliance of Associations for Rheumatology (EULAR)
recommendations for the management of RA and PsA, a conventional synthetic disease modifying anti-rheumatic drug (csDMARD), preferably methotrexate, should be part of the first treatment strategy (27, 28). Methotrexate is usually administered as a weekly dose of 15-30 mg (30). In the NORD-STAR trial, remission was achieved in 42.7% (95% confidence interval (CI) 36.1 to 49.3) of early RA-patients on a conventional treatment strategy including methotrexate, which was comparable to three different biologic DMARDs (bDMARDs) (31).
Sulfasalazine or leflunomide are alternatives if methotrexate is contraindicated or not tolerated. Non-steroidal anti-inflammatory drugs may be considered to relieve symptoms.
Systemic glucocorticoids are often used when therapy is initiated due to their rapid anti- inflammatory effects, but rapid tapering is recommended owing to their potential side- effects. Intra-articular administration of steroids can be used in mono- or oligoarthritis and as adjunctive therapy. In RA and PsA, a bDMARD (i.e. biopharmaceutical) is often added if the treatment target is not achieved with a csDMARD. In RA, a targeted synthetic DMARD, i.e. a Janus kinase inhibitor, may alternatively be considered (27). TNFi are usually the first choice when starting therapy with a bDMARD. Data from the Norwegian (NOR-) DMARD study has shown that in patients for whom methotrexate monotherapy has failed,
methotrexate pluss TNFi was found to be more effective than combining methotrexate with other csDMARDs (32). However, treatment strategies vary across centers and different countries because access to biopharmaceuticals varies due to high costs (33). Other non- TNFi bDMARDs used in RA are abatacept (anti T-cell costimulator fusion protein), rituximab (anti-CD20 monoclonal antibody (mAb)) and tocilizumab (anti interleukin (IL) 6 receptor
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mAb). In PsA sekukinumab and iksekizumab (anti-IL17A mAbs) and ustekinumab (anti- IL12/23 mAb) are alternatives to TNFi.
1.3.2 AxSpA
In treatment of axSpA, non-steroidal anti-inflammatory drugs are first-line therapies but bDMARDs should be considered in patients with inadequate effect of conventional therapy (29). According to the 2019 update of the American College of Rheumatology
recommendations, TNFi are recommended as the first bDMARD (34). TNFi are effective in controlling disease activity and improving quality of life in patients with axSpA (35), however the protective effect on structural changes remains unclear (36). Sekukinumab, iksekizumab and Janus kinase inhibitors are non-TNFi treatment options in axSpA.
1.4 Biopharmaceuticals
Biopharmaceuticals (commonly called biologic drugs or biologic DMARDs in rheumatology) are drugs that are produced in or extracted from biological sources, in contrast to
conventional chemically synthesised pharmaceuticals. Furthermore, they differ from conventional pharmaceuticals in structure, administration and pharmacokinetics.
Most biopharmaceuticals, including most TNFi, are therapeutic mAbs, usually of the IgG1
subclass. Therapeutic monoclonal antibodies have considerable therapeutic benefits, such as high specificity for their targets and prolonged half-lives compared to other therapeutic proteins. IgGs are large hydrophilic proteins with a molecular weight of around 150
kilodalton. The structure of an IgG1 molecule is shown in Figure 1. The molecule consists of two heavy chains and two light chains joined together by disulfide bridges, each of them including constant and variable domains. The variable antigen-binding regions are located on the antigen-binding fragments (Fabs). The complementarity determining regions are small hypervariable regions located within the variable domain, and determine the specificity and affinity for a particular antigen (37). The fragment crystallizable (Fc) portion interacts with effector systems, such as complement and the cell-surface Fc gamma receptor (FcγR). The Fc also binds to the neonatal Fc receptor (FcRn), which protects IgG from intracellular
catabolism (37, 38). Based on characteristics the Fc-region, four subclasses with different properties, IgG 1-4, have been described.
