WO2014110438A1 - Compounds and methods related to the c5d domain of complement component c5 - Google Patents

Abstract

The present disclosure relates to, among other things, compounds that bind to the C5d domain of complement component C5 (e.g., human complement component C5) and compounds that comprises all or part of human C5d. In some embodiments, the compounds described herein inhibit the interaction between C5b and C6 and, thus, inhibit formation of the terminal complement complex (e.g., the assembly and/or activity of the C5b-9 TCC). Also featured are methods for producing and/or screening for such compounds as methods therapeutic applications in which the compounds can be used.

Description

COMPOUNDS AND METHODS RELATED TO THE C5d DOMAIN OF

COMPLEMENT COMPONENT C5

Cross-Reference to Related Applications

This application claims priority to and the benefit of U.S. provisional patent application serial number 61/751,555, filed on January 11, 2013, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The field of the invention is medicine, immunology, molecular biology, and protein chemistry.

Background

The complement system acts in conjunction with other immunological systems of the body to defend against intrusion of cellular and viral pathogens. There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane co factors. The plasma proteins make up about 10% of the globulins in vertebrate serum. Complement components achieve their immune defensive functions by interacting in a series of intricate but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic,

immunoregulatory, and lytic functions. A concise summary of the biologic activities associated with complement activation is provided, for example, in The Merck Manual, 16^th Edition.

The complement cascade can progress via the classical pathway (CP), the lectin pathway, or the alternative pathway (AP). The lectin pathway is typically initiated with binding of mannose-binding lectin (MBL) to high mannose substrates. The AP can be antibody independent, and can be initiated by certain molecules on pathogen surfaces. The CP is typically initiated by antibody recognition of, and binding to, an antigenic site on a target cell. These pathways converge at the C3 convertase - the point where complement component C3 is cleaved by an active protease to yield C3a and C3b.

The AP C3 convertase is initiated by the spontaneous hydrolysis of complement component C3, which is abundant in the plasma fraction of blood. This process, also known as "tickover," occurs through the spontaneous cleavage of a thioester bond in C3 to form C3i or C3(H₂0). Tickover is facilitated by the presence of surfaces that support the binding of activated C3 and/or have neutral or positive charge characteristics (e.g., bacterial cell surfaces). This formation of C3(H₂0) allows for the binding of plasma protein Factor B, which in turn allows Factor D to cleave Factor B into Ba and Bb. The Bb fragment remains bound to C3 to form a complex containing C3(H₂0)Bb - the "fluid-phase" or "initiation" C3 convertase. Although only produced in small amounts, the fluid-phase C3 convertase can cleave multiple C3 proteins into C3a and C3b and results in the generation of C3b and its subsequent covalent binding to a surface (e.g., a bacterial surface). Factor B bound to the surface-bound C3b is cleaved by Factor D to thus form the surface-bound AP C3 convertase complex containing C3b,Bb. (See, e.g., Muller-Eberhard (1988) Ann Rev Biochem 57:321- 347.)

The AP C5 convertase - (C3b)₂,Bb - is formed upon addition of a second C3b monomer to the AP C3 convertase. (See, e.g., Medicus et al. (1976) J Exp Med 144: 1076- 1093 and Fearon et al. (1975) J Exp Med 142:856-863.) The role of the second C3b molecule is to bind C5 and present it for cleavage by Bb. (See, e.g., Isenman et al. (1980) J Immunol 124:326-331.) The AP C3 and C5 convertases are stabilized by the addition of the trimeric protein properdin as described in, e.g., Medicus et al. (1976), supra. However, properdin binding is not required to form a functioning alternative pathway C3 or C5 convertase. (See, e.g., Schreiber et al. (1978) Proc Natl Acad Sci USA 75: 3948-3952 and Sissons et al. (1980) Proc Natl Acad Sci USA 77: 559-562).

The CP C3 convertase is formed upon interaction of complement component CI, which is a complex of CI q, CI r, and Cls, with an antibody that is bound to a target antigen (e.g., a microbial antigen). The binding of the Clq portion of CI to the antibody-antigen complex causes a conformational change in CI that activates Clr. Active Clr then cleaves the CI -associated Cls to thereby generate an active serine protease. Active Cls cleaves complement component C4 into C4b and C4a. Like C3b, the newly generated C4b fragment contains a highly reactive thioester that readily forms amide or ester bonds with suitable molecules on a target surface (e.g., a microbial cell surface). Cls also cleaves complement component C2 into C2b and C2a. The complex formed by C4b and C2a is the CP C3 convertase, which is capable of processing C3 into C3a and C3b. The CP C5 convertase - C4b,C2a,C3b - is formed upon addition of a C3b monomer to the CP C3 convertase. (See, e.g., Muller-Eberhard (1988), supra and Cooper et al. (1970) J Exp Med 132:775-793.)

In addition to its role in C3 and C5 convertases, C3b also functions as an opsonin through its interaction with complement receptors present on the surfaces of antigen- presenting cells such as macrophages and dendritic cells. The opsonic function of C3b is generally considered to be one of the most important anti-infective functions of the complement system. Patients with genetic lesions that block C3b function are prone to infection by a broad variety of pathogenic organisms, while patients with lesions later in the complement cascade sequence, i.e., patients with lesions that block C5 functions, are found to be more prone only to Neisseria infection, and then only somewhat more prone.

The AP and CP C5 convertases cleave C5 into C5a and C5b. Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotactic factor, and C5b, which allows for the formation of the lytic terminal complement complex, C5b-9. C5b combines with C6, C7, and C8 to form the C5b-8 complex at the surface of the target cell. Upon binding of several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal complement complex - TCC) is formed. When sufficient numbers of MACs insert into target cell membranes the openings they create (MAC pores) mediate rapid osmotic lysis of the target cells.

While a properly functioning complement system provides a robust defense against infecting microbes, inappropriate regulation or activation of the complement pathways has been implicated in the pathogenesis of a variety of conditions including, e.g., rheumatoid arthritis (RA); lupus nephritis; asthma; ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); paroxysmal nocturnal

hemoglobinuria (PNH); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome;

thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; multiple sclerosis (MS); traumatic brain injury; and injury resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis. (See, e.g., Holers et al. (2008) Immunological Reviews 223:300-316.) The down-regulation of complement activation has been demonstrated to be effective in treating several disease indications in a variety of animal models. See, e.g., Rother et al. (2007) Nature Biotechnology 25(11): 1256-1264; Wang et al. (1996) Proc Natl Acad Sci USA 93:8563-8568; Wang et al. (1995) Proc Natl Acad Sci USA 92:8955-8959; Rinder et al. ( 1995) J Clin Invest 96 : 1564- 1572; Kroshus et al. (1995) Transplantation 60 : 1194- 1202; Homeister et al. (1993) J Immunol 150: 1055-1064; Weisman et al. (1990) Science 249: 146- 151; Amsterdam et al. (1995) Am J Physiol 268:H448-H457; and Rabinovici et al. (1992) J Immunol 149: 1744 1750. Summary

The present disclosure relates to, among other things, compounds that bind to the C5d domain of complement component C5 (e.g., human complement component C5). In some embodiments, the C5d-binding compounds described herein inhibit the interaction between C5b and C6 and, thus, inhibit formation of the terminal complement complex (e.g., the assembly and/or activity of the C5b-9 TCC). In some embodiments, the compound is an anti- C5d antibody or C5d-binding fragment thereof. In some embodiments, the compound is a small molecule or a nucleic acid such as, e.g., an aptamer that binds to C5d and, optionally, inhibits the interaction between C5b and C6. Also featured are methods for producing and/or screening for such compounds.

Through study of the structure of C5 and related structures for C3b, the inventors have determined that, upon cleavage by the C5 convertase (discussed above), C5 undergoes a major conformational rearrangement. One of the domains of C5 that translocates upon cleavage is "C5d", the domain of C5 equivalent to C3d of C3. C5d translocates as a rigid body - that is, the C5d domain remains structurally unchanged during its translocation. In the process of translocation, exposed are certain surfaces of C5d that otherwise remain occluded in C5. While the disclosure is in no way limited by any particular theory or mechanism of action, these newly exposed surfaces (e.g., comprising one or more

neoepitopes) constitute an interface that is involved in forming C5bC6, the nucleus of the terminal complement complex. The inventors believe that interfering at the stage of C5bC6 formation, by way of a compound that binds to one or more neoepitopes of C5d and inhibits the C5b-C6 interaction, has a potential to inhibit C5b9 formation and therefore MAC activity.

However, one of skill in the art would recognize that the generation of compounds that bind to C5d neoepitopes is complicated, in part, by the transient nature of free C5b itself. Once generated, C5b quickly forms a complex with C6 thereby rendering the putative neoepitopes inaccessible. The instant inventors solve this problem by using the C5d domain itself as an immunogen and/or as the antigen against which to screen putative C5d-binding compounds such as anti-C5d antibodies. The inventors determined that the isolated C5d domain can be expressed in vitro and that the isolated domain, unlike C5b as a whole, does not as readily dimerize with C6, thus allowing the isolated C5d domain to be used as an immunogen and/or as a screening reagent. Skilled artisans would recognize that an isolated domain may not always be so useful. For example, isolated C5d does not exist in nature and the discovery that C5d can adopt a native conformation as an independent entity is unexpected; generally, the structure and stability of a constituent domain of a protein often turn upon context - its position within the whole C5 or C5b structure.

Thus, in one aspect, the disclosure features an isolated polypeptide comprising the C5d domain of human complement component C5, or an antigenic peptide fragment of the polypeptide, wherein the C5d domain has the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16. In some embodiments, the polypeptide is not a full-length C5b polypeptide or a full-length, uncleaved C5 polypeptide. In some embodiments, the antigenic peptide fragment comprises the amino acid sequence depicted in any one of SEQ ID NOs:3- 8. In some embodiments, the isolated polypeptide or the antigenic peptide fragment thereof binds to complement component C6. In some embodiments, the isolated polypeptide or the antigenic peptide fragment thereof inhibits the interaction between C5b and C6.

In some embodiments, any of the isolated polypeptides, or antigenic peptide fragments thereof described herein can comprise a heterologous moiety such as a protein or any other heterologous moieties described herein or known in the art. A heterologous protein moiety can include or be, e.g., all or part (e.g., CHI, CH2, CH3, or CH4 domain) of the Fc region of an antibody.

In another aspect, the disclosure features a pharmaceutical composition comprising one or more of any of the isolated polypeptides, or antigenic peptide fragments thereof described herein and a pharmaceutically acceptable carrier. For example, in some embodiments, the pharmaceutical composition comprises an isolated polypeptide or antigenic peptide fragment thereof that binds to complement component C6 and/or inhibits the interaction between C5b and C6. In some embodiments, the pharmaceutical composition comprises one or more of the an isolated polypeptide or antigenic peptide fragment thereof that is capable of inducing an immune response to itself in a mammal.

Furthermore, the disclosure features: (i) a nucleic acid encoding any of the isolated polypeptides or antigenic peptide fragments described herein; (ii) a vector comprising the nucleic acid of (i); (iii) an expression vector comprising the nucleic acid of (i); (iv) a cell (e.g., a non-human mammalian cell, insect cell, yeast cell, bacterial cell, or a human cell) comprising the vector of (ii) or (iii); and/or (v) a method for producing/expressing a polypeptide in a cell. The method comprises culturing the cell of (iv) under conditions suitable for expression of the polypeptide or antigenic peptide fragment thereof from the nucleic acid in the cell. The method can further comprise isolating the polypeptide or antigenic peptide fragment from the cultured cell or the media in which the cell is cultured. Additional details related to making and using the nucleic acids, vectors, and cell culture methods provided herein are described in detail below. In another aspect, the disclosure also features a method for generating an immune response in a non-human mammal (e.g., a rodent such as a mouse, rat, rabbit, or gerbil), the method comprising administering to a mammal any of the C5d-containing polypeptides or antigenic peptide fragments thereof in an amount effective to induce an immune response in the mammal against the polypeptide or antigenic peptide fragment thereof. In some embodiments, the polypeptide or antigenic peptide fragment is administered with an adjuvant. In some embodiments, more than one (e.g., two, three, four, five, six, seven, or eight or more) antigenic peptide fragment(s) is administered to the non-human mammal. In some embodiments, the polypeptide and one or more antigenic peptide fragments thereof are administered to the non-human mammal. In some embodiments, the non-human mammal does not express an endogenous complement component C5 protein.

In some embodiments, the method can further include, after the induction of the immune response, isolating from the non-human mammal: (a) an antibody that binds to the polypeptide or antigenic peptide fragment thereof or (b) a nucleic acid encoding the antibody. Also featured is an antibody generated by the method, or an antigen-binding fragment of such an antibody.

And in yet another aspect, the disclosure features a method for identifying a compound that binds to the C5d domain of human complement component C5. The method includes the steps of: contacting a polypeptide or antigenic peptide fragment thereof described herein with a compound to be screened under physiological conditions; and determining whether the compound binds to the polypeptide or antigenic peptide fragment thereof. The compound can be, e.g., an antibody or antigen-binding fragment thereof. The compound can be, e.g., a small molecule, a polypeptide, or an aptamer.

In yet another aspect, the disclosure features a method for identifying an inhibitor of the interaction between C5b and C6, which method includes: (i) mixing C6, or a C5b-binding fragment thereof, with C5b under conditions that allow the binding of C6, or a C5b-binding fragment thereof, to C5b to form a complex; (ii) measuring the amount of C6:C5b complex or C5b-binding fragment of C6:C5b complex; (iii) mixing C6, or a C5b-binding fragment thereof, with C5b and with a candidate inhibitor under the same conditions in step (i); (iv) measuring the amount of C6:C5b complex or C5b-binding fragment of C6:C5b complex; and (v) comparing the amount of complex formed in step (ii) with the amount of complex formed in step (iv). If the amount of complex formed in step (iv) is less than the amount of complex formed in step (ii) then the candidate inhibitor is identified as an inhibitor of the interaction between C5b and C6.

In yet another aspect, the disclosure features a method for identifying an inhibitor of the interaction between C5b and C6, which method includes: (i) mixing an isolated C5d polypeptide, or a C6-binding fragment thereof, with C6 under conditions that allow the binding of the C5d polypeptide, or a C6-binding fragment thereof, to C6 to form a complex;

(ii) measuring the amount of C6:C5b complex or C6-binding fragment of C5d:C6 complex;

(iii) mixing C5d, or a C6-binding fragment thereof, with C6 and with a candidate inhibitor under the same conditions in step (i); (iv) measuring the amount of C6:C5b complex or C6- binding fragment of C5d:C6 complex; and (v) comparing the amount of complex formed in step (ii) with the amount of complex formed in step (iv). If the amount of complex formed in step (iv) is less than the amount of complex formed in step (ii) then the candidate inhibitor is identified as an inhibitor of the interaction between C5b and C6.

In some embodiments of any of the methods described herein, the method can include determining whether an identified inhibitor reduces complement activation in an in vitro assay. In some embodiments, the C5d polypeptide comprises the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16. In some embodiments, the C6-binding fragment of C5d comprises the amino acid sequence depicted in any one of SEQ ID NOs:3-8.

In some embodiments, the C5d polypeptide, C6-binding fragment, C6 polypeptide, or C5b-binding fragment thereof comprises a heterologous moiety. The heterologous moiety can be, e.g., an Fc domain of an antibody. The heterologous moiety can be, e.g., one member of a specific binding pair such as, but not limited to, streptavidin and biotin. The

heterologous moiety can be, e.g., a detectable label.

Compounds, such as antibodies or antigen-binding fragments thereof, which bind to C5d neoepitopes and/or antagonize the C5b-C6 interaction, identified through the

aforementioned methods would offer a number of advantages, e.g., advantages over antibodies that bind to, and inhibit cleavage of, full-length or mature C5. Like such anti-C5 antibodies, the antagonist compounds described herein are capable of inhibiting the downstream effects of C5 activation. That is, in some embodiments, the compounds described herein can inhibit the C5b (MAC)-dependent cell lysis that results from cleavage of C5. However, as the concentration of C5 in human plasma is approximately 0.37 μΜ (Rawal and Pangburn (2001) J Immunol 166(4):2635-2642), the use of high concentrations and/or frequent administration of C5 antagonists (e.g., anti-C5 antibodies) is often necessary to effectively inhibit C5 in a human. Unlike C5, fragment C5b, specifically the one or more neoepitopes of the C5d domain thereof, is present in blood at much lower concentrations and are often restricted to specific areas of local complement activation such as, e.g., the surface of red blood cells in patients with PNH, the joints of RA patients, or the drusen in the eyes of patients with AMD.

In addition, while anti-C5 antibodies are highly effective at inhibiting complement in vitro and in vivo (see, e.g., Hillmen et al. (2004) N EnglJ Med 3_50(6}:552), the antibodies are particularly susceptible to target-mediated clearance because of the high concentration of C5 in blood (see International application publication no. WO 2010/151526). This indicates that compounds (e.g., anti-C5d antibodies or antigen-binding fragments thereof) which specifically target C5b, by way of specifically binding to the neoepitopes present on C5d, are very likely to have a longer half-life, as compared to C5 -binding inhibitors (such as anti-C5 antibodies), in blood due to a reduced contribution of antigen-mediated clearance. As described above, fragment C5b is present at a much lower concentration than C5 and generally produced at specific areas of complement activation. Thus, in view of their longer half-life, the compounds (e.g., anti-C5d antibodies or antigen-binding fragments thereof) described herein can be administered to a human at a much lower dose and/or less frequently than an anti-C5 antibody and effectively provide the same or greater inhibition of terminal complement in a human. The ability to administer a lower dose of the compounds, as compared to the dose of an anti-C5 antibody, also allows for additional delivery routes such as, e.g., subcutaneous administration, intramuscular administration, intrapulmonary delivery, and administration via the use of biologically degradable microspheres.

Accordingly, in one aspect, the disclosure features a compound that binds to the C5d domain of complement component C5 (e.g., human C5). The compound can bind, e.g., to one or more neoepitopes within the C5d domain.

In another aspect, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5, but does not bind to uncleaved, native human C5, wherein the C5d domain has the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16.

In another aspect, the disclosure features an isolated antibody, or an antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5 and uncleaved, native human C5, wherein the affinity of the antibody for C5d is at least 10-fold greater than the affinity of the antibody for uncleaved, native human C5, wherein the C5d domain has the amino acid sequence depicted in in any one of SEQ ID NOs:2 or 14-16.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the affinity of the antibody for C5d is at least 20 (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10000, or 20000 or more)-fold greater than the affinity of the antibody for uncleaved, native human C5.

