Anti-c5 Antibodies Having Improved Pharmacokinetics Patent Application (2025)

U.S. patent application number 16/246842 was filed with the patent office on 2019-08-29 for anti-c5 antibodies having improved pharmacokinetics.The applicant listed for this patent is Alexion Pharmaceuticals, Inc.. Invention is credited to Bruce A. Andrien, JR., Douglas L. Sheridan, Paul P. Tamburini, Yi Wang.

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ANTI-C5 ANTIBODIES HAVING IMPROVED PHARMACOKINETICS

Abstract

The disclosure provides antibodies that are useful for, amongother things, inhibiting terminal complement (e.g., the assemblyand/or activity of the C5b-9 TCC) and C5a anaphylatoxin-mediatedinflammation and, thus, treating complement-associated disorders.The antibodies have a number of improved properties relative toeculizumab, including, e.g., increased serum half-life in ahuman.

Related U.S. Patent Documents

Claims

1-7. (canceled)

8. A method for treating Atypical Hemolytic Uremic Syndrome (aHUS)in a patient, the method comprising administering to the patient anantibody, or antigen-binding fragment, thereof, in an amounteffective to treat aHUS, wherein the antibody, or antigen-bindingfragment thereof, binds to complement component human C5, inhibitsthe cleavage of C5 into fragments C5a and C5b and comprises a heavychain CDR1 comprising the amino acid sequence set forth in SEQ IDNO:23, a heavy chain CDR2 comprising the amino acid sequence setforth in SEQ ID NO:19, a heavy chain CDR3 comprising the amino acidsequence set forth in SEQ ID NO:3, a light chain CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:4, a light chainCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5,and a light chain CDR3 comprising the amino acid sequence set forthin SEQ ID NO:6.

9. A method for treating paroxysmal nocturnal hemoglobinuria (PNH)in a patient, the method comprising administering to the patient anantibody, or antigen-binding fragment, thereof in an amounteffective to treat aHUS, wherein the antibody, or antigen-bindingfragment thereof, binds to complement component human C5, inhibitsthe cleavage of C5 into fragments C5a and C5b and comprises a heavychain CDR1 comprising the amino acid sequence set forth in SEQ IDNO:23, a heavy chain CDR2 comprising the amino acid sequence setforth in SEQ ID NO:19, a heavy chain CDR3 comprising the amino acidsequence set forth in SEQ ID NO:3, a light chain CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:4, a light chainCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5,and a light chain CDR3 comprising the amino acid sequence set forthin SEQ ID NO:6.

10. The method of claim 8, wherein the isolated antibody, orantigen-binding fragment thereof, binds to human C5 at pH 7.4 and25.degree. C. with an affinity dissociation constant (K.sub.D) thatis in the range 0.1 nM.ltoreq.K.sub.D.ltoreq.1 nM.

11. The method of claim 9, wherein the isolated antibody, orantigen-binding fragment thereof, binds to human C5 at pH 7.4 and25.degree. C. with an affinity dissociation constant (K.sub.D) thatis in the range 0.1 nM.ltoreq.K.sub.D.ltoreq.1 nM.

12. The method of claim 8, wherein the antibody, or antigen-bindingfragment thereof, binds to human C5 at pH 6.0 and 25.degree. C.with a K.sub.D.gtoreq.10 nM.

13. The method of claim 9, wherein the antibody, or antigen-bindingfragment thereof, binds to human C5 at pH 6.0 and 25.degree. C.with a K.sub.D.gtoreq.10 nM.

14. The method of claim 8, wherein the [(K.sub.D of the antibody,or antigen-binding fragment thereof, for human C5 at pH 6.0 and at25.degree. C)/(K.sub.D of the antibody, or antigen-binding fragmentthereof, for human C5 at pH 7.4 and at 25.degree. C.)] is greaterthan 25.

15. The method of claim 9, wherein the [(K.sub.D of the antibody,or antigen-binding fragment thereof, for human C5 at pH 6.0 and at25.degree. C)/(K.sub.D of the antibody, or antigen-binding fragmentthereof, for human C5 at pH 7.4 and at 25.degree. C.)] is greaterthan 25.

16. The method of claim 9, wherein the antibody, or antigen-bindingfragment thereof, has a serum half-life in humans that is at least30 days.

17. The method of claim 10, wherein the antibody, orantigen-binding fragment thereof, has a serum half-life in humansthat is at least 30 days.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patentapplication Ser. No. 15/708,658 (filed Sep. 19, 2017), which is acontinuation of U.S. patent application Ser. No. 15/492,622 (filedApr. 20, 2017, now U.S. Pat. No. 9,803,007), which is acontinuation of U.S. patent application Ser. No. 15/160,364 (filedMay 20, 2016, now U.S. Pat. No. 9,663,574), which is a continuationof U.S. patent application Ser. No. 14/923,879 (filed Oct. 27,2015, now U.S. Pat. No. 9,371,377), which is a continuation of U.S.patent application Ser. No. 14/789,329 (filed Jul. 1, 2015, nowU.S. Pat. No. 9,206,251), which is a divisional of U.S. patentapplication Ser. No. 14/727,313 (filed Jun. 1, 2015, now U.S. Pat.No. 9,107,861), which is a divisional of U.S. patent applicationSer. No. 14/641,026 (filed on Mar. 6, 2015, now U.S. Pat. No.9,079,949), which claims priority to and the benefit of U.S.provisional patent application No. 61/949,932 (filed on Mar. 7,2014), the disclosures of which are incorporated herein byreference in their entireties.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing whichhas been submitted electronically in ASCII format and is herebyincorporated by reference in its entirety. Said ASCII copy, createdon Jan. 14, 2019 is named AXJ_186DV2CN5_SEQ.txt and is 67,429 bytesin size.

TECHNICAL FIELD

[0003] The field of the invention is medicine, immunology,molecular biology, and protein chemistry.

BACKGROUND

[0004] The complement system acts in conjunction with otherimmunological systems of the body to defend against intrusion ofcellular and viral pathogens. There are at least 25 complementproteins, which are found as a complex collection of plasmaproteins and membrane cofactors. The plasma proteins make up about10% of the globulins in vertebrate serum. Complement componentsachieve their immune defensive functions by interacting in a seriesof intricate but precise enzymatic cleavage and membrane bindingevents. The resulting complement cascade leads to the production ofproducts with opsonic, immunoregulatory, and lytic functions. Aconcise summary of the biologic activities associated withcomplement activation is provided, for example, in The MerckManual, 16.sup.th Edition.

[0005] The complement cascade can progress via the classicalpathway (CP), the lectin pathway, or the alternative pathway (AP).The lectin pathway is typically initiated with binding ofmannose-binding lectin (MBL) to high mannose substrates. The AP canbe antibody independent, and can be initiated by certain moleculeson pathogen surfaces. The CP is typically initiated by antibodyrecognition of, and binding to, an antigenic site on a target cell.These pathways converge at the C3 convertase--the point wherecomplement component C3 is cleaved by an active protease to yieldC3a and C3b.

[0006] The AP C3 convertase is initiated by the spontaneoushydrolysis of complement component C3, which is abundant in theplasma fraction of blood. This process, also known as "tickover,"occurs through the spontaneous cleavage of a thioester bond in C3to form C3i or C3(H.sub.2O). Tickover is facilitated by thepresence of surfaces that support the binding of activated C3and/or have neutral or positive charge characteristics (e.g.,bacterial cell surfaces). This formation of C3(H.sub.2O) allows forthe binding of plasma protein Factor B, which in turn allows FactorD to cleave Factor B into Ba and Bb. The Bb fragment remains boundto C3 to form a complex containing C3(H.sub.2O)Bb--the"fluid-phase" or "initiation" C3 convertase. Although only producedin small amounts, the fluid-phase C3 convertase can cleave multipleC3 proteins into C3a and C3b and results in the generation of C3band its subsequent covalent binding to a surface (e.g., a bacterialsurface). Factor B bound to the surface-bound C3b is cleaved byFactor D to thus form the surface-bound AP C3 convertase complexcontaining C3b,Bb. (See, e.g., Muller-Eberhard (1988) Ann RevBiochem 57:321 347.)

[0007] The AP C5 convertase--(C3b)2,Bb--is formed upon addition ofa second C3b monomer to the AP C3 convertase. (See, e.g., Medicuset al. (1976) J Exp Med 144:1076-1093 and Fearon et al. (1975) JExp Med 142:856-863.) The role of the second C3b molecule is tobind C5 and present it for cleavage by Bb. (See, e.g., Isenman etal. (1980) J Immunol 124:326-331.) The AP C3 and C5 convertases arestabilized by the addition of the trimeric protein properdin asdescribed in, e.g., Medicus et al. (1976), supra. However,properdin binding is not required to form a functioning alternativepathway C3 or C5 convertase. (See, e.g., Schreiber et al. (1978)Proc Natl Acad Sci USA 75: 3948-3952 and Sissons et al. (1980) ProcNatl Acad Sci USA 77: 559-562).

[0008] The CP C3 convertase is formed upon interaction ofcomplement component C1, which is a complex of C1q, C1r, and C1s,with an antibody that is bound to a target antigen (e.g., amicrobial antigen). The binding of the C1q portion of C1 to theantibody-antigen complex causes a conformational change in C1 thatactivates C1r. Active C1r then cleaves the C1-associated C1s tothereby generate an active serine protease. Active C1s cleavescomplement component C4 into C4b and C4a. Like C3b, the newlygenerated C4b fragment contains a highly reactive thiol thatreadily forms amide or ester bonds with suitable molecules on atarget surface (e.g., a microbial cell surface). C1s also cleavescomplement component C2 into C2b and C2a. The complex formed by C4band C2a is the CP C3 convertase, which is capable of processing C3into C3a and C3b. The CP C5 convertase--C4b,C2a,C3b--is formed uponaddition of a C3b monomer to the CP C3 convertase. (See, e.g.,Muller-Eberhard (1988), supra and Cooper et al. (1970) J Exp Med132:775-793.)

[0009] In addition to its role in C3 and C5 convertases, C3b alsofunctions as an opsonin through its interaction with complementreceptors present on the surfaces of antigen-presenting cells suchas macrophages and dendritic cells. The opsonic function of C3b isgenerally considered to be one of the most important anti-infectivefunctions of the complement system. Patients with genetic lesionsthat block C3b function are prone to infection by a broad varietyof pathogenic organisms, while patients with lesions later in thecomplement cascade sequence, i.e., patients with lesions that blockC5 functions, are found to be more prone only to Neisseriainfection, and then only somewhat more prone.

[0010] The AP and CP C5 convertases cleave C5 into C5a and C5b.Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotacticfactor, and C5b, which allows for the formation of the lyticterminal complement complex, C5b-9. C5b combines with C6, C7, andC8 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. Whensufficient numbers of MACs insert into target cell membranes theopenings they create (MAC pores) mediate rapid osmotic lysis of thetarget cells.

[0011] While a properly functioning complement system provides arobust defense against infecting microbes, inappropriate regulationor activation of the complement pathways has been implicated in thepathogenesis of a variety of disorders including, e.g., rheumatoidarthritis (RA); lupus nephritis; asthma; ischemia-reperfusioninjury; atypical hemolytic uremic syndrome (aHUS); dense depositdisease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); maculardegeneration (e.g., age-related macular degeneration (AMD));hemolysis, elevated liver enzymes, and low platelets (HELLP)syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneousfetal loss; Pauci-immune vasculitis; epidermolysis bullosa;recurrent fetal loss; multiple sclerosis (MS); traumatic braininjury; 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 ofcomplement activation has been demonstrated to be effective intreating several disease indications in a variety of animal models.See, e.g., Rother t al. (2007) Nature Biotechnology25(11):1256-1264; Wang et al. (1996) Proc Natl Acad Sci USA93:8563-8568; Wang et al. (1995) Proc Natl Acad Sci USA92:8955-8959; Rinder et al. (1995) J Clin Invest 96:1564-1572;Kroshus et al. (1995) Transplantation 60:1194-1202; Homeister etal. (1993) J Immunol 150:1055-1064; Weisman et al. (1990) Science249:146-151; Amsterdam et al. (1995) Am J Physiol 268:H448-H457;and Rabinovici et al. (1992) J Immunol 149:1744 1750.

SUMMARY

[0012] The disclosure relates to anti-C5 antibodies that have oneof more improved characteristics, e.g., relative to known anti-C5antibodies used for therapeutic purposes. For example, the anti-C5antibodies described herein exhibit increased serum-life relativeto the serum elimination half-life of eculizumab. Because of theirimproved pharmacokinetic properties, the antibodies describedherein feature a number of advantages, e.g., advantages over otheranti-C5 antibodies that bind to, and inhibit cleavage of,full-length or mature C5. Like such anti-C5 antibodies, theantibodies described herein can inhibit the C5a-mediatedinflammatory response and the C5b (MAC)-dependent cell lysis thatresults from cleavage of C5. However, as the concentration of C5 inhuman plasma is approximately 0.37 .mu.M (Rawal and Pangburn (2001)J Immunol 166(4):2635-2642), the use of high concentrations and/orfrequent administration of anti-C5 antibodies, such as eculizumab,is often necessary to effectively inhibit C5 in a human. Thedisclosure sets forth in the working examples experimental dataevidencing that while anti-C5 antibodies are highly effective atinhibiting complement in vitro and in vivo (see, e.g., Hillmen etal. (2004) N Engl J Med 350(6):552), the antibodies areparticularly susceptible to target-mediated clearance because ofthe high concentration of C5 in blood. The disclosure also showsthat the new antibodies described herein have reducedsusceptibility to the target-mediated clearance and thus have alonger serum elimination half-life (half-life), as compared topreviously known anti-C5 antibodies, in blood.

[0013] In view of their longer half-life, the antibodies describedherein can be administered to a human at a much lower dose and/orless frequently than previously known anti-C5 antibodies (such as,eculizumab) and effectively provide the same or greater inhibitionof C5 in a human. The ability to administer a lower dose of theantibody, as compared to, e.g., the dose of eculizumab, also allowsfor additional delivery routes such as, e.g., subcutaneousadministration, intramuscular administration, intrapulmonarydelivery, and administration via the use of biologically degradablemicrospheres.

[0014] Accordingly, in one aspect, the disclosure features ananti-C5 antibody having one or more improved properties (e.g.,improved pharmacokinetic properties) relative to eculizumab. Theantibody or C5-binding fragment thereof is one that: (a) binds tocomplement component C5; (b) inhibits the cleavage of C5 intofragments C5a and C5b; and (c) comprises: (i) a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:1, (ii) aheavy chain CDR2 comprising the amino acid sequence depicted in SEQID NO:2, (iii) a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:3, (iv) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:4, (v) alight chain CDR2 comprising the amino acid sequence depicted in SEQID NO:5, and (vi) a light chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:6, in which at least one (e.g., atleast two, at least three, at least four, at least five, at leastsix, at least seven, or at least eight) amino acid(s) of (i)-(vi)is substituted with a different amino acid. In some embodiments,the C5 is human C5.

[0015] In some embodiments of any of the antibodies or fragmentsdescribed herein, at least one amino acid of heavy chain CDR1 issubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, at least one aminoacid of heavy chain CDR2 is substituted with a different aminoacid. In some embodiments of any of the antibodies or fragmentsdescribed herein, at least one amino acid of heavy chain CDR3 issubstituted with a different amino acid.

[0016] In some embodiments of any of the antibodies or fragmentsdescribed herein at least one amino acid of light chain CDR1 issubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, the glycine atposition 8 relative to SEQ ID NO:4 is substituted with a differentamino acid (e.g., a histidine).

[0017] In some embodiments of any of the antibodies or fragmentsdescribed herein, at least one amino acid of light chain CDR2 issubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, at least one aminoacid of light chain CDR3 is substituted with a different aminoacid.

[0018] In some embodiments of any of the antibodies or fragmentsdescribed herein, a substitution is made at an amino acid positionselected from the group consisting of: glycine at position 1relative to SEQ ID NO:1, tyrosine at position 2 relative to SEQ IDNO:1, isoleucine at position 3 relative to SEQ ID NO:1,phenylalanine at position 4 relative to SEQ ID NO:1, serine atposition 5 relative to SEQ ID NO:1, asparagine at position 6relative to SEQ ID NO:1, tyrosine at position 7 relative to SEQ IDNO:1, tryptophan at position 8 relative to SEQ ID NO:1, isoleucineat position 9 relative to SEQ ID NO:1, glutamine at position 10relative to SEQ ID NO:1, glutamic acid at position 1 relative toSEQ ID NO:2, isoleucine at position 2 relative to SEQ ID NO:2,leucine at position 3 relative to SEQ ID NO:2, proline at position4 relative to SEQ ID NO:2, glycine at position 5 relative to SEQ IDNO:2, serine at position 6 relative to SEQ ID NO:2, glycine atposition 7 relative to SEQ ID NO:2, serine at position 8 relativeto SEQ ID NO:2, threonine at position 9 relative to SEQ ID NO:2,glutamic acid at position 10 relative to SEQ ID NO:2, tyrosine atposition 11 relative to SEQ ID NO:2, threonine at position 12relative to SEQ ID NO:2, glutamic acid at position 13 relative toSEQ ID NO:2, asparagine at position 14 relative to SEQ ID NO:2,phenylalanine at position 15 relative to SEQ ID NO:2, lysine atposition 16 relative to SEQ ID NO:2, aspartic acid at position 17relative to SEQ ID NO:2, tyrosine at position 1 relative to SEQ IDNO:3, phenylalanine at position 2 relative to SEQ ID NO:3,phenylalanine at position 3 relative to SEQ ID NO:3, glycine atposition 4 relative to SEQ ID NO:3, serine at position 5 relativeto SEQ ID NO:3, serine at position 6 relative to SEQ ID NO:3,proline at position 7 relative to SEQ ID NO:3, asparagine atposition 8 relative to SEQ ID NO:3, tryptophan at position 9relative to SEQ ID NO:3, tyrosine at position 10 relative to SEQ IDNO:3, phenylalanine at position 11 relative to SEQ ID NO:3,aspartic acid at position 12 relative to SEQ ID NO:3, and valine atposition 13 relative to SEQ ID NO:3.

[0019] In some embodiments of any of the antibodies or fragmentsdescribed herein, a substitution is made at an amino acid positionselected from the group consisting of: glycine at position 8relative to SEQ ID NO:4, leucine at position 10 relative to SEQ IDNO:4, valine at position 3 relative to SEQ ID NO:6, and threonineat position 6 relative to SEQ ID NO:6.

[0020] In some embodiments of any of the antibodies or fragmentsdescribed herein, a substitution is made at an amino acid positionselected from the group consisting of: tyrosine at position 2relative to SEQ ID NO:1, isoleucine at position 9 relative to SEQID NO:1, leucine at position 3 relative to SEQ ID NO:2, and serineat position 8 relative to SEQ ID NO:2.

[0021] In some embodiments of any of the antibodies or fragmentsdescribed herein, both tyrosine at position 2 relative to SEQ IDNO:1 and leucine at position 3 relative to SEQ ID NO:2 aresubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, the differentamino acid is a histidine.

[0022] In some embodiments of any of the antibodies or fragmentsdescribed herein, both isoleucine at position 9 relative to SEQ IDNO:1 and serine at position 8 relative to SEQ ID NO:2 aresubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, both isoleucine atposition 9 relative to SEQ ID NO:1 and leucine at position 3relative to SEQ ID NO:2 are substituted with a different aminoacid. In some embodiments of any of the antibodies or fragmentsdescribed herein, the different amino acid is a histidine.

[0023] In some embodiments of any of the antibodies or fragmentsdescribed herein, both tyrosine at position 2 relative to SEQ IDNO:1 and serine at position 8 relative to SEQ ID NO:2 aresubstituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, the antibody orantigen-binding fragment comprises a combination of amino acidsubstitutions selected from Table 1. In some embodiments of any ofthe antibodies or fragments described herein, the different aminoacid is a histidine.

[0024] In some embodiments of any of the antibodies or fragmentsdescribed herein, the combination of amino acid substitutionscomprises: (i) a substitution of a different amino acid for glycineat position 8 relative to SEQ ID NO:4 in the light chainpolypeptide of the antibody or antigen-binding fragment thereof;(ii) a substitution of a different amino acid for glycine atposition 2 relative to SEQ ID NO:1 of the heavy chain polypeptideof the antibody or antigen-binding fragment thereof; and (iii) asubstitution of a different amino acid for serine at position 8relative to SEQ ID NO:2 of the heavy chain polypeptide of theantibody or antigen-binding fragment thereof. In some embodimentsof any of the antibodies or fragments described herein, thedifferent amino acid is a histidine.

[0025] In some embodiments of any of the antibodies or fragmentsdescribed herein, tyrosine at position 2 relative to SEQ ID NO:1and serine at position 8 relative to SEQID NO:2 are substitutedwith histidine. In some embodiments of any of the antibodies orfragments described herein, the different amino acid is ahistidine.

[0026] In some embodiments, any of the antibodies or fragmentsdescribed herein bind to C5 at pH 7.4 and 25.degree. C. with anaffinity dissociation constant (K.sub.D) that is in the range 0.1nM.ltoreq.K.sub.D.ltoreq.1 nM. In some embodiments, any of theantibodies or fragments described herein bind to C5 at pH 7.4 and25.degree. C. with a K.sub.D that is in the range 0.2nM.ltoreq.K.sub.D.ltoreq.1 nM. In some embodiments, any of theantibodies or fragments described herein bind to C5 at pH 7.4 and25.degree. C. with a K.sub.D that is in the range 0.5nM.ltoreq.K.sub.D.ltoreq.1 nM.

[0027] In some embodiments, any of the antibodies or fragmentsdescribed herein bind to C5 at pH 6.0 and 25.degree. C. with aK.sub.D that is .gtoreq.1 nM (e.g., .gtoreq.50 nM, .gtoreq.100 nM,or .gtoreq.1 .mu.M).

[0028] In some embodiments of any of the antibodies or fragmentsdescribed herein, the [(K.sub.D of the antibody or antigen-bindingfragment thereof for C5 at pH 6.0 and at 25.degree. C)/(K.sub.D ofthe antibody or antigen-binding fragment thereof for C5 at pH 7.4and at 25.degree. C.)] is greater than 25. In some embodiments ofany of the antibodies or fragments described herein, the [(K.sub.Dof the antibody or antigen-binding fragment thereof for C5 at pH6.0 and at 25.degree. C)/(K.sub.D of the antibody orantigen-binding fragment thereof for C5 at pH 7.4 and at 25.degree.C.)] is greater than 100 (e.g., greater than 200, 300, 400, 500,600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,or 8500).

[0029] In some embodiments of any of the antibodies or fragmentsdescribed herein, the K.sub.D of the antibody or antigen-bindingfragment thereof for C5 at pH 7.4 and at 25.degree. C. is less than1 (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1)nM.

[0030] Based on the characterization of several variant eculizumabmolecules as described in the working examples, the inventorsdiscovered a new genus of antibodies having improvedpharmacokinetic properties as compared to eculizumab. Antibodieswithin this genus have an affinity for C5 that is weaker than theaffinity of eculizumab for C5 at pH 7.4. Yet the antibodies have anaffinity dissociation constant (K.sub.D) for C5 at pH 7.4 that isequal to or less than 1 nM. While the disclosure is not bound byany particular theory or mechanism of action, the inventors believethat slightly reducing the affinity of eculizumab for C5 at pH 7.4,and its subsequent effect on the affinity of the antibody for C5 atpH 6.0 while maintaining the same/similar ratio of affinity atpH7.4 and pH 6.0, will substantially reduce the C5-mediatedclearance of the antibody without substantially affecting thecomplement inhibitory function of the resultant antibody inpatients. Thus, the inventors have defined an optimal affinityrange for anti-C5 antibodies giving rise to improvedpharmacokinetic properties while preserving the requiredpharmacodynamic properties, each relative to eculizumab.Accordingly, in another aspect, the disclosure features an isolatedantibody, or antigen-binding fragment thereof, that: (a) binds tocomplement component C5 at pH 7.4 and 25.degree. C. with anaffinity dissociation constant (K.sub.D) that is .ltoreq.1 nM; (b)binds to C5 at pH 6.0 and 25.degree. C. with a K.sub.D that is nolower than 10 nM; (c) inhibits the cleavage of C5 into fragmentsC5a and C5 b, wherein the [(K.sub.D of the antibody orantigen-binding fragment thereof for C5 at pH 6.0 and 25.degree.C)/(K.sub.D of the antibody or antigen-binding fragment thereof forC5 at pH 7.4 and 25.degree. C.)] is greater than or equal to25.

[0031] In some embodiments, the antibody or antigen-bindingfragment thereof binds to C5 at pH 7.4 and 25.degree. C. with anaffinity dissociation constant (K.sub.D) that is in the range 0.1nM .ltoreq.K.sub.D.ltoreq.1 nM. In some embodiments, the antibodyor antigen-binding fragment thereof binds to C5 at pH 7.4 and25.degree. C. with a K.sub.D that is in the range 0.2 nM.ltoreq.K.sub.D.ltoreq.1 nM. In some embodiments, the antibody orantigen-binding fragment thereof binds to C5 at pH 7.4 and25.degree. C. with a K.sub.D that is in the range 0.5nM.ltoreq.K.sub.D.ltoreq.1 nM. In some embodiments, the antibody orantigen-binding fragment thereof binds to C5 at pH 6.0 and25.degree. C. with a K.sub.D that is .gtoreq.1 nM. In someembodiments, the antibody or antigen-binding fragment thereof bindsto C5 at pH 6.0 and 25.degree. C. with a K.sub.D that is .gtoreq.50nM. In some embodiments, the antibody or antigen-binding fragmentthereof binds to C5 at pH 6.0 and 25.degree. C. with a K.sub.D thatis .gtoreq.100 nM. In some embodiments, the antibody orantigen-binding fragment thereof binds to C5 at pH 6.0 and25.degree. C. with a K.sub.D that is .gtoreq.1 .mu.M.

[0032] In some embodiments, the [(K.sub.D of the antibody orantigen-binding fragment thereof for C5 at pH 6.0 and at 25.degree.C)/(K.sub.D of the antibody or antigen-binding fragment thereof forC5 at pH 7.4 and at 25.degree. C.)] is greater than 50 (e.g.,greater than 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, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,7500, 8000, or 8500).

[0033] In some embodiments, the antibody or antigen-bindingfragment thereof binds to C5 at pH 7.4 and at 25.degree. C. with aK.sub.D<1 nM. In some embodiments, the antibody orantigen-binding fragment thereof binds to C5 at pH 7.4 and at25.degree. C. with a K.sub.D<0.8 nM. In some embodiments, theantibody or antigen-binding fragment thereof binds to C5 at pH 7.4and at 25.degree. C. with a K.sub.D<0.5 nM. In some embodiments,the antibody or antigen-binding fragment thereof binds to C5 at pH7.4 and at 25.degree. C. with a K.sub.D<0.2 nM.

[0034] In some embodiments, the antibody or antigen-bindingfragment thereof comprises: (i) a heavy chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:1, (ii) a heavy chainCDR2 comprising the amino acid sequence depicted in SEQ ID NO:2,(iii) a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:3, (iv) a light chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:4, (v) a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:5, and(vi) a light chain CDR3 comprising the amino acid sequence depictedin SEQ ID NO:6, in which at least one amino acid of (i)-(vi) issubstituted with a different amino acid. The different amino acidcan be any amino acid (e.g., a histidine). In some embodiments, atleast one amino acid of heavy chain CDR1 is substituted with adifferent amino acid. In some embodiments, at least one amino acidof heavy chain CDR2 is substituted with a different amino acid. Insome embodiments, at least one amino acid of heavy chain CDR3 issubstituted with a different amino acid. In some embodiments, atleast one amino acid of light chain CDR1 is substituted with adifferent amino acid. In some embodiments, at least one amino acidof light chain CDR2 is substituted with a different amino acid. Insome embodiments, at least one amino acid of light chain CDR3 issubstituted with a different amino acid.

[0035] In some embodiments, a substitution is made at an amino acidposition selected from the group consisting of: glycine at position8 relative to SEQ ID NO:4, leucine at position 10 relative to SEQID NO:4, valine at position 3 relative to SEQ ID NO:6, andthreonine at position 6 relative to SEQ ID NO:6. In someembodiments, a substitution is made at an amino acid positionselected from the group consisting of: tyrosine at position 2relative to SEQ ID NO:1, isoleucine at position 9 relative to SEQID NO:1, leucine at position 3 relative to SEQ ID NO:2, and serineat position 8 relative to SEQ ID NO:2. In some embodiments, theantibody or antigen-binding fragment comprises a combination ofamino acid substitutions selected from Table 1.

[0036] In some embodiments, a combination of amino acidsubstitutions introduced into the CDRs comprises: (i) asubstitution a different amino acid for glycine at position 8relative to SEQ ID NO:4 in the light chain polypeptide of theantibody or antigen-binding fragment thereof; (ii) a substitutionof a different amino acid for glycine at position 2 relative to SEQID NO:1 of the heavy chain polypeptide of the antibody orantigen-binding fragment thereof; and (iii) a substitution of adifferent amino acid for serine at position 8 relative to SEQ IDNO:2 of the heavy chain polypeptide of the antibody orantigen-binding fragment thereof.

[0037] In some embodiments, a combination of amino acidsubstitutions comprises: (i) a substitution of a different aminoacid for glycine at position 2 relative to SEQ ID NO:1 of the heavychain polypeptide of the antibody or antigen-binding fragmentthereof; and (ii) a substitution of a different amino acid forserine at position 8 relative to SEQ ID NO:2 of the heavy chainpolypeptide of the antibody or antigen-binding fragmentthereof.

[0038] In some embodiments, tyrosine at position 2 relative to SEQID NO:1 and serine at position 8 relative to SEQID NO:2 aresubstituted (e.g., with histidine).

[0039] In some embodiments, any of the antibodies or fragmentthereof comprise a variant human Fc constant region (e.g., avariant human IgG Fc constant region) that binds to human neonatalFc receptor (FcRn) with greater affinity than that of the nativehuman Fc constant region from which the variant human Fc constantregion was derived. The variant Fc constant region can comprise oneor more (e.g., two, three, four, or five or more) amino acidsubstitutions relative to the native human Fc constant region fromwhich the variant human Fc constant region was derived. Thesubstitution can be at, e.g., amino acid position 237, 238, 239,248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297,298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332,334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433,434, or 436 (EU numbering) relative to the native human Fc constantregion. The substitution can be one selected from the groupconsisting of: methionine for glycine at position 237; alanine forproline at position 238; lysine for serine at position 239;isoleucine for lysine at position 248; alanine, phenylalanine,isoleucine, methionine, glutamine, serine, valine, tryptophan, ortyrosine for threonine at position 250; phenylalanine, tryptophan,or tyrosine for methionine at position 252; threonine for serine atposition 254; glutamic acid for arginine at position 255; asparticacid, glutamic acid, or glutamine for threonine at position 256;alanine, glycine, isoleucine, leucine, methionine, asparagine,serine, threonine, or valine for proline at position 257; histidinefor glutamic acid at position 258; alanine for aspartic acid atposition 265; phenylalanine for aspartic acid at position 270;alanine, or glutamic acid for asparagine at position 286; histidinefor threonine at position 289; alanine for asparagine at position297; glycine for serine at position 298; alanine for valine atposition 303; alanine for valine at position 305; alanine, asparticacid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine,serine, valine, tryptophan, or tyrosine for threonine at position307; alanine, phenylalanine, isoleucine, leucine, methionine,proline, glutamine, or threonine for valine at position 308;alanine, aspartic acid, glutamic acid, proline, or arginine forleucine or valine at position 309; alanine, histidine, orisoleucine for glutamine at position 311; alanine, or histidine foraspartic acid at position 312; lysine, or arginine for leucine atposition 314; alanine, or histidine for asparagine at position 315;alanine for lysine at position 317; glycine for asparagine atposition 325; valine for isoleucine at position 332; leucine forlysine at position 334; histidine for lysine at position 360;alanine for aspartic acid at position 376; alanine for glutamicacid at position 380; alanine for glutamic acid at position 382;alanine for asparagine or serine at position 384; aspartic acid, orhistidine for glycine at position 385; proline for glutamine atposition 386; glutamic acid for proline at position 387; alanine,or serine for asparagine at position 389; alanine for serine atposition 424; alanine, aspartic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, asparagine, proline,glutamine, serine, threonine, valine, tryptophan, or tyrosine formethionine at position 428; lysine for histidine at position 433;alanine, phenylalanine, histidine, serine, tryptophan, or tyrosinefor asparagine at position 434; and histidine for tyrosine orphenylalanine at position 436, all in EU numbering.

