Aetiology and pathology

Crohn’s disease can affect adults, adolescents or children. CD manifests mainly during late adolescence or early adulthood. The first onset most commonly occurs between the ages of 16 and 30 years, with a second peak between the ages of 60 and 80 years. Women are slightly more frequently affected than men, but in children it is seen more often in boys than in girls. CD is most common in white people in westernised countries and has its highest prevalence among Jewish people of European descent.⁴

Crohn’s disease follows a pattern of acute disease (relapse) interspersed with periods of remission (lack of symptoms). CD causes inflammation of the lining of the digestive tract, which, depending on the individual, occurs at any location from the mouth to the rectum, but most commonly affects the end of the small intestine (terminal ileum; 35%) or the connection between the small intestine and large intestine (ileocaecal region; 40%).⁵ Within individuals the disease location is fairly constant.

Fistulising CD describes the condition in patients who have developed complications in the form of abnormal connections between the bowel and other organs known as fistulae. Fistulae develop in between 17% and 43% of people with CD.⁶ Active luminal CD describes the condition in patients who have inflammation in the tube of the intestine.

The main symptoms of CD depend on the location of disease. They include abdominal pain, chronic or nocturnal diarrhoea, anal lesions, rectal bleeding, weight loss and swelling of the abdomen with tenderness. Complications include strictures, perforations, abdominal obstructions and development of fistulae. Extraintestinal symptoms related to intestinal inflammation include inflammation of the joints, skin, liver and the eyes.⁷ CD in children is often noticed because of growth failure.⁸ Symptoms range in severity and the assessment of severity is used to classify CD into mild, moderate or severe disease according to disease activity scales. A response is defined as a reduction in symptoms.

Measurement of disease activity

Crohn’s disease can be difficult to diagnose because symptoms overlap with those of other gastrointestinal disorders, such as ulcerative colitis (UC) and irritable bowel syndrome. Investigations to aid diagnosis include taking the patient’s medical history, physical examination, blood and stool tests and, finally, endoscopy to confirm diagnosis. As the treatment for CD depends on the location and severity of disease, an assessment of disease activity once disease is confirmed is important. However, disease activity is difficult to assess, and a global measure which includes clinical, endoscopic, biochemical and pathological features to define the heterogeneous disease pattern of CD is not available.⁹ This means that there is no ‘reference standard’ for the assessment of disease severity, which has important implications for this assessment. For example, there is no standardised definition for when remission has been achieved. The two most commonly used measures of disease activity are the Crohn’s Disease Activity Index (CDAI) and the Harvey–Bradshaw Index (HBI) (a simplified version of the CDAI), which are based on the patient’s history, physical features and laboratory data. A paediatric CDAI that emphasises the less subjective laboratory parameters has been developed.¹⁰ Additional measures include the Perianal Disease Activity Index (PDAI), the Inflammatory Bowel Disease Questionnaire (IBDQ) and the Crohn’s Disease Endoscopic Index of Severity.⁵ However, these tools have been primarily developed for clinical trials rather than clinical practice. In clinical practice, the use of endoscopy to assess mucosal healing as an indicator of response and remission is becoming increasingly important, and the potential of objective laboratory markers, such as C-reactive protein (CRP) and faecal calprotectin (FC), for the assessment of disease activity, risk of complications and prediction of relapse, and for monitoring the effect of therapy has been recognised.¹¹

The variables measured by the CDAI measures include number of liquid stools, abdominal pain, general well-being, extraintestinal complications, use of anti-diarrhoeal drugs, abdominal mass, haematocrit and body weight.¹² These are weighted according to their ability to predict disease activity, leading to an individual score ranging from 0 to 600. The CDAI has been criticised for giving too much weight to relatively subjective items;¹² however, more objective measures, such as mucosal healing on endoscopy, are not infallible either because of the patchy distribution of inflammation in CD. Samples taken for examination may not necessarily be representative of the whole bowel.²

Although the CDAI uses a symptom diary of the patient over 7 days for the assessment, the HBI uses only a 1-day diary entry for assessment. Furthermore, the HBI does not take into consideration body weight, haematocrit and use of drugs for diarrhoea for the measurement of disease activity. HBI scores range from 0 to 20.¹³

In the absence of standardised definitions in which scores correspond to the different disease severity stages, this review adopts the definitions from the NICE guidance technology appraisal 187:⁶

• Remission is defined as a CDAI score of < 150 points.
• Moderate to severe disease is defined as a CDAI score of > 220 points.
• Severe disease is defined as a CDAI score of > 300 points.