In yet another aspect, the disclosure features an isolated antibody, or antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5, wherein the antibody or antigen-binding fragment thereof binds to an isolated C5d polypeptide in vitro with a K_D that is less than 5 x 10^"9 M in the presence of a molar excess of uncleaved, native human C5 and under physiological conditions, and wherein the isolated C5d polypeptide comprises at least five (e.g., at least six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 or more) consecutive amino acids depicted in any one of SEQ ID NOs:2 or 14-16. In some embodiments, the uncleaved, native human C5 can be from human plasma.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 2-fold and 20- fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 2-fold and 15 -fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen- binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 2-fold and 30-fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 2-fold and 50-fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 3 -fold and 15 -fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen- binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 3-fold and 20-fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 5 -fold and 15 -fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 5 -fold and 20-fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen- binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 5-fold and 50-fold greater than the concentration of the isolated C5d polypeptide. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the concentration of uncleaved, native human C5 is between 10-fold and 50-fold greater than the concentration of the isolated C5d polypeptide.

In another aspect, the disclosure features an isolated antibody or antigen-binding fragment thereof that binds to the C5d domain of human complement component C5, wherein the antibody or antigen-binding fragment thereof binds to the C5d domain with a KD that is less than 5 x 10^"9 M, wherein the antibody or antigen-binding fragment thereof binds to the C5d domain with an affinity that is at least 100-fold greater than its corresponding affinity for uncleaved human C5, and wherein the C5d domain has the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof inhibits the interaction between C5b and C6. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof inhibits the formation of the terminal complement complex. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen- binding fragment thereof inhibits hemolysis in vitro.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof binds to the C5d domain of a non-human mammalian species. That is, in some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, an antibody or antigen-binding fragment thereof described herein is capable of binding to a human C5d domain and (dual- specificity )/or a C5 domain of a C5 protein expressed in a non-human mammalian species (e.g., a non-human primate, a dog, a cat, a goat, a horse, or a rodent). In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the non- human mammalian species is a non-human primate and the non-human primate is a cynomolgus macaque, rhesus macaque, or baboon. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen- binding fragment thereof binds to human C5d with an affinity no greater than 100-fold its corresponding affinity for C5d from the non-human mammalian species. In some

embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof binds to human C5d with an affinity no greater than 50-fold its corresponding affinity for C5d from the non-human mammalian species. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody is a bispecific antibody. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the bispecific antibody binds to C5a. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 7 x 10^"10 M, less than 5 x 10^"10 M, less than 2.5 x 10^"10 M, less than 1.5 x 10^"10 M, or less than 8.0 x 10^"11 M.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody, or antigen-binding fragment thereof is humanized, fully human, or chimeric. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof is selected from the group consisting of a recombinant antibody, a single chain antibody, a diabody, an intrabody, a chimerized or chimeric antibody, a deimmunized antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, and an F(ab')₂ fragment.

In some embodiments, any of the antibodies or antigen-binding fragments thereof described herein further comprise a heterologous moiety. The heterologous moiety can be, e.g., a sugar. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof is glycosylated. The heterologous moiety can be, e.g., a detectable label such as a fluorescent label, a luminescent label, a heavy metal label, a radioactive label, or an enzymatic label.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof is modified with a moiety that improves one or both of: (a) the stabilization of the antibody or antigen-binding fragment thereof in circulation and (b) the retention of the antibody or antigen-binding fragment thereof in circulation. The modification can be, e.g., PEGylation.

In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the antibody comprises an altered heavy chain constant region that has reduced effector function as compared to the effector function of the unaltered form of the heavy chain constant region.

In yet another aspect, the disclosure features an isolated antibody, or antigen-binding fragment thereof, that crossblocks the binding of any one of the antibodies or antigen-binding fragments thereof disclosed herein. In another aspect, the disclosure features a pharmaceutical composition comprising one or more of any of the antibodies or antigen-binding fragments thereof described herein and a pharmaceutically-acceptable carrier.

In further aspects, the disclosure provides: (i) a nucleic acid encoding one or more of any of the antibodies or antigen-binding fragments thereof described herein or (ii) a nucleic acid encoded a heavy chain polypeptide and/or a light chain polypeptide of one or more of any of the antibodies or antigen-binding fragments thereof described herein. Also featured is a vector comprising such a nucleic acid as (i) or (ii). The vector can be, in some

embodiments, an expression vector. In addition, the disclosure features a cell (e.g., a eukaryotic cell or a prokaryotic cell) comprising above-referenced the vector and/or a composition comprising a plurality of cells comprising the above-referenced vector. The cell can be, e.g., a yeast cell, a bacterial cell, an insect cell, a rodent cell, a cell from a non-human primate, or a human cell. The disclosure further provides a method for producing any of the antibodies or antigen-binding fragments thereof described herein. The method includes culturing the cell or culture of cells referenced above under conditions and for a time sufficient to allow expression by the cell or cells of the antibody or antigen-binding fragment encoded by the nucleic acid. The method can also include isolating the antibody or antigen- binding fragment thereof, e.g., from the cultured cell or cells or the media in which the cell or cells are cultured.

Moreover, the disclosure provides diagnostic and therapeutic applications in which the aforementioned compounds are useful. For example, featured herein are methods for using the compounds to treat or prevent complement-associated conditions. Complement- associated conditions include any medical condition in a human, the treatment of which would benefit directly or indirectly from inhibition of the complement system. Such conditions are generally characterized by inappropriate regulation of the complement system such as inappropriate: (i) activation of the complement system or (ii) duration of an activated complement system in a subject. In some embodiments, a complement-associated condition is one in which normal operation or function of the complement system is detrimental, e.g., in the case of liver regeneration or repair. Complement-associated conditions include, without limitation, inflammatory and autoimmune disorders. A complement-associated condition can be, e.g., rheumatoid arthritis (RA); antiphospholipid antibody syndrome (APS); lupus nephritis; ischemia-reperfusion injury; PNH; atypical hemolytic uremic syndrome (aHUS); typical (also referred to as diarrheal or infectious) hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); transplant rejection; multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); HELLP syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; and traumatic brain injury. In some embodiments, the complement-associated condition is a complement-associated vascular disorder such as a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral (e.g., musculoskeletal) vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated

cardiomyopathy, diabetic angiopathy, Kawasaki's disease (arteritis), venous gas embolus (VGE), and restenosis following stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA). Additional complement-associated disorders include, without limitation, myasthenia gravis (MG), cold agglutinin disease (CAD), dermatomyositis, paroxysmal cold hemoglobinuria (PCH), Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome, Degos disease, and catastrophic APS (CAPS).

Accordingly, in another aspect, the disclosure features a method for treating a patient afflicted with a complement-associated condition. The method includes administering to the subject a compound in an amount effective to treat the complement-associated condition, wherein the compound binds to the C5d domain of complement component C5 and inhibits the interaction between C5b and C6. In some embodiments, the compound is a small molecule. In some embodiments, the compound is any one of the isolated antibodies or antigen-binding fragments thereof described herein. In some embodiments, the compound is any of the isolated polypeptides or antigenic peptide fragments thereof (e.g., an isolated polypeptide or antigenic peptide fragment thereof that inhibits the interaction between C5d and C6 and/or inhibits terminal complement formation or activity).

In some embodiments, the complement-associated condition is selected from the group consisting of rheumatoid arthritis, antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous fetal loss, thrombotic thrombocytopenic purpura, Pauci- immune vasculitis, epidermolysis bullosa, recurrent fetal loss, traumatic brain injury, myocarditis, a cerebrovascular disorder, a peripheral vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated

cardiomyopathy, diabetic angiopathy, Kawasaki's disease, venous gas embolus, restenosis following stent placement, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury,

glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpastures syndrome, Degos disease, and catastrophic antiphospholipid syndrome. In some

embodiments, the complement-associated condition is transplant rejection. In some embodiments, the complement-associated condition is liver damage and administration of the compound promotes liver regeneration. In some embodiments, the complement-associated condition is a complement-associated inflammatory condition. The complement-associated inflammatory condition can be, e.g., one selected from the group consisting of atypical hemolytic uremic syndrome, age-related macular degeneration, severe burn, rheumatoid arthritis, sepsis, lupus nephritis, and antiphospholipid syndrome.

In yet another aspect, the disclosure features a therapeutic kit comprising: (i) one or more of any of the isolated antibodies or antigen-binding fragments thereof described herein and/or one or more of any of the isolated polypeptides or antigenic peptide fragments thereof described herein and (ii) means for delivery of the antibody, antigen-binding fragment, polypeptide, or antigenic peptide fragment to a human. The means can be, e.g., a syringe.

In another aspect, the disclosure features an article of manufacture comprising: a container comprising a label; and a composition comprising: (i) one or more of any one of the isolated antibodies or antigen-binding fragments thereof described herein or (ii) one or more of any of the isolated polypeptides or antigenic peptide fragments thereof (e.g., an isolated polypeptide or antigenic peptide fragment thereof that inhibits the interaction between C5d and C6 and/or inhibits terminal complement formation or activity), wherein the label indicates that the composition is to be administered to a human having, suspected of having, or at risk for developing, a complement-associated condition. The article of manufacture may further contain one or more additional active therapeutic agents for use in treating a human having, suspected of having, or at risk for developing, a complement-associated condition.

"Polypeptide," "peptide," and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The C5d polypeptides, or fragments thereof, described herein can contain or be wild-type proteins or can be variants that have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

As used herein, "antigenic peptide fragments" are shorter than the full-length proteins from which they are derived, but retain at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% or more) of the ability of the full-length proteins to induce an antigenic response in a mammal (see below under "Methods for Producing an Antibody"). Antigenic peptide fragments of a C5d polypeptide include terminal as well internal deletion variants of the protein. Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Antigenic peptide fragments can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues in length (e.g., at least 6 contiguous amino acid residues in any one of SEQ ID NOS:2-8). In some embodiments, an antigenic peptide fragment of a human C5d polypeptide has fewer than 400 (e.g., fewer than 375, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues (e.g., fewer than 400 contiguous amino acid residues in any one of SEQ ID NOs:2 or 14-16). In some embodiments, an antigenic peptide fragment of C5d has at least 6, but fewer than 400, amino acid residues in length.

In some embodiments, the human C5d polypeptide can have an amino acid sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human C5d polypeptide having the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16 (see below).

Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods. Amino acid sequences for exemplary human C5d polypeptides as well as antigenic peptide fragments thereof are set forth below.

As used throughout the present disclosure, the term "antibody" refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that is generated by any one of a variety of methods that are known in the art and described herein. The term "antibody" includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.

As used herein, the term "antibody fragment," "antigen-binding fragment," or similar terms refer to a fragment of an antibody that retains the ability to bind to an antigen (e.g., an epitope present in C5d, but not in the alpha chain of uncleaved, native C5 protein), e.g., a single chain antibody (scFv), an Fd fragment, an Fab fragment, an Fab' fragment, or an F(ab')₂ fragment. An scFv is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, diabodies (Poljak (1994) Structure 202}: 1121-1123; Hudson et al. (1999) J Immunol Methods 23(1-2 : 177-189, the disclosures of both of which are incorporated herein by reference in their entirety), minibodies, triabodies (Schoonooghe et al. (2009) BMC

Biotechnol 9:70), domain antibodies (also known as "heavy chain immunoglobulins" or camelids; Holt et al. (2003) Trends Biotechnol 21(11):484-490); and intrabodies (Huston et al. (2001) Hum Antibodies 100^4): 127-142; Wheeler et al. (2003) Mol Ther 80}:355-366; Stocks (2004) Drug Discov Today 9(22): 960-966, the disclosures of each of which are incorporated herein by reference in their entirety) are included in the definition of antibody fragments and can be incorporated into the compositions, and used in the methods, described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Other features and advantages of the present disclosure, e.g., methods for treating or preventing a complement-associated condition, will be apparent from the following description, the examples, and from the claims.

Brief Description of the Sequences

SEQ ID NO:l is an exemplary amino acid sequence for full-length human

complement component C5.

SEQ ID NO:2 is an exemplary amino acid sequence for the C5d domain of human complement component C5 (residues 982 to 1310 of SEQ ID NO: l).

SEQ ID NOs:3-8 are amino acid sequences of exemplary peptide fragments of the C5d domain of human complement component C5.

SEQ ID NO:9 is an amino acid sequence for the so-called "FLAG" immunogenic tag.

SEQ ID NO: 10 is an exemplary polyhistidine amino acid sequence.

SEQ ID NO: 11 is an amino acid sequence for the so-called hemagglutinin or "HA" immunogenic tag.

SEQ ID NO: 12 is an exemplary amino acid sequence for human factor H protein.

SEQ ID NO: 13 is an exemplary amino acid sequence for human complement receptor 2 (CR2).

SEQ ID NO: 14 is an exemplary amino acid sequence for the C5d domain of human complement component C5 (residues 984 to 1309 of SEQ ID NO: l).

SEQ ID NO: 15 is an exemplary amino acid sequence for the C5d domain of human complement component C5 (residues 992 to 1310 of SEQ ID NO: l).

SEQ ID NO: 16 is an exemplary amino acid sequence for the C5d domain of human complement component C5 (residues 993 to 1310 of SEQ ID NO: l).

SEQ ID NO: 17 is an exemplary amino acid sequence for a fusion construct comprising a glutathione-S-transferase amino acid sequence and a human C5d domain amino acid sequence of human complement component C5 (residues 992 to 1310 of SEQ ID NO: l).

SEQ ID NO: 18 is an exemplary amino acid sequence for a fusion construct comprising a histidine tag (six histidines) sequence, an enterokinase cleavage site, and a human C5d protein portion (amino acids 993-1310 of SEQ ID NO:l). Detailed Description

As noted above, the present disclosure provides, among other things, compounds that bind to the C5d domain of complement component C5. In some embodiments, the compounds inhibit the interaction between C5b and C6 and, thus, inhibit formation of the terminal complement complex (e.g., the assembly and/or activity of the C5b-9 TCC). In some embodiments, the compound is an anti-C5d antibody or C5d-binding fragment thereof. Also featured are methods for producing and/or screening for such compounds. Methods for using any of the foregoing to treat or prevent complement-associated conditions such as, but not limited to, atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) are further described. While in no way intended to be limiting, exemplary compounds (including antibodies and antigen-binding fragments), compositions (e.g., pharmaceutical compositions and formulations) comprising the compositions, and methods for using the compounds and compositions are elaborated on below.

Compounds The disclosure features compounds that bind to the C5d domain of complement component C5 (e.g., the C5d domain of human C5 protein) and inhibit the interaction between C5b and C6. As discussed above, the proform of C5, a 1676 amino acid residue precursor protein, is processed by a series of proteolytic cleavage events. The first 18 peptides (numbered -18 to -1) constitute a signal peptide that is cleaved from the precursor protein. The remaining 1658 amino acid protein is cleaved in two places to form the alpha and beta chains. The first cleavage event occurs between amino acid residues 655 and 656. The second cleavage occurs between amino acid residues 659 and 660. The two cleavage events result in the formation of three distinct polypeptide fragments: (i) a fragment comprising amino acids 1 to 655, which is referred to as the beta chain; (ii) a fragment comprising amino acids 660 to 1658, which is referred to as the alpha chain; and (iii) a tetrapeptide fragment consisting of amino acids 656 to 659. The alpha chain and the beta chain polypeptide fragments are connected to each other via disulfide bond and constitute the mature C5 protein. The CP or AP C5 convertase activates mature C5 by cleaving the alpha chain between residues 733 and 734, which results in the liberation of C5a fragment (amino acids 660 to 733). The remaining portion of mature C5 is fragment C5b, which contains the residues 734 to 1658 of the alpha chain disulfide bonded to the beta chain. Within C5b is the C5d domain, comprising residues 964 to 1291 of the C5 alpha chain. Upon cleavage of C5 into fragment C5a and C5b, this domain translocates as a rigid body thereby exposing one or more neoepitopes, which interact with C6.

Compounds that bind to C5d, particularly the neoepitopes of C5d, and inhibit the interaction between C5b and C6, can inhibit formation of the membrane attack complex (MAC). In some embodiments, a compound described herein does not inhibit the cleavage of C5 into fragments C5a and C5b. In some embodiments, a compound described herein does not inhibit the production or activity of the C5a cleavage product of C5.

The C5d-binding compound can be, e.g., a small molecule, a polypeptide, a polypeptide analog, a nucleic acid, or a nucleic acid analog. "Small molecule" as used herein, is meant to refer to an agent, which has a molecular weight of less than about 6 kDa and most preferably less than about 2.5 kDa. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the application. This application contemplates using, among other things, small chemical libraries, peptide libraries, or collections of natural products. Tan et al. described a library with over two million synthetic compounds that is compatible with miniaturized cell- based assays (J Am Chem Soc (1998) 120:8565-8566). It is within the scope of this application that such a library may be used to screen for compounds that bind to C5d. There are numerous commercially available compound libraries, such as the Chembridge

DIVERSet. Libraries are also available from academic investigators, such as the Diversity set from the NCI developmental therapeutics program. Rational drug design may also be employed. For example, rational drug design can employ the use of crystal or solution structural information on the human complement component C5 protein. See, e.g., the structures described in Hagemann et al. (2008) J Biol Chem 283(12):7763-75 and Zuiderweg et al. (1989) Biochemistry 280}: 172-85.

Peptidomimetics can be compounds in which at least a portion of a subject polypeptide is modified, and the three dimensional structure of the peptidomimetic remains substantially the same as that of the subject polypeptide. Peptidomimetics may be analogues of a subject polypeptide of the disclosure that are, themselves, polypeptides containing one or more substitutions or other modifications within the subject polypeptide sequence.

Alternatively, at least a portion of the subject polypeptide sequence may be replaced with a non-peptide structure, such that the three-dimensional structure of the subject polypeptide is substantially retained. In other words, e.g., one, two or three amino acid residues within the subject polypeptide sequence may be replaced by a non-peptide structure. In addition, other peptide portions of the subject polypeptide may, but need not, be replaced with a non-peptide structure. Peptidomimetics (both peptide and non-peptidyl analogues) may have improved properties (e.g., decreased proteolysis, increased retention or increased bioavailability). Peptidomimetics generally have improved oral availability, which makes them especially suited to treatment of conditions in a human or animal. It should be noted that

peptidomimetics may or may not have similar two-dimensional chemical structures, but share common three-dimensional structural features and geometry. Each peptidomimetic may further have one or more unique additional binding elements.

Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, including cell surface proteins. The systematic evolution of ligands by exponential enrichment (SELEX) process is powerful and can be used to readily identify such aptamers. Aptamers can be made for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do (see, e.g., Ulrich (2006) Handb Exp Pharmacol 173:305-326). The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases.

Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5- iodouracil, 5-iodocytosine, and C₅- propynyl pyrimidines such as Cs-propynylcytosine and C₅-propynyluracil. Other suitable modified nucleobases include, e.g., 7-substituted-8-aza-7- deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines, and 7-aminocarbonyl-7-deazapurines. Examples of these include 6- amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines, 6- amino-7-aminocarbonyl- 7-deazapurines, 2-amino-6-hydroxy-7-iodo-7-deazapurines, 2- amino-6-hydroxy-7-cyano-7- deazapurines, and 2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. See, e.g., U.S. Patent Nos. 4,987,071; 5,116,742; and 5,093,246; "Antisense R A and DNA," D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); Haselhoff and Gerlach (1988) Nature 334:585-59; Helene (1991) Anticancer Drug D 6:569-84; Helene (1992) Ann NY Acad Sci 660:27-36; and Maher (1992) Bioassays 14:807-15.

In some embodiments, a compound described herein is a protein or protein fragment. For example, in some embodiments the compounds can be or contain antibodies, or antigen- binding fragments thereof, specific for the C5d domain of complement component C5.

(Herein, such an antibody may sometimes be referred to as an "anti-C5d antibody.")

In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with the C5d domain of human complement component C5, e.g., the C5d domain comprising amino acids 982 to 1310 of the following full-length human C5 amino acid sequence:

MGLLGILCFLIFLGKTWGQEQTYVISAPKIFRVGASENIVIQVYGYTEAFDATISIKSYP

DKKFSYSSGHVHLSSENKFQNSAILTIQPKQLPGGQNPVSYVYLEVVSKHFSKSKRMP

ITYDNGFLFIHTDKPVYTPDQSVKVRVYSLNDDLKPAKRETVLTFIDPEGSEVDMVEE

IDHIGIISFPDFKIPSNPRYGMWTIKAKYKEDFSTTGTAYFEVKEYVLPHFSVSIEPEYN

FIGYK FKNFEITIKARYFYNKVVTEADVYITFGIREDLKDDQKEMMQTAMQNTMLI

NGIAQVTFDSETAVKELSYYSLEDLNNKYLYIAVTVIESTGGFSEEAEIPGIKYVLSPY

KLNLVATPLFLKPGIPYPIKVQVKDSLDQLVGGVPVTLNAQTIDVNQETSDLDPSKSV

TRVDDGVASFVLNLPSGVTVLEFNVKTDAPDLPEENQAREGYRAIAYSSLSQSYLYI

DWTDNHKALLVGEHLNIIVTPKSPYIDKITHYNYLILSKGKIIHFGTREKFSDASYQSI

NIPVTQNMVPSSRLLVYYIVTGEQTAELVSDSVWLNIEEKCGNQLQVHLSPDADAYS

PGQTVSLNMATGMDSWVALAAVDSAVYGVQRGAKKPLERVFQFLEKSDLGCGAG

GGL NANVFHLAGLTFLTNANADDSQENDEPCKEILRPRRTLQKKIEEIAAKYKHSV

VK CCYDGACV NDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLG

RLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGVGISNTG

ICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSGMQFCVKMSAVEG

ICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILV

KTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLL

VGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEK

QKLKKKLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQN

QNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRK

AFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKR

EALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVI

KWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLSMDIDVSYKHKGALHNYK

MTDK FLGRPVEVLLNDDLIVSTGFGSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQ

DIEASHYRGYGNSDYKRIVACASYKPSREESSSGSSHAVMDISLPTGISANEEDLKAL

VEGVDQLFTDYQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRP

DKQCTMFYSTSNIKIQKVCEGAACKCVEADCGQMQEELDLTISAETRKQTACKPEIA YAYKVSITSITVENVFVKYKATLLDIYKTGEAVAEKDSEITFIK VTCTNAELVKGRQ YLIMGKEALQIKYNFSFRYIYPLDSLTWIEYWPRDTTCSSCQAFLANLDEFAEDIFLN GC (SEQ ID NO: l) (NCBI Accession No. AAA51925 and Haviland et al, supra).

That is, in some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with a C5d polypeptide comprising or consisting of the following amino acid sequence:

LLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLI

EKQKLKKKLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQ

NQNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIR

KAFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALK

REALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNP

VIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLS (SEQ ID NO:2).

In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with the C5d domain of human complement component C5, e.g., the C5d domain comprising amino acids 984 to 1309 of the human C5 amino acid sequence depicted in SEQ ID NO: 1. In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with a C5d polypeptide comprising or consisting of the following amino acid sequence:

VGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEK

QKLKKKLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQN

QNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRK

AFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKR

EALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVI

KWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRL (SEQ ID NO:14).

In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with the C5d domain of human complement component C5, e.g., the C5d domain comprising amino acids 992 to 1310 of the human C5 amino acid sequence depicted in SEQ ID NO: 1. In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with a C5d polypeptide comprising or consisting of the following amino acid sequence:

LSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKL

KEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICNSL

LWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLV KIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGN PPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVIKWLSEEQ RYGGGFYSTQDTINAIEGLTEYSLLVKQLRLS (SEQ ID NO:15).

In some embodiments, the anti-C5d antibody binds to an epitope within or

overlapping with the C5d domain of human complement component C5, e.g., the C5d domain comprising amino acids 993 to 1310 of the human C5 amino acid sequence depicted in SEQ ID NO: 1. In some embodiments, the anti-C5d antibody binds to an epitope within or overlapping with a C5d polypeptide comprising or consisting of the following amino acid sequence:

SQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLK

EGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICNSLL

WLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLVK

IDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGNP

PIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVIKWLSEEQR

YGGGFYSTQDTINAIEGLTEYSLLVKQLRLS (SEQ ID NO:16).

An "epitope" refers to the site on a protein (e.g., the C5d domain of human

complement component C5) that is bound by an antibody. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residue(s).

In some embodiments, the anti-C5d antibody can bind to an epitope within, or overlapping with, an antigenic peptide fragment of a human complement component C5 protein. For example, the anti-C5d antibody can bind to an epitope within, or overlapping with, an antigenic peptide fragment of the C5d domain of human C5 protein, the fragment containing, or consisting of, the following amino acid sequence: HLPKGSAEAEL (SEQ ID NO:3).

In some embodiments, the anti-C5 antibody can bind to an epitope within, or overlapping with, an antigenic peptide fragment of the C5d domain of the human C5 protein, the fragment containing, or consisting of, any one of the following amino acid sequences (which are exemplary antigenic fragments of SEQ ID NO:2):

KEGMLSIMSYRNADY (SEQ ID NO:4); QYQPIKLQGTLPVE (SEQ ID NO:5);

HKDSSVPNTGTARM (SEQ ID NO:6); NPVIKWLSEEQRYGGGFYSTQ (SEQ ID NO:7); or YSLLVKQLRL (SEQ ID NO:8). In some embodiments, the anti-C5d antibody, or C5d-binding fragment thereof, specifically binds to C5d (e.g., human C5d). The terms "specific binding" or "specifically binds" refer to two molecules forming a complex (e.g., a complex between an antibody and human C5d) that is relatively stable under physiologic conditions. Typically, binding is considered specific when the association rate constant (k_a) is higher than 10⁶ M^'V¹. Thus, an antibody can specifically bind to the C5d domain with a k_a of at least (or greater than) 10⁶ (e.g., at least or greater than 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹) M^'V¹. In some embodiments, an anti-C5d antibody described herein has a dissociation constant (k_d) of less than or equal to 10^{" 3} (e.g., 8 x 10^"4, 5 x 10^"4, 2 x 10^"4, 10^"4, or 10^"5) s^"1. In some embodiments, an anti-C5d antibody, antigen-binding fragment thereof, described herein has a K_D of less than 10^"8, 10^~9, 10^"10, 10^"11, or 10^"12 M. The equilibrium constant K_D is the ratio of the kinetic rate constants - k_d/k_a. In some embodiments, an anti-C5d antibody described herein has a K_D of less than 5 x 10^"9 M.

Methods for determining whether an antibody binds to a protein antigen and/or the affinity for an antibody to a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assay (ELISA). See, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo (2004) "Antibody Engineering: Methods and

Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," W.H. Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2^nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) J Immunol Meth 160: 191-198; Jonsson et al. (1993) Ann Biol Clin 51 : 19-26; and Jonsson et al. (1991)

Biotechniques 11 :620-627.

In some embodiments, the anti-C5d antibody can crossblock binding of another antibody that binds to an epitope within, or overlapping with, the C5d domain of C5 (e.g., the C5d domain of human C5). In some embodiments, the anti-C5d antibody can crossblock binding of an antibody that binds to an epitope within, or overlapping with, a peptide fragment of C5d. The peptide fragment can be a fragment of a human complement component C5 protein having the amino acid sequence depicted in any one of SEQ ID NOS:3-8. As used herein, the term "crossblocking antibody" refers to an antibody that lowers the amount of binding of anti-C5d antibody to an epitope on a complement component C5d protein relative to the amount of binding of the anti-C5d antibody to the epitope in the absence of the antibody. Suitable methods for determining whether a first antibody crossblocks binding of a second antibody to an epitope are known in the art. For example, crossblocking antibodies can be identified by comparing the binding of a first anti-C5d antibody in the presence and absence of a test antibody. Decreased binding of the first anti- C5d antibody in the presence of the test antibody as compared to binding of the first anti-C5d antibody in the absence of the test antibody indicates the test antibody is a crossblocking antibody with respect to the first anti-C5d antibody.

Methods for identifying the epitope to which a particular antibody (e.g., an anti-C5d antibody) binds are also known in the art. For example, the binding epitope of an anti-C5d antibody can be identified by measuring the binding of the antibody to several (e.g., three, four, five, six, seven, eight, nine, 10, 15, 20, or 30 or more) overlapping peptide fragments of a complement component C5d protein (e.g., several overlapping fragments of a protein having the amino acid sequence depicted in any one of SEQ ID NOs:3-8). Each of the different overlapping peptides is then bound to a unique address on a solid support, e.g., separate wells of a multi-well assay plate. Next, the anti-C5d antibody is interrogated by contacting it to each of the peptides in the assay plate for an amount of time and under conditions that allow for the antibody to bind to its epitope. Unbound anti-C5d antibody is removed by washing each of the wells. Next, a detectably-labeled secondary antibody that binds to the anti-C5d antibody, if present in a well of the plate, is contacted to each of the wells, and unbound secondary antibody is removed by washing steps. The presence or amount of the detectable signal produced by the detectably-labeled secondary antibody in a well is an indication that the anti-C5d antibody binds to the particular peptide fragment associated with the well. See, e.g., Harlow and Lane {supra), Benny K. C. Lo {supra), and U.S. Patent Application Publication No. 20060153836, the disclosure of which is

incorporated by reference in its entirety. A particular epitope to which an antibody binds can also be identified using BIAcore chromatographic techniques (see, e.g., Pharmacia

BIAtechnology Handbook, "Epitope Mapping," Section 6.3.2, (May 1994); and Johne et al.

( 1993) J Immunol Methods 160:20191-8).

In some embodiments, a C5d-binding compound (e.g., an anti-C5d antibody or antigen-binding fragment thereof) described herein can reduce the ability of a C5b protein to bind to human complement component C6 by greater than 50 (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more) %. As the binding of C5b to C6 is an initial step in the assembly of the MAC, which promotes the lysis of cells to which the MAC is inserted, the anti-C5d antibody, or C5d-binding fragment thereof, can also inhibit in vitro lysis (e.g., lysis of rabbit or chicken red blood cells) by greater than 50 (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more) %. Suitable in vitro hemolysis assays for evaluating any of the compounds (e.g., anti-C5d antibodies or antigen-binding fragments thereof) described herein are known in the art and described in, e.g., Rinder et al. (1995) J Clin Invest 96: 1564-1572.

In some embodiments, inhibition of terminal complement activity can be measured using an in vitro CH50eq assay. Inhibition, e.g., as it pertains to terminal complement activity, includes at least a 5 (e.g., at least a 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60) % decrease in the activity of terminal complement in, e.g., a hemolytic assay or CH50eq assay as compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and at an equimolar concentration. Substantial inhibition, as used herein, refers to inhibition of a given activity (e.g., terminal complement activity) of at least 40 (e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 or greater) %.

In some embodiments, the anti-C5d antibody or C5d-binding fragment thereof does not block the cleavage of C5 into fragments C5a and C5b. Methods for determining whether, in the presence of an anti-C5d antibody or a C5d-binding fragment thereof, complement component C5 can be cleaved by an AP or CP C5 convertase are known in the art and described in, e.g., U.S. Patent No. 6,355,245 and Wurzner et al. (1991) Complement Inflamm 8:328-340. See also below.

Methods for Producing the Anti-C5d Antibodies and Antigen-binding Fragments Thereof

The disclosure also features methods for producing any of the anti-C5d antibodies or antigen-binding fragments thereof described herein. In some embodiments, methods for preparing an antibody described herein can include immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. Suitable immunogens for generating any of the antibodies described herein are set forth herein. For example, to generate an antibody that binds to the C5d domain of complement component C5 (e.g., human complement component C5), a skilled artisan can immunize a suitable subject (e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with a C5d polypeptide such as the C5d polypeptide comprising the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16. In some embodiments, the antigen is an antigenic peptide fragment of C5d (e.g., a C6-binding fragment of C5d), e.g., having the amino acid sequence depicted in any one of SEQ ID NOs:3-8.

In some embodiments, the antigen comprises, or consists of, the following amino acid sequence:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWR K FELGLEFPNLPY

YIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDF

ETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCL

DAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSLS

QEGINILTHLPKGSAEAELMSWPVFYVFHYLETGNHWNIFHSDPLIEKQKLKK

KLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSI

CNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKA

FDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSAL

KREALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDIN

YVNPVIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLS (SEQ ID

NO: 17), which comprises a glutathione-S-transferase sequence (normal font) and a human

C5d protein portion (amino acids 992-1310 of SEQ ID NO: 1) in bold.

In some embodiments, the antigen comprises, or consists of, the following amino acid sequence:

MGGSHHHHHHGMASMTGGOOMGRTLYDDDDiOjRWGSOEGINILTHLPKGSAEA ELMSWPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNAD YSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICNSLLWLVENYQLDNG SFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLVKIDTALIKAD NFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGNPPIYRF WKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVIKWLSEEQRY GGGFYSTQDTINAIEGLTEYSLLVKQLRLS (SEQ ID NO: 18), which comprises a histidine tag (six histidines) sequence (underlined), an enterokinase cleavage site (italics), and a human C5d protein portion (amino acids 993-1310 of SEQ ID NO: 1) in bold.

In some embodiments, the non-human mammal is C5 deficient, e.g., a C5-deficient mouse described in, e.g., Levy and Ladda (1971) Nat New Biol 229(2):51-52; Crocker et al. (1974) J Clin Pathol 27(2): 122-124; Wetsel et al. (1990) J Biol Chem 265:2435-2440; and Jungi and Pepys (1981) Immunology 43£2):271-279. Methods for producing a purified recombinant human C5d protein are described herein. A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen along with subsequent booster immunizations a number of times sufficient to elicit the production of an antibody by the mammal. The immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corymb acterium parvum or Salmonella Minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in the methods for inducing an immune response include, e.g., cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(l): 15-24. See also, e.g., Lodmell et al. (2000) Vaccine 18: 1059-1066; Johnson et al. (1999) J Med Chem 42:4640-4649; Baldridge et al. (1999) Methods 19: 103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, methods for immunizing a non-human mammal (e.g., a rodent) can include administering to the non-human mammal a nucleic acid (e.g., DNA or messenger RNA) that encodes the C5d domain of C5 (e.g., the C5d domain of human C5). For example, a eukaryotic expression vector construct encoding the human C5d domain (e.g., having the amino acid sequence depicted in SEQ ID NO:2) can be created using standard molecular biology techniques and a suitable amount of the purified construct can be injected (e.g., intramuscular, intravenous, subcutaneous, or intraperitoneal injection) into the mammal. See, e.g., Anderson et al. (1996) Infection and Immunity 64(8):3168-3173 and Angus et al. (2000) J Infectious Disease 181 :317-324. Alternatively, the construct can be delivered to the non-human mammal by topical application (e.g., oral or intravaginal administration). Nucleic acid-based immunizations can also include, optionally, one or more additional injections/applications of the construct as a booster.

In some embodiments, the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal such as a laboratory mouse is immunized with a C5d polypeptide as described above. Antibody-producing cells (e.g., B cells of the spleen) of the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter such as, e.g., vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb/c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. After the fusion, the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The obtained hybrid cells are then screened for secretion of the desired antibodies, e.g., an antibody that binds to C5d and inhibits the interaction between C5b and C6.

In some embodiments, a skilled artisan can identify an anti-C5d antibody from a non- immune biased library as described in, e.g., U.S. patent no. 6,300,064 (to Knappik et al; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

In some embodiments, the methods described herein can involve, or be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (see, e.g., Etz et al. (2001,⁾ J Bacteriol 183:6924-6935;

Cornells (2000) Curr Opin Biotechnol U_:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10: 1303-1310; Yeung et al. (2002)

Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444;

Grabherr et al. (2001) Comb Chem High Throughput Screen 4: 185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231 : 119-135; and Hanes et al. (2000) Nat Biotechnol 18: 1287-1292).

Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide- bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage used in these methods are typically filamentous phage such as fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to any of the phage coat proteins pill, pVIII, or pIX. See, e.g., Shi et al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to make the

immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184: 177- 186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9- 18; Burton et al. (1994) Advances in Immunology 57: 191-280; and PCT publication nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401. Suitable methods are also described in, e.g., U.S. patent nos. 5,698,426;

5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

In some embodiments, the phage display antibody libraries can be generated using mRNA collected from B cells from the immunized mammals. For example, a splenic cell sample comprising B cells can be isolated from mice immunized with C5d polypeptide, or an antigenic peptide fragment thereof, as described above. mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. See, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2^nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995), supra. The cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins are used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995) JNeurosci Methods 62(1 -2 :213-9; Di Niro et al. (2005) Biochem J 388(Pt 3 :889- 894; and Engberg et al. (1995) Methods Mol Biol 51 :355-376.

In some embodiments, a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library. Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pill, pVIII, or pIX of M13 phage) and a different antigen-combining region are produced using standard molecular biology

techniques and then introduced into a population of bacteria (e.g., E. coli). Expression of the bacteriophage in bacteria can, in some embodiments, require use of a helper phage. In some embodiments, no helper phage is required (see, e.g., Chasteen et al. (2006) Nucleic Acids Res 34(21):el45). Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen (e.g., a C5d polypeptide or an antigenic peptide fragment thereof) bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support.

