Antibodies that bind human c6 and uses thereof

Abstract

Isolated monoclonal antibodies that bind to human complement component C6 and related antibody-based compositions and molecules are described. Additionally, a therapeutic method for using said antibody is described.

Description

Technology Field

[0001] This application claims priority to U.S. provisional application No. 62 / 094,649 (filed Dec. 19, 2014), the contents of which are incorporated herein by reference. Background Technology

[0002] The complement system is part of the innate immune system that assists or "complements" antibodies and phagocytes in the elimination of pathogens in an organism. Upon activation of the system, activated cells, organic matter, or particles become targets for destruction as a result of a catalytic set of reactions and interactions. The complement system comprises a set of more than 30 plasma and membrane proteins that work together in a cascade system regulated to attack extracellular pathogens (e.g., bacteria). The complement system includes a typical pathway and an alternative pathway, both activating cascades by two distinct enzymes that converge on a common-terminal non-enzymatic pathway known as the membrane attack pathway.

[0003] The first enzymatically activated cascade, known as the typical pathway, involves several components of C1, C4, C2, C3, and C5 (listed in the order of the pathway). The initiation of the typical pathway of the complement system occurs after the binding and activation of the first complement component (C1) by both immuno- and non-immuno-activators. C1 comprises a calcium-dependent complex of components C1q, C1r, and C1s, and is activated through the binding of the C1q component. C1q contains six identical subunits, each containing three chains (chains A, B, and C). Each chain has a spherical head region connected to a collagen-like tail. The binding and activation of C1q by antigen-antibody complexes occurs through the C1q head group region. Numerous non-antibody C1q activators, including proteins, lipids, and nucleic acids, bind to and activate C1q through prominent regions of the collagen-like stem region. Subsequently, the C1qrs complex promotes the activation of complement components C4 and C2, which form the C4b2a complex acting as a C3 converting enzyme.

[0004] The second enzymatically-activated cascade, known as the alternative pathway, is a rapid, antibody-independent pathway for the activation and amplification of the complement system. The alternative pathway involves C3, Factor B, and Factor D (listed in the order of the pathway). Activation of the alternative pathway occurs when C3b, a proteolytic cleavage form of C3, binds to an activated surface agent such as a bacteriologist. Subsequently, Factor B binds to C3b and is cleaved by Factor D to produce the active enzyme, Ba. Enzyme Ba then cleaves more C3 to produce more C3b, creating extensive deposits of the C3b-Ba complex on the activated surface.

[0005] Therefore, both the typical and alternative pathways generate C3 convertases that split factor C3 into C3a and C3b. At this point, the two C3 convertases are further assembled into C5 convertases (C4b2a3b and C3b3bBb). These complexes then cleave complement component C5 into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide (170 kDa). The C5a polypeptide binds to the 7-membrane transmembrane G-protein coupled receptor associated with leukocytes and is now known to be expressed in various tissues, including hepatocytes and neurons. The C5a molecule is a major chemotactic component of the human complement system and can induce various biological responses, including leukocyte chemotaxis, smooth muscle contraction, activation of intracellular signaling pathways, neutrophil-endothelial adhesion, release of cytokines and lipid mediators, and oxidant formation.

[0006] The larger C5b fragment sequentially binds to subsequent complement cascade components C6, C7, C8, and C9 to form the C5b-9 Membrane Attack Complex ("MAC"). The lipophilic C5b-9 MAC can directly lyse red blood cells, is soluble to leukocytes, and damages tissues such as muscle, epithelial, and endothelial cells. At low lytic doses, the C5b-9 MAC can stimulate the upregulation of adhesion molecules, increase intracellular calcium, and stimulate cytokine release. Additionally, at low lytic concentrations, the C5b-9 MAC can stimulate cells such as endothelial cells and platelets without causing cell lysis. The non-lytic effects of C5a and the C5b-9 MAC are similar and interchangeable.

[0007] While the complement system plays a crucial role in maintaining health, it also has the potential to cause or contribute to disease. For example, studies have shown that inhibition of the complement cascade or depletion of complement components reduces damage from degenerative diseases of the central nervous system or experimental brain injury (Feasby, TE et al. (1987) Brain Res. 419: 97-103; Vriesendorp, FJ et al. (1995) J. Neuroimmunol. 58: 157-165; Jung, S. et al. (1995) Neurosci. Lett. 200: 167-170; Dailey, AT et al.; Leinhase, I. et al. (2006) BMS Neurosci. 14: 7: 55). In particular, rats deficient in C6 and unable to form the Membrane Attack Complex (MAC) showed neither demyelination nor axonal damage compared to rats with sufficient C6, and exhibited significantly reduced clinical scores in an antibody-mediated experimental autoimmune encephalomyelitis model for multiple sclerosis (Mead, RJ et al. (2002) J. Immunol. 168:458-465). However, the level of mononuclear cell infiltration was the same as that observed in rats with sufficient C6. Mead et al. (2002) concluded that demyelination and axonal damage occur in the presence of antibodies and require the activation of the entire complement cascade, including MAC precipitation, which can be inhibited by the depletion of C6.

[0008] Therefore, a reagent to inhibit C6 is required, and such a reagent is desirable for various therapeutic purposes.

[0009] The present invention provides an anti-C6 antibody having desirable functional characteristics for therapeutic purposes, wherein the formation of membrane attack complexes (MACs) is not only in vitro and in vivo but also exhibits very low KD (e.g., 1×10⁻⁶). -8 M, 1×10 -9M, 5×10 -10 It includes the ability to effectively inhibit the functional activity of C6, which is inhibited at M or less and very long half-lives (e.g., 40 hours or more). A panel of 38 anti-human antibodies was generated by rats immunized with purified human C6 protein. Two of the 38 were found to inhibit the formation of the membrane attack complex (MAC). A specific rat anti-human C6 antibody (referred to herein as 7E5) possessing these desired functional characteristics was produced, and a panel of humanized antibodies possessing the CDR of 7E5 was prepared, including humanized mAbs 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02. The heavy and light chain variable regions of these eight humanized mAbs, as well as the heavy chain variable region of humanized mAb 7C02 and the light chain variable region of mAb 7G08, were expressed in all 81 possible "mix and match" combinations, and all 81 pairs were found to effectively inhibit C6 functional activity. In addition, the 7E5 epitope was mapped within human C6.

[0010] Accordingly, in one aspect, the present invention relates to an isolated antibody that binds to human C6,

[0011] The above antibody exhibits at least three of the following characteristics:

[0012] (a) IC50 in hemolysis analysis of 0.5 µg / ml or less 50 Having;

[0013] (b) K measured by surface plasmon resonance D 1×10 -8 M or less;

[0014] (c) antibody-C6 binding half-life measured by surface plasmon resonance is 40 hours or more; and

[0015] (d) Cross-reacts with C6 of cynomolgus monkeys.