Response (i.e. relief of symptoms) has often been defined as a reduction in the CDAI score of at least 70 points from baseline.¹⁴

Severe active CD was defined for the purpose of the indication of IFX or ADA treatment as:

Very poor general health and one or more symptoms such as weight loss, fever, severe abdominal pain and usually frequent (3–4 or more) diarrhoeal stools daily. People with severe active Crohn’s disease may or may not develop new fistulae or have extra-intestinal manifestations of the disease. This clinical definition normally, but not exclusively, corresponds to a Crohn’s Disease Activity Index (CDAI) score of 300 or more, or a Harvey–Bradshaw score of 8 to 9 or above.

NICE (2010) technology appraisal number 187. Infliximab (Review) and Adalimumab for the Treatment of Crohn’s Disease. London: NICE. Available from www.nice.org.uk/guidance/TA187.⁶ Reproduced with permission. This information is accurate at time of publication

Furthermore, the Practice Parameter Committee of the American College of Gastroenterology has produced definitions of disease severity.⁷ These are as follows.

Management and care pathway

The treatment of CD is complex; in general, it aims at (1) reducing symptoms through induction and maintenance of remission, (2) minimising drug-related toxicity and (3) reducing the risk of surgery.¹⁵ The management options for CD include drug therapy [e.g. glucocorticosteroids, 5-aminosalicylic acid (5-ASA), antibiotics, immunosuppressants, TNF-α inhibitors], enteral nutrition, smoking cessation and, in severe or chronic active disease, surgery. The choice of treatment among the available drugs is influenced by patient age, site and activity of disease, previous drug tolerance and response to treatment, and the presence of extraintestinal manifestations.^16,17 Enteral nutrition is widely used as a first-line treatment to facilitate growth and development in children and young people. Adjuvant therapy commonly coexists and includes management of extraintestinal manifestations, antibiotics, corticosteroids or immunosuppressant therapy. Between 50% and 80% of people with CD require surgery because of complications such as strictures causing symptoms of obstruction, fistula formation, perforation or failure of medical therapy.²

Once remission has been achieved, maintenance therapy can be considered following assessment of the course and extent of CD, effectiveness and tolerance of previous treatments, presence of biological or endoscopic signs of inflammation, and potential for complications.¹⁵

National Institute for Health and Care Excellence guidelines

The NICE guideline technology appraisal 187⁶ describes when IFX or ADA should be used to treat patients with severe active or fistulising CD in the NHS in England and Wales.

Infliximab

The guideline states:

Infliximab has a UK marketing authorisation for the treatment of:

• severe, active Crohn’s disease in people whose disease has not responded despite a full and adequate course of therapy with a corticosteroid and/or an immunosuppressant, or who are intolerant to or have medical contraindications for such therapies
• fistulising, active Crohn’s disease in people whose disease has not responded despite a full and adequate course of therapy with conventional treatment (including antibiotics, drainage and immunosuppressive therapy)
• severe, active Crohn’s disease in people aged 6–17 years whose disease has not responded to conventional therapy, including a corticosteroid, an immunomodulator and primary nutrition therapy, or who are intolerant to or have contraindications for such therapies.

NICE (2010) technology appraisal number 187. Infliximab (Review) and Adalimumab for the Treatment of Crohn’s Disease. London: NICE. Available from www.nice.org.uk/guidance/TA187.⁶ Reproduced with permission. This information is accurate at time of publication

Administration of IFX should follow this pattern:

. . . 5-mg/kg intravenous infusion over a 2-hour period followed by another 5-mg/kg infusion 2 weeks after the first. If a person’s disease does not respond after two doses, no additional treatment with infliximab should be given. In people whose disease responds, infliximab regimens include maintenance treatment (another 5-mg/kg infusion at 6 weeks after the initial dose, followed by infusions every 8 weeks) or re-administration, otherwise known as episodic treatment (an infusion of 5-mg/kg if signs and symptoms of the disease recur).