In some embodiments, the immobilized phage are the phage of interest. Accordingly, the unbound phage are removed by washing the support. Following the wash step, bound phage are then eluted from the solid support, e.g., using a low pH buffer or a free target antigen competitor, and recovered by infecting bacteria. In some embodiments, the phage that are not immobilized are the phage of interest. In such embodiments, the population of phage can be contacted to the antigen two or more times to deplete from the population any of the phage that bind to the support. Unbound phage are then collected and used for subsequent screening steps.

To enrich the phage population for phage particles that contain antibodies having a higher affinity for the target antigen (while reducing the proportion of phage that may bind to the antigen non-specifically), the eluted phage (described above) can be used to re-infect a population of bacterial host cells. The expressed phage are then isolated from the bacteria and again contacted to a target antigen. The concentration of antigen, pH, temperature and inclusion of detergents and adjuvants during contact can be modulated to enrich for higher affinity antibody fragments. The unbound phage are removed by washing the solid support. The number or cycles, duration, pH, temperature and inclusion of detergents and adjuvants during washing can also be modulated to enrich for higher affinity antibody fragments. Following the wash step, bound phage are then eluted from the solid support. Anywhere from one to six iterative cycles of panning may be used to enrich for phage containing antibodies having higher affinity for the target antigen. In some embodiments, a deselection step can also be performed in conjunction with any of the panning approaches described herein.

Individual phage of the population can be isolated by infecting bacteria and then plating at a density to allow formation of monoclonal antibodies.

For example, to identify using phage display techniques an antibody that binds to C5d, but not to C5, or an antibody that binds to C5d with a higher affinity than to C5, the following panning approach can be employed. The population of phage can first be contacted to a surface containing bound native, full-length human C5. The process can be repeated two or more times, each time collecting the unbound phage. The population can also be contacted to a solid support containing surface-bound C4 and/or C3 proteins.

Unbound phage from the foregoing steps are then contacted to a surface containing bound C5d or an antigenic peptide fragment thereof. Phage that bind to C5d (or the antigenic peptide fragments) are eluted from the surface and recovered by infecting bacteria. Iterative rounds of phage selection may be performed. After one to six rounds of selection, individual recovered phagemid can be screened for expression of antibody fragments with the desired specificity and affinity. A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular immunogen (e.g., C5d) using any immunological or biochemical based method known in the art. For example, specific binding of an antibody to C5d, as compared to native, full-length C5, may be determined for example using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above. Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays,

immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

Antibodies can also be assayed using any SPR-based assays known in the art for characterizing the kinetic parameters of the interaction of the antibody with C5d. Any SPR instrument commercially available including, but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden); lAsys instruments (Affinity Sensors; Franklin,

Massachusetts); IBIS system (Windsor Scientific Limited; Berks, UK), SPR-CELLIA systems (Nippon Laser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta (Texas Instruments; Dallas, Texas) can be used in the methods described herein. See, e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al. (2002) Reviews in Mol Biotech 82: 303- 323; Fivash et al. (1998) Curr Opin Biotechnol 9: 97-101; and Rich et al. (2000) Curr Opin Biotechnol 11 : 54-61.

It is understood that the above methods can also be used to determine if, e.g., an anti- C5d antibody does not bind to full-length, native C5, C4, or C3 proteins or fragments thereof such as C3b, C3d, and/or C4b proteins. The above methods can also be used to determine if an antibody that binds to C5d also inhibits the interaction between C5b and C6. The above methods can also be used to determine if an antibody that binds to C5d also inhibits formation of terminal complement and subsequent lysis of cells (e.g., hemolysis).

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')₂ fragments can also be employed using methods known in the art such as those disclosed in PCT publication no. WO 92/22324; Mullinax et al. (1992) BioTechniques 12(6}: 864-869; Sawai et al. (1995) Am J Repr Immunol 34:26-34; and Better et al. (1988) Science 240: 1041-1043.

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. patent nos. 4,946,778 and 5,258,498; Huston et al. (1991)

Methods in Enzymology 203:46-88; Shu et al. (1993) Proc Nat Acad Sci USA 90:7995-7999; and Skerra et al. (1988) Science 240: 1038-1040.

Phage display technology can also be used to, e.g., increase the affinity of an antibody for its cognate antigen. The technology, referred to as affinity maturation, can employ mutagenesis or CDR walking and re-selection to identify antibodies that bind with higher affinity to an antigen as compared to the initial or parental antibody. See, e.g., Glaser et al.

(1992) J Immunol 149:3903-3913. Libraries can be constructed consisting of a pool of variant clones, each differing by one or more amino acid substitutions. Mutants with increased binding affinity for the antigen can be selected for by contacting the immobilized mutants with labeled antigen or any combination of methods described above. Any screening method known in the art can be used to identify mutant antibodies with increased affinity to the antigen (e.g., SPR or ELISA techniques).

In some embodiments, epitope mapping can be used to identify, e.g., the region of

C5d that interacts with an antibody, e.g., a region of C5d that binds to C6. Methods for identifying the epitope to which a particular antibody binds are also known in the art and are described above.

The antibodies and fragments thereof identified herein can be or can be made

"chimeric." Chimeric antibodies and antigen-binding fragments thereof comprise portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced with mouse variable regions of desired specificity fused to human constant domains (for example, U.S. Patent No. 4,816,567). In this manner, non-human antibodies can be modified to make them more suitable for human clinical application (e.g., methods for treating or preventing a complement-mediated condition in a subject).

The monoclonal antibodies of the present disclosure include "humanized" forms of the non-human (e.g., mouse) antibodies. Humanized or CDR-grafted mAbs are particularly useful as therapeutic agents for humans because they are not cleared from the circulation as rapidly as mouse antibodies and do not typically provoke an adverse immune reaction.

Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Methods of preparing humanized antibodies are generally well known in the art. For example, humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting rodent frameworks or CDR sequences for the corresponding sequences of a human antibody. Also see, e.g., Staelens et al. (2006) Mo I Immunol 43: 1243-1257. In some embodiments, humanized forms of non-human (e.g., mouse) antibodies are human antibodies (recipient antibody) in which the CDR region amino acid residues of the non-human antibody (e.g., mouse, rat, rabbit, or non-human primate antibody) having the desired specificity, affinity, and binding capacity are grafted onto the framework scaffold of a human antibody. Additional humanization methods are described below in the working examples.

Methods for grafting CDR sequences from a donor antibody (e.g., a non-human antibody) to the framework regions of an acceptor antibody (e.g., a human antibody) are well known in the art and are described in, e.g., Jones et al. (1986) Nature 321 :522-525;

Verhoeyen et al. (1988) Science 239(4847 : 1534-1536; Riechmann et al. (1988) Nature 332:323-327; Queen et al. (1989) Proc Natl Acad Sci USA 86:10029-10033; PCT publication no. WO 93/011237; Kettleborough et al. (1991) Protein Engineering, Design and Selection 4:773-783; Benny K. C. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," W.H. Freeman and Co., NY; and Borrebaek (1995) "Antibody Engineering," 2^nd Edition, Oxford University Press, NY, Oxford. For example, CDRs from a donor antibody can be grafted onto framework regions of an acceptor antibody using overlap extension polymerase chain reaction (PCR) techniques as described in, e.g., Daugherty et al. (1991) Nucleic Acids Res 19(9}:2471-2476; Roguska et al. (1996) Protein Engineering 9(10 :895-904; and Yazaki et al. (2004) Protein Engineering, Design & Selection 17(5}:481-489.

In embodiments where the selected CDR amino acid sequences are short sequences (e.g., fewer than 10-15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1 : 129-130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody.

In some instances, one or more framework region amino acid residues of the human immunoglobulin are also replaced by corresponding amino acid residues of the non-human antibody (so called "back mutations"). In addition, phage display libraries can be used to vary amino acids at chosen positions within the antibody sequence. The properties of a humanized antibody are also affected by the choice of the human framework. Furthermore, humanized and chimerized antibodies can be modified to comprise residues that are not found in the recipient antibody or in the donor antibody in order to further improve antibody properties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human immunoglobulin sequences, preferably human germline sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human or human antibodies may be derived from transgenic mice carrying human antibody genes (carrying the variable (V), diversity (D), joining (J), and constant (C) exons) or from human cells. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al. (1993) Proc Natl Acad Sci USA 90:2551; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355:258. Transgenic mouse strains can be engineered to contain gene sequences from unrearranged human immunoglobulin genes. One example of such a mouse is the HuMAb Mouse® (Medarex, Inc.), which contains human immunoglobulin transgene miniloci that encode unrearranged human μ heavy and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci. See, e.g., Lonberg, et al. (1994) Nature 368(6474):856-859. The preparation and use of HuMab mice, and the genomic modifications carried by such mice, are further described in Taylor et al. (1992) Nucleic Acids Res 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc Natl Acad Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4: 1 17-123; Tuaillon et al. (1994) J Immunol 152:2912-2920; Taylor et al. (1994) International Immunology 6:579-591; and Fishwild et al. (1996) Nature Biotechnol 14:845- 851. An alternative transgenic mouse system for expressing human immunoglobulin genes is referred to as the Xenomouse (Abgenix, Inc.) and is described in, e.g., U.S. patent nos.

6,075,181; 6,114,598; 6,150,584; and 6,162,963. Like the HuMAb Mouse® system, the Xenomouse system involves disruption of the endogenous mouse heavy and light chain genes and insertion into the genome of the mouse transgenes carrying unrearranged human heavy and light chain immunoglobulin loci that contain human variable and constant region sequences. Other systems known in the art for expressing human immunoglobulin genes include the KM Mouse® system, described in detail in PCT Publication WO 02/43478 and the TC mouse system described in Tomizuka et al. (2000) Proc Natl Acad Sci USA 97:722- 727.

The human sequences may code for both the heavy and light chains of human antibodies and would function correctly in the mice, undergoing rearrangement to provide a wide antibody repertoire similar to that in humans. The transgenic mice can be immunized with the target protein immunogen to create a diverse array of specific antibodies and their encoding R A. Nucleic acids encoding the antibody chain components of such antibodies may then be cloned from the animal into a display vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and the separate populations then recombined on insertion into the vector, such that any given copy of the vector receives a random combination of a heavy and a light chain. The vector is designed to express antibody chains so that they can be assembled and displayed on the outer surface of a display package containing the vector. For example, antibody chains can be expressed as fusion proteins with a phage coat protein from the outer surface of the phage. Thereafter, display packages can be selected and screened for display of antibodies binding to a target.

In addition, the phage-display libraries screened above can include human antibodies (Hoogenboom et al. (1992) JMol Biol 227:381; Marks et al. (1991) JMol Biol 222:581-597; and Vaughan et al. (1996) Nature Biotech 14:309). Synthetic phage libraries can be created which use randomized combinations of synthetic human antibody V-regions. By selection on antigen, fully human antibodies can be made in which the V-regions are very human-like in nature. See, e.g., U.S. Patent Nos. 6,794,132; 6,680,209; 4,634,666; and Ostberg et al. (1983) Hybridoma 2:361-367, the contents of each of which are incorporated herein by reference in their entirety. For the generation of human antibodies, also see Mendez et al. (1998) Nature

Genetics 15: 146-156 and Green and Jakobovits (1998) J Exp Med 188:483-495, the disclosures of which are hereby incorporated by reference in their entirety. Human antibodies are further discussed and delineated in U.S. Patent Nos.: 5,939,598; 6,673,986; 6,114,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. Patent Publication Nos. 20030229905 Al, 20040010810 Al, 20040093622 Al, 20060040363 Al, 20050054055 Al, 20050076395 Al, and 20050287630 Al . See also International

Publication Nos. WO 94/02602, WO 96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 Bl . The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more ½ genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in, e.g., U.S. Patent Nos.: 5,545,807; 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the disclosures of each of which are hereby

incorporated by reference in their entirety. See also European Patent No. 0 546 073 Bl, International Patent Application Publication Nos. WO 92/03918, WO 92/22645, WO

92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, the disclosures of each of which are hereby incorporated by reference in their entirety. See further Taylor et al. (1992) Nucleic Acids Res 20: 6287; Chen et al. (1993) Int Immunol 5: 647; TuaiUon et al. (1993) Proc Natl Acad Sci USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor et al. (1994) International Immunology 6: 579-591; TuaiUon et al. (1995) J. Immunol 154: 6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and TuaiUon et al. (2000) Eur J Immunol. 10: 2998-3005, the disclosures of each of which are hereby incorporated by reference in their entirety.

In certain embodiments, de-immunized forms of the antibodies, or antigen-binding fragments described herein are provided. De-immunized antibodies or antigen-binding fragments thereof are antibodies that have been modified so as to render the antibody or antigen-binding fragment thereof non-immunogenic, or less immunogenic, to a given species. De-immunization can be achieved by modifying the antibody or antigen-binding fragment thereof utilizing any of a variety of techniques known to those skilled in the art (see, e.g., PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, an antibody or antigen-binding fragment thereof may be de -immunized by identifying potential T cell epitopes and/or B cell epitopes within the amino acid sequence of the antibody or antigen- binding fragment thereof and removing one or more of the potential T cell epitopes and/or B cell epitopes from the antibody or antigen-binding fragment thereof, for example, using recombinant techniques. The modified antibody or antigen-binding fragment thereof may then optionally be produced and tested to identify antibodies or antigen-binding fragments thereof that have retained one or more desired biological activities, such as, for example, binding affinity, but have reduced immunogenicity. Methods for identifying potential T cell epitopes and/or B cell epitopes may be carried out using techniques known in the art, such as, for example, computational methods (see e.g., PCT Publication No. WO 02/069232), in vitro or in silico techniques, and biological assays or physical methods (such as, for example, determination of the binding of peptides to MHC molecules, determination of the binding of peptide:MHC complexes to the T cell receptors from the species to receive the antibody or antigen-binding fragment thereof, testing of the protein or peptide parts thereof using transgenic animals with the MHC molecules of the species to receive the antibody or antigen- binding fragment thereof, or testing with transgenic animals reconstituted with immune system cells from the species to receive the antibody or antigen-binding fragment thereof, etc.). In various embodiments, the de -immunized antibodies described herein include de- immunized antigen-binding fragments, Fab, Fv, scFv, Fab' and F(ab')₂, monoclonal antibodies, murine antibodies, engineered antibodies (such as, for example, chimeric, single chain, CDR-grafted, humanized, and artificially selected antibodies), synthetic antibodies and semi-synthetic antibodies.

In the therapeutic embodiments of the present disclosure, bispecific antibodies are contemplated. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for C5d, the other one is for any other antigen (e.g., C5a). Suitable anti-C5a antibodies useful as constituents of the bispecific antibodies described herein include, e.g., those anti-C5a antibodies disclosed in International patent application publication no. WO 2011/137395.

Methods for making bispecific antibodies are within the purview of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co- expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/ light-chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537- 539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, C_H2, and C_H3 regions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of illustrative currently known methods for generating bispecific antibodies see, e.g., Suresh et al. (1986) Methods in Enzymology 121 :210; PCT Publication No. WO 96/27011; Brennan et al. (1985) Science 229:81; Shalaby et al. J Exp Med (1992) 175:217-225; Kostelny et al. (1992) J Immunol 148(5}: 1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also include cross-linked or

heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol

148(5 : 1547-1553. The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers and then re -oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See, e.g.,

Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies can be "linear antibodies" as described in, e.g., Zapata et al. (1995) Protein Eng. 8(10 : 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V_H-C_H1-V_H-C_H1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies) are

contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60.

The disclosure also embraces variant forms of multi-specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297. The DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain C_HI and Fc region.

Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715.

The disclosure also provides came lid or dromedary antibodies (e.g., antibodies derived from Camelus bactrianus, Calelus dromaderius, or lama paccos). Such antibodies, unlike the typical two-chain (fragment) or four-chain (whole antibody) antibodies from most mammals, generally lack light chains. See U.S. patent no. 5,759,808; Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al. (2003) Nature 424:783-788; and Pleschberger et al. (2003) Bioconjugate Chem 14:440-448.

Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx (Ghent, Belgium). As with other antibodies of non- human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the camelid can be "humanized" to thereby further reduce the potential immunogenicity of the antibody.

In some embodiments, the anti-C5d antibodies described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its corresponding unaltered constant region. Effector functions involving the constant region of the anti-C5d antibody may be modulated by altering properties of the constant or Fc region. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular embodiments, modulation includes situations in which an activity is abolished or completely absent.

An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide which has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region. An altered constant region which displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region which displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region. Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%>) of the binding to the FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to the FcR. Similarly, an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region. For example, in some embodiments, the anti-C5d antibody comprising an altered constant region can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activity of the unaltered form of the constant region. An anti-C5d antibody described herein comprising an altered constant region displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDC activity as exemplified herein.

In certain embodiments, the altered constant region has at least one amino acid substitution, insertion, and/or deletion, compared to a native sequence constant region or to the unaltered constant region, e.g. from about one to about one hundred amino acid substitutions, insertions, and/or deletions in a native sequence constant region or in the constant region of the parent polypeptide. In some embodiments, the altered constant region herein will possess at least about 70% homology (similarity) or identity with the unaltered constant region and in some instances at least about 75% and in other instances at least about

80% homology or identity therewith, and in other embodiments at least about 85%, 90% or

95% homology or identity therewith. The altered constant region may also contain one or more amino acid deletions or insertions. Additionally, the altered constant region may contain one or more amino acid substitutions, deletions, or insertions that result in altered post-translational modifications, including, for example, an altered glycosylation pattern (e.g., the addition of one or more sugar components, the loss of one or more sugar components, or a change in composition of one or more sugar components relative to the unaltered constant region).

Antibodies with altered or no effector functions may be generated by engineering or producing antibodies with variant constant, Fc, or heavy chain regions; recombinant DNA technology and/or cell culture and expression conditions may be used to produce antibodies with altered function and/or activity. For example, recombinant DNA technology may be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (such as, for example, Fc or constant regions) that affect antibody function including effector functions. Alternatively, changes in post-translational modifications, such as, e.g., glycosylation patterns, may be achieved by manipulating the cell culture and expression conditions by which the antibody is produced. Suitable methods for introducing one or more substitutions, additions, or deletions into an Fc region of an antibody are well known in the art and include, e.g., standard DNA mutagenesis techniques as described in, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2^nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT publication no. WO 06/53301; and U.S. patent no. 7,704,497.

In some embodiments, an anti-C5d antibody described herein exhibits reduced or no effector function. For example, the antibodies described herein can contain a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) Adv lmmun 51 : 1-18; Canfield et al. (1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mol Immunol 34(6}:441-452). In some embodiments (and in accordance with Kabat numbering), the IgGl and IgG4 constant regions comprise G249G250 residues whereas the IgG2 constant region does not comprise residue 249, but does comprise G250. In a G2/G4 hybrid constant region, where the 249-250 region comes from the G2 sequence, the constant region can be further modified to introduce a glycine residue at position 249 to produce a G2/G4 fusion having G249/G250. Other constant domain hybrids that comprise G249/G250 can also be part of engineered antibodies in accordance with the disclosure.

