Abstract
Abstract 3021
Poster Board II-997
The complement system is activated through three pathways: classical, lectin/mannose and alternative. Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases including atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). The complement system is also centrally involved in many hemolytic disorders, including paroxysmal nocturnal hemoglobinuria (PNH) where the CAP initiates complement activation resulting in intravascular hemolysis (IVH) after engagement of C5 and formation of the membrane attack complex (MAC). Systemic neutralization of C5 with the anti-C5 monoclonal antibody, eculizumab, abrogates IVH when plasma concentrations are maintained above the minimal efficacious concentration (Cmin = 35 μg/mL). However, because eculizumab does not inhibit CAP activity prior to C5, C3 fragments (C3frag) continue to covalently bind to and accumulate on PNH red blood cells (RBCs). Clearance by the reticuloendothelial system of PNH RBCs that are C3frag-coated is a putative cause of extravascular hemolysis (EVH) in eculizumab-treated patients.
In order to selectively modulate CAP activity, we developed TT30, a novel therapeutic 65kD fusion protein linking the first four short consensus repeat (SCR) domains of human complement receptor type 2 (CR2/CD21) with the first five SCR of human factor H (fH). CR2 SCR1-4 encompasses the antigen-fixed C3frag (iC3b, C3dg and C3d) binding domain. Factor H is the primary soluble phase, negative regulator of CAP activity functioning via the SCR1-5 domains. The unique mechanism of TT30 utilizes CR2 SCR1-4 to recognize and bind to C3frag on cells in which complement activation is occurring, thus delivering cell surface-targeted inhibition of CAP activity via fH SCR 1-5. TT30 both prevents CAP-dependent hemolysis of rabbit RBCs in human serum and blocks accumulation of C3frag on the RBC surface.
By design, TT30 should also be a potent inhibitor of the CAP, but with minimal inhibition of the complement classical (CCP) and mannose (lectin; CMP) pathways. To test this hypothesis, we utilized sensitive pharmacodynamic assays that allow in vitro or ex vivo assessment in an ELISA format of individual complement pathway activity present in human serum. In this format, TT30 is a potent and selective inhibitor of CAP activity in normal human complement-preserved serum, with EC50 and EC100 values of ∼0.1 and 1 μg/mL serum. As predicted by the use of fH in its construction, TT30 is a much less potent inhibitor of the CCP and CMP, with EC100 values of ∼65 μg/mL. By contrast, in these assays a monoclonal and polyclonal anti-C5 antibody each demonstrate non-selective inhibition of CAP and CCP activity at all effective concentrations.
TT30 activity is dependent upon CR2 binding to C3frag, as an anti-CR2 monoclonal antibody reverses the surface inhibition of CAP activity. This surface-targeting approach to delivering fH SCR1-5 results in a molecule with a 10-fold potency gain in CAP inhibition relative to added purified fH and an ∼30-fold potency gain relative to the total fH present in the serum used in the assay.
TT30 administered as a single IV injection at 20 mg/kg to rats, rabbits and monkeys results in Cmax values of ∼400, 500 and 300 μg/mL and concentration-dependent inhibition of CAP activity. At serum concentrations of TT30 that induced maximal (100%) inhibition of systemic CAP activity for up to 12 hours, CCP activity is modestly (∼35-60%) inhibited for only 2 hours. CAP activity returns to baseline levels in a predictable fashion. Pharmacokinetic analysis indicates no gender-related differences and the expected scaling of parameters across species. TT30 is pharmacologically active in monkeys, rabbits and mice. TT30 administered as a single subcutaneous injection at 20 mg/kg to monkeys results in Cmax values of ∼25 μg/mL, and EC100 values identical to those observed with IV administration, but with a 3-fold prolongation of the maximal pharmacodynamic effect.
The novel therapeutic TT30 has been shown in vitro and ex vivo to deliver cell surface-targeted control of CAP activation with minimal CCP and CMP inhibition and effective blockade of C3frag accumulation and MAC formation. As a result, TT30 has potential utility for the treatment of complement-mediated diseases such as PNH, AMD and aHUS, in which cell surface-targeted control of CAP activation may be clinically beneficial.
Disclosures
Holers: Taligen Therapeutics: Employment, Equity Ownership, Patents & Royalties, Research Funding. Mazsaroff:Taligen Therapeutics: Employment. Akana:Taligen Therapeutics: Employment. Smith:Taligen Therapeutics: Employment. Emlen:Taligen Therapeutics: Employment, Equity Ownership. Marians:Taligen Therapeutics: Employment. Horvath:Taligen Therapeutics: Employment.
New functional and structural insights from updated mutational databases for complement factor H, Factor I, membrane cofactor protein and C3