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Nanomethods to understand what makes an endogenous protein immunogenic

Periodic Reporting for period 4 - PredicTOOL (Nanomethods to understand what makes an endogenous protein immunogenic)

Reporting period: 2018-08-01 to 2020-09-30

Immunology has unraveled many aspects of the complex immune system. However, it is still unknown what mechanisms and structures trigger the immune system to recognize an endogenous protein (i.e. self-protein) as foreign, and to drive an autoimmune response. To close this gap in immunology, this project applied biophysical tools and molecular dynamic simulation studies to predict protein immunogenicity (antigenicity). In contrast to existing immunological methods (mostly observational-based), the advanced nanotechnologies (based on spectroscopic and imaging techniques with increased sensitivity and the use of human antibodies) employed and developed in this project allowed identification of common mechanisms and patterns which helped to clarify how and when an endogenous protein becomes immunogenic.

To verify whether the identified features are general to any autoimmune disease, we have addressed blood- and non-blood proteins, soluble- and transmembrane proteins, all of them involved in autoimmune diseases. We also investigated proteins modified via mutations or posttranslational modifications, which are more vulnerable to stress conditions (e.g. pH, temperature, ions, drugs). Such modifications may determine protein conformational changes which result in exposure of antibody-binding sites which were hidden under normal circumstances. This leads to binding of antibodies leading to autoimmunity.

With more than 80 autoimmune diseases known, autoimmunity is a very important aspect for the society. The project has an enormous impact on the society because it allows to identify triggers of diseases and to understand the mechanisms of autoimmunity. Moreover, the project helps towards development of new ways of prevention and treatment and development of safer biotherapeutics.

The project aimed: i) to identify a pattern (e.g. protein conformational changes) that is expressed in proteins to which autoantibodies bind; ii) to investigate whether mutations or post-translational changes of proteins induce or facilitate conformational changes which lead to expression of certain patterns under stress factors (e.g. pH, salt, temperature); iii) to assess and quantify the binding affinities of autoantibodies to endogenous proteins; iv) to study the interaction of native/wild type and conformationally changed proteins with immune cells.
We have shown by atomic force microscopy that blood protein beta2-glycoprotein I (β2GPI, involved in antiphospholipid syndrome) opens up after chemical acetylation of lysine residues (Buchholz et al, Phys. Chem. Chem. Phys., 2018). The conformational dynamics did not lead to significant protein structural changes, as shown by circular dichroism spectroscopy, although dynamic light scattering showed a change in the protein size upon acetylation, suggesting a conformational transition.
We also found that reduced β2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein. The binding of pathogenic antiphospholipid syndrome (APS) autoantibodies to reduced β2GPI (Buchholz, I. et al, submitted) was increased compared to antibody binding to untreated protein, as shown by enzyme-linked immunosorbent assay.

We found that environmental pH induces structural changes in both SPINK1 and its N34S mutant in a different manner (Buchholz et al., BBA – Prot. Proteomics, 2020). Although similar binding forces between monoclonal anti-SPINK1 antibodies and SPINK1 or its N34S mutant were revealed by force spectroscopy measurements, more specific interaction events were found for SPINK1 than for N34S mutant, indicating, as expected, a higher affinity of the antibodies to the wild type protein.

Our project allowed the establishment of a platform to investigate the activation of αIIbβ3 by stress factors using biomimetic membrane systems (i.e. αIIbβ3-containing liposomes). We found that Mn2+ induces activation of αIIbβ3 (change from the bent conformation to the extended conformation), as indicated by the binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. We also showed that Mn2+- and quinine treatments led to the activation of αIIbβ3 without significant changes in protein secondary structure as revealed by circular dichroism spectroscopy and molecular dynamics simulation studies (Janke et al, PLoS ONE, 2019).

We monitored platelet activation levels by force spectroscopy and found significantly reduced rupture forces between platelets when the platelet was immobilized on collagen and on fibronectin, but less reduced when it was immobilized on poly-L-lysine as compared to native platelets (Nguyen et al, Sci. Rep., 2016). However, to avoid the activation of platelets, we established an αIIbβ3 expression platform in HEK cells. This platform allowed to address questions which are difficult to answer using directly platelets (e.g. mechanical properties of cells with mutated αIIbβ3, impact of mutations on cytoskeleton reorganization induced by stress conditions).

By force spectroscopy studies identified three features for the binding of anti-PF4/heparin antibodies isolated from heparin-induced thrombocytopenia patients to PF4/heparin complexes. i) anti-PF4/heparin antibodies with binding forces to PF4/heparin complexes <60 pN do not activate platelets, even in the presence of polyanions, ii) antibodies with binding forces between 60-100 pN activate platelets in the presence of polyanions, and iii) antibodies with binding forces >100 pN bind to PF4 and activate platelets even in the absence of polyanions (Nguyen at al, Nat. Commun., 2017).

We also explored the interaction of nanoparticles (NPs) with blood proteins such as fibrinogen and with cell-mimicking systems (e.g. lipidic systems) containing αIIbβ3 and found that fibrinogen corona formed around maghemite nanoparticles functionalized with citrate-, dextran- and polyethylene glycol (PEG) coatings provides colloidal stability to the nanoparticles. It was demonstrated that bioconjugates of fibrinogen with dextran- and citrate-coated NPs interact with αIIbβ3 integrin-containing lipid bilayer, especially upon treatment with divalent ions, whereas PEG-coating reveals minor interaction (Martens et al, Nanoscale, 2020).
The project contributes to closing the gap in immunology by providing reliable tools to predict why and when the immune system attacks an endogenous protein (i.e. self-proteins).

By high sensitivity nanotechnological tools based on spectroscopic and imaging techniques, we are able to identify common patterns that characterize the interaction of autoantibodies with their antigens which may be applied to other proteins characteristic to autoimmune diseases.

The project has an enormous impact on the society by allowing to identify triggers of diseases and to understand the mechanisms of autoimmunity. There are two major areas of application with great socio-economic impact: i) in medicine – for elucidating the mechanisms of autoimmunity, for new ways for prevention and treatment; and ii) in pharmaceutical industry for development of safer biotherapeutic drugs.

In addition, the methods for immunogenicity prediction, which we describe, are for in vitro studies and therefore, beneficial because animal studies are not required.