Homogeneous Time Resolved Fluorescence Marisa TM...deteção e quantificação dos agentes...
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HTRF – A New Technique for Detection and Quantification of Infliximab in Autoimmune Patients
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Index
Abstract 1
1. Introduction 3
1.1. Tumor necrosis factor-alpha Inhibitors 4
1.2. Immunogenicity 5
1.2.1. Anti-drug antibodies 6
1.2.2. Pharmacokinetics 7
1.3. Bridging ELISA 9
2. Aims 10
3. Material and Methods 12
3.1. HTRF principals 13
3.2. Materials 14
3.3. Procedure 15
4. Results 16
4.1. Standard curve 17
4.2. Cut point determination 18
4.3. Infliximab quantification 20
4.4. HTRF and bridging ELISA correlation 21
5. Discussion and Conclusions 23
6. References 27
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Abstract
Biologic therapies revolutionized the treatment of autoimmune diseases in the last
years. Typically, they target important disease mediators. Tumor necrosis factor-alpha
(TNF-α) antagonists constitute a very prescribed group of biologic agents as they are
indicated for the treatment of common immune-mediated diseases, such as
rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing
spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α
inhibitors it has been noticed that they have an important immunogenic potential that
can compromise long-term outcomes in chronically treated patients. The production of
anti-drug antibodies seems to cause secondary therapeutic failure in many patients.
One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug
clearance, in turn, varies among individuals, reflecting different pharmacokinetic
profiles. Determination of serum anti-TNF-α drug trough levels is though very
informative and could support treatment decisions. However, immunologic assays to
determine drug serum concentrations are not readily available in clinical practice.
In order to investigate a potentially reliable and practical new technique for detection
and quantification of anti-TNF-α biologic agents, homogeneous time-resolved
fluorescence resonance energy transfer (HTRF) technique was tested for
determination of serum infliximab concentrations. Although presenting some
limitations related with fluorescence reading conditions, this technique proved to give
results close to the concentrations obtained by the widely used bridging enzyme-linked
immunosorbent assay (ELISA). In addition, it has the advantage of being much easier
and faster to perform. Thus, HTRF technique can be optimized and become a valuable
laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α
therapy failure.
Key-words: TNF-α inhibitors, immunogenicity, pharmacokinetics, fluorescence.
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Resumo
As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos
últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das
doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de
agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças
imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil,
artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o
uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm
um potencial imunogénico importante, que pode comprometer o prognóstico a longo
prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco
parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos
anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do
fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis
farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é
assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes
imunológicos para determinar as concentrações séricas do fármaco não estão
facilmente disponíveis na prática clínica.
De forma a investigar uma nova técnica potencialmente fidedigna e prática para a
deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por
HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a
determinação de concentrações séricas de infliximab. Apesar de apresentar algumas
limitações relacionadas com as condições de leitura da fluorescência, esta técnica
provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked
immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução
muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e
tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas
em doentes autoimunes com falência da terapêutica anti-TNF-α.
Palavras-chave: inibidores do TNF-α, imunogenicidade, farmacocinética, fluorescência.
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Introduction
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1. Introduction
1.1. Tumor necrosis factor-alpha inhibitors
Inflammatory rheumatic diseases comprise a variety of conditions and syndromes with
diverse degrees of severity, overall provoking poor quality of life and premature death.
They are more prevalent in developed countries and constitute a huge burden to
health care systems (Helmick 2008, Jacobs 2011). The incidence of inflammatory
rheumatic diseases in Western countries is estimated to be 5-7% (Kuek 2007). Biologic
therapies brought new perspectives in the treatment of immune-mediated diseases as
they block specific targets that are important players in the disease physiopathology.
Monoclonal antibodies in particular, used in the treatment of many refractory
inflammatory rheumatic diseases, have proved to be effective in the reduction of
disease-associated activity scores.
Amongst the biologic therapies, tumor necrosis factor-alpha (TNF-α) blocking drugs are
the most prescribed as they constitute a therapeutic option in many immune-
mediated diseases, namely rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic
arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis (Saad 2010).
