Homogeneous Time Resolved Fluorescence Marisa TM...deteção e quantificação dos agentes...

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


1

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.


2

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.


3

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


Inhibits TNF-α/β and cell surface receptors bounding; inhibits LT-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


25

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


26

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.


27

References


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