[0040] In some embodiments of any of the antibodies orantigen-binding fragments described herein, the variant human Fcconstant region comprises a methionine at position 428 and anasparagine at position 434, each in EU numbering.

[0041] In some embodiments, any of the antibodies orantigen-binding fragments thereof can comprise, or consist of, aheavy chain polypeptide comprising the amino acid sequence depictedin SEQ ID NO:12 or 14 and a light chain polypeptide comprising theamino acid sequence depicted in SEQ ID NO:8 or 11.

[0042] The disclosure also features an antibody comprising theheavy chain variable region of eculizumab (SEQ ID NO:7) or the CDRsof the heavy chain region of eculizumab (SEQ ID NOs:1-3) and any ofthe variant human Fc constant regions described herein, e.g., thevariant human Fc constant region comprising a methionine atposition 428 and an asparagine at position 434, each in EUnumbering.

[0043] In one embodiment, the antibody or antigen binding fragmenthas an increased half-life in humans relative to half-life in serumof eculizumab. The half-life as used herein is defined as the timeit takes for the plasma concentration of the antibody drug in thebody to be reduced by one half or 50%. This 50% reduction in serumconcentration reflects the amount of drug circulating and notremoved by the natural methods of antibody clearance. The half-lifeof eculizumab has been determined to be 272+82 hours or 11.3 daysin PNH patients and 12.1 days in aHUS patients (See SolirisPrescribing information). The half-life in humans of antibodies orfragments described herein may be increased relative to thehalf-life in humans of eculizumab. The half-life increase relativeto eculizumab may be at least 1.5 times the half life eculizumab,at least 2 times the half life eculizumab, at least 2.5 times thehalf-life of eculizumab or at least 3 times the half-life ofeculizumab.

[0044] In some embodiments of any of the antibodies or fragmentsdescribed herein, the antibody has a serum half-life in a humanthat is greater than, or at least, 10 (e.g., greater than, or atleast, 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 or 40) days.This half-life (or extension of half-life relative to eculizumab)can, in some embodiments, be achieved by an antibody describedherein containing a naturally-occurring human Fc constant region.In some embodiments, the half-life is measured relative to anantibody comprising a variant human Fc constant region describedherein. The half-life in humans of antibodies or fragmentsdescribed herein may be increased relative to the half-life inhumans of eculizumab. The half-life in humans of the antibodydescribed herein is at least 25 days, at least 26 days, at least 27days, at least 28 days, at least 29 days, at least 30 days, atleast 31 days, at least 32 days, at least 33 days, at least 34days, or at least 35 days.

[0045] In some embodiments, any of the antibodies or fragmentsdescribed herein are humanized, fully human, deimmunized, orchimeric. In some embodiments, an antibody or fragment thereofdescribed herein can be, e.g., a recombinant antibody, a singlechain antibody, a diabody, an intrabody, an Fv fragment, an Fdfragment, an Fab fragment, an Fab' fragment, and an F(ab').sub.2fragment.

[0046] In some embodiments, any of the antibodies or fragmentsthereof described herein can comprise a heterologous moiety, e.g.,a sugar. For example, the antibody or fragment thereof can beglycosylated. The heterologous moiety can also be a detectablelabel, e.g., a fluorescent label, a luminescent label, a heavymetal label, a radioactive label, or an enzymatic label.

[0047] In some embodiments, any of the antibodies orantigen-binding fragments thereof described herein can bemanufactured in a CHO cell. In some embodiments, the antibodies orantigen-binding fragments thereof do not contain detectable sialicacid residues.

[0048] In some embodiments, any of the antibodies orantigen-binding fragments thereof described herein can be modifiedwith a moiety that improves one or both of: (a) the stabilizationof the antibody or antigen-binding fragment thereof in circulationand (b) the retention of the antibody or antigen-binding fragmentthereof in circulation. Such a moiety can be PEG (PEGylation).

[0049] In yet another aspect, the disclosure features a nucleicacid that encodes one or both of the heavy and light chainpolypeptides of any of the antibodies or antigen-binding fragmentsdescribed herein. Also featured is a vector (e.g., a cloning orexpression vector) comprising the nucleic acid and a cell (e.g., aninsect cell, bacterial cell, fungal cell, or mammalian cell)comprising the vector. The disclosure further provides a method forproducing any of the antibodies or antigen-binding fragmentsthereof described herein. The methods include, optionally,providing the above described cell (or culture of cells) containingan expression vector (integrated or extrachromosomal), which vectorcontains a nucleic acid that encodes one or both of the heavy andlight chain polypeptides of any of the antibodies orantigen-binding fragments described herein. The cell or culture ofcells is cultured under conditions and for a time sufficient toallow expression by the cell (or culture of cells) of the antibodyor antigen-binding fragment thereof encoded by the nucleic acid.The method can also include isolating the antibody orantigen-binding fragment thereof from the cell (or cells of theculture) or from the media in which the cell or cells werecultured.

[0050] In another aspect, the disclosure features a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier andone or more of any of the antibodies or antigen-binding fragmentsthereof described herein.

[0051] In another aspect, the disclosure features a therapeutic kitcomprising: (i) one or more of any of the antibodies orantigen-binding fragments thereof described herein and (ii) meansfor delivery of the antibody or antigen-binding fragment thereof toa human. The means can be, e.g., a syringe or a pump.

[0052] In yet another aspect, the disclosure features an article ofmanufacture comprising: a container comprising a label and one ormore of any of the antibodies or antigen-binding fragments thereofdescribed herein, wherein the label indicates that the compositionis to be administered to a human having, suspected of having, or atrisk for developing, a complement-associated condition. The articleof manufacture can further comprise one or more additional activetherapeutic agents for use in treating a human having, suspected ofhaving, or at risk for developing, a complement-associatedcondition.

[0053] In another aspect, the disclosure features a method fortreating a patient afflicted with a complement-associatedcondition, the method comprising administering to the subject oneor more of any of the antibodies or antigen-binding fragmentsthereof described herein in an amount effective to treat thecomplement-associated condition. The complement-associatedcondition can be, e.g., one selected from the group consisting ofrheumatoid arthritis, antiphospholipid antibody syndrome, lupusnephritis, ischemia-reperfusion injury, atypical hemolytic uremicsyndrome, typical hemolytic uremic syndrome, paroxysmal nocturnalhemoglobinuria, dense deposit disease, neuromyelitis optica,multifocal motor neuropathy, multiple sclerosis, maculardegeneration, HELLP syndrome, spontaneous fetal loss, thromboticthrombocytopenic purpura, Pauci-immune vasculitis, epidermolysisbullosa, recurrent fetal loss, traumatic brain injury, myocarditis,a cerebrovascular disorder, a peripheral vascular disorder, arenovascular disorder, a mesenteric/enteric vascular disorder,vasculitis, Henoch-Schonlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, Takayasu'sdisease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki'sdisease, venous gas embolus, restenosis following stent placement,rotational atherectomy, percutaneous transluminal coronaryangioplasty, myasthenia gravis, cold agglutinin disease,dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipidsyndrome, Graves' disease, atherosclerosis, Alzheimer's disease,systemic inflammatory response sepsis, septic shock, spinal cordinjury, glomerulonephritis, transplant rejection (e.g., kidneytransplant), Hashimoto's thyroiditis, type I diabetes, psoriasis,pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenicpurpura, Goodpasture's syndrome, Degos disease, and catastrophicantiphospholipid syndrome.

[0054] As used herein, the term "antibody" refers to a wholeantibody comprising two light chain polypeptides and two heavychain polypeptides. Whole antibodies include different antibodyisotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term"antibody" includes a polyclonal antibody, a monoclonal antibody, achimerized or chimeric antibody, a humanized antibody, a primatizedantibody, a deimmunized antibody, and a fully human antibody. Theantibody can be made in or derived from any of a variety ofspecies, e.g., mammals such as humans, non-human primates (e.g.,orangutan, 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 recombinantantibody.

[0055] As used herein, the term "antibody fragment,""antigen-binding fragment," or similar terms refer to a fragment ofan antibody that retains the ability to bind to a target antigen(e.g., human C5) and inhibit the activity of the target antigen.Such fragments include, e.g., a single chain antibody, a singlechain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab'fragment, or an F(ab').sub.2 fragment. An scFv fragment is a singlepolypeptide chain that includes both the heavy and light chainvariable regions of the antibody from which the scFv is derived. Inaddition, intrabodies, minibodies, triabodies, and diabodies arealso included in the definition of antibody and are compatible foruse in the methods described herein. See, e.g., Todorovska et al.(2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) JImmunol Methods 231(1):177-189; Poljak (1994) Structure2(12):1121-1123; Rondon and Marasco (1997) Annual Review ofMicrobiology 51:257-283, the disclosures of each of which areincorporated herein by reference in their entirety.

[0056] As used herein, the term "antibody fragment" also includes,e.g., single domain antibodies such as camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem Sci26:230-235; Nuttall et al. (2000) Curr Pharm Biotech 1:253-263;Reichmann et al. (1999) J Immunol Meth 231:25-38; PCT applicationpublication nos. WO 94/04678 and WO 94/25591; and U.S. Pat. No.6,005,079, all of which are incorporated herein by reference intheir entireties. In some embodiments, the disclosure providessingle domain antibodies comprising two VH domains withmodifications such that single domain antibodies are formed.

[0057] In some embodiment, an antigen-binding fragment includes thevariable region of a heavy chain polypeptide and the variableregion of a light chain polypeptide. In some embodiments, anantigen-binding fragment described herein comprises the CDRs of thelight chain and heavy chain polypeptide of an antibody.

[0058] Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure pertains.Preferred methods and materials are described below, althoughmethods and materials similar or equivalent to those describedherein can also be used in the practice or testing of the presentlydisclosed methods and compositions. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

[0059] Other features and advantages of the present disclosure,e.g., methods for treating or preventing a complement-associatedcondition, will be apparent from the following description, theexamples, and from the claims.

Brief Description of the Sequences

[0060] SEQ ID NO:1 depicts the amino acid sequence of the heavychain CDR1 of eculizumab (as defined under the combinedKabat-Chothia definition).

[0061] SEQ ID NO:2 depicts the amino acid sequence of the heavychain CDR2 of eculizumab (as defined under the Kabatdefinition).

[0062] SEQ ID NO:3 depicts the amino acid sequence of the heavychain CDR3 of eculizumab (as defined under the combined Kabatdefinition).

[0063] SEQ ID NO:4 depicts the amino acid sequence of the lightchain CDR1 of eculizumab (as defined under the Kabatdefinition).

[0064] SEQ ID NO:5 depicts the amino acid sequence of the lightchain CDR2 of eculizumab (as defined under the Kabatdefinition).

[0065] SEQ ID NO:6 depicts the amino acid sequence of the lightchain CDR3 of eculizumab (as defined under the Kabatdefinition).

[0066] SEQ ID NO:7 depicts the amino acid sequence of the heavychain variable region of eculizumab.

[0067] SEQ ID NO:8 depicts the amino acid sequence of the lightchain variable region of eculizumab and the BNJ441 antibody.

[0068] SEQ ID NO:9 depicts the amino acid sequence of the heavychain constant region of eculizumab.

[0069] SEQ ID NO:10 depicts the amino acid sequence of the entireheavy chain of eculizumab.

[0070] SEQ ID NO:11 depicts the amino acid sequence of the entirelight chain of eculizumab and the BNJ441 antibody.

[0071] SEQ ID NO:12 depicts the amino acid sequence of the heavychain variable region of the BNJ441 antibody.

[0072] SEQ ID NO:13 depicts the amino acid sequence of the heavychain constant region of the BNJ441 antibody.

[0073] SEQ ID NO:14 depicts the amino acid sequence of the entireheavy chain of the BNJ441 antibody.

[0074] SEQ ID NO:15 depicts the amino acid sequence of an IgG2heavy chain constant region variant comprising the YTEsubstitutions.

[0075] SEQ ID NO:16: depicts the amino acid sequence of the entireheavy chain of an eculizumab variant comprising the heavy chainconstant region depicted in SEQ ID NO:15 (above).

[0076] SEQ ID NO:17 depicts the amino acid sequence of the lightchain CDR1 of eculizumab (as defined under the Kabat definition)with a glycine to histidine substitution at position 8 relative toSEQ ID NO:4.

[0077] SEQ ID NO:18 depicts the amino acid sequence of the lightchain variable region of the EHG303 antibody.

[0078] SEQ ID NO:19 depicts the amino acid sequence of the heavychain CDR2 of eculizumab in which serine at position 8 relative toSEQ ID NO:2 is substituted with a histidine.

[0079] SEQ ID NO:20 depicts the amino acid sequence of theso-called "FLAG" tag.

[0080] SEQ ID NO:21 depicts a polyhistidine sequence commonly usedas an antigenic tag.

[0081] SEQ ID NO:22 depicts the amino acid sequence of theso-called hemagglutinin tag.

[0082] SEQ ID NO:23 depicts the amino acid sequence of the heavychain CDR1 of eculizumab in which the tyrosine at position 2(relative to SEQ ID NO:1) is substituted with histidine.

[0083] SEQ IDNO:24 depicts the heavy chain polypeptide amino acidsequence of the EHG303 antibody.

[0084] SEQ ID NO:25 depicts the light chain polypeptide amino acidsequence of the EHG303 antibody.

[0085] SEQ ID NO: 26 depicts the amino acid sequence of the heavychain polypeptide of the EHL049 antibody.

[0086] SEQ ID NO: 27 depicts the amino acid sequence of the lightchain polypeptide of the EHL049 antibody.

[0087] SEQ ID NO:28 depicts the EHL000 heavy chain polypeptideamino acid sequence.

[0088] SEQ ID NO:29 depicts the amino acid sequence of the lightchain polypeptide of the EHL000 antibody.

[0089] SEQ ID NO:30 depicts the light chain polypeptide amino acidsequence of BHL006.

[0090] SEQ ID NO:31 depicts the amino acid sequence of the heavychain polypeptide of the BHL006 antibody.

[0091] SEQ ID NO:32 depicts the amino acid sequence of the lightchain polypeptide of the BHL009 antibody.

[0092] SEQ ID NO:33 depicts the amino acid sequence of the heavychain of the BHL009 antibody.

[0093] SEQ ID NO:34 depicts the amino acid sequence of the lightchain of the BHL0011 antibody.

[0094] SEQ ID NO:35 depicts the amino acid sequence of the heavychain of the BHL011 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 is a line graph depicting the clearance of eculizumabfrom the serum of human FcRn transgenic mice in the presence orabsence of exogenous human C5. The Y-axis represents the percentageof antibody remaining in the serum and the X-axis represents thetime in days.

[0096] FIG. 2 is a line graph depicting the clearance of aneculizumab variant having an IgG2 constant region (Ecu-IgG2) andthe Ecu-IgG2 antibody containing the YTE substitutions(Ecu-IgG2(YTE)) from the serum of mice. The Y-axis represents thepercentage of antibody remaining in the serum and the X-axisrepresents the time in days.

[0097] FIG. 3 is a line graph depicting the clearance of aneculizumab variant having an IgG2 constant region (Ecu-IgG2) andthe Ecu-IgG2 antibody containing the YTE substitutions(Ecu-IgG2(YTE)) from the serum of mice. The experiments wereperformed in the presence or absence of exogenous human C5. TheY-axis represents the percentage of antibody remaining in the serumand the X-axis represents the time in days.

[0098] FIG. 4 is a sensorgram plot depicting the kinetics ofassociation (at pH 7.4) and dissociation (at pH 7.4 and pH 6.0) forthree anti-C5 antibodies: EHL000, EHG303, and EHL049. The Y-axis isin arbitrary units, whereas the X-axis represents time (inseconds).

[0099] FIG. 5A is a sensorgram plot depicting the kinetics ofdissociation at pH 7.4 and pH 6.0 for the EHG303 (Y27H-S57H doublesubstitution) antibody, the Y27H single substitution variant ofeculizumab, and eculizumab (ecu; Ec293F). The Y-axis is innanometers (nm), whereas the X-axis represents time (inseconds).

[0100] FIG. 5B is a sensorgram plot depicting the kinetics ofdissociation at pH 7.4 and pH 6.0 for the EHG304 (I34H-L52H doublesubstitution) antibody, the I34H single substitution variant ofeculizumab, and eculizumab (ecu; Ec293F). The Y-axis is innanometers (nm), whereas the X-axis represents time (in seconds).The EHG304 antibody did not meet the second threshold forselection--namely it exceeded the maximum tolerated variance (fromeculizumab) for dissociation at pH7.4.

[0101] FIG. 5C is a sensorgram plot depicting the kinetics ofdissociation at pH 7.4 and pH 6.0 for the EHG303 (Y27H-S57H doublesubstitution) antibody and eculizumab (ecu; Ec293F). The Y-axis isin nanometers (nm), whereas the X-axis represents time (inseconds).

[0102] FIG. 5D is a sensorgram plot depicting the kinetics ofdissociation at pH 7.4 and pH 6.0 for the EHL049 [G31H (lightchain)/Y27H-S57H double substitution (heavy chain)] antibody, theY27H-S57H (EHG303) double substitution variant of eculizumab, andeculizumab (ecu). The Y-axis is in nanometers (nm), whereas theX-axis represents time (in seconds).

[0103] FIG. 5E is a sensorgram plot depicting the kinetics ofdissociation at pH 7.4 and pH 6.0 for the EHL058 [G31H (lightchain)/L52H-S57H double substitution (heavy chain)] antibody, theL52H-S57H double substitution (heavy chain) variant of eculizumab,and eculizumab (ecu). The Y-axis is in nanometers (nm), whereas theX-axis represents time (in seconds). The EHL058 antibody did notmeet the second threshold for selection--namely it exceeded themaximum tolerated variance (from eculizumab) for dissociation atpH7.4.

[0104] FIG. 6 is a line graph depicting the clearance of EHL000,BNJ421, and BNJ423 from the serum of NOD/scid/C5-deficient mice.The Y-axis represents the percentage of antibody remaining in theserum and the X-axis represents the time in days.

[0105] FIG. 7 is a line graph depicting the clearance of EHL000,BNJ421, and BNJ423 from the serum of NOD/scid/C5-deficient mice inthe presence or absence of human C5. The Y-axis represents thepercentage of antibody remaining in the serum and the X-axisrepresents the time in days.

[0106] FIG. 8 is a line graph depicting the activity of the EHL000,BNJ423, and BNJ421 antibodies in an ex vivo hemolytic assay. TheY-axis represents the percentage of hemolysis and the X-axisrepresents the time in days.

[0107] FIG. 9A is a line graph depicting the pharmacokinetics ofthe BHL011 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the concentration ofantibody in ug/mL. The X-axis represents time in days.

[0108] FIG. 9B is a line graph depicting the pharmacokinetics ofthe BHL011 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the % of theconcentration of antibody at day 1 remaining in the serum at eachtime point. The X-axis represents time in days.

[0109] FIG. 10A is a line graph depicting the pharmacokinetics ofthe BHL006 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the concentration ofantibody in ug/mL. The X-axis represents time in days.

[0110] FIG. 10B is a line graph depicting the pharmacokinetics ofthe BHL006 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the % of theconcentration of antibody at day 1 remaining in the serum at eachtime point. The X-axis represents time in days.

[0111] FIG. 11A is a line graph depicting the pharmacokinetics ofthe BHL009 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the concentration ofantibody in ug/mL. The X-axis represents time in days.

[0112] FIG. 11B is a line graph depicting the pharmacokinetics ofthe BHL009 antibody in hFcRn-transgenic mice. Each line representsa different animal. The Y-axis represents the % of theconcentration of antibody at day 1 remaining in the serum at eachtime point. The X-axis represents time in days.

[0113] FIG. 12 is a line graph depicting a log plot of the meanpharmacokinetics of the BHL011, BHL006, and BHL009 antibodies inhFcRn-transgenic mice. Each line represents a different antibody asindicated. The Y-axis represents the % of the concentration ofantibody at day 1 remaining in the serum at each time point. TheX-axis represents time in days.

[0114] FIG. 13 is a line graph depicting a linear plot of the meanpharmacokinetics of the BHL011, BHL006, and BHL009 antibodies inhFcRn-transgenic mice. Each line represents a different antibody asindicated. The Y-axis represents the % of the concentration ofantibody at day 1 remaining in the serum at each time point. TheX-axis represents time in days.

[0115] FIG. 14 is a line graph depicting the blocking ability ofthe BHL011 antibody in an ex vivo serum hemolytic assay after asingle dose. The Y-axis represents the percentage of hemolysis(relative to pre-dose levels) and the X-axis represents the time indays.

[0116] FIG. 15 is a line graph depicting the blocking ability ofthe BHL006 antibody in an ex vivo serum hemolytic assay after asingle dose. The Y-axis represents the percentage of hemolysis(relative to pre-dose levels) and the X-axis represents the time indays.

[0117] FIG. 16 is a line graph depicting the blocking ability ofthe BHL009 antibody in an ex vivo serum hemolytic assay after asingle dose. The Y-axis represents the percentage of hemolysis(relative to pre-dose levels) and the X-axis represents the time indays.

[0118] FIG. 17 is a graph depicting the correlation of BHL011 serumconcentration and ex vivo serum hemolytic activity after a singledose. The Y-axis represents the percentage of hemolysis (relativeto pre-dose levels) and the X-axis represents antibodyconcentration in .mu.g/mL.

[0119] FIG. 18 is a graph depicting the correlation of BHL006 serumconcentration and ex vivo serum hemolytic activity after a singledose. The Y-axis represents the percentage of hemolysis (relativeto pre-dose levels) and the X-axis represents antibodyconcentration in .mu.g/mL.

[0120] FIG. 19 is a graph depicting the correlation of BHL009 serumconcentration and ex vivo serum hemolytic activity after a singledose. The Y-axis represents the percentage of hemolysis (relativeto pre-dose levels) and the X-axis represents antibodyconcentration in .mu.g/mL.

[0121] FIG. 20 is a line graph depicting the mean ex vivo hemolyticactivity after a single dose of BHL011, BHL009, or BHL006 inhFcRn-transgenic mice. Each line represents a different antibody asindicated. The Y-axis represents the percentage of hemolysis(relative to pre-dose levels) and the X-axis represents time indays.

[0122] FIGS. 21A and 21B are a pair of line graphs depicting thesemi-log (FIG. 21A) and linear (FIG. 21B) plots of the affinity ofBNJ441 and eculizumab as a function of pH. The Y axis represents %dissociation and the X-axis is pH.

[0123] FIG. 22 is a line graph depicting the pharmacokinetics ofBNJ441 and eculizumab in the NOD/scid mice and in the absence ofhuman C5. The Y-axis represents the concentration of antibody in.mu.g/mL. The X-axis represents time in days.

[0124] FIG. 23 is a line graph depicting the pharmacokinetics ofBNJ441 and eculizumab in the NOD/scid mice and in the presence ofhuman C5. The Y-axis represents the concentration of antibody in.mu.g/mL. The X-axis represents time in days.

[0125] FIG. 24 is a line graph depicting the percentage of BNJ441and eculizumab remaining in the serum of NOD/scid mice in thepresence of human C5 as a function of time. The Y-axis representsthe concentration of antibody in .mu.g/mL. The X-axis representstime in days.

[0126] FIG. 25 is a line graph depicting the ex vivo serumhemolytic blocking activity of the BNJ441 antibody and eculizumabafter a single dose as a function of time. The Y-axis representsthe percentage of hemolysis (relative to pre-dose levels) and theX-axis represents the time in days.

[0127] FIG. 26 depicts mean serum BNJ441 concentration-timeprofiles following intravenous administration of a 200 mg or 400 mgDose to Healthy Volunteers (top panel--linear scale; bottompanel--log-linear scale).

[0128] FIG. 27 depicts mean chicken red blood cell hemolysis--timeprofiles following intravenous administration of placebo, 200 mgBNJ441, or 400 mg BNJ441 to Healthy Volunteers.

[0129] FIG. 28 depicts the relationship between BNJ441concentration and percent chicken red blood cell hemolysisfollowing intravenous administration of BNJ441 to healthy humanvolunteers.

[0130] FIG. 29 depicts the potency of BNJ441 compared toecculizumab in terminal complement activity assays.

[0131] FIG. 30 depicts the structure of BNJ441.

[0132] FIG. 31 depcits the inter-chain disulfide bonds ofBNJ441.

DETAILED DESCRIPTION

[0133] The disclosure provides antibodies that are useful for,among other things, inhibiting terminal complement (e.g., theassembly and/or activity of the C5b-9 TCC) and C5aanaphylatoxin-mediated inflammation and, thus, treatingcomplement-associated disorders. The antibodies have a number ofimproved properties relative to eculizumab, including, e.g.,increased serum half-life in a human. While in no way intended tobe limiting, exemplary antibodies, conjugates, pharmaceuticalcompositions and formulations, and methods for using any of theforegoing are elaborated on below and are exemplified in theworking Examples.

Antibodies

[0134] The anti-C5 antibodies described herein bind to complementcomponent C5 (e.g., human C5) and inhibit the cleavage of C5 intofragments C5a and C5b. As described above, such antibodies alsohave, for example, improved pharmacokinetic properties relative toother anti-C5 antibodies (e.g., eculizumab) used for therapeuticpurposes.

[0135] In some embodiments, an anti-C5 antibody described hereincomprises: (i) a heavy chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:1, (ii) a heavy chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:2, (iii) aheavy chain CDR3 comprising the amino acid sequence depicted in SEQID NO:3, (iv) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:4, (v) a light chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:S, and (vi) a light chainCDR3 comprising the amino acid sequence depicted in SEQ ID NO:6, inwhich at least one (e.g., at least two, three, four, five, six,seven, eight, nine, or 10 or more) amino acid(s) of (i)-(vi) issubstituted with a different amino acid.

[0136] The exact boundaries of CDRs have been defined differentlyaccording to different methods. In some embodiments, the positionsof the CDRs or framework regions within a light or heavy chainvariable domain can be as defined by Kabat et al. [(1991)"Sequences of Proteins of Immunological Interest." NIH PublicationNo. 91-3242, U.S. Department of Health and Human Services,Bethesda, Md]. In such cases, the CDRs can be referred to as "KabatCDRs" (e.g., "Kabat LCDR2" or "Kabat HCDR1"). In some embodiments,the positions of the CDRs of a light or heavy chain variable regioncan be as defined by Chothia et al. (1989) Nature 342:877-883.Accordingly, these regions can be referred to as "Chothia CDRs"(e.g., "Chothia LCDR2" or "Chothia HCDR3"). In some embodiments,the positions of the CDRs of the light and heavy chain variableregions can be as defined by a Kabat-Chothia combined definition.In such embodiments, these regions can be referred to as "combinedKabat-Chothia CDRs". Thomas et al. [(1996) Mol Immunol33(17/18):1389-1401] exemplifies the identification of CDRboundaries according to Kabat and Chothia definitions.

[0137] Any amino acid can be substituted with any other amino acid.In some embodiments, the substitution is a conservativesubstitution. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine;valine, isoleucine, and leucine; aspartic acid and glutamic acid;asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine. In some embodiments, oneor more amino acids are substituted with histidine.

[0138] In some embodiments, at least one (e.g., at least two,three, four, or five) amino acid of heavy chain CDR1 is substitutedwith a different amino acid. In some embodiments, at least one(e.g., at least two, three, four, or five) amino acid of heavychain CDR2 is substituted with a different amino acid. In someembodiments, at least one (e.g., at least two, three, four, orfive) amino acid of heavy chain CDR3 is substituted with adifferent amino acid.

[0139] In some embodiments, at least one (e.g., at least two,three, four, or five) amino acid of light chain CDR1 is substitutedwith a different amino acid. In some embodiments, at least one(e.g., at least two, three, four, or five) amino acid of lightchain CDR2 is substituted with a different amino acid. In someembodiments, at least one (e.g., at least two, three, four, orfive) amino acid of light chain CDR3 is substituted with adifferent amino acid.

[0140] In some embodiments, a substitution is made at an amino acidposition selected from the group consisting of: glycine at position1 relative to SEQ ID NO:1, tyrosine at position 2 relative to SEQID NO:1, isoleucine at position 3 relative to SEQ ID NO:1,phenylalanine at position 4 relative to SEQ ID NO:1, serine atposition 5 relative to SEQ ID NO:1, asparagine at position 6relative to SEQ ID NO:1, tyrosine at position 7 relative to SEQ IDNO:1, tryptophan at position 8 relative to SEQ ID NO:1, isoleucineat position 9 relative to SEQ ID NO:1, glutamine at position 10relative to SEQ ID NO:1, glutamic acid at position 1 relative toSEQ ID NO:2, isoleucine at position 2 relative to SEQ ID NO:2,leucine at position 3 relative to SEQ ID NO:2, proline at position4 relative to SEQ ID NO:2, glycine at position 5 relative to SEQ IDNO:2, serine at position 6 relative to SEQ ID NO:2, glycine atposition 7 relative to SEQ ID NO:2, serine at position 8 relativeto SEQ ID NO:2, threonine at position 9 relative to SEQ ID NO:2,glutamic acid at position 10 relative to SEQ ID NO:2, tyrosine atposition 11 relative to SEQ ID NO:2, threonine at position 12relative to SEQ ID NO:2, glutamic acid at position 13 relative toSEQ ID NO:2, asparagine at position 14 relative to SEQ ID NO:2,phenylalanine at position 15 relative to SEQ ID NO:2, lysine atposition 16 relative to SEQ ID NO:2, aspartic acid at position 17relative to SEQ ID NO:2, tyrosine at position 1 relative to SEQ IDNO:3, phenylalanine at position 2 relative to SEQ ID NO:3,phenylalanine at position 3 relative to SEQ ID NO:3, glycine atposition 4 relative to SEQ ID NO:3, serine at position 5 relativeto SEQ ID NO:3, serine at position 6 relative to SEQ ID NO:3,proline at position 7 relative to SEQ ID NO:3, asparagine atposition 8 relative to SEQ ID NO:3, tryptophan at position 9relative to SEQ ID NO:3, tyrosine at position 10 relative to SEQ IDNO:3, phenylalanine at position 11 relative to SEQ ID NO:3,aspartic acid at position 12 relative to SEQ ID NO:3, and valine atposition 13 relative to SEQ ID NO:3.

[0141] In some embodiments, the glycine at position 31 relative toSEQ ID NO:8 is substituted with a different amino acid. Forexample, the underlined glycine in CDR1 of the light chain ofeculizumab can be substituted with a different amino acid:GASENIYGALN (SEQ ID NO:4). The substitution can be a histidine forglycine, i.e., GASENIYHALN (SEQ ID NO:17).

[0142] In some embodiments, an anti-C5 antibody described hereincomprises an amino acid substitution at an amino acid positionselected from the group consisting of: glycine at position 26relative to SEQ ID NO:7, tyrosine at position 27 relative to SEQ IDNO:7, isoleucine at position 28 relative to SEQ ID NO:7,phenylalanine at position 29 relative to SEQ ID NO:7, serine atposition 30 relative to SEQ ID NO:7, asparagine at position 31relative to SEQ ID NO:7, tyrosine at position 32 relative to SEQ IDNO:7, tryptophan at position 33 relative to SEQ ID NO:7, isoleucineat position 34 relative to SEQ ID NO:7, glutamine at position 35relative to SEQ ID NO:7, glutamic acid at position 50 relative toSEQ ID NO:7, isoleucine at position 51 relative to SEQ ID NO:7,leucine at position 52 relative to SEQ ID NO:7, proline at position53 relative to SEQ ID NO:7, glycine at position 54 relative to SEQID NO:7, serine at position 55 relative to SEQ ID NO:7, glycine atposition 56 relative to SEQ ID NO:7, serine at position 57 relativeto SEQ ID NO:7, threonine at position 58 relative to SEQ ID NO:7,glutamic acid at position 59 relative to SEQ ID NO:7, tyrosine atposition 60 relative to SEQ ID NO:7, threonine at position 61relative to SEQ ID NO:7, glutamic acid at position 62 relative toSEQ ID NO:7, asparagine at position 63 relative to SEQ ID NO:7,phenylalanine at position 64 relative to SEQ ID NO:7, lysine atposition 65 relative to SEQ ID NO:7, aspartic acid at position 66relative to SEQ ID NO:7, tyrosine at position 99 relative to SEQ IDNO:7, phenylalanine at position 100 relative to SEQ ID NO:7,phenylalanine at position 101 relative to SEQ ID NO:7, glycine atposition 102 relative to SEQ ID NO:7, serine at position 103relative to SEQ ID NO:7, serine at position 104 relative to SEQ IDNO:7, proline at position 105 relative to SEQ ID NO:7, asparagineat position 106 relative to SEQ ID NO:7, tryptophan at position 107relative to SEQ ID NO:7, tyrosine at position 108 relative to SEQID NO:7, phenylalanine at position 109 relative to SEQ ID NO:7,aspartic acid at position 110 relative to SEQ ID NO:7, and valineat position 111 relative to SEQ ID NO:7. In some embodiments, theanti-C5 antibody comprises two or more (e.g., at least two, three,four, five, six, seven, eight, nine, or 10 or more) of any of theforegoing substitutions and in any combination.