There are five TNF-α inhibitors currently in the Portuguese market (Table 1): 1)
infliximab is a chimeric anti-TNF-α immunoglobulin (Ig) G1, 25% murine and 75%
human, administered intravenously; 2) adalimumab is a fully human anti-TNF-α IgG1,
administered by subcutaneous injection; 3) etanercept is a fully human fusion protein
constituted by the fragment crystallisable (Fc) of human IgG1 and the soluble TNF-α
receptor, administered by subcutaneous injection; 4) certolizumab is a pegylated
fragment antigen binding (Fab) of a humanized anti-TNFα antibody, administered
subcutaneously or intravenously; 5) golimumab is a fully human anti-TNF-α IgG1,
administered by subcutaneous injection. Infliximab (IFX) was the first of its group
getting market approval (Taylor 2010).
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IFX ADA ETA CZP GLM Structure Chimeric IgG1 Fully human
IgG1 Fully human fusion protein of IgG1 Fc and soluble TNF-α receptor 2
Pegylated Fab of a humanized anti-TNF-α antibody
Fully human IgG1
Mechanism of action
Neutralizes tmTNF-α and sTNF-α
Neutralizes tmTNF-α and sTNF-α
Inhibits TNF-α/β and cell surface receptors bounding; inhibits LT-α
Neutralizes tmTNF-α and sTNF-α
Neutralizes tmTNF-α and sTNF-α
Half-life 8-10 days 10-20 days 3-6 days 11-14 days 7-20 days
Administration route
i.v. s.c. s.c. i.v. or s.c. s.c.
Dosage for autoimmune diseases
3-10mg/Kg at 0, 2 and 4 weeks and then every 8 weeks
40mg every one or two weeks
50mg once weekly or 25mg twice weekly
400mg at 0, 2 and 4 weeks and then 200mg every 2 weeks or 400mg every 4 weeks
3mg/Kg once monthly
Table 1: Properties of TNF-α inhibitors. IFX – infliximab; ADA – adalimumab; ETA – etanercept;
CZP – certolizumab pegol; GLM – golimumab; TNF – tumor necrosis factor; tmTNF-α –
transmembrane TNF-α; sTNF-α – soluble TNF-α; LT – lymphotoxine; i.v. – intravenous; s.c.
subcutaneous.
1.2. Immunogenicity
Although TNF-α blockers dramatically improved the above mentioned disease-related
outcomes, some patients do not respond to therapy (primary therapeutic failure) and
others experience a loss of efficacy over time (secondary therapeutic failure). Loss of
response has been attributed mainly to immunogenicity and drug pharmacokinetics
(Pascual-Salcedo 2011, Bartelds 2011). Immunogenicity is also responsible for other
adverse events, namely infusion reactions in the case of intravenously administrated
drugs and injection site reactions in subcutaneous formulations, thus influencing both
efficacy and safety of anti-TNF-α drugs.
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The core mechanism of immunogenicity is the production of antibodies against TNF-α
antagonists. To reduce the immunogenic potential of anti-TNF-α agents, companies
decreased its murine content: chimeric anti-TNF-α monoclonal antibody is more
immunogenic than fully human anti-TNF-α monoclonal antibodies, and this is more
immunogenic than the soluble TNF-α receptor-Fc fusion protein (de Vries 2009, Aikawa
2010, Krieckaert 2012). However, many other factors may contribute to
immunogenicity. These factors can be categorized as drug-related and patient/disease-
related factors. The main drug-related factors are previous exposure to similar or
related proteins, dose and duration of treatment, route of administration (intravenous
administration reduces the likelihood of an immune response), and product related
drivers (drug post-translational modification, excipients formulation, production and
purification processes, storage conditions, structural alterations, immune-complex
formation). Patient and disease-related factors are poorly understood, but certain
concomitant medications also seem to influence immunogenicity (Kriechaert 2012,
Mulleman 2011).
1.2.1. Anti-drug antibodies
Anti-drug antibodies (ADAb) can be neutralizing or non-neutralizing. Neutralizing ADAb
reduce drug efficacy through interference with TNF-α binding site (van Schouwenburg
2013). Non-neutralizing ADAb bind to anti-TNF-α agents without interfering with the
TNF-α binding site. In this manner they do not directly reduce drug efficacy but they
can contribute to therapeutic failure through immune-complex formation. Drug-ADAb
immune-complexes also increase the drug clearance rates (van der Laken 2007). In
rare cases, conversely, ADAb can prolong drug half-life. Globally, the main effects of
ADAb are enhanced clearance and neutralization of therapeutic antibodies, allergic
reactions and cross-reactivity with endogenous proteins. Screening of ADAb has been
used in preclinical, clinical and post-marketing studies to address immunogenicity.
