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Monitoring of TNF-alpha inhibitors (TNF-α-i)

Clinical Use

TNFα-i's are excellent tools for treatment of severe chronic inflammatory diseases. Experience shows that a large proportion of patients (60-70%) with various autoimmune diseases such as rheumatic disorders and inflammatory bowel disease have an excellent primary response to treatment with TNF-α inhibitors.

Treatment with TNFα-i show a large variation between patients, in terms of treatment dose and interval between dosing. Consequently, many patients may benefit from an individualized treatment approach depending on their response to the biological drug.

There is a significant percentage of patients for whom TNF-α inhibitors fail to demonstrate efficacy (primary failure) or where the effect of the TNF-α inhibitors decreases over time despite an initial good response (secondary failure) (Bendtzen 2011 and 2013, Vincent 2013). Tests measuring drug activity (concentration) of TNF-α inhibitors and NAbs (neutralising anti-drug-antibodies) can be used as a tool to establish why the patient has no effect of their treatment.

Measuring biological activity of a drug

Wieslab Diagnostic Services uses a test technology that measures the biological activity of the drug which gives a high therapeutic significance. If the test shows that the patient has low concentrations of the TNF-α inhibitors and/or has developed NAbs, it is both clinically and economically correct to adjust the treatment regime.

Testing inhibitor concentration

The assay is performed with an in vitro reporter cell line (iLite) which measures the amount of drug in the serum that can inhibit TNF-α binding to the TNF receptor on the cells. The specific TNF-α binding activates a reporter gene (Firefly Luciferase) and the effect of the produced enzyme can be measured.
 
The method is very stable and reproducible and is based on an arrest of the normal cell cycle. The cellular activity assay has a very good correlation to classical concentration measurement by radioimmunoassay (RIA) but measures in contrary to binding assays (RIA, ELISA, HMSA etc.) the activity of the drug, which is of a high therapeutic significance and clinical value. 

Testing neutralising anti-drug antibodies

NAbs - Testing for NAbs against TNF-α inhibitors are performed only when the concentration of TNF-α inhibitors activity in serum is < 0.65 µg/mL.

Test results for neutralising anti-drug antibodies (NAbs) to TNF-α inhibitors are reported as negative or positive with titer.

Testing anti-drug antibodies

ADA - When the patient has measurable, but low concentration of drug in the blood (0.65-10 µg/mL), the sample can be further tested to evaluate if the patient has developed ADAs (anti-drug antibodies).

Test results for anti-drug antibodies (ADAs) to TNF-α inhibitors are reported as negative or positive (qualitative assay).

Clinical Relevance

  • At detectable levels of the drug the presence of NAbs are of low therapeutic significance.
  • Normal or high concentration of active drug but no treatment effect is observed in patients with a profile of the disease that is not driven by TNF-α.
  • When secondary failure is suspected despite the presence of normal or high concentrations of active drug, the sample may have been collected not at Cmin.
  • When the concentration of the TNF inhibitors is below the detection level, this may be caused by NAbs against the TNF-α inhibitor which prevents the drug to bind to TNF-α. This in turn causes the TNF-α to bind normally to the TNF receptor.
  • Patient compliance problems may be the reason when the serum concentration of TNF inhibitors is lower than expected in combination with undetectable levels of NAbs. Low drug concentration and no treatment effect may also suggest that the patient needs a higher drug dose to achieve the desired therapeutic effect.
  • Low concentrations of the drug can also be due to non-neutralizing ADA. Non-neutralizing ADA can both limit the bioavailability (with local administration of the drug) and increase the elimination of the drug in the circulation.
  • ADA´s or NAbs do not cross-react between the different TNF inhibitors.

Personalized Medicine

Monitoring biologic levels in individual patients allows optimization of treatment efficacy by adjusting therapy depending on the specific response of the patient. When drug levels are decreased in a non-responder, this may reflect formation of inactivating antibodies directed to the biologic treatment. Immunogenicity testing identifies presence or absence of inactivating antibodies, which guides the next treatment decision – switch to other biological treatment, lowering of dose or intensify dosing. Reducing drug dosage in well-responding patients with relatively high drug levels cuts down cost.  A personalized approach can improve therapy efficacy and quality of life.

METHOD - iLite® Cell-Based Solutions

The assay for TNF-α inhibitor NAbs is based on an in vitro reporter cell line (iLite) which is used to measure the activity (concentration) of the drug (Lallemand 2011). In short, the NAbs tests show if antibodies in patient serum can prevent TNFα-inhibitor activity in an in vitro cell model.