In addition to using a G2/G4 construct as described above, an anti-C5d antibody described herein having reduced effector function may be produced by introducing other types of changes in the amino acid sequence of certain regions of the antibody. Such amino acid sequence changes include but are not limited to the Ala- Ala mutation described in, e.g., PCT Publication nos. WO 94/28027 and WO 98/47531; and Xu et al. (2000) Cell Immunol 200: 16-26. Thus, in some embodiments, an anti-C5d antibody with one or more mutations within the constant region including the Ala- Ala mutation has reduced or no effector function. According to these embodiments, the constant region of the antibody can comprise a substitution to an alanine at position 234 or a substitution to an alanine at position 235. Additionally, the altered constant region may contain a double mutation: a mutation to an alanine at position 234 and a second mutation to an alanine at position 235. In one embodiment, an anti-C5d antibody comprises an IgG4 framework, wherein the Ala- Ala mutation would describe a mutation(s) from phenylalanine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. In another embodiment, the anti-C5d antibody comprises an IgGl framework, wherein the Ala-Ala mutation would describe a mutation(s) from leucine to alanine at position 234 and/or a mutation from leucine to alanine at position 235. An anti-C5d antibody may alternatively or additionally carry other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. (2001) J Virol 75: 12161-12168). An antibody with said mutation(s) in the constant region may furthermore be a blocking or non-blocking antibody.

Additional substitutions that, when introduced into a heavy chain constant region, result in decreased effector function are set forth in, e.g., Shields et al. (2001) J Biol Chem 276(9}:6591-6604. See particularly Table 1 ("Binding of human IgGl variants to human FcRn and FcyR) of Shields et al., the disclosure of which is incorporated herein by reference in its entirety. By screening a library of anti-IgE antibodies, each antibody of the library differing by one or more substitutions in the heavy chain constant region, for binding to a panel of Fc receptors (including FcRn, FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA), the authors identified a number of substitutions that modulate specific Fc-Fc receptor interactions. For example, a variant IgG2a heavy chain constant region in which the CH2 domain contains a D265A substitution (heavy chain amino acid numbering according to Kabat et al. {supra)) results in a complete loss of interaction between the variant constant region and IgG Fc receptors FcyRIIB, FcyRIII, FcyRI, and FcyRIV. Shields et al. (2001) at page 6595, Table 1. See also Baudino et al. (2008) J Immunol 181 :6664-6669 {supra).

Changes within the hinge region also affect effector functions. For example, deletion of the hinge region may reduce affinity for Fc receptors and may reduce complement activation (Klein et al. (1981) Proc Natl Acad Sci USA 78: 524-528). The present disclosure therefore also relates to antibodies with alterations in the hinge region. In some embodiments, an anti-C5d antibody may contain an altered constant region exhibiting enhanced or reduced complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region of the antibody. See, e.g., U.S. patent no. 6,194,551. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See, e.g., Caron et al. (1992) J Exp Med 176: 1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCT publication nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988) Nature 322:738-40; and U.S. Patent Nos. 5,648,260 and 5,624,821.

Another potential means of modulating effector function of antibodies includes changes in glycosylation, which is summarized in, e.g., Raju (2003) BioProcess International l(4):44-53. According to Wright and Morrison, the microheterogeneity of human IgG oligosaccharides can affect biological functions such as CDC and ADCC, binding to various Fc receptors, and binding to Clq protein. (1997) TIBTECH 15:26-32. Glycosylation patterns of antibodies can differ depending on the producing cell and the cell culture conditions (Raju, supra). Such differences can lead to changes in both effector function and pharmacokinetics. See, e.g., Israel et al. (1996) Immunology 89(4 :573-578; Newkirk et al. (1996) Clin Exp Immunol 106(2):259-264. Differences in effector function may be related to the IgG's ability to bind to the Fey receptors (FcyRs) on the effector cells. Shields et al. have shown that IgG, with alterations in amino acid sequence that have improved binding to FcyR, can exhibit up to 100% enhanced ADCC using human effector cells. (2001) J Biol Chem 276(9 :6591-6604. While these alterations include changes in amino acids not found at the binding interface, both the nature of the sugar component as well as its structural pattern may also contribute to the differences observed. In addition, the presence or absence of fucose in the

oligosaccharide component of an IgG can improve binding and ADCC. See, e.g., Shields et al. (2002) J Biol Chem 2770O}:26733-2674O. An IgG that lacked a fucosylated carbohydrate linked to Asn²⁹⁷ exhibited normal receptor binding to the FcyRI receptor. In contrast, binding to the FcyRIIIA receptor was improved 50-fold and accompanied by enhanced ADCC, especially at lower antibody concentrations.

Shinkawa et al. demonstrated that an antibody to the human IL-5 receptor produced in a rat hybridoma showed more than 50% higher ADCC when compared to the antibody produced in Chinese hamster ovary cells (CHO) (Shinkawa et al. (2003) J Biol Chem 278(5):3466-73). Monosaccharide composition and oligosaccharide profiling showed that the rat hybridoma-produced IgG had a lower content of fucose than the CHO-produced protein. The authors concluded that the lack of fucosylation of an IgGl has a critical role in enhancement of ADCC activity.

A different approach was taken by Umana et al. who changed the glycosylation pattern of chCE7, a chimeric IgGl anti-neuroblastoma antibody. (1999) Nat Biotechnol 17(2): 176- 180). Using tetracycline, they regulated the activity of a glycosyltransferase enzyme (GnTIII) which bisects oligosaccharides that have been implicated in ADCC activity. The ADCC activity of the parent antibody was barely above background level. Measurement of ADCC activity of the chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity. This activity correlated with the level of constant region-associated, bisected complex oligosaccharide. Newly optimized variants exhibited substantial ADCC activity. Similarly, Wright and Morrison produced antibodies in a CHO cell line deficient in glycosylation and showed that antibodies produced in this cell line were incapable of complement-mediated cyto lysis.

(1994) J Exp Med 180: 1087-1096. Thus, as known alterations that affect effector function include modifications in the glycosylation pattern or a change in the number of glycosylated residues, the present disclosure relates to an anti-C5d antibody wherein glycosylation is altered to either enhance or decrease effector function(s) including ADCC and CDC. Altered glycosylation includes a decrease or increase in the number of glycosylated residues as well as a change in the pattern or location of glycosylated residues.

Still other approaches exist for altering the effector function of antibodies. For example, antibody-producing cells can be hypermutagenic, thereby generating antibodies with randomly altered polypeptide residues throughout an entire antibody molecule. See, e.g., PCT publication no. WO 05/011735. Hypermutagenic host cells include cells deficient in DNA mismatch repair. Antibodies produced in this manner may be less antigenic and/or have beneficial pharmacokinetic properties. Additionally, such antibodies may be selected for properties such as enhanced or decreased effector function(s). Additional details of molecular biology techniques useful for preparing an antibody or antigen-binding fragment thereof described herein are set forth below.

Isolated C5d-containing Polypeptides and Fragments Thereof

As featured herein are isolated C5d-containing polypeptides and fragments thereof

(e.g., antigenic peptide fragments thereof). The isolated polypeptides can, in some embodiments, comprise the C5d domain of human complement component C5 having the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16. Such polypeptides are not, e.g., full-length C5b polypeptides or full-length, uncleaved C5 polypeptides.

In some embodiments, the C5d-containing polypeptides are fragments, e.g., antigenic peptide fragments, comprising at least six (e.g., at least seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 or more) consecutive amino acids of any one of SEQ ID NOs:2 or 14-16. In some embodiments, a fragment of a C5d-containing polypeptide can consist of, or comprise, the amino acid sequence depicted in any one of SEQ ID NOs:3-8. In some embodiments, the C5d-containing polypeptides, or fragments thereof, bind to complement component C6 (e.g., human complement component C6). Methods for detecting and/or quantifying the interaction between two proteins are known in the art and described herein. Suitable methods for detecting the interaction between C5b and C6 are described in, e.g., Discipio et al. (1999) J Biol Chem 274:31811-31818. In some embodiments, the C5d- containing polypeptide, or peptide fragment thereof, can inhibit the interaction between C5b and C6. Methods for determining whether a compound inhibits the interaction between C5b and C6 are described herein and can include, e.g., binding assays as well as functional assays measuring inhibition of terminal complement activity.

In some embodiments, any of the C5d-containing polypeptides, or fragments thereof (e.g., antigenic peptide fragments, C6-binding fragments, or fragments that inhibit the interaction between C5b and C6) can further include a heterologous moiety. The

heterologous moiety can be, e.g., any of those described herein or any others known in the art. One exemplary heterologous moiety useful in connection with the C5d-containing polypeptides described herein is the Fc domain of an antibody. In some embodiments, the C5d-containing polypeptide, or fragment thereof, can be a fusion protein comprising the C5d-containing polypepeptide (or fragment thereof) and a corresponding heterologous moiety described herein. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, or a luminescent label. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG, polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), or maltose- binding protein (MBP)) for use in purifying the antibodies. Heterologous polypeptides also include polypeptides that are useful as diagnostic or detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). Where the heterologous moiety is a polypeptide, the moiety can be incorporated into a fusion protein described herein, resulting in a fusion protein. Heterologous polypeptides also include, e.g., growth factors, cytokines, and chemokines. Growth factors can include, e.g., vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), bone morphogenic protein (BMP), granulocyte-colony stimulating factor (G-CSF), granulocyte- macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF); a

neurotrophin, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), basic fibroblast growth factor (bFGF or FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and a neuregulin (e.g., NRG1, NRG2, NRG3, or NRG4). Cytokines include, e.g., interferons (e.g., IFNy), tumor necrosis factor (e.g., TNFa or TNF ), and the interleukins (e.g., IL-1 to IL-33 (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, or IL- 15)). Chemokines include, e.g., 1-309, TCA-3, MCP-I, ΜΙΡ-Ια, ΜΙΡ-Ιβ, RANTES, CIO, MRP-2, MARC, MCP-3, MCP-2, MRP-2, CCF18, Eotaxin, MCP-5, MCP-4, NCC-I, HCC-I, leukotactin-1, LEC, NCC-4, TARC, PARC, or Eotaxin-2.

In some embodiments, the heterologous moiety is a targeting moiety. For example, in some embodiments, a fusion protein can comprise in any order all or part of a factor H polypeptide, e.g., a human factor H polypeptide having the following amino acid sequence: MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRS LGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYT CNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHF GQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYK ENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHR TGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRR PYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGY NQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVKTCSKSSIDI ENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSCDIPV FMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYEREC ELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPICKEQV QSCGPPPELLNGNVKEKTKEEYGHSEVVEYYCNPRFLMKGPNKIQCVDGEWTTLPV CIVEESTCGDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLP QCVAIDKLK CKSSNLIILEEHLK KKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPE

VNCSMAQIQLCPPPPQIPNSHNMTTTLNYRDGEKVSVLCQENYLIQEGEEITCKDGR

WQSIPLCVEKIPCSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTCYM

GKWSSPPQCEGLPCKSPPEISHGVVAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCL

GEKWSHPPSCIKTDCLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNV

TCINSRWTGRPTCRDTSCVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDE

EVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQL

EGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYSRTGESVEFVC

KRGYRLSSRSHTLRTTCWDGKLEYPTCAKR (SEQ ID NO: 12). In some embodiments, a fusion protein described herein can include exemplary targeting fragments of factor H such as amino acids 265 to 507 of SEQ ID NO: 12 and/or amino acids 1107 to 1230 of SEQ ID

NO: 12.

In some embodiments, a fusion protein can comprise in any order all or part of a targeting polypeptide such as CR2, e.g., a human CR2 polypeptide comprising the following amino acid sequence:

MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGT

FRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPY

RHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLPTCVSVFPLECPALPMIHNG

HHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSSGKWSAVPPTCEEARCKSLGRFP

NGKVKEPPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPP

PILNGRHIGNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWSGPA

PRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQ

GTWEPSAPVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQC

TSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYKLSGSVYQEC

QGTIPWFMEIRLCKEITCPPPPVIYNGAHTGSSLEDFPYGTTVTYTCNPGPERGVEFSLI

GESTIRCTSNDQERGTWSGPAPLCKLSLLAVQCSHVHIANGYKISGKEAPYFYNDTV

TFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKETCQHVRQSLQELPAGSRVELVNTS

CQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCHPPPVIVNGKHTGMMAENFLY

GNEVSYECDQGFYLLGEK LQCRSDSKGHGSWSGPSPQCLRSPPVTRCPNPEVKHG

YKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIGCPPP

PKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTWSQPAPHCKEVN

CSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGSPQSQCQSDHQWNPPLA

VCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRARNYYTDTSQKEAFHLEAREVYS

VDPYNPAS (SEQ ID NO:13). Exemplary fragments of CR2 useful for targeting a polypeptide to the site of complement activation include, e.g., amino acids 21 to 148 of SEQ ID NO: 13, amino acids 21 to 273 of SEQ ID NO: 13, or amino acids 21 to 344 of SEQ ID NO: 13.

In some embodiments, a fusion protein can comprise an antibody that binds to a natural CR2 ligand (e.g., C3d). That is, in some embodiments, the targeting polypeptide is an antibody that binds to a natural ligand of CR2, e.g., C3d, iC3b, and C3dg. The targeting polypeptide can be, e.g., an antigen-binding fragment of an antibody that binds to a natural ligand of CR2. In some embodiments, the antibody binds to a human C3d, iC3b, or C3dg polypeptide. For example, the targeting moiety may comprise antibodies which bind to C3 or C3 fragments such as iC3b, C3d and C3dg, or an antigen-binding fragment thereof.

Antibodies that bind to C3 and to cleavage fragments C3b, iC3b and C3d, are known. For example, see U.S. Patent No. 6,572,856, Taylor; Tosic et al. (1989) J Immunol Methods 120:241-249; Sokoloff et al. (2000) Cancer Immunol Immunother 49:551-562 (2000);

Mastellos et al. (2004) Mol Immunol 40: 1213-1221; Dilillo et al. (2006) Mo I Immunol 43: 1010-1019; U.S. patent application publication nos. 20090081211, and 20090175875; and Aguado et al. (1985) J Clin Invest 76: 1418-1426. The disclosure of these documents is hereby incorporated herein by reference. Such antibodies, and functional fragments thereof, may be useful in the present invention as the targeting moiety for directing therapeutic fragments to tissue experiencing complement activation, and thus expressing C3 or its fragments. Additional fragments of CR2, factor H, or anti-C3d antibodies useful for targeting a C5d-containing polypeptide are described in International patent application publication nos. WO 2011/163412 and WO 2007/149567, the disclosures of each of which are incorporated herein by reference in their entirety.

The C5d-containing polypeptides, or peptide fragments thereof, are used in a variety of applications including, but not limited to, methods for generating an immune response in a non-human mammal and thus producing an anti-C5d antibody. The polypeptides and fragments, particularly those that inhibit the interaction between C5b and C6, are further useful in therapeutic applications for treating human disease (e.g., complement-associated conditions). Recombinant Protein Expression and Purification

The antibodies or antigen-binding fragments thereof described herein, or the C5d polypeptides (or fusion proteins comprising the C5d polypeptides), can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

Several possible vector systems are available for the expression of recombinant polypeptides, e.g., cloned heavy chain and light chain polypeptides or C5d-containing polypeptides, from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo

(Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81 : 1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaP0₄ precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated trans fection, protoplast fusion, and direct microinjection.

Appropriate host cells for the expression of recombinant proteins (e.g., antibodies or C5d-containing polypeptides) include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.

In some embodiments, a recombinant protein such as an antibody or C5d-containing polypeptide can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2 : 155-159; and Pollock et al. (1999) J Immunol Methods 23 K1-2 147-157.

The C5d-containing polypeptides, antibodies, and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al.

(1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—A Laboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

Following expression, the recombinant proteins (e.g., C5d-containing polypeptides or antibodies and antigen-binding fragments thereof) can be isolated. The term "purified" or

"isolated" as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryotic or eukaryotic cell expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.

The recombinant proteins (e.g., C5d-containing polypeptides or antibody or fragment thereof) can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic

techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) "Protein Purification, 3^rd edition," Springer- Verlag, New York City, New York. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed proteins (e.g., antibody or fragments or C5d-containing polypeptides) will be necessary.

Methods for determining the yield or purity of a purified protein are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

In some embodiments, endotoxin can be removed from the protein preparations.

Methods for removing endotoxin from a protein sample are known in the art and exemplified in the working examples. For example, endotoxin can be removed from a protein sample using a variety of commercially available reagents including, without limitation, the

ProteoSpin™ Endotoxin Removal Kits (Norgen Biotek Corporation), Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific; Pierce Protein Research Products), MiraCLEAN®

Endotoxin Removal Kit (Minis), or Acrodisc™ - Mustang® E membrane (Pall Corporation).

Methods for detecting and/or measuring the amount of endotoxin present in a sample (both before and after purification) are known in the art and commercial kits are available. For example, the concentration of endotoxin in a protein sample can be determined using the QCL-1000 Chromogenic kit (BioWhittaker), the limulus amebocyte lysate (LAL)-based kits such as the Pyrotell®, Pyrotell®-T, Pyrochrome®, Chromo-LAL, and CSE kits available from the Associates of Cape Cod Incorporated. Modification of Proteins

The proteins described herein, e.g., anti-C5d antibodies, antigen-binding fragments thereof, or C5d-containing polypeptides, can be modified following their expression and purification. The modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the proteins by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.

In some embodiments, the proteins described herein can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK (SEQ ID NO:9)), polyhistidine (6- His; HHHHHH (SEQ ID NO: 10), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: l 1)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g., ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹1, ³⁵S, and ³H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7.

Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

Two molecules (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those which link two amino acid residues via a linkage that includes a "hindered" disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4- succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1 ,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m- maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the antibody, antigen-binding fragment thereof, or C5d-containing polypeptide. Alternatively, the radioactive label can be included as part of a larger molecule (e.g., ¹²⁵I in meta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds to free amino groups to form meta-iodophenyl (mlP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) JNucl Med 38: 1221-1229) or chelate (e.g., to DOTA or DTP A) which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules/chelates containing them to the proteins described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a

"fluorophore") to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein, or fragment thereof, with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications," John Wiley and Sons (ISBN 0471495603). In some embodiments, the proteins described herein (e.g., the anti-C5d antibodies, C5d-binding fragments thereof, or C5d-containing polypeptides) can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisic et al. (2010) IntJ Pharm 387(1-2): ! 10-119). The stabilization moiety can improve the stability, or retention of, the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the proteins described herein can be glycosylated. In some embodiments, recombinant proteins described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation. Methods for producing proteins (e.g., antibodies) with reduced glycosylation are known in the art and described in, e.g., U.S. patent no. 6,933,368; Wright et al. ( 1991 ) EMBO J 10(10):2717-2723 ; and Co et al. (1993) Mol Immunol 30: 1361.

