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Purpose Polymorphisms in factor H (fH), an inhibitor of the alternative pathway (AP) of complement activation, are associated with increased risk for age-related macular degeneration (AMD). The authors investigated the therapeutic use of a novel recombinant form of fH, CR2-fH, which is targeted to sites of complement activation, in mouse choroidal neovascularization (CNV). CR2-fH consists of the N terminus of mouse fH, which contains the AP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. Methods Laser-induced CNV was analyzed in factor-B–deficient mice or in mice treated with CR2-fH, soluble CR2 (targeting domain), or PBS. CNV progression was analyzed by molecular, histologic, and electrophysiological readouts. Results Intravenously administered CR2-fH reduced CNV size, preserved retina function, and abrogated the injury-associated expression of C3 and VEGF mRNA. CR2 and PBS treatment was without effect. In therapeutically relevant paradigms involving delayed treatment after injury, CR2-fH was effective in reducing CNV and provided approximately 60% of the amount of protection of that seen in factor B–deficient mice that lacked functional AP. After intravenous injection, CR2-fH localized to sites of C3 deposition in RPE-choroid. Conclusions Specific inhibition of the AP reduces angiogenesis in mouse CNV. Of note, intravenous injection of C3d-targeted CR2-fH is protective even though endogenous fH is present in serum at a higher relative concentration, and serum fH contains native C3d and cell surface binding domains that target it to cell surfaces. The most common AMD-associated variant of fH resides within a native cell-binding region of fH (Tyr402His). These data may open new avenues for AMD treatment strategies.

Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1–5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm.

CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (␣ chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8␣ was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8␣ and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies.Complement activation leads ultimately to the generation and membrane insertion of a cytolytic protein assembly termed the membrane attack complex (MAC).2 The MAC is formed by the self-assembly of complement proteins C5b, C6, C7, C8, and from 1 to 18 molecules of C9. Host cells are protected from MAC-mediated lysis by CD59, an 18 -21-kDa glycophosphatidylinositol-linked membrane glycoprotein. The MAC appears to play an important role in causing tissue injury following inappropriate complement activation in various ischemic and inflammatory conditions (reviewed in Refs. 1-5), and soluble forms of CD59 have been shown to be therapeutic in rodent models of disease (6, 7). Furthermore, CD59 is sometimes overexpressed on tumor cells, and its expression has been linked to promoting tumor growth and the protection of tumor cells from mAb therapy (8 -10). It is therefore of interest to understand the molecular interaction between CD59 and its complement ligands for the goal of engineering effective soluble CD59-based therapeutics for treating inflammatory conditions, or for designing CD59 inhibitory molecules to enhance tumor cell susceptibility to complement.CD59 functions by binding to the ␣-subunit of C8 in the C5b-8 complex and preventing subsequent binding of C9, and/or by binding to C9 in the C5b-9 complex and preventing recruitment of additional C9 molecules (11-13). CD59 can only bind to C8␣ and C9 in the nascent complex after conformation rearrangements of the two proteins that occur during MAC assembly. The activity of CD59 is species selective, and previous characterization of chimeric human/rabbit C8␣-and C9-identified regions within the primary sequence of these proteins that interact with human CD59 (14, 15). There is considerable sequence homology between C8␣ and C9, and the identified CD59 binding sequence in the two proteins aligned to a region just C-terminal to the proposed membrane binding domain that encompassed residues 320 -415 in human C8␣, and 334 -415 in human C9. Pe...

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