Enzyme-linked immunosorbent assay (ELISA) has been the preferred technique for this
purpose. Standard direct and indirect ELISAs were improved and nowadays bridging
ELISA is a widely used method. However, there is high variability of ADAb detection
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among studies and it is not possible to monitor ADAb formation in routine clinical
practice. In fact, the method is not yet standardized and has important limitations such
as the levels of rheumatoid factor in the serum and the concomitant presence of the
anti-TNF-α drug (Aarden 2008, Hart 2011).
1.2.2. Pharmacokinetics
Once ADAb detection techniques are not completely reliable, immunogenicity studies
usually include pharmacokinetic data. In fact, several studies showed an inverse
relationship between drug levels and ADAb formation: trough levels of TNF-α
inhibitors may be lower in patients with ADAb (Wolbink 2006, Bartelds 2007, van Kuijk
2010).
Immunogenicity can have a big impact in long-term outcomes and patients with
chronic inflammatory diseases are the most likely to be affected as they are exposed to
repeated administrations of the drug, over long periods. Ideally, there should be an
individualized treatment planned in accordance with different tools that would allow
to assess the likelihood and severity of drug-related immunogenicity, as well as its
expected side effects. Such a regimen would allow to determine changes in drug
dosage and/or frequency of administration, and even the switch to an alternative
biological therapy, either another TNF-α inhibitor or a drug with a different target
(Keystone 2009, Jamnitski 2011). Since drug immunogenicity is closely related to its
pharmacokinetics, trough serum drug levels are very informative. Additionally, other
factors than immunogenicity, such as gender and body size, influence TNF-α blockers
pharmacokinetics (Ordás 2012).
Regarding infliximab, for example, the optimal trough level is not known. The existent
evidence shows that infliximab must be always detectable to be therapeutic and some
studies suggest that trough levels inferior to 4µg/mL may not be in the therapeutic
window (Takeuchi 2009, Seow 2010). Having the limitations of the analytical methods
in mind, the available pharmacokinetic data for infliximab can be summarized as
follows. After administration of 5 mg/kg of infliximab, the plasma concentration of free
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infliximab remained in the same order as Cmax (118 µg/ml, range: 71-283 µg/ml) for
approximately 24 hours. The concentrations then declined exponentially with a half-
life of 8-10 days. Detectable concentrations of free infliximab have been observed for
up to 28 weeks (mean 12 weeks) after the recommended dose regimen. Clearance of
free infliximab was about 11 ml/h (range 3-40 ml/h). No clinically significant dose or
time dependencies have been observed in the pharmacokinetics of free infliximab.
There are no specific studies regarding metabolism or excretion of infliximab in
humans. Since infliximab can be expected to be eliminated in a similar manner as
native antibodies, this is considered to be acceptable. Furthermore, no studies of the
pharmacokinetics of infliximab have been performed in patients with impaired organ
functions (Zhu 2005, Nestorov 2005, EMEA 2012).
Figure 1: example of concentration vs. time curve of infliximab (adapted from
Ordás 2012). Serum infliximab concentrations below the horizontal line are
probably out of the therapeutic window.
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Since pharmacokinetics and immunogenicity are not predictable, trough drug levels
should be measured in each patient serum immediately before periodical drug
administration (Rutgeerts 2010). Many authors argue they should be routinely
determined (Afif 2010, Ducourau 2011, Plasencia 2012) because it would be benefic
for the patients who receive a personalized therapeutic scheme and, given the high
costs of biologic therapies, serum drug level monitoring would probably be cost-
effective (Krieckaert 2012).
1.3. Bridging ELISA
Bridging ELISA is the method most frequently used to detect anti-TNF-α drugs and to
quantify its levels. It has several advantages as it uses widely available equipment and
expertise, and many centers even have automated systems. It is also sensitive and
specific. However, bridging ELISA also presents some relevant disadvantages: a) it is
not standardized among laboratories (Casteele 2012); b) involves repetitive steps of
washing and incubation which are time-consuming and may remove low affinity
antibodies. Therefore its use is limited outside research centers.
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Aims
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2. Aims
In order to have optimized therapeutic regimens, pharmacokinetic profile of TNF-α
antagonists should be obtained in every patient chronically treated. While this is not a
generalized practice in the clinical context, research laboratories conduct
pharmacokinetic studies using mainly bridging ELISA to measure serum drug levels.
This is a multi-step and time-consuming technique that requires technology expertise.
These are probably the reasons why it is not readily available in routine clinical
practice.