[0016] At least three combinations of the above characteristics are included. In one embodiment, the antibody exhibits characteristics (a), (b), and (c). In another embodiment, the antibody exhibits properties (a), (b), (c), and (d). In other embodiments, the antibody is 1 x 10⁻⁶ as determined by surface plasmon resonance. -9 M or less or 5 x 10 -10 K less than or equal to M D has

[0017] In another aspect, the present invention relates to an isolated antibody that binds to a region of human C6 containing all or part of residues 835-854 of SEQ ID NO. 52 (i.e., the antibody binds to one or more residues within residues 835-854). In another aspect, the antibody binds to an epitope comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 (i.e., the antibody binds to one or more amino acid residues within SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3). In a specific aspect, the epitope is part of a discontinuous epitope recognized by the antibody. For example, in one aspect, the antibody binds to an epitope comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, said epitope is discontinuous.

[0018] In another aspect, the present invention provides an antibody that cross-competes with an antibody comprising the heavy chain variable region shown in SEQ ID NO. 5 and the light chain variable region shown in SEQ ID NO. 10 for binding to human C6 (i.e., the heavy chain and light chain variable regions of mAb 7E5). In another aspect, the present invention provides an antibody that cross-competes with an mAb selected from the group consisting of 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 for binding to human C6.

[0019] In various embodiments, the antibody of the present invention is a human, humanized, or chimeric antibody.

[0020] In various embodiments, the antibody of the present invention comprises heavy chain CDR1, 2, and 3 sequences shown in SEQ ID NOs 6, 7, and 8, respectively, and light chain CDR1, 2, and 3 sequences shown in SEQ ID NOs 11, 12, and 13, respectively. For example, the antibody may be a humanized antibody comprising the aforementioned CDRs.

[0021] In another aspect, the present invention provides an isolated antibody that binds to human C6, comprising a heavy chain CDR3 according to SEQ ID NO. 8; and a light chain CDR3 according to SEQ ID NO. 13. The antibody may further comprise a heavy chain CDR2 according to SEQ ID NO. 7; and a light chain CDR2 according to SEQ ID NO. 12. The antibody may further comprise a heavy chain CDR1 according to SEQ ID NO. 6; and a light chain CDR1 according to SEQ ID NO. 11. Representative examples of antibodies comprising the CDRs include the following:

[0022] (a) An antibody comprising the heavy chain variable region of SEQ ID NO. 30 and the light chain variable region of SEQ ID NO. 31;

[0023] (b) an antibody comprising the heavy chain variable region of SEQ ID NO. 32 and the light chain variable region of SEQ ID NO. 33;

[0024] (c) An antibody comprising the heavy chain variable region of SEQ ID NO. 34 and the light chain variable region of SEQ ID NO. 35;

[0025] (d) an antibody comprising the heavy chain variable region of SEQ ID NO. 36 and the light chain variable region of SEQ ID NO. 37;

[0026] (e) an antibody comprising the heavy chain variable region of SEQ ID NO. 38 and the light chain variable region of SEQ ID NO. 39;

[0027] (f) an antibody comprising the heavy chain variable region of SEQ ID NO. 40 and the light chain variable region of SEQ ID NO. 41;

[0028] (g) an antibody comprising the heavy chain variable region of SEQ ID NO. 42 and the light chain variable region of SEQ ID NO. 43; and

[0029] (h) An antibody comprising the heavy chain variable region of SEQ ID NO. 44 and the light chain variable region of SEQ ID NO. 45.

[0030] In another aspect, the present invention provides an isolated antibody that binds to human C6, comprising:

[0031] (a) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46; and

[0032] (b) A light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of sequence numbers 31, 33, 35, 37, 39, 41, 43, 45 and 47.

[0033] In another embodiment, the heavy chain and light chain variable regions are 95%, 96%, 97%, 98%, or 99% identical to the aforementioned amino acid sequence. In yet another embodiment, the isolated antibody is:

[0034] (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46; and

[0035] (b) The light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45 and 47.

[0036] The present invention also includes an expression vector comprising a nucleotide sequence encoding a light chain, a heavy chain, or a variable region of the light chain and heavy chain of the antibody according to the present invention, as well as a host cell transformed by said expression vector, and a method for recombinantly expressing the antibody using the transformed host cell. In another embodiment, the antibody of the present invention is expressed as a Fab fragment. In yet another embodiment, the antibody of the present invention is expressed as a full-length antibody, such as an IgG4 isotype antibody, e.g., an IgG4 isotype antibody having an IgG4 (S228P) constant region.

[0037] Compositions comprising the antibody of the present invention, such as a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier, are also included.

[0038] In another aspect, the present invention relates to a method of using the antibody of the present invention. In one aspect, the present invention provides a method for inhibiting the formation or activity of a membrane-forming complex (MAC) in a patient, the method comprising administering the antibody of the present invention to the patient in an amount effective for inhibiting the formation or activity of the MAC in the patient. In another aspect, the present invention provides a method for treating, preventing, or reducing symptoms of a disease mediated by undesirable activity of the complement system in a patient, the method comprising administering an effective amount of the antibody of the present invention to the patient.

[0039] In another aspect, the present invention provides a method for regenerating a patient's nerve, wherein the method comprises administering a therapeutically effective amount of the antibody of the present invention to the patient. In another aspect, the present invention provides a method for promoting the recovery of a patient's damaged or degenerated nerve, wherein the method comprises administering a therapeutically effective amount of the antibody of the present invention to the patient. In another aspect, the present invention provides a method for reducing or delaying a patient's nerve degeneration, wherein the method comprises administering a therapeutically effective amount of the antibody of the present invention to the patient.

[0040] In one embodiment, the patient suffers from physical damage to a nerve, such as a traumatic injury (e.g., due to an accident), a surgical injury, or a non-traumatic injury (e.g., nerve compression). In one embodiment, the damage is to the peripheral nervous system (PNS). In another embodiment, the damage is to the central nervous system (CNS). In one embodiment, the antibody is administered at or near the site of damage.

[0041] In another embodiment, the patient suffers from an immune-mediated inflammatory disease or a progressive neurodegenerative disease. In another embodiment, the disease is acquired. In another embodiment, the disease is hereditary. In one embodiment, the disease is a chronic demyelinating neuropathy such as multiple sclerosis (MS). In another embodiment, the disease is a neurodegenerative disease such as myasthenia gravis or amyotrophic lateral sclerosis (ALS). Brief explanation of the drawing