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Köhler and Millstein developed the first in vitro mAb in 1975, and their discovery was the first step toward the development of therapeutic mAbs (39). Later, OKT3, a fully murine mAb targeting the CD3 receptor on T cells, was the first therapeutic mAb to be approved by the United States Food and Drug Administration in 1985 (40). OKT3 was used as an anti-rejection agent in renal transplantation. In common with OKT3, all first-generation mAbs in clinical use were of murine origin. The clinical use of murine antibodies was limited by frequent
formation of human anti-murine antibodies and short half-life (1-2 days), attributed to low affinity to human FcRns, when administered in humans (38, 41). In addition, low affinity for human FcγR was associated with low recruitment of effector cells (42). The next generation of therapeutic mAbs were chimeric antibodies in which human sequences replaced murine sequences in the constant domains, while retaining murine variable regions. Following advances in antibody engineering, humanised (<5% murine regions) and fully human antibodies became available. Since then, therapeutic mAbs have revolutionised the
management of chronic inflammatory diseases and are also increasingly used in treatment of cancer. TNFi, a class of biopharmaceuticals frequently used in treatment of IJD and other chronic inflammatory diseases will be the focus of this thesis.
1.5 Tumour necrosis factor alpha inhibitors
In parallel with the developments in production of therapeutic mAbs, it was discovered by Brennan et al. in 1989 that blocking TNFα inhibits the production of IL-1 in synovial cell cultures (43). This was confirmed and extended to a range of key pro-inflammatory cytokines in subsequent experiments (24). The discovery of the TNFα-cytokine cascade,
Figure 1. Structure of an IgG1 antibody. IgG1 consists of two identical antigen-binding fragments (Fab) and one fragment crystallisable (Fc) region which mediates effector functions.
CH, constant heavy; CL, constant light; VH, variable heavy; VL, variable light.
Created with BioRender.
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identified TNFα as a potential target in treatment of inflammatory diseases. Following the ATTRACT trial (4), infliximab was the first TNFi to be approved by the United States Food and Drug Administration for the treatment of RA in 1999. The other TNFi were proven efficacious in subsequent trials (5, 6, 44, 45). Etanercept was approved for treatment of RA in 1999, adalimumab in 2003, and golimumab and certolizumab pegol in 2009 (42). The patent for originator infliximab expired in 2015 and the first infliximab biosimilar was approved by the European Medicines Agency in 2013 and by the United States Food and Drug Administration in 2016. Biosimilars are similar to the originator drugs in terms of quality, safety and efficacy, but in contrast to generic drugs they are not identical due to the complex manufacturing process of biopharmaceuticals. The PLANETRA and the PLANETAS studies demonstrated equivalence of the infliximab biosimilar CT-P13 to originator infliximab in RA and ankylosing spondylitis (46, 47). Thereafter, the NOR-SWITCH trial, a landmark trial conducted by members of our research group, demonstrated that switching patients on stable infliximab treatment from originator to biosimilar infliximab was safe (48, 49). The introduction of biosimilars has reduced treatment costs considerably and led to better access to
biopharmaceuticals (50). Currently, infliximab, adalimumab and etanercept biosimilars are in clinical use.
TNFi are expensive treatments. In Norway, the total expenses for TNFi have increased
considerably since their introduction in the late 1990s. In 2018 approximately 2.0 billion NOK were spent on TNF-inhibitors (51). In 2019, the total expenses declined for the first time (to approximately 1.5 billion NOK), due to the increased use of less expensive biosimilar TNFi and substantial discounts provided through the annual tender system.
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Figure 2. Schematic structure of the tumour necrosis factor alpha inhibitors infliximab, adalimumab, golimumab, etanercept and certolizumab pegol. Infliximab is a mouse/human chimeric IgG1 anti-TNF mAb.
Adalimumab and golimumab are both fully human IgG1 anti-TNF mAbs. Etanercept is a recombinant fusion protein consisting of human TNF receptor 2 (p75) coupled to Fc of human IgG1. Certolizumab pegol is a PEGylated humanised Fab’ fragment of an IgG1 anti-TNF mAb. Created with BioRender.
Due to their different molecular structures (Figure 2), the TNFi exhibit differences in properties with regard to immunogenicity (further explained in section 1.6), binding specificities and cytotoxicity (52). For example, certolizumab pegol (PEGylated Fab’
fragment) cannot elicit Fc-mediated effector functions. However, other beneficial
pharmacokinetic and pharmacodynamic properties have been attributed to its structure, including effective penetration into inflamed tissues and lack of active transport across the placenta during pregnancy (53, 54).
