Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - PredicTOOL (Nanomethods to understand what makes an endogenous protein immunogenic)

Teaser

Although immunology has unraveled many aspects of the complex immune system, it is still unknown what drives an autoimmune response. PredicTOOL focuses by applying biophysical tools to identify the mechanisms and structures that trigger the immune system to recognize an...

Summary

Although immunology has unraveled many aspects of the complex immune system, it is still unknown what drives an autoimmune response. PredicTOOL focuses by applying biophysical tools to identify the mechanisms and structures that trigger the immune system to recognize an endogenous protein (i.e. self-protein) as foreign.
We are addressing proteins characteristic for autoimmune diseases. This is of great relevance for society because, on one hand, it helps to understand autoimmunity and to develop new ways of prevention and treatment and, on the other hand, it allows to develop safer biotherapeutics.

The objectives of PredicTOOL are:
• To identify if there is a pattern (e.g. conformational changes in the protein structure) that is expressed in proteins to which autoantibodies bind.

• To investigate whether certain degrees of mutations or post-translational changes in the proteins induce or facilitate conformational changes which lead to expression of certain patterns under stress factors (e.g. pH, salt, temperature).

• To assess and quantify the binding affinities of autoantibodies to endogenous proteins.

• To develop sensitive microstructured arrays to study the interaction of native/wild type and conformationally changed proteins with immune cells.

Work performed

We have investigated using biophysical tools those features that make endogenous proteins immunogenic (i.e. to induce an immune response). The following proteins have been investigated: platelet factor 4 (PF4)-involved in heparin-induced thrombocytopenia, beta2-glycoprotein I (β2GPI)- involved in antiphospholipid syndrome, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)-involved in thrombotic thrombocytopenia, alphaIIbbetaIII (αIIbβ3)-involved in immune thrombocytopenia and serine protease inhibitor Kazal-type 1 (SPINK1)-involved in chronic pancreatitis.

Using single molecule force spectroscopy and isothermal titration calorimetry, we have studied the interaction of anti-PF4/heparin antibodies with PF4/heparin complexes isolated from heparin-induced thrombocytopenia patients. We have shown that the biological activity of anti-PF4/heparin antibodies depends on their binding affinity, as measured by binding forces of highly purified antibodies. Most antibodies with binding forces to PF4/heparin complexes <60 pN do not activate platelets, even in the presence of polyanions; antibodies with binding forces between 60-100 pN activate platelets in the presence of polyanions; while antibodies with binding forces >100 pN bind to PF4 and activate platelets even in the absence of polyanions.

We have studied by circular dichroism spectroscopy, fluorescence spectroscopy and atomic force microscopy the impact of extreme pH on the conformational changes of β2GPI. The two forms (open and closed) of the proteins were further correlated with the binding of autoantibody isolated from antiphospholipid syndrome patients using an enzyme-immunoassay. The results indicate that the open form binds antibodies with higher affinity than the closed one. This indicate that under extreme pH conditions, the protein becomes immunogenic.

We have shown differences in the secondary structure of wild type and mutant SPINK1 proteins. In addition, they also present different isoelectric points which involves that the two proteins will interact differently under various conditions (calcium ions, pH, temperature). These characteristics suggest differences in thermal stability of the wild type and mutant proteins and subsequent different antibody binding.

Platelet activation levels by different materials (e.g. collagen, fibronectin, and poly-L-lysine) has been monitored. Using single force spectroscopy we measured the rupture forces among single platelets at different activation states. Our approach was successfully applied to native and modified platelets. Further, we have blocked using abciximab the αIIbβ3 receptor which is the major receptor for fibrinogen and plays the most important role in platelet-platelet interactions, platelet-plug stability in hemostasis and platelet aggregation. We found that the rupture forces between two platelets after blocking αIIbβ3 receptors, were significantly reduced, when the basal platelet was immobilized on collagen and on fibronectin, but less reduced when it was immobilized on poly-L-lysine as compared to native platelets.

In view of identifying point mutations in the αIIbβ3 protein from immune thrombocytopenia patients and insertion of such mutations in the wild type of αIIbβ3 to monitor whether these mutations induce conformational changes in the protein structure under stress conditions, a protocol to recombinantly express αIIbβ3 into cells has been established.

Final results

To answer when and why the defense system attacks endogenous proteins (i.e. self-proteins), we provide reliable tools to predict what induces such an immune response with a major impact in medicine by causing autoimmune diseases, and in biotechnology by inducing adverse reactions towards new drugs.

By high sensitivity nanotechnological tools based on spectroscopic and imaging techniques (e.g. Circular Dichroism Spectroscopy, Single Molecule Force Spectroscopy, Isothermal Titration Calorimetry, Fluorescence Microscopy), we are able to identify common patterns that characterize the interaction of autoantibodies with their antigens and further interaction with cells of the immune system. The proposed technologies will allow applying the rules of classic mechanics to identify the pattern of autoantibody-antigen interactions and will lead to better understand why an endogenous protein induces an immune response.

There are two major areas of application with great socio-economic impact. In medicine, it can lead to better understand autoimmunity and to develop new ways for prevention and treatment. In the development of biotherapeutics, it can help to finally produce safer biotherapeutic drugs. The methods of immunogenicity prediction which we describe are for in vitro studies and therefore beneficial because animal studies are not required.