[0042] Figure 1a is a bar graph showing the results of a hemolysis analysis using supernatants from 38 hybridomas from two different rats immunized with human C6, illustrating that supernatant 11 (which produced 7E5 mAb) has the strongest inhibitory effect. Figure 1b is a bar graph showing the results of a mannan-activated ELISA using supernatants from 38 hybridomas from two different rats immunized with human C6, illustrating that supernatant 11 (which produced 7E5 mAb) had the most potent inhibitory effect. Figure 2 is a graph showing biacore kinetics for recombinant rat 7E5 binding to human C6. Figures 3A-D are graphs showing the results of the epitope cross-blocking experiment between 27B1 mAb and 7E5 mAb, indicating that 7E5 occupies a different epitope than 27B1. Figure 4a is an alignment of the rat C6 partial amino acid sequence (Sequence No. 51) and the human C6 partial amino acid sequence (Sequence No. 50) showing the position of peptide 418. Figure 4b is a schematic diagram of the human C6 protein showing the location of peptide 418. Figure 5 is a bar graph showing that 7E5 blocks C6 in vivo in C6-deficient rats supplemented with human C6, as measured by hemolysis analysis. Rats received the indicated amounts of human C6 and antibody 7E5. Complement activity is plotted on the Y-axis, where OD 1.0 indicates maximum lysis of sensitized red blood cells and OD 0 indicates no lysis. Figure 6a shows the alignment of the heavy chain variable region amino acid sequences of rat anti-C5 7E5 mAb (SEQ No. 5) and human VH3_1 germ cell (SEQ No. 48) showing differences. The amino acid exchanges aimed at humanization are indicated below the alignment. Figure 6b shows the alignment of the amino acid sequences of the light chain variable region of rat anti-C5 7E5 mAb (SEQ No. 10) and human Vk2_5 germ cell (SEQ No. 49) showing differences. The amino acid exchanges aimed at humanization are indicated below the alignment. Figure 7a is a bar graph showing the results of a hemolytic analysis illustrating the inhibitory activity of all 81 possible combinations (shown in Figures 7a and 7b, respectively) of 9 humanized 7E5 variant VH chains and 9 humanized 7E5 variant VL chains. Figure 7b is a bar graph showing the MAC ELISA analysis results illustrating the inhibitory activity of all 81 possible combinations (shown in Figures 7a and 7b, respectively) of the humanized 7E5 variant VH chain and 9 humanized 7E5 variant VL chains. FIG. 8a is an alignment of the amino acid sequence of the 7E5 heavy chain variable region (SEQ No. 5) and the amino acid sequences of the heavy chain variable region of humanized 7E5 variants, 7C02 (SEQ No. 46), 7E11 (SEQ No. 42), 7E12 (SEQ No. 32), 7F02 (SEQ No. 44), 7F06 (SEQ No. 38), 7F11 (SEQ No. 40), 7G09 (SEQ No. 34), 8F07 (SEQ No. 36), and 8G09 (SEQ No. 30). Conserved CDR1, 2, and 3 regions are shown. FIG. 8b is an alignment of the amino acid sequence of the 7E5 light chain variable region (SEQ No. 10) and the amino acid sequences of the light chain variable region of humanized 7E5 variants, 7E11 (SEQ No. 43), 7E12 (SEQ No. 33), 7F02 (SEQ No. 45), 7F06 (SEQ No. 39), 7F11 (SEQ No. 41), 7G08 (SEQ No. 47), 7G09 (SEQ No. 35), 8F07 (SEQ No. 37), and 8G09 (SEQ No. 31). Conserved CDR1, 2, and 3 regions are shown. Figure 9 shows the affinity (Biacore) of 8 humanized F'Abs to human C6 compared to the affinity of wild-type 7E5 rat F'Abs. Figure 10 shows the results of an in vivo nerve crush experiment. Specific details for implementing the invention

[0043] The present invention provides an anti-C6 antibody exhibiting beneficial functional properties. These functional properties are, for example: (a) IC50 in a hemolytic assay of 0.5 μg / ml or less. 50 ; (b) K measured by surface plasmon resonance D 5×10 -10M or less; (c) an antibody-C6 binding half-life of 40 hours or more as measured by surface plasmon resonance; and / or (d) cross-reactivity with C6 of cynomolgus monkeys. In another embodiment, the antibody comprises specific heavy chain and light chain variable regions and / or CDR sequences. For example, nine humanized heavy chain and nine humanized light chain variable regions are provided that exhibit effective C6 inhibitory activity in a "mix and match" combination of all 81 possible chains. In yet another embodiment, the anti-C6 antibody binds to an epitope that is identical to or competes to bind to C6 with a specific anti-C6 antibody disclosed herein, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, or 7F02.

[0044] To make the present invention easier to understand, specific terms are defined first. Additional definitions are explained through the detailed description.

[0045] The term "C6" (also referred to as "Complement C6" or "Complement Component C6") refers to a component of the complement cascade that combines with components C5b, C7, C8, and C9 to form the C5b-C9 Membrane Attack Complex (MAC). The term "C6" includes any variant or isoform of naturally expressed C6. The antibody of the present invention may be specific to human CD6 and may not exhibit cross-reactivity with other species. Alternatively, the antibody of the present invention may cross-react with C6 from non-human species, such as cynomolgus monkeys. Alternatively, the antibody of the present invention may cross-react with C6 from primates, such as cynomolgus monkeys, but may not cross-react with non-primate C6, such as mouse or rat C6. C6 or any variants and isoforms thereof may be isolated from naturally expressing cells or tissues, or may be produced recombinantly using techniques known in the art. GenBank® (Accession No. NP_00110860.3) reports the amino acid sequence of human C6 as follows (Sequence No. 52):

[0046] 1 marrsvlyfi llnalinkgq acfcdhyawt qwtscsktcn sgtqsrhrqi vvdkyyqenf

[0047] 61 ceqicskqet recnwqrcpi ncllgdfgpw sdcdpciekq skvrsvlrps qfggqpctap

[0048] 121 lvafqpcips klckieeadc knkfrcdsgr ciarklecng endcgdnsde rdcgrtkavc

[0049] 181 trkynpipsv qlmgngfhfl ageprgevld nsftggickt vkssrtsnpy rvpanlenvg

[0050] 241 fevqtaeddl ktdfykdlts lghnenqqgs fssqggssfs vpifysskrs eninhnsafk

[0051] 301 qaiqashkkd ssfirihkvm kvlnfttkak dlhlsdvflk alnhlpleyn salysrifdd

[0052] 361 fgthyftsgs lggvydllyq fsseelknsg lteeeakhcv rietkkrvlf akktkvehrc

[0053] 421 ttnklsekhe gsfiqgaeks islirggrse ygaalawekg ssgleektfs ewlesvkenp

[0054] 481 avidfelapi vdlvrnipca vtkrnnlrka lqeyaakfdp cqcapcpnng rptlsgtecl

[0055] 541 cvcqsgtyge ncekqspdyk snavdgqwgc wsswstcdat ykrsrtrecn npapqrggkr

[0056] 601 cegekrqeed ctfsimenng qpcinddeem kevdlpeiea dsgcpqpvpp engfirnekq

[0057] 661 lylvgedvei scltgfetvg yqyfrclpdg twrqgdvecq rtecikpvvq evltitpfqr

[0058] 721 lyrigesiel tcpkgfvvag psrytcqgns wtppisnslt cekdtltklk ghcqlgqkqs

[0059] 781 gsecicmspe edcshhsedl cvfdtdsndy ftspackfla ekclnnqqlh flhigscqdg

[0060] 841 rqlewglert rlssnstkke scgydtcydw ekcsastskc vcllppqcfk ggnqlycvkm

[0061] 901 gsstsektln icevgtirca nrkmeilhpg kcla

[0062] The term “antibody” as used herein comprises the whole antibody and any antigen-binding fragment (i.e., “antigen-binding portion”) or a single chain thereof. In one preferred embodiment, “antibody” refers to a glycoprotein comprising two or more heavy chains (H) and two light chains (L) or their antigen-binding portions interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions may be further subdivided into hypervariability regions interspersed with regions called framework regions (FR), which are more conserved than regions called complementarity determining regions (CDR). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, and has the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding sites that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., primary cells) and the first component (C1q) of the classical complement system.