All TNFi on the market are administered parenterally. Standard treatment with TNFi is based on fixed doses and intervals for all patients on subcutaneous TNFi, whilst intravenously administered infliximab is dosed according to weight (3 mg/kg in RA and 5 mg/kg in PsA and axSpA, every 6th-8th weeks). The other TNFi are administered subcutaneously and usually given by the following fixed doses: adalimumab 40 mg every 2nd week, etanercept 50 mg weekly (or 25 mg twice a week), golimumab 50 mg monthly and certolizumab pegol 200 mg every 2nd week.
1.5.1 Pharmacodynamics of TNFi
Pharmacodynamics can be defined as how the effects of the drug are manifested in the body.
All five TNFi exert their anti-inflammatory effects as competitive inhibitors of TNFα ligand- receptor binding. Etanercept also has the ability to bind to lymphotoxin α3 and α2β1, but the in vivo consequences of this binding remain uncertain (55, 56). All TNFi, except
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certolizumab pegol, can induce a number of cellular functions mediated by FcγR, including complement activation and antibody-dependent cell-mediated cytotoxicity (55, 57). The TNFi may also act as agonists by binding to transmembrane TNFα through the process of
“reversed-signaling”(22). The significance of “reversed-signaling” remains to be elucidated, but it may include suppression of cytokine production (57). The anti-inflammatory effects of TNFi are associated with a rapid reduction in cellularity in inflammatory tissues (55, 57). This has been attributed to macrophage and lymphocyte apoptosis, but the mechanism remains unclear. Furthermore, as explained in section 1.2, TNFi probably influence the production of multiple pro-inflammatory cytokines (24).
1.5.1.1 Clinical non-response
TNFi are effective in treatment of IJDs, but not all patients respond adequately to treatment and some patients lose efficacy over time (4, 5). Understanding the mechanisms underlying non-response to treatment is important in order to optimise treatment strategies. Non- response is commonly divided into primary and secondary non-response, and the
mechanisms underlying these differ. The commonly used clinical definition separates non- responses in relation to timing; a primary non-response occurs within the first 12 weeks after treatment initiation, whereas a secondary non-response is a loss of initial response occurring after 12 weeks of treatment. In pharmacology, one commonly refers to a pharmacodynamic failure, i.e. mechanistic failure due to a mismatch between the drug target (e.g. TNFα) and key disease mediators, or a pharmacokinetic failure (non-immune mediated- or immune mediated pharmacokinetic failure)(58). Sometimes, the term
pharmacodynamic failure is used synonymously with primary failure, whilst pharmacokinetic failure refers to a secondary failure. However, a pharmacokinetic failure can occur within the first 12 weeks, e.g. in the context of accelerated drug clearance in patients with high disease activity or in the context of early development of immunogenicity. Currently, there are no established clinical tools or biomarkers to predict responses vs. non-responses to TNFi treatment, but the employment of TDM is increasingly recognised as helpful in
understanding the mechanism underlying a non-response. These concepts will be the focus later in this thesis (section 1.7.3 and 6.5.2)
24 1.5.1.2 Adverse events in TNFi treatment
The safety of TNFi has been monitored closely since their introduction. Overall, TNFis have demonstrated acceptable safety profiles, but adverse events are relatively common during treatment with these agents. The adverse events can be attributed to either
pharmacodynamic effects of the drug, mediated trough immune-modulatory effects of TNFi, or immunological reactions to the drug. A meta-analysis from 2014 showed that TNFi
treatment was associated with an increased risk of infections, OR 1.42 (95% CI 1.13-1.78) (59) and another meta-analysis from 2015 showed that the risk was dose dependent (60).
Two meta-analyses addressing the risk of malignancies in RA patients treated with bDMARDs demonstrated no increase in the overall risk of malignancies (61, 62). Other less frequent, but serious, adverse events associated with TNFi treatment include demyelinating disorders (63). Furthermore, induction of rare autoimmune manifestations such as lupus-like
syndromes, cutaneous vasculitis and interstitial lung disease have been reported in TNFi treated patients, mostly in RA (64, 65). Paradoxical immune-mediated adverse events, such as psoriatic skin-lesions and arthritis have also been reported in patients with inflammatory bowel disease receiving TNFi (66). An increased risk of venous thromboembolism related to TNFi treatment has been a concern among RA patients, but has not been confirmed in prospective observational studies (67, 68).
Immunological reactions related to anti-drug antibody (ADAb) formation are described in section 1.6.
