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Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies.

ABSTRACT: The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8-86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.

SUBMITTER: Kohlberger M

PROVIDER: S-EPMC7644011 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies.

Kohlberger Michael M Wildner Sabrina S Regl Christof C Huber Christian G CG Gadermaier Gabriele G

PloS one 20201105 11

The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, w ...[more]

PMID: 33151990

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Project description:ObjectiveThe TrialNet Study Group evaluated rituximab, a B-cell-depleting monoclonal antibody, for its effect in new-onset patients with type 1A diabetes. Rituximab decreased the loss of C-peptide over the first year of follow-up and markedly depleted B lymphocytes for 6 months after administration. This article analyzes the specific effect of rituximab on multiple islet autoantibodies.Research design and methodsA total of 87 patients between the ages of 8 and 40 years received either rituximab or a placebo infusion weekly for four doses close to the onset of diabetes. Autoantibodies to insulin (IAAs), GAD65 (GADAs), insulinoma-associated protein 2 (IA2As), and ZnT8 (ZnT8As) were measured with radioimmunoassays. The primary outcome for this autoantibody analysis was the mean level of autoantibodies during follow-up.ResultsRituximab markedly suppressed IAAs compared with the placebo injection but had a much smaller effect on GADAs, IA2As, and ZnT8As. A total of 40% (19 of 48) of rituximab-treated patients who were IAA positive became IAA negative versus 0 of 29 placebo-treated patients (P < 0.0001). In the subgroup (n = 6) treated within 50 days of diabetes, IAAs were markedly suppressed by rituximab in all patients for 1 year and for four patients as long as 3 years despite continuing insulin therapy. Independent of rituximab treatment, the mean level of IAAs at study entry was markedly lower (P = 0.035) for patients who maintained C-peptide levels during the first year of follow-up in both rituximab-treated and placebo groups.ConclusionsA single course of rituximab differentially suppresses IAAs, clearly blocking IAAs for >1 year in insulin-treated patients. For the patients receiving insulin for >2 weeks prior to rituximab administration, we cannot assess whether rituximab not only blocks the acquisition of insulin antibodies induced by insulin administration and/or also suppresses preformed insulin autoantibodies. Studies in prediabetic non-insulin-treated patients will likely be needed to evaluate the specific effects of rituximab on levels of IAAs.

Project description:Membranous Nephropathy (MN) is an autoimmune disease associated with antibodies against podocyte proteins: M-type phospholipase A2 receptor (PLA2R1) or thrombospondin type-1 domain-containing 7A (THSD7A) in 70 and 3% of patients, respectively. Antibody titer is correlated with disease activity: rising during active disease and decreasing before remission. Therefore, decreasing PLA2R1-Antibodies titer has become an important goal of therapy. Rituximab a chimeric monoclonal antibody induces remission in 60-80% of primary MN patients. All monoclonal antibodies such as rituximab can elicit antidrug antibodies, which may interfere with therapeutic response. We aim to analyze the relevance of anti-rituximab antibodies on the outcome of MN after a first course of rituximab. Forty-four MN patients were included and treated with two 1 g infusions of rituximab at 2-weeks interval. Anti-rituximab antibodies, CD19 count, and clinical response were analyzed. Then, we (i) analyzed the association of anti-rituximab antibodies at month-6 with response to treatment: remission, relapse and the need for another rituximab course; (ii) confirmed if anti-rituximab antibodies could neutralize rituximab B-cells depletion; and (iii) tested whether anti-rituximab antibodies could cross-inhibit new humanized anti-CD20 therapies. Anti-rituximab antibodies were detected in 10 patients (23%). Seventeen patients received a second rituximab course after a median time of 12 months (7-12), following nine cases of resistance and eight relapses. Anti-rituximab antibodies were significantly associated with faster B-cell reconstitution at month-6 (75 [57-89] vs. 2 [0-41] cells/μl, p = 0.006), higher proteinuria 12 months after rituximab infusion (1.7 [0.7; 5.8] vs. 0.6 [0.2; 3.4], p = 0.03) and before treatment modification (3.5 [1.6; 7.1] vs. 1.7 [0.2; 1.7] p = 0.0004). Remission rate 6 months after rituximab was not different according to anti-rituximab status (p > 0.99) but the rate of relapse was significantly higher for patients with anti-rituximab antibodies (p < 0.001). These patients required more frequently a second course of rituximab infusions (7/10 vs. 10/34, p = 0.03). Anti-rituximab antibodies neutralized rituximab activity in 8/10 patients and cross-reacted with other humanized monoclonal antibodies in only two patients. Three patients with anti-rituximab antibodies were successfully treated with ofatumumab. Anti-rituximab antibodies could neutralize rituximab B cells cytotoxicity and impact clinical outcome of MN patients. Humanized anti-CD20 seems to be a satisfying therapeutic alternative for patients with anti-rituximab antibodies and resistant or relapsing MN.

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Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies.

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Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies.

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