[0063] As used herein, the term “antigen-binding site” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., human C6). Such “fragments” have, for example, a length of about 8 to about 1500 amino acids, suitably about 8 to about 745 amino acids, suitably about 8 to about 300 amino acids, about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term “antigen-binding site” of an antibody include (i) a Fab fragment which is a monovalent fragment composed of VL, VH, CL, and CH1 domains; and (ii) a F(ab’)2 fragment which is a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region. (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); and (vi) a separate complementarity determining region (CDR) or (vii) a combination of two or more separate CDRs that can be optionally combined by a synthetic linker. In addition, even if the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined by a recombinant method using a synthetic linker, and the synthetic linker enables them to be prepared as a single protein chain (known as single-strand Fv) in which the VL and VH domains are paired to form a monovalent molecule; e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) See.These single-strand antibodies are also intended to be included within the term "antigen-binding site" of the antibody. These antibody fragments are obtained using conventional techniques known to a person skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding sites may be generated by recombinant DNA technology, or by enzymatic or chemical cleavage of complete immunoglobulin.

[0064] "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods involving the fusion of hybridomas or the linkage of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0065] As used herein, the term “monoclonal antibody” refers to an antibody that exhibits single-binding specificity and affinity for a specific epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody that exhibits single-binding specificity and has a variable and arbitrary constant region derived from a human germline immunoglobulin sequence. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse having a genome containing a human heavy chain transgenic gene and a light chain transgenic gene fused to an immortalized cell.

[0066] As used herein, the term “recombinant antibody” comprises: (a) antibodies isolated from animals (e.g., mice) to which a human immunoglobulin gene has been transplanted or a chromosome has been transplanted, or from hybridomas produced therefrom; (b) antibodies isolated from host cells transformed to express antibodies, e.g., transfectomas; (c) antibodies isolated from a library of recombinant, combined human or humanized antibodies; and (d) chimeric, humanized, and human antibodies produced, expressed, generated, or isolated by recombinant means such as any other means including splicing of a human immunoglobulin gene sequence into another DNA sequence.

[0067] The term “humanized antibody” refers to an antibody having a framework region from a human germline sequence and a CDR from a non-human species (e.g., mouse, rat, rabbit), and includes, for example, a human framework region and / or CDR that has undergone mutagenesis to specify a particular site to optimize binding. An exemplary technique for the preparation of a humanized anti-C6 antibody is described in Example 8.

[0068] The term "human antibody" includes antibodies having variable and constant regions (if present) of a human germline immunoglobulin sequence. The human antibodies of the present invention may comprise amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis caused by somatic mutations in vitro or in vivo) (Lonberg, N. et al. (1994) Nature 368 (6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci 764: 536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, are transplanted onto human framework sequences (i.e., humanized antibodies).

[0069] As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to human C6 is substantially free of antibodies that specifically bind to antigens other than human C6). However, an isolated antibody that specifically binds to an epitope may be cross-reactive with other C6 proteins from different species. However, the antibody always binds to human C6 whenever possible. Additionally, the isolated antibody is typically substantially free of other cellular material and / or chemical substances. In an embodiment of the invention, a combination of "isolated" antibodies having different C6 specificities is combined in a well-defined composition.

[0070] The terms "epitope" or "antigenic determinant" refer to a region on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from either adjacent amino acids or non-adjacent amino acids placed side-by-side by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are generally lost upon treatment with a denaturing solvent. Epitopes generally contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a distinct spatial structure. Methods for determining which epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, wherein overlapping or adjacent peptides from C6 are tested for reactivity with a given anti-C6 antibody. Methods for determining the spatial structure of an epitope include techniques in the art and those described herein, e.g., X-ray crystallography and two-dimensional nuclear magnetic resonance (e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0071] The term "discontinuous epitope" refers to an epitope composed of non-contiguous amino acids. For example, the epitope may include residues from multiple regions of human C6 that, when structurally folded, are linked (closely) so that an antibody binds to one or more residues within each region.

[0072] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0073] With respect to two or more antibodies, the term “binds to the same epitope” means that the antibodies compete for binding to the antigen and bind to a continuous or discontinuous segment of amino acids that is identical, overlaps, or encompasses. A person skilled in the art understands that the phrase “binds to the same epitope” does not mean that the antibodies bind to exactly the same amino acids. The exact amino acids to which the antibodies bind may differ. For example, the first antibody may bind to an amino acid segment that is completely surrounded by an amino acid segment bound by the second antibody. In another example, the first antibody binds to one or more amino acid segments that significantly overlap with one or more segments bound by the second antibody. For the purposes of this invention, such antibodies are considered to “bind to the same epitope.”

[0074] As used herein, the terms “specific binding,” “selective binding,” “selective binding,” and “binds specifically” refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is about 10 when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 device using recombinant human C6 as the analyte and the antibody as the ligand. -7 Less than M, about 10 -8 Less than M, about 10 -9 M or about 10 -10 M or even a lower equilibrium dissociation constant (K D It binds to ) and binds with an affinity at least twice as great as its affinity for binding to non-specific antigens (e.g., BSA, casein) other than a predetermined antigen or closely related antigen. The phrases “antibody recognizing an antigen” and “antibody specific to an antigen” are used interchangeably with the term “antibody specifically binding to an antigen” in this specification.

[0075] The term "K" used in this document D" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibody of the present invention is approximately 10 when determined by surface plasmon resonance (SPR) technology in a BIACORE 2000 device using recombinant human C6 as the analyte and the antibody as the ligand. -8 M or less, or 10 -9 M or less, or 10 -10 Dissociation equilibrium constant less than or equal to M (K D Combines with C6.

[0076] The term "kd" as used herein refers to the off-rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0077] The term "ka" as used herein refers to the on rate constant for the binding of antibodies and antigens.

[0078] The term "IC" used in this document 50 "IQ" refers to the concentration of an antibody or its antigen-binding portion required in an in vivo or in vitro assay to inhibit a given biological response by half. In other words, it is the minimum (50%) inhibition concentration (IC) of the antibody or its antigen-binding portion.

[0079] As used herein, "isotype" refers to a class of antibody (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. In one embodiment, the human monoclonal antibody of the present invention is an IgG1 isotype. In another embodiment, the human monoclonal antibody of the present invention is an IgG2 isotype. In another embodiment, the human monoclonal antibody of the present invention is an IgG4 isotype. In another embodiment, the human monoclonal antibody of the present invention is an IgG4 (S228P) isotype (i.e., an IgG4 isotype having a proline substitution of a wild-type serine residue at amino acid position 228).

[0080] The term "binding to immobilized C6" refers to the ability of the human antibody of the present invention to bind to C6, for example, to bind to C6 expressed on a cell surface or attached to a solid support.