NICE (2010) technology appraisal number 187. Infliximab (Review) and Adalimumab for the Treatment of Crohn’s Disease. London: NICE. Available from www.nice.org.uk/guidance/TA187.⁶ Reproduced with permission. This information is accurate at time of publication

For fistulising disease, the first three doses at weeks 0, 2 and 6 are considered as induction therapy and additional IFX therapy should not be given if the first three doses have not induced a response. The patient pathway for people responding to IFX induction therapy and moving onto maintenance therapy is given in Figure 1.

FIGURE 1

Patient pathway of patients with CD on IFX therapy.

Adalimumab

Adalimumab can be used to treat severe active CD in adults whose disease has not responded to treatment with an immunosuppressant and/or corticosteroid, or who are intolerant to or have contraindications to such therapies.

Administration of ADA should follow this pattern:

The adalimumab induction treatment dose regimen for adults with severe Crohn’s disease is 80 mg via subcutaneous injection, followed by 40 mg 2 weeks later. After induction treatment the recommended dose is 40 mg every other week. This can be increased to 40 mg every week in people whose disease shows a decrease in response to treatment.

NICE (2010) technology appraisal number 187. Infliximab (Review) and Adalimumab for the Treatment of Crohn’s Disease. London: NICE. Available from www.nice.org.uk/guidance/TA187.⁶ Reproduced with permission. This information is accurate at time of publication

Anti-tumour necrosis factor alpha agents

Crohn’s disease is associated with elevated levels of the immune regulatory protein, TNF-α. The reasons for this elevation in CD are still largely unknown. TNF-α is a small cell signalling protein (cytokine) involved in inflammatory responses primarily by influencing regulation of various effector cells of the immune system. TNF-α has been shown to have a role in several inflammatory diseases including CD, UC, rheumatoid arthritis and ankylosing spondylitis. Anti-TNF-α agents bind to cell surface TNF-α and free TNF-α, and block their activity. Blocking of TNF-α with anti-TNF-α drugs has been shown to be successful for some patients with inflammatory diseases, including CD. Anti-TNF-α agents recommended by NICE for the treatment of CD are IFX and ADA. These monoclonal antibodies are introduced into the human body to bind and block TNF-α. They are classed as monoclonal antibodies because they are derived from genetically engineered immune cells, which are all daughters of a single parent cell, so that in culture they generate and secrete antibodies that are all of identical structure to and affinity for TNF-α.¹⁵

Infliximab

Infliximab is a chimeric (mouse–human) monoclonal antibody. It is said to be chimeric because the genetic code determining its amino acid sequences is partly derived from the mouse genome and partly from the human genome. IFX belongs to the immunoglobulin gamma type 1 (IgG1) group of antibody molecules (Figure 2). It should be borne in mind that IgG1 molecules are globular (not linear as in the diagram) and that they are glycoproteins, which have carbohydrate chains attached (not shown in Figure 2). As IFX is generated from cultured mouse cells, the carbohydrate part of the molecule corresponds to that of mouse rather than human glycoproteins.¹⁵

FIGURE 2

Diagrammatic representation of the structure of an IgG1 antibody molecule. The molecule has two heavy chains and two light chains; the heavy chains are joined by disulphide bonds (S–S) and each light chain is joined to a heavy chain by S–S (more...)

Infliximab is composed of human IgG1 heavy-chain constant regions and human kappa light-chain constant regions (together representing 70% of the genetic make-up of the molecule), plus mouse-derived heavy- and light-chain variable regions (30% of the genetic make-up, 4/12 domains), which carry the binding sites with high affinity and specificity to TNF-α (see Figure 2). IFX was the first anti-TNF-α agent that was approved and licensed for treating severe active CD and active fistulising CD in adults and children aged > 6 years. It is administered intravenously over 1–2 hours.

Side effects of IFX include:

• allergic reaction to the infusion (or IFX) apparent by:
  • hives (red, raised, itchy patches of skin) or other skin rashes
  • difficulty swallowing or breathing
  • pains in the chest or muscle, or joint pain, fever or chills
  • swelling of the face or hands
  • headaches or a sore throat.
• serious viral or bacterial infections including tuberculosis, especially in people aged > 65 years
• skin reactions including psoriasis (red scaly patches), rashes, skin lesions, ulcers and hives, and swollen face and lips
• worsening of heart problems
• increased risk of cancer or lymphoma
• liver inflammation.