[0143] In some embodiments, the anti-C5 antibody comprises at leastone substitution that meets the following criteria with respect toeculizumab: [0144] (1) a maximum variation for association kineticsat pH 7.4 of a 33% smaller peak phase shift at 800 seconds ascompared to the averaged peak phase shift at 800 seconds observedfor eculizumab; [0145] (2) a maximum variation for dissociationkinetics at pH 7.4 of no more than 3-fold reduction in peak phaseshift over 800 seconds as compared to the averaged peak phase shiftat 800 seconds observed for eculizumab; and [0146] (3) a minimumvariation for dissociation kinetics at pH 6.0 of at least a 3-foldreduction in the peak phase shift over 800 seconds as compared tothe averaged peak phase shift at 800 seconds observed foreculizumab.

[0147] For example, with respect to the criterion (1) above, if theaverage peak phase shift after 800 seconds of association witheculizumab is approximately 0.75 nm, a test antibody that has aphase shift of less than 0.5 nm (e.g., reproduced two or moretimes) would not meet the above criteria. By contrast, an anti-C5antibody with greater than a 0.5 nm peak phase shift at 800 secondsmeets the first criterion. Such substitutions give rise to anti-C5antibodies that only deviate from the k.sub.a and k.sub.d ofeculizumab at pH 7.4 to a minor degree, but deviate from thek.sub.d of eculizumab at pH 6.0 more significantly.

[0148] In some embodiments, an anti-C5 antibody described hereincomprises at least one (e.g., at least two, three, or four) aminoacid substitution at an amino acid position selected from the groupconsisting of: glycine at position 31 relative to SEQ ID NO:8,leucine at position 33 relative to SEQ ID NO:8, valine at position91 relative to SEQ ID NO:8, and threonine at position 94 relativeto SEQ ID NO:8. In some embodiments, an anti-C5 antibody describedherein comprises at least one (e.g., two, three, four or five)amino acid substitution(s) at an amino acid position selected fromthe group consisting of: tyrosine at position 27 relative to SEQ IDNO:7, isoleucine at position 34 relative to SEQ ID NO:7, leucine atposition 52 relative to SEQ ID NO:7, and serine at position 57relative to SEQ ID NO:7.

[0149] In some embodiments, an anti-C5 antibody described hereincontains in its light chain variable region at least onesubstitution selected from the following: glycine at position 31relative to SEQ ID NO:8, leucine at position 33 relative to SEQ IDNO:8, valine at position 91 relative to SEQ ID NO:8, and threonineat position 94 relative to SEQ ID NO:8. See Table 1 below. In someembodiments, an anti-C5 antibody described herein contains in itsheavy chain variable region at least one substitution selected fromthe following: tyrosine at position 27 relative to SEQ ID NO:7,isoleucine at position 34 relative to SEQ ID NO:7, leucine atposition 52 relative to SEQ ID NO:7, and serine at position 57relative to SEQ ID NO:7. See Table 1 below.

[0150] In some embodiments, an antibody comprises at least two(e.g., at least three, four, five, six, seven, eight, nine, or 10)amino acid substitutions relative to the CDR set defined by SEQ IDNOs:1-6. Thus, in some embodiments, an anti-C5 antibody describedherein comprises two or more substitutions in the combinations andat the amino acid positions set forth in Table 1.

TABLE-US-00001 TABLE 1 Amino Acid Substitution Combinations AminoSubstitutions within Substitutions within Acid the Light ChainVariable the Heavy Chain Variable Position/ Region CDRs ofEculizumab Region CDRs of Eculizumab Ab Cmb (relative to SEQ ID NO:8). (relative to SEQ ID NO: 7). No.: G31 L33 V91 T94 Y27 I34 L52S57 1 .circle-solid. .circle-solid. 2 .circle-solid. .circle-solid.3 .circle-solid. .circle-solid. 4 .circle-solid. .circle-solid. 5.circle-solid. .circle-solid. 6 .circle-solid. .circle-solid. 7.circle-solid. .circle-solid. 8 .circle-solid. .circle-solid. 9.circle-solid. .circle-solid. 10 .circle-solid. .circle-solid. 11.circle-solid. .circle-solid. 12 .circle-solid. .circle-solid. 13.circle-solid. .circle-solid. .circle-solid. .circle-solid. 14.circle-solid. .circle-solid. .circle-solid. .circle-solid. 15.circle-solid. .circle-solid. .circle-solid. .circle-solid. 16.circle-solid. .circle-solid. .circle-solid. .circle-solid. 17.circle-solid. .circle-solid. .circle-solid. .circle-solid. 18.circle-solid. .circle-solid. .circle-solid. .circle-solid. 19.circle-solid. .circle-solid. .circle-solid. .circle-solid. 20.circle-solid. .circle-solid. .circle-solid. .circle-solid. 21.circle-solid. .circle-solid. .circle-solid. .circle-solid. 22.circle-solid. .circle-solid. .circle-solid. .circle-solid. 23.circle-solid. .circle-solid. .circle-solid. .circle-solid. 24.circle-solid. .circle-solid. .circle-solid. .circle-solid. 25.circle-solid. .circle-solid. .circle-solid. .circle-solid. 26.circle-solid. .circle-solid. .circle-solid. .circle-solid. 27.circle-solid. .circle-solid. .circle-solid. .circle-solid. 28.circle-solid. .circle-solid. .circle-solid. .circle-solid. 29.circle-solid. .circle-solid. .circle-solid. .circle-solid. 30.circle-solid. .circle-solid. .circle-solid. .circle-solid. 31.circle-solid. .circle-solid. .circle-solid. 32 .circle-solid..circle-solid. .circle-solid. 33 .circle-solid. .circle-solid..circle-solid. 34 .circle-solid. .circle-solid. .circle-solid. 35.circle-solid. .circle-solid. .circle-solid. 36 .circle-solid..circle-solid. .circle-solid. 37 .circle-solid. .circle-solid..circle-solid. 38 .circle-solid. .circle-solid. .circle-solid. 39.circle-solid. .circle-solid. .circle-solid. 40 .circle-solid..circle-solid. .circle-solid. 41 .circle-solid. .circle-solid..circle-solid. 42 .circle-solid. .circle-solid. .circle-solid. 43.circle-solid. .circle-solid. .circle-solid. 44 .circle-solid..circle-solid. .circle-solid. 45 .circle-solid. .circle-solid..circle-solid. 46 .circle-solid. .circle-solid. .circle-solid. 47.circle-solid. .circle-solid. .circle-solid. 48 .circle-solid..circle-solid. .circle-solid. 49 .circle-solid. .circle-solid..circle-solid. 50 .circle-solid. .circle-solid. .circle-solid. 51.circle-solid. .circle-solid. .circle-solid. 52 .circle-solid..circle-solid. .circle-solid. 53 .circle-solid. .circle-solid..circle-solid. 54 .circle-solid. .circle-solid. .circle-solid. 55.circle-solid. .circle-solid. .circle-solid. 56 .circle-solid..circle-solid. .circle-solid. 57 .circle-solid. .circle-solid..circle-solid. 58 .circle-solid. .circle-solid. .circle-solid. 59.circle-solid. .circle-solid. .circle-solid. 60 .circle-solid..circle-solid. .circle-solid. 61 .circle-solid. .circle-solid..circle-solid. .circle-solid. 62 .circle-solid. .circle-solid..circle-solid. .circle-solid. 63 .circle-solid. .circle-solid..circle-solid. .circle-solid. 64 .circle-solid. .circle-solid..circle-solid. .circle-solid. 65 .circle-solid. .circle-solid..circle-solid. .circle-solid. .circle-solid. 66 .circle-solid..circle-solid. .circle-solid. .circle-solid. .circle-solid. 67.circle-solid. .circle-solid. .circle-solid. .circle-solid..circle-solid. 68 .circle-solid. .circle-solid. .circle-solid..circle-solid. 69 .circle-solid. .circle-solid. .circle-solid..circle-solid. 70 .circle-solid. .circle-solid. .circle-solid..circle-solid. 71 .circle-solid. .circle-solid. .circle-solid..circle-solid. 72 .circle-solid. .circle-solid. .circle-solid..circle-solid. 73 .circle-solid. .circle-solid. .circle-solid..circle-solid. 74 .circle-solid. .circle-solid. .circle-solid..circle-solid. 75 .circle-solid. .circle-solid. .circle-solid..circle-solid. 76 .circle-solid. .circle-solid. .circle-solid..circle-solid. .circle-solid. .circle-solid. .circle-solid.".circle-solid." indicates which of the amino acids are substitutedin a given antibody. For example, Ab Cmb. No. 76 defines anantibody comprising the six CDRs of eculizumab, in which the lightchain CDRs comprise substitutions at positions 31, 33, and 91,relative to SEQ ID NO: 8 and the heavy chain CDRs comprisesubstitutions at positions 27, 34, 52, and 57, relative to SEQ IDNO: 7. "Ab Comb. No." refers to a numerical designation given to aparticular variant anti-CS antibody referred to in the table. To beclear, the variant anti-CS antibodies referred to in Table 1 needonly have the amino acid sequences of the six (6) CDRs ofeculizumab in which the given, indicated amino acid substitutionsare made. The variant antibodies may, optionally, include theframework regions of SEQ ID NO: 7 or SEQ ID NO: 8.

The substitutions described in Table 1 can be for any amino acidthat is different from the indicated amino acid residue. In someembodiments, the different amino acid is a histidine.

[0151] In some embodiments, an anti-C5 antibody described hereincomprises a substitution made at an amino acid position selectedfrom the group consisting of: tyrosine at position 27 relative toSEQ ID NO:7, isoleucine at position 34 relative to SEQ ID NO:7,leucine at position 52 relative to SEQ ID NO:7, and serine atposition 57 relative to SEQ ID NO:7. In some embodiments, bothtyrosine at position 27 relative to SEQ ID NO:7 and leucine atposition 52 relative to SEQ ID NO:7 are each substituted with adifferent amino acid. In some embodiments, both isoleucine atposition 34 relative to SEQ ID NO:7 and serine at position 57relative to SEQ ID NO:7 are each substituted with a different aminoacid. In some embodiments, both isoleucine at position 34 relativeto SEQ ID NO:7 and leucine at position 52 relative to SEQ ID NO:7are each substituted with a different amino acid. In someembodiments, both tyrosine at position 27 relative to SEQ ID NO:7and serine at position 57 relative to SEQ ID NO:7 are eachsubstituted with a different amino acid. In some embodiments of anyof the anti-C5 antibodies described herein, the different aminoacid is a histidine. For example, tyrosine at position 27 andserine at position 57 can each be substituted with histidine.

[0152] In some embodiments, an anti-C5 antibody described hereincomprises a heavy chain CDR1 comprising, or consisting of, thefollowing amino acid sequence: GHIFSNYWIQ (SEQ ID NO:23). In someembodiments, an anti-C5 antibody described herein comprises a heavychain CDR2 comprising, or consisting of, the following amino acidsequence: EILPGSGHTEYTENFK.sub.D (SEQ ID NO:19). In someembodiments, an anti-C5 antibody described herein comprises a heavychain variable region comprising the following amino acidsequence:

TABLE-US-00002 (SEQ ID NO: 12)QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.

[0153] In some embodiments, an anti-C5 antibody described hereincomprises a light chain variable region comprising the followingamino acid sequence:

TABLE-US-00003 (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

[0154] An anti-C5 antibody described herein can bind to C5 at pH7.4 and 25.degree. C. (and, otherwise, under physiologicconditions) with an affinity dissociation constant (K.sub.D) thatis at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3,0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55,0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8,0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM. In someembodiments, the K.sub.D of the anti-C5 antibody is no greater than1 (e.g., no greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2)nM.

[0155] In some embodiments of any anti-C5 antibody describedherein, the [(K.sub.D of the antibody for C5 at pH 6.0 atC)/(K.sub.D of the antibody for C5 at pH 7.4 at 25.degree. C.)] isgreater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, or 8000).

[0156] Methods for determining whether an antibody binds to aprotein antigen and/or the affinity for an antibody to a proteinantigen are known in the art. For example, the binding of anantibody to a protein antigen can be detected and/or quantifiedusing a variety of techniques such as, but not limited to, Westernblot, dot blot, surface plasmon resonance (SPR) method (e.g.,BlAcore system; Pharmacia Biosensor AB, Uppsala, Sweden andPiscataway, 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 andProtocols," Humana Press (ISBN: 1588290921); Borrebaek (1992)"Antibody Engineering, A Practical Guide," W. H. Freeman and Co.,NY; Borrebaek (1995) "Antibody Engineering," 2.sup.nd Edition,Oxford University Press, NY, Oxford; Johne et al. (1993) J ImmunolMeth 160:191-198; Jonsson et al. (1993) Ann Biol Clin 51:19-26; andJonsson et al. (1991) Biotechniques 11:620-627. In addition,methods for measuring the affinity (e.g., dissociation andassociation constants) are set forth in the working examples.

[0157] As used herein, the term "k.sub.a" refers to the rateconstant for association of an antibody to an antigen. The term"k.sub.d" refers to the rate constant for dissociation of anantibody from the antibody/antigen complex. And the term "K.sub.D"refers to the equilibrium dissociation constant of anantibody-antigen interaction. The equilibrium dissociation constantis deduced from the ratio of the kinetic rate constants,K.sub.D=k.sub.a/k.sub.d. Such determinations preferably aremeasured at 25.degree. C. or 37.degree. C. (see the workingexamples). For example, the kinetics of antibody binding to humanC5 can be determined at pH 8.0, 7.4, 7.0, 6.5 and 6.0 via surfaceplasmon resonance (SPR) on a BIAcore 3000 instrument using ananti-Fc capture method to immobilize the antibody.

[0158] The anti-C5 antibody described herein can have activity inblocking the generation or activity of the C5a and/or C5b activefragments of a C5 protein (e.g., a human C5 protein). Through thisblocking effect, the antibodies inhibit, e.g., the proinflammatoryeffects of C5a and the generation of the C5b-9 membrane attackcomplex (MAC) at the surface of a cell.

[0159] Methods for determining whether a particular antibodydescribed herein inhibits C5 cleavage are known in the art.Inhibition of human complement component C5 can reduce thecell-lysing ability of complement in a subject's body fluids. Suchreductions of the cell-lysing ability of complement present in thebody fluid(s) can be measured by methods well known in the art suchas, for example, by a conventional hemolytic assay such as thehemolysis assay described by Kabat and Mayer (eds.), "ExperimentalImmunochemistry, 2.sup.nd Edition," 135-240, Springfield, Ill., C CThomas (1961), pages 135-139, or a conventional variation of thatassay such as the chicken erythrocyte hemolysis method as describedin, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552. Methodsfor determining whether a candidate compound inhibits the cleavageof human C5 into forms C5a and C5b are known in the art anddescribed in, e.g., Moongkarndi et al. (1982) Immunobiol 162:397;Moongkarndi et al. (1983) Immunobiol 165:323; Isenman et al. (1980)J Immunol 124(1):326-31; Thomas et al. (1996) Mol Immunol33(17-18):1389-401; and Evans et al. (1995) Mol Immunol32(16):1183-95. For example, the concentration and/or physiologicactivity of C5a and C5b in a body fluid can be measured by methodswell known in the art. Methods for measuring C5a concentration oractivity include, e.g., chemotaxis assays, RIAs, or ELISAs (see,e.g., Ward and Zvaifler (1971) J Clin Invest 50(3):606-16 andWurzner et al. (1991) Complement Inflamm 8:328-340). For C5b,hemolytic assays or assays for soluble C5b-9 as discussed hereincan be used. Other assays known in the art can also be used. Usingassays of these or other suitable types, candidate agents capableof inhibiting human complement component C5 can be screened.

[0160] Immunological techniques such as, but not limited to, ELISAcan be used to measure the protein concentration of C5 and/or itssplit products to determine the ability of an anti-C5 antibody toinhibit conversion of C5 into biologically active products. In someembodiments, C5a generation is measured. In some embodiments, C5b-9neoepitope-specific antibodies are used to detect the formation ofterminal complement.

[0161] Hemolytic assays can be used to determine the inhibitoryactivity of an anti-C5 antibody on complement activation. In orderto determine the effect of an anti-C5 antibody on classicalcomplement pathway-mediated hemolysis in a serum test solution invitro, for example, sheep erythrocytes coated with hemolysin orchicken erythrocytes sensitized with anti-chicken erythrocyteantibody are used as target cells. The percentage of lysis isnormalized by considering 100% lysis equal to the lysis occurringin the absence of the inhibitor. In some embodiments, the classicalcomplement pathway is activated by a human IgM antibody, forexample, as utilized in the Wieslab.RTM. Classical PathwayComplement Kit (Wieslab.RTM. COMPL CP310, Euro-Diagnostica,Sweden). Briefly, the test serum is incubated with an anti-C5antibody in the presence of a human IgM antibody. The amount ofC5b-9 that is generated is measured by contacting the mixture withan enzyme conjugated anti-C5b-9 antibody and a fluorogenicsubstrate and measuring the absorbance at the appropriatewavelength. As a control, the test serum is incubated in theabsence of the anti-C5 antibody. In some embodiments, the testserum is a C5-deficient serum reconstituted with a C5polypeptide.

[0162] To determine the effect of anti-C5 antibody on alternativepathway-mediated hemolysis, unsensitized rabbit or guinea pigerythrocytes are used as the target cells. In some embodiments, theserum test solution is a C5-deficient serum reconstituted with a C5polypeptide. The percentage of lysis is normalized by considering100% lysis equal to the lysis occurring in the absence of theinhibitor. In some embodiments, the alternative complement pathwayis activated by lipopolysaccharide molecules, for example, asutilized in the Wieslab.RTM. Alternative Pathway Complement Kit(Wieslab.RTM. COMPL AP330, Euro-Diagnostica, Sweden). Briefly, thetest serum is incubated with an anti-C5 antibody in the presence oflipopolysaccharide. The amount of C5b-9 that is generated ismeasured by contacting the mixture with an enzyme conjugatedanti-C5b-9 antibody and a fluorogenic substrate and measuring thefluorescence at the appropriate wavelength. As a control, the testserum is incubated in the absence of the anti-C5 antibody.

[0163] In some embodiments, C5 activity, or inhibition thereof, isquantified using a CH50eq assay. The CH50eq assay is a method formeasuring the total classical complement activity in serum. Thistest is a lytic assay, which uses antibody-sensitized erythrocytesas the activator of the classical complement pathway and variousdilutions of the test serum to determine the amount required togive 50% lysis (CH50). The percent hemolysis can be determined, forexample, using a spectrophotometer. The CH50eq assay provides anindirect measure of terminal complement complex (TCC) formation,since the TCC themselves are directly responsible for the hemolysisthat is measured.

[0164] The assay is well known and commonly practiced by those ofskill in the art. Briefly, to activate the classical complementpathway, undiluted serum samples (e.g., reconstituted human serumsamples) are added to microassay wells containing theantibody-sensitized erythrocytes to thereby generate TCC. Next, theactivated sera are diluted in microassay wells, which are coatedwith a capture reagent (e.g., an antibody that binds to one or morecomponents of the TCC). The TCC present in the activated samplesbind to the monoclonal antibodies coating the surface of themicroassay wells. The wells are washed and to each well is added adetection reagent that is detectably labeled and recognizes thebound TCC. The detectable label can be, e.g., a fluorescent labelor an enzymatic label. The assay results are expressed in CH50 unitequivalents per milliliter (CH50 U Eq/mL).

[0165] Inhibition, e.g., as it pertains to terminal complementactivity, 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 theactivity of terminal complement in, e.g., a hemolytic assay orCH50eq assay as compared to the effect of a control antibody (orantigen-binding fragment thereof) under similar conditions and atan equimolar concentration. Substantial inhibition, as used herein,refers to inhibition of a given activity (e.g., terminal complementactivity) 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, ananti-C5 antibody described herein contains one or more amino acidsubstitutions relative to the CDRs of eculizumab (i.e., SEQ IDNOs:1-6), yet retains at least 30 (e.g., at least 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, or 95) % of the complement inhibitoryactivity of eculizumab in a hemolytic assay or CH50eq assay.

[0166] An anti-C5 antibody described herein has a serum half-lifein humans that is at least 20 (e.g., at least 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) days. Methods formeasuring the serum half-life of an antibody are known in the artand exemplified in the working examples. See, e.g., Dall'Acqua etal. (2006) J Biol Chem 281: 23514-23524; Hinton et al. (2004) JBlot Chem 279:6213-6216; Hinton et al. (2006) J Immunol176:346-356; and Petkova et al. (2006) Int Immunol 18(12):1759-69,the disclosures of each of which are incorporated herein byreference in their entirety. In some embodiments, an anti-C5antibody described herein has a serum half-life that is at least 20(e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 125, 150, 175, 200, 250, 300, 400, 500) % greater than theserum half-life of eculizumab, e.g., as measured in one of themouse model systems described in the working examples (e.g., theC5-deficient/NOD/scid mouse or hFcRn transgenic mouse modelsystem).

[0167] Modifications to the Fc Region

[0168] An anti-C5 antibody described herein can, in someembodiments, comprise a variant human Fc constant region that bindsto human neonatal Fc receptor (FcRn) with greater affinity thanthat of the native human Fc constant region from which the varianthuman Fc constant region was derived. For example, the Fc constantregion can comprise one or more (e.g., two, three, four, five, six,seven, or eight or more) amino acid substitutions relative to thenative human Fc constant region from which the variant human Fcconstant region was derived. The substitutions can increase thebinding affinity of an IgG antibody containing the variant Fcconstant region to FcRn at pH 6.0, while maintaining the pHdependence of the interaction. See, e.g., Hinton et al. (2004) JBiol Chem 279:6213-6216 and Datta-Mannan et al. (2007) Drug MetabDispos 35:1-9. Methods for testing whether one or moresubstitutions in the Fc constant region of an antibody increase theaffinity of the Fc constant region for FcRn at pH 6.0 (whilemaintaining pH dependence of the interaction) are known in the artand exemplified in the working examples. See, e.g., Datta-Mannan etal. (2007) J Biol Chem 282(3):1709-1717; International PublicationNo. WO 98/23289; International Publication No. WO 97/34631; andU.S. Pat. No. 6,277,375, the disclosures of each of which areincorporated herein by reference in their entirety.

[0169] Substitutions that enhance the binding affinity of anantibody Fc constant region for FcRn are known in the art andinclude, e.g., (1) the M252Y/S254T/T256E triple substitutiondescribed by Dall'Acqua et al. (2006) J Biol Chem 281: 23514-23524;(2) the M428L or T250Q/M428L substitutions described in Hinton etal. (2004) J Biol Chem 279:6213-6216 and Hinton et al. (2006) JImmunol 176:346-356; and (3) the N434A or T307/E380A/N434Asubstitutions described in Petkova et al. (2006) Int Immunol18(12):1759-69. The additional substitution pairings: P257I/Q3111,P257I/N434H, and D376V/N434H are described in, e.g., Datta-Mannanet al. (2007) J Biol Chem 282(3):1709-1717, the disclosure of whichis incorporated herein by reference in its entirety.

[0170] In some embodiments, the variant constant region has asubstitution at EU amino acid residue 255 for valine. In someembodiments, the variant constant region has a substitution at EUamino acid residue 309 for asparagine. In some embodiments, thevariant constant region has a substitution at EU amino acid residue312 for isoleucine. In some embodiments, the variant constantregion has a substitution at EU amino acid residue 386.

[0171] In some embodiments, the variant Fc constant regioncomprises no more than 30 (e.g., no more than 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine,eight, seven, six, five, four, three, or two) amino acidsubstitutions, insertions, or deletions relative to the nativeconstant region from which it was derived. In some embodiments, thevariant Fc constant region comprises one or more amino acidsubstitutions selected from the group consisting of: M252Y, S254T,T256E, N434S, M428L, V259I, T2501, and V308F. In some embodiments,the variant human Fc constant region comprises a methionine atposition 428 and an asparagine at position 434, each in EUnumbering. In some embodiments, the variant Fc constant regioncomprises a 428L/434S double substitution as described in, e.g.,U.S. Pat. No. 8.088,376.

[0172] In some embodiments, the variant constant region comprises asubstitution at amino acid position 237, 238, 239, 248, 250, 252,254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305,307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376,380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EUnumbering) relative to the native human Fc constant region. In someembodiments, the substitution is selected from the group consistingof: methionine for glycine at position 237; alanine for proline atposition 238; lysine for serine at position 239; isoleucine forlysine at position 248; alanine, phenylalanine, isoleucine,methionine, glutamine, serine, valine, tryptophan, or tyrosine forthreonine at position 250; phenylalanine, tryptophan, or tyrosinefor methionine at position 252; threonine for serine at position254; glutamic acid for arginine at position 255; aspartic acid,glutamic acid, or glutamine for threonine at position 256; alanine,glycine, isoleucine, leucine, methionine, asparagine, serine,threonine, or valine for proline at position 257; histidine forglutamic acid at position 258; alanine for aspartic acid atposition 265; phenylalanine for aspartic acid at position 270;alanine, or glutamic acid for asparagine at position 286; histidinefor threonine at position 289; alanine for asparagine at position297; glycine for serine at position 298; alanine for valine atposition 303; alanine for valine at position 305; alanine, asparticacid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine,serine, valine, tryptophan, or tyrosine for threonine at position307; alanine, phenylalanine, isoleucine, leucine, methionine,proline, glutamine, or threonine for valine at position 308;alanine, aspartic acid, glutamic acid, proline, or arginine forleucine or valine at position 309; alanine, histidine, orisoleucine for glutamine at position 311; alanine or histidine foraspartic acid at position 312; lysine or arginine for leucine atposition 314; alanine or histidine for asparagine at position 315;alanine for lysine at position 317; glycine for asparagine atposition 325; valine for isoleucine at position 332; leucine forlysine at position 334; histidine for lysine at position 360;alanine for aspartic acid at position 376; alanine for glutamicacid at position 380; alanine for glutamic acid at position 382;alanine for asparagine or serine at position 384; aspartic acid orhistidine for glycine at position 385; proline for glutamine atposition 386; glutamic acid for proline at position 387; alanine orserine for asparagine at position 389; alanine for serine atposition 424; alanine, aspartic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, asparagine, proline,glutamine, serine, threonine, valine, tryptophan, or tyrosine formethionine at position 428; lysine for histidine at position 433;alanine, phenylalanine, histidine, serine, tryptophan, or tyrosinefor asparagine at position 434; and histidine for tyrosine orphenylalanine at position 436, all in EU numbering.

[0173] An anti-C5 antibody described herein can, in someembodiments, comprise a heavy chain polypeptide comprising theamino acid sequence depicted in SEQ ID NO:12 or 14 and/or a lightchain polypeptide comprising the amino acid sequence depicted inSEQ ID NO:8 or 11.

[0174] Methods for Producing the Anti-C5 Antibodies andAntigen-Binding Fragments thereof

[0175] The disclosure also features methods for producing any ofthe anti-C5 antibodies or antigen-binding fragments thereofdescribed herein. In some embodiments, methods for preparing anantibody described herein can include immunizing a subject (e.g., anon-human mammal) with an appropriate immunogen. Suitableimmunogens for generating any of the antibodies described hereinare set forth herein. For example, to generate an antibody thatbinds to C5, a skilled artisan can immunize a suitable subject(e.g., a non-human mammal such as a rat, a mouse, a gerbil, ahamster, a dog, a cat, a pig, a goat, a horse, or a non-humanprimate) with a full-length C5 polypeptide such as a full-lengthhuman C5 polypeptide. In some embodiments, the non-human mammal isC5 deficient, e.g., a C5-deficient mouse described in, e.g., Levyand Ladda (1971) Nat New Biot 229(2):51-52; Crocker et al. (1974) JClin Pathol 27(2):122-124; Wetsel et al. (1990) J Blot Chem265:2435-2440; and Jungi and Pepys (1981) Immunology 43(2):271-279.

[0176] A suitable subject (e.g., a non-human mammal) can beimmunized with the appropriate antigen along with subsequentbooster immunizations a number of times sufficient to elicit theproduction of an antibody by the mammal. The immunogen can beadministered to a subject (e.g., a non-human mammal) with anadjuvant. Adjuvants useful in producing an antibody in a subjectinclude, but are not limited to, protein adjuvants; bacterialadjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum orSalmonella minnesota) and bacterial components including cell wallskeleton, trehalose dimycolate, monophosphoryl lipid A, methanolextractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants,e.g., aluminum hydroxide, and iodoacetate and cholesterylhemisuccinate. Other adjuvants that can be used in the methods forinducing an immune response include, e.g., cholera toxin andparapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity31(1):15-24. See also, e.g., Lodmell et al. (2000) Vaccine18:1059-1066; 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.

[0177] In some embodiments, the methods include preparing ahybridoma cell line that secretes a monoclonal antibody that bindsto the immunogen. For example, a suitable mammal such as alaboratory mouse is immunized with a C5 polypeptide as describedabove. Antibody-producing cells (e.g., B cells of the spleen) ofthe immunized mammal can be isolated two to four days after atleast one booster immunization of the immunogen and then grownbriefly in culture before fusion with cells of a suitable myelomacell line. The cells can be fused in the presence of a fusionpromoter such as, e.g., vaccinia virus or polyethylene glycol. Thehybrid cells obtained in the fusion are cloned, and cell clonessecreting the desired antibodies are selected. For example, spleencells of Balb/c mice immunized with a suitable immunogen can befused with cells of the myeloma cell line PAI or the myeloma cellline Sp2/0-Ag 14. After the fusion, the cells are expanded insuitable culture medium, which is supplemented with a selectionmedium, for example HAT medium, at regular intervals in order toprevent normal myeloma cells from overgrowing the desired hybridomacells. The obtained hybrid cells are then screened for secretion ofthe desired antibodies, e.g., an antibody that binds to C5 andinhibits cleavage of C5 into fragments C5a and C5b.

[0178] In some embodiments, any of the antibodies orantigen-binding fragments thereof described herein can bemanufactured in a CHO cell. In some embodiments, the antibodies orantigen-binding fragments thereof do not contain detectable sialicacid residues.

[0179] In some embodiments, a skilled artisan can identify ananti-C5 antibody from a non-immune biased library as described in,e.g., U.S. Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) andSchoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0180] A subpopulation of antibodies screened using the abovemethods can be characterized for their specificity and bindingaffinity for a particular immunogen (e.g., C5) using anyimmunological or biochemical based method known in the art. Forexample, specific binding of an antibody to native, full-length C5,as compared to C5a, may be determined for example usingimmunological or biochemical based methods such as, but not limitedto, an ELISA assay, SPR assays, immunoprecipitation assay, affinitychromatography, and equilibrium dialysis as described above.Immunoassays which can be used to analyze immunospecific bindingand cross-reactivity of the antibodies include, but are not limitedto, competitive and non-competitive assay systems using techniquessuch as Western blots, RIA, ELISA (enzyme linked immunosorbentassay), "sandwich" immunoassays, immunoprecipitation assays,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, andprotein A immunoassays. Such assays are routine and well known inthe art.

[0181] Antibodies can also be assayed using any SPR-based assaysknown in the art for characterizing the kinetic parameters of theinteraction of the antibody with C5. Any SPR instrumentcommercially available including, but not limited to, BlAcoreInstruments (Biacore AB; Uppsala, Sweden); lAsys instruments(Affinity Sensors; Franklin, Mass.); IBIS system (WindsorScientific Limited; Berks, UK), SPR-CELLIA systems (Nippon Laserand Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta(Texas Instruments; Dallas, Tex.) can be used in the methodsdescribed 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 etal. (2000) Curr Opin Biotechnol 11: 54-61.

[0182] It is understood that the above methods can also be used todetermine if, e.g., an anti-C5 antibody does not bind tofull-length, native C3 and/or C4 proteins.