Principle for the analysis of TNF-alpha (TNF-α) monitoring

1. The reporter cells in the test carry a TNF-α-induceable, NFkB-regulated Firefly lucifer's reporter gene construct. When TNF-α is added to the reporter cell, the reporter gene is activated via NFkB to express Firefly luciferase. The level of Firefly luciferase is normalized relative to the level of the Renilla luciferase gene, which is under the control of a constitutive promoter in the same cell.

2. Serum from a patient treated with TNF-α inhibitor is mixed with a predetermined amount of TNF-α. The amount of TNF-α-inhibitor correlates inversely with the amount of luminescence (light).

3. The patient's serum is mixed with a certain amount of TNF-α-i (the TNF-α-i used for the treatment of the individual patient) and incubated. Then this mix is added to a predetermined amount of TNF-α and cells.

4. In neutralising anti-drug antibodies (NAbs) against TNF-α-i: NAbs bind TNF-α-i which thus cannot bind to TNF-α. This means that TNF-α can bind to the TNF-α receptor and provide normal activity in the cell.

METHOD - ACID EIA

Before analysis, the sample is treated with acid and diluted to allow dissolution of immune complexes. This process describes an EIA (enzyme immunoassay) designed to determine the presence of binding antibodies to TNF-α inhibitors. The process can be described as a sandwich EIA.

Want to know more about the iLite® Technology?

Learn more about the dual reporter gene system and
the unique features of the iLite Cell-Based Solutions

Read more

In need of cell-based bioanalytical services?

Our Bioanalytical Services offer a range of cell-based assay services using iLite and can help accelerate and optimize your drug development program.

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Relevant literature

  • Bendtzen K. Personalized Medicine: Theranostics (therapeutics diagnostics) essential for rational use of tumor necrosis factor-alpha antagonists. Discovery Medicine, 15(83), 201-211 (2013)
  • Bendtzen K. Is there a need for immunopharmacologic guidance of anti-tumor necrosis factor therapies? Arthritis Rheum. 63(4):867-870 (2011)
  • Bendtzen K, Ainsworth M, Steenholdt C, Thomsen OO, Brynskov J. Individual medicine in inflammatory bowel disease: monitoring bioavailability, pharmacokinetics and immunogenicity of anti-tumour necrosis factor-alpha antibodies. Scand J Gastroenterol. 44, 774-781 (2009)
  • Labels information of individual TNF-inhibitors - http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm
  • EMA Committee for Medicinal Products for Human Use (CHMP). Guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use. EMA/CHMP/BMWP/86289/2010 (2012)
  • EMA Committee for Medicinal Products for Human Use (CHMP). Guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins EMEA/CHMP/BMWP/14327/2006. (2008)
  • FDA Guidance for Industry. CDER/CBER. Assay Development for Immunogenicity Testing of Therapeutic Proteins. Draft guidance (2009)
  • Jani M, Barton A, Warren RB, Griffiths CE, Chinoy H. The role of DMARDs in reducing the immunogenicity of TNF inhibitors in chronic inflammatory diseases. Rheumatology (Oxford). 2013 Aug 14
  • Lallemand C, Kavrochorianou A, Steenholdt C, Bendtzen K, Ainsworth MA, Meritet J-F, Blanchard B, Lebon P, Taylor P, Charles P, Alzabin S, Tovey MG. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFalpha antagonists. J Immunol Meth. 373, 229-239 (2011)
  • Garcês S, Demengeot J, Benito-Garcia E. The immunogenicity of anti-TNF therapy in immune-mediated inflammatory diseases: a systematic review of the literature with a meta-analysis. Ann Rheum Dis. Dec 1;72(12):1947-55 (2013)
  • Garcês S, Antunes M, Benito-Garcia E, da Silva JC, Aarden L, Demengeot J. A preliminary algorithm introducing immunogenicity assessment in the management of patients with RA receiving tumour necrosis factor inhibitor therapies. Ann Rheum Dis. May 11. (2013)
  • Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. Pharmacol Ther. Feb;117(2):244-79. (2008)
  • Vincent FB, Morand EF, Murphy K, Mackay F, Mariette X, Marcelli C. Antidrug antibodies (ADAb) to tumour necrosis factor (TNF)-specific neutralising agents in chronic inflammatory diseases: a real issue, a clinical perspective. Ann Rheum Dis. 72:165–178 (2013)