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Immunogenicity of Anti-TNF-α Biotherapies: II. Clinical Relevance of Methods Used for Anti-Drug Antibody Detection.

Bendtzen K - Front Immunol (2015)

Bottom Line: This paper discusses immunogenicity of genetically engineered immunoglobulins directed against tumor-necrosis factor-α (TNF).Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies.The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists.

View Article: PubMed Central - PubMed

Affiliation: Institute for Inflammation Research (IIR 7521), Rigshospitalet University Hospital , Copenhagen , Denmark.

ABSTRACT
Immunogenicity of biopharmaceuticals is complex and influenced by both structural and pharmacological factors, and by patient-related conditions such as disease being treated, previous and concomitant therapies, and individual immune responsiveness. Essential for tailored therapeutic strategies based on immunopharmacological evidence from individual patients (personalized medicine) is the use of assays for anti-drug antibodies (ADA) that are accurate and relevant in the clinical setting. This paper discusses immunogenicity of genetically engineered immunoglobulins directed against tumor-necrosis factor-α (TNF). Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies. The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists.

No MeSH data available.


Related in: MedlinePlus

Methods for ADA detection – and shortcomings. (A) bELISA for drug-binding ADA. bELISA depends on the bivalency of IgG ADA (and multivalency of IgA and IgM ADA) and therefore the ability of these immunoglobulins to “bridge” drug molecules preadsorbed to a plastic well with an added enzyme-labeled drug molecule (left panel). Note that IgG4 antibodies are usually bispecific because half molecules are exchanged after synthesis. They are therefore “invisible” in bELISA (right panel). (B) HMSA for drug-binding ADA. HMSA depends on association of fluorescence-labeled drug added to serum and subsequent chromatographic separation of ADA-bound and free tagged drug (left panel). Note that functionally inactive ADA, bound to drug in vivo, may be split during assay and reassociated with tagged drug before or during chromatography (right panel), thus reporting similar data as visualized in the left panel. (C) RGA for neutralizing ADA. RGA reports functional levels of all classes of drug-neutralizing ADA and, in addition, functional levels of all currently used anti-TNF drugs. When human recombinant TNF is added to the target cells, the cytokine initiates intracellular signaling through the surface TNF-receptor, type 1 (TNF-R1), thus activating the cytoplasmic nuclear factor (NF)-κB. The active components of this transcription factor are then transported into the nucleus where they bind to NF-κB response elements (NF-κB-REs) in the genome. This activates more than a hundred genes, including an inserted reporter-gene construct encoding the enzyme Firefly luciferase. After cell lysis and addition of substrate, luciferase-catalyzed light emission can be quantified. When TNF is preincubated with patient serum containing an anti-TNF drug and then added to the cells (step 1 mid), the drug, if functional, neutralizes the effect of TNF, and no intracellular signal is initiated. When TNF is preincubated with patient serum containing drug-neutralizing ADA and then added to the cells (step 1 right), the drug no longer interferes with TNF-mediated signaling, resulting in a luminescence signal.
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Figure 2: Methods for ADA detection – and shortcomings. (A) bELISA for drug-binding ADA. bELISA depends on the bivalency of IgG ADA (and multivalency of IgA and IgM ADA) and therefore the ability of these immunoglobulins to “bridge” drug molecules preadsorbed to a plastic well with an added enzyme-labeled drug molecule (left panel). Note that IgG4 antibodies are usually bispecific because half molecules are exchanged after synthesis. They are therefore “invisible” in bELISA (right panel). (B) HMSA for drug-binding ADA. HMSA depends on association of fluorescence-labeled drug added to serum and subsequent chromatographic separation of ADA-bound and free tagged drug (left panel). Note that functionally inactive ADA, bound to drug in vivo, may be split during assay and reassociated with tagged drug before or during chromatography (right panel), thus reporting similar data as visualized in the left panel. (C) RGA for neutralizing ADA. RGA reports functional levels of all classes of drug-neutralizing ADA and, in addition, functional levels of all currently used anti-TNF drugs. When human recombinant TNF is added to the target cells, the cytokine initiates intracellular signaling through the surface TNF-receptor, type 1 (TNF-R1), thus activating the cytoplasmic nuclear factor (NF)-κB. The active components of this transcription factor are then transported into the nucleus where they bind to NF-κB response elements (NF-κB-REs) in the genome. This activates more than a hundred genes, including an inserted reporter-gene construct encoding the enzyme Firefly luciferase. After cell lysis and addition of substrate, luciferase-catalyzed light emission can be quantified. When TNF is preincubated with patient serum containing an anti-TNF drug and then added to the cells (step 1 mid), the drug, if functional, neutralizes the effect of TNF, and no intracellular signal is initiated. When TNF is preincubated with patient serum containing drug-neutralizing ADA and then added to the cells (step 1 right), the drug no longer interferes with TNF-mediated signaling, resulting in a luminescence signal.

Mentions: Assessing ADA is especially difficult when testing binding of ADA directed against drugs that are also antibodies. In these cases, standard laboratory techniques may fail to provide accurate and clinically useful results (8). The most commonly used assays include various modifications of the enzyme-linked immunosorbent assay (ELISA). Unfortunately, however, rheumatoid factors, anti-allotypic antibodies, and heterophilic antibodies may interfere with readout in these assays. In addition, the frequently used bridging-type ELISA (bELISA) fail to detect IgG4 ADA, which may dominate after prolonged immunizations (Figure 2). Finally, all solid-phase techniques are sensitive to artifacts such as epitope shielding and neoepitope formation because protein drugs, including anti-TNF biopharmaceuticals, may aggregate on plastic surfaces (7).