[0183] As described in the above references, after phage selection,the antibody coding regions from the phage can be isolated and usedto generate whole antibodies, including human antibodies, or anydesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria,e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab' and F(ab').sub.2 fragments can alsobe employed using methods known in the art such as those disclosedin PCT publication no. WO 92/22324; Mullinax et al. (1992)BioTechniques 12(6):864-869; and Sawai et al. (1995) Am J ReprImmunol 34:26-34; and Better et al. (1988) Science 240:1041-1043.Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology203:46-88; Shu et al. (1993) Proc Nat Acad Sci USA 90:7995-7999;and Skerra et al. (1988) Science 240:1038-1040.

[0184] In some embodiments, epitope mapping can be used toidentify, e.g., the region of C5 that interacts with an antibody.Methods for identifying the epitope to which a particular antibodybinds are also known in the art and are described above.

[0185] The antibodies and fragments thereof identified herein canbe or can be made "chimeric." Chimeric antibodies andantigen-binding fragments thereof comprise portions from two ormore different species (e.g., mouse and human). Chimeric antibodiescan be produced with mouse variable regions of desired specificityfused to human constant domains (for example, U.S. Pat. No.4,816,567). In this manner, non-human antibodies can be modified tomake them more suitable for human clinical application (e.g.,methods for treating or preventing a complement-mediated disorderin a subject).

[0186] 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 astherapeutic agents for humans because they are not cleared from thecirculation as rapidly as mouse antibodies and do not typicallyprovoke an adverse immune reaction. Generally, a humanized antibodyhas one or more amino acid residues introduced into it from anon-human source. These non-human amino acid residues are oftenreferred to as "import" residues, which are typically taken from an"import" variable domain. Methods of preparing humanized antibodiesare generally well known in the art. For example, humanization canbe essentially performed following the method of Winter andco-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 orCDR sequences for the corresponding sequences of a human antibody.Also see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. Insome embodiments, humanized forms of non-human (e.g., mouse)antibodies are human antibodies (recipient antibody) in which theCDR region amino acid residues of the non-human antibody (e.g.,mouse, rat, rabbit, or non-human primate antibody) having thedesired specificity, affinity, and binding capacity are graftedonto the framework scaffold of a human antibody.

[0187] In some instances, one or more framework region amino acidresidues of the human immunoglobulin are also replaced bycorresponding amino acid residues of the non-human antibody (socalled "back mutations"). In addition, phage display libraries canbe used to vary amino acids at chosen positions within the antibodysequence. The properties of a humanized antibody are also affectedby the choice of the human framework. Furthermore, humanized andchimerized antibodies can be modified to comprise residues that arenot found in the recipient antibody or in the donor antibody inorder to further improve antibody properties, such as, for example,affinity or effector function.

[0188] Fully human antibodies are also provided in the disclosure.The term "human antibody" includes antibodies having variable andconstant regions (if present) derived from human immunoglobulinsequences, preferably human germline sequences. Human antibodiescan include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term "human antibody" does not include antibodies inwhich CDR sequences derived from another mammalian species, such asa mouse, have been grafted onto human framework sequences (i.e.,humanized antibodies). Fully human or human antibodies may bederived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), andconstant (C) exons) or from human cells.

[0189] The human sequences may code for both the heavy and lightchains of human antibodies and would function correctly in themice, undergoing rearrangement to provide a wide antibodyrepertoire similar to that in humans. The transgenic mice can beimmunized with the target protein immunogen to create a diversearray of specific antibodies and their encoding RNA. Nucleic acidsencoding the antibody chain components of such antibodies may thenbe cloned from the animal into a display vector. Typically,separate populations of nucleic acids encoding heavy and lightchain sequences are cloned, and the separate populations thenrecombined on insertion into the vector, such that any given copyof the vector receives a random combination of a heavy and a lightchain. The vector is designed to express antibody chains so thatthey can be assembled and displayed on the outer surface of adisplay package containing the vector. For example, antibody chainscan be expressed as fusion proteins with a phage coat protein fromthe outer surface of the phage. Thereafter, display packages can beselected and screened for display of antibodies binding to atarget.

[0190] In some embodiments, the anti-C5 antibodies described hereincomprise an altered heavy chain constant region that has reduced(or no) effector function relative to its corresponding unalteredconstant region. Effector functions involving the constant regionof the anti-C5 antibody may be modulated by altering properties ofthe constant or Fc region. Altered effector functions include, forexample, a modulation in one or more of the following activities:antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC), apoptosis, binding to oneor more Fc-receptors, and pro-inflammatory responses. Modulationrefers to an increase, decrease, or elimination of an effectorfunction activity exhibited by a subject antibody containing analtered constant region as compared to the activity of theunaltered form of the constant region. In particular embodiments,modulation includes situations in which an activity is abolished orcompletely absent.

[0191] An altered constant region with altered FcR binding affinityand/or ADCC activity and/or altered CDC activity is a polypeptidewhich has either an enhanced or diminished FcR binding activityand/or ADCC activity and/or CDC activity compared to the unalteredform of the constant region. An altered constant region whichdisplays increased binding to an FcR binds at least one FcR withgreater affinity than the unaltered polypeptide. An alteredconstant region which displays decreased binding to an FcR binds atleast one FcR with lower affinity than the unaltered form of theconstant region. Such variants which display decreased binding toan 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 tothe level of binding of a native sequence immunoglobulin constantor Fc region to the FcR. Similarly, an altered constant region thatdisplays modulated ADCC and/or CDC activity may exhibit eitherincreased or reduced ADCC and/or CDC activity compared to theunaltered constant region. For example, in some embodiments, theanti-C5 antibody comprising an altered constant region can exhibitapproximately 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 ofthe unaltered form of the constant region. An anti-C5 antibodydescribed herein comprising an altered constant region displayingreduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDCactivity.

[0192] In certain embodiments, the altered constant region has atleast one amino acid substitution, insertion, and/or deletion,compared to a native sequence constant region or to the unalteredconstant region, e.g. from about one to about one hundred aminoacid substitutions, insertions, and/or deletions in a nativesequence constant region or in the constant region of the parentpolypeptide. In some embodiments, the altered constant regionherein will possess at least about 70% homology (similarity) oridentity with the unaltered constant region and in some instancesat least about 75% and in other instances at least about 80%homology or identity therewith, and in other embodiments at leastabout 85%, 90% or 95% homology or identity therewith. The alteredconstant region may also contain one or more amino acid deletionsor insertions. Additionally, the altered constant region maycontain one or more amino acid substitutions, deletions, orinsertions that results in altered post-translationalmodifications, including, for example, an altered glycosylationpattern (e.g., the addition of one or more sugar components, theloss of one or more sugar components, or a change in composition ofone or more sugar components relative to the unaltered constantregion).

[0193] Antibodies with altered or no effector functions may begenerated by engineering or producing antibodies with variantconstant, Fc, or heavy chain regions; recombinant DNA technologyand/or cell culture and expression conditions may be used toproduce antibodies with altered function and/or activity. Forexample, recombinant DNA technology may be used to engineer one ormore amino acid substitutions, deletions, or insertions in regions(such as, for example, Fc or constant regions) that affect antibodyfunction including effector functions. Alternatively, changes inpost-translational modifications, such as, e.g., glycosylationpatterns, may be achieved by manipulating the cell culture andexpression conditions by which the antibody is produced. Suitablemethods for introducing one or more substitutions, additions, ordeletions into an Fc region of an antibody are well known in theart and include, e.g., standard DNA mutagenesis techniques asdescribed in, e.g., Sambrook et al. (1989) "Molecular Cloning: ALaboratory Manual, 2.sup.nd Edition," Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra;Borrebaek (1992), supra; Johne et al. (1993), supra; PCTpublication no. WO 06/53301; and U.S. Pat. No. 7,704,497.

[0194] In some embodiments, an anti-C5 antibody described hereinexhibits reduced or no effector function. In some embodiments, ananti-C5 antibody comprises a hybrid constant region, or a portionthereof, such as a G2/G4 hybrid constant region (see e.g., Burtonet al. (1992) Adv Immun 51:1-18; Canfield et al. (1991) J Exp Med173:1483-1491; and Mueller et al. (1997) Mol Immunol34(6):441-452). See above.

[0195] In addition to using a G2/G4 construct as described above,an anti-C5 antibody described herein having reduced effectorfunction may be produced by introducing other types of changes inthe amino acid sequence of certain regions of the antibody. Suchamino acid sequence changes include but are not limited to theAla-Ala mutation described in, e.g., PCT Publication nos. WO94/28027 and WO 98/47531; and Xu et al. (2000) Cell Immunol200:16-26. Thus, in some embodiments, an anti-C5 antibody with oneor more mutations within the constant region including the Ala-Alamutation has reduced or no effector function. According to theseembodiments, the constant region of the antibody can comprise asubstitution to an alanine at position 234 or a mutation to analanine at position 235. Additionally, the altered constant regionmay contain a double mutation: a mutation to an alanine at position234 and a second mutation to an alanine at position 235. In oneembodiment, an anti-C5 antibody comprises an IgG4 framework,wherein the Ala-Ala mutation would describe a mutation(s) fromphenylalanine to alanine at position 234 and/or a mutation fromleucine to alanine at position 235. In another embodiment, theanti-C5 antibody comprises an IgG1 framework, wherein the Ala-Alamutation would describe a mutation(s) from leucine to alanine atposition 234 and/or a mutation from leucine to alanine at position235. An anti-C5 antibody may alternatively or additionally carryother mutations, including the point mutation K322A in the CH2domain (Hezareh et al. (2001) J Virol 75:12161-12168). An antibodywith said mutation(s) in the constant region may furthermore be ablocking or non-blocking antibody.

[0196] Additional substitutions that, when introduced into a heavychain constant region, result in decreased effector function areset forth in, e.g., Shields et al. (2001) J Biol Chem276(9):6591-6604. See particularly Table 1 ("Binding of human IgG1variants to human FcRn and FcyR) of Shields et al., the disclosureof which is incorporated herein by reference in its entirety. Byscreening a library of anti-IgE antibodies, each antibody of thelibrary differing by one or more substitutions in the heavy chainconstant region, for binding to a panel of Fc receptors (includingFcRn, Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIB, andFc.gamma.RIIIA), the authors identified a number of substitutionsthat modulate specific Fc-Fc receptor interactions. For example, avariant IgG2a heavy chain constant region in which the CH2 domaincontains a D265A substitution (heavy chain amino acid numberingaccording to Kabat et al. (supra)) results in a complete loss ofinteraction between the variant constant region and IgG Fcreceptors Fc.gamma.RIIB, Fc.gamma.RIII, Fc.gamma.RI, andFc.gamma.RIV. Shields et al. (2001) at page 6595, Table 1. See alsoBaudino et al. (2008) J Immunol 181:6664-6669 (supra).

[0197] Changes within the hinge region also affect effectorfunctions. For example, deletion of the hinge region may reduceaffinity for Fc receptors and may reduce complement activation(Klein et al. (1981) Proc Natl Acad Sci USA 78: 524-528). Thepresent disclosure therefore also relates to antibodies withalterations in the hinge region.

[0198] In some embodiments, an anti-C5 antibody may contain analtered constant region exhibiting enhanced or reduced complementdependent cytotoxicity (CDC). Modulated CDC activity may beachieved by introducing one or more amino acid substitutions,insertions, or deletions in an Fc region of the antibody. See,e.g., U.S. Pat. No. 6,194,551. Alternatively or additionally,cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved or reducedinternalization capability and/or increased or decreasedcomplement-mediated cell killing. See, e.g., Caron et al. (1992) JExp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCTpublication nos. WO 99/51642 and WO 94/29351; Duncan and Winter(1988) Nature 322:738-40; and U.S. Pat. Nos. 5,648,260 and5,624,821.

[0199] Another potential means of modulating effector function ofantibodies includes changes in glycosylation, which is summarizedin, e.g., Raju (2003) BioProcess International 1(4):44-53.According to Wright and Morrison, the microheterogeneity of humanIgG oligosaccharides can affect biological functions such as CDCand ADCC, binding to various Fc receptors, and binding to Clqprotein. (1997) TIBTECH 15:26-32. Glycosylation patterns ofantibodies can differ depending on the producing cell and the cellculture conditions (Raju, supra). Such differences can lead tochanges in both effector function and pharmacokinetics. See, e.g.,Israel et al. (1996) Immunology 89(4):573-578; and Newkirk et al.(1996) Clin Exp Immunol 106(2):259-264. Differences in effectorfunction may be related to the IgG's ability to bind to theFc.gamma. receptors (Fc.gamma.Rs) on the effector cells. Shields etal. have shown that IgG, with alterations in amino acid sequencethat have improved binding to Fc.gamma.R, can exhibit up to 100%enhanced ADCC using human effector cells. (2001) J Biol Chem276(9):6591-6604. While these alterations include changes in aminoacids not found at the binding interface, both the nature of thesugar component as well as its structural pattern may alsocontribute to the differences observed. In addition, the presenceor absence of fucose in the oligosaccharide component of an IgG canimprove binding and ADCC. See, e.g., Shields et al. (2002) J BiolChem 277(30):26733-26740. An IgG that lacked a fucosylatedcarbohydrate linked to Asn.sup.297 exhibited normal receptorbinding to the Fc.gamma.RI receptor. In contrast, binding to theFc.gamma.RIIIA receptor was improved 50-fold and accompanied byenhanced ADCC, especially at lower antibody concentrations. Stillother approaches exist for altering the effector function ofantibodies. For example, antibody-producing cells can behypermutagenic, thereby generating antibodies with randomly alteredpolypeptide residues throughout an entire antibody molecule. See,e.g., PCT publication no. WO 05/011735. Hypermutagenic host cellsinclude cells deficient in DNA mismatch repair. Antibodies producedin this manner may be less antigenic and/or have beneficialpharmacokinetic properties. Additionally, such antibodies may beselected for properties such as enhanced or decreased effectorfunction(s). Additional details of molecular biology techniquesuseful for preparing an antibody or antigen-binding fragmentthereof described herein are set forth below.

[0200] Recombinant Antibody Expression and Purification

[0201] The antibodies or antigen-binding fragments thereofdescribed herein can be produced using a variety of techniquesknown in the art of molecular biology and protein chemistry. Forexample, a nucleic acid encoding one or both of the heavy and lightchain polypeptides of an antibody can be inserted into anexpression vector that contains transcriptional and translationalregulatory sequences, which include, e.g., promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, transcription terminatorsignals, polyadenylation signals, and enhancer or activatorsequences. The regulatory sequences include a promoter andtranscriptional start and stop sequences. In addition, theexpression vector can include more than one replication system suchthat it can be maintained in two different organisms, for examplein mammalian or insect cells for expression and in a prokaryotichost for cloning and amplification.

[0202] Various modifications, e.g., substitutions, can beintroduced into the DNA sequences encoding the heavy and/or lightchain polypeptides described herein using standard methods known tothose of skill in the art. For example, introduction of a histidinesubstitution at one or more CDR positions of an antibody can becarried out using standard methods, such as PCR-mediatedmutagenesis, in which the mutated nucleotides are incorporated intothe PCR primers such that the PCR product contains the desiredmutations or site-directed mutagenesis. A substitution may beintroduced into one or more of the CDR regions to increase ordecrease the K.sub.D of the antibody for antigen, e.g., at pH 7.4or pH 6.0. Techniques in site-directed mutagenesis are well-knownin the art. See, e.g., Sambrook et al., supra.

[0203] Several possible vector systems are available for theexpression of cloned heavy chain and light chain polypeptides fromnucleic acids in mammalian cells. One class of vectors relies uponthe integration of the desired gene sequences into the host cellgenome. Cells which have stably integrated DNA can be selected bysimultaneously introducing drug resistance genes such as E. coligpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) orTn5 neo (Southern and Berg (1982) Mot Appl Genet 1:327). Theselectable marker gene can be either linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection (Wigler et al. (1979) Cell 16:77). A second classof vectors utilizes DNA elements which confer autonomouslyreplicating capabilities to an extrachromosomal plasmid. Thesevectors can be derived from animal viruses, such as bovinepapillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA,79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) ProcNatl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981)Nature 293:79).

[0204] The expression vectors can be introduced into cells in amanner suitable for subsequent expression of the nucleic acid. Themethod of introduction is largely dictated by the targeted celltype, discussed below. Exemplary methods include CaPO.sub.4precipitation, liposome fusion, cationic liposomes,electroporation, viral infection, dextran-mediated transfection,polybrene-mediated transfection, protoplast fusion, and directmicroinjection.

[0205] Appropriate host cells for the expression of antibodies orantigen-binding fragments thereof include yeast, bacteria, insect,plant, and mammalian cells. Of particular interest are bacteriasuch as E. coli, fungi such as Saccharomyces cerevisiae and Pichiapastoris, insect cells such as SF9, mammalian cell lines (e.g.,human cell lines), as well as primary cell lines.

[0206] In some embodiments, an antibody or fragment thereof can beexpressed in, and purified from, transgenic animals (e.g.,transgenic mammals). For example, an antibody can be produced intransgenic non-human mammals (e.g., rodents) and isolated from milkas described in, e.g., Houdebine (2002) Curr Opin Biotechnol13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res9(2):155-159; and Pollock et al. (1999) J Immunol Methods231(1-2):147-157.

[0207] The antibodies and fragments thereof can be produced fromthe cells by culturing a host cell transformed with the expressionvector containing nucleic acid encoding the antibodies orfragments, under conditions, and for an amount of time, sufficientto allow expression of the proteins. Such conditions for proteinexpression will vary with the choice of the expression vector andthe host cell, and will be easily ascertained by one skilled in theart through routine experimentation. For example, antibodiesexpressed in E. coli can be refolded from inclusion bodies (see,e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expressionsystems and methods for their use are well known in the art (seeCurrent Protocols in Molecular Biology, Wiley & Sons, andMolecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring HarborLaboratory Press, New York (2001)). The choice of codons, suitableexpression vectors and suitable host cells will vary depending on anumber of factors, and may be easily optimized as needed. Anantibody (or fragment thereof) described herein can be expressed inmammalian cells or in other expression systems including but notlimited to yeast, baculovirus, and in vitro expression systems(see, e.g., Kaszubska et al. (2000) Protein Expression andPurification 18:213-220).

[0208] Following expression, the antibodies and fragments thereofcan be isolated. The term "purified" or "isolated" as applied toany of the proteins (antibodies or fragments) described hereinrefers to a polypeptide that has been separated or purified fromcomponents (e.g., proteins or other naturally-occurring biologicalor organic molecules) which naturally accompany it, e.g., otherproteins, lipids, and nucleic acid in a prokaryote expressing theproteins. Typically, a polypeptide is purified when it constitutesat least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or99) %, by weight, of the total protein in a sample.

[0209] An antibody or fragment thereof can be isolated or purifiedin a variety of ways known to those skilled in the art depending onwhat other components are present in the sample. Standardpurification methods include electrophoretic, molecular,immunological, and chromatographic techniques, including ionexchange, hydrophobic, affinity, and reverse-phase HPLCchromatography. For example, an antibody can be purified using astandard anti-antibody column (e.g., a protein-A or protein-Gcolumn). Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. See, e.g.,Scopes (1994) "Protein Purification, 3r.sup.d edition,"Springer-Verlag, New York City, N.Y. The degree of purificationnecessary will vary depending on the desired use. In someinstances, no purification of the expressed antibody or fragmentsthereof will be necessary.

[0210] Methods for determining the yield or purity of a purifiedantibody or fragment thereof are known in the art and include,e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowryprotein assay, amido black protein assay, high pressure liquidchromatography (HPLC), mass spectrometry (MS), and gelelectrophoretic methods (e.g., using a protein stain such asCoomassie Blue or colloidal silver stain).

[0211] In some embodiments, endotoxin can be removed from theantibodies or fragments. Methods for removing endotoxin from aprotein sample are known in the art. For example, endotoxin can beremoved from a protein sample using a variety of commerciallyavailable reagents including, without limitation, theProteoSpin.TM. Endotoxin Removal Kits (Norgen Biotek Corporation),Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific; Pierce ProteinResearch Products), MiraCLEAN.RTM. Endotoxin Removal Kit (Mirus),or Acrodisc.TM.-Mustang.RTM. E membrane (Pall Corporation).

[0212] Methods for detecting and/or measuring the amount ofendotoxin present in a sample (both before and after purification)are known in the art and commercial kits are available. Forexample, the concentration of endotoxin in a protein sample can bedetermined using the QCL-1000 Chromogenic kit (BioWhittaker) or thelimulus amebocyte lysate (LAL)-based kits such as thePyrotell.RTM., Pyrotell.RTM.-T, Pyrochrome.RTM., Chromo-LAL, andCSE kits available from the Associates of Cape CodIncorporated.

Modification of the Antibodies or Antigen-Binding Fragmentsthereof

[0213] The antibodies or antigen-binding fragments thereof can bemodified following their expression and purification. Themodifications can be covalent or non-covalent modifications.

[0214] Such modifications can be introduced into the antibodies orfragments by, e.g., reacting targeted amino acid residues of thepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. Suitablesites for modification can be chosen using any of a variety ofcriteria including, e.g., structural analysis or amino acidsequence analysis of the antibodies or fragments.

[0215] In some embodiments, the antibodies or antigen-bindingfragments thereof can be conjugated to a heterologous moiety. Theheterologous moiety can be, e.g., a heterologous polypeptide, atherapeutic agent (e.g., a toxin or a drug), or a detectable labelsuch as, but not limited to, a radioactive label, an enzymaticlabel, a fluorescent label, a heavy metal label, a luminescentlabel, or an affinity tag such as biotin or streptavidin. Suitableheterologous polypeptides include, e.g., an antigenic tag (e.g.,FLAG (DYK.sub.D DDDK (SEQ ID NO:20)), polyhistidine (6-His; HHHHHH(SEQ ID NO:21)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO:22)),glutathione-S-transferase (GST), or maltose-binding protein (MBP))for use in purifying the antibodies or fragments. Heterologouspolypeptides also include polypeptides (e.g., enzymes) that areuseful as diagnostic or detectable markers, for example,luciferase, a fluorescent protein (e.g., green fluorescent protein(GFP)), or chloramphenicol acetyl transferase (CAT). Suitableradioactive labels include, e.g., .sup.32P, .sup.33P, .sup.14C,.sup.125I, .sup.131I, .sup.35S, and .sup.3H. Suitable fluorescentlabels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), green fluorescent protein (GFP), DyLight.TM.488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-AlexaFluor.RTM. 700, Cy5, allophycocyanin, and Cy7. Luminescent labelsinclude, e.g., any of a variety of luminescent lanthanide (e.g.,europium or terbium) chelates. For example, suitable europiumchelates include the europium chelate of diethylene triaminepentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymaticlabels include, e.g., alkaline phosphatase, CAT, luciferase, andhorseradish peroxidase.

[0216] Two proteins (e.g., an antibody and a heterologous moiety)can be cross-linked using any of a number of known chemical crosslinkers. Examples of such cross linkers are those which link twoamino acid residues via a linkage that includes a "hindered"disulfide bond. In these linkages, a disulfide bond within thecross-linking unit is protected (by hindering groups on either sideof the disulfide bond) from reduction by the action, for example,of reduced glutathione or the enzyme disulfide reductase. Onesuitable reagent,4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.(2-pyridyldithio)toluene (SMPT), forms such a linkage between two proteins utilizinga terminal lysine on one of the proteins and a terminal cysteine onthe other. Heterobifunctional reagents that cross-link by adifferent coupling moiety on each protein can also be used. Otheruseful cross-linkers include, without limitation, reagents whichlink two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups(e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydrylgroup (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), anamino group and a carboxyl group (e.g.,4-[p-azidosalicylamido]butylamine), and an amino group and aguanidinium group that is present in the side chain of arginine(e.g., p-azidophenyl glyoxal monohydrate).

[0217] In some embodiments, a radioactive label can be directlyconjugated to the amino acid backbone of the antibody.Alternatively, the radioactive label can be included as part of alarger molecule (e.g., .sup.125I inmeta-[.sup.125I]iodophenyl-N-hydroxysuccinimide ([.sup.125I]mIPNHS)which binds to free amino groups to form meta-iodophenyl (mIP)derivatives of relevant proteins (see, e.g., Rogers et al. (1997) JNucl Med 38:1221-1229) or chelate (e.g., to DOTA or DTPA) which isin turn bound to the protein backbone. Methods of conjugating theradioactive labels or larger molecules/chelates containing them tothe antibodies or antigen-binding fragments described herein areknown in the art. Such methods involve incubating the proteins withthe radioactive label under conditions (e.g., pH, saltconcentration, and/or temperature) that facilitate binding of theradioactive label or chelate to the protein (see, e.g., U.S. Pat.No. 6,001,329).

[0218] Methods for conjugating a fluorescent label (sometimesreferred 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., oflysines) or sulfhydryl groups (e.g., cysteines) of proteins usingsuccinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moietiesattached to the fluorophores. In some embodiments, the fluorophorescan be conjugated to a heterobifunctional cross-linker moiety suchas sulfo-SMCC. Suitable conjugation methods involve incubating anantibody protein, or fragment thereof, with the fluorophore underconditions that facilitate binding of the fluorophore to theprotein. See, e.g., Welch and Redvanly (2003) "Handbook ofRadiopharmaceuticals: Radiochemistry and Applications," John Wileyand Sons (ISBN 0471495603).

[0219] In some embodiments, the antibodies or fragments can bemodified, e.g., with a moiety that improves the stabilizationand/or retention of the antibodies in circulation, e.g., in blood,serum, or other tissues. For example, the antibody or fragment canbe PEGylated as described in, e.g., Lee et al. (1999) BioconjugChem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug DeliveriesReviews 54:477-485; and Roberts et al. (2002) Advanced DrugDelivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany;see, e.g., Pavisie et al. (2010) Int J Pharm 387(1-2):110-119). Thestabilization 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.

[0220] In some embodiments, the antibodies or antigen-bindingfragments thereof described herein can be glycosylated. In someembodiments, an antibody or antigen-binding fragment thereofdescribed herein can be subjected to enzymatic or chemicaltreatment, or produced from a cell, such that the antibody orfragment has reduced or absent glycosylation. Methods for producingantibodies with reduced glycosylation are known in the art anddescribed in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991)EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol30:1361.

[0221] Pharmaceutical Compositions and Formulations

[0222] The compositions described herein can be formulated as apharmaceutical solution, e.g., for administration to a subject forthe treatment or prevention of a complement-associated disorder.The pharmaceutical compositions will generally include apharmaceutically acceptable carrier. As used herein, a"pharmaceutically acceptable carrier" refers to, and includes, anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. The compositions caninclude a pharmaceutically acceptable salt, e.g., an acid additionsalt or a base addition salt (see, e.g., Berge et al. (1977) JPharm Sci 66:1-19).

[0223] The compositions can be formulated according to standardmethods. Pharmaceutical formulation is a well-established art, andis further described in, e.g., Gennaro (2000) "Remington: TheScience and Practice of Pharmacy," 20.sup.th Edition, Lippincott,Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999)"Pharmaceutical Dosage Forms and Drug Delivery Systems," 7.sup.thEdition, Lippincott Williams & Wilkins Publishers (ISBN:0683305727); and Kibbe (2000) "Handbook of PharmaceuticalExcipients American Pharmaceutical Association," 3.sup.rd Edition(ISBN: 091733096X). In some embodiments, a composition can beformulated, for example, as a buffered solution at a suitableconcentration and suitable for storage at 2-8.degree. C. (e.g.,4.degree. C.). In some embodiments, a composition can be formulatedfor storage at a temperature below 0.degree. C. (e.g., -20.degree.C. or -80.degree. C.). In some embodiments, the composition can beformulated for storage for up to 2 years (e.g., one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, 10 months, 11 months, 1 year, 11/2 years, or 2 years) at 2-8.degree. C. (e.g., 4.degree. C.).Thus, in some embodiments, the compositions described herein arestable in storage for at least 1 year at 2-8.degree. C. (e.g.,4.degree. C.).

[0224] The pharmaceutical compositions can be in a variety offorms. These forms include, e.g., liquid, semi-solid and soliddosage forms, such as liquid solutions (e.g., injectable andinfusible solutions), dispersions or suspensions, tablets, pills,powders, liposomes and suppositories. The preferred form depends,in part, on the intended mode of administration and therapeuticapplication. For example, compositions containing a compositionintended for systemic or local delivery can be in the form ofinjectable or infusible solutions. Accordingly, the compositionscan be formulated for administration by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscularinjection). "Parenteral administration," "administeredparenterally," and other grammatically equivalent phrases, as usedherein, refer to modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intranasal, intraocular, pulmonary,intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intrapulmonary,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intracerebral, intracranial, intracarotid and intrasternalinjection and infusion (see below).

[0225] The compositions can be formulated as a solution,microemulsion, dispersion, liposome, or other ordered structuresuitable for stable storage at high concentration. Sterileinjectable solutions can be prepared by incorporating a compositiondescribed herein in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, asrequired, followed by filter sterilization. Generally, dispersionsare prepared by incorporating a composition described herein into asterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectablesolutions, methods for preparation include vacuum drying andfreeze-drying that yield a powder of a composition described hereinplus any additional desired ingredient (see below) from apreviously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use ofsurfactants. Prolonged absorption of injectable compositions can bebrought about by including in the composition a reagent that delaysabsorption, for example, monostearate salts, and gelatin.

[0226] The compositions described herein can also be formulated inimmunoliposome compositions. Such formulations can be prepared bymethods known in the art such as, e.g., the methods described inEpstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al.(1980) Proc Natl Acad Sci USA 77:4030; and U.S. Pat. Nos. 4,485,045and 4,544,545. Liposomes with enhanced circulation time aredisclosed in, e.g., U.S. Pat. No. 5,013,556.

[0227] In certain embodiments, compositions can be formulated witha carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of suchformulations are known in the art. See, e.g., J.R. Robinson (1978)"Sustained and Controlled Release Drug Delivery Systems," MarcelDekker, Inc., New York.

[0228] In some embodiments, compositions can be formulated in acomposition suitable for intrapulmonary administration (e.g., foradministration via an inhaler or nebulizer) to a mammal such as ahuman. Methods for formulating such compositions are well known inthe art and described in, e.g., U.S. Patent Application PublicationNo. 20080202513; U.S. Pat. Nos. 7,112,341 and 6,019,968; and PCTPublication Nos. WO 00/061178 and WO 06/122257, the disclosures ofeach of which are incorporated herein by reference in theirentirety. Dry powder inhaler formulations and suitable systems foradministration of the formulations are described in, e.g., U.S.Patent Application Publication No. 20070235029, PCT Publication No.WO 00/69887; and U.S. Pat. No. 5,997,848. Additional formulationssuitable for intrapulmonary administration (as well as methods forformulating polypeptides) are set forth in, e.g., U.S. PatentApplication Publication Nos. 20050271660 and 20090110679.

[0229] In some embodiments, compositions can be formulated fordelivery to the eye. As used herein, the term "eye" refers to anyand all anatomical tissues and structures associated with an eye.The eye has a wall composed of three distinct layers: the outersclera, the middle choroid layer, and the inner retina. The chamberbehind the lens is filled with a gelatinous fluid referred to asthe vitreous humor. At the back of the eye is the retina, whichdetects light. The cornea is an optically transparent tissue, whichconveys images to the back of the eye. The cornea includes onepathway for the permeation of drugs into the eye. Other anatomicaltissue structures associated with the eye include the lacrimaldrainage system, which includes a secretory system, a distributivesystem and an excretory system. The secretory system comprisessecretors that are stimulated by blinking and temperature changedue to tear evaporation and reflex secretors that have an efferentparasympathetic nerve supply and secrete tears in response tophysical or emotional stimulation. The distributive system includesthe eyelids and the tear meniscus around the lid edges of an openeye, which spread tears over the ocular surface by blinking, thusreducing dry areas from developing.

[0230] In some embodiments, compositions can be administeredlocally, for example, by way of topical application or intravitrealinjection. For example, in some embodiments, the compositions canbe formulated for administration by way of an eye drop.

[0231] The therapeutic preparation for treating the eye can containone or more active agents in a concentration from about 0.01 toabout 1% by weight, preferably from about 0.05 to about 0.5% in apharmaceutically acceptable solution, suspension or ointment. Thepreparation will preferably be in the form of a sterile aqueoussolution containing, e.g., additional ingredients such as, but notlimited to, preservatives, buffers, tonicity agents, antioxidantsand stabilizers, nonionic wetting or clarifying agents, andviscosity-increasing agents.

