Gertjan Wolbink

A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells

The acute phase response is initiated in response to infection or physical trauma and is characterized by an increase in the levels of some plasma proteins. Here, Erik Hack and colleagues suggest that the combined actions of two of these acute phase proteins, secretory phospholipase A2 and C-reactive protein, may serve to promote phagocytosis of injured cells and tissue debris, thereby enhancing inflammation and tissue damage.

Immunogenicity of anti-tumor necrosis factor antibodies—toward improved methods of anti-antibody measurement

To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab)2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

Development of the anti–citrullinated protein antibody repertoire prior to the onset of rheumatoid arthritis

OBJECTIVEnTo examine how anti-citrullinated protein antibody (ACPA) epitope spreading takes place prior to the onset of clinical rheumatoid arthritis (RA), and to analyze the pattern of autoantigen reactivity at the beginning of the immune response.nnnMETHODSnMultiple consecutive serum samples from 79 RA patients who had donated blood before disease onset were available for analysis. Fifty-three patients tested positive for ACPAs prior to the onset of clinical RA. For these patients, a median of 6 (interquartile range 4-9) sequential pre-RA serum samples obtained 1-2 years apart were tested. Reactivity to 5 distinct citrullinated peptides was measured by enzyme-linked immunosorbent assay. Two peptides were derived from fibrinogen, 1 from vimentin, 1 from α-enolase, and 1 from filaggrin.nnnRESULTSnIn 25 of 53 ACPA-positive patients, seroconversion from ACPA absence to ACPA presence was observed. In 72% of these patients, the immune response started with reactivity to 1 peptide, without preference for a particular peptide. The number of peptides recognized increased over time, without a dominant epitope-spreading pattern. ACPAs appeared in low levels several years prior to the diagnosis of RA. Antibody titers increased markedly ∼2-4 years before diagnosis.nnnCONCLUSIONnOur findings indicate that ACPA epitope spreading occurs over several years prior to the onset of clinical RA. The initial autoimmune response is mostly directed toward only 1 autoantigen, but this is not always the same antigen. The marked increase in ACPA titers a few years prior to the diagnosis of RA suggests a second stage in disease development, which might be due to a variety of factors.

The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region

Background In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising. Objectives To study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising. Methods Neutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department. Results In 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope). Conclusions Our study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.

Detection of soluble human granzyme K in vitro and in vivo

Granzymes are serine proteases released from the granules of cytotoxic lymphocytes during the induction of apoptosis. To evaluate the physiologic role of human granzymeu2004K (GzmK), we developed a sensitive ELISA which was shown to specifically detect human GzmK in its active as well as its inactive conformation. Analysis of the lysate of lymphokine‐activated killer (LAK) cells by gel filtration revealed that GzmK seems to be complexed to proteoglycans within these cells. While the expression of GzmA andu2004B by cytotoxic lymphocytes was strongly up‐regulated in response to several activating stimuli, GzmK expression did not increase significantly above constitutive levels, indicating differential regulation of these granzymes. However, low levels of GzmK were detected in plasma samples of healthy volunteers, which were in the same range as levels of GzmA andu2004B. Furthermore, circulating levels of GzmK as well as of GzmA andu2004B were significantly elevated in patients suffering from viral infections. We conclude that GzmK protein is produced by cytotoxic cells, and just as GzmA andu2004B it can be released in a soluble form into the extracellular space. Furthermore, our data suggest that despite a more restricted cellular expression pattern, GzmK seems to participate in immune responses against several viruses.

Methotrexate Normalizes Up-Regulated Folate Pathway Genes in Rheumatoid Arthritis

OBJECTIVEnThe folate antagonist methotrexate (MTX) is an anchor drug in the treatment of rheumatoid arthritis (RA), but its mechanism of action with regard to the impact on folate metabolism remains elusive. The aim of the present study was to investigate the cellular pharmacologic impact of MTX on peripheral blood cells, by comparing MTX-treated RA patients to MTX-naive RA patients and healthy controls.nnnMETHODSnGene expression microarray data were used to investigate the expression of 17 folate pathway genes by peripheral blood cells from a cohort of 25 RA patients treated with MTX, 10 MTX-naive RA patients starting treatment with MTX, and 15 healthy controls (test cohort). Multiplex real-time polymerase chain reaction was used to validate the results in an independent cohort, consisting of 151 RA patients treated with MTX, 28 MTX-naive RA patients starting treatment with MTX, and 24 healthy controls (validation cohort).nnnRESULTSnMultiple folate metabolism-related genes were consistently and significantly altered between the 3 groups in both cohorts. Concurrent with evidence of an immune-activation gene signature in MTX-naive RA patients, significant up-regulation of the folate-metabolizing enzymes γ-glutamyl hydrolase and dihydrofolate reductase, as well as the MTX/folate efflux transporters ABCC2 and ABCC5, was observed in the MTX-naive RA group compared to healthy controls. Strikingly, MTX treatment of RA patients normalized these differential gene expression levels to the levels observed in healthy controls.nnnCONCLUSIONnThese results suggest that under inflammatory conditions, basal folate metabolism in the peripheral blood cells of RA patients is markedly up-regulated, and treatment with MTX restores folate metabolism to normal levels. Identification of this novel gene signature provides insight into the mechanism of action of MTX, thus paving the way for development of novel folate metabolism-targeted therapies.

Antibodies to IgG4 hinge can be found in rheumatoid arthritis patients during all stages of disease and may exacerbate chronic antibody-mediated inflammation.

In rheumatoid arthritis (RA), autoantibodies such as anti–citrullinated protein antibodies (ACPAs) develop in response to neoepitopes that are formed under conditions of chronic inflammation. These autoantibodies may subsequently be fragmented by inflammation‐associated proteases, leading to the formation of F(ab′)2 fragments. The hinge of F(ab′)2 fragments can serve as a neoepitope, and so‐called antihinge antibodies (AHAs) can be found in RA patients, which might modulate the function of (fragmented) autoantibodies. We undertook this study to investigate the presence and specificities of AHAs in different stages of RA and to study their function.

Nanomolar to sub-picomolar affinity measurements of antibody–antigen interactions and protein multimerizations: Fluorescence-assisted high-performance liquid chromatography

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.

Neutralizing capacity of monoclonal and polyclonal anti-natalizumab antibodies: The immune response to antibody therapeutics preferentially targets the antigen-binding site

This study shows that the immune response towards natalizumab, an IgG4 antibody targeting alpha4-integrins, is directed towards the antigen binding site of the drug. This extends previous observations for TNF inhibitors, suggesting that antibodies towards therapeutic antibodies may be inherently neutralizing

Response to: 'The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region’ by Rinaudo-Gaujous et al

Assessment of clinically relevant immunogenicity is a much-debated topic. With respect to the neutralising capacity of antidrug antibodies (ADAs) to tumour necrosis factor (TNF) blockers, we recently demonstrated that the vast majority of antibodies in fact compete with TNF binding.1 Therefore, we are pleased to find this notion further supported by the letter of Rinaudo-Gaujous et al .2nnThe aim of our study was to quantify to which extent ADAs were neutralising. Using the TNF competition assay, we concluded that …