Considering the pros and cons of bridging ELISA, other methods may prove to be more
useful to measure anti-TNF-α drugs in the serum of patients with immune-mediated
diseases. HTRF is a technique widely used to study biomolecules interaction, single
molecules conformation and cells organization. To date, there is no evidence in the
literature of HTRF use for monoclonal antibodies quantification. It is here hypothesized
that HTRF can be a new technique for biologic TNF-α antagonists quantification.
The main aim of this work is to demonstrate the proof-of-concept that HTRF can
detect and measure infliximab trough levels in the serum of autoimmune patients.
Additionally, it is probably easier to perform than the currently used techniques.
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Materials and Methods
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3. Materials and Methods
3.1. HTRF principals
Fluorescence (or Fӧrster) resonance energy transfer (FRET) is a nonradiative energy
transfer that occurs between two fluorescent molecules, a donor and an acceptor.
Energy transfer occurs only when the donor and acceptor are close and the energy
transfer efficiency (E) is given by the following formula:
E = 1/1+(R/Ro)6
where Ro is the Fӧrster distance (or critical transfer distance) and R is the distance
between donor and acceptor.
The energy goes from donor molecules to acceptor molecules through dipole-dipole
coupling. The donor is initially in its electronic excited state after being exposed to an
energy source, secondly the energy transfer occurs, and finally the acceptor emits
flurescence at a given wavelength. The donor element must consist is a macrocyclic
lanthanide ion, Europium cryptate (Eu3+cryptate) or Terbium cryptate (Tb2+cryptate).
Acceptors consist in small organic structures derived from algae pigments (d2) or in
synthetic molecules. The maximum distance that allows the fluorophores to react is
dependent on the molecular size and conformation of the labeled molecules. As such,
the techniques employing FRET are referred as sensitive, because they are able to
detect molecules in close proximity, safe, because they use a nonradiative reaction,
and fast to perform, because there is no need for separation steps (e.g. washing). The
FRET reaction was further ameliorated and nowadays investigators may use time-
resolved FRET (TR-FRET) and homogeneous TR-FRET (HTRF). These improved
techniques brought the possibility of eliminating the short-lived background
fluorescence related to other sample components, by delaying the fluorescence
measurement after excitation.
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HTRF is currently used, for example, for detection of biomolecular interactions and for
single-molecule studies to elucidate conformational changes (Schuler 2008, Piston
2007, Sahoo 2011). It has also being increasingly used in drug development studies due
to its potential to discover new therapeutic targets (Degorce 2009).
Figure 2: HTRF for detection and quantification of infliximab uses a TNF-α cryptate
donor and a anti-IgG d2 aceptor.
Our assay will be developed for the quantitative determination of anti-IFX antibodies.
It is based on the principle of HTRF technology, where assays yield a distance-related
signal. Anti-IFX antibodies are detected by a system of an anti-human IgG monoclonal
antibody (mouse-anti human IgG MAb) and TNF-α, one labeled with d2, the other one
with Eu3+
cryptate.
3.2. Materials
Commercial infliximab (RemicadeTM) was used in successive dilutions to obtain a
standard calibration curve. Serum samples from drug-naïve healthy controls and from
patients with an autoimmune disease treated with IFX were analysed. The reagents
TNF-α cryptate Anti-IgG d2
Infliximab
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were provided by Cisbio International (France), and were reconstituted and diluted
according to the instructions of the manufacturer (Cisbio: www.htrf.com) in dilution
buffer (provided with the kit). For fluorescence readings a Tecan spectrophotometer
was used. Tecan’s Infinite F200 is equipped with a sophisticated and ingenious mirror
system containing a dichroic mirror with a break point at 510nm, which permits
optimal excitation in the UV range and thus enables HTRF measurements in the Infinite
F200.
3.3. Procedure
1- In a 96 well white plate, 5uL of sample, 10 uL of TNF-α cryptate at
50nM concentration, and 10uL of anti-IgG d2 at 50nM concentration
were pipetted to each well. Before pipetting, samples were kept cold
in an ice recipient and reagents were at room temperature.
2- The plate was maintained at room temperature and protected from
direct light until reading. The liquid in the wells remain colorless.
3- Fluorescence was read ten minutes after the addition of reagents by
Tecan Infinite F200. HTRF measurements were set up using the
‘multi labelling’ function of Tecan i-control software. The Eu3+
cryptate (donor) was excited at 320 nm (bandwidth 25 nm). The
cryptate and d2 (acceptor) emissions were detected at a wavelenght
near 600 nm.