[0232] Suitable preservatives for use in such a solution includebenzalkonium chloride, benzethonium chloride, chlorobutanol,thimerosal and the like. Suitable buffers include, e.g., boricacid, sodium and potassium bicarbonate, sodium and potassiumborates, sodium and potassium carbonate, sodium acetate, and sodiumbiphosphate, in amounts sufficient to maintain the pH at betweenabout 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 sodiumchloride.

[0233] Suitable antioxidants and stabilizers include sodiumbisulfite, sodium metabisulfite, sodium thiosulfite, and thiourea.Suitable wetting and clarifying agents include polysorbate 80,polysorbate 20, poloxamer 282 and tyloxapol. Suitableviscosity-increasing agents include dextran 40, dextran 70,gelatin, glycerin, hydroxyethylcellulose,hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, andcarboxymethylcellulose. The preparation can be administeredtopically to the eye of the subject in need of treatment (e.g., asubject afflicted with AMD) by conventional methods, e.g., in theform of drops, or by bathing the eye in a therapeutic solution,containing one or more compositions.

[0234] In addition, a variety of devices have been developed forintroducing drugs into the vitreal cavity of the eye. For example,U.S. patent application publication no. 20020026176 describes apharmaceutical-containing plug that can be inserted through thesclera such that it projects into the vitreous cavity to deliverthe pharmaceutical agent into the vitreous cavity. In anotherexample, U.S. Pat. No. 5,443,505 describes an implantable devicefor introduction into a suprachoroidal space or an avascular regionfor sustained release of drug into the interior of the eye. U.S.Pat. Nos. 5,773,019 and 6,001,386 each disclose an implantable drugdelivery device attachable to the scleral surface of an eye. Thedevice comprises an inner core containing an effective amount of alow solubility agent covered by a non-bioerodible polymer that ispermeable to the low solubility agent. During operation, the lowsolubility agent permeates the bioerodible polymer cover forsustained release out of the device. Additional methods and devices(e.g., a transscleral patch and delivery via contact lenses) fordelivery of a therapeutic agent to the eye are described in, e.g.,Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Rantaand Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocasand 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 publicationno. WO 04/073551, the disclosures of which are incorporated hereinby reference in their entirety.

[0235] As described above, relatively high concentrationcompositions can be made. For example, the compositions can beformulated at a concentration of between about 10 mg/mL to 100mg/mL (e.g., between about 9 mg/mL and 90 mg/mL; between about 9mg/mL and 50 mg/mL; between about 10 mg/mL and 50 mg/mL; betweenabout 15 mg/mL and 50 mg/mL; between about 15 mg/mL and 110 mg/mL;between about 15 mg/mL and 100 mg/mL; between about 20 mg/mL and100 mg/mL; between about 20 mg/mL and 80 mg/mL; between about 25mg/mL and 100 mg/mL; between about 25 mg/mL and 85 mg/mL; betweenabout 20 mg/mL and 50 mg/mL; between about 25 mg/mL and 50 mg/mL;between about 30 mg/mL and 100 mg/mL; between about 30 mg/mL and 50mg/mL; between about 40 mg/mL and 100 mg/mL; or between about 50mg/mL and 100 mg/mL). In some embodiments, compositions can beformulated at a concentration of greater than 5 mg/mL and less than50 mg/mL. Methods for formulating a protein in an aqueous solutionare known in the art and are described in, e.g., U.S. Pat. No.7,390,786; McNally and Hastedt (2007), "Protein Formulation andDelivery," Second Edition, Drugs and the Pharmaceutical Sciences,Volume 175, CRC Press; and Banga (2005), "Therapeutic peptides andproteins: formulation, processing, and delivery systems, SecondEdition" CRC Press. In some embodiments, the aqueous solution has aneutral pH, e.g., a pH between, e.g., 6.5 and 8 (e.g., between andinclusive of 7 and 8). In some embodiments, the aqueous solutionhas a pH of about 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the aqueoussolution has a pH of greater than (or equal to) 6 (e.g., greaterthan or equal to 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9), but less than pH8.

[0236] Nucleic acids encoding a therapeutic polypeptide can beincorporated into a gene construct to be used as a part of a genetherapy protocol to deliver nucleic acids that can be used toexpress and produce agents within cells. Expression constructs ofsuch components may be administered in any therapeuticallyeffective carrier, e.g. any formulation or composition capable ofeffectively delivering the component gene to cells in vivo.Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associatedvirus, lentivirus, and herpes simplex virus-1 (HSV-1), orrecombinant bacterial or eukaryotic plasmids. Viral vectors cantransfect cells directly; plasmid DNA can be delivered with thehelp of, for example, cationic liposomes (lipofectin) orderivatized, polylysine conjugates, gramicidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO.sub.4 precipitation (see,e.g., WO04/060407) carried out in vivo. (See also, "Ex vivoApproaches," 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 andMulligan (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 NatlAcad Sci USA 88:8039-8043; Ferry et al. (1991) Proc Natl Acad SciUSA 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 Immunol150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; and PCTPublication Nos. WO89/07136, WO89/02468, WO89/05345, andWO92/07573). Another viral gene delivery system utilizesadenovirus-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 adenoviralvectors derived from the adenovirus strain Ad type 5 d1324 or otherstrains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known tothose skilled in the art. Yet another viral vector system usefulfor delivery of the subject gene is the adeno-associated virus(AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Blot7:349-356; Samulski et al. (1989) J Virol 63:3822-3828; andMcLaughlin et al. (1989) J Virol 62:1963-1973.

[0237] In some embodiments, compositions can be formulated with oneor more additional therapeutic agents, e.g., additional therapiesfor treating or preventing a complement-associated disorder (e.g.,an AP-associated disorder or a CP-associated disorder) in asubject. Additional agents for treating a complement-associateddisorder in a subject will vary depending on the particulardisorder being treated, but can include, without limitation, anantihypertensive (e.g., an angiotensin-converting enzyme inhibitor)[for use in treating, e.g., HELLP syndrome], an anticoagulant, acorticosteroid (e.g., prednisone), or an immunosuppressive agent(e.g., vincristine or cyclosporine A). Examples of anticoagulantsinclude, e.g., warfarin (Coumadin), aspirin, heparin, phenindione,fondaparinux, idraparinux, and thrombin inhibitors (e.g.,argatroban, lepirudin, bivalirudin, or dabigatran). A compositiondescribed herein can also be formulated with a fibrinolytic agent(e.g., ancrod, .epsilon.-aminocaproic acid, antiplasmin-a.sub.1,prostacyclin, and defibrotide) for the treatment of acomplement-associated disorder. In some embodiments, a compositioncan be formulated with a lipid-lowering agent such as an inhibitorof hydroxymethylglutaryl CoA reductase. In some embodiments, acomposition can be formulated with, or for use with, an anti-CD20agent such as rituximab (Rituxan.TM.; Biogen Idec, Cambridge,Mass.). In some embodiments, e.g., for the treatment of RA, thecomposition can be formulated with one or both of infliximab(Remicade.RTM.; Centocor, Inc.) and methotrexate (Rheumatrex.RTM.,Trexall.RTM.). In some embodiments, a composition described hereincan be formulated with a non-steroidal anti-inflammatory drug(NSAID). Many different NSAIDS are available, some over the counterincluding ibuprofen (Advil.RTM., Motrin.RTM., Nuprin.RTM.) andnaproxen (Aleve.RTM.) and many others are available by prescriptionincluding meloxicam (Mobic.RTM.), etodolac (Lodine.RTM.),nabumetone (Relafen.RTM.), sulindac (Clinoril.RTM.), tolementin(Tolectin.RTM.), choline magnesium salicylate (Trilisate.RTM.),diclofenac (Cataflam.RTM., Voltaren.RTM., Arthrotec.RTM.),Diflunisal (Dolobid.RTM.), indomethacin (Indocin.RTM.), Ketoprofen(Orudis.RTM., Oruvail.RTM.), Oxaprozin (Daypro.RTM.), and piroxicam(Feldene.RTM.). In some embodiments a composition can be formulatedfor use with an anti-hypertensive, an anti-seizure agent (e.g.,magnesium sulfate), or an anti-thrombotic agent. Anti-hypertensivesinclude, e.g., labetalol, hydralazine, nifedipine, calcium channelantagonists, nitroglycerin, or sodium nitroprussiate. (See, e.g.,Mihu et al. (2007) J Gastrointestin Liver Dis 16(4):419-424.)Anti-thrombotic agents include, e.g., heparin, antithrombin,prostacyclin, or low dose aspirin.

[0238] In some embodiments, compositions formulated forintrapulmonary administration can include at least one additionalactive agent for treating a pulmonary disorder. The at least oneactive agent can be, e.g., an anti-IgE antibody (e.g., omalizumab),an anti-IL-4 antibody or an anti-IL-5 antibody, an anti-IgEinhibitor (e.g., montelukast sodium), a sympathomimetic (e.g.,albuterol), an antibiotic (e.g., tobramycin), a deoxyribonuclease(e.g., Pulmozyme.RTM.), an anticholinergic drug (e.g., ipratropiumbromide), a corticosteroid (e.g., dexamethasone), a.beta.-adrenoreceptor agonist, a leukotriene inhibitor (e.g.,zileuton), a 5-lipoxygenase inhibitor, a PDE inhibitor, a CD23antagonist, an IL-13 antagonist, a cytokine release inhibitor, ahistamine H1 receptor antagonist, an anti-histamine, ananti-inflammatory agent (e.g., cromolyn sodium), or a histaminerelease inhibitor.

[0239] In some embodiments, compositions can be formulated foradministration with one or more additional therapeutic agents foruse in treating a complement-associated disorder of the eye. Suchadditional therapeutic agents can be, e.g., bevacizumab or the Fabfragment of bevacizumab or ranibizumab, both sold by RochePharmaceuticals, Inc., and pegaptanib sodium (Mucogen.RTM.; Pfizer,Inc.). Such a kit can also, optionally, include instructions foradministering the composition to a subject.

[0240] In some embodiments, compositions can be formulated foradministration to a subject along with intravenous gamma globulintherapy (IVIG), plasmapheresis, plasma replacement, or plasmaexchange. In some embodiments, compositions can be formulated foruse before, during, or after, a kidney transplant.

[0241] When compositions are to be used in combination with asecond active agent, the compositions can be coformulated with thesecond agent or the compositions can be formulated separately fromthe second agent formulation. For example, the respectivepharmaceutical compositions can be mixed, e.g., just prior toadministration, and administered together or can be administeredseparately, e.g., at the same or different times (see below).

Applications

[0242] The compositions described herein can be used in a number ofdiagnostic and therapeutic applications. For example,detectably-labeled antigen-binding molecules can be used in assaysto detect the presence or amount of the target antigens in a sample(e.g., a biological sample). The compositions can be used in invitro assays for studying inhibition of target antigen function. Insome embodiments, e.g., in which the compositions bind to andinhibit a complement protein, the compositions can be used aspositive controls in assays designed to identify additional novelcompounds that inhibit complement activity or otherwise are usefulfor treating a complement-associated disorder. For example, aC5-inhibiting composition can be used as a positive control in anassay to identify additional compounds (e.g., small molecules,aptamers, or antibodies) that reduce or abrogate C5 production orformation of MAC. The compositions can also be used in therapeuticmethods as elaborated on below.

[0243] Methods for Treatment

[0244] The compositions described herein can be administered to asubject, e.g., a human subject, using a variety of methods thatdepend, in part, on the route of administration. The route can be,e.g., intravenous injection or infusion (IV), subcutaneousinjection (SC), intraperitoneal (IP) injection, or intramuscularinjection (IM).

[0245] Subcutaneous administration can be accomplished by means ofa device. The device means may be a syringe, a prefilled syring, anauto-injector either disposable or reusable, a pen injector, apatch injector, a wearable injector, an ambulatory syringe infusionpump with subcutaneous infusion sets or other device for combiningwith the antibody drug for subcutaneous injection.

[0246] Administration can be achieved by, e.g., local infusion,injection, or by means of an implant. The implant can be of aporous, non-porous, or gelatinous material, including membranes,such as sialastic membranes, or fibers. The implant can beconfigured for sustained or periodic release of the composition tothe subject. See, e.g., U.S. Patent Application Publication No.20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795;EP488401; and EP 430539, the disclosures of each of which areincorporated herein by reference in their entirety. A compositiondescribed herein can be delivered to the subject by way of animplantable device based on, e.g., diffusive, erodible, orconvective systems, e.g., osmotic pumps, biodegradable implants,electrodiffusion systems, electroosmosis systems, vapor pressurepumps, electrolytic pumps, effervescent pumps, piezoelectric pumps,erosion-based systems, or electromechanical systems.

[0247] In some embodiments, a composition described herein istherapeutically delivered to a subject by way of localadministration. As used herein, "local administration" or "localdelivery," refers to delivery that does not rely upon transport ofthe composition or agent to its intended target tissue or site viathe vascular system. For example, the composition may be deliveredby injection or implantation of the composition or agent or byinjection or implantation of a device containing the composition oragent. Following local administration in the vicinity of a targettissue or site, the composition or agent, or one or more componentsthereof, may diffuse to the intended target tissue or site.

[0248] In some embodiments, a composition described herein can belocally administered to a joint (e.g., an articulated joint). Forexample, in embodiments where the disorder is arthritis, atherapeutically appropriate composition can be administereddirectly to a joint (e.g., into a joint space) or in the vicinityof a joint. Examples of intraarticular joints to which acomposition described herein can be locally administered include,e.g., the hip, knee, elbow, wrist, sternoclavicular,temperomandibular, carpal, tarsal, ankle, and any other jointsubject to arthritic conditions. A composition described herein canalso be administered to bursa such as, e.g., acromial,bicipitoradial, cubitoradial, deltoid, infrapatellar, ischial, andany other bursa known in the art of medicine.

[0249] In some embodiments, a composition described herein can belocally administered to the eye. As used herein, the term "eye"refers to any and all anatomical tissues and structures associatedwith 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 fluidreferred to as the vitreous humor. At the back of the eye is theretina, which detects light. The cornea is an optically transparenttissue, which conveys images to the back of the eye. The corneaincludes one pathway for the permeation of drugs into the eye.Other anatomical tissue structures associated with the eye includethe lacrimal drainage system, which includes a secretory system, adistributive system and an excretory system. The secretory systemcomprises secretors that are stimulated by blinking and temperaturechange due to tear evaporation and reflex secretors that have anefferent parasympathetic nerve supply and secrete tears in responseto physical or emotional stimulation. The distributive systemincludes the eyelids and the tear meniscus around the lid edges ofan open eye, which spread tears over the ocular surface byblinking, thus reducing dry areas from developing.

[0250] In some embodiments, a composition described herein isadministered to the posterior chamber of the eye. In someembodiments, a composition described herein is administeredintravitreally. In some embodiments, a composition described hereinis administered trans-sclerally.

[0251] In some embodiments, e.g., in embodiments for treatment orprevention of a disorder such as COPD or asthma, a compositiondescribed herein can be administered to a subject by way of thelung. Pulmonary drug delivery may be achieved by inhalation, andadministration by inhalation herein may be oral and/or nasal.Examples of pharmaceutical devices for pulmonary delivery includemetered dose inhalers, dry powder inhalers (DPIs), and nebulizers.For example, a composition described herein can be administered tothe lungs of a subject by way of a dry powder inhaler. Theseinhalers are propellant-free devices that deliver dispersible andstable dry powder formulations to the lungs. Dry powder inhalersare well known in the art of medicine and include, withoutlimitation: the TurboHaler.RTM. (AstraZeneca; London, England) theAIR.RTM. inhaler (Alkermes.RTM.; Cambridge, Mass.); Rotahaler.RTM.(GlaxoSmithKline; London, England); and Eclipse.TM.(Sanofi-Aventis; Paris, France). See also, e.g., PCT PublicationNos. WO 04/026380, WO 04/024156, and WO 01/78693. DPI devices havebeen used for pulmonary administration of polypeptides such asinsulin and growth hormone. In some embodiments, a compositiondescribed herein can be intrapulmonary administered by way of ametered dose inhaler. These inhalers rely on a propellant todeliver a discrete dose of a compound to the lungs. Examples ofcompounds administered by metered dose inhalers include, e.g.,Atrovent.RTM. (Boehringer-Ingelheim; Ridgefield, Conn.) andFlovent.RTM. (GlaxoSmithKline). See also, e.g., U.S. Pat. Nos.6,170,717; 5,447,150; and 6,095,141.

[0252] In some embodiments, a composition described herein can beadministered to the lungs of a subject by way of a nebulizer.Nebulizers use compressed air to deliver a compound as a liquefiedaerosol or mist. A nebulizer can be, e.g., a jet nebulizer (e.g.,air or liquid-jet nebulizers) or an ultrasonic nebulizer.Additional devices and intrapulmonary administration methods areset forth in, e.g., U.S. Patent Application Publication Nos.20050271660 and 20090110679, the disclosures of each of which areincorporated herein by reference in their entirety.

[0253] In some embodiments, the compositions provided herein arepresent in unit dosage form, which can be particularly suitable forself-administration. A formulated product of the present disclosurecan be included within a container, typically, for example, a vial,cartridge, prefilled syringe or disposable pen. A doser such as thedoser device described in U.S. Pat. No. 6,302,855 may also be used,for example, with an injection system of the presentdisclosure.

[0254] An injection system of the present disclosure may employ adelivery pen as described in U.S. Pat. No. 5,308,341. Pen devices,most commonly used for self-delivery of insulin to patients withdiabetes, are well known in the art. Such devices can comprise atleast one injection needle (e.g., a 31 gauge needle of about 5 to8mm in length), are typically pre-filled with one or moretherapeutic unit doses of a therapeutic solution, and are usefulfor rapidly delivering the solution to a subject with as littlepain as possible.

[0255] One medication delivery pen includes a vial holder intowhich a vial of insulin or other medication may be received. Thevial holder is an elongate generally tubular structure withproximal and distal ends. The distal end of the vial holderincludes mounting means for engaging a double-ended needle cannula.The proximal end also includes mounting means for engaging a penbody which includes a driver and dose setting apparatus. Adisposable medication (e.g., a high concentration solution of acomposition described herein) containing vial for use with theprior art vial holder includes a distal end having a pierceableelastomeric septum that can be pierced by one end of a double-endedneedle cannula. The proximal end of this vial includes a stopperslidably disposed in fluid tight engagement with the cylindricalwall of the vial. This medication delivery pen is used by insertingthe vial of medication into the vial holder. A pen body then isconnected to the proximal end of the vial holder. The pen bodyincludes a dose setting apparatus for designating a dose ofmedication to be delivered by the pen and a driving apparatus forurging the stopper of the vial distally for a distancecorresponding to the selected dose. The user of the pen mounts adouble-ended needle cannula to the distal end of the vial holdersuch that the proximal point of the needle cannula pierces theseptum on the vial. The patient then selects a dose and operatesthe pen to urge the stopper distally to deliver the selected dose.The dose selecting apparatus returns to zero upon injection of theselected dose. The patient then removes and discards the needlecannula, and keeps the medication delivery pen in a convenientlocation for the next required medication administration. Themedication in the vial will become exhausted after several suchadministrations of medication. The patient then separates the vialholder from the pen body. The empty vial may then be removed anddiscarded. A new vial can be inserted into the vial holder, and thevial holder and pen body can be reassembled and used as explainedabove. Accordingly, a medication delivery pen generally has a drivemechanism for accurate dosing and ease of use.

[0256] A dosage mechanism such as a rotatable knob allows the userto accurately adjust the amount of medication that will be injectedby the pen from a prepackaged vial of medication. To inject thedose of medication, the user inserts the needle under the skin anddepresses the knob once as far as it will depress. The pen may bean entirely mechanical device or it may be combined with electroniccircuitry to accurately set and/or indicate the dosage ofmedication that is injected into the user. See, e.g., U.S. Pat. No.6,192,891.

[0257] In some embodiments, the needle of the pen device isdisposable and the kits include one or more disposable replacementneedles. Pen devices suitable for delivery of any one of thepresently featured compositions are also described in, e.g., U.S.Pat. Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures ofeach of which are incorporated herein by reference in theirentirety. A microneedle-based pen device is described in, e.g.,U.S. Pat. No. 7,556,615, the disclosure of which is incorporatedherein by reference in its entirety. See also the Precision PenInjector (PPI) device, Molly.TM., manufactured by ScandinavianHealth Ltd.

[0258] The present disclosure also presents controlled-release orextended-release formulations suitable for chronic and/orself-administration of a medication such as a composition describedherein. The various formulations can be administered to a patientin need of treatment with the medication as a bolus or bycontinuous infusion over a period of time.

[0259] In some embodiments, a high concentration compositiondescribed herein is formulated for sustained-release,extended-release, timed-release, controlled-release, orcontinuous-release administration. In some embodiments, depotformulations are used to administer the composition to the subjectin need thereof. In this method, the composition is formulated withone or more carriers providing a gradual release of active agentover a period of a number of hours or days. Such formulations areoften based upon a degrading matrix which gradually disperses inthe body to release the active agent.

[0260] In some embodiments, a composition described herein isadministered by way of intrapulmonary administration to a subjectin need thereof. For example, a composition described herein can bedelivered by way of a nebulizer or an inhaler to a subject (e.g., ahuman) afflicted with a disorder such as asthma or COPD.

[0261] A suitable dose of a composition described herein, whichdose is capable of treating or preventing a disorder 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 inhibitorcompound used. For example, a different dose of one composition(e.g., an anti-C5 composition) may be required to treat a subjectwith RA as compared to the dose of a different composition (e.g.,an anti-TNF.gamma. composition) required to treat the same subject.Other factors affecting the dose administered to the subjectinclude, e.g., the type or severity of the disorder. For example, asubject having RA may require administration of a different dosageof an anti-C5 composition described herein than a subject with PNH.Other factors can include, e.g., other medical disordersconcurrently or previously affecting the subject, the generalhealth 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 thesubject. It should also be understood that a specific dosage andtreatment regimen for any particular subject will also depend uponthe judgment of the treating medical practitioner (e.g., doctor ornurse).

[0262] A composition described herein can be administered as afixed dose, or in a milligram per kilogram (mg/kg) dose. In someembodiments, the dose can also be chosen to reduce or avoidproduction of antibodies or other host immune responses against oneor more of the antigen-binding molecules in the composition. Whilein no way intended to be limiting, exemplary dosages of anantibody, such as a composition described herein include, e.g.,1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages of acomposition described herein include, without limitation, 0.1mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, or 20mg/kg.

[0263] A pharmaceutical solution can include a therapeuticallyeffective amount of a composition described herein. Such effectiveamounts can be readily determined by one of ordinary skill in theart based, in part, on the effect of the administered composition,or the combinatorial effect of the composition and one or moreadditional active agents, if more than one agent is used. Atherapeutically effective amount of a composition described hereincan also vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thecomposition (and one or more additional active agents) to elicit adesired response in the individual, e.g., amelioration of at leastone condition parameter, e.g., amelioration of at least one symptomof the complement-mediated disorder. For example, a therapeuticallyeffective amount of a composition described herein can inhibit(lessen the severity of or eliminate the occurrence of) and/orprevent a particular disorder, and/or any one of the symptoms ofthe particular disorder known in the art or described herein. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

[0264] Suitable human doses of any of the compositions describedherein can further be evaluated in, e.g., Phase I dose escalationstudies. See, e.g., van Gurp et al. (2008) Am J Transplantation8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part1):523-531; and Hetherington et al. (2006) Antimicrobial Agents andChemotherapy 50(10): 3499-3500.

[0265] The terms "therapeutically effective amount" or"therapeutically effective dose," or similar terms used herein areintended to mean an amount of an agent (e.g., a compositiondescribed herein) that will elicit the desired biological ormedical response (e.g., an improvement in one or more symptoms of acomplement-associated disorder). In some embodiments, apharmaceutical solution described herein contains a therapeuticallyeffective amount of at least one of said compositions. In someembodiments, the solutions contain one or more compositions and oneor more (e.g., two, three, four, five, six, seven, eight, nine, 10,or 11 or more) additional therapeutic agents such that thecomposition as a whole is therapeutically effective. For example, asolution can contain an anti-C5 composition described herein and animmunosuppressive agent, wherein the composition and agent are eachat a concentration that when combined are therapeutically effectivefor treating or preventing a complement-associated disorder (e.g.,a complement-associated inflammatory disorder such as COPD, asthma,sepsis, or RA) in a subject.

[0266] Toxicity and therapeutic efficacy of such compositions canbe determined by known pharmaceutical procedures in cell culturesor experimental animals (e.g., animal models of any of thecomplement-mediated disorders described herein). These procedurescan be used, e.g., for determining the LD50 (the dose lethal to 50%of the population) and the ED.sub.50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxicand therapeutic effects is the therapeutic index and it can beexpressed as the ratio LD.sub.50/ED.sub.50. A composition describedherein that exhibits a high therapeutic index is preferred. Whilecompositions that exhibit toxic side effects may be used, careshould be taken to design a delivery system that targets suchcompounds to the site of affected tissue and to minimize potentialdamage to normal cells and, thereby, reduce side effects.

[0267] Data obtained from cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of the composition described herein lies generally within arange of circulating concentrations of the compositions thatinclude the ED.sub.50 with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed andthe route of administration utilized. For a composition describedherein, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated inanimal models to achieve a circulating plasma concentration rangethat includes the IC.sub.50 (i.e., the concentration of theantibody which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquidchromatography. In some embodiments, e.g., where localadministration (e.g., to the eye or a joint) is desired, cellculture or animal modeling can be used to determine a dose requiredto achieve a therapeutically effective concentration within thelocal site.

[0268] In some embodiments, the methods can be performed inconjunction with other therapies for complement-associateddisorders. For example, the composition can be administered to asubject at the same time, prior to, or after, plasmapheresis, IVIGtherapy, or plasma exchange. See, e.g., Appel et al. (2005) J AmSoc Nephrol 16:1392-1404. In some embodiments, the composition canbe administered to a subject at the same time, prior to, or after,a kidney transplant.

[0269] A "subject," as used herein, can be any mammal. For example,a subject can be a human, a non-human primate (e.g., orangutan,gorilla, macaque, baboon, or chimpanzee), a horse, a cow, a pig, asheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, ahamster, a rat, or a mouse. In some embodiments, the subject is aninfant (e.g., a human infant).

[0270] As used herein, a subject "in need of prevention," "in needof treatment," or "in need thereof," refers to one, who by thejudgment of an appropriate medical practitioner (e.g., a doctor, anurse, or a nurse practitioner in the case of humans; aveterinarian in the case of non-human mammals), would reasonablybenefit from a given treatment.

[0271] The term "preventing" is art-recognized, and when used inrelation to a condition, is well understood in the art, andincludes administration of a composition which reduces thefrequency of, or delays the onset of, symptoms of a medicalcondition in a subject relative to a subject which does not receivea composition described herein. Thus, prevention of acomplement-associated disorder such as asthma includes, forexample, reducing the extent or frequency of coughing, wheezing, orchest pain in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/ordelaying the occurrence of coughing or wheezing in a treatedpopulation versus an untreated control population, e.g., by astatistically and/or clinically significant amount.

[0272] As described above, the compositions described herein (e.g.,anti-C5 compositions) can be used to treat a variety ofcomplement-associated disorders such as, but not limited to:rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusioninjury; atypical hemolytic uremic syndrome (aHUS); typical orinfectious hemolytic uremic syndrome (tHUS); dense deposit disease(DDD); paroxysmal nocturnal hemoglobinuria (PNH); multiplesclerosis (MS); macular degeneration (e.g., age-related maculardegeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; sepsis; dermatomyositis; diabeticretinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneousfetal loss; Pauci-immune vasculitis; epidermolysis bullosa;recurrent fetal loss; multiple sclerosis (MS); and traumatic braininjury. See, e.g., Holers (2008) Immunological Reviews 223:300-316and Holers and Thurman (2004) Molecular Immunology 41:147-152. Insome embodiments, the complement-mediated disorder is acomplement-mediated vascular disorder such as, but not limited to,a cardiovascular disorder, myocarditis, a cerebrovascular disorder,a peripheral (e.g., musculoskeletal) vascular disorder, arenovascular disorder, a mesenteric/enteric vascular disorder,revascularization to transplants and/or replants, vasculitis,Henoch-Schonlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, organ or tissuetransplantation, Takayasu's disease, capillary leak syndrome,dilated cardiomyopathy, diabetic angiopathy, thoracic-abdominalaortic aneurysm, Kawasaki's disease (arteritis), venous gas embolus(VGE), and restenosis following stent placement, rotationalatherectomy, and percutaneous transluminal coronary angioplasty(PTCA). (See, e.g., U.S. patent application publication no.20070172483.) In some embodiments, the complement-associateddisorder is myasthenia gravis, cold-agglutinin disease (CAD),paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma,warm autoimmune hemolytic anemia, Graves' disease, Hashimoto'sthyroiditis, type I diabetes, psoriasis, pemphigus, autoimmunehemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP),Goodpasture syndrome, antiphospholipid syndrome (APS), Degosdisease, and catastrophic APS (CAPS).

[0273] In some embodiments, a composition described herein, aloneor in combination with a second anti-inflammatory agent, can beused to treat an inflammatory disorder such as, but not limited to,RA (above), inflammatory bowel disease, sepsis (above), septicshock, acute lung injury, disseminated intravascular coagulation(DIC), or Crohn's disease. In some embodiments, the secondanti-inflammatory agent can be one selected from the groupconsisting of NSAIDs, corticosteroids, methotrexate,hydroxychloroquine, anti-TNF agents such as etanercept andinfliximab, a B cell depleting agent such as rituximab, aninterleukin-1 antagonist, or a T cell costimulatory blocking agentsuch as abatacept.

[0274] In some embodiments, the complement-associated disorder is acomplement-associated neurological disorder such as, but notlimited to, amyotrophic lateral sclerosis (ALS), brain injury,Alzheimer's disease, and chronic inflammatory demyelinatingneuropathy.

[0275] Complement-associated disorders also includecomplement-associated pulmonary disorders such as, but not limitedto, 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 vasculardisorders.

[0276] In some embodiments, a composition described herein isadministered to a subject to treat, prevent, or ameliorate at leastone symptom of a complement-associated inflammatory response (e.g.,the complement-associated inflammatory response aspect of acomplement-associated disorder) in a subject. For example, acomposition can be used to treat, prevent, and/or ameliorate one ormore symptoms associated with a complement-associated inflammatoryresponse such as graft rejection/graft-versus-host disease (GVHD),reperfusion injuries (e.g., following cardiopulmonary bypass or atissue transplant), and tissue damage following other forms oftraumatic injury such as a burn (e.g., a severe burn), blunttrauma, spinal injury, or frostbite. See, e.g., Park et al. (1999)Anesth Analg 99(1):42-48; Tofukuji et al. (1998) J ThoracCardiovasc Surg 116(6):1060-1068; Schmid et al. (1997) Shock8(2):119-124; and Bless et al. (1999) Am J Physiol 27 6(1):L57-L63.

[0277] In some embodiments, a composition described herein can beadministered to a subject as a monotherapy. Alternatively, asdescribed above, the composition can be administered to a subjectas a combination therapy with another treatment, e.g., anothertreatment for a complement-associated disorder or acomplement-associated inflammatory response. For example, thecombination 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 subjectwho has, or is at risk of developing, sepsis. In another example,the combination therapy can include administering to the subjectone or more additional agents (e.g., an anti-IgE antibody, ananti-IL-4 antibody, an anti-IL-5 antibody, or an anti-histamine)that provide therapeutic benefit to a subject who has, is at riskof developing, or is suspected of having a complement-associatedpulmonary disorder such as COPD or asthma. In some embodiments, acomposition and the one or more additional active agents areadministered at the same time. In other embodiments, thecomposition is administered first in time and the one or moreadditional active agents are administered second in time. In someembodiments, the one or more additional active agents areadministered first in time and the composition is administeredsecond in time.

[0278] A composition described herein can replace or augment apreviously or currently administered therapy. For example, upontreating with a composition described herein, administration of theone or more additional active agents can cease or diminish, e.g.,be administered at lower levels, e.g., lower levels of eculizumabfollowing administration of an anti-C5 composition describedherein. In some embodiments, administration of the previous therapycan be maintained. In some embodiments, a previous therapy will bemaintained until the level of the composition reaches a levelsufficient to provide a therapeutic effect. The two therapies canbe administered in combination.