Many of the side effects are reversible if the drug is stopped.¹⁵

Adalimumab

Adalimumab is a purely human IgG1 monoclonal antibody. ADA is a more recent anti-TNF-α therapy that was approved for treating CD in adults only. It is administered as a subcutaneous injection by a doctor or nurse, or can be self-injected by the patient or a family member.¹⁵

Side effects of ADA include:

• reactions to the injection including pain, swelling, redness, bruising and itching
• allergic reaction to ADA including:
  • rashes or hives
  • swollen face, hands and feet
  • trouble breathing.
• greater susceptibility to infections such as colds, influenza, pneumonia, sepsis and tuberculosis
• skin reactions including psoriasis (scaly patches), eczema, other skin rashes and ulcers
• skin cancer, lymphoma or leukaemia
• damage to nerves (demyelination)
• lupus.

Many of the side effects are reversible if the drug is stopped.¹⁵

Significance to the NHS and current service cost

The aim of successful therapies in CD is to prolong remission and to minimise relapse. Patients’ quality of life (QoL) fluctuates through time and, unsurprisingly, has been found to be better during remission. Studies using various disease-specific health-related QoL measures (such as McMaster’s IBDQ, IBDQ-36, short IBDQ, Rating Form of IBD Patient Concerns, Cleveland Clinic questionnaire and Gastrointestinal Quality of Life Index) show a clear correlation between health-related QoL and symptoms. These measures in patients with CD have allowed utility estimates to be developed for patients in various clinical states.²⁰ QoL has been found to be somewhat worse in CD than in UC, and substantially worse in relapse than in healthy matched individuals. It has also been found to be similar or worse to that experienced in many other medical conditions.²¹ Gastroenterologists tend to rely on global clinical judgement, which tends to be less reproducible than QoL assessment tools, but is, of course, simpler for decision-making in everyday clinical practice.⁹

Patients with CD can be cared for in primary or secondary care depending on symptom severity. Although general practitioners manage patients in remission or with mild symptoms, patients with more severe active disease are managed in secondary care. These are patients who are likely to be steroid dependent, on immunosuppressants or anti-TNF-αs, or requiring surgery. It has been estimated that about 50% of patients with CD experience at least one flare per year. Of these, 20% of patients will require hospitalisation.²² Disease flares have been found to be associated with a two- to threefold increase in hospitalisation and a 20-fold increase in cost compared with managing patients in remission.²³ Audit data show that anti-TNF-α agents are potentially cost-saving by successfully maintaining patients in remission and reducing hospital admissions. Cost reductions of £138 per patient at 6 months²⁴ and £2750 per patient at 12 months (excluding IFX costs) have been demonstrated in a before-and-after study of IFX therapy.²⁵ However, in that study both non-responders (£3608) as well as responders (£1656) incurred a considerably higher annual cost than the mean annual costs of long-term care in CD of £631/£762 (UK) and £838/£796 (Europe) estimated in another study using decision modelling.²⁶ Using 2008 prevalence figures, the total annual cost to the NHS for the approximate 60,000 patients with CD was estimated at £38M. Updating this figure with more recent prevalence data (115,000), but still using 2008 prices, would double that cost to £73M. This, however, might be a modest estimate when considering the wide range of measured 6-month costs for individual patients with CD (£73–£33,254).²³ The main drivers of costs were hospital admission, surgery and anti-TNF-α treatment.²⁶ In the 2003 Health Technology Assessment (HTA) publication by Clark et al.,³ the average cost of a single 5-mg/kg IFX infusion for a 70-kg patient was reported to be £1804.80. No comparable data for ADA are available.

The significance of CD to the NHS is increased by the fact that the prevalence of CD is increasing and the disease affects many people at a young age. The lifetime care costs for patients with CD are now comparable to the costs of caring for patients with other major chronic diseases, such as diabetes mellitus and cancer.²² This argues not only clinically but also economically for interventions that keep patients in remission and out of hospital. This review focuses on whether or not monitoring anti-TNF-α agents and their antibodies with enzyme-linked immunosorbent assays (ELISAs) could potentially contribute to this aim.

Responders and non-responders definitions and incidence rates

Similar to other treatment regimens for CD, anti-TNF-α treatment aims to induce remission (defined as < 150 points on CDAI and no draining fistulae), in which case it is described as induction therapy, and to prevent relapse (maintenance therapy). However, failure to induce a response and LOR to anti-TNF-α are common problems in clinical practice. The lack of consensus regarding clear definitions for response and remission result in inconsistencies in the reported incidence rates of non-response and LOR covered in this section.