1.5.2 Pharmacokinetics of therapeutic mAbs, including TNFi
Pharmacokinetics can be defined as what the body does to the drug and includes absorption, distribution, metabolism and excretion. These are complex processes, but the following will provide an overview of the principles of pharmacokinetics of therapeutic mAbs, including TNFi. Admittedly, less data exists for receptor fusion proteins and antibody fragments (i.e.
etanercept and certolizumab pegol), but many of the principles of mAb pharmacokinetics can probably be extrapolated.
25 1.5.2.1 Absorption
Therapeutic mAbs must be administered parenterally, as absorption in the gastrointestinal tract is limited by degradation and inefficient passage through the gastrointestinal
epithelium, due to their size and hydrophilicity. The mechanism behind systemic absorption of subcutaneous mAbs is not fully understood, but is thought to include convection through lymphatic vessels to blood and diffusion directly across blood vessels located near the site of injection (37, 38). The flow through lymphatic vessels is usually slow, leading to slow
absorption and large variability between individuals (42). The first order rate-constant of absorption for mAbs has been estimated between 0.15 and 0.30 /day in RA (37). Therapeutic mAbs are usually exposed to some proteolytic degradation at the site of injection or during lymphatic convection, which reduces the bioavalability of subcutaneously dosed mAbs (38).
The absolute bioavailability has been estimated around 50-70% for mAbs and 80% for certolizumab pegol (42).
1.5.2.2 Distribution
Owing to the limited ability of mAbs to cross cell membranes due to their large molecular weight and hydrophilicity, their volumes of distribution are restricted to the vascular and interstitial spaces (37). The central volumes of distribution range between 3-4 liters for mAbs (42). The mechanisms of distribution of IgG mAbs is not fully understood, but the slow transport of mAbs from blood to interstitial fluid is believed to occur by convection (38).
Mechanisms for penetration into cells include endocytosis. A study in mice using a
biofluorescense labeling method, showed that infliximab, adalimumab and certolizumab are all distributed more effectively into inflamed than non-inflamed tissue (54).
1.5.2.3 Elimination
In contrast to small molecular drugs, mAbs do not undergo metabolism by liver enzymes or renal elimination, but are eliminated by receptor-mediated endocytosis and proteolytic catabolism within cells of the mononuclear phagocyte system (reticuloendothelial system).
Two different mechanisms have been described; I) non-specific linear, FcγR mediated, intracellular elimination (phagocytosis and antibody-dependent cellular cytotoxicity) and II) non-linear specific target-mediated elimination (37, 42). Due to target-mediated elimination,
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there is a particular pharmacodynamics-pharmacokinetics interaction for mAbs. Elimination is also thought to be enhanced by the formation of drug-anti-drug antibody (ADAb)
complexes (further discussed in section 1.6). In an imaging study of patients infused with radiolabelled infliximab, van der Laken at al. found a trend towards faster blood clearance and of higher liver/spleen uptake, in an ADAb-positive non-responding patient. The finding was likely to be attributed to accumulation of drug-antibody complexes in the mononuclear phagocyte system (69). Whilst the FcγR is involved in mAb degradation, the FcRn increases the half-life of mAbs by salvage of IgG from lysosomal degradation mediated through pH- dependent recycling of mAbs from inside the cell to the the cell surface (38, 70). The FcRn- mediated salvage pathway explains the long half-lives of IgG compared to other Ig isotypes and other proteins. Although with large variations, the elimination half-life of human(ised) mAbs is typically around 14-21 days, but 9 days for infliximab and 12 days for certolizumab pegol (42). Etanercept has a shorter half-life of around 90 hours (71).
1.5.2.4 Factors influencing TNFi pharmacokinetics
The inter-individual variation in pharmacokinetics of TNFi is considerable and the following section will provide an overview of factors that affect pharmacokinetics. Factors can be divided into drug-related or patient-related factors (Figure 3).
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Figure 3. Factors influencing tumour necrosis factor alpha-inhibitor (TNFi) pharmacokinetics. CsDMARDs, Conventional synthetic disease modifying anti-rheumatic drugs; FcRn, Neonatal Fc receptor. Created with Microsoft PowerPoint.
1.5.2.4.1 Drug related factors Drug characteristics
The structure and molecular weight of the particular TNFi (i.e. mAb, mAb fragment, or receptor fusion protein), as well as binding capacity to FcRn, are among factors influencing pharmacokinetics (52).