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Results
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4. Results
4.1. Standard curve
Every method that uses spectrophotometry technology to determine concentrations
of a given molecule starts by obtaining a standard curve. This is created through
standard concentrations of the study molecule and respective optical density (OD)
values. The data is then computed to verify linearity. The resultant formula permits to
calculate concentrations from OD values.
To obtain the standard curve, eight successive dilutions of infliximab in the
concentration range of 0.39-50 µg/mL were tested (figure 3). The procedure was
repeated three times. The OD values obtained show some variability that increase with
concentration.
Figure 3: Standard curve of infliximab concentrations. From the graph it can be
seen that doubling infliximab concentration doubles the optical density (OD)
values obtained by HTRF. The variation in OD values reflects the three-fold
measurements.
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Using a linear regression model, a graph was generated to show the relationship
between OD values and infliximab concentrations (figure 4). The linear regression
curve and the r2 value show linearity.
Figure 4: Linear regression curve. The data were analyzed by a linear regression
model. A positive correlation exists between infliximab concentrations and
optical density (OD) values.
4.2. Cut point determination
Ideally, HTRF technique does not emit signal in the absence of infliximab. However,
background fluorescence can exist and give positive results. Hence, it is necessary to
determine the cut point above which the result is considered positive, using negative
controls.
The serum of 31 drug-naïve healthy donors was analyzed in three repetitions and the
results are shown in figures 5 and 6. Although HTRF signal is absent, there is also a
variation that is probably related with the adaptations made in the fluorescence
reader to perform HTRF.
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Figure 5: Optical density (OD) values of negative controls. Negative controls show OD
values below zero although some variation exists between the three measurements of
each sample.
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Figure 6: Infliximab detection in negative controls. The cut-off value of infliximab
concentration for HTRF detection is zero, as indicated by the dashed line.
4.3. Infliximab quantification
Serum samples containing infliximab previously quantified through bridging ELISA were
analyzed by the HTRF technique. The analysis of 34 samples of serum from patients
with immune-mediated diseases treated with infliximab gave the trough drug levels
(Figure 7). Serums 1-4 are internal positive controls created by dilution of infliximab in
infliximab-naïve serum. All the samples were tested three times. As in previous
observations, the variation of results is greater in higher infliximab concentrations.
Serum 5 showed one variant measurement (zero) that was excluded from calculations.
Figure 7: Infliximab concentrations in positive serums. Sampes 1-4 are internal controls.
Infliximab trough levels vary among patients.
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4.4. HTRF and bridging ELISA correlation
Globally, HTRF and bridging ELISA infliximab concentrations show a positive correlation
(p value < 0.01). The mean levels of serum IFX were slightly higher with bridging ELISA
than with HTRF (mean ± SD = 6.13 ± 5.44 vs. 6.63 ± 5.44). Interestingly, the tendency in
the positive control samples (samples 1-4) seems to be different from the other
samples (Figure 8).
Figure 8: Correlation between HTRF and bridging ELISA measurements of serum
infliximab levels. HTRF – homogeneous time-resolved fluorescence resonance
energy transfer. ELISA – enzyme-linked immunosorbent assay.
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HTRF ELISA Sample 1 1,55 2,14
Sample 2 1,48 1,1
Sample 3 1,56 1,8
Sample 4 20,12 17
Sample 5 1,49 1,7
Sample 6 1,67 2,8
Sample 7 20,8 25
Sample 8 1,54 1
Sample 9 1,74 2,5
Sample 10 1,58 1,91
Sample 11 2,29 4,5
Sample 12 1,98 11,2
Sample 13 1,46 1,7
Sample 14 1,52 2,3
Sample 15 1,47 2,1
Sample 16 1,51 1,3
Sample 17 4,99 3,2
Sample 18 9,59 7,32
Sample 19 5,54 6,1
Sample 20 21,45 15,2
Sample 21 4,99 6,1
Sample 22 7,10 6,9
Sample 23 8,75 9,88
Sample 24 14,27 15,1
Sample 25 8,92 9,6
Sample 26 7,36 8,2
Sample 27 8,45 7,8
Sample 28 4,62 5,5
Sample 29 6,69 6,1
Sample 30 7,22 7,8
Sample 31 10,48 14,6
Sample 32 3,86 3,8
Sample 33 7,29 7,3
Sample 34 4,38 4,2
Sample 35 4,32 3,8
Sample 36 3,82 3,1
Sample 37 4,55 5,6
Sample 38 10,44 14,7
Table 2: Serum infliximab concentrations (µg/mL) obtained with HTRF and
bridging ELISA methods. HTRF - homogeneous time-resolved fluorescence
resonance energy transfer. ELISA – enzyme-linked immunosorbent assay.