Screening Methods

The disclosure also provides methods for identifying a compound that binds to the C5d domain of complement component C5 (e.g., human complement component C5). The methods can include use of one or more isolated, soluble C5d-containing polypeptides, or C6-binding fragments thereof. Such polypeptides and fragments are described in detail herein. Compounds that bind to C5d polypeptides, or C6-binding fragments thereof, can be identified by numerous methods known to those of skill in the art, for example, binding assays (e.g., Western blot or SPR methods), competition studies, or ligand panning.

Antibody-related screening methods (e.g., using phage display) are described above. For example, a candidate compound, such as, for example, an antibody, can be contacted to a C5d-containing polypeptide or C6-binding fragment thereof under conditions suitable for interaction. The binding of the compound to the C5d-containing polypeptide, or C6-binding fragment thereof, can be detected by way of, e.g., detectable label. In the case of antibodies, detection can be accomplished by way of a detectably-labeled second antibody that binds to the candidate antibody compound. Suitable detectable labels are known in the art and described herein. Appropriate labels include, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I,

35 S, 3 H, 32 P, 33 P, or 14 C), fluorescent moieties (e.g., fluorescein, rhodamine, or phycoerythrin), luminescent moieties (e.g., Qdot™ nanoparticles), compounds that absorb light of a defined wavelength, or enzymes (e.g., alkaline phosphatase or horseradish peroxidase), or any other labels known in the art and/or described herein. The products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Methods of detecting and/or for quantifying a detectable label depend on the nature of the label and are known in the art. Examples of detectors include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

In some embodiments, the C5d-containing polypeptides, or C6-binding fragments thereof, can be bound to a solid support or substrate. Suitable solid substrates to which the capture antibody can be bound include, without limitation, the plastic bottoms and sides of wells of microtiter plates, membranes such as nylon or nitrocellulose membranes, polymeric (e.g., without limitation, agarose, cellulose, or polyacrylamide) beads or particles.

A compound (e.g., an antibody or antigen-binding fragment thereof) identified as one binding to the C5d domain of complement component C5 can also be evaluated for its ability to selectively bind to C5d over full-length, native, uncleaved C5 or for a lack of binding to full-length, native, uncleaved C5. The compound may be further evaluated for its ability to inhibit the interaction between C5b and C6 and/or inhibit terminal complement activity. It is understood that these methods can be used in conjunction with the methods for generating anti-C5d antibodies described herein. In some embodiments, the candidate compound used in the screening method is not itself a C6-containing polypeptide (or C5b-binding fragment thereof) nor is it a C5b-containing polypeptide or C6-binding fragment thereof.

The C5d-containing polypeptides, or C6-binding fragment thereof, described herein can also be used to screen for compounds which inhibit the interaction between C5b and C6. Such screening methods can include, e.g., determining the inhibition of binding of ligand to a C6 polypeptide (or C5b-binding fragment thereof) in the presence of a candidate compound under conditions that permit binding between the ligand and the C6 polypeptide (or C5b- binding fragment thereof) in the absence of the candidate compound, wherein the ligand is an isolated C5d polypeptide or a C6-binding fragment thereof; and determining the amount of ligand bound to the C6 polypeptide (or C5b-binding fragment thereof), such that a compound capable of causing reduction of binding of the ligand is an inhibitor. Alternatively, the methods can include determining the inhibition of binding of ligand to an isolated C5d polypeptide, or a C6-binding fragment thereof, in the presence of a candidate compound under conditions to permit binding between the ligand and the C5d polypeptide, or a C6- binding fragment thereof, in the absence of the candidate compound, wherein the ligand is C6 polypeptide or C5b-binding fragment thereof; and determining the amount of ligand bound to the C5d polypeptide, or C6-binding fragment thereof, such that a compound capable of causing reduction of binding of the ligand is an inhibitor.

In some embodiments, a C5d-containing polypeptide, or C6-binding fragment thereof, can be immobilized on a solid substrate. Alternatively, the polypeptide or fragment can be bound to a plastic substrate (e.g., the plastic bottom of an ELISA (enzyme-linked

immunosorbent assay) plate well) using methods known in the art. The substrate-bound polypeptide or fragment is then exposed to a detectably-labeled C6 polypeptide, or C5b- binding fragment thereof, in the presence and absence of the test compound. After incubating the resulting mixture for a period of time and at temperature optimized for the system of interest, the presence and/or amount of C6 polypeptide, or C5b-binding fragment thereof, bound to the immobilized C5d polypeptide (or C6-binding fragment thereof) is then assayed by detecting the amount of detectable label present on the substrate. It will be appreciated that instead of binding the C5d polypeptides to the solid substrate, the C6 polypeptide can be bound to it. In that case, binding of the C5d polypeptide, or C6-binding fragment thereof, to the substrate-bound C6 polypeptide is tested by obvious adaptions of the method described above for substrate-bound C5d polypeptides. In some embodiments, the screening methods can include determining whether a candidate compound (so determined to bind to C5d and inhibit the interaction between C5b and C6) inhibits terminal complement activity. In some embodiments, the screening methods described herein can include determining whether or not a candidate compound binds to full- length, native, unc leaved C5. In some embodiments, compounds that do not bind to full- length, native, uncleaved C5 are selected. In some embodiments, compounds that

preferentially bind to C5d (a neoepitope within C5d) over full-length, native, uncleaved C5 are selected.

In some embodiments, the methods can include determining whether or not a candidate compound (so determined to bind to C5d and inhibit the interaction between C5b and C6) does or does not inhibit formation or activity of C5a. In some embodiments, the methods can include determining whether or not a candidate compound (so determined to bind to C5d and inhibit the interaction between C5b and C6) binds to C3, C4, C3b, C3d, or

C4b. In some embodiments, compounds that do not bind to C3, C4, C3b, C3d, or C4b are selected. In some embodiments, compounds that preferentially bind to C5d (a neoepitope within C5d) over C3, C4, C3b, C3d, or C4b are selected.

Compounds identified in any of the methods described herein, or any compound with appropriate activity useful in any of the methods described herein can include various chemical classes, though typically small organic molecules. Potential compounds, as noted above, include small organic molecules, peptides, polypeptides, antibodies, and antigen- binding antibody fragments.

Pharmaceutical Compositions

Compositions containing any of the compounds described herein (e.g., a compound that inhibits the interaction between C5b and C6 such as an antagonist anti-C5d antibody or antigen-binding fragment thereof or C5d-containing polypeptide described herein) can be formulated as a pharmaceutical composition, e.g., for administration to a subject for the treatment or prevention of a complement-associated condition. The pharmaceutical compositions will generally include a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a

pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) JPharm Sci 66: 1-19).

The compositions can be formulated according to standard methods. Pharmaceutical formulation is a well-established art, and is further described in, e.g., Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20^th Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems," 7^th Edition, Lippincott Williams & Wilkins Publishers (ISBN:

0683305727); and Kibbe (2000) "Handbook of Pharmaceutical Excipients American

Pharmaceutical Association," 3^rd Edition (ISBN: 091733096X). In some embodiments, a composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8°C (e.g., 4°C). In some embodiments, a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1½ years, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

The pharmaceutical compositions can be in a variety of forms. These forms include, e.g., liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends, in part, on the intended mode of administration and therapeutic application. For example, compositions containing an antibody, antigen- binding fragment thereof, or C5d-containing polypeptide intended for systemic or local delivery can be in the form of injectable or infusible solutions. Accordingly, the

compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). "Parenteral administration," "administered parenterally," and other grammatically equivalent phrases, as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

The compositions can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an antibody (or a fragment of the antibody) described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an antibody or fragment described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze- drying that yield a powder of an antibody, or an antigen-binding fragment thereof, described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. The anti-C5d antibodies, antigen-binding fragments thereof, or C5d-containing polypeptides described herein can also be formulated in immunoliposome compositions. Liposomes containing an antibody, e.g., can be prepared by methods known in the art such as, e.g., the methods described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in, e.g., U.S. Patent No. 5,013,556.

In certain embodiments, therapeutic protein described herein (e.g., an anti-C5d antibody, C5d-binding fragment thereof, or a C5d-containing polypeptide) can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e.g., J.R.

Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

In some embodiments, a therapeutic protein described herein can be formulated in a composition suitable for intrapulmonary administration (e.g., for administration via nebulizer; see below) to a mammal such as a human. Methods for preparing such compositions are well known in the art and described in, e.g., U.S. patent application publication no. 20080202513; U.S. patent nos. 7,112,341 and 6,019,968; and PCT application publication nos. WO

00/061178 and WO 06/122257, the disclosures of each of which are incorporated herein by reference in their entirety. Dry powder inhaler formulations and suitable systems for administration of the formulations are described in, e.g., U.S. patent application publication no. 20070235029, PCT Publication No. WO 00/69887; and U.S. patent no. 5,997,848.

In some embodiments, therapeutic protein described herein can be formulated in a composition suitable for delivery to the eye. In some embodiments, one or more of the anti- C5d antibodies (or antigen-binding fragments thereof) or C5d-containing polypeptides described herein can be administered locally, for example, by way of topical application or intravitreal injection. For example, in some embodiments, the anti-C5d antibodies, C5d- binding fragments, or C5d-containing polypeptides can be formulated for administration by way of an eye drop.

The therapeutic preparation for treating the eye can contain one or more of therapeutic proteins described herein in a concentration from about 0.01 to about 1% by weight, preferably from about 0.05 to about 0.5% in a pharmaceutically acceptable solution, suspension or ointment. The preparation will preferably be in the form of a sterile aqueous solution containing, e.g., additional ingredients such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, and viscosity-increasing agents.

Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include, e.g., boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, and sodium biphosphate, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, and sodium chloride.

Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfite, and thiourea. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose,

hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethylcellulose. The preparation can be administered topically to the eye of the subject in need of treatment (e.g., a subject afflicted with AMD) by conventional methods, e.g., in the form of drops, or by bathing the eye in a therapeutic solution, containing one or more anti-C5d antibodies.

In addition, a variety of devices have been developed for introducing drugs into the vitreal cavity of the eye. For example, U.S. patent application publication no. 20020026176 describes a pharmaceutical-containing plug that can be inserted through the sclera such that it projects into the vitreous cavity to deliver the pharmaceutical agent into the vitreous cavity. In another example, U.S. patent no. 5,443,505 describes an implantable device for introduction into a suprachoroidal space or an avascular region for sustained release of drug into the interior of the eye. U.S. patent nos. 5,773, 019 and 6,001,386 each disclose an implantable drug delivery device attachable to the scleral surface of an eye. The device comprises an inner core containing an effective amount of a low solubility agent covered by a non-bioerodible polymer that is permeable to the low solubility agent. During operation, the low solubility agent permeates the bioerodible polymer cover for sustained release out of the device. Additional methods and devices (e.g., a transscleral patch and delivery via contact lenses) for delivery of a therapeutic agent to the eye are described in, e.g., Ambati and Adamis (2002) Prog Retin Eye Res 21(2): 145-151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11): 1164- 1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1): 1 - 10(10); Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al.

(2007) Ophthalmic Res 39:244-254; and PCT publication no. WO 04/073551, the disclosures of which are incorporated herein by reference in their entirety.

Nucleic acids encoding a therapeutic protein described herein (e.g., an anti-C5d antibody, an antigen-binding fragment thereof, or a C5d-containing polypeptide) can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce agents within cells (see below).

Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant bacterial or eukaryotic plasmids. Viral vectors can transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or by being derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaP0₄ precipitation (see, e.g., WO04/060407) carried out in vivo. (See also, "Ex vivo Approaches," below.) Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art (see, e.g., Eglitis et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85 :6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA

85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89: 10892-10895; Hwu et al. (1993) J Immunol. 150:4104-4115; U.S. Patent Nos. 4,868,116 and 4,980,286; PCT Publication Nos.

WO89/07136, WO89/02468, WO89/05345, and WO92/07573). Another viral gene delivery system utilizes adenovirus-derived vectors (see, e.g., Berkner et al. (1988) BioTechniques

6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68: 143-

155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Yet another viral vector system useful for delivery of the subject gene is the adeno-associated virus (AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356; Samulski et al. (1989) J Virol 63:3822-3828; and McLaughlin et al. (1989) J Virol 62: 1963-1973.

In some embodiments, a therapeutic protein described herein can be formulated with one or more additional active agents useful for treating or preventing a complement- associated condition in a subject. Additional agents for treating a complement-associated condition in a subject will vary depending on the particular condition being treated, but can include, without limitation, an antihypertensive (e.g., an angiotensin-converting enzyme inhibitor), an anticoagulant, a corticosteroid (e.g., prednisone), or an immunosuppressive agent (e.g., vincristine or cyclosporine A). Examples of anticoagulants include, e.g., warfarin (Coumadin), heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran). An antibody or fragment thereof described herein can also be formulated with a fibrinolytic agent (e.g., ancrod, ε-aminocaproic acid, antiplasmin-al, prostacyclin, and defibrotide) for the treatment of a complement-mediated condition. In some embodiments, an antibody can be formulated with a lipid-lowering agent such as an inhibitor of hydroxymethylglutaryl CoA reductase. In some embodiments, an antibody can be formulated with, or for use with, an anti-CD20 agent such as rituximab (Rituxan™; Biogen Idee, Cambridge, MA). In some embodiments, e.g., for the treatment of RA, the antibody or antigen-binding fragment thereof can be formulated with one or both of infliximab (Remicade®; Centocor, Inc.) and methotrexate (Rheumatrex®, Trexall®). In some embodiments, an antibody or an antigen-binding fragment thereof described herein can be formulated with a non-steroidal anti-inflammatory drug (NSAID). Many different NSAIDs are available, some over the counter including ibuprofen (Advil ®, Motrin®, Nuprin ®) and naproxen (Alleve®) and many others are available by prescription including meloxicam (Mobic®), etodolac (Lodine®), nabumetone (Relafen®), sulindac (Clinoril®), tolementin (Tolectin®), choline magnesium salicylate (Trilasate®), diclofenac (Cataflam®, Voltaren®, Arthrotec®), Diflusinal (Dolobid®), indomethicin (Indocin®), Ketoprofen (Orudis®, Oruvail®), oxaprozin (Daypro®), and piroxicam (Feldene®). In some

embodiments an antibody or a fragment thereof can be formulated for use with an antihypertensive, an anti-seizure agent (e.g., magnesium sulfate), or an anti-thrombotic agent. Anti-hypertensives include, e.g., labetalol, hydralazine, nifedipine, calcium channel antagonists, nitroglycerin, or sodium nitroprussiate. See, e.g., Mihu et al. (2007) J

Gasrointestin Liver Dis 16(4):419-424. Anti-thrombotic agents include, e.g., heparin, antithrombin, prostacyclin, or low dose aspirin. In some embodiments, a therapeutic antibody described herein can be formulated for administration to a subject along with intravenous gamma globulin therapy (IVIG), plasmapheresis, or plasma exchange. In some embodiments, a therapeutic protein can be formulated for use before, during, or after, an organ transplant, e.g., a kidney transplant or a liver transplant. In some embodiments, a therapeutic protein described herein can be formulated for use in promoting liver regeneration (e.g., supportive therapy during and after a liver transplant or for use treating a patient's own damaged liver).

When a therapeutic protein described herein is to be used in combination with a second active agent, the agents can be formulated separately or together. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times (see below).

As described above, a composition can be formulated such that it includes a therapeutically effective amount of a therapeutic protein (e.g., anti-C5d antibody, antigen- binding fragment thereof, or C5d-containing polypeptide) described herein. In some embodiments, a composition can be formulated to include a sub-therapeutic amount of the therapeutic protein and a sub-therapeutic amount of one or more additional active agents such that the components in total are therapeutically effective for treating or preventing a complement-associated condition. Methods for determining a therapeutically effective dose of an agent such as a therapeutic antibody are known in the art and described herein.

Applications

Inhibitors of the interaction between C5b and C6 (e.g., anti-C5d antibodies, antigen- binding fragments thereof, C5d-containing polypeptides, conjugates, and compositions of any of the foregoing) can be used in a number of diagnostic and therapeutic applications. For example, detectably-labeled anti-C5d antibodies (e.g., anti-human C5d antibodies or anti- mouse C5d antibodies) can be used in assays to detect the presence or amount of C5b present in a biological sample. Determining the amount of C5b in a sample, e.g., a patient blood sample, can be useful to evaluate the level of complement activation in the sample. Suitable methods for using the antibodies in diagnostic assays are known in the art and include, without limitation, ELISA, fluorescence resonance energy transfer applications, Western blot, and dot blot techniques. See, e.g., Sambrook et al, supra and Ausubel et al, supra.

In some embodiments, the inhibitors described herein can be used as positive controls in assays designed to identify additional novel compounds for treating complement-mediated conditions. For example, an anti-C5d antibody that inhibits formation of terminal complement and thus complement activity can be used as a positive control in an assay to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that inhibit the interaction between C5b and C6.

In some embodiments, the cross-reactive anti-C5d antibodies or antigen-binding fragments thereof (e.g., cross-reactive with human C5d and, e.g., cynomolgus macaque C5d) described herein can be used for pre-clinical testing in non-human mammals, e.g., pharmacokinetic or pharmacodynamic studies in non-human primates. Accordingly, a researcher wishing to evaluate the efficacy of an anti-C5d antibody in treating a complement- associated condition of interest (e.g., liver regeneration) can use a cross-reactive anti-C5d antibody described herein in an appropriate non-human primate model of the disease. If the researcher, for example, establishes efficacy of the antibody in the non-human primate model, these results may provide sufficient proof-of-concept support for regulatory approval for use of the antibody in treating humans. Alternatively, or in addition, a researcher may administer the cross-reactive antibody to a non-human primate to study, e.g., antibody clearance and/or pharmacodynamics properties. Based on such studies using the cross- reactive antibody, the researcher can better approximate the dose required to treat human disease.