Dose and interval
As expected, studies in RA patients treated with infliximab or adalimumab, have shown that serum drug concentration is dependent on dose and interval (72-75).
Route of administration
The TNFi in clinical use are administered either intravenously (infliximab) or subcutaneously (adalimumab, etanercept, golimumab, certolizumab pegol). Other non-TNFi bDMARDs, such as tocilizumab and abatacept are administered both intravenously and subcutaneously. For these bDMARDs, phase III-studies have shown that the variation in drug levels during the administration interval is considerably larger for intravenous than subcutaneous
administration and peak serum concentration is higher for intravenous than subcutaneous,
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whereas the trough serum concentration is higher for subcutaneous than intravenous (76, 77).
Timing of sampling
For intravenously administered TNFi, such as infliximab, pharmacokinetic studies have demonstrated large intra-individual variations between peak and trough concentration, ranging from >100 mg/L to undetectable concentrations (78, 79). Conversely,
pharmacokinetic testing- and simulation studies have suggested relatively low peak-to- trough variations for subcutaneous TNFi (55, 80). Statistically significant differences between peak and trough concentrations have been demonstrated for adalimumab and etanercept (81, 82), however, the absolute differences were unlikely to be of clinical relevance. The relatively small variation in peak-to-trough serum concentrations for subcutaneous TNFi can probably be attributed to the relatively frequent dosing combined with the slow absorption from the peripheral to the central compartment (42), as described in section 1.5.2.1 above.
Immunogenicity
Formation of ADAb influences TNFi pharmacokinetics and is associated with lower drug concentrations (83, 84), as further described in section 1.6 below.
Concomitant csDMARDs
In RA patients, concomitant treatment with csDMARDs (particularly methotrexate) is associated with higher serum levels of infliximab and adalimumab (85, 86). Robust data are lacking for the other TNFi. The effect of methotrexate on pharmacokinetics is probably mediated both through a synergistic inhibition of inflammatory mediators and a subsequent reduction in target-mediated clearance, as well as through protecting against formation of ADAb (83, 84). The FcRn has also been suggested to play a role in the interaction (87).
Reports have indicated that the effect of methotrexate on TNFi levels is dose-dependent (86, 88-90). Data for other csDMARDs, such as salazopyrin and leflunomide, are limited.
However, in a study comparing RA patients treated with TNFi monotherapy, TNFi and concomitant methotrexate and TNFi and concomitant other sDMARDs, TNFi and
concomitant methotrexate was associated with the highest probability of detectable TNFi serum level as well as clinical response after one year of treatment, compared to both other
29
groups (86). The result was confirmed in another study including RA and PsA patients (85). In contrast to RA, the relationship between use of methotrexate and TNFi serum
concentrations is less clear for PsA and axSpA. The same relationship as seen in RA has been suggested in PsA (91), but data are conflicting in axSpA (79, 92).
1.5.2.4.2 Patient related factors Gender
The influence of gender on pharmacokinetic variability remains unclear. A longer half-life for adalimumab in women compared to men, was found in a study in RA patients (93). Lower weight and volume of distribution among women, compared to men, is an important
confounder when assessing gender differences, but this was accounted for in the final model which included weight as a covariate. Hormonal factors could potentially influence TNFi clearance as well.
Body mass index
A few studies have shown that adalimumab and etanercept serum concentrations are
inversely correlated with body mass index (94-96). Pharmacokinetic studies of infliximab and golimumab have shown that the central volume of distribution increased with body weight (78, 97). Adalimumab clearance is also associated with bodyweight (93). In addition to an increase in central volume of distribution, obesity has been linked to production of pro- inflammatory cytokines, including TNFα (98). Furthermore, as subcutaneous TNFi are subjected to some proteolytic degradation near the site of injection (38), it seems intuitive that other features of the adipose tissue could also contribute to pharmacokinetic
variability.
Inflammation
An inverse relationship between CRP and TNFi serum concentrations has been demonstrated in RA (93, 99-101). Furthermore, the same inverse relationship has been demonstrated between TNFi serum concentrations and inflammation assessed by clinical disease activity scores (100-103). Active inflammation is associated with an increase in pro-inflammatory cytokines, including TNFα, and the increase in antigenic burden accelerates target-mediated
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clearance of TNFi. Also, as most assays measure free drug, an increase in binding to target will lead to lower quantities of detectable drug.