[0279] Monitoring a subject (e.g., a human patient) for animprovement in a disorder (e.g., sepsis, severe burn, RA, lupusnephritis, Goodpasture syndrome, or asthma), 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 disorder.The symptoms of many of the above disorders (e.g.,complement-associated disorders) are well known in the art ofmedicine. In some embodiments, the evaluation is performed at leastone (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or atleast 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, orat least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks ormore, after an administration of a composition described herein.The subject can be evaluated in one or more of the followingperiods: prior to beginning of treatment; during the treatment; orafter 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, orduration of treatment should be altered. It can also includeevaluating the need to add or drop a selected therapeutic modality,e.g., adding or dropping any of the treatments for acomplement-associated disorder described herein.

[0280] The following examples are merely illustrative and shouldnot be construed as limiting the scope of this disclosure in anyway as many variations and equivalents will become apparent tothose skilled in the art upon reading the present disclosure. Allpatents, patent applications and publications cited herein areincorporated herein by reference in their entireties.

EXAMPLE S

Example 1

Half-Life of Eculizumab is a Combination of Several ClearanceRates

[0281] The average half-life of eculizumab in PNH and aHUS patientsreceiving the prescribed dosing regimen is approximately 11-12days, whereas the expected half-life for a humanized monoclonalantibody having an IgG2/4 Fc is predicted to be similar to that ofan antibody containing an IgG2 or IgG4 Fc, approximately 21-28days. Morell et al. (1970) J Clin Invest 49(4):673-680. Tounderstand the potential impact of antigen-mediated clearance onthe overall clearance rate of eculizumab, the following experimentswere performed using the human neonatal Fc receptor (hFcRn) mousemodel (the mice lack endogenous FcRn but are transgenic for hFcRn(B6.Cg-Fcgrt.sup.tm1Dcr Tg(FCGRT)32Dcr/DcrJ; Stock Number 014565,Jackson Laboratories, Bar Harbor, Me.)). The transgenic FcRn modelhas been described in, e.g., Petkova et al. (2006) Int Immunology18(12):1759-1769; Oiao et al. (2008) Proc Natl Acad Sci USA105(27):9337-9342; and Roopenian et al. (2010) Methods Mol Biol602:93-104.

[0282] A single dose of 100 .mu.g of eculizumab in 200 .mu.L ofphosphate buffered saline (PBS) was administered by intravenous(i.v.) injection to each of five hFcRn transgenic mice. Bloodsamples of approximately 100 .mu.L were collected from each of themice at days one, three, seven, 14, 21, 28, and 35 following theadministration. The concentration of eculizumab in serum wasmeasured by ELISA. Briefly, assay plates were coated with a sheepanti-human Ig.kappa. light chain capture antibody and blocked. Thewells of the plate were then contacted with the serum samples underconditions that allow eculizumab, if present in the serum, to bindto the capture antibody. The relative amount of eculizumab bound toeach well was detected using a detectably-labeled anti-human IgG4antibody and quantified relative to a standard curve generated fromnaive mouse serum containing known quantities of eculizumab.

[0283] Antibody serum half-life was calculated using the followingformula:

Halflife = T .times. ln 2 ln A 0 A t ##EQU00001##

Where: T=Time elapsed, A.sub.o=Original serum concentration of theantibody (concentration at day 1 in the present study) andA.sub.t=Amount of the antibody remaining after elapsed time T(minimal detectable concentration or the last bleeding time point(day 35) in the present study).

[0284] The results of the experiment are depicted in FIG. 1. Thehalf-life of eculizumab in the hFcRn mouse model was 13.49.+-.0.93days.

[0285] To determine the effect of human C5 on the half-life ofeculizumab using the hFcRn model, antibody was premixed with a 4:1molar ratio of human C5 (Complement Technology Inc., CatalogNumber: A120) prior to dosing, A dose of 100 .mu.g of eculizumabwas intravenously (i.v.) administered on day 0. Approximately 100.mu.L blood was collected into 1.5 mL Eppendorf tubes for serum viaretro-orbital bleeding at 1, 3, 7, 14, 21, 28 and 35 days.

[0286] As shown in FIG. 1, the half-life of eculizumab in the hFcRnmouse model in the presence of C5 was 4.55.+-.1.02 days. Theseresults indicate that, in addition to endocytosis-mediated antibodyclearance mechanisms in which a long half-life is governed largelyby FcRn-mediated recycling, the half-life of eculizumab may besignificantly impacted by antigen-mediated clearance through humanC5.

Example 2

Amino Acid Substitutions in the Fc Domain of Eculizumab Increasethe Half-Life of Eculizumab but are Not Sufficient to Overcome theEffect of C5 on Eculizumab Clearance

[0287] Certain amino acid substitutions in the Fc region of an IgGantibody have been shown to lessen the rate of elimination of theantibody from circulation. Substitutions that increase the bindingaffinity of an IgG antibody for FcRn at pH 6.0 are examples of sucha biological effect. See, e.g., Dall'Acqua et al. (2006) J Immunol117:1129-1138 and Ghetie et al. (1997) Nat Biotech 15: 637-640.Zalevsky et al. [(2010) Nat Biotech 28:157-159] describe a numberof amino acid substitutions, e.g., M428L/N434S, capable ofincreasing the half-life of an IgG antibody in serum. Otherhalf-life extending amino acid substitutions include, e.g.,T250Q/M428L and M252Y/S254T/T256E. See, e.g., International patentapplication publication no. WO 2008/048545 and Dall'Acqua et al.(2006) J Biol Chem 281:23514-23524. To determine whether one ormore amino acid substitutions in the Fc constant region ofeculizumab are capable of extending the half-life of eculizumab inserum, the following substitutions were introduced into eculizumab:M252Y/S254T/T256E, based on the EU numbering index (herein afterthis variant of eculizumab is referred to as the YTE variant). Theheavy chain constant region consisted of the following amino acidsequence:

TABLE-US-00004 (SEQ ID NO: 15)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The amino acid sequence for the full-length heavy chain polypeptideof the YTE variant of eculizumab is depicted in SEQ ID NO:16.

[0288] The YTE variant was evaluated alongside eculizumab in thehFcRn mouse model described in Example 1. That is, 100 .mu.g ofeculizumab (IgG2/4 Fc region), a variant of eculizumab containingan Fc or the YTE variant of eculizumab in 200 .mu.L of phosphatebuffered saline (PBS) was administered by intravenous (i.v.)injection to each of eight hFcRn transgenic mice. Serum wascollected from each of the mice at days one, three, seven, 14, 21,28, and 35 following the administration. The concentration of eachantibody in the serum was measured by ELISA and the half-lifecalculated as described in Example 1. The results are depicted inFIG. 2 and Table 2.

TABLE-US-00005 TABLE 2 Standard Antibody Tested Half-Life Error(SE) Eculizumab 13.49 0.93 Eculizumab-IgG2 14.28 1 Eculizumab-IgG2-29.07 4.7 YTE

[0289] As shown in FIG. 2 and Table 2, the YTE substitutionincreased the mean half-life of eculizumab more than 2-fold from14.28.+-.1 days to 29.07.+-.4.7 days.

[0290] To determine the effect of human C5 on the half-life of theYTE variant of eculizumab, mice were administered human C5 asdescribed above in Example 1. A dose of 100 .mu.g of eculizumab,the eculizumab-IgG2 variant, or the eculizumab-IgG2 YTE variant wasintravenously administered on day 0. As shown in FIG. 3 and Table3, the half-life of eculizumab, the eculizumab-IgG2 variant, andthe eculizumab-IgG2 YTE variant decreased significantly in thepresence of a molar excess of human C5. Thus, amino acidsubstitutions in FcRn-binding domain of eculizumab wereinsufficient to overcome the contribution of C5-mediated clearanceon the half-life of eculizumab.

TABLE-US-00006 TABLE 3 Standard Antibody Tested T1/2 Error (SE)Eculizumab 13.49 0.93 Eculizumab-IgG2 14.28 1 Eculizumab-IgG2(YTE)29.07 4.7 Eculizumab + hC5 4.55 1.02 Eculizumab-IgG2 + C5 2.11 0.31Eculizumab-IgG2(YTE) + hC5 4.28 1.09

Example 3

The Effect of Amino Acid Substitutions in the CDRs of Eculizumab onHalf-Life

[0291] As described above, the half-life of eculizumab in mice issignificantly shorter in the presence of its antigen, human C5(hC5). While not being bound by any particular theory or mechanismof action, it is hypothesized that the accelerated clearance in thepresence of antigen is, in part, the result of the very highaffinity of eculizumab for C5 (K.sub.D.about.30 pM at pH 7.4 and.about.600 pM at pH 6.0) which does not allow efficientdissociation of the antibody:C5 complex in the early endosomalcompartments after pinocytosis. Without dissociation, theantibody:antigen complex is either recycled to the extracellularcompartment via the neonatal Fc receptor (FcRn) or targeted forlysosomal degradation. In either case the antibody is incapable ofbinding more than two C5 molecules in its lifetime.

[0292] The strong affinity of eculizumab for C5 (K.sub.D.about.30pM) allows for near complete binding of all C5 in blood, ensuringthat very little C5 is activated to form C5a and TCC. The affinityof eculizumab for C5 is therefore directly connected to the in vivoefficacy of the antibody in patients treated with the antibody. Theinventors set out to weaken the affinity of eculizumab for C5,without compromising the efficacy of eculizumab in vivo. While thedisclosure is not limited to such an approach, this was achieved byintroducing histidine into one or more positions in the CDRs ofeculizumab. Histidine has a pKa of 6.04. This means that as pHvalues drop from 7.4 (blood) to less than 6.0 (early endosomes),histidines gain a proton. Thus, in the endosome, histidines becomemore positively-charged. The inventors hypothesized thatintroducing histidines at or near the binding site for C5 ineculizumab, the charge shift in the endosome may disrupt binding inthe endosome, whilst preserving the high affinity for C5 at neutralpH in the blood. Such substitutions are hypothesized to increasethe half-life by facilitating the dissociation of antibody from theantibody:C5 complex in the acidic environment of the endosome,allowing free antibody to be recycled while the C5 is degraded inthe lysosome.

[0293] Using eculizumab as the parent antibody, a series of variantantibodies was generated in which every CDR position wassubstituted with a histidine. The heavy chain variable region ofeculizumab has the following amino acid sequence:

TABLE-US-00007 (SEQ ID NO: 7)QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.

(The CDR regions of the heavy chain variable region areunderlined.)

[0294] The light chain variable region of eculizumab has thefollowing amino acid sequence:

TABLE-US-00008 (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

[0295] The result of this histidine-scanning effort was 66 singlehistidine substitution variants of eculizumab. The light chain andheavy chain coding sequences for these antibody variants werecloned into separate "single gene construct" plasmids suitable forexpression in mammalian cells and sequence confirmed. Antibodiescontaining a single amino acid substitution were expressedtransiently in HEK293F cells by co-transfection of single geneconstructs encoding a single light chain or heavy chain. Aco-transfection of "wildtype" heavy and light chains representingunmodified eculizumab CDR sequences was also performed (EHL000).Tissue culture supernatants were normalized for antibody expressionlevel and use to evaluate antibody binding to human C5, relative toEHL000, using biolayer interferometry on an Octet Red instrument(ForteBio Inc.). Briefly, antibodies were captured on an anti-humanIgG Fc biosensor (ForteBio, cat # 18-5001). Loaded tips were thenexposed to a pH 7.4 buffered solution containing 12.5 nM of nativepurified human C5 for 800 seconds to assess the kinetics ofassociation relative to the parental antibody. Dissociationkinetics were assessed by transferring the tip to a pH 7.4 bufferedsolution or pH 6.0 buffered solution for 800 seconds. Allmeasurements were repeated to ensure consistency of readings.

[0296] Single histidine substitution variants of eculizumab wereselected based on a series of three properties relative toeculizumab. Preferred histidine variants only deviated from thek.sub.a and k.sub.d of eculizumab at pH 7.4 to a minor degree, butdeviated from the k.sub.d of eculizumab at pH 6.0 moresignificantly. The relative threshold selection criteria were asfollows: [0297] (1) a maximum variation for association kinetics atpH 7.4 of a 33% smaller peak phase shift at 800 seconds as comparedto the averaged peak phase shift at 800 seconds observed foreculizumab; [0298] (2) a maximum variation for dissociationkinetics at pH 7.4 of no more than 3-fold reduction in peak phaseshift over 800 seconds as compared to the averaged peak phase shiftat 800 seconds observed for eculizumab; and [0299] (3) a minimumvariation for dissociation kinetics at pH 6.0 of at least a 3-foldreduction in the peak phase shift over 800 seconds as compared tothe averaged peak phase shift at 800 seconds observed foreculizumab. For example, with respect to prong (1) above, if theaverage peak phase shift after 800 seconds of association witheculizumab is approximately 0.75 nm, a test antibody that has aphase shift of less than 0.5 nm (e.g., reproduced two or moretimes) would not meet the above criteria. By contrast, a testantibody with greater than a 0.5 nm peak phase shift at 800 secondsmeets the first criterion.

[0300] Single substitutions in the light chain variable region thatmet these thresholds were the following: G31H, L33H, V91H, andT94H, all relative to SEQ ID NO:8. Single substitutions in theheavy chain variable region that met these thresholds were thefollowing: Y27H, I34H, L52H, and S57H, all relative to SEQ ID NO:7.See FIGS. 5A, 5B, 5C, and 5D.

[0301] A second series of antibodies was generated containing allpossible combinations of two histidine substitutions at positionswhere single substitutions met threshold criteria. See Table 1.These association and dissociation kinetics were analyzed via thesame methods and compared to both the original parental antibodyand the single histidine substitutions. Likewise, a third andfourth series of antibodies containing three or four histidinesubstitutions, respectively, were generated and association anddissociation kinetics were analyzed compared to the relevant two orthree histidine substitution predecessors. See Table 1. At eachstage the same criteria were used for minimum thresholds forassociation kinetics at pH 7.4, maximum thresholds for dissociationkinetics at pH 7.4 and minimum thresholds for dissociation kineticsat pH 6. Eight substitution combinations met the above criteria andselected for affinity determination at pH 7.4 and pH 6.0 via SPR.The affinities are set forth in Table 4.

TABLE-US-00009 TABLE 4 ratio of KD pH KD pH KD at Clone VL VH 7.46.0 pH 6.0/pH Designation Sequence Sequence (nM) (nM) 7.4eculizumab SIN: 8 SIN: 7 0.033 0.685 21 EHL000 SIN: 8 SIN: 7 0.0180.419 24 EHL001 G31H, SIN: 7 0.330 1900 5758 relative to SIN: 8.EHL004 G31H, S57H 0.135 374 2770 relative to SIN: 8. EHL046 G31H,SIN: 7, 1.150 ND NA relative with: to SIN: 8. Y27H, L52H EHL049G31H, SIN: 7, 0.573 ND NA relative with: to SIN: 8. Y27H, S57HEHL055 G31H, SIN: 7, 0.623 2550 4093 relative with: to SIN: 8.I34H, S57H EHG302 SIN: 8 SIN: 7, 0.289 10.0 35 with: Y27H, L52HEHG303 SIN: 8 SIN: 7, 0.146 1190 8151 with: Y27H, S57H EHG305 SIN:8 SIN: 7, 0.160 10.8 68 with: I34H, S57H *SIN refers to SEQ IDNO.

[0302] For these combinations of substitutions, the affinity ofeculizumab for C5 was reduced by greater than 1000 fold at pH 6.0,while the affinity suffered no greater than a 20-fold reduction inaffinity at pH 7.0. From these, EHG303 (Table 4) was selected forfurther analysis due to its high affinity at pH 7.4 (0.146 nM) andthe ratio of (K.sub.D at pH 6.0)/(K.sub.D at pH 7.4) of over 8,000.The heavy chain polypeptide of the EHG303 antibody comprises thefollowing amino acid sequence:

TABLE-US-00010 (SEQ ID NO: 24)MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The light chain polypeptide of the EHG303 antibody comprises thefollowing amino acid sequence:

TABLE-US-00011 (SEQ ID NO: 25)MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In the above sequences, the underlined portions correspond to theleader sequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

[0303] Also selected was the EHL049 antibody. Its heavy chainpolypeptide comprises the following amino acid sequence:

TABLE-US-00012 (SEQ ID NO: 26)MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The light chain polypeptide of the EHL049 antibody comprises thefollowing amino acid sequence:

TABLE-US-00013 (SEQ ID NO: 27)MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In the above sequences, the underlined portions correspond to theleader sequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

[0304] Finally, the EHL000 heavy chain polypeptide comprises thefollowing amino acid sequence:

TABLE-US-00014 (SEQ ID NO: 28)MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The light chain polypeptide of the EHL000 antibody comprises thefollowing amino acid sequence:

TABLE-US-00015 (SEQ ID NO: 29)MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In the above sequences, the underlined portions correspond to theleader sequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

Example 4

Histidine Substitutions Prolong the Half-Life of Eculizumab inSerum

[0305] The light chain polypeptide and heavy chain polypeptide ofeach of the EHL and EHG antibodies above, were expressed fromsingle gene constructs. Heavy and light chain coding sequences fromEHG303 were combined into a double gene expression vector, as werethe light and heavy chain sequences for the EHL049 antibody. Theresulting EHG303 clone was designated as BNJ421 and the resultingEHL049 clone was designated as BNJ423. The amino acid sequence ofthe heavy chain variable region of BNJ421 is as follows:

TABLE-US-00016 (SEQ ID NO: 12)QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.

The light chain variable region amino acid sequence for BNJ421 isas follows:

TABLE-US-00017 (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

The heavy chain variable region of the BNJ423 antibody comprisesthe following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFG-SSPNWYFDVWGQG TLVTVSS (SEQ ID NO:12). The light chain amino acidsequence for BNJ423 is as follows:

TABLE-US-00018 (SEQ ID NO: 18)DIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

[0306] These two molecules were evaluated alongside EHL000 in micethat were immunodeficient (NOD/scid) and C5 deficient. A singledose of 100 .sub.j--Ig of EHL000, BNJ421, or BNJ423 in 200 .sub.IALof phosphate buffered saline (PBS) was administered by intravenous(i.v.) injection to each of eight mice. Serum was collected fromeach of the mice at days one, three, seven, 14, 21, 28, and 35following the administration. The concentration of each antibody inthe serum was measured by ELISA. Antibody serum half-life wascalculated using Pharsight Phoenix.RTM. WinNonlin.RTM. version 6.3software by using the non-compartmental analysis (NCA) and directresponse Emax. The percentage of the antibody remaining in theserum was calculated as follows:

% of antibody remaining=C.sub.t/C.sub.1.times.100

wherein, C.sub.t=Antibody concentration on a given day; andC.sub.1=Antibody concentration on day 1. The results are depictedin FIG. 6 and Table 5.

TABLE-US-00019 TABLE 5 Serum T 1/2 Standard Ab Tested (days) Error(SE) EHL000 22.18 1.01 BNJ421 25.29 0.81 BNJ423 24.69 2.16

[0307] To determine the effect of human C5 on the half-life ofthese antibodies using the same mouse model, mice were administeredhuman C5 subcutaneously at a loading dose of 250 .mu.g at day-1(the day before the antibodies were administered to the mice),followed by twice daily doses of 50 .mu.g of C5 to maintain theserum C5 concentration at approximately 20 .mu.g/mL (as describedin Example 1).

[0308] As shown in FIG. 7 (and Table 6, below), the half-life ofEHL000 (eculizumab-IgG1) in the mouse model in the presence ofhuman (hC5) (at a concentration that was greater than a 1:1 molarratio of C5 to eculizumab) was 2.49.+-.0.34 days, whereas thehalf-life of the BNJ421 and BNJ423 antibodies (containing thehistidine substitutions) was substantially greater at 15.25.+-.0.90days and 22.71.+-.0.71 days, respectively. These results indicatethat histidine substitutions in the CDRs of eculizumab, and theresultant pH-dependent affinity for C5, significantly decrease therate of clearance of the eculizumab variants from serum relative toeculizumab.

TABLE-US-00020 TABLE 6 Serum T 1/2 Ab Tested (days) SE EHL000 22.181.01 BNJ421 25.29 0.81 BNJ423 24.69 2.16 EHL000 + hC5 2.49 0.34BNJ421 + hC5 15.25* 0.90 BNJ423 + hC5 22.71 1.19 *Significantrelative to EHL000 + hC5.

Example 5

Histidine Substituted-Eculizumab Variants do not LoseComplement-Inhibitory Activity

[0309] In addition, the serum hemolytic activity in each of thesamples containing human C5 from the experiments described inExample 4 were also evaluated. Terminal complement activity inmouse sera was determined by assessing its ability to lyse chickenerythrocytes. Since the mice used were C5 deficient, the hemolyticactivity directly reflects the activity of human C5 in the sample.Briefly, antibodies at 50, 3, and 0 .mu.g/mL in GelatinVeronal-Buffered Saline (GVBS) (Comptech Catalog # B100) containing0.1% gelatin, 141 mM NaCl, 0.5 mM MgCl.sub.2, 0.15 mM CaCl2, and1.8 mM sodium barbital were used as low, medium and 100% lysiscontrol, respectively. Experimental samples were prepared bydiluting the murine test serum 1:10 in GVBS. Sample aliquots (50.mu.L) were dispensed to corresponding triplicate wells of a96-well plate (Corning; Tewksbury, MA Catalog # 3799) containing anequal volume of 20% mouse C5-deficient serum and 20% human serum(Bioreclamation, Catalog# HMSRM-COMP+) in GVBS in control wells andan equal volume of 20% mouse C5-deficient serum and 20% humanC5-depleted serum (Complement Technologies, Catalog number A320) inGVBS in test sample wells. EDTA (2 .mu.L at 500 mM, Sigma, catalognumber E-9884) was added into the third well of both control andsample triplicates to generate "no hemolysis" serum color control.Chicken erythrocytes were washed in GVBS, sensitized to activatethe complement classical pathway by incubation with an anti-chickenRBC polyclonal antibody (Intercell Technologies; 0.1% v/v) at4.degree. C. for 15 minutes, washed again, and re-suspended in GVBSat a final concentration of .about.7.5.times.10.sup.7 cells/mL. Thesensitized chicken erythrocytes (.about.2.5.times.10.sup.6 cells)were added to the plate containing the controls and samples, mixedbriefly on a plate shaker, and incubated at 37.degree. C. for 30min. The reagents were mixed again, centrifuged at 845.times.g for3 min, and 85 .mu.L of the supernatant was transferred to wells ofa 96-well flat-bottom microtiter plate (Nunc, Penfield, N.Y.,Catalog# 439454). Absorbance was measured at 415 nm using amicroplate reader and the percentage of hemolysis was determinedusing the following formula:

% of hemolysis = Sample OD - Sample color control OD 100 % lysiscontrol OD - 100 % lysis color control OD .times. 100##EQU00002##

where, OD=optical density.

[0310] As shown in FIG. 8, despite the slight reduction in affinityat pH 7.4 relative to eculizumab, both BNJ421 and BNJ423 were stillcapable of binding nearly all of the human C5 present incirculation and inhibiting hemolysis. These results indicate theaffinity of eculizumab for C5 can be weakened without compromisingthe efficacy of the antibody in vivo, and conferring upon theantibody an increase serum half-life.

Example 6

pH-Dependent Binding to C5 and Enhanced FcRn-Mediated Recycling areAdditive for Serum Half-Life of Eculizumab Variants

[0311] As shown above, in the presence of human C5, the half-lifeof a histidine-substituted eculizumab variant was significantlyextended in transgenic mice. To assess the potential additiveeffects of pH-dependent binding to C5 and to FcRn on thepharmacokinetics (PK) and pharmacodynamics (PD) of anti-C5antibodies in the presence of constitutive C5 synthesis and humanFcRn, a series of PK/PD experiments were performed using anti-mouseC5 antibodies with human constant regions in transgenic miceexpressing human FcRn. These murine anti-C5 antibodies wereengineered from the variable region of BB5.1, a murine antibodythat serves as a pharmacologic surrogate for eculizumab as it bindsmouse C5 and prevents its cleavage into the active metabolicfragments C5a and C5b [De Vries et al. (2003) Transplantation3:375-382]. A high affinity anti-mouse C5 antibody (designated as:BHL011) was engineered with an affinity-optimized variant of theBB5.1 murine variable regions and human Igic and human IgG2/G4constant regions. A pH-dependent variant of BHL011 lwas engineeredby incorporating three histidine substitutions into the murinevariable regions (this variant was designated as: BHL006).

[0312] A third antibody was engineered by incorporating two aminoacid substitutions into the human constant region heavy chain(M428L, N434S) to increase the affinity for hFcRn (this variant wasdesignated as:BHL009).

[0313] The amino acid sequence of the light chain polypeptide ofBHL006 is as follows:

TABLE-US-00021 (SEQ ID NO: 30)NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The amino acid sequence of the heavy chain polypeptide of theBHL006 antibody is as follows:

TABLE-US-00022 (SEQ ID NO: 31)QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0314] The amino acid sequence of the light chain polypeptide ofBHL009 is as follows:

TABLE-US-00023 (SEQ ID NO: 32)NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The amino acid sequence of the heavy chain polypeptide of BHL009 isas follows:

TABLE-US-00024 (SEQ ID NO: 33)QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK.

[0315] The amino acid sequence of the light chain polypeptide ofBHL011 is as follows:

TABLE-US-00025 (SEQ ID NO: 34)NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQYLSSRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The amino acid sequence of the heavy chain polypeptide of BHL011 isas follows:

TABLE-US-00026 (SEQ ID NO: 35)QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0316] The kinetics of BHL011, BHL006 and BHL009 binding topurified mouse were determined via SPR on a BIACore 3000 instrumentusing an anti-Fc human capture method. Briefly, anti-human Fc (KPL,catalogue number: 01-10-20) diluted to 0.1 mg/mL in 10 mM sodiumacetate pH 5.0, was immobilized on two flow cells of a CMS chip for8 minutes by amine coupling. The antibodies were diluted to 0.25.mu.g/mL in running buffer (HBS-EP; 0.01 M HEPES pH 7.4, 0.15 MNaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE Life Sciences,catalogue number: BR1001-88). The diluted antibody was theninjected on one flow cell followed by an injection of 6 nM mouse C5on both cells. The second flow cell was used as a referencesurface. The binding was evaluated at pH 7.4 and pH 6.0. Thesurface was regenerated each time with 20 mM HCl, 0.01% P20. Thedata was processed with a 1:1 Langmuir model using BIAevaluation4.1 software with `double referencing`. The dissociation of BHL011,BHL006 and BHL009 complexed to mouse C5 at pH 6.0 were evaluatedsimilarly, with an injection of 6 nM mouse C5 (pH 7.4) followed byan injection of HBS-EP buffer (pH 6.0). The results of theseexperiments are shown in Table 7.

TABLE-US-00027 TABLE 7 Dissociation Association Rate: DissociationRate: Constant: % Diss'n K.sub.a (1/M*s) K.sub.d (1/s) K.sub.D (nM)Chi.sup.2 in 300 sec Ab pH 7.4 pH 6.0 pH 7.4 pH 6.0 pH 7.4 pH 6.0pH 7.4 pH 6.0 pH 7.4 pH 6.0 BHL011 6.44 .times. 10.sup.5 2.39.times. 10.sup.3 6.13 .times. 10.sup.-5 1.28 .times. 10.sup.-40.0952 53.6 0.0194 0.048 1 7 BHL006 2.93 .times. 10.sup.5 NB 1.02.times. 10.sup.-3 NB 3.49 NB 0.021 NB 28 100 BHL009 2.61 .times.10.sup.5 NB 1.09 .times. 10.sup.-3 NB 4.19 NB 0.0234 NB 28 100*"NB" no specific binding was observed; "Ab" refers to antibodydesignation.

[0317] In order to determine the effects of pH-dependent binding toC5 on the pharmacokinetics (PK) of an anti-C5 antibody in thepresence of constitutive C5 synthesis and the potential forenhanced FcRn recycling to confer additive increases in half-life,the total serum concentration of BHL011, BHL006 and BHL009 wereanalyzed using the transgenic FcRn mouse model described in Example1. Total antibody serum concentration and serum concentration as apercentage of the day 1 concentration are shown in FIGS. 9-11. Malemice are represented as solid lines and females as dashed lines.Total antibody serum concentrations at day 1 were higher forfemales than for males, proportional to the differences in bodymass and the volume of distribution. This gender differencecontributed the inter-animal variability for BHL011pharmacokinetics, possibly due to dose-dependent non-linearityresulting from C5-mediated clearance (FIGS. 9A and 9B). Generallythe inter-animal variability was low for BHL006 (FIGS. 10A and 10B)and BHL009 (FIGS. 11A and 11B) with the exception of one female inthe BHL006 dose cohort (2939) which displayed acceleratedclearance. The reasons for accelerated clearance in animal 2939 areunknown.

[0318] In the presence of constitutive synthesis of C5 and hFcRn,the high affinity IgG2/4 anti- C5 antibody (BHL011) had a meanterminal half-life of 6 days and was cleared from circulation by.about.98% at 21 days (FIGS. 12 and 13; Table 8). The meanclearance rate for a pH-dependent anti-C5 antibody with an IgG2/4Fc region (BHL006) was attenuated, with a mean beta-phase half-lifeof 16-19 days. An additional .about.2-fold increase in half-lifewas observed for a pH-dependent anti-C5 antibody with an IgG2/4 Fcregion with improved affinity for hFcRn (BHL009 half-life .about.36days). These parameters are consistent with those observed forIgG2/4 antibodies with and without M428L, N434S substitutions inthe absence of antigen in hFcRn mice. These results demonstratethat pH-dependent C5 binding and increased affinity for FcRn conferadditive effects to extend the PK exposure of anti-C5antibodies.

TABLE-US-00028 TABLE 8 Animal Body Weight C.sub.MAX Half-lifeAntibody Designation Gender (g) (.mu.g/mL) (days) BHL011 2929 M37.8 519.8 7.2 2930 M 33.5 512.2 7.1 2963 F 23.2 805.0 6.2 2964 F20.2 814.6 5.0 2965 F 23.4 823.5 4.4 Mean = 6.0 BHL006 2905 M 37.5361.6 15.4 2906 M 36.1 378.8 19.1 2939 F 21.8 836.0 4.6 2940 F 23.9635.3 21.6 2941 F 20.0 906.9 20.1 Mean = 16.2 BHL009 2913 M 31.2402.6 45.8 2914 M 31.0 606.7 45.0 2947 F 21.3 724.9 33.2 2948 F22.3 590.1 22.8 2949 F 20.9 652.8 33.1 Mean = 36.0

[0319] Pharmacodynamics of Anti-Mouse C5 Antibodies in Human FcRnTransgenic Mice

[0320] The pharmacologic activity of the anti-mouse C5 antibodiesin serum samples was evaluated ex vivo in a complement classicalpathway-mediated chicken erythrocyte (chicken red blood cells;cRBC) hemolysis assay. Hemolytic activity was calculated as apercentage of the activity in pre-dose samples and are shown inFIGS. 14-16. Males are represented as solid lines and females asdashed lines. Antagonism of ex vivo hemolytic activity isproportional to the concentration of total antibody in the sample.The gender difference in the duration of antagonism of hemolyticactivity was pronounced for BHL011 (FIG. 14) corresponding to thebody mass-dependent inter-animal variability for BHL011 PK (FIG.9). Generally the inter-animal variability was low for BHL006 (FIG.15) and BHL 009 (FIG. 16) with the exception of the female in theBHL006 dose cohort (2939) which displayed accelerated antibodyclearance (FIG. 10).

[0321] Differences in the correlation between total antibody serumconcentration and antagonism of ex vivo hemolytic activity areproportional to the affinity of the antibody for C5. The highaffinity antibody (BHL011) nearly completely suppressed hemolyticactivity at .about.200 .mu.g/mL (FIG. 17) while the weakeraffinity, pH-dependent anti-C5 antibodies require 2 to 3-foldhigher concentrations to achieve full antagonism ex vivo (FIGS. 18and 19).

[0322] Despite this loss in potency in the pH-dependent anti-C5antibodies, mean activity levels for cRBC hemolysis across animalsfrom each cohort suggest that they could support an extended dosinginterval. At day 14 the high affinity anti-C5 (BHL011) treatedanimals had mean hemolytic activity levels of >40%, while thepH-dependent anti-C5 (BHL006 and BHL009) treated animals maintainedmean hemolytic activity levels <40% through day 21 and 28,respectively (FIG. 20).

