Multimolekulare Membran-Mikrodomänen in antigenpräsentierenden Zellen

Die vorliegende Arbeit hatte zum Ziel, Einsichten in die Funktion der als CDw78 bezeichneten Membran-Mikrodomänen zu gewinnen. Bisher war lediglich bekannt, dass die CDw78 Mikrodomänen aus HLA und Tetraspan Molekülen zusammengesetzt sind und auf der Zelloberfläche von humanen antigenpräsentierenden Zellen exprimiert werden. Ungeklärt war hingegen unter anderem die Verwandtschaft von CDw78 Mikrodomänen zu Detergenz-resistenten Membran-Mikrodomänen (DRMs). Untersuchungen an DRMs und CDw78 Mikrodomänen mittels Saccharose-Gradienten sowie die Analyse der HLAgebundenen Peptide zeigten, dass die beiden Formen von Mikrodomänen zwar verschieden voneinander sind, jedoch durch schwache Interaktionen auch miteinander in Verbindung stehen können. Interessanterweise ergab die Analyse der HLA-Peptide, dass CDw78 Mikrodomänen im Gegensatz zur Gesamtheit der HLA-DR-präsentierten Peptide und im Gegensatz zu den DRMs nur ein sehr selektives Repertoire an Peptiden präsentieren. Dies konnte nicht nur für B Zellinien und Dendritische Zellen (DCs) gezeigt werden, sondern auch für PBMCs. Weiterführende Experimente zeigten, dass allein die Aminosäuresequenz über die Lokalisation eines Peptids in CDw78 Mikrodomänen bestimmen kann. In diesem Zusammenhang erwies sich der Austausch einer einzelnen Aminosäure als hinreichend für die Anreicherung eines definierten Peptids in CDw78 Mikrodomänen. Eine mögliche Erklärung für diesen Befund liefert die Beobachtung, dass HLA-DM, welches für seine Peptid-Editor Funktion bekannt ist, mit diesen Mikrodomänen assoziiert vorliegt. Der genaue Mechanismus bleibt jedoch noch zu ergründen. Bezüglich der Funktion der CDw78 Mikrodomänen für die Antigenpräsentation konnte gezeigt werden, dass CDw78 Mikrodomänen im Zentrum des supramolecular activation cluster (cSMAC) der immunologischen Synapse, die sich im Laufe einer spezifischen Interaktion von DCs und T Zellen ausbildet, stark angereichert werden. Ferner resultierte die Zerstörung der CDw78 Mikrodomänen auf B Zellen in der Unfähigkeit, T Zellen zu aktivieren, welche spezifisch für ein Antigen waren, welches in CDw78 angereichert wurde. Ebenso wenig waren T / B Hybridzellen, welche zwar mit HLA Molekülen transfiziert worden waren, jedoch keine CDw78 Mikrodomänen exprimierten, in der Lage diese T Zellen zu stimulieren, obwohl sie das entsprechende Peptid präsentierten. Zusammengenommen ergibt sich demnach, dass CDw78 Mikrodomänen zumindest für bestimmte Antigene eine essentielle Rolle während der Aktivierung von T Zellen einnehmen. Da sich die Hinweise häufen, dass Interferon-a (IFNa) während der Differenzierung und der Maturierung von DCs in vivo eine Rolle spielt, wurde dessen Einfluss auf die Expression von CDw78 analysiert. Es konnten jedoch bezüglich CDw78 keine signifikanten Unterschiede zwischen Differenzierung mit GM-CSF und IL-4 oder GM-CSF und IFNa festgestellt werden. Ebenso wenig zeigten sich Unterschiede für die Maturierung durch IFNa im Vergleich zu anderen Maturierungs-Stimuli, wie TNFa oder LPS

Secukinumab immunogenicity over 52 weeks in patients with psoriatic arthritis and ankylosing spondylitis