Adherence
Poor adherence should be considered in patients with low drug concentrations, particularly in the absence of ADAb.
FcRn polymorphisms
In line with the important role of the FcRn in protecting IgG from catabolism, FcRn gene polymorphisms are associated with lower TNFi serum concentrations (104).
1.6 Immunogenicity of TNFi
1.6.1 Concept of immunogenicity
Therapeutic mAbs, including TNFi, are large allogenic molecules that have the ability to elicit an immune response in the host, including the formation of ADAb. ADAb formation to the first murine mAb in clinical use, OKT3, was recognised by researchers in the 1980s (105, 106). Later, ADAb formation was noted in early infliximab trials in RA (107). The incidence of ADAb to TNFi has been extensively studied, but varies in different studies. In a meta-analysis from 2015, including patients with RA, spondyloarthritis and inflammatory bowel disease, ADAb development was found in 25.3% (95% CI 19.5-32.3) of patients receiving infliximab, 14.1% (95% CI 8.6-22.3) receiving adalimumab, 1.2% (95% CI 0.4-3.8) receiving etanercept, 3.8% (95% CI 2.1-6.6) receiving golimumab and 6.9% (95% CI 3.4-13.5) receiving
certolizumab pegol (84). In a more recent systematic literature review published in 2017, ADAb were reported in 0-83% with infliximab, 0-54% with adalimumab, 0-13% with etanercept, 0-19% with golimumab and 3-37% with certolizumab pegol (108). The wide variations in the reported frequencies of ADAb illustrate the challenges in interpreting immunogenicity results, and can be attributed to heterogeneity in study designs and immunoassay technologies (further described in section 1.8.2).
Most ADAb against TNFi are neutralising, i.e. anti-idiotype antibodies with affinity for the antigen-binding domain of the Fab portion of the antibody (109, 110). Neutralising ADAb are
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clinically important as they prevent binding of TNFi to their target molecule (TNFα) (Figure 4). Of note, no studies have reported neutralising anti-etanercept antibodies (84, 108), which can be attributed to its less immunogenic structure (receptor fusion protein). Non- neutralising ADAb, often referred to as binding ADAb can theoretically accelerate clearance of TNFi by forming immune-complexes (52).
Figure 4. ADAb –neutralising vs binding. A) TNFi binding to target TNFα. B) Neutralising (anti-idiotype) ADAb binds and blocks target-binding site of TFNi. C) TNFi-ADAb immune-complex formation can lead to accelerated clearance via binding to Fcγ receptors and subsequent elimination by phagocytosis and antibody-dependent cellular cytotoxicity.
ADAb, Anti-drug antibody; Fcγ receptor, Fc gamma receptor; TNFα, tumour necrosis factor alpha; TNFi, Tumour necrosis factor inhibitor. Created with BioRender.
ADAb usually develop early in the treatment course. Bartelds et al. showed that the majority of ADAb developed within the first 6 months in RA patients (111). Other studies have
reported development of ADAb against TNFi during the first 4-52 weeks after treatment initiation (112-115). Incipient ADAb are expected to be of IgM isotype, followed by T-helper cell dependent stimulation of B-cells leading to isotype switching to the IgG isotype. In the mature ADAb-response IgG1 and IgG4 are the dominating isotypes (109). Van
Schouwenbourg et al. found ADAb of IgG4 isotype in a considerable proportion of RA patients during long term treatment with adalimumab (116). ADAb of the IgE isotype occur less commonly (117).
Factors that influence the immunogenicity of TNFi, as well as the consequences of immunogenicity, are summarised in Figure 5 and described in the following sections.
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Figure 5. Summary of factors influencing immunogenicity and consequences of immunogenicity. Created with Microsoft PowerPoint.
1.6.2 Factors that influence the immunogenicity of TNFi
Drug- and treatment-related factors have been extensively researched, but the evidence with regard to predisposing patient demographic and disease characteristics is less robust.
