A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an Anti-C5aR1 Antibody and C5 Small Interfering RNA

Abstract

Rheumatoid Arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated antibodies or siRNAs or small molecule inhibitors against C5 and C5aR1 (CD88) which have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. Here we test in CAIA a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that while C5aR2 or C5L2 (GPR77) plays no role in collagen antibody induced arthritis (CAIA), C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking either C5, C5aR1 or the combination decreased clinical disease activity (CDA) in mice with CAIA by 45%, 51% and 58%, respectively. Anti-C5 antibody (BB5.1) has only limited efficacy nonetheless significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1ab-protamine-C5siRNA conjugate. Here we show for the first time that while unconjugated antibody plus siRNAs reduce arthritis by 19%, our an anti-C5aR1ab - protamine - C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Mannan-Binding Lectin–Associated Serine Protease 1/3 Cleavage of Pro–Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference–Mediated Gene Silencing

“…CAIA was induced in C57BL/6 WT male mice using a cocktail of 5 mAbs to bovine CII (ArthritoMab™ antibody) suspended in sterile 1xPBS as previously described (5). Briefly, WT mice were injected i.p.…”

“…Briefly, WT mice were injected i.p. with 4 mg/mouse of ArthritoMab on day 0 and 50 µg/mouse of LPS from E. coli strain 0111B4 on day 3 to synchronize the development of arthritis (5). All mice started to develop arthritis at day 4 and were sacrificed at day 10.…”

“…All mice started to develop arthritis at day 4 and were sacrificed at day 10. Clinical disease activity (CDA) was examined daily until day 10 as per our previously published studies (5–7). CDA was examined by a blinded observer.…”

“…The complement system (CS) is also thought to play an important role in the development of RA, but the optimal target within the system is uncertain (4). We and others have developed complement-based therapeutics which have shown efficacy in mouse models of RA (5). One of the major goals of our current study was to design and develop a RNA interference-based therapy to inhibit mannan-binding lectin (MBL)-associated serine proteases (MASPs) in order to determine whether these proteins would play an important role in the pathogenesis of experimental RA and might be appropriate therapeutic targets for human RA (6).…”

“…Additionally, viral gene delivery methods such as lentiviral vectors and adeno-associated virus type 5 expressing short hairpin RNA (shRNA) for the silencing of B-cell activating factor or galectin-3 or TNF-α, respectively, have shown efficacy in mouse models of arthritis (30–32). Recently, we have shown that the conjugates of anti-C5aR1 siRNA-protamine-C5siRNA ameliorated CAIA in mice (5). …”