Objective. Secukinumab, a fully human antiinterleukin 17A monoclonal antibody, is efficacious for the treatment of psoriatic arthritis (PsA) and ankylosing spondylitis (AS). This study examined the immunogenicity of secukinumab in patients with PsA and AS exposed to secukinumab for up to 52 weeks. Methods. Antibody bridging assays were used to assess the immunogenicity of secukinumab in patients with PsA [FUTURE 1–3 studies, and AS (MEASURE 1–4 studies)]. Evaluations were at baseline and at weeks 16 (AS only), 24, and 52. Treatment-emergent antidrug antibodies (TE-ADA) were defined as a positive ADA signal in ≥ 1 posttreatment sample in patients negative at baseline. Positive samples were analyzed for drug-neutralizing potential, and effect of TE-ADA on secukinumab pharmacokinetics, immunogenicity-related adverse events (AE), and efficacy through Week 52 were assessed. Results. Of 1414 treated PsA and 1164 treated AS patients with samples available for immunogenicity evaluation, 5 (0.35%) and 8 (0.69%), respectively, developed TE-ADA. All but 1 PsA patient were biologic-naive; two of the 5 PsA and one of the 8 AS patients received concomitant methotrexate, and two of the 8 AS patients received concomitant sulfasalazine. Associations between TE-ADA and secukinumab dose, frequency, or administration mode were not observed. Other than one PsA patient, all TE-ADA were non-neutralizing. No TE-ADA were associated with any AE. All TE-ADA were associated with normal secukinumab pharmacokinetics and none were associated with loss of secukinumab efficacy. Conclusion. Secukinumab treatment was associated with a low (< 1%) incidence of immunogenicity in patients with PsA or AS. (clinicaltrials.gov: NCT01392326; NCT01752634; NCT01989468; NCT01358175; NCT01649375; NCT02008916; NCT02159053)

Low Percentage of Signal Regulatory Protein α/β+ Memory B Cells in Blood Predicts Development of Anti-drug Antibodies (ADA) in Adalimumab-Treated Rheumatoid Arthritis Patients

An important goal for personalized treatment is predicting response to a particular therapeutic. A drawback of biological treatment is immunogenicity and the development of antibodies directed against the drug [anti-drug antibodies (ADA)], which are associated with a poorer clinical outcome. Here we set out to identify a predictive biomarker that discriminates rheumatoid arthritis (RA) patients who are more likely to develop ADA in response to adalimumab, a human monoclonal antibody against tumor necrosis factor (TNF)α. By taking advantage of an immune-phenotyping platform, LEGENDScreen™, we measured the expression of 332 cell surface markers on B and T cells in a cross-sectional adalimumab-treated RA patient cohort with a defined ADA response. The analysis revealed seven differentially expressed markers (DEMs) between the ADA+ and ADA− patients. Validation of the DEMs in an independent prospective European cohort of adalimumab treated RA patients, revealed a significant and consistent reduced frequency of signal regulatory protein (SIRP)α/β-expressing memory B cells in ADA+ vs. ADA− RA patients. We also assessed the predictive value of SIRPα/β expression in a longitudinal RA cohort prior to the initiation of adalimumab treatment. We show that a frequency of < 9.4% of SIRPα/β-expressing memory B cells predicts patients that will develop ADA, and consequentially fail to respond to treatment, with a receiver operating characteristic (ROC) area under the curve (AUC) score of 0.92. Thus, measuring the frequency of SIRPα/β-expressing memory B cells in patients prior to adalimumab treatment may be clinically useful to identify a subgroup of active RA subjects who are going to develop an ADA response and not gain substantial clinical benefit from this treatment

Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?

Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an in vivo ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response

Tregitopes and impaired antigen presentation: Drivers of the immunomodulatory effects of IVIg?