[0081] As used herein, the term “cross-reactivity” refers to the ability of the antibody of the present invention to bind to C6 of other species. For example, the antibody of the present invention that binds to human C6 may also bind to other species of C6, such as cynomolgus monkeys. As used herein, cross-reactivity is measured by detecting specific reactivity with a purified antigen in binding assays (e.g., SPR, ELISA), binding to cells physiologically expressing C6, or otherwise functionally interacting. Methods for determining cross-reactivity include standard binding assays described herein, e.g., Biacore TM Biacore using the 2000 SPR device (Biacore AB, Uppsala, Sweden) or flow cytometry technology TM Includes surface plasmon resonance (SPR) analysis.

[0082] As used herein, the “glycosylation pattern” is defined as a pattern of carbohydrate units covalently bonded to a protein, more specifically, an immunoglobulin protein. When a person skilled in the art recognizes that the glycosylation pattern of a heterologous antibody is more similar to the glycosylation pattern in a non-human transgenic animal species than in the species from which the CH gene of the transgenic gene originated, the glycosylation pattern of the heterologous antibody may be characterized as substantially similar to the glycosylation pattern that occurs naturally on antibodies produced by a non-human transgenic animal species.

[0083] The term “naturally occurring” as used herein to apply to objects means the fact that objects can be found in nature. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring.

[0084] As used herein, the term “rearranged” refers to an arrangement of heavy or light chain immunoglobulin loci located immediately adjacent to a DJ or J segment in a form where the V segment essentially encodes a complete VH or VL domain, respectively. The rearranged immunoglobulin loci can be identified by comparison with germline DNA; the rearranged loci will have at least one recombinant heptamer / nonamer homology element.

[0085] With respect to the V segment, the terms “unrearranged” or “germline arrangement” as used herein refer to an arrangement in which the V segment is not rearranged to be immediately adjacent to the D or J segment.

[0086] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0087] With respect to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to C6, the term “isolated nucleic acid molecule” as used herein is intended to refer to a nucleic acid molecule in which the nucleotide sequence encoding the antibody or antibody portion does not have another nucleotide sequence encoding the antibody or does not contain an antibody portion binding to an antigen other than C6, and other sequences may naturally encounter nucleic acids in human genomic DNA. For example, SEQ ID NOs 14, 16, 18, 20, 22, 24, 26, and 28 correspond to nucleotide sequences encoding the heavy chain (VH) variable region of an anti-C6 monoclonal antibody, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02, respectively. Sequence numbers 15, 17, 19, 21, 23, 25, 27 and 29 correspond to nucleotide sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02, respectively, encoding the variable region of the light chain (VH) of the anti-C6 monoclonal antibody.

[0088] The present invention also comprises "conservative sequence modifications" of the sequences described in SEQ ID NOs. 4 to 47, namely modifications of nucleotide and amino acid sequences that do not exclude binding to an antigen of an antibody encoded by a nucleotide sequence or an antibody containing an amino acid sequence. Such conservative sequence modifications include additions and deletions of nucleotides and amino acids, as well as conservative nucleotide and amino acid substitutions. For example, modifications to SEQ ID NOs. 4 to 47 may be introduced by standard techniques known in the art, such as site-specific mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the substitution of an amino acid residue with an amino acid residue having a similar side chain. A series of amino acid residues having a similar side chain is defined in the art. This series includes amino acids with basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; non-charged polar side chains such as vitamin C (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., phenylalanine, tryptophan, histidine). Accordingly, non-essential amino acid residues predicted in human anti-C6 antibodies are preferably replaced with other amino acid residues from the same side chain series. Methods for identifying nucleotide and amino acid conservative substitutions that do not remove antigen binding are well known in the art (e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999) and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0089] In addition, in another embodiment, mutations may be randomly introduced along all or part of the anti-C6 antibody coding sequence, such as saturation mutagenesis, and the resulting modified anti-C6 antibody may be screened for binding activity.

[0090] In the case of nucleic acids, the term "substantial homology" indicates that when two nucleic acids or their specified sequences are optimally aligned and compared, they are identical in at least about 80% of the nucleotides, typically at least about 90% to 95%, and more preferably about 98% to 99.5%, with appropriate nucleotide insertions or deletions. Additionally, substantial homology exists when a segment is hybridized to a segment complementary to a strand under selective hybridization conditions.

[0091] The homology ratio between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of homology between two sequences can be achieved using a mathematical algorithm as described in the following non-limiting examples.

[0092] Identity between two nucleotide sequences can be determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, and 80 and length weights of 1, 2, 3, 4, 5, or 6. Identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) integrated into the ALIGN program (version 2.0) using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48) : 444-453 (1970)) algorithm integrated into the GAP program using the Blossum 62 matrix or PAM250 matrix of the GCG software package (available at http: / / www.gcg.com) and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6.

[0093] The nucleic acid and protein sequences of the present invention may also be used as "query sequences" to perform a search on public databases to identify related sequences, for example. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. A BLAST nucleotide search may be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search may be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences corresponding to the protein molecule of the present invention. To obtain gap alignment for comparison purposes, Gapped BLAST may be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. Refer to http: / / www.ncbi.nlm.nih.gov.

[0094] The nucleic acid or protein of the present invention may exist in whole cells, cell lysates, or in a partially purified or substantially pure form. By standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and those widely known in the art, the nucleic acid or protein is “separated” or “substantially purified” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins.

[0095] The nucleic acid compositions of the present invention may often be mutated from the original sequences (excluding modification restriction sites, etc.) from cDNA, genomes, or mixtures thereof, according to standard techniques for providing gene sequences. In the case of coding sequences, these mutations may affect the amino acid sequence as desired. In particular, DNA sequences derived from or substantially homologous to the original V, D, J, constants, switches, and other such sequences described herein are considered ("derived from" indicates that the sequence is identical or modified from another sequence).

[0096] Nucleic acids are "operably linked" when they are placed in a functional relationship with other nucleic acid sequences. For example, a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence. In the context of transcriptional regulatory sequences, operably linked means that the linked DNA sequence is continuous and located in a continuous reading frame, which is necessary to link two protein-coding regions. In the case of switch sequences, operably linked indicates that the sequence is capable of performing switch recombination.

[0097] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying other nucleic acids to which it is connected. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be connected. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Certain vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors of bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Additionally, certain vectors can direct the expression of operably connected genes. Such vectors are referred herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors of utility in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably because plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors that perform equivalent functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0098] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. This term should be understood to mean not only specific target cells but also the offspring of such cells. Because specific modifications may occur in the next generation due to mutations or environmental influences, these offspring are not actually identical to the parent cells, but are included within the scope of the term "host cell" as used herein.

[0099] As used herein, the term “linked” means the combination of two or more molecules. The combination may be covalent or non-covalent. The combination may also be genetic (i.e., recombinantly fused). Such combinations can be achieved using various recognized techniques, such as chemical conjugation and recombinant protein production.