Anti-drug antibodies

Anti-drug antibodies can be elicited by IFX or ADA during therapy as a response by the human immune system to these foreign proteins; this is termed immunogenicity of anti-TNF-α agents. These anti-drug antibodies bind to the anti-TNF-α agent and neutralise its action. If sufficient amounts of antibodies are present, the individual loses response to the drug treatment. During scheduled maintenance therapy, the incidence of anti-drug antibodies is 5–18%^27,38,39 and 3–17%³⁹ for IFX and ADA, respectively. The similar rates for IFX and ADA might initially appear counterintuitive as ADA is a fully human recombinant protein, whereas IFX is partly human and partly mouse protein and, therefore, ‘more’ foreign. However, ADA, similar to IFX, is a foreign protein that will prompt a response when coming into contact with the immune system. This indicates that the degree of ‘human-ness’ is not the main determinant of immunogenicity (i.e. formation of antibodies to a foreign protein).³⁹

Levels of antibodies have been found to be higher during episodic treatment, at 36–61%,^39–41 than levels found during maintenance therapy. This indicates that other factors may influence immunogenicity. The true incidence of antibodies in anti-TNF-α-treated patients is, therefore, unknown. The ability to mount an immune response and measurement of that response depends on a number of factors, including the method of measuring antibody levels and age, and also depends on concomitant treatment with immunosuppressants.^{14,27,40–44} For that reason, concomitant immunosuppressants might be given to patients to prevent or reduce the formation of antibodies. This effect was not observed in one study for ADA,¹⁴ and Vermeire et al.⁴⁵ reported that increasing anti-TNF-α above antibody-binding capacity might have similar effects to immunosuppressants, by neutralising free antibodies. Vande Casteele et al.⁴⁶ made a similar observation for transient antibodies (antibodies detectable for a short period during a series of follow-up test assays conducted during a course of infusions), while sustained antibodies did not disappear after dose optimisation and were associated with LOR.

The clinical importance of antibodies can be presented as:³⁹

• positive/negative/inconclusive
• high/low
• above/below a threshold in arbitrary units or in µg/ml
• drug concentration
• clinical effect (duration of response, need for dose intensification or switch drug)
• impact on safety (infusion reactions).

The clinical relevance of antibodies has been debated. However, numerous studies report the correlation between presence of antibodies with low or absent drug levels and consequent response.^{27,38,40,43,45,47,48} This can be explained by antibodies binding to the epitope of anti-TNF-α and neutralising the drug (i.e. making it unable to bind to TNF-α and inhibiting the working mechanism of anti-TNF-α) or by forming immune complexes with the drug (non-neutralising antibodies), which are subsequently cleared from the circulation (reducing the drug’s bioavailability).⁴⁹

Although the importance of the neutralising antibodies has been universally acknowledged,^14,34,49,50 reviewers seem to disagree in their conclusion about the importance of non-neutralising antibodies.^14,34 Over 90% of antibodies to IFX and ADA are neutralising.⁵¹ In a meta-analysis, Garces et al.⁴⁹ estimated that detectable antibodies can decrease response to anti-TNF-α by as much as 80%.

An interesting additional observation was made by Steenholdt et al.,⁵² who showed that immunoglobulin gamma (IgG) antibodies reacting with the fragment antigen-binding portion (see Figure 2) of IFX exist in IFX-naive IBD patients prior to treatment. The presence of pre-existing antibodies affected response and safety of IFX treatment in patients with CD, and the study concluded that the clinical utility of measuring pre-treatment antibodies should be assessed.

Drug levels

Although anti-drug antibodies have been shown to reduce anti-TNF-α drug levels, there are other known mechanisms that affect drug levels. These include dose and dosing interval, body mass index, sex, serum albumin levels (serum albumin transports drugs and can affect the half-life of drugs), concomitant immunosuppressants, severity of inflammation, mode of administration (intravenous vs. subcutaneous) and drug half-life.^14,44

As a consequence, drug levels vary considerably between patients and within individuals over time.³⁴ Following administration of the anti-TNF-α agent, circulating drug concentration will be at its peak level; the concentration just before the next round of treatment is classed as ‘trough level’. The optimal time of testing drug levels within this cycle has been debated⁴⁴ and it is largely unknown what the optimal drug levels would be at the different time points. Although a threshold trough level is thought to be needed for effectiveness, it is also known that supratherapeutic levels can cause infections and other adverse events.¹⁴

