Rheumatoid factor autoantibody repertoire profiling reveals distinct binding epitopes in health and autoimmunity.

BACKGROUND: Rheumatoid factors (RF) are one of the hallmark autoantibodies characteristic of rheumatoid arthritis (RA), and are frequently observed in other diseases and in healthy individuals. RFs comprise multiple subtypes with different specificities towards the constant region of human IgG. Studies indicate that these patterns differ between naturally occurring RFs and RFs associated with disease. However, individual specificities characteristic of either have not been clearly defined. METHODS: In this study, we developed an extended set of engineered IgG-fragment crystallisable (Fc) targets with preferential RF binding to specific (conformational) epitopes, which was subsequently used for profiling of RF binding patterns in a compiled exploration cohort, consisting of sera from healthy donors with measurable RF and patients with RA, primary Sjögren's syndrome (pSS) and seropositive arthralgia. RESULTS: We identified an epitope that is strongly associated with RA, which was targeted by both IgM-RF and IgA-RF. We also identified an epitope that is preferentially targeted by healthy donor (IgM) RFs. IgM-RFs derived from healthy donors and patients with RA and pSS all target distinct regions on the IgG-Fc, whereas overall, the IgA-RF repertoire is largely restricted to pathology-associated specificities. Using monoclonal RFs with different specificities, we furthermore demonstrate that the capacity to activate complement or even inhibit IgG-mediated complement activation varies according to the epitopes to which RFs bind. CONCLUSIONS: Our results demonstrate both the need and feasibility to redefine 'RF' into pathological and physiological autoantibody subtypes.

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants.

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

Belimumab concentration measurements using a homologous anti-idiotype immunoassay.

Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.

Early Postnatal Cardiac Stress Does Not Influence Ventricular Cardiomyocyte Cell-Cycle Withdrawal.

Congenital heart disease (CHD) is the most common birth defect. After birth, patients with CHD may suffer from cardiac stress resulting from abnormal loading conditions. However, it is not known how this cardiac burden influences postnatal development and adaptation of the ventricles. To study the transcriptional and cell-cycle response of neonatal cardiomyocytes to cardiac stress, we used a genetic mouse model that develops left ventricular volume overload within 2 weeks after birth. The increased volume load caused upregulation of the cardiac stress marker Nppa in the left ventricle and interventricular septum as early as 12 days after birth. Transcriptome analysis revealed that cardiac stress induced the expression of cell-cycle genes. This did not influence postnatal cell-cycle withdrawal of cardiomyocytes and other cell types in the ventricles as measured by Ki-67 immunostaining.

Biased anti-idiotype response in rabbits leads to high-affinity monoclonal antibodies to biologics.

Antibody formation to human(ized) therapeutic antibodies in humans is highly skewed toward anti-idiotype responses, probably because the idiotype is the only 'foreign' part of the antibody molecule. Here, we analyzed antibody responses to F(ab')2 fragments of a panel of 17 human(ized) therapeutic antibodies in rabbits. Homology between the rabbit germline and the human(ized) antibodies is moderate not only for the variable domains (both the complementarity-determining regions and the framework regions), but also for the constant domains (66% or less). Nevertheless, we observed a highly skewed anti-idiotype response in all cases, with up to >90% of the antibodies directed toward the idiotype. These results indicate that the idiotype may be inherently immunodominant. We used these biased responses to raise monoclonal rabbit anti-idiotype antibodies against secukinumab, ustekinumab, reslizumab, mepolizumab, palivizumab, and dupilumab and demonstrate the potential to develop sensitive pharmacokinetic assays with these antibodies.