[0100] As used herein, the terms “inhibition” or “blockade” (e.g., referring to the inhibition / blockade of MAC formation by an anti-C6 antibody) are used interchangeably and include partial and complete inhibition / blockade. Inhibition / blockade of C6 preferably reduces or alters the normal level or type of activity that occurs when C6 is not blocked or inhibited. Inhibition and blocking are also intended to include a measurable reduction in the binding or activity of C6 upon contact with the anti-C6 antibody compared to C6 not in contact with the anti-C6 antibody, for example, inhibiting the binding or activity of C6 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the anti-C6 antibody inhibits the binding or activity of C6 by at least about 70%. In another embodiment, the anti-C6 antibody inhibits the binding or activity of C6 by at least 80%.

[0101] As used herein, the terms “treat,” “treating,” and “treatment” refer to the treatment or preventive measures described herein. A method of “treatment” means administering the antibody of the present invention to a subject requiring treatment, for example, a patient requiring such treatment.

[0102] The terms "effective dose" or "effective dosage" are defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutic effective dose" is defined as an amount sufficient to treat, or at least partially suppress, the disease and its complications in patients already suffering from the disease. The effective dose for such use will depend on the severity of the disorder being treated and the general state of the patient's own immune system.

[0103] The term "patient" includes human and other mammalian individuals receiving preventive or therapeutic treatment.

[0104] As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention may be used to treat an individual with an immune disorder. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0105] Various aspects of the present invention are described in more detail in the following details.

[0106] I. Production of antibodies against C6

[0107] The present invention comprises antibodies that bind to C6 (e.g., human C6), e.g., humanized antibodies. Exemplary monoclonal antibodies that bind to C6 include 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02, the heavy chain variable regions thereof represented by SEQ ID NOs 5, 30, 32, 34, 36, 38, 40, 42 and 44, respectively, and light chain variable regions thereof represented by SEQ ID NOs 10, 31, 33, 35, 37, 39, 41, 43 and 45, respectively. The heavy chains CDR1, 2, and 3 of these antibodies appear in sequence numbers 6, 7, and 8, respectively, while the light chains CDR1, 2, and 3 of these antibodies appear in sequence numbers 11, 12, and 13, respectively.

[0108] The monoclonal antibody of the present invention can be produced using various known techniques, such as the standard somatic hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While the somatic hybridization procedure is preferred, in principle, other techniques for producing monoclonal antibodies, such as viral or oncogenic modification of B lymphocytes, phage display techniques using a library of human antibody genes, and humanization techniques such as those described in Example 6 may also be used.

[0109] Accordingly, in one embodiment, a hybridoma method is used to produce antibodies that bind to human C6. In this method, rats, mice, or other suitable host animals may be immunized with a suitable antigen to produce antibodies that specifically bind to the antigen used for immunization, or to induce lymphocytes capable of producing such antibodies. As described in Example 1, a particularly suitable host animal for growing anti-human C6 antibodies is a rat deficient in C6 (C6- / - rats), and thus immunization with human C6 will be considered entirely "foreign." The supernatant from the immunized host animal may be tested with a suitable assay for detecting anti-CD activity, such as a hemolytic assay or MAC ELISA as described in detail in Example 1, to identify host animals expressing antibodies with C6 inhibitory activity.

[0110] Lymphocytes from selected host animals can be fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). An exemplary fusion partner is Y3-Ag1.2.3 cells, but other myeloma cells known in the art, such as SP2 / 0-Ag8.653 cells (ATCC, CRL 1580), are also suitable. The culture medium in which the hybridoma cells are grown is analyzed for the production of monoclonal antibodies against the antigen, along with hemolysis assays and / or MAC ELISA. Once hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Culture media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells can be grown in vivo as ascites tumors in animals. Monoclonal antibodies secreted by a subclone can be separated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification processes such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. An exemplary and non-limiting example of the preparation of a hybridoma secreting an anti-C6 antibody is described in detail in Example 1.

[0111] Non-human monoclonal antibodies, such as rat or mouse antibodies, can be humanized using methods known in the art. For example, as described in detail in Example 6, a rat anti-human C6 mAb can be humanized using the approach described in the literature Hwang, WY et al. (2205) Methods 36:35-42. This approach is based on the principle that if non-human and human antibodies have similarly structured CDRs, the human framework also supports the non-human CDR while maintaining affinity. Thus, in this method, a human framework sequence is selected from a set of human germline genes based on the structural similarity of the human CDR to the antibody to be humanized. A phage display library of Fab variant sequences containing deviated FR residues is generated. After affinity-driven selection, individual clones are screened for binding, off-rate, and sequence human identity, and homology is determined. Other approaches and methodologies for CDR transplantation and humanization well established in the industry can also be used to generate the humanized anti-C6 antibody of the present invention.

[0112] In another embodiment, the antibody and antibody portion binding to human C6 are, for example, McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); and Hoet et al. (2005) Nature Biotechnology 23, 344-348; US Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; US Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; US Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and US Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths can be isolated from antibody phage libraries generated using the disclosed techniques. Additionally, in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)) can be used as a strategy for constructing combinatorial infections and large phage libraries, as well as for producing high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)).

[0113] In one embodiment, antibodies binding to human C6 are produced using the phage display technology disclosed in Hoet et al., supra. This technology involves generating a human Fab library having synthetic diversity in heavy chain CDRs, which has a unique combination of immunoglobulin sequences isolated from human donors. The library is screened for Fabs binding to human C6.

[0114] In one embodiment, antibodies induced against C6 are generated using transgenic or chromosomal transplant mice carrying a part of the human immune system rather than a mouse system. In one embodiment, the present invention uses transgenic mice referred to as "HuMAb mice" containing human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with target mutations that inactivate endogenous μ and γ chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunity, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci 764:536-546). The preparation of HuMAb mice is described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al (1993) International Immunology 5: 647-656; Tuaillon et al (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al (1993) Nature Genetics 4:117-123; Chen, J. et al (1993) EMBO J.12: 821-830; Tuaillon 외 (1994) J. Immunol. 152:2912-2920; Lonberg 외, (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L. 외 (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D. 외 (1996) Nature Biotechnology 14: 845-851. See further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharm International; U.S. Patent No. 5,545,807 to Surani 외; International Publication Nos. WO 98 / 24884, published on June 11, 1998; WO 94 / 25585, published November 10, 1994; WO 93 / 1227, published June 24, 1993; WO 92 / 22645, published December 23, 1992; WO 92 / 03918, published March 19, 1992).

[0115] In another aspect, the human antibody of the present invention may be produced using a mouse having a human immunoglobulin sequence on a transgene and a transchromosome, such as a mouse possessing a human heavy chain transgene and a human light chain transchromosome. Such a mouse, referred to as a "KM mouse" in the art, is described in detail in PCT Publication WO 02 / 43478 Ishida et al.

[0116] In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-C6 antibody of the present invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) may be used; such mice are described, for example, in U.S. Patents No. 5,939,598; 6,075,181; 6,114,598; and U.S. Patents No. 6,150,584 and 6,162,963 of Kucherlapati et al.

