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Development of a universal anti-adalimumab antibody standard for interlaboratory harmonization.

Gils A, Vande Casteele N, Poppe R, Van de Wouwer M, Compernolle G, Peeters M, Brouwers E, Vermeire S, Geukens N, Declerck PJ - Ther Drug Monit (2014)

Bottom Line: When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated.Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity.The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

View Article: PubMed Central - PubMed

Affiliation: *Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven; †PharmAbs, The KU Leuven Antibody Center, University of Leuven; and ‡Department of Gastroenterology, University Hospitals Leuven, Belgium.

ABSTRACT

Background: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients.

Methods: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti-tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response.

Results: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity.

Conclusions: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

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Related in: MedlinePlus

Dose–response curve of TNF (A), ADM (B), and MA-ADM6A10 (C) in CBA. HT1080 cells were incubated with TNF. A, IL-6 was measured and expressed as log TNF concentration versus IL-6 response (mean ± SD, n = 2). B, Inhibition of TNF by ADM was determined using 7.5 ng/mL TNF (441.2 pmol/L) and ADM (0–240 ng/mL) (mean ± SD, n = 2). C, To determine the inhibitory effect of MA-ADM6A10, different doses (0–240 ng/mL) of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) (mean ± SD, n = 5).
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Figure 2: Dose–response curve of TNF (A), ADM (B), and MA-ADM6A10 (C) in CBA. HT1080 cells were incubated with TNF. A, IL-6 was measured and expressed as log TNF concentration versus IL-6 response (mean ± SD, n = 2). B, Inhibition of TNF by ADM was determined using 7.5 ng/mL TNF (441.2 pmol/L) and ADM (0–240 ng/mL) (mean ± SD, n = 2). C, To determine the inhibitory effect of MA-ADM6A10, different doses (0–240 ng/mL) of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) (mean ± SD, n = 5).

Mentions: HT1080 cells, a human fibrosarcoma cell line that expresses IL-6, was used to develop a functional CBA. Basal IL-6 concentrations [2.2 ± 1.5 ng/mL (mean ± SD), n = 6] were set at 0%. IL-6 concentrations increased on addition of TNF (maximum response was set at 100%), revealing an EC50 of 3.78 ± 0.02 ng/mL (217.7 ± 1.2 pmol/L) and an EC80 of 11.1 ± 0.02 ng/mL (637.7 ± 1.3 pmol/L) (Fig. 2A). Based on the obtained TNF EC50/80, a concentration of 7.5 ng/mL TNF (441.2 pmol/L) was chosen to determine the reduced IL-6 production because of the inhibition of TNF by ADM. ADM was able to neutralize 7.5 ng/mL TNF with an IC50 of 8.07 ± 0.18 ng/mL (53.8 ± 1.2 pmol/L) and an IC80 of 21.2 ± 0.2 ng/mL (141.9 ± 1.4 pmol/L) (Fig. 2B). To determine if MA-ADM6A10 is able to inhibit the inhibitory effect of ADM on TNF, different doses of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) to HT1080 cells using a similar setup as described above. On addition of 7.5 ng/mL TNF and 15 ng/mL ADM, an IC50 of 9.1 ± 0.20 ng/mL (61.0 ± 1.3 pmol/L) was determined for MA-ADM6A10 (Fig. 2C).


Development of a universal anti-adalimumab antibody standard for interlaboratory harmonization.

Gils A, Vande Casteele N, Poppe R, Van de Wouwer M, Compernolle G, Peeters M, Brouwers E, Vermeire S, Geukens N, Declerck PJ - Ther Drug Monit (2014)

Dose–response curve of TNF (A), ADM (B), and MA-ADM6A10 (C) in CBA. HT1080 cells were incubated with TNF. A, IL-6 was measured and expressed as log TNF concentration versus IL-6 response (mean ± SD, n = 2). B, Inhibition of TNF by ADM was determined using 7.5 ng/mL TNF (441.2 pmol/L) and ADM (0–240 ng/mL) (mean ± SD, n = 2). C, To determine the inhibitory effect of MA-ADM6A10, different doses (0–240 ng/mL) of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) (mean ± SD, n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4218762&req=5

Figure 2: Dose–response curve of TNF (A), ADM (B), and MA-ADM6A10 (C) in CBA. HT1080 cells were incubated with TNF. A, IL-6 was measured and expressed as log TNF concentration versus IL-6 response (mean ± SD, n = 2). B, Inhibition of TNF by ADM was determined using 7.5 ng/mL TNF (441.2 pmol/L) and ADM (0–240 ng/mL) (mean ± SD, n = 2). C, To determine the inhibitory effect of MA-ADM6A10, different doses (0–240 ng/mL) of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) (mean ± SD, n = 5).
Mentions: HT1080 cells, a human fibrosarcoma cell line that expresses IL-6, was used to develop a functional CBA. Basal IL-6 concentrations [2.2 ± 1.5 ng/mL (mean ± SD), n = 6] were set at 0%. IL-6 concentrations increased on addition of TNF (maximum response was set at 100%), revealing an EC50 of 3.78 ± 0.02 ng/mL (217.7 ± 1.2 pmol/L) and an EC80 of 11.1 ± 0.02 ng/mL (637.7 ± 1.3 pmol/L) (Fig. 2A). Based on the obtained TNF EC50/80, a concentration of 7.5 ng/mL TNF (441.2 pmol/L) was chosen to determine the reduced IL-6 production because of the inhibition of TNF by ADM. ADM was able to neutralize 7.5 ng/mL TNF with an IC50 of 8.07 ± 0.18 ng/mL (53.8 ± 1.2 pmol/L) and an IC80 of 21.2 ± 0.2 ng/mL (141.9 ± 1.4 pmol/L) (Fig. 2B). To determine if MA-ADM6A10 is able to inhibit the inhibitory effect of ADM on TNF, different doses of MA-ADM6A10 were added to sera supplemented with 7.5 ng/mL TNF (441.2 pmol/L) and 15 ng/mL ADM (100 pmol/L) to HT1080 cells using a similar setup as described above. On addition of 7.5 ng/mL TNF and 15 ng/mL ADM, an IC50 of 9.1 ± 0.20 ng/mL (61.0 ± 1.3 pmol/L) was determined for MA-ADM6A10 (Fig. 2C).

Bottom Line: When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated.Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity.The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

View Article: PubMed Central - PubMed

Affiliation: *Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven; †PharmAbs, The KU Leuven Antibody Center, University of Leuven; and ‡Department of Gastroenterology, University Hospitals Leuven, Belgium.

ABSTRACT

Background: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients.

Methods: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti-tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response.

Results: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity.

Conclusions: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

Show MeSH
Related in: MedlinePlus