[0323] The significant extension in the half-life and correspondingduration of antagonism of the antibodies with pH-dependent bindingto mouse C5 (BHL006 and BHL009) relative to the high affinityanti-mouse C5 antibody (BHL011) was consistent with studiesdescribed in Examples 4 and 7 in which a pH-dependent anti-human C5antibody (BNJ421, BNJ423 or BNJ441) exhibited a similar increasehalf-life relative to its high affinity counterpart (EHL000 oreculizumab) in mice co-administered human C5. These findingsfurther substantiate the notion that engineering pH-dependentantigen binding through select histidine substitutions in the CDRscan significantly attenuate antigen-mediated clearance though C5,enabling the free antibody to be recycled back to the circulation.Furthermore, the combination of pH-dependent antigen binding andenhanced affinity for FcRn in BHL009 was additive in the effects onPK properties, doubling the half-life over pH-dependent bindingalone (BHL006). These observations are consistent with thehypothesis that pH-dependent binding to C5 in combination withimproved affinity for FcRn may provide a significant extension inthe PK parameters and duration of therapeutic PD observed foreculizumab to enable.gtoreq.monthly dosing.

Example 7

Generation of a Variant Eculizumab with pH-Dependent Binding to C5and Enhanced FcRn-Mediated Recycling

[0324] An antibody was generated using eculizumab as a parentmolecule. Relative to eculizumab, the variant antibody (designatedBNJ441) contained four amino acid substitutions in the heavy chain,Tyr-27-His, Ser-57-His, Met-429-Leu and Asn-435-Ser (note thatpositions 429 and 435 of BNJ441 correspond to positions 428 and 434under the EU numbering system). The amino acid sequence for theheavy chain polypeptide is depicted in SEQ ID NO:14. The amino acidsequence for the light chain polypeptide is depicted in SEQ IDNO:11. These mutations were engineered to enable an extended dosinginterval with BNJ441 (cf. eculizumab) by increasing the circulatinghalf-life through two distinct mechanisms: (1) reducing antibodyclearance through target-mediated antibody clearance and (2)increasing the efficiency of FcRn-mediated antibody recycling.

[0325] The two amino acid substitutions in the first and secondcomplementarity determining regions (CDRs) of the heavy chainvariable region, Tyr-27-His and Ser-57-His, weaken the affinitydissociation constant (K.sub.D) of BNJ441 for C5 by .about.17-foldat pH 7.4 and .about.36-fold at pH 6.0 compared with eculizumab.The two mutations in the third heavy chain constant region domain(CH3), Met-429-Leu and Asn-435-Ser, increase the affinity of BNJ441for FcRn by 10-fold at pH 6.0 compared to eculizumab.

[0326] Binding Kinetics (Antibodies to C5)

[0327] The kinetics of BNJ441 or eculizumab binding to C5 weredetermined via surface plasmon resonance (SPR) on a BlAcore 3000instrument using an anti-Fc capture method at pH 8.0, 7.4, 7.0, 6.5and 6.0. Goat anti-human IgG (Fc) polyclonal antibody (KPL#01-10-20) was diluted to 0.1 mg/mL in 10 mM sodium acetate pH 5.0and immobilized on two flow cells of a CM5 chip for 8 min by aminecoupling. The test antibody (BNJ441 or eculizumab) was diluted to0.20 pg/mL in running buffer (HBS-EP; 0.01 M HEPES pH 7.4, 0.15 MNaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE Life Sciences,catalogue number: BR1001-88). The diluted antibody was theninjected on one flow cell (20 .mu.L for pH 7.4 experiment and 40.mu.l for pH 6.0 experiment) followed by injections of varyingconcentrations of C5 on both cells. The running buffer was titratedwith 3M HCl for pH 7.0, 6.5 and 6.0 kinetics and with 0.5M NaOH forthe pH 8.0 kinetics. The surface was regenerated each cycle with 20mM HCl, 0.01% P20. The data were processed with a 1:1 Langmuirmodel using BlAevaluation 4.1 software (BlAcore AB, Uppsala,Sweden) with `double referencing`.

[0328] The dissociation rates of C5 from BNJ441 or eculizumab at pH8.0, 7.4, 7.0, 6.5 and 6.0 were determined via SPR on a BlAcore3000 instrument using the anti-Fc capture method described abovewith the following modifications. The diluted test antibodies wereinjected on one flow cell followed by an injection of 6 nM C5 onboth cells. Immediately following the C5 injection, 250 .mu.L ofrunning buffer at various pH's were injected. Running buffers wereprepared as described above. The data were processed usingBIAevaluation 4.1 software (BlAcore AB, Uppsala, Sweden) with`double referencing`. The % dissociation of C5 from BNJ441 andeculizumab was calculated by taking the difference in dissociationat t=0 and t=300 seconds.

[0329] Binding Kinetics (Antibodies to FcRn)

[0330] The kinetics of BNJ441 or eculizumab binding to human FcRnwere determined via SPR on a BlAcore 3000 instrument using anF(ab').sub.2 capture method at pH 7.4, and 6.0. Goat F(ab').sub.2anti-human IgG F(ab').sub.2 (Rockland Immunochemicals, Cataloguenumber: 709-1118) diluted to 0.04 mg/mL in 10 mM sodium acetate pH5.0, was immobilized on two flow cells of a CMS chip for 7 minutesby amine coupling. The test antibody (BNJ441 or eculizumab) wasdiluted to 2 .mu.g/mL in running buffer ((HBS-EP; 0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE LifeSciences, Cat. # BR1001-88). The diluted antibody was then injectedon one flow cell followed by injections of FcRn on both cells. Therunning buffer was titrated with 3M HCl for pH 6.0 kinetics. Thesurface was regenerated each cycle with 10 mM Glycine HCl, pH 1.5).The data were processed with a 1:1 Langmuir model usingBIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden) with`double referencing`.

[0331] Results of Binding Studies

[0332] The kinetics of antibody:C5 binding were found to bepH-dependent with effects on both association and dissociationrates are shown in Table 9.

TABLE-US-00029 TABLE 9 Association Rate: Dissociation Rate:Dissociation Constant: pH K.sub.a (1/M*s) K.sub.d (1/s) K.sub.D (M)Chi.sup.2 BNJ441 8.0 6.25 * 10.sup.5 1.33 * 10.sup.-4 2.13 *10.sup.-10 0.055 7.4 4.62 * 10.sup.5 2.27 * 10.sup.-4 4.91 *10.sup.-10 0.045 7.0 4.28 * 10.sup.5 3.90 * 10.sup.-4 9.11 *10.sup.-10 0.028 6.5 4.08 * 10.sup.5 8.94 * 10.sup.-4 2.19 *10.sup.-9 0.172 6.0 1.63 * 10.sup.5 3.54 * 10.sup.-3 2.18 *10.sup.-8 0.373 Eculizumab 8.0 1.39 * 10.sup.6 2.04 * 10.sup.-51.47 * 10.sup.-11 0.104 7.4 1.10 * 10.sup.6 3.23 * 10.sup.-5 2.93 *10.sup.-11 0.094 7.0 8.86 * 10.sup.5 6.34 * 10.sup.-5 7.15 *10.sup.-11 0.032 6.5 8.41 * 10.sup.5 1.73 * 10.sup.-4 2.06 *10.sup.-10 0.037 6.0 7.05 * 10.sup.5 4.28 * 10.sup.-4 6.06 *10.sup.-10 0.092

[0333] In an attempt to model the relative rates of dissociation ofantibody:C5 complexes after pinocytosis and acidification of theearly endosome, antibody:C5 complexes were allowed to form in a pH7.4 buffer, then the buffer pH conditions were switched duringdissociation. The percent of antibody complex dissociation(estimated by the decrease in resonance units [RUs]) after 300seconds was calculated for each pH condition (Table 10). OnlyBNJ441 at pH 6.0 resulted in greater than 50% antibody:C5 complexdissociation after 5 minutes.

TABLE-US-00030 TABLE 10 RU RU pH 0 sec 300 sec % DissociationBNJ441 8.0 55.4 53.5 3.4 7.4 55.7 52.0 6.6 7.0 55.2 49.1 11.0 6.555.2 39.4 28.6 6.0 55.8 22.3 60.0 Eculizumab 8.0 70.2 69.7 0.8 7.470.0 69.5 0.7 7.0 71.3 69.9 2.0 6.5 71.2 67.8 4.7 6.0 71.6 62.912.2

[0334] FIGS. 21A and 21B depict semi-log and linear plots of thepercentage of dissociation of BNJ441:C5 complexes or eculizumab:C5complexes as a function of pH.

[0335] The two amino acid substitutions in the first and secondcomplementarity determining regions (CDRs) of the heavy chainvariable region, Tyr-27-His and Ser-57-His, weaken the affinitydissociation constant (K.sub.D) of BNJ441 for C5 by .about.17-foldat pH 7.4 and .about.36-fold at pH 6.0 compared with eculizumab. Itis unclear if the pH-dependence in the affinity of BNJ441 for C5 isthe result of changes in the protonation state of the histidinesintroduced at positions 27 and/or 57, or simply an overallweakening of the affinity for C5. It has been observed in otheranti-C5 antibodies, however, that these mutations in combinationwith additional histidine substitutions, resulted in much morepronounced losses of affinity at pH levels below 6.5. The twomutations in the third heavy chain constant region domain (CH3),Met-429-Leu and Asn-435-Ser, strengthen the affinity of BNJ441 forFcRn by .about.10-fold at pH 6.0 compared to eculizumab.

[0336] PK Properties of the BNJ441 Antibody

[0337] The BNJ441 antibody and eculizumab were evaluated in micethat were immunodeficient (NOD/scid) and C5 deficient. A singledose of 100 .mu.g of BNJ441 or eculizumab in 200 .mu.L of phosphatebuffered saline (PBS) was administered by intravenous (i.v.)injection to each of eight mice. Serum was collected from each ofthe mice at days one, three, seven, 14, 21, 28, and 35 followingthe administration. The concentration of each antibody in the serumwas measured by ELISA.

[0338] As shown in FIG. 22, in the absence of human C5, the serumantibody concentrations declined similarly in mice dosed withBNJ441 and eculizumab over a 35 day period. However, in thepresence of human C5, eculizumab serum concentrations declinedrapidly to undetectable levels after day 14 while serumconcentration of BNJ441 decayed more slowly and at a consistentrate through duration of study (FIG. 23).

[0339] Comparing the PK profiles of the two antibodies in thepresence and absence of human C5, the clearance of eculizumab wasaccelerated in the presence of human C5 compared to that in theabsence of human C5, while the PK profile of BNJ441 in the presenceof human C5 was similar to that of BNJ441 in the absence of humanC5 through day 28, and clearance was only accelerated between days28 and 35 (FIG. 24). The half-life of BNJ441 and the half-life ofeculizumab were comparable in the absence of human C5(25.37.+-.1.02 days for BNJ441 and 27.65.+-.2.28 days foreculizumab). However, in the presence of human C5, BNJ441demonstrated more than three-fold increase in half-life incomparison with eculizumab (13.40.+-.2.18 days for BNJ441 vs.3.93.+-.0.54 days for eculizumab). It should be noted that theclearance rate of BNJ441 did not differ significantly in thepresence or absence of human C5 through day 28. See Table 11.

TABLE-US-00031 TABLE 11 Treatment Group Animal # Half-life (days)BNJ441 2009 26.99 2011 25.55 2212 24.5 2213 20.34 2214 27.18 221524.35 2216 28.65 Mean = 25.37 SE = 1.02 Eculizumab 2201 30.65 220216.85 2203 27.02 2204 28.54 2205 19.7 2206 35.47 2207 33.77 220829.18 Mean = 27.65 SE = 2.28 BNJ441 + Human C5 2225 24.31 222613.45 2227 N/A 2228 13.48 2229 16.09 2230 8.55 2231 11.25 2232 6.66Mean = 13.40 SE = 2.18 Eculizumab + Human C5 2217 3.35 2218 2.722219 7.45 2220 3.26 2221 2.74 2222 3.93 2223 4.5 2224 3.51 Mean =3.93 SE = 0.54

[0340] Serum Hemolytic Activity

[0341] To determine the effect of the histidine substitutions onhemolytic activity of the antibody, an ex vivo hemolytic assay wasperformed as described in Example 6. In the presence of BNJ441, oreculizumab, terminal complement activity was consistent with therespective PK profiles of each antibody (FIG. 25)--that is, thelevel of inhibition of serum hemolytic activity was proportional tothe concentration of each antibody remaining in the serum. Bothantibodies conferred near total inhibition of hemolysis through day3. However, eculizumab showed no antagonism by day 14, whereasBNJ441 retained about 83% inhibition by day 14 and partialcomplement inhibition through day 28.

[0342] Conclusion

[0343] The findings from this study suggest that in the presence ofhuman C5, BNJ441 showed more than three-fold extension in half-lifecompared with eculizumab. In addition, the serum half-life ofBNJ441 relative to eculizumab translated into an extendedpharmacodynamic profile, as evidenced by prolonged hemolyticinhibition.

Example 8

Safety, Tolerability PK and PD of BNJ441 in Healthy HumanSubjects

[0344] The safety, tolerability, PK and PD of BNJ441 was assessedin a Phase 1, randomized, blinded, placebo-controlled, singleascending dose (SAD) human clinical study, wherein BNJ441 wasadministered intravenously to healthy subjects.

[0345] BNJ441 was formulated in a sterile, preservative-free,aqueous solution with formulation excipients. The BNJ441formulation did not contain any unusual excipients, or excipientsof animal or human origin. The formulation was phosphate-bufferedto a pH of 7.0. The components included BNJ441 10 mg/ml, sodiumphosphate monobasic 3.34 mM, sodium phosphate dibasic 6.63 mM,sodium chloride 150 mM, polysorbate 80 0.02% and Q.S. water.

[0346] The BNJ441 formulation was supplied as a 10 mg/mL antibodysolution in a 20 mL single-use vial, and was designed for infusionby diluting it into commercially available saline (0.9% sodiumchloride injection, Ph Eur) for IV administration.

TABLE-US-00032 TABLE 12 Phase 1 Clinical Trial in HealthyVolunteers Protocol Popu- Dosing Number Title Study Design lationRegimen BNJ441- Phase 1, randomized, First-in-human, healthy Cohort1: HV-101 blinded, placebo- randomized, volun- 200 mg controlled,single placebo- teers BNJ441 ascending-dose study controlled, (4active, to evaluate BNJ441 double-blind, 2 placebo) safety,tolerability, single Cohort 2: PK, and PD as a singleascending-dose 400 mg dose administered BNJ441 IV to healthy (6active, subjects 2 placebo)

[0347] Ten healthy subjects received a single dose of BNJ441. Foursubjects received a dose of 200 mg and six subjects received a doseof 400 mg. The PK and safety data for this study were determinedand discussed below.

Pharmacokinetics

[0348] Serum BNJ441 concentration-time profiles following IVadministration of 200 mg and 400 mg doses are depicted in FIG. 26.Concentration-time data were available for up to Day 90 (2136hours) and Day 57 (1344 hours), following 200 mg and 400 mg doses,respectively. Mean serum concentrations remained above 50 .mu.g/mLfor 2 to 4 days (48 to 96 hours) after the 200 mg dose, and 14 to21 days (336 to 504 hours) after the 400 mg dose.

[0349] A summary of BNJ441 PK parameters is reported in Table 12below. The geometric mean (CV) C.sub.max of BNJ441 was 78.5 (10.2%).mu.g/mL following the 200 mg dose, and 139 (16.2%) .mu.g/mLfollowing the 400 mg dose. The observed median (range) t.sub.maxwas 2.4 (0.79 to 8.0) hours for the 200 mg dose, and 0.58 (0.58 to1.1) hours for the 400 mg dose after the start of infusion.Geometric mean (CV) AUC.sub.(0-56 days) is 32,800 (8.6%) .mu.-hr/mLfor the 200 mg dose, and 58,100 (18.9%) .mu.g-hr/mL for the 400 mgdose. Geometric mean C.sub.max and AUC.sub.(0-56 days) indicatethat exposure increased in an apparent dose-proportional manner.The geometric mean t.sub.1/2 (CV) is 38.5 (18.4%) days, and 32.9(13.3%) days for the 200 mg and 400 mg doses, respectively.

[0350] In summary, the PK data suggest mean BNJ441 C.sub.max andAUC.sub.(0-56 days) increased in a dose proportional manner, andsupport a mean (standard deviation [SD]) t.sub.1/2 of 35.5.+-.6.1days following IV administration. Analysis of chicken red bloodcell (cRBC) hemolysis data indicate terminal complement wascompletely inhibited for up to 2 days after a single 400 mg IVdose, when BNJ441 concentrations were greater than 100.mu.g/mL.

TABLE-US-00033 TABLE 12 Summary of Pharmacokinetic Parameters forBNJ441 Following IV Administration of 200 mg or 400 mg to HealthyVolunteers Dose Descriptive C.sub.max C.sub.max /Dose t.sub.maxAUC.tau..sup.a AUC.tau./Dose t.sub.1/2 (mg) Statistic (ug/mL)(ug/mL/mg) (h) ( /mL) (( /mL)/mg) (day) 200 N 4 4 4 4 4 4 GeometricMean 78.5 0.392 2.40.sup.b 32,800 164 38.5 CV % Geometric 10.2 10.20.79-8.0.sup.c 8.6 8.6 18.4 Mean 400 N 6 6 6 6 6 6 Geometric Mean139 0.348 0.58 .sup.b 58,100 145 32.9 CV % Geometric 16.2 16.20.58-1.1 .sup.c 18.9 18.9 13.3 Mean .sup.aAUC.tau. = AUC.sub.(0-56days) .sup.bmedian .sup.crange

[0351] Pharmacodynamics

[0352] The ability of BNJ441 to inhibit cRBC hemolysis over timewas also assessed, as illustrated in FIG. 27. Mean cRBC hemolysisactivity was relatively stable in subjects who received placebo.The onset of cRBC hemolysis inhibition was rapid, with completeterminal complement inhibition observed at the end of infusion(0.29 hours for the 200 mg dose, and 0.58 hours for the 400 mgdose). BNJ441 had a dose-dependent duration of action, which lastedfor 4 to 14 days.

[0353] The relationship between BNJ441 concentration and cRBChemolysis were plotted and are depicted in FIG. 28. As shown inFIG. 28, complete terminal complement inhibition occurred at BNJ441concentrations above 50 .mu.g/mL, with no inhibition was observedat BNJ441 concentrations below 25 .mu.g/mL.

Example 9

Single Dose Study in Cynomologous Monkeys

[0354] A single IV dose of BNJ441 was administered to cynomolgusmonkeys at doses of 60 or 150 mg/kg (n=4 for each dose group; 2males and 2 females per dose group) as a 2-hour infusion. Bloodsamples for BNJ441 analysis were collected from Day 1 to Day112.

[0355] All BNJ441-treated monkeys were screened for the presence ofCynomologous anti-human antibodies (CAHA) before dosing (0 hour),and on Days 8, 14, 28, 56, 84, and 112.

[0356] All monkeys in the 60 and 150 mg/kg dose group wereconfirmed positive on at least a single occasion, except Animal2002 in the 150 mg/kg dose group. The presence of CAHA in Animal2002, or at non-positive time points for the other animals, cannotbe excluded, due to possible interference of the administeredBNJ441 with the biotinylated-BNJ441 and ruthenylated-BNJ441bridging assay. The positive CAHA results were observed in the 60mg/kg dose group from Day 56 to 112 after dosing, and in the 150mg/kg dose group from Day 28 to 112 after dosing. The firstconfirmed CAHA-positive sample in the 60 mg/kg was on Day 56(Animals 1002 and 1503), 2 on Day 84 (Animals 1002 and 1503), and 3on Day 112 (Animals 1001, 1002, and 1502). Animal 1503, who wasCAHA positive on Days 56 and 84, was no longer CAHA-positive on Day112. The first confirmed CAHA-positive sample in the 150 mg/kg dosegroup was Animal 2502 on Day 28, followed by 2 monkeys on Day 56(Animals 2001 and 2502), 3 monkeys on Day 84 (Animals 2001, 2501,and 2502), and 3 monkeys on Day 112 (Animals 2001, 2501 and2502).

[0357] Individual BNJ441 concentration-time profiles werecalculated. In the 60 mg/kg dose group, all monkeys hadquantifiable plasma BNJ441 concentrations through the Day 112 PKsample, whereas in the 150 mg/kg dose group, only 1 monkey (Animal2002) had quantifiable plasma BNJ441 concentrations through Day112. Concentration-time data indicated a prolonged residence ofBNJ441 in the systemic circulation of monkeys.

[0358] Noncompartmental PK parameters and summary statistics forBNJ441 were calculated for all monkeys by dose level, and shown inTables 13 and 14 for the 60 mg/kg and 150 mg/kg dose levels,respectively. Consistent with duration of infusion, mediant.sub.max was 2 hours for the 60 mg/kg and 150 mg/kg dose levels.One monkey in the 150 mg/kg dose group, Animal 2501, had at.sub.max of 12 hours after dosing, and had a relatively flatprofile from 2 to 12 hours after dosing, with the 12-hour post dosesample concentration approximately 5% greater than that observed at2 hours after dosing. Geometric mean C.sub.max, AUC., andAUC.sub.last all increased with increasing dose. Geometric meandose-normalized C.sub.max values were similar across the 2 doses,indicating a dose-proportional increase in peak BNJ441concentration with an increase in dose, but geometric meandose-normalized AUCs values were different between the dose groups.This difference is likely due to CAHA-mediated increase in BNJ441CL in the 150 mg/kg dose group; clearance of BNJ441 wasapproximately 37% greater in monkeys dosed with 150 mg/kg comparedto the monkeys dosed with 60 mg/kg. Geometric mean V.sub.ss wassimilar (within 12%) between the 2 dose groups.

TABLE-US-00034 TABLE 13 Summary of Noncompartmental PharmacokineticParameters of BNJ441 (60 mg/kg Dose) Dose C.sub.max C.sub.max/t.sub.max AUC.sub.last AUC.sub..infin. AUC.sub..infin./ V.sub.ss CLt.sub.1/2 t.sub.1/2 Animal (mg/kg) (mg/mL) Dose.sup.1) (hr) (hr.times. mg/mL) (hr .times. mg/mL) Dose.sup.2) (mL/kg) (mL/h/kg)(hr) (day) 1001 60 1.92 0.0320 2.0 546 555 9.25 63.4 0.108 479 20.01002 60 1.90 0.0317 2.0 470 475 7.92 55.3 0.126 474 19.8 1502 601.45 0.0242 2.0 598 614 10.2 64.9 0.0977 547 22.8 1503 60 1.440.0240 2.0 701 745 12.4 73.7 0.0806 649 27.1 N 4 4 4 4 4 4 4 4 4 4Mean 1.68 0.0280 2.00 579 597 9.95 64.3 0.103 537 22.4 SD 0.2690.00448 NA 97.0 113 1.89 7.53 0.0191 81.7 3.40 Min 1.44 0.0240 2.00470 475 7.92 55.3 0.0806 474 19.8 Median 1.68 0.0279 2.00 572 5859.74 64.2 0.103 513 21.4 Max 1.92 0.0320 2.00 701 745 12.4 73.70.126 649 27.1 CV % 16.0 16.0 NA 16.8 19.0 19.0 11.7 18.5 15.2 15.2Geometric Mean 1.66 0.0277 NA 573 589 9.82 64.0 0.102 533 22.2 CV %Geometric Mean 16.2 16.2 NA 16.9 19.0 19.0 11.8 19.0 14.8 14.8.sup.1)Units are mg/mL/mg/kg .sup.2)Units are h .times. mg/mL/mg/kghr = hour; NA = not applicable;

TABLE-US-00035 TABLE 14 Summary of Noncompartmental PharmacokineticParameters of BNJ441 (150 mg/kg Dose) Dose C.sub.max C.sub.max/t.sub.max AUC.sub.last AUC.sub..infin. AUC.sub..infin./ V.sub.ss CLt.sub.1/2 t.sub.1/2 Animal (mg/kg) (mg/mL) Dose.sup.1) (hr) (hr.times. mg/mL) (hr .times. mg/mL) Dose.sup.2) (mL/kg) (mL/h/kg)(hr) (day) 2001 150 3.79 0.0253 2.0 787 787 5.25 52.6 0.191 61.02.54 2002 150 4.51 0.0301 2.0 1160 1220 8.15 89.8 0.123 759 31.62501 150 4.48 0.0299 12.0 1460 1460 9.71 58.8 0.103 87.6 3.65 2502150 4.40 0.0293 2.0 1030 1030 6.86 37.5 0.146 54.1 2.25 N 4 4 4 4 44 4 4 4 4 Mean 4.30 0.0286 4.50 1110 1120 7.49 59.7 0.141 240 10.0SD 0.340 0.00227 NA 279 285 1.90 22.0 0.0377 346 14.4 Min 3.790.0253 2.00 787 787 5.25 37.5 0.103 54.1 2.25 Median 4.44 0.02962.00 1100 1130 7.50 55.7 0.134 74.3 3.09 Max 4.51 0.0301 12.0 14601460 9.71 89.8 0.191 759 31.6 CV % 7.91 7.91 NA 25.2 25.3 25.3 36.926.8 144 144 Geometric 4.28 0.0286 NA 1080 1100 7.30 56.8 0.137 1225.07 Mean CV % 8.25 8.25 NA 26.1 26.7 26.7 37.2 26.7 190 190Geometric Mean .sup.1)Units are mg/mL/mg/kg .sup.2)Units are h.times. mg/mL/mg/kg hr = hour; NA = not applicable

Example 10

A Comparative Assessment of BNJ441, Eculizumab and h5G1.1 Bindingto Fc-Gamma Receptors C1q In Vitro

[0359] The binding of three humanized antibodies, BNJ441,eculizumab and h5G1.1-IgG1 to molecules known to be mediators ofantibody effector function was examined. BNJ441, eculizumab, andh5G1.1-IgG1 each have unique functional and therapeutic profiles.However, all three are humanized antibody antagonists of terminalcomplement, which bind a very similar epitope on human complementcomponent C5 and prevent its cleavage during complement activationinto its active metabolites, C5a and C5 b.

[0360] BNJ441, eculizumab, and h5G1.1-IgG1 are identical in theirlight chain sequences, each having a humanized variable region andhuman IgKappa constant region. BNJ441 and eculizumab both contain ahuman hybrid IgG2-G4 Fc, which includes the CH1 region, hinge andfirst 29 amino acids of the CH2 region from human IgG2 fused to theremainder of the CH2 and CH3 regions of human IgG4. This chimericFc combines the stable disulfide bond pairing of an IgG2 with theeffector less properties of an IgG4. Since BNJ441 and eculizumabare directed against a soluble antigen, it was not possible todirectly assess their capacity to initiate antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-dependentcytotoxicity (CDC). Instead, direct measurements of BNJ441 oreculizumab binding to Fc gamma receptors (FcyRs) and complementcomponent C1q were performed and it was inferred that in theabsence of binding they cannot mediate ADCC or CDC, respectively.h5G1.1-IgG1 (an IgG1 isotype antibody with the same humanizedvariable region as eculizumab) was included as a control. The IgG1isotype Fc region is expected to bind effector function moleculesfully, though h5G1.1-IgG1 itself would not elicit ADCC or CDC inthe absence of a cell associated antigen.

[0361] As dicussed above in Example 7, BNJ441 was engineered fromeculizumab to increase its half life in vivo by introducing 4 aminoacids substitutions in the heavy chain. Two amino acid changes inthe humanized heavy chain variable region, Tyr-27-His andSer-57-His respectively (heavy chain amino acid numbering accordingto Kabat et al.), were introduced to destabilize binding to C5 atpH 6.0 with minimal impact on binding to C5 at pH 7.4. Mutations inthe third heavy chain constant region domain (CH3), Met-428-Leu andAsn-434-Ser, were introduced to enhance binding to the humanneonatal Fc receptor (FcRn). Taken together these mutations weredesigned to significantly attenuate antigen-mediated drug clearanceby increasing dissociation of antibody:C5 complexes to freeantibody in the acidified environment of the early endosome afterpinocytosis, and to increase the fraction of antibody recycled fromthe early endosome back into the vascular compartment by FcRn.

[0362] In these studies, multimeric interactions of the Fc.gamma.Rsubclasses (Fc.gamma.R1, Fc.gamma.RIIa, Fc.gamma.RIIb,Fc.gamma.RIIb/c, Fc.gamma.RIIIa and Fc.gamma.RIIIb) with all threeantibodies were evaluated in an enzyme linked immunosorbent assay(ELISA) and monomeric interactions with Fc.gamma.Rs were evaluatedusing surface plasmon resonance (SPR). Biolayer interferometry andSPR was used to examine the binding of C1q to the three antibodies.The reagents used to conduct these analyses are shown in Table15.

TABLE-US-00036 TABLE 15 Antibodies and Protein Reagents ReagentSource Concentration BNJ441 Alexion 10 mg/mL Eculizumab Alexion 10mg/mL Goat anti-human Jackson 1.1 mg/mL F(ab')2-biotin ImmunolabsHRP-streptavidin Invitrogen 1.25 mg/mL h5G1.1-IgG1 Alexion 1.43mg/mL 8.11 mg/mL C1q Complement 1 mg/mL Technology HumanFc.gamma.RI R&D systems 100 .mu.g/mL (CD64) Human Fc.gamma.RIIaR&D systems 100 .mu.g/mL (CD32a) Human Fc.gamma.RIIb/c R&Dsystems 100 .mu.g/mL (CD32b/c) Human Fc.gamma.RIIIa R&D systems100 .mu.g/mL (CD16a) Human Fc.gamma.RIIIb R&D systems 100.mu.g/mL (CD16b)

[0363] Binding of Multivalent Antibody Complexes to Fc.gamma.Rs

[0364] Antibody complexes were prepared by incubating BNJ441,Eculizumab or h5G1.1-hG1 overnight with goat-anti-human F(ab')2-biotin (Jackson Immunolabs), at a 2:1 antibody: F(ab')2 molarratio in phosphate buffered saline (PBS) in a 1.5 mL microfugetube.

[0365] Microtiter plates pre-coated with Ni-NTA (Qiagen) wereincubated with 50 .mu.L/well of 6.times. histidine-tagged humanFc.gamma.Rs (Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb/c,Fc.gamma.RIIIa or Fc.gamma.RIIIb), at a receptor concentration of 5.mu.g/mL in PBS, overnight at 4.degree. C. The plate was thenwashed 3 times with PBS/0.05% Tween-20. After washing, 50 .mu.L ofantibody complexes in PBS/0.05% Tween-20 were incubated in theplate for 60 min at room temperature (RT). After washing the platewith PBS/0.05% Tween-20, 50 .mu.L of streptavidin-HRP (Invitrogen)in PBS/0.05% Tween-20 was added to the plate and incubated for 60min at RT. Following this incubation and washes, 75 .mu.L ofTMB-ELISA substrate (3, 3', 5, 5'-tetramethylbenzidine, ThermoScientific) was added. The reaction was stopped with 75 .mu.L of 2M H.sub.2SO.sub.4, and the absorbance read at 450 nm.

[0366] Samples were run in duplicate and data were presented asmean values. Results were entered into a spreadsheet program. Theabsorbance at 450 nm of each concentration of antibody immunecomplex or in the absence of antibody immune complexes plotted as agraphical representation. The key dissociation constants werecalculated and are summarized in Table 16 and discussed below.

[0367] Binding of Monovalent Antibodies to Fc.gamma.Rs

[0368] The kinetics of BNJ441, eculizumab, and h5G1.1-IgG bindingto Fc.gamma.Rs were determined via SPR on a BlAcore 3000 instrumentusing direct immobilization. BNJ441, eculizumab, and h5G1.1 werediluted in 10 mM sodium acetate pH 5.0, was immobilized on one flowcell of a CMS chip by amine coupling. A second flow cell was usedas a reference surface. Concentrations of Fc.gamma.Rs diluted inrunning buffer (HBS-EP, pH 7.4) were injected on both cells. Thesurface was regenerated each cycle with 20 mM HCl, 0.01% P20. Thedata was analyzed using a steady state affinity model inBlAevaluation 4.1 software (BlAcore AB, Uppsala, Sweden) with`double referencing`.

[0369] The kinetics of h5G1.1-IgG1 binding to Fc.gamma.RI wasassessed via single cycle kinetics due to its stronger affinity.The antibody was diluted in 10 mM sodium acetate at pH 5.0 anddirectly immobilized on one flow cell of a CMS chip by aminecoupling. A second flow cell was used as a reference surface.Concentrations of Fc.gamma.R1 diluted in running buffer (HBS-EP, pH7.4) were injected on both cells. This assay required noregeneration. The data was analyzed using a titration kinetics 1:1model in BlAevaluation 4.1(Biacore AB, Uppsala, Sweden) softwarewith `double referencing`.