Although intravenous immunoglobulin (IVIg) is commonly used in the clinic to treat various autoimmune and severe inflammatory diseases, the mode of action is not fully elucidated. This work investigates two proposed mechanisms: (1) the potential role of regulatory T-cell epitopes (Tregitopes) from the constant domain of IgG in the immunosuppressive function of IVIg, and (2) a potential impact of IVIg on the ability of antigen presenting cells (APCs) to present peptides. Investigation of the HLA class II peptide repertoire from IVIg-loaded dendritic cells (DCs) via MHC-associated peptide proteomics (MAPPs) revealed that numerous IgG-derived peptides were strongly presented all along the antibody sequence. Surprisingly, Tregitopes 167 and 289 did not show efficient natural presentation although they both bound to HLA class II when directly loaded as ‘naked’ peptides on human DCs. In addition, both Tregitopes could not reproduce the inhibitory effect of IVIg in a human in vitro T-cell proliferation assay as well as in vivo in mice. MAPPs data demonstrate that presentation of peptides from several antigens remained unchanged even when competed with high doses of IVIg, in both human and mouse. These data suggest that the effects mediated by IVIg are not caused by Tregitopes nor by impaired antigen presentation

A cell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by tisagenlecleucel

The use of T-cells expressing Chimeric Antigen Receptors (CARs) offers new opportunities for cancer treatment, as well as new challenges for the bioanalysis of this new class of drugs. The analysis of humoral immunogenicity (anti-drug antibodies) against CARs could be performed with a bridging ELISA, using labeled CAR fragments. However, outside of its native cell membrane environment and without potential interaction partners on the cell surface, a labeled or coated recombinant CAR fragment may structurally differ from the membrane-bound CAR expressed on CAR-T cells. Consequently, immunogenicity to CARs may be missed due to the artificial nature of a ligand binding assay setup. T-cell lines expressing the CAR offer the opportunity to measure anti-drug antibodies to the CAR in its natural cell environment, as an alternative to ligand-binding assays. Here we describe a novel, flow cytometry-based humoral immunogenicity assay for tisagenlecleucel (CTL019, Kymriah®) using a human T-cell line that expresses murine CAR19. The assay described here was fully validated according to health authority guidelines for the development and validation of immunogenicity assays and has a sensitivity of 100 ng/mL. A good correlation of screening assay signal strengths to titer assay results was observed while exploring options to increase titration assay throughput. Pre-existing antibodies against the cell line used in the assay as well as against the CAR itself complicate the assay and data interpretation

STABILITY TESTING OF ANTI-VACCINE ANTIBODIES IN HUMAN SERUM

Background: To generate exhaustive data on the stability of human anti-vaccine or anti-drug antibodies, we designed a GLP study using anti-vaccine antibodies (AVA) samples collected from over 100 different subjects. Samples were selected from various time points after vaccination to cover the heterogeneity of the polyclonal human immune response. Samples were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze-thaw (FT). The general acceptance criteria for incurred sample reanalysis (ISR) for ligand binding assays (LBA) were applied, as well as some alternative stricter acceptance criteria promoted by various white papers. Results: Anti-vaccine antibodies are stable in undiluted serum for at least three cycles of freeze-thaw and at least 3.5 years storage at a nominal temperature of -80°C. Conclusion: The nature of the humoral immune response allows extending the conclusion of this study to all anti-vaccine and anti-drug antibodies, making it unnecessary to test long-term stability for each new biological therapeutics or vaccine program

STABILITY OF ANTI-IMMUNOTHERAPEUTIC ANTIBODIES IN FROZEN HUMAN SERUM SAMPLES

Background: To generate exhaustive data on the stability of human anti-vaccine or anti-drug antibodies, we designed a GLP study using anti-vaccine antibodies (AVA) samples collected from over 100 different subjects. Samples were selected from various time points after vaccination to cover the heterogeneity of the polyclonal human immune response. Samples were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze-thaw (FT). The general acceptance criteria for incurred sample reanalysis (ISR) for ligand binding assays (LBA) were applied, as well as some alternative stricter acceptance criteria promoted by various white papers. Results: Anti-vaccine antibodies are stable in undiluted serum for at least three cycles of freeze-thaw and 3.5 years storage at a nominal temperature of -80°C. Conclusion: The nature of the humoral immune response allows extending the conclusion of this study to all anti-vaccine and anti-drug antibodies, making it unnecessary to test long-term stability for each new biological therapeutics or vaccine program

Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?

<div><p>Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an <i>in vivo</i> ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.</p></div