The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

Abstract

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1–4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1–5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.

Knee synovial fluid complement C3‐β chain levels correlate with clinical symptoms of knee osteoarthritis

“…Similar improvements of the arthritic disease state were found in knockout mice of other complement factors, including factor B, factor D, mannose‐associated serine protease 1/3, and C5 . In experimental arthritis studies, systemic administration of soluble complement receptor 1 (sCR1), sCR2, and complement receptor of the immunoglobulin family limited the complement‐mediated tissue damage and inflammation in disease models . Moreover, systemic complement inhibition by C3aR and C5aR antagonists and an anti‐C5 Ab were found to be effective in animal studies by ameliorating arthritis.…”

C2 IgM Natural Antibody Enhances Inflammation and Its Use in the Recombinant Single Chain Antibody-Fused Complement Inhibitor C2-Crry to Target Therapeutics to Joints Attenuates Arthritis in Mice

“…All mice were sacrificed at day 35. CDA was examined according to our published methods (58). At day 35, all four limbs (two forepaws, two hind paws with knee joint and ankle) were surgically removed and fixed in 10% neutral buffered formalin (NBF) for histopathological analysis (58).…”

“…From CAIA mice, no limbs were processed for histopathology and C3 deposition. Histopathology with scores for inflammation, pannus, cartilage damage and bone damage was assessed by using Toluidine-blue (T-blue) according to our published criteria (5,10,15,27,58). C3 deposition was assessed by using a primary polyclonal goat anti-mouse C3 Ab (dilution 1:10,000; ICN Pharmaceuticals, Costa Mesa, CA, United States) and detected by goat anti-HRP polymer kit per manufacturer's instructions (Biocare Medical, Concord, CA, United States).…”

“…Serum was collected from each mouse with CIA treated with C2-Crry or controls by retro-orbital bleeding under anesthesia on day 0 (right eye), on day 21 (left eye), and on day 35 (right eye). Samples were used in triplicate to measure IgG1 and IgG2a anti-mouse CII antibodies by ELISA at a dilution of 1:2000 in 1 × PBS as previously describe (27,58). A standard pool of anti-CII antibodies was made by mixing sera from several mice with severe arthritis and used to create a standard curve.…”

Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

“…We and others have extensively probed the role of the complement protein network in RA pathogenesis to identify targets within the pathway that are suitable for therapeutic development (11,(13)(14)(15)(16)(17)(18). Inhibition of complement in virtually every RA mouse model tested leads to decreased disease progression (12).…”