Functional characterization of protective antibodies in murine models of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease, characterized by chronic, erosive polyarthritis and by the presence of various autoantibodies in serum and synovial fluid. There are many different types of arthritogenic antibodies present in patients who suffer from RA. Among them, there are RFs (rheumatoid factors), ACPAs (anti-citrullinated protein antibodies) and anti-CarP (anti-carbamylated protein antibodies). Additionally, antibodies against collagen type II (COL2) are also found in some patients. However, we will see in this thesis that not all autoantibodies have arthritogenic properties. Some of them can have regulatory properties and even confer protection.

In paper I, recombinant ACPA antibodies derived from human RA patient B cell clones were generated using Expi293F cells and we efficacy characterized them in different mouse models of RA. We found that some of these ACPA clones were non-reactive when administered individually to mouse or in combination with other known anti-COL2 pathogenic antibodies. To our surprise, one clone (E4) not only was non-reactive, but also conferred protection in collagen antibody induced arthritis (CAIA) and glucose-6-phosphate isomerase arthritis (GPIA) mouse models. Furthermore, we observed that E4 forms immune complexes with alpha enolase (ENO1) in the synovium interacting with synovial macrophages promoting IL10 secretion through FCGR2B. This is of vital importance because it is the first time that an ACPA antibody isolated from RA patients shows outstanding protection in several murine models of rheumatoid arthritis.

In paper II, antibodies against the F4 specific epitope on COL2 were generated using a phage display library, and a group of candidate antibodies with high affinity for the F4 epitope were selected (together with other criteria). Some of these protective antibodies have been chosen and validated in the collagen antibody induced arthritis (CAIA) mouse model. Furthermore, we observed that R69-4 binds to a wide range of targets in the synovium (including C1q) and dampens the neutrophil signal by downregulating neutrophil FCGR3. We selected a good candidate antibody (R69-4) that could be used for prospective clinical trials in humans and possibly diminish the acute phase inflammation in patients suffering from RA.

In paper III, we discovered that Fcgr2b and Fcgr3 are important regulators of collagen antibody induced arthritis in mice. Using FCGR2B knock-out mice, we observed that it induces severe arthritis very rapidly, even outweighing the resistance from the deficiency of complement C5. On the other hand, when there is deficiency of FCGR3, mice are completely resistant to arthritis.

In paper IV, the protective antibody candidate described in paper II (R69-4) was tested in different mouse models of rheumatoid arthritis in the widely used strain DBA/1 with promising results. Interestingly, it protects against the T cell dependent and COL2 independent glucose-6-phosphate isomerase (GPIA) mouse model during early time points. Furthermore, we found that R69-4 antibody follows an alternative pathway for protection by decreasing activating FCGRs in macrophages and increasing the surface expression of FCGR2B, thus promoting a macrophage anti-inflammatory phenotype compared to isotype and pathogenic antibody controls.

In paper V, we studied the effect of Fc sialylation in anti-COL2 pathogenic antibodies. Fc sialylation has been associated with anti-inflammation and some studies indicate that it might have a potential as a treatment. We have engineered known arthritogenic anti-COL2 antibodies so that they have a high degree of sialylation in the Fc glycan fraction. These engineered antibodies were injected into mice, but they were only effective in attenuating the signs of arthritis in collagen-induced arthritis (CIA) and not in collagen antibody-induced arthritis (CAIA). Additionally, their efficacy depended on the antibody Fc isotype.