[0117] Furthermore, alternative chromosomal transplant animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-C6 antibody of the present invention. For example, mice carrying both human heavy-chain transchromosomes and human light-chain transchromosomes may be referred to in the art as "TC mice"; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, dairy cows possessing human heavy-chain and light-chain transchromosomes are described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894) and can be used to produce the anti-C6 antibody of the present invention.

[0118] Additional mouse systems described in the art for producing human antibodies may also be applied to induce the anti-C6 antibody of the present invention, which comprises (i) VelocImmune® mice (Regeneron Pharmaceuticals, Inc.) in which the endogenous mouse chimeric antibody (human V / mouse C) is grown in mice in which the human heavy and light chain variable regions are replaced by homologous recombination with the human heavy and light chain variable regions operably linked to the endogenous mouse constant region, and subsequently converted into a fully human antibody using standard recombinant DNA technology; and (ii) MeMo® mice (Merus Biopharmaceuticals, Inc.) in which the mouse comprises, but is not limited to, a human heavy chain variable region that has not been relocated, but contains a relocated human common light chain variable region. The use of such mice and their use in producing antibodies is described, for example, in WO 2009 / 15777, US 2010 / 0069614, WO 2011 / 072204, WO 2011 / 097603, WO 2011 / 163311, WO 2011 / 163314, 148873, US 2012 / 0070861 and US 2012 / 0073004.

[0119] The human monoclonal antibody of the present invention may also be prepared using SCID mice that have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patents No. 5,476,996 and No. 5,698,767 in Wilson et al.

[0120] Generation of transfectomas that produce monoclonal antibodies against C6

[0121] The antibodies of the present invention can also be produced in host cell transspectomas using, for example, a combination of recombinant DNA technology and gene transformation methods widely known in the art (Morrison, S. (1985) Science 229: 1202). Exemplary embodiments of the recombinant expression of anti-C6 antibodies are further described in Example 5 (pMQR expression vector used in HEK-293 host cells), Example 6 (Fab expression using pCB4 expression vector in E. coli host cells), and Example 7 (CHO cell expression).

[0122] Additionally, in one embodiment, the gene of interest (e.g., antibody gene) may be linked into an expression vector, such as a eukaryotic expression plasmid, as used by GS gene expression systems disclosed in WO 87 / 04462, WO 89 / 4462, WO 89 / 0136 and EP338,841 or other expression systems widely known in the art, including WO 01036 and EP 338,841 or other expression systems widely known in the art. The purified plasmid having the cloned antibody gene may be introduced into eukaryotic host cells, such as CHO- cells or NSO- cells, or alternatively into other eukaryotic cells, such as plant-derived cells, fungal or yeast cells. The method used to introduce these genes may be a method described in the art, such as electroporation, lipofectine, lipofectamine, or others. After introducing these antibody genes into host cells, cells expressing the antibody may be identified and selected. These cells exhibit transspectomas, and their expression levels can be amplified, and antibody production can be increased. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0123] Alternatively, these cloned antibody genes can be expressed in other expression systems, such as E. coli or complete organisms, or synthetically expressed.

[0124] Cross-competitive and identical epitope-binding antibodies

[0125] As described in detail in Example 3, the epitope to which the 7E5 antibody binds is mapped by alanine scanning mutagenesis to residues within the region of Human C6 corresponding to amino acids 835-854 of SEQ ID NO. 52 (Human C6). Specific peptides shown in SEQ ID NOs. 1, 2, and 3 were also shown to contain residues that form part of the epitope to which 7E5 binds. Accordingly, in one embodiment, the present invention provides an antibody that binds to an epitope of Human C6 comprising all or part of residues 835-854 of SEQ ID NO. 52. In another embodiment, the present invention provides an antibody that binds to an epitope of Human C6 comprising all or part of residues 835-854 of SEQ ID NO. 52, said epitope being discontinuous. In another aspect, the present invention provides an antibody that binds to an epitope of human C6 comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, and 3. In another aspect, the present invention provides an antibody that binds to an epitope of human C6 comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, and 3, said epitope being discontinuous. In another aspect, the present invention provides an antibody that cross-competes to bind to human C6 with an antibody comprising a heavy chain variable region shown in SEQ ID NO. 5 and a light chain variable region shown in SEQ ID NO. 10 (VH and VL sequences of 7E5). In another aspect, the antibody of the present invention cross-competes to bind to C6 with other anti-C6 antibodies such as 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, or 7F02 described herein.

[0126] These competitive antibodies can be identified based on the ability of one or more of mAbs 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 to competitively inhibit binding to C6 in a standard C6 binding assay. An exemplary test for cross-competition for binding to C6 is the epitope sandwich ELISA described in detail in Example 3. Additionally, antibodies that recognize the same epitope or compete for binding can be identified using other conventional techniques. These techniques include, for example, immunoassays that indicate the ability of one antibody to block the binding of another antibody to a target antigen, namely competitive binding assays. Competitive binding is determined in an assay where the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as C6. Several types of competitive binding assays are known, for example: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect immunoassay (EIA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase directly labeled assay, solid-phase directly labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeled RIA using I-125 label (see Morel et al., Mol. Immunol. 25 (1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).Typically, this analysis involves the use of cells containing either a purified antigen bound to a solid surface or unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Generally, when the competitive antibody is present in excess, the antibody will inhibit the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, and 70-75% or more.

[0127] Accordingly, antibodies binding to an epitope on C6 comprising all or part of the epitope recognized by the specific antibody described herein are also included in the present invention (e.g., identical or overlapping regions or regions between regions).

[0128] In one embodiment, an antibody that competes for binding to C6 and / or binds to the same epitope on human C6 is a humanized antibody. Such humanized monoclonal antibodies can be prepared and isolated, for example, as described in Example 6.

[0129] Other techniques for determining the epitopes to which antibodies bind include epitope mapping methods, such as X-ray analysis of antigen-antibody complexes that provide atomic resolution of the epitopes. Other methods monitor the binding of antibodies to antigen fragments or variants of mutated antigens, where the loss of binding due to modifications in amino acid residues within the antigen sequence is often considered an indicator of the epitope component. Additionally, computational combinatorial methods for epitope mapping may also be used. These methods rely on the ability of the antibody of interest to separate specific short peptides by affinity from a combinatorial phage display peptide library. The peptide is considered a lead for defining the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map structurally discontinuous antigen epitopes.

[0130] Once a single typical anti-C6 mAb having the desired properties described herein is isolated, it is simple to generate another mAb having the same properties, e.g., the same epitope, by using methods known in the art. For example, mice or rats can be immunized with a C6 hybridoma as described herein, and the resulting monoclonal antibody screens for the ability to compete with the typical mAb for binding to C6. Rats or mice can be immunized with a smaller fragment of C6 containing the epitope to which the typical mAb binds. The epitope can be limited, for example, by screening for binding to a series of overlapping peptides across C6. Alternatively, the method of Jespers et al., Biotechnology 12: 899, 1994 can be used to induce the selection of an mAb having the same epitope and properties similar to the corresponding typical mAb. Using phage display, the heavy chain of the first typical antibody is paired with a repertoire of (preferably human) light chains for selecting a C6-binding mAb, and the new light chain is paired with a repertoire of heavy chains for selecting a (preferably human) C6-binding mAb having the same epitope as the (preferably human) typical mAb. Alternatively, variants of the typical mAb can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of the antibody.