In some embodiments, the anti-mouse C5d antibodies or antigen-binding fragments thereof described herein, as well as antibodies that crossreact with human and mouse C5d, can be used as a surrogate antibody in mouse models of human disease. This can be especially useful where a humanized anti-human C5d antibody does not cross-react with mouse C5d and/or is likely to cause an anti-human antibody response in a mouse to which the humanized antibody is administered. Accordingly, a researcher wishing to study the effect of an anti-C5d antibody in treating a condition (e.g., promoting regeneration of a damaged liver) can use an anti-mouse C5d antibody described herein in an appropriate mouse model of the disease. If the researcher can establish efficacy in the mouse model of disease using the anti- mouse C5d antibody, the results may establish proof-of-concept for use of an anti-human C5d antibody in treating the disease in humans.

The anti-C5d antibodies described herein can also be used in methods for purifying free C5b from a sample (e.g., a biological sample). In some embodiments, an anti-C5d antibody can be immobilized on a solid phase support using methods well known in the art. A sample containing the antigen to be purified, in this case C5b, is contacted to the antibody on the solid support under conditions and for a time sufficient to allow the antigen to bind to the antibody. The solid support is then washed one or more times with a suitable buffer to remove unbound material. The solid support can be then contacted with a second buffer that results in the release of the antigen from the antibody. The released antigen is then collected and characterized (e.g., for purity and activity) using well known methods in the art.

Inhibitors of the C5b-C6 interaction, e.g., anti-C5d antibodies, C5d-binding fragments thereof, and C5d-containing polypeptides described herein can also be used in therapeutic methods as elaborated on below.

Methods for Treatment

The above-described compositions are useful in, inter alia, methods for treating or preventing a variety of complement-associated conditions in a subject. The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or intramuscular injection (IM).

Administration can be achieved by, e.g., local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion- based systems, or electromechanical systems.

In some embodiments, a therapeutic polypeptide described herein (e.g., an anti-C5d antibody, antigen-binding fragment thereof, or C5d-containing polypeptide) is therapeutically delivered to a subject by way of local administration. As used herein, "local administration" or "local delivery," refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. Following local administration in the vicinity of a target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site.

In some embodiments, a therapeutic protein described herein can be locally administered to a joint (e.g., an articulated joint). For example, in embodiments where the complement-associated condition is arthritis, the complement inhibitor can be administered directly to a joint (e.g., into a joint space) or in the vicinity of a joint. Examples of intraarticular joints to which an anti-C5d antibody or antigen-binding fragment thereof can be locally administered include, e.g., the hip, knee, elbow, wrist, sternoclavicular,

temperomandibular, carpal, tarsal, ankle, and any other joint subject to arthritic conditions. A therapeutic protein described herein (e.g., anti-C5d antibody or antigen-binding fragment thereof) can also be administered to bursa such as, e.g., acromial, bicipitoradial, cubitoradial, deltoid, infrapatellar, ischial, and any other bursa known in the art of medicine.

In some embodiments, a therapeutic protein described herein (e.g., an antagonist anti- C5d antibody or antigen-binding fragment thereof or a C5d-containing polypeptide) can be locally administered to the eye. As used herein, the term "eye" refers to any and all anatomical tissues and structures associated with an eye. The eye has a wall composed of three distinct layers: the outer sclera, the middle choroid layer, and the inner retina. The chamber behind the lens is filled with a gelatinous fluid referred to as the vitreous humor. At the back of the eye is the retina, which detects light. The cornea is an optically transparent tissue, which conveys images to the back of the eye. The cornea includes one pathway for the permeation of drugs into the eye. Other anatomical tissue structures associated with the eye include the lacrimal drainage system, which includes a secretory system, a distributive system and an excretory system. The secretory system comprises secretors that are stimulated by blinking and temperature change due to tear evaporation and reflex secretors that have an efferent parasympathetic nerve supply and secrete tears in response to physical or emotional stimulation. The distributive system includes the eyelids and the tear meniscus around the lid edges of an open eye, which spread tears over the ocular surface by blinking, thus reducing dry areas from developing.

In some embodiments, a therapeutic protein described herein can be administered to the posterior chamber of the eye. In some embodiments, an anti-C5d antibody or antigen- binding fragment thereof is administered intravitreally. In some embodiments, an anti-C5d antibody or antigen-binding fragment thereof is administered trans-sclerally.

In some embodiments, the therapeutic proteins (e.g., antibodies or antigen-binding fragments thereof or C5d-containing polypeptides) provided herein are present in unit dosage form, which can be particularly suitable for self-administration. A formulated product of the present disclosure can be included within a container, typically, for example, a vial, cartridge, prefilled syringe or disposable pen. A doser such as the doser device described in U.S. Patent No. 6,302,855 may also be used, for example, with an injection system of the present disclosure.

An injection system of the present disclosure may employ a delivery pen as described in U.S. Patent No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can comprise at least one injection needle (e.g., a 31 gauge needle of about 5 to 8 mm in length), are typically pre-filled with one or more therapeutic unit doses of a therapeutic solution, and are useful for rapidly delivering the solution to a subject with as little pain as possible.

One medication delivery pen includes a vial holder into which a vial of insulin or other medication may be received. The vial holder is an elongate generally tubular structure with proximal and distal ends. The distal end of the vial holder includes mounting means for engaging a double-ended needle cannula. The proximal end also includes mounting means for engaging a pen body which includes a driver and dose setting apparatus. A disposable medication (e.g., a high concentration solution of an anti-C5d antibody or antigen-binding fragment thereof) containing vial for use with the prior art vial holder includes a distal end having a pierceable elastomeric septum that can be pierced by one end of a double-ended needle cannula. The proximal end of this vial includes a stopper slidably disposed in fluid tight engagement with the cylindrical wall of the vial. This medication delivery pen is used by inserting the vial of medication into the vial holder. A pen body then is connected to the proximal end of the vial holder. The pen body includes a dose setting apparatus for designating a dose of medication to be delivered by the pen and a driving apparatus for urging the stopper of the vial distally for a distance corresponding to the selected dose. The user of the pen mounts a double-ended needle cannula to the distal end of the vial holder such that the proximal point of the needle cannula pierces the septum on the vial. The patient then selects a dose and operates the pen to urge the stopper distally to deliver the selected dose. The dose selecting apparatus returns to zero upon injection of the selected dose. The patient then removes and discards the needle cannula, and keeps the medication delivery pen in a convenient location for the next required medication administration. The medication in the vial will become exhausted after several such administrations of medication. The patient then separates the vial holder from the pen body. The empty vial may then be removed and discarded. A new vial can be inserted into the vial holder, and the vial holder and pen body can be reassembled and used as explained above. Accordingly, a medication delivery pen generally has a drive mechanism for accurate dosing and ease of use.

A dosage mechanism such as a rotatable knob allows the user to accurately adjust the amount of medication that will be injected by the pen from a prepackaged vial of medication. To inject the dose of medication, the user inserts the needle under the skin and depresses the knob once as far as it will depress. The pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and/or indicate the dosage of medication that is injected into the user. See U.S. Patent No. 6,192,891.

In some embodiments, the needle of the pen device is disposable and the kits include one or more disposable replacement needles. Pen devices suitable for delivery of any one of the presently featured antibodies or antigen-binding fragments thereof are also described in, e.g., U.S. patent nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g., U.S. patent no. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, Molly™, manufactured by Scandinavian Health Ltd.

The present disclosure also presents controlled-release or extended-release formulations suitable for chronic and/or self-administration of a medication such as an anti- C5d antibody or an antigen-binding fragment thereof described herein. The various formulations can be administered to a patient in need of treatment with the medication as a bolus or by continuous infusion over a period of time.

In some embodiments, a high concentration therapeutic protein described herein (e.g., anti-C5d antibody, antigen-binding fragment thereof, or C5d-containing polypeptide) is formulated for sustained-release, extended-release, timed-release, controlled-release, or continuous-release administration. In some embodiments, depot formulations are used to administer the therapeutic protein to the subject in need thereof. In this method, the therapeutic protein is formulated with one or more carriers providing a gradual release of active agent over a period of a number of hours or days. Such formulations are often based upon a degrading matrix which gradually disperses in the body to release the active agent.

A suitable dose of therapeutic protein described herein (e.g., an antibody or fragment thereof described herein), which dose is capable of treating or preventing a complement- associated condition in a subject, can depend on a variety of factors including, e.g., the age, sex, and weight of a subject to be treated and the particular inhibitor compound used. For example, a different dose of a whole anti-C5d antibody may be required to treat a subject with PNH as compared to the dose of a C5d-binding Fab' antibody fragment required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the complement-mediated condition. For example, a subject having MD may require administration of a different dosage of an anti-C5d antibody than a subject with AMD. Other factors can include, e.g., other medical conditions concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will also depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse).

A therapeutic protein described herein (e.g., an antibody, antigen-binding fragment thereof, or a C5d-containing polypeptide described herein) can be administered as a fixed dose, or in a milligram per kilogram (mg/kg) dose. In some embodiments, the dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against one or more of the active antibodies in the composition. While in no way intended to be limiting, exemplary dosages of therapeutic protein described herein include, e.g., 1-1000 %, 1-100 μ , 0.5-50 μ , 0.1-100 μ , 0.5-25 μ , 1-20 μ , and 1-10 μ , 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, 0.100-1 mg/kg, and 1-10 mg/kg. Exemplary dosages of an antibody or antigen-binding fragment thereof described herein include, without limitation, 0.1 μg/kg, 0.5 μg/kg, 1.0 μg/kg, 2.0 μg/kg, 4 μg/kg, and 8 μg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, and 20 mg/kg.

A pharmaceutical composition can include, e.g., a therapeutically effective amount of a therapeutic protein described herein (e.g., an anti-C5d antibody or antigen-binding fragment thereof described herein). Such effective amounts can be readily determined by one of ordinary skill in the art based, in part, on the effect of the administered antibody, or the combinatorial effect of the antibody and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of an antibody or fragment thereof described herein can also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (and one or more additional active agents) to elicit a desired response in the individual, e.g., amelioration of at least one condition parameter, e.g., amelioration of at least one symptom of the complement-mediated condition. For example, a therapeutically effective amount of an anti-C5d antibody can inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a particular condition, and/or any one of the symptoms of the particular condition known in the art or described herein. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

Suitable human doses of any of the antibodies or fragments thereof described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

The terms "therapeutically effective amount" or "therapeutically effective dose," or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-C5d antibody or an antigen-binding fragment thereof or C5d-containing polypeptide) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a complement-associated condition). In some embodiments, a composition described herein contains a therapeutically effective amount of an antibody, or antigen- binding fragment thereof, which specifically binds to a neo-epitope present in C5d. In some embodiments, the composition contains any of the antibodies or antigen-binding fragments thereof described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, a composition can contain an anti-C5d antibody described herein and an immunosuppressive agent, wherein the antibody and agent are each at a concentration that when combined are therapeutically effective for treating or preventing a complement-associated condition in a subject.

Toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the complement-mediated conditions described herein). These procedures can be used, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.

An antibody or antigen-binding fragment thereof that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such antibodies, antigen- binding fragments thereof, or C5d-containing polypeptides lies generally within a range of circulating concentrations of the antibodies or fragments that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a therapeutic protein (e.g., anti-C5d antibody or a C5d-containing polypeptide) described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the antibody which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration (e.g., to the eye or a joint) is desired, cell culture or animal modeling can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

In some embodiments, the methods can be performed in conjunction with other therapies for complement-associated conditions. For example, the composition can be administered to a subject at the same time, prior to, or after, plasmapheresis, IVIG therapy, or plasma exchange. See, e.g., Appel et al. (2005) J Am Soc Nephrol 16: 1392-1404. In some embodiments, the composition can be administered to a subject at the same time, prior to, or after, a kidney transplant.

A "subject," as used herein, can be any mammal. For example, a subject can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In some embodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof," refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-C5d antibody).

The term "preventing" is art-recognized, and when used in relation to a condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of a complement- associated condition such as asthma includes, for example, reducing the extent or frequency of coughing, wheezing, or chest pain in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the occurrence of coughing or wheezing in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

As described above, the therapeutic proteins (e.g., antagonist anti-C5d antibodies, antigen-binding fragments thereof, or C5d-containing polypeptides) described herein can be used to treat a variety of complement-associated conditions such as, but not limited to:

rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS); typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; sepsis; dermatomyositis; diabetic retinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; multiple sclerosis (MS); and traumatic brain injury. See, e.g., Holers (2008) Immunological Reviews 223:300-316 and Holers and Thurman (2004) Molecular Immunology 4J_: 147-152. In some embodiments, the complement-mediated condition is a complement-mediated vascular condition such as, but not limited to, a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral (e.g., musculoskeletal) vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, revascularization to transplants and/or replants, vasculitis, Henoch- Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy, thoracic-abdominal aortic aneurysm, Kawasaki's disease (arteritis), venous gas embolus (VGE), and restenosis following stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA). (See, e.g., U.S. patent application publication no. 20070172483.) In some embodiments, the complement-associated disorder is myasthenia gravis, cold- agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome, antiphospholipid syndrome (APS), Degos disease, and catastrophic APS (CAPS). In some embodiments, a therapeutic protein (e.g., anti-C5d antibody or antigen- binding fragment thereof) described herein, alone or in combination with a second antiinflammatory agent, can be used to treat an inflammatory disorder such as, but not limited to, RA (above), inflammatory bowel disease, sepsis (above), septic shock, acute lung injury, disseminated intravascular coagulation (DIC), or Crohn's disease. In some embodiments, the second anti-inflammatory agent can be one selected from the group consisting of NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, a B cell depleting agent such as rituximab, an interleukin-1 antagonist, or a T cell costimulatory blocking agent such as abatacept.

In some embodiments, the complement-associated condition is a complement- associated neurological condition such as, but not limited to, amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease, and chronic inflammatory demyelinating neuropathy.

Complement-associated conditions also include complement-associated pulmonary conditions such as, but not limited to, asthma, bronchitis, a chronic obstructive pulmonary disease (COPD), an interstitial lung disease, a-1 anti-trypsin deficiency, emphysema, bronchiectasis, bronchiolitis obliterans, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular conditions.

In some embodiments, the therapeutic proteins described herein, which can selectively inhibit the terminal complement arm of C5, are useful for treating diseases in which inhibition of terminal complement is therapeutic, but in which the activity of C5a should be preserved. For example, International patent application publication no. WO 2011/003098 describes a role for C5a in priming stages of liver regeneration and thus indicates that preservation of C5a is useful for treating patients in need of liver regeneration (e.g., patients who have received a liver transplant, have undergone partial liver resection, or who have liver injury resulting from, e.g., cirrhosis or viral infection (hepatitis infection)).

In some embodiments, a therapeutic protein described herein (e.g., an anti-C5d antibody or an antigen-binding fragment thereof described herein) can be administered to a subject as a monotherapy. Alternatively, as described above, the antibody or fragment thereof can be administered to a subject as a combination therapy with another treatment, e.g., another treatment for a complement-associated condition or a complement-associated inflammatory response. For example, the combination therapy can include administering to the subject (e.g., a human patient) one or more additional agents (e.g., anti-coagulants, anti- hypertensives, or anti-inflammatory drugs (e.g., steroids)) that provide a therapeutic benefit to a subject who has, or is at risk of developing, sepsis. In some embodiments, an anti-C5d antibody, or a C5d-containing polypeptide, described herein and the one or more additional active agents are administered at the same time. In other embodiments, the anti-C5d

5 antibody, or C5d-containing polypeptide, is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the anti-C5d antibody, or C5d-containing polypeptide is administered second in time.

The therapeutic proteins described herein (e.g., an anti-C5d antibody or an antigenic) binding fragment thereof or C5d-containng polypeptide) can replace or augment a previously or currently administered therapy. For example, upon treating with an anti-C5d antibody or antigen-binding fragment thereof, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous 15 therapy will be maintained until the level of the anti-C5d antibody reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in a complement- associated condition, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., an improvement in one or more symptoms of a given condition. The

20 symptoms of complement-associated condition are well known in the art of medicine. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an

administration. The subject can be evaluated in one or more of the following periods: prior to 25 beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a complement- 30 associated condition described herein. Therapeutic and Diagnostic Kits

The disclosure also features therapeutic and diagnostic kits containing, among other things, one or more of the compounds described herein (e.g., anti-C5d antibodies, C5d- binding fragments thereof, or C5d-containing polypeptides). The therapeutic kits can contain, e.g., a suitable means for delivery of a therapeutic protein described herein to a subject. In some embodiments, the means is suitable for subcutaneous delivery of the protein (e.g., anti-C5d antibody, antigen-binding fragment thereof to the subject. The means can be, e.g., a syringe or an osmotic pump. That is, a therapeutic kit described herein can contain a syringe pre-filled with an anti-C5d antibody or antigen-binding fragment thereof (e.g., a pen device containing the antibody or fragment) described herein or the kit can contain a pump (e.g., an osmotic pump) and one or more disposable cassettes configured for use with the pump, the cassettes pre-filled with an anti-C5d antibody or antigen-binding fragment thereof described herein (e.g., prefilled with an aqueous solution containing the anti-C5d antibody or antigen-binding fragment thereof). In another example, the kit can contain an implantable delivery device (e.g., a plug) that is pre-filled with (or otherwise contains) a solution containing an anti-C5d antibody or antigen-binding fragment thereof described herein.

In some embodiments, the means for delivering an anti-C5d antibody or antigen- binding fragment thereof is a pen device for drug delivery.

The therapeutic kits can include, e.g., one or more additional active agents for treating or preventing a complement-associated condition and/or ameliorating a symptom thereof. For example, therapeutic kits designed for use in treating or preventing a complement- associated pulmonary condition can include one or more additional active agents including, but not limited to, another antibody therapeutic (e.g., an anti-IgE antibody, an anti-IL-4 antibody, or an anti-IL-5 antibody), a small molecule IgE inhibitor (e.g., montelukast sodium), a sympathomimetic (e.g., albuterol), an antibiotic (e.g., tobramycin), a

deoxyribonuclease (e.g., pulmozyme), an anticholinergic drug (e.g., ipratropium bromide), a corticosteroid (e.g., dexamethasone), a β-adrenoreceptor agonist, a leukotriene inhibitor (e.g., zileuton), a 5 -lipoxygenase inhibitor, a phosphodiesterase (PDE) inhibitor, a CD23 antagonist, an IL-13 antagonist, a cytokine release inhibitor, a histamine HI receptor antagonist, an anti-histamine, an anti-inflammatory agent (e.g., cromolyn sodium or any other anti-inflammatory agent known in the art or described herein), or a histamine release inhibitor. In some embodiments, the means can be suitable for intraocular administration of an anti-C5d antibody, or an antigen-binding fragment thereof, described herein to a subject in need thereof, e.g., a subject afflicted with AMD or any other complement-associated ocular condition. The means can be, e.g., a syringe, a trans-scleral patch, or even a contact lens containing the antibody or fragment. The means can, in some embodiments, be an eye dropper, wherein the anti-C5d antibody or antigen-binding fragment thereof is formulated for such administration. Such therapeutic kits can also include, e.g., one or more additional therapeutic agents for use in treating a complement-associated condition of the eye. The therapeutic agents can be, e.g., bevacizumab or the Fab fragment of bevacizumab, ranibizimab, both sold by Roche Pharmaceuticals, Inc., or pegaptanib sodium (Mucogen®; Pfizer, Inc.). Such a kit can also, optionally, include instructions for administering a therapeutic protein (e.g., an anti-C5d antibody or antigen-binding fragment thereof or C5d- containing polypeptide) to a subject.