1.6.2.1 Drug related factors The biopharmaceutical molecule
The propensity of a therapeutic mAb to provoke an immune response, largely depends on the fraction of allogenic sequences within the mAb. Murine mAbs, which are rarely in clinical use, are particularly immunogenic, as they contain numerous foreign epitopes. Chimeric mAbs are the most immunogenic among the therapeutic mAbs in common clinical use, while humanised and fully human mAbs are less immunogenic (52). However, even fully human mAbs are allogenic molecules and contain potentially immunogenic epitopes within the complementarity determining regions (118). Soluble receptors (such as etanercept) are less immunogenic than mAbs (108). Immunogenic potential can also be affected by post-
translational modifications, such as glycosylation or pegylation. Factors such as impurities, formulation and storage might also lead to alterations in immunogenic potential. Although not systematically studied, the pharmacodynamics of the biologic molecule is likely to affect
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its immunogenic potential, i.e. drugs inhibiting B-cells directly (e.g. rituximab) or via IL-6 pathways (e.g. tocilizumab), likely have lower immunogenic potential (108).
Immune complex formation
The size of immune complexes formed between the biopharmaceutical and their target molecules (e.g. TNFα) may also influence the immunogenic potential of biopharmaceuticals, as large immune complexes are likely to be associated with enhanced uptake by antigen- presenting cells (119). This could influence TNFi immunogenicity profiles, as Kohno et al.
have demonstrated that the different TNFi exhibit different binding characteristics;
etanercept typically forms small complexes of one or two etanercept molecules bound to each TNFα trimer (180 and 300 kilodalton), whereas adalimumab and infliximab form a variety of larger complexes with TNFα (4,000-14,000 kilodalton) (120).
1.6.2.2 Treatment related factors Route and mode of administration
In general, subcutaneous administration has a higher immunogenic potential than
intravenous administration. Furthermore, irregular/on-demand administration of TNFi has been suggested to be associated with a higher potential for immunogenicity than
regular/scheduled administration (108). Drug-holidays are also associated with an increased risk of immunogenicity (121, 122).
Low TNFi levels
Low TNFi levels have been suggested to be associated with an increased risk of ADAb development (108, 122). Although higher levels could be protective, it is complicated to explore the causality of this association because it is likely to be bidirectional, as low TNFi levels could also be an effect of an emerging ADAb development. Moreover, ADAb are less likely to be detected in the presence of circulating drug, as most ADAb assays are drug sensitive (discussed in section 1.8.2).
34 Concomitant csDMARDs
Concomitant administration of csDMARD reduces the risk of ADAb development. The odds ratio of ADAb formation was 0.26 (95% CI 0.21-0.32) with immunosuppression vs. TNFi monotherapy in a meta-analysis including patients with chronic immune-mediated
inflammatory diseases on TNFi treatment (84). Similar to the association between csDMARD and TNFi concentrations, discussed in section 1.5.2, the majority of data are derived from methotrexate studies in RA patients, but was also suggested in axSpA (92). The inverse relationship between methotrexate and ADAb-development, has also been suggested to be dose-dependent (89, 90)
1.6.2.3 Patient related factors Disease characteristics
The risk of ADAb development has been shown to differ between diagnoses.
Immunogenicity was found in 16% of patients with inflammatory bowel disease, 12% of RA and 9% of SpA in a meta-analysis (84). Longer disease duration and higher baseline disease activity have also been associated with an increased risk of ADAb development (111, 122).
Genetic predisposition
Limited evidence exists regarding a genetic predisposition to ADAb development and most has been derived from individuals with inflammatory bowel disease. Observational studies have, however, suggested that certain HLA-haplotypes are associated with an increased risk of ADAb development. The HLA haplotype HLA-DRB1 was more prevalent among patients with ADAb against infliximab and adalimumab, compared to no ADAb, in inflammatory bowel disease, RA and hidradenitis suppurativa (123, 124). Another observational study in Crohn’s patients found a significant association between HLA-DQA1*5 and ADAb against infliximab and adalimumab (125). Furthermore, one study has indicated that certain IL-10 polymorphisms are associated with an increased risk of anti-adalimumab antibodies in RA patients (126).
35 Smoking
Smoking was identified as a risk factor of ADAb development against infliximab in subanalysis of data from the NOR-DRUM A trial (122).
1.6.3 Consequences of immunogenicity
Consequences of immunogenicity are summarised in Figure 5 and described in the following section.