Anti-tumour necrosis factor alpha and antibody level monitoring in Crohn’s disease

One of the key studies to demonstrate and quantify the link between drug and anti-drug antibody levels, immunosuppressant therapy and response in patients with CD on anti-TNF-α agents was the study by Baert et al.⁴³

Baert et al.⁴³ was an early, and influential, study of the development of anti-drug antibodies to IFX in patients with CD; this study stimulated numerous subsequent investigations. The study enrolled 125 consecutive patients (38 with fistulising and 87 with luminal disease) who received 5 mg/kg of IFX at 0, 2 and 6 weeks. Responders (89/125, 71%) were retreated with this regimen if they required restart of IFX therapy according to clinical judgement. Mean treatment period was 10 months and median follow-up was 36 months. Anti-drug antibodies and IFX serum levels were measured before and at 4, 8 and 12 weeks after each infusion, using ELISA (PROMETHEUS^® ELISA, Prometheus Laboratories Inc., San Diego, CA, USA). After five infusions, 76 out of 125 (61%) patients were classified as positive for anti-drug antibodies.

When a level of 8 µg of anti-drug antibodies/ml serum was selected, it was found that concentrations of anti-drug antibodies were > 8 µg/ml in 24 out of 56 (43%) patients taking immunosuppressants, compared with 52 out of 69 (75%) not taking immune suppressants. The relative risk (RR) of anti-drug antibodies concentration > 8 µg/ml in patients taking compared with those not taking suppressive therapy was 2.40 [95% confidence interval (CI) 1.65 to 3.66; p < 0.001]. Infusion reactions had occurred in 27% of patients by the fifth infusion. The median anti-drug antibody level in patients experiencing infusion reactions was higher than in those with no reactions (p < 0.001). Reactions were significantly more common in patients not taking immunosuppressant therapy than in those who were taking it. When time to next infusion was taken as a measure of response duration, it was found that response duration was reduced in those with anti-drug antibodies levels > 8 µg/ml relative to those with levels < 8 µg/ml (median 35 days vs. 71 days; p < 0.001).

The level of IFX at 4 weeks after an infusion was correlated with the level of anti-drug antibodies prior to the infusion (R² = 0.34; p < 0.001) and was positively correlated with duration of response. IFX level and anti-drug antibodies level were independent variables influencing response duration. Logistic regression indicated that the only variable that influenced a 4-week level of IFX > 12 µg/ml was the use of immunosuppressant therapy. IFX level was higher in those without an infusion reaction than those with one.

In summary, the results of this study suggest that production of anti-drug antibodies is common during IFX therapy; anti-drug antibodies are associated with reduced IFX levels; duration of response is reduced by the presence of anti-drug antibodies; and production of anti-drug antibodies may be reduced when concomitant immunosuppressant therapy is employed.

Further evidence steadily accumulated from retrospective analyses of multiple clinical trials and case series,^38,53–55 and the observation that detectable trough levels of drug are associated with greater clinical efficacy is now well established.⁴⁴

This accumulating evidence has formed the basis of investigations into drug and anti-drug antibody monitoring in anti-TNF-α-treated patients with CD.

Without monitoring, the options for a clinician if the anti-TNF-α agent fails are to wait and see, to intensify drug treatment, to switch drug within its class or to switch to a different class of drugs.³⁴ Measuring drug and anti-drug antibodies, however, could enable clinicians and patients to make informed choices on management. A number of studies have investigated the clinical utility of measuring drug and anti-drug antibody levels in sera by translating the clinical management decision following a test outcome into a treatment algorithm stipulating the management pathways for patients with a specific test outcome in clinical practice.^56–59