In some embodiments, the means can be suitable for intraarticular administration of an anti-C5d antibody, or antigen-binding fragment thereof, described herein to a subject in need thereof, e.g., a subject afflicted with RA. The means can be, e.g., a syringe or a double- barreled syringe. See, e.g., U.S. Patent Nos. 6,065,645 and 6,698,622. A double-barreled syringe is useful for administering to a joint two different compositions with only one injection. Two separate syringes may be incorporated for use in administering the therapeutic while drawing off knee fluid for analysis (tapping) in a push-pull fashion. Additional therapeutic agents that can be administered with the therapeutic proteins described herein (e.g., anti-C5d antibodies or antigen-binding fragments thereof or C5d-containing

polypeptides) in conjunction with the double-barreled syringe, or which can otherwise be generally included in the therapeutic kits described herein, include, e.g., NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, a B cell depleting agent such as rituximab, an interleukin-1 antagonist, or a T cell costimulatory blocking agent such as abatacept. Such a kit can also, optionally, include instructions for administering the anti-C5d antibody or antigen-binding fragment thereof to a subject. It will be appreciated that the disclosure embraces kits comprising one or more of the anti-C5d antibodies described herein and one or more anti-inflammatory agents selected from the group consisting of NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, a B cell depleting agent such as rituximab, an interleukin-1 antagonist, or a T cell costimulatory blocking agent such as abatacept. The antibodies and agents can be, e.g., formulated separately or together. The kits can be used to treat an inflammatory condition such as RA, Crohn's disease, inflammatory bowel disease, or any other inflammatory condition known in the art or recited herein.

Also featured are diagnostic kits containing the anti-C5d antibodies or antigen- binding fragments thereof described herein. For example, the kits can contain a detectably- labeled form of an anti-C5d antibody (e.g., an anti-C5d antibody or an anti-mouse C5d antibody) described herein for use in, e.g., detecting or quantitating the amount of C5b in a biological sample. In some embodiments, the kits can contain isolated C5b protein or recombinant C5d-containing polypeptide (e.g., one or both of human and mouse C5b protein) and/or a control sample comprising one or both of human and mouse C5b protein. In some embodiments, the kit contains a multi-well plate coated with a first anti-C5d antibody having a first specificity. The kit also contains a second anti-C5d antibody (e.g., a detectably-labeled second anti-C5d antibody) having a second specificity. Such a kit is designed for use in capturing, with the first antibody bound to the plate, C5d protein (e.g., human C5d protein) in a sample (e.g., a biological sample) contacted to the plate and then detecting the captured C5d protein using the second antibody. In some embodiments, diagnostic kits include both an anti-mouse C5d antibody and an anti-human C5d antibody described herein. In some embodiments, the diagnostic kits include an anti-C5d antibody that binds to both mouse C5d and human C5d.

The following examples are intended to illustrate, not limit, the invention.

Example 1. Treatment of thrombotic thrombocytopenic purpura using an anti-C5d antibody A human patient is identified by a medical practitioner as having an inherited form of TTP. Once a week for four weeks the patient is administered a composition containing an anti-C5d antibody by intravenous infusion. The patient and medical practitioner observe a substantial improvement in at least two known symptoms of TTP during the initial treatment. One week after the initial four week treatment, the patient receives intravenously

administered "maintenance doses" of the antibody every two weeks until the medical practitioner determines that the TTP is in remission.

Example 2. Treatment of dense deposit disease using a C5d polypeptide

A human patient presenting with DDD is intravenously administered every two weeks a composition containing a fusion protein comprising a C5d polypeptide and an antibody Fc constant region. The patient and medical practitioner observe a substantial reduction in overall severity of the patient's DDD symptoms during the initial treatment. The patient is maintained on the same treatment regimen until the medical practitioner determines that the DDD is in remission.

Example 3. Treatment of PNH using an anti-C5d antibody

A human patient is identified by a medical practitioner as having PNH. Once a week for four weeks the patient is administered a composition containing an anti-C5d antibody by intravenous infusion. The patient and medical practitioner observe a substantial improvement in at least two known symptoms of PNH during the initial treatment. One week after the initial four week treatment, the patient receives intravenously administered "maintenance doses" of the antibody every three to four weeks until the medical practitioner determines that patient's condition has stabilized.

While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

Claims

What we claim is:

1. An isolated antibody, or an antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5, but does not bind to uncleaved, native human C5, wherein the C5d domain has the amino acid sequence depicted in SEQ ID NO:2.

2. An isolated antibody, or an antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5 and uncleaved, native human C5, wherein the affinity of the antibody for C5d is at least 10-fold greater than the affinity of the antibody for uncleaved, native human C5, wherein the C5d domain has the amino acid sequence depicted in SEQ ID NO:2.

3. The isolated antibody, or antigen-binding fragment thereof, of claim 2, wherein the affinity of the antibody for C5d is at least 100-fold greater than the affinity of the antibody for uncleaved, native human C5.

4. The isolated antibody, or antigen-binding fragment thereof, of claim 2, wherein the affinity of the antibody for C5d is at least 1000-fold greater than the affinity of the antibody for uncleaved, native human C5.

5. An isolated antibody, or antigen-binding fragment thereof, that binds to the C5d domain of human complement component C5, wherein the antibody or antigen-binding fragment thereof binds to an isolated C5d polypeptide in vitro with a K_D that is less than 5 x 10^"9 M in the presence of a molar excess of uncleaved, native human C5 and under physiological conditions, and wherein the isolated C5d polypeptide comprises at least six (6) consecutive amino acids depicted in SEQ ID NO:2.

6. The isolated antibody, or antigen-binding fragment thereof, of claim 5, wherein the concentration of uncleaved, native human C5 is between 2-fold and 20-fold greater than the concentration of the isolated C5d polypeptide.

7. The isolated antibody, or antigen-binding fragment thereof, of claim 5, wherein the concentration of uncleaved, native human C5 is between 2-fold and 15-fold greater than the concentration of the isolated C5d polypeptide.

8. The isolated antibody, or antigen-binding fragment thereof, of claim 5, wherein the uncleaved, native human C5 is from human plasma.

9. An isolated antibody or antigen-binding fragment thereof that binds to the C5d domain of human complement component C5, wherein the antibody or antigen-binding fragment thereof binds to the C5d domain with a K_D that is less than 5 x 10^~9 M, wherein the antibody or antigen-binding fragment thereof binds to the C5d domain with an affinity that is at least 100-fold greater than its corresponding affinity for uncleaved human C5, and wherein the C5d domain has the amino acid sequence depicted in SEQ ID NO:2.

10. The isolated antibody, or antigen-binding fragment thereof, according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof inhibits the interaction between C5b and C6.

11. The isolated antibody, or antigen-binding fragment thereof, according to any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof inhibits the formation of the terminal complement complex.

12. The isolated antibody, or antigen-binding fragment thereof, according to any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof inhibits hemolysis in vitro.

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof binds to the C5d domain of a non- human mammalian species.

14. The isolated antibody, or antigen-binding fragment thereof, of claim 13, wherein the non-human mammalian species is a non-human primate.

15. The isolated antibody, or antigen-binding fragment thereof, of claim 14, wherein the non-human primate is a cynomolgus macaque, rhesus macaque, or baboon.

16. The isolated antibody, or antigen-binding fragment thereof, of claim 13, wherein the antibody, or antigen-binding fragment thereof, binds to human C5d with an affinity no greater than 100-fold its corresponding affinity for C5d from the non-human mammalian species.

17. The isolated antibody, or antigen-binding fragment thereof, of claim 13, wherein the antibody or antigen-binding fragment thereof binds to human C5d with an affinity no greater than 50-fold its corresponding affinity for C5d from the non-human mammalian species.

18. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-17, wherein the antibody is a bispecific antibody.

19. The isolated antibody, or antigen-binding fragment thereof, of claim 18, wherein the bispecific antibody binds to C5a.

20. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 7 x 10^"10 M.

21. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 5 x 10^"10 M.

22. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 2.5 x 10^"10 M.

23. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 1.5 x 10^"10 M.

24. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human C5d with a K_D that is less than 8.0 x 10^"11 M.

25. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-24, wherein the antibody, or antigen-binding fragment thereof is humanized, fully human, or chimeric.

26. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a recombinant antibody, a single chain antibody, a diabody, an intrabody, a chimerized or chimeric antibody, a deimmunized antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, and an F(ab')₂ fragment.

27. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-25, further comprising a heterologous moiety.

28. The isolated antibody, or antigen-binding fragment thereof, of claim 27, wherein the heterologous moiety is a sugar.

29. The isolated antibody, or antigen-binding fragment thereof, of claim 28, wherein the antibody or antigen-binding fragment thereof is glycosylated.

30. The isolated antibody, or antigen-binding fragment thereof, of claim 27, wherein the heterologous moiety is a detectable label.

31. The isolated antibody, or antigen-binding fragment thereof, of claim 30, wherein the detectable label is a fluorescent label, a luminescent label, a heavy metal label, a radioactive label, or an enzymatic label.

32. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-31, wherein the antibody or antigen-binding fragment thereof is modified with a moiety that improves one or both of: (a) the stabilization of the antibody or antigen-binding fragment thereof in circulation and (b) the retention of the antibody or antigen-binding fragment thereof in circulation.

33. The isolated antibody, or antigen-binding fragment thereof, of claim 32, wherein the modification is PEGylation.

34. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-33, wherein the antibody comprises an altered heavy chain constant region that has reduced effector function as compared to the effector function of the unaltered form of the heavy chain constant region.

35. An isolated antibody, or antigen-binding fragment thereof, that crossb locks the binding of the antibody or antigen-binding fragment thereof of any one of claims 1-34.

36. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-35 and a pharmaceutically-acceptable carrier.

37. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-26.

38. A vector comprising the nucleic acid of claim 37.

39. An expression vector comprising the nucleic acid of claim 37.

40. A cell comprising the expression vector of claim 39.

41. A method for producing an antibody or antigen-binding fragment thereof, the method comprising culturing the cell of claim 40 under conditions and for a time sufficient to allow expression by the cell of the antibody or antigen-binding fragment encoded by the nucleic acid.

42. The method of claim 41, further comprising isolating the antibody or antigen-binding fragment thereof.

43. An isolated polypeptide comprising the C5d domain of human complement component C5, or an antigenic peptide fragment of the polypeptide, wherein the C5d domain has the amino acid sequence depicted in SEQ ID NO:2, and wherein the polypeptide is not a full-length C5b polypeptide or a full-length, uncleaved C5 polypeptide.

44. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 43, wherein the antigenic peptide fragment thereof comprises the amino acid sequence depicted in any one of SEQ ID NOs:3-8.

45. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 43, wherein the isolated polypeptide or the antigenic peptide fragment thereof binds to complement component C6.

46. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 45, wherein the isolated polypeptide or the antigenic peptide fragment thereof inhibits the interaction between C5b and C6.

47. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 43, further comprising a heterologous moiety.

48. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 47, wherein the heterologous moiety is a protein.

49. The isolated polypeptide, or antigenic peptide fragment thereof, of claim 48, wherein the heterologous moiety comprises the Fc region of an antibody.

50. A pharmaceutical composition comprising the isolated polypeptide or antigenic peptide fragment thereof of claim 46 and a pharmaceutically acceptable carrier.

51. A nucleic acid encoding the isolated polypeptide or antigenic peptide fragment of any one of claims 43-49.

52. A vector comprising the nucleic acid of claim 51.

53. An expression vector comprising the nucleic acid of claim 51.

54. A cell comprising the vector of claim 52 or the expression vector of claim 53.

55. A method for generating an immune response in a non-human mammal, the method comprising administering to a mammal the polypeptide, or antigenic fragment thereof, of claim 43 in an amount effective to induce an immune response in the mammal against the polypeptide or antigenic peptide fragment thereof.

56. The method of claim 55, wherein the polypeptide or antigenic peptide fragment is administered with an adjuvant.

57. The method of claim 55, wherein more than one antigenic peptide fragment is administered to the non-human mammal.

58. The method of claim 55, wherein the polypeptide and one or more antigenic peptide fragments thereof are administered to the non-human mammal.

59. The method of claim 55, wherein the non-human mammal does not express an endogenous complement component C5 protein.

60. The method of claim 55, wherein the non-human mammal is a rodent.

61. The method of claim 60, wherein the rodent is a mouse.

62. The method of claim 55, further comprising, after the induction of the immune response, isolating from the non-human mammal: (a) an antibody that binds to the

polypeptide or antigenic peptide fragment thereof or (b) a nucleic acid encoding the antibody.

63. The isolated antibody generated by the method of claim 62, or an antigen-binding fragment of the antibody.

64. A method for identifying a compound that binds to the C5d domain of human complement component C5, the method comprising:

contacting the polypeptide or antigenic peptide fragment thereof of claim 43 with a compound to be screened under physiological conditions; and

determining whether the compound binds to the polypeptide or antigenic peptide fragment thereof.

65. The method of claim 64, wherein the compound is an antibody or antigen-binding fragment thereof.

66. The method of claim 64, wherein the compound is a small molecule, a polypeptide, or an aptamer.

67. A method for identifying an inhibitor of the interaction between C5b and C6, the method comprising:

I.

(i) mixing C6, or a C5b-binding fragment thereof, with C5b under conditions that allow the binding of C6, or a C5b-binding fragment thereof, to C5b to form a complex;

(ii) measuring the amount of C6:C5b complex or C5b-binding fragment C6:C5b complex; (iii) mixing C6, or a C5b-binding fragment thereof, with C5b and with a candidate inhibitor under the same conditions in step (i);

(iv) measuring the amount of C6:C5b complex or C5b-binding fragment of C6:C5b complex; and

(v) comparing the amount of complex formed in step (ii) with the amount of complex formed in step (iv), wherein if the amount of complex formed in step (iv) is less than the amount of complex formed in step (ii) then the candidate inhibitor is identified as an inhibitor of the interaction between C5b and C6; or

II. (a) mixing C5d, or a C6-binding fragment thereof, with C6 under conditions that allow the binding of C5d, or a C6-binding fragment thereof, to C6 to form a complex;

(b) measuring the amount of C6:C5b complex or C6-binding fragment of C5d:C6 complex; (c) mixing C5d, or a C6-binding fragment thereof, with C6 and with a candidate inhibitor under the same conditions in step (a);

(d) measuring the amount of C6:C5b complex or C6-binding fragment of C5d:C6 complex; and

(e) comparing the amount of complex formed in step (b) with the amount of complex formed in step (d), wherein if the amount of complex formed in step (d) is less than the amount of complex formed in step (b) then the candidate inhibitor is identified as an inhibitor of the interaction between C5b and C6.

68. The method of claim 67, further comprising determining whether an identified inhibitor reduces complement activation in an in vitro assay.

69. The method of claim 67, wherein the C5d polypeptide comprises the amino acid sequence depicted in any one of SEQ ID NOs:2 or 14-16.

70. The method of claim 67, wherein the C6-binding fragment comprises the amino acid sequence depicted in any one of SEQ ID NOs:3-8.

71. The method of claim 67, wherein the isolated C5d polypeptide, C6-binding fragment, C6 polypeptide, or C5b-binding fragment thereof comprises a heterologous moiety.

72. The method of claim 71, wherein the heterologous moiety is an Fc domain of an antibody.

73. The method of claim 71 , wherein the heterologous moiety is one member of a specific binding pair.

74. The method of claim 73, wherein the specific binding pair is streptavidin and biotin.

75. The method of claim 71, wherein the heterologous moiety is a detectable label.

76. A therapeutic kit comprising: (i) the isolated antibody or antigen-binding fragment of any one of claims 1-35 or 63 or the isolated polypeptide or antigenic peptide fragment thereof of claim 46 and (ii) means for delivery of the antibody, antigen-binding fragment, polypeptide, or antigenic peptide fragment to a human.

77. The therapeutic kit of claim 76, wherein the means is a syringe.

78. An article of manufacture comprising:

a container comprising a label; and

a composition comprising: (i) the isolated antibody or antigen-binding fragment of any one of claims 1-35 or 63 or (ii) the isolated polypeptide or antigenic peptide fragment thereof of claim 46, wherein the label indicates that the composition is to be administered to a human having, suspected of having, or at risk for developing, a complement-associated condition.

79. The article of manufacture of claim 78, further comprising one or more additional active therapeutic agents for use in treating a human having, suspected of having, or at risk for developing, a complement-associated condition.

80. A method for treating a patient afflicted with a complement-associated condition, the method comprising administering to the subject a compound in an amount effective to treat the complement-associated condition, wherein the compound binds to the C5d domain of complement component C5 and inhibits the interaction between C5b and C6.

81. The method of claim 80, wherein the compound is a small molecule.

82. The method of claim 80, wherein the compound is the isolated antibody or antigen- binding fragment thereof of any one of claims 1-35 or 63.

83. The method of claim 80, wherein the compound is the isolated polypeptide or antigenic peptide fragment thereof of claim 43.

84. The method of claim 80, wherein the complement-associated condition is selected from the group consisting of rheumatoid arthritis, antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous fetal loss, thrombotic thrombocytopenic purpura, Pauci- immune vasculitis, epidermolysis bullosa, recurrent fetal loss, traumatic brain injury, myocarditis, a cerebrovascular disorder, a peripheral vascular disorder, a renovascular disorder, a mesenteric/enteric vascular disorder, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated

cardiomyopathy, diabetic angiopathy, Kawasaki's disease, venous gas embolus, restenosis following stent placement, rotational atherectomy, percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospho lipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury,

glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpastures syndrome, Degos disease, and catastrophic antiphospholipid syndrome.

85. The method of claim 80, wherein the complement-associated condition is transplant rejection.

86. The method of claim 80, wherein the complement-associated condition is liver damage and administration of the compound promotes liver regeneration.

87. The method of claim 80, wherein the complement-associated condition is a complement-associated inflammatory condition.

88. The method of claim 87, wherein the complement-associated inflammatory condition is selected from the group consisting of atypical hemolytic uremic syndrome, age-related macular degeneration, severe burn, rheumatoid arthritis, sepsis, lupus nephritis, and antiphospholipid syndrome.