TABLE-US-00037 TABLE 16 Dissociation constants for BNJ441,eculizumab and h5G1.1-IgG1 binding to monomeric FcyRs BNJ441,Eculizumab, h5G1.1-IgG1, Fc.gamma.R K.sub.D [.mu.M] K.sub.D [.mu.M]K.sub.D [.mu.M] RI 3.75 3.78 0.123 RIIa 2.31 2.58 0.8 RIIb/c 8.099.84 3.06 RIIIa 7.23 6.78 0.85 RIIIb 3.33 3.49 1.89

[0370] ELISA assays to detect avidity-driven multimericinteractions of antibody immune complexes and Fc.gamma.Rs wereperformed. The results are summarized in Table 16. BNJ441 andeculizumab displayed no detectable binding to Fc.gamma.RI,Fc.gamma.RIIb/c, Fc.gamma.RIIIa or Fc.gamma.RIIIb and a 4-fold to8-fold weaker association with Fc.gamma.RIIa, respectively.Dissociation constants (K.sub.D) for monomeric Fc.gamma.R bindingto BNJ441 and eculizumab derived by SPR confirmed that FcyRinteractions are very weak and nearly indistinguishable between thetwo antibodies: Fc.gamma.RI (.about.4 .mu.M), Fc.gamma.RIIa(.about.2 .mu.M), Fc.gamma.RIIb (.about.9 .mu.M), Fc.gamma.RIIIa(.about.7 .mu.M) and Fc.gamma.RIIIb (.about.3 .mu.M). Dissociationconstants for the IgG1 isotype control (h5G1.1-IgG1) wereconsistent with high affinity interactions with Fc.gamma.R1 (123pM) and modest increases in binding to the low affinity Fc.gamma.Rsrelative to the IgG2-G4 isotype antibodies: Fc.gamma.RIIa (.about.1.mu.M), Fc.gamma.RIIb (.about.3 .mu.M), Fc.gamma.RIIIa (.about.1.mu.M) and Fc.gamma.RIIIb (.about.2 .mu.M). See Table 16. Nointeractions between C1q and BNJ441 or eculizumab were detectablevia biolayer interferometry. These results are consistent with theidea that the chimeric human IgG2-G4 Fc of eculizumab has little tono capacity to elicit effector function through Fc.gamma.Rs or C1qto mediate ADCC or CDC, respectively. Furthermore, these resultsshow that the heavy chain amino acid substitutions incorporated inBNJ441 do not significantly alter binding to these, relative toeculizumab.

Example 11

Tissue Cross Reactive Studies

[0371] 1. GLP Human Cross-Reativity Studies

[0372] Potential cross reactivity with human tissues was determinedusing fluoresceinated BNJ441 (designated BNJ441-FITC) and a controlantibody (OX-90G2G4-FITC) with a different antigenicspecificity.

[0373] BNJ441-FITC produced moderate to intense staining of thepositive control material (purified human complement protein C5ultraviolet [UV]-resin spot slides, designated hC5) but did notspecifically react with the negative control material (humanhypercalcemia of malignancy peptide, amino acid residues 1-34,UV-resin spot slides, designated PTHrP 1-34). The control article,OX-90G2G4-FITC, did not specifically react with either the positiveor negative control materials. The excellent specific reactions ofBNJ441-FITC with the positive control material and the lack ofspecific reactivity with the negative control material, as well asthe lack of reactivity of the control article, indicated that theassay was sensitive, specific, and reproducible.

Staining with BNJ441-FITC was observed in the human tissue panel,as summarized below: [0374] Proteinaceous material in most humantissues [0375] Cytoplasm and/or cytoplasmic granules in thefollowing tissue elements: [0376] mononuclear cells in the colon,esophagus, lymph node, parathyroid, spleen, and tonsil [0377]platelets in blood smears and bone marrow [0378] megakaryocytes inthe bone marrow [0379] epithelium in the fallopian tube, liver(hepatocytes), pancreatic ducts, and cervix [0380] mesothelium inthe lung

[0381] Because C5 is a circulating serum protein, the staining ofproteinaceous material was expected. Mononuclear cells such asmonocytes, macrophages, and dendritic cells, as well as platelets,have been reported to secrete C5; therefore, the staining of thesecell types with BNJ441-FITC was also expected. Additionally,mesothelial cell lines have been shown to produce C5. However, noliterature was available describing the expression of C5 by theepithelial cell types stained with BNJ441-FITC in the currentstudy, or megakaryocytes, although platelets, which have been shownto produce C5, are derived from megakaryocytes. Therefore, stainingof epithelial cell types might represent either previouslyunrecognized sites of C5 expression, or tissue cross-reactivitywith a protein sequence or structure from a similar but unrelatedprotein or other constituent(s) of the tissue sections. However,with the exception of staining of proteinaceous material, allstaining observed in this study was cytoplasmic in nature, and itis unlikely that the cytoplasm and cytoplasmic structures would beaccessible to the test article in vivo. In summary, no specificcross-reactivity of BNJ441-FITC staining was observed that wouldlead to the expectation of treatment-related toxicity.

[0382] 2. GLP Cynomolgus Monkey Tissue Cross-Reactivity Studies

[0383] A standard GLP tissue cross-reactivity study was also doneusing a panel of cynomolgus monkey tissues to examine bothoff-target and on-target binding, with the same reagents used inthe human tissue binding studies.

[0384] Some staining with BNJ441-FITC was observed in thecynomolgus monkey tissue panel, as summarized below: [0385]Proteinaceous material in most cynomolgus monkey tissues [0386]Cytoplasm and/or cytoplasmic granules in the following tissueelements: [0387] mononuclear cells in the lymph node, spleen, andtonsil [0388] epithelium in the fallopian tube

[0389] The BNJ441-FITC staining pattern observed in the cynomolgusmonkey tissue panel was overall less intense and less frequent thanthat observed in the human tissue panel in the companion humantissue cross-reactivity study. Further, in the human tissue panel,staining of platelets, megakaryocytes, pancreatic ductalepithelium, cervical epithelium, hepatocytes, and mesothelium wasobserved, although these tissue elements were not stained in thecynomolgus monkey tissue panel. Moreover, with the exception ofstaining of proteinaceous material, the staining observed in thisstudy was cytoplasmic in nature, and it is unlikely that thecytoplasm and cytoplasmic structures would be accessible to thetest article in vivo. Because BNJ441 has been shown to beexquisitely specific for human C5 (and is not cross-reactive withC5 from nonhuman primates), it is likely that the limited bindingobserved in this study was due to nonspecific binding with anunidentified cross-reactive material

Example 12

Potency of BNJ441 Compared to Eculizumab in Terminal ComplementActivity Assays

[0390] The mutations engineered in BNJ441 to yield pH-dependentbinding to C5 weaken its affinity at pH 7.4 (approximately 491 pM)by approximately 17-fold relative to eculizumab (approximately 29.3pM) and might be expected to reduce BNJ441 inhibition potency ofC5-mediated terminal complement activity compared to eculizumab. Toestimate the potencies of BNJ441 and eculizumab underphysiologically relevant conditions, antagonism ofcomplement-mediated hemolysis of red blood cells (RBCs) from 3commonly used animal models (chicken, sheep, and rabbits) wasassessed in 90% normal human serum.

[0391] RBCs and sheep red blood cells (sRBCs) were pre-sensitizedwith antibodies to initiate activation of the complement classicalpathway (CCP). Rabbit red blood cells (rRBCs) were notpre-sensitized and are used as a model of complement alternativepathway (CAP) activation. Antibodies were pre-incubated in serum at100, 200, and 400 nM to yield molar ratios of antigen binding sitesto C5 of approximately 0.5:1, 1:1, and 2:1, respectively. AntibodyBNJ430 contains the same Fc region as BNJ441, but does not bindhuman C5, and was included as a negative control. Percent hemolysiswas measured at 0, 1, 2, 3, 4, 5, 6, and 8 minutes to ensure thatreactions were observed under initial velocity conditions.

[0392] As shown in FIG. 29, neither BNJ441 nor eculizumab displayedantagonism at 100 nM in cRBC hemolysis. Both antibodies exhibitedpartial antagonism at 200 nM (approximately 1:1molar ratio ofantigen binding sites to C5), with BNJ441 having reduced potencyrelative to eculizumab. Inhibition of hemolysis was nearly completefor either antibody when incubated at a 2:1 molar ratio of antigenbinding sites to C5 (400 nM). Results of sRBC hemolysis assays weresimilar, showing less than 20% hemolysis in the presence of BNJ441at 200 nM, and near complete inhibition with each antibody at 400nM (data not shown). The CAP-mediated rRBC hemolysis assays exhibithigher levels of hemolysis in the presence of anti-C5 antibodies,with no detectable inhibition at 200 nM, and only partialinhibition at 400 nM (data not shown).

[0393] In conclusion, the modest loss in potency of BNJ441 relativeto eculizumab in these in vitro complement activity assays isconsistent with its weaker affinity for C5. The affinity of BNJ441for C5 is still approximately 1000-fold lower than theconcentrations of C5 in vivo and targeted therapeutic levels ofBNJ441, and is therefore unlikely to compromise its therapeuticefficacy.

Example 13

Selectivity of BNJ441 Compared to Eculizumab in Terminal ComplementActivity Assays

[0394] To assess the pharmacologic activity of BNJ441 in non-humananimal models the ability of BNJ441 to antagonizecomplement-mediated hemolysis of antibody-sensitized cRBCs in serumfrom chimpanzee, baboon, rhesus macaque, cynomolgus macaque,beagle, rabbit, guinea pig, rat and mouse were measured. Eculizumaband an anti-mouse-05 antibody with a human IgG2/G4 Fc (BNJ430) wereused as isotype controls.

[0395] Sensitized cRBCs were prepared for each assay from 400 .mu.Lof chicken whole blood in Alsever's (Lampire Biologicals) andwashed 4 times with 1 mL of GVBS at 4.degree. C. and re-suspendedin GVBS at 5.times.10.sup.7 cells/mL. To sensitize chickenerythrocytes, a polyclonal anti-chicken RBC antibody (Rockland) wasadded to the cells at 150 .mu.g/mL and incubated for 15 min on ice.After washing with GVBS once, the cells were re-suspended in GVBSto a final volume of 3.6 mL.

[0396] Complement preserved sera were obtained from Bioreclamationincluding serum from the following mammals: human; chimpanzee;baboon; rhesus macaque; cynomolgous macaque; beagle; rabbit; guineapig; and rat. Antibodies BNJ441 at 10 mg/ml; eculizumab (10 mg/ml);BNJ430 at 0.873 mg/ml were diluted to a final concentration of 0,60, 300 and 600 nM in 30% serum in GVBS and incubated at roomtemperature for 30 min. Sensitized cRBCs were added to theantibody/serum mixture at 30 .mu.L per well (2.5.times.10.sup.6cells), incubated at 37.degree. C. for 30 min and reactions werestopped by adding 30 .mu.L of 0.5M EDTA to each well. The plateswere centrifuged at 1800.times.g for 3 min and 80 .mu.L of thesupernatant was transferred to a new flat-bottom 96-well plate. Theabsorbance was measured at 415 nm.

[0397] As mouse serum is a poor source of classical pathwaycomplement activity, mouse serum was mixed 1:1 with C5-depletedhuman serum to assess potential BNJ441 pharmacologic activity inmice. Antibodies were diluted to a final concentration of 0, 60,300 and 600 nM in 50% total serum (25% mouse serum, 25% C5 depletedhuman serum) in GVBS and incubated at room temperature for 30 min.Sensitized cRBCs were added to the antibody/serum mixture at 30 perwell (2.5.times.10.sup.6 cells), incubated at 37.degree. C. for 30min and reactions were stopped by adding 30 .mu.L of 0.5M EDTA toeach well. The plates were centrifuged at 1800.times.g for 3 minand 80 of the supernatant was transferred to a new flat-bottom96-well plate. The absorbance was measured at 415 nm.

[0398] Samples containing serum without anti-C5 antibodies with orwithout 10 mM EDTA were used as no lysis or complete lysiscontrols, respectively. Sample conditions were run in triplicate orduplicate.

[0399] Results were entered into a spreadsheet to allow backgroundsubtraction of no lysis controls and normalization of percenthemolysis relative to complete lysis controls, calculation of meanvalues (.+-.s.d.) and graphical representation of the data.Absorbance values for mean background from no lysis controls weresubtracted from each replicate and sample absorbance was expressedas the percent of lysis in complete lysis controls according to thefollowing equation: % of cRBC hemolysis equals (A415 value in eachsample replicate sample--mean A415 value in no lysis control) /(mean A415 value in complete lysis control - mean A415 value in nolysis control).times.100.

[0400] The mean and standard deviation of the % cRBC hemolysis forsample replicates were plotted as a graphical representation (datanot shown).

[0401] BNJ441 was shown to have no detectable binding to native C5from cynomolgus macaque and no pharmacologic activity in vitro inany non-human sera tested at an 8-fold molar excess of antigenbinding sites to C5. Taken together, these data are consistent withthe conclusion that BNJ441 does not have any relevant pharmacologicactivity in any readily accessible non-human species suitable formodeling the pharmacokinetics or pharmacodynamics in humans.

Example 14

Physicochemical Characterization of BNJ441

[0402] The BNJ441 antibody is a recombinant, humanized antibody,and consists of two identical 448 amino acid heavy chains and twoidentical 214 amino acid light chains. See FIG. 30. The constantregions of BNJ441 include the human kappa light chain constantregion and the hybrid human IgG2-IgG4 heavy chain constant region(also referred to as "G2/G4"). The IgG2/G4 constant region wasrationally designed to reduce the effector function activation,complement activation, and immunogenicity of the antibody. Theheavy chain CH1 domain, hinge region and the first 5 amino acids ofthe CH2 domain match human IgG2 amino acid sequence, residues 6 to36 in the CH2 region and common to both human IgG2 and IgG4 aminoacid sequence, while the remainder of the CH2 domain and the CH3domain match human IgG4 amino acid sequence. The heavy and lightchain variable regions which form the human C5 binding site consistof human framework regions were grafted to murinecomplementarity-determining regions. The inter-chain disulfidebonds in the BNJ441 antibody are depicted in FIG. 31. The residuenumbers are shown in FIG. 31 for all the disulfide bond pairing andN-linked glycan sites.

[0403] Table 17 lists the general properties of the BNJ441antibody. The theoretical chemical formula and theoretical averagemolecular weight for the main component presented below assume thatthe antibody contains eighteen disulfide bonds, two heavy chainN-terminal pyroglutamations, the clipping of two heavy chain Cterminal lysines, and the addition of two GOF glycan residues. Thenumber of amino acid residues in BNJ441 has been predicted by aminoacid analysis.

TABLE-US-00038 TABLE 17 General Properties of the BNJ441 AntibodyProperty Value Theoretical Chemical Formula C.sub.6542H.sub.10072N.sub.1704 O.sub.2106 S.sub.48 Theoretical AverageMolecular Weight 147,827.62 Da Number of Amino Acids 1324

[0404] A stable Chinese hamster ovary (CHO) cell line expressingBNJ441 was developed for the manufacture of BNJ441. The sourceCHOK1SV cells used to generate this cell line were obtained fromLonza Biologics CHOK1SV master cell bank 269-M. This cell sourcewas verified to be free of bacterial and fungal contaminants andall detectable viruses other than cell endogenous retroviralparticles that are not infectious. Host CHOK1SV cells weretransfected with plasmid pBNJ441.1 and stable clones were selectedwith the MSX. Primary clone 3A5 was selected as the production cellline for the manufacture of BNJ441.

[0405] Engineering and GMP batches of BNJ441 bulk drug substancebatches were prepared and physicochemically characterized by thetests listed in Table 18. The engineering batch was produced in apilot plant using CHO cells grown a 200 L bioreactor and thepurified material was used in the PK study. The GMP batch wasproduced using CHO cells grown in the pilot plant using a 200 Lbioreactor. The BNJ441 engineering and GMP bulk drug substancebatches were formulated and tested at approximately 10 mg/mL. Thephysicochemical properties for the batches are summarized in Table19.

TABLE-US-00039 TABLE 18 BNJ441 Physicochemical characterizationTest Category Test Purity Analytical Ultracentrifugation SizeIntact Molecular Weight Analysis (MALDI-ToF-MS) Size IntactMolecular Weight Analysis (ESI-ToF-MS) Identity N-TerminalSequencing Primary structure Amino Acid Analysis Higher orderstructure Circular Dichroism Spectrometry Glycosylation patternN-Linked Oligosaccharides Mass Profiling (MALDI-ToF-MS)Glycosylation pattern Oligosaccharides Glycosylation patternMonosaccharides Glycosylation pattern Sialic Acid ThermostabilityDifferential Scanning Calorimetry Kinetics and Self BiacoreKinetics and Self-Association Association

TABLE-US-00040 TABLE 19 BNJ441 Physicochemical Summary EngineeringBatch GMP Test BNJ441 BNJ441 Analytical 99.3% 99.0%Ultracentrifugation % monomer Molecular Weight 148,484 148,522Analysis MALDI-ToF- MS (Da) Molecular Weight Major isoform Majorisoform Analysis ESI-ToF-MS 147830.80 147830.72 (Da) Range 147,000-Range 147,000- 149,500 149,500 N-Terminal Sequencing PyroQ PyroQHeavy Chain V V Q Q L L V V Q Q S S G G A A E E V V K K K K P P G GA A S S V V K K V V S S N-Terminal Sequencing D D Light Chain I I QQ M M T T Q Q S S P P S S S S L L S S A A S S V V G G D D R R V V TT residues per residues per Amino Acid Analysis (#) moleculemolecule ASX (106) 105 102 GLX (138) 137 135 SER (166) 170 167 GLY(84) 89 88 HIS (22) 26 26 ARG (36) 42 42 THR (110) 106 105 ALA (64)68 67 PRO (88) 93 92 TYR (54) 51 53 VAL (128) 127 129 MET (12) 1111 ILE (28) 26 27 LEU (94) 92 94 PHE (50) 51 51 LYS (82) 68 73Circular Dichroism Near UV Feature NearUV (nm) Near UV (nm) max 295295 min 269 269 max 266 266 min 262 262 negative deflection 250 250Far UV Feature Far UV (nm) Far UV (nm) shoulder 239-231 239-231 max218 218 min 201 202 Deconvolution Decon Decon .alpha.-helix 0.0300.030 3/10 helix 0.026 0.026 .beta.-sheet 0.328 0.334 Turns 0.1560.158 Poly (Pro) II 0.059 0.061 Unordered 0.397 0.388 Total.sup.10.996 0.997 Oligosaccharides (MALDI-ToF-MS) m/z (M + Na)+ m/z (M +Na)+ G1F 1647.61 1647.55 G1 1501.52 1501.49 G0F 1485.56 1485.51 G01339.47 1339.49 G0F-GN 1282.46 1282.39 Man-5 1257.43 1257.48Oligosaccharide % % M3N2F 0.00 0.00 G0F-GN 0.66 0.93 G0F 90.4591.26 G1F 8.79 7.7 G2F 0.00 0.00 Man-5 0.09 0.12 aGal1 0.00 0.00Man-6 0.00 0.00 aGal2 0.00 0.00 Man-7 0.00 0.00 aGal3 0.00 0.00SA1-1 0.00 0.00 SA1-2 0.00 0.00 SA1/aGal4 0.00 0.00 SA1-3 0.00 0.00SA1-4 0.00 0.00 SA2-1 0.00 0.00 SA2-2 0.00 0.00 Total G0F, G1F, G2F99.24 98.96 Acidic 0.00 0.00 High Mannose 0.09 0.12 aGal 0.00 0.00Neutral 99.99 100.01 Monosialylated 0.00 0.00 Disialylated 0.000.00 (nmol mono/ (nmol mono/ Monosaccharide mg protein) mg protein)GlcNAc 22.14 29.26 GalNAc 0.00 0.00 Galactose 0.66 0.82 Mannose20.25 23.24 Fucose 5.38 6.53 Total 48 60 % Glycosylation 0.93%1.16% Sialic Acid (mmol/mol) (mmol/mol) NGNA ND ND NANA <LoQ<LoQ Calorimetry T.sub.m 67.0.degree. C. 67.0.degree. C. BiacoreKinetics k.sub.a (1/MS) 4.44e.sup.5 4.86e.sup.5 K.sub.d (1/s)2.05e.sup.-4 2.04e.sup.-4 K.sub.D (M) 4.61e.sup.-10 4.21e.sup.-10Chi.sup.2 0.0257 0.0347 Biacore Self-Association K.sub.D(M)7.12e.sup.-3 2.71e.sup.-4 Chi.sup.2 0.147 0.359

[0406] Table 19 shows the intact molecular weight determined forthe engineering batch was 147830.80 Da and GMP batch was 147830.72Da. The values were consistent with the calculated major componentmolecular weight value for BNJ441 of 147,827.62 Da in Table 17, andwithin the 100 ppm mass accuracy of the externally calibratedESI-ToF-MS. No major peaks were observed beyond the 147,000-149,500Da range. This method identified the molecule on the basis ofintact molecular weight. Test samples were injected onto a C4RP-HPLC column and eluted with an aqueous:organic solvent gradient.The eluate was then electrosprayed into a ToF mass spectrometer anda spectrum from the upper half of the chromatographic peak wasdeconvoluted to provide the intact molecular weight.

[0407] Table 19 shows the N-Terminal sequence determined for theBNJ441 batches. The determined N-Terminal sequences of the heavychain and light chain were consistent with the amino acid sequencefor BNJ441 batches. The heavy chain was found to be blocked with aPyroQ, as expected, and was de-blocked with pyroglutamateaminopeptidase (PGAP). We determined the primary sequence of theprotein at the N-terminus of the polypeptide chain by sequentialEdman degradation and HPLC analysis.

[0408] Table 19 shows the Amino Acid Analysis residues per moleculedetermined for the BNJ441 batches. These values were all consistentwith the calculated number of residues per molecule for BNJ441based on the primary sequence, shown in the first column of Table19. The Amino Acid Analysis data were acquired in triplicate. Thismethod assesses the primary structure of the molecule by acidichydrolysis of the protein into its individual amino acidconstituents. This method does not detect cysteine or tryptophan.Asparagine and aspartate were detected in a single peak and labeledAsx. Glutamine and glutamate are also detected in a single peak andlabeled Glx. Of the 20 standard amino acids, fourteen are uniquelydetected by this method plus the Asx and Glx groups for a total ofsixteen amino acids. Of those represented, BNJ441 has a total of1262 residues that can be detected by these methods.

[0409] Table 19 shows the circular dichroism (CD) Near UV LocalFeature, Far UV Local Feature and Deconvolution results for theBNJ441 batches. The deconvolution describes the amounts of a-helix,3/10 helix, (3-sheet, Turns, Poly (Pro) II and unordered structuresdetermined by CDPro software against a given reference set. The CDspectra for Near UV (tertiary structure) and for Far UV (secondarystructure) for each batch were determined. This method assessedhigher order molecular structure (2.degree. and)3.degree. in themolecule by the differential absorption of left and rightcircularly polarized light exhibited in the absorption bands ofoptically active (chiral) molecules, such as proteins.Deconvolution of the CD spectra was performed and the results areshown in Table 19.

[0410] Table 19 shows the mean molecular weight for each glycandetermined. The observed N-Linked Oligosaccharide or glycanmolecular weights for the BNJ441 batches were consistent with thetheoretical glycan molecular weights shown in Table 20. The freeglycan molecular weight spectra were determined by MALDI-TOF massspectrometry. This method identified the glycans associated withthe drug molecule by molecular weight. The glycans were previouslyenzymatically cleaved from the antibody with PNGase F. The glycanswere then solid phase extracted and mixed with the3,4-dihydroxybenzoic acid matrix solution and co-precipitated onthe MALDI target. This dried sample was ionized with a nitrogenlaser into a TOF mass spectrometer. An m/z (M+Na).sup.+ spectrumwas collected.

TABLE-US-00041 TABLE 20 Theoretical Glycan Molecular Weight GlycanTheoretical Structure m/z (M + Na)+ G1F 1647.58 G1 1501.53 G0F1485.53 G0 1339.47 G0F-GN 1282.45 Man-5 1257.41

[0411] The oligosaccharide percentages determined for the BNJ441batches are shown in Table 19. The totals for various types ofN-linked oligosaccharides were calculated: (Total GOF, G1F),Acidic, High Mannose, Neutral, Monosialylated and Disialylated. TheN-linked oligosaccharides only contained neutral oligosaccharides.The level of neutral oligosaccharides was 99.99 and 100.0% for theengineering and GMP batches respectively. The oligosaccharides weredetected using HPLC and the chromatograms were evaluatedquantitatively. This method evaluates the glycosylation pattern byidentifying the N-linked oligosaccharides associated with the drugmolecule on the basis of the retention time of the enzymaticallyreleased and fluorescently tagged oligosaccharides. This methodprovided the relative abundance of each oligosaccharide species.Briefly, the oligosaccharides were enzymatically cleaved from theantibody with PNGase F and tagged with anthranilic acid. Excessanthranilic acid was removed using a HILIC filtration step. Sampleswere injected on to a wAX-HPLC system with a Showa Denko AsahipakAmino Column and the tagged oligosaccharides were detected with afluorescence detector; 360 nm excitation and 420 nm emission.

[0412] The monosaccharide percentages were determined for theBNJ441 batches and are shown in Table 19. The monosaccharidepercentages were determine for the five monosaccharides (GlcNAc,GalNAc, Galactose, Mannose, Fucose) using fluorescence labellingfollowed by reverse phase high pressure chromatography (RP-HPLC).This assay characterizes the glycosylation pattern by determiningthe monosaccharides associated with the drug molecule on the basisof the retention time of the fluorescently labelledmonosaccharides. Briefly, acid hydrolysis removed theoligosaccharides from the protein and into its constituentmonosaccharides. The free monosaccharides were then labelled withanthranilic acid (AA) by reductive amination. Samples were theninjected on to an RP-HPLC system with a Waters Symmetry.RTM. C-18column and the AA tagged monosaccharides were detected with afluorescence detector; 360 nm excitation 420 nm emission. Sampleswere tested in duplicate and the value reported was the mean of thetwo results.

[0413] Next we determined the sialic acids N-acetylneuraminic acid(NANA), and N-glycolylneuraminic acid (NGNA). In each case, thedetermined NANA and NGNA sialic acid content of the BNJ441 batcheswere below the limit of quantitation (<6 mmol/mol) as shown inTable 19. No NGNA was observed for either batch. The sialic acidswere measured separated on RP-HPLC following fluorencelabelling.and using multi-point calibration. This method assessesthe glycosylation pattern by determining the type and relativeamount of the sialic acids associated with the drug molecule. Thesialic acids were chemically cleaved from the antibody byincubation with sodium bisulfate then tagged withO-phenylenediamine. Samples were injected on to an RP-HPLC systemwith a Beckman C18 Ultrasphere column and the tagged sialic acidswere detected with a fluorescence detector (230 nm excitation; 425nm emission). Samples were tested in duplicates and the mean of thetwo results was reported.

[0414] The determined T.sub.m value of each BNJ441 batch was67.0.degree. C., as shown in Table 19. Differential scanningcalorimetry (DSC) scans were performed and calorimetry dataacquired using the Micro-Cal VP-DSC by up-scanning at a rate of75.degree. C/hr from 20.degree. C. to 95.degree. C. The Y-axis andtemperature calibrations were performed prior to sample testing.The Y-axis deflection % error was <1% and transition mid-pointswere within the accepted range of .+-.0.2.degree. C. of both28.2.degree. C. and 75.9.degree. C. Samples were scanned againstblanks of the same buffer composition and volume. DSC measures theenthalpy (.DELTA.H) of unfolding due to heat denaturation. Abiomolecule in solution is in equilibrium between the native(folded) conformation and its denatured (unfolded) state. Thetransition midpoint (T.sub.m) is the temperature where 50% of theprotein is in its native conformation and 50% is denatured. TheT.sub.m for each sample is determined by measuring .DELTA.H acrossa temperature gradient in the sample cell compared to that of theblank cell.

[0415] The affinity (KD) for BNJ441 engineering and GMP batchmaterials were 461 pM and 421 pM respectively with good fits.Binding kinetics of each BNJ441 batch are shown in Table 19.Surface plasmon resonance (Biacore 3000) was used to evaluate thebinding kinetics of anti-C5 antibody (BNJ441) to human C5.Sensorgrams not shown. The kinetics of BNJ441 to C5 were determinedusing an anti-Fc human capture method. Anti-Fc-Human (KPL #01-10-20) diluted to 0.1 mg/mL in 10 mM sodium acetate pH 5.0 wasimmobilized on two flow cells of a CM5 chip for 8 minutes by aminecoupling. The anti-C5 antibody (BNJ441) was diluted to 0.35.mu.g/mL in running buffer (HBS-EP, 0.01 M HEPES, 0.15 M NaCl, 3 mMEDTA, 0.005% P20, pH 7.4). Diluted antibody was then injected onthe other flow cell, followed by injections of C5 (0.19-6 nM) onboth flow cells. The secondary flow cell was used as a reference.The surface was regenerated each time with 20 mM HCl, 0.01% P20(100 .mu.L/min, 200 .mu.L injection). The data was processed with a1:1 Langmuir model using BIAevaluation 4.1 with `doublereferencing`.

[0416] The affinity (KD) for self association of BNJ441 engineeringand GMP batch materials were 7.1 mM and 0.27 mM respectively. SeeTable 19. Poor fits were due to low levels of binding observed forboth BNJ441 engineering and GMP batch materials, self associationand the measured affinity were below the level of limits ofdetection of the instrument. A low level of self-association isadvantageous for manufacturability and ultimately foradministration to patients. Sensorgrams not shown. Surface plasmonresonance (Biacore 3000) was used to evaluate the self-associationkinetics of anti-C5 antibody (BNJ441). The self-associationkinetics of BNJ441 were determined by direct immobilization of theantibody (BNJ441). BNJ441 was diluted to approximately 31 .mu.g/mLin 10 mM sodium acetate pH 5.0 was immobilized on one flow cell ofa CM5 chip to obtain 2000RU's by amine coupling. A secondary flowcell was used as a reference. Dilutions of anti-C5 antibody, BNJ441(1.6-50 .mu.M in running buffer, HBS-EP, 0.01 M HEPES, 0.15 M NaCl,3 mM EDTA, 0.005% P20, pH 7.4) was then injected on both flowcells. No regeneration was necessary due to poor binding. The datawas processed with a steady state affinity model usingBlAevaluation 4.1 with `double referencing`.

[0417] The physicochemical characterization of BNJ441 has beenconducted using the engineering and GMP batches and has been shownto be consistent with the amino acid sequence for the antibody. Thephysicochemical data summarized in this example encompass a rangeof properties including purity, molecular size, identity,structure, glycosylation, thermostability, kinetics andself-association, and are expected to serve as a basis for thecharacterization of BNJ441 bulk drug substance.

[0418] While the present disclosure has been described withreference to the specific embodiments thereof, it should beunderstood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from thetrue spirit and scope of the disclosure. In addition, manymodifications may be made to adapt a particular situation,material, composition of matter, process, process step or steps, tothe objective, spirit and scope of the present disclosure. All suchmodifications are intended to be within the scope of thedisclosure.

TABLE-US-00042 SEQUENCES REFERENCED IN DISCLOSURE SEQ ID NO: 1GYIFSNYWIQ SEQ ID NO: 2 EILPGSGSTEYTENFKD SEQ ID NO: 3YFFGSSPNWYFDV SEQ ID NO: 4 GASENIYGALN SEQ ID NO: 5 GATNLAD SEQ IDNO: 6 QNVLNTPLT SEQ ID NO: 7QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS SEQ ID NO: 8DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK SEQ IDNO: 9 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 10QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 11DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 12 QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS SEQ ID NO: 13ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK SEQ ID NO: 14QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV HEALH HYTQKSLSLSLGK SEQ ID NO: 15ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16:QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17GASENIYHALN SEQ ID NO: 18DIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK SEQ IDNO: 19 EILPGSGHTEYTENFKD SEQ ID NO: 20 DYKDDDDK SEQ ID NO: 21HHHHHH SEQ ID NO: 22 YPYDVPDYA SEQ ID NO: 23 GHIFSNYWIQ SEQ ID NO:24 MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 25MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 27MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 29MGWSCIILFLVATATGVHSRDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 31QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 32NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 33QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK SEQ ID NO: 34NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQYLSSRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 35QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

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