Drug and anti-drug antibody monitoring could be undertaken in good responders (i.e. those responding to an initial induction course of anti-TNF-α treatment), as well as in patients with LOR (i.e. those initially responding to anti-TNF-α treatment but losing this response over time). The use of these technologies provides a clinician with potentially useful information that may guide an individual patient’s future treatment. Such information may aid in anticipating the LOR in responders or allow drug optimisation, whereas for non-responders such analyses may help in estimating the likelihood of various candidate reasons for LOR. In non-responders with low levels of drug and high levels of anti-drug antibodies, for example, the loss or lack of response may be surmised to be because of rapid clearance of the drug as a result of the action of anti-drug antibodies. On the other hand, a low level of anti-TNF-α in the absence of anti-drug antibodies may be suggestive of non-immune mechanisms of rapid drug clearance, whereas high levels of drug in the absence of antibodies in non-responders may be suggestive of a pathology for the condition independent of TNF-α in a particular patient. Algorithms for future treatment based on anti-TNF-α and anti-drug antibody estimates have been published.^56–59

In theory, the application of the tests in conjunction with an appropriate algorithm for treatment based on test results may:

• improve QoL and other outcomes (e.g. faster healing of flare-ups, reduced abdominal pain and associated diarrhoea)
• optimise the treatment plan (facilitate adoption of the most suitable future treatment for individual patients; this might involve a switch to an alternative anti-TNF-α or a biologic with an alternative mechanism of action)
• minimise the risk of drug overdose and associated adverse events
• allow earlier de-escalation of therapy, leading to a reduction in the overall drug used
• help to reduce the amount of drugs used inappropriately, unnecessary hospital visits, risk of surgery and associated costs.¹⁵

Intervention technologies

Various assay procedures for anti-TNF-α agents and for anti-drug antibodies have been developed in the belief that the levels of circulating anti-TNF-α and of anti-drug antibodies can provide information useful to clinicians in indicating potential reasons for treatment failure, and for dosage or treatment adjustment.

Commercially available ELISA kits (the LISA-TRACKER^® ELISA kits, Theradiag, Marne La Vallee, France, or Alpha Laboratories, Heriot, UK; the TNF-α-Blocker ELISA kits, Immundiagnostik AG, Bensheim, Germany; and the Promonitor^® ELISA kits, Proteomika, Progenika Biopharma, Bizkaia, Spain) are the intervention technologies designed to measure IFX and ADA levels, and their antibodies and are investigated in this review.

These are all particular examples of solid-phase ELISAs. They estimate the following molecules in patient blood sera:

• IFX
• ADA
• anti-IFX antibodies
• anti-ADA antibodies.

Details of the ELISA kits available from these companies are summarised in Appendix 1.

Other ELISAs commercially available for measuring these molecules in sera include the SHIKARI^® ELISA kits (Matriks Biotechnology Co. Ltd, Ankara, Turkey). These are not included as index tests in the NICE scope.

In the UK a number of non-commercial kits are also available, but these are not the focus of this report.

Other methodologies based on alternative principles of detection and measurement include (1) radioimmunoassays (RIAs) (liquid-phase assays using the radioisotope ¹²⁵I to label TNF); (2) cell reporter assays based on genetically engineered cells incubated in culture medium; and (3) mobility shift assays (liquid-phase assays using size-exclusion high-performance liquid chromatography with fluorescent dye detection). The differences in these assays may have an effect on their individual performance, and describing and contrasting them will help the reader to understand the abilities and limitations of the assays.

Enzyme-linked immunosorbent assays for infliximab and adalimumab

For details of the ELISA kits, refer to Appendix 1. All three specified ELISA methods employ similar principles in which, typically, microtitre plates with 96 wells coated with reagent receive the patient serum samples or various standards and calibrators. Reagents are added with wash steps between additions. The final step involves quantifying the amount of a peroxidase label in the titre well, this amount being proportional to the amount of anti-TNF-α or anti-drug antibody in the patient’s sample or in the calibrator standard.¹⁵

The amount of peroxidase present in the well is quantified using a timed incubation with excess substrates [hydrogen peroxide + 3,3’,5,5’-tetramethylbenzidine (3,3’,5,5’-tetramethylbiphenyl-4,4’-diamine)]. Peroxidase catalyses the following reaction:

$$\text{tetramethylbenzidine}+\text{hydrogen\hspace{0.17em}peroxide}\to \text{chromogen}+\text{water}.$$

(1)

The incubation is stopped after an appropriate time by the addition of acid and the accumulated chromogen quantified by measuring optical density with a spectrophotometer.

The reagents used for coating the microtitre plate wells and the reagents used in subsequent steps of the assay procedure differ in detail according to manufacturer. The LISA-TRACKER assays for IFX and for ADA are illustrated in Figure 3.