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Discussion and Conclusions
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5. Discussion and Conclusions
Despite being very immunogenic, infliximab is benefic for many patients with immune-
mediated diseases. Furthermore, adverse effects associated with immunogenicity are
still a matter of debate. Secondary therapeutic failure of long-term treatments seems
to be the most challenging problem as it is influenced by many factors and there is still
no way to predict it. It is therefore understandable the boom that has characterized
research in the immunogenicity of TNF-α inhibitors in the last few years.
To better understand the factors implicated, immunogenicity should be searched for
every patient receiving anti-TNF-α biologic therapies. However, this is far from reality.
Several authors tried to determine the relationship between immunogenicity and
clinical outcomes by performing assays to detect ADAb or through pharmacokinetic
studies. The majority of them used bridging ELISA techniques, which are currently only
available for use in research laboratories.
For all the reasons stated above, assays for ADAb detection are not reliable predictors
of immunogenicity, but pharmacokinetic profiles may be a useful tool to clarify
secondary therapeutic failures of anti-TNF-α agents. Thus, it would be very
advantageous to find a technique at least as sensitive and specific as bridging ELISA,
but faster, simpler to perform, and preferentially cheaper, that would allow
periodically to determine drug trough levels in the serum, in every-day clinical practice.
Our experiment demonstrates the proof-of-concept for HTRF as a new technique to
detect infliximab. Although widely used to study protein-protein interactions and
single protein conformations, HTRF was never applied as a monoclonal antibody
quantification test. Additionally, it could improve the throughput of pharmacokinetic
studies.
In accordance with the criteria for the HTRF Reader Control Kit, the Infinite F200 in
combination with the 510 dichroic mirror meets all performance parameters that are
relevant for HTRF-compatible readers and is thus regarded as HTRF-certified. In
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addition, IFX can be reliably and sensitively quantified in the Infinite F200. In summary,
the results of the Reader Control Kit, show that the Infinite F200 with an integrated
510 dichroic mirror is a perfectly suited detection instrument for the measurement of
various HTRF assays formats.
HTRF results were close to those obtained through bridging ELISA, and the HTRF
technique is much easier to perform. However, some unexpected difficulties were
found. The main disadvantage of the HTRF technique is the need for fluorescence
measurements within 10 minutes after the mixture of the sample with the reagents.
This increases the variability of the results and limits the number of samples tested
that can be measured in one reading, unless an automated system is available.
Another problem initially found was the variability amongst different commercial
infliximab samples that were previously submitted to a dialysis procedure. This issue
was overcome by using untreated commercial infliximab. Finally, once the correlation
between bridging ELISA and HTRF infliximab concentration in the internal positive
controls (known dilutions of infliximab in infliximab-naïve serum) seems to vary, there
is a possibility that other serum components interfere with HTRF results.
Some of the above mentioned difficulties could be overcome, for example through
automated systems, hopefully in a near future. In this way, a HTRF test could give
results only minutes after blood collection and easily help in clinical decisions.
Exemplifying, if the values of infliximab concentration obtained in this experiment
represent infliximab trough levels in patients with therapeutic failure, samples 25-38
could reflect an inefficacy of treatment related with such low values. In this case,
infliximab dose escalation would be an option. Conversely, if the remaining samples
were from patients with therapeutic failure, dose escalation strategy would not be
grounded since infliximab trough levels are high. In this situation the switch to another
biologic drug should be equated.
This work is not the first trying to find alternative techniques to bridging ELISA (Wang
2012). However, HTRF had never been experimented for quantification of a
monoclonal antibody and seems to be the simplest and the fastest method. As such, it
is worth trying to improve HTRF conditions. If optimized and available for routine
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clinical practice, rapid HTRF tests would help to find efficient drug regimens, to
enhance therapeutic success, to reduce adverse events, and eventually to decrease
therapeutic-associated costs.
HTRF – A New Technique for Detection and Quantification of Infliximab in Autoimmune Patients
27
References
HTRF – A New Technique for Detection and Quantification of Infliximab in Autoimmune Patients
28
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