1.6.3.1 Influence of immunogenicity on pharmacokinetics and clinical effect TNFi immunogenicity is associated with lower TNFi serum levels and reduced therapeutic effects (83, 84, 108, 111), attributed to neutralisation and increased clearance. In a meta- analysis from 2015, the likelihood of a reduced response was 67% in ADAb positive
compared to ADAb negative patients with chronic immune-mediated inflammatory diseases treated with TNFi (84). Furthermore, the odds ratio of a TNFi response in ADAb positive, compared to ADAb negative, was 0.27 (95% CI 0.20-0.36) in RA and 0.18 (95% CI 0.09-0.37) in SpA. Most data were derived from studies on infliximab or adalimumab. A model of the relationship between ADAb development, decrease in TNFi serum concentration and subsequent loss of clinical response is shown in Figure 6.
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Figure 6. Model of the relationship between anti-drug antibody (ADAb) development, decrease in tumour necrosis factor-inhibitor serum concentration and subsequent loss of clinical response.
1. Development of ADAb and formation of TNFi-ADAb complexes.
2. The amount of free TNFi declines as a result of accelerated clearance of TNFi-ADAb complexes.
3. The concentration of ADAb continues to increase in parallel with reduced TNFi concentration.
4. ADAb in complex with TNFi only detectable using drug-tolerant ADAb assays.
5. Free ADAb becomes detectable using drug-sensitive assays (when TNFi undetectable/very low).
6. Manifestation of clinical non-response when free TNFi is unmeasurable/very low (the relative amount of ADAb is higher than free TNFi).
ADAb, Anti-drug antibodies; TNFi, Tumour necrosis factor alpha inhibitor.
Adapted from [Long-term measurement of anti-adalimumab using pH-shift-anti idiotype antigen binding test shows predictive value and transient antibody formation, Schouwenburg PA, Krieckaert CL, Rispens T, Aarden L, Wolbink GJ, Wouters D, Ann Rheum Dis 2013;72:1680–1686 (115) ©2013, with permission from BMJ
Publishing Group Ltd; license number 5021891336046. Created with BioRender.
The consequences of ADAb formation vary among individuals and particularly depend on the relative amounts of ADAb and TNFi (127). ADAb might only influence clinical response when the amount of free active drug is lowered to subtherapeutic concentrations.
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In some cases, ADAb formation is an irreversible process, with a subsequent reduction in TNFi levels and treatment failure. In other cases, low levels of ADAb might be transient (115). Low to moderate levels of ADAb can sometimes be overcome, at least temporary, by increasing TNFi exposure by increasing dose or reducing the dosing interval. It is, however, unclear whether the apparently transient ADAb actually disappear due to induction of tolerance (which has been described in patients with haemophilia A treated with exogenous factor VIII (128, 129)), or whether it can be attributed to false negative results due to drug interference in ADAb measurements. Van Schouwenburg et al. investigated this issue by employing a drug sensitive assay in parallel with a drug tolerant ADAb assay in adalimumab treated RA patients. They concluded that the ADAb response was truly transient in 4 out of 10 patients, whereas in the remaining 6 patients ADAb were hidden in the drug sensitive assay due to higher drug levels (115). The effect of adding or increasing the dose of concomitant csDMARD during an incipient ADAb-response is debated.
1.6.3.2 Infusion-/hypersensitivity reactions
Another important consequence of ADAb development is the increased risk of infusion reactions in patients treated with intravenous biopharmaceuticals, such as infliximab. In the meta-analysis by Thomas et al., the odds ratio of developing an infusion reaction during infliximab treatment was three times higher in ADAb positive vs. ADAb negative patients (84). Infusion reactions range from mild hypersensitivity reactions, including arthralgia and malaise, to more severe infusion reactions, serum sickness, and rare anaphylactic reactions.
In contrast, there is no evidence that ADAb increases the risk of injection-site reactions in patients treated with subcutaneous TNFi. Some efforts have been made to elucidate
predictors of infusion reactions. Results are contradictory with regard to the role of ADAb of IgE isotype in the development of infusion reactions (117, 130). Van Schie et al. investigated the role of the relative amounts of ADAb vs. TNFi and ADAb-TNFi complex formation. They found that large complexes were formed at high ADAb:TNFi ratios, whilst dimers were the predominant complex at low ratios. Furthermore, their results implied that only large complexes were efficiently cleared from the circulation by FcγR-mediated phagocytosis and only complexes larger than hexamers had the ability to activate complement (131). These