Immunogenicity of Anti-TNF-α Biotherapies: II. Clinical Relevance of Methods Used for Anti-Drug Antibody Detection.

Bendtzen K - Front Immunol (2015)

Methods for ADA detection – and shortcomings. (A) bELISA for drug-binding ADA. bELISA depends on the bivalency of IgG ADA (and multivalency of IgA and IgM ADA) and therefore the ability of these immunoglobulins to “bridge” drug molecules preadsorbed to a plastic well with an added enzyme-labeled drug molecule (left panel). Note that IgG4 antibodies are usually bispecific because half molecules are exchanged after synthesis. They are therefore “invisible” in bELISA (right panel). (B) HMSA for drug-binding ADA. HMSA depends on association of fluorescence-labeled drug added to serum and subsequent chromatographic separation of ADA-bound and free tagged drug (left panel). Note that functionally inactive ADA, bound to drug in vivo, may be split during assay and reassociated with tagged drug before or during chromatography (right panel), thus reporting similar data as visualized in the left panel. (C) RGA for neutralizing ADA. RGA reports functional levels of all classes of drug-neutralizing ADA and, in addition, functional levels of all currently used anti-TNF drugs. When human recombinant TNF is added to the target cells, the cytokine initiates intracellular signaling through the surface TNF-receptor, type 1 (TNF-R1), thus activating the cytoplasmic nuclear factor (NF)-κB. The active components of this transcription factor are then transported into the nucleus where they bind to NF-κB response elements (NF-κB-REs) in the genome. This activates more than a hundred genes, including an inserted reporter-gene construct encoding the enzyme Firefly luciferase. After cell lysis and addition of substrate, luciferase-catalyzed light emission can be quantified. When TNF is preincubated with patient serum containing an anti-TNF drug and then added to the cells (step 1 mid), the drug, if functional, neutralizes the effect of TNF, and no intracellular signal is initiated. When TNF is preincubated with patient serum containing drug-neutralizing ADA and then added to the cells (step 1 right), the drug no longer interferes with TNF-mediated signaling, resulting in a luminescence signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Methods for ADA detection – and shortcomings. (A) bELISA for drug-binding ADA. bELISA depends on the bivalency of IgG ADA (and multivalency of IgA and IgM ADA) and therefore the ability of these immunoglobulins to “bridge” drug molecules preadsorbed to a plastic well with an added enzyme-labeled drug molecule (left panel). Note that IgG4 antibodies are usually bispecific because half molecules are exchanged after synthesis. They are therefore “invisible” in bELISA (right panel). (B) HMSA for drug-binding ADA. HMSA depends on association of fluorescence-labeled drug added to serum and subsequent chromatographic separation of ADA-bound and free tagged drug (left panel). Note that functionally inactive ADA, bound to drug in vivo, may be split during assay and reassociated with tagged drug before or during chromatography (right panel), thus reporting similar data as visualized in the left panel. (C) RGA for neutralizing ADA. RGA reports functional levels of all classes of drug-neutralizing ADA and, in addition, functional levels of all currently used anti-TNF drugs. When human recombinant TNF is added to the target cells, the cytokine initiates intracellular signaling through the surface TNF-receptor, type 1 (TNF-R1), thus activating the cytoplasmic nuclear factor (NF)-κB. The active components of this transcription factor are then transported into the nucleus where they bind to NF-κB response elements (NF-κB-REs) in the genome. This activates more than a hundred genes, including an inserted reporter-gene construct encoding the enzyme Firefly luciferase. After cell lysis and addition of substrate, luciferase-catalyzed light emission can be quantified. When TNF is preincubated with patient serum containing an anti-TNF drug and then added to the cells (step 1 mid), the drug, if functional, neutralizes the effect of TNF, and no intracellular signal is initiated. When TNF is preincubated with patient serum containing drug-neutralizing ADA and then added to the cells (step 1 right), the drug no longer interferes with TNF-mediated signaling, resulting in a luminescence signal.
Mentions: Assessing ADA is especially difficult when testing binding of ADA directed against drugs that are also antibodies. In these cases, standard laboratory techniques may fail to provide accurate and clinically useful results (8). The most commonly used assays include various modifications of the enzyme-linked immunosorbent assay (ELISA). Unfortunately, however, rheumatoid factors, anti-allotypic antibodies, and heterophilic antibodies may interfere with readout in these assays. In addition, the frequently used bridging-type ELISA (bELISA) fail to detect IgG4 ADA, which may dominate after prolonged immunizations (Figure 2). Finally, all solid-phase techniques are sensitive to artifacts such as epitope shielding and neoepitope formation because protein drugs, including anti-TNF biopharmaceuticals, may aggregate on plastic surfaces (7).

Bottom Line: This paper discusses immunogenicity of genetically engineered immunoglobulins directed against tumor-necrosis factor-α (TNF).Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies.The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists.

View Article: PubMed Central - PubMed

Affiliation: Institute for Inflammation Research (IIR 7521), Rigshospitalet University Hospital , Copenhagen , Denmark.

ABSTRACT
Immunogenicity of biopharmaceuticals is complex and influenced by both structural and pharmacological factors, and by patient-related conditions such as disease being treated, previous and concomitant therapies, and individual immune responsiveness. Essential for tailored therapeutic strategies based on immunopharmacological evidence from individual patients (personalized medicine) is the use of assays for anti-drug antibodies (ADA) that are accurate and relevant in the clinical setting. This paper discusses immunogenicity of genetically engineered immunoglobulins directed against tumor-necrosis factor-α (TNF). Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies. The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists.

No MeSH data available.


Related in: MedlinePlus