FIGURE 3

Diagrammatic representation of the LISA-TRACKER assay for IFX and ADA. Procedural steps C and D are detection steps that function to detect the anti-TNF-α that is bound to the microtitre well surface via TNF-α, ensuring a quantitative (more...)

Serum samples from patients may contain soluble TNF-α receptors; these could compete with anti-TNF-α for the immobilised TNF-α on the microtitre well plate and may potentially interfere with the assay. The assay quantifies free anti-TNF-α. Samples may contain anti-TNF-α bound to antibodies to anti-TNF-α, especially in patients who have lost a response to treatment. These anti-TNF-α–antibody complexes will be washed away at the first wash step, leaving only free anti-TNF-α bound to immobilised TNF-α. The amount of anti-TNF-α lost at the wash step is likely to vary between patients and is unknown; the practical implications of this are uncertain.¹⁵

TNF-α-Blocker and Promonitor assays for anti-TNF-α drugs differ from the LISA-TRACKER assay in that the well coat is not TNF-α, but rather a reagent (antibody or antibody fragment) able to bind specifically to the TNF-α binding site of IFX or of ADA that is added to the microtitre well in the patient’s sample (or calibrator). After washing, the second reagent is a peroxidase-labelled antibody able to bind the fragment crystallising (Fc) region of the anti-TNF-α antibody (Figure 4). Thus, fewer steps and a single reagent are used to detect well-bound anti-TNF-α drug. Table 1 summarises the information describing the mechanisms underlying these assays.

FIGURE 4

Diagrammatic representation of TNF-α-Blocker and Promonitor assays for IFX and ADA. Procedural steps C and D are detection steps that function to detect the anti-TNF-α (e.g. drugs IFX or ADA) that has been bound to the well surface via (more...)

TABLE 1

Summary of ELISAs to be considered in this review for detection of IFX and ADA

Enzyme-linked immunosorbent assays for anti-drug antibodies

The LISA-TRACKER assays for antibodies to IFX and to ADA are illustrated in Figure 5.

FIGURE 5

Diagrammatic representation of the LISA-TRACKER assay for antibodies to IFX or to ADA. This is a bridging assay in which antibodies to anti-TNF-α in the patient sample bridge the immobilised anti-TNF-α to the biotinylated anti-TNF-α. (more...)

This assay quantitatively estimates only free antibodies to anti-TNF-α. Therefore, anti-drug antibodies bound to the drug are lost at the first wash. The amount of bound anti-drug antibody is likely to vary between patients and is unknown. Whether anti-drug antibodies directed at non-idiotypic regions of the drugs (e.g. glycoprotein moieties, variable non-idiotypic mouse regions of IFX, etc.) are detectable or present in samples appears to be insufficiently investigated to date and is therefore uncertain. However, in vitro tests indicate that about 90% of anti-drug antibodies bind to the TNF-binding region of anti-TNF-α drugs.⁵¹ These, and the other anti-drug antibodies, may hasten clearance of drug from the circulation as well as neutralising its binding capacity.

Tumour necrosis factor alpha and Promonitor assays differ from LISA-TRACKER assays in employing a single reagent for detecting well-bound anti-drug antibodies rather than two (biotinylated IFX or biotinylated ADA, plus avidin-conjugated peroxidase). Table 2 summarises the information describing the mechanisms underlying these assays.¹⁵

TABLE 2

Summary of ELISAs to be considered in this review for detection of antibodies to IFX and ADA

Current usage of assays in the NHS

Current practice for monitoring TNF-α inhibitor antibody and drug levels in the UK is patchy because of the lack of agreed consensus and evidence for its cost-effectiveness. In-house tests are performed in a few laboratories in England. However, demand is low, analyses are often undertaken in batches and it can be weeks (in some cases) before a clinician receives a result on which to act.

Although some centres have local monitoring protocols in conjunction with their link laboratory, there is, as yet, no agreed algorithm for clinicians to refer to which allows for the translation of the results of the tests into coherent plans for patient management according to test outcome.

However, recent emerging evidence to support anecdotal practice that such monitoring could be useful in managing patients with TNF-α inhibitors, has encouraged a cautious increase in uptake.

It is expected that therapeutic monitoring of TNF-α inhibitors might be useful in a number of clinical scenarios in the treatment of CD in the NHS, including for primary and LOR to anti-TNF-α therapy and in the optimisation of dosages for those who are already responding.