[0131] For example, epitope mapping as described in the literature Champe et al. (1995) J. Biol. Chem. 270: 1388-1394 can be performed to determine whether an antibody binds to an epitope of interest. "Alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science 244: 1081-1085, or other forms of point mutagenesis of human C6 amino acid residues, can also be used to determine functional epitopes for the anti-C6 antibody of the present invention. However, since mutagenesis studies may reveal amino acid residues that are important to the overall three-dimensional structure of C6 but are not directly involved in antibody-antigen contact, other methods may be required to confirm the functional epitopes determined using this method.

[0132] Epitopes bound by specific antibodies can also be determined by evaluating the binding of antibodies to peptides containing fragments of human C6. A series of overlapping peptides containing the sequence of C6 can be synthesized, screened for binding, or present on a chip, for example, in direct ELISA or competitive ELISA (where the ability of the peptide to prevent the binding of antibodies to C6 bound to wells of a microtiter plate is evaluated). These peptide screening methods cannot detect some discontinuous functional epitopes, namely functional epitopes containing non-contiguous amino acid residues along the primary sequence of the C6 polypeptide chain.

[0133] The epitope bound to the antibody of the present invention can also be determined by structural methods such as X-ray crystal structure determination (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR measurement of the HD exchange rate of unstable amide hydrogens at C6 when bound to a complex with a free antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347), Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

[0134] In relation to X-ray crystallography, crystallization may be performed using any method known in the art (e.g., Giege et al. (1994) Acta Crystallogr. D50: 339-350; McPherson (1990) Eur.J.Biochem.189:1-23), including microbatch (e.g., Chayen (1997) Structure 5: 1269-1274), hanging-drop vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251: 6300-6303), seeding, and dialysis. It is preferable to use a protein preparation having a concentration of at least about 1 mg / mL, preferably about 10 mg / mL to about 20 mg / mL. Crystallization occurs at about ± 10% to 30% (w / v) in a precipitating agent solution containing 1,000-20,000 polyethylene glycol (PEG; average molecular weight in the range of about 1,000 to about 20,000 Da), preferably about 5,000 to about 7,000 Da, more preferably about 6,000 Da. It may be desirable to include a protein stabilizer, for example, glycerol at a concentration in the range of about 0.5% to about 20%. Suitable salts such as sodium chloride, lithium chloride, or sodium citrate may also be present in the precipitating agent solution, preferably at a concentration in the range of about 1 mM to about 1,000 mM. The precipitating agent is preferably buffered to a pH of about 3.0 to about 5.0, preferably about 4.0. Specific buffers useful for precipitating agent solutions can vary and are well known in the field (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York).Examples of useful buffers include, but are not limited to, HEPES, Tris, MES, and acetate. Crystals can be grown over a wide range of temperatures, such as 2°C, 4°C, 8°C, and 26°C.

[0135] Antibody: Antigen determination can be studied using well-known X-ray diffraction techniques and can be defined using computer software such as X-PLOR (distributed by Yale University, 1992, Molecular Simulations, Inc., e.g. Blundell & Johnson (1985) Meth. Enzymol. 114&115, HW Wyckoff et al., eds., Academic Press; US Patent Application Publication No. 2004 / 0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49: 37-60, Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosure of which is incorporated herein by reference in its entirety.

[0136] Use of partial antibody sequences to express whole antibodies

[0137] In a specific embodiment, the anti-C6 antibody of the present invention comprises the heavy chain CDR3 shown in SEQ ID NO. 8; and the light chain CDR3 shown in SEQ ID NO. 13. The antibody further comprises the heavy chain CDR2 shown in SEQ ID NO. 7; and the light chain CDR2 shown in SEQ ID NO. 12. The antibody still further comprises the heavy chain CDR1 shown in SEQ ID NO. 6; and the light chain CDR1 shown in SEQ ID NO. 11. An exemplary antibody of the present invention utilizing the said CDRs comprises the following:

[0138] (a) An antibody comprising the heavy chain variable region of SEQ ID NO. 30 and the light chain variable region of SEQ ID NO. 31;

[0139] (b) an antibody comprising the heavy chain variable region of SEQ ID NO. 32 and the light chain variable region of SEQ ID NO. 33;

[0140] (c) An antibody comprising the heavy chain variable region of SEQ ID NO. 34 and the light chain variable region of SEQ ID NO. 35;

[0141] (d) an antibody comprising the heavy chain variable region of SEQ ID NO. 36 and the light chain variable region of SEQ ID NO. 37;

[0142] (e) an antibody comprising the heavy chain variable region of SEQ ID NO. 38 and the heavy chain variable region of SEQ ID NO. 39;

[0143] (f) an antibody comprising the heavy chain variable region of SEQ ID NO. 40 and the light chain variable region of SEQ ID NO. 41;

[0144] (g) an antibody comprising the heavy chain variable region of SEQ ID NO. 42 and the heavy chain variable region of SEQ ID NO. 43; and

[0145] (h) An antibody comprising the heavy chain variable region of SEQ ID NO. 44 and the light chain variable region of SEQ ID NO. 45.

[0146] Antibodies interact with target antigens primarily through amino acid residues located in six heavy and light chain complementarity determining regions (CDRs). For this reason, amino acid sequences within CDRs vary more among individual antibodies than sequences outside of CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the characteristics of specific naturally occurring antibodies by constructing an expression vector containing CDR sequences from specifically occurring antibodies grafted onto the backbone sequences of different antibodies of different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332: 323-327; Jones, P. et al., 1986, Nature 321: 522-525; and Queen, C. et al., 1989, Proc. Natl. Acad., USA 86: 10029-10033). These framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences because they do not contain the fully assembled variable genes formed by V(D)J binding during B cell maturation. Germline gene sequences will also differ individually from the sequences of second-repertoire antibodies that are evenly affinity across the variable region. For example, somatic mutations are relatively rare in the amino-terminal portion of the framework region. For instance, somatic mutations are relatively rare in the amino-terminal portion of Framework Region 1 and the carboxy-terminal portion of Framework Region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a specific antibody to regenerate an intact recombinant antibody with binding properties similar to those of the original antibody (see PCT / US99 / 05535). For this purpose, partial heavy and light chain sequences across the CDR region are generally sufficient.Partial sequences are used to determine the germline variable and binding gene segments that contribute to the recombinant antibody variable gene. Subsequently, the germline sequences are used to fill in the missing portions of the variable region. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the characteristics of the final antibody. To add missing sequences, the cloned cDNA sequence can be combined with synthetic oligonucleotides via ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short overlapping oligonucleotides and combined via PCR amplification to generate a full synthetic variable region clone. This process offers specific advantages, such as the ability to remove or include specific restriction enzymes or optimize specific codons.

[0147] The nucleotide sequences of heavy and light chain transcriptions derived from hybridomas are used to design a set of overlapping synthetic oligonucleotides to generate a synthetic V sequence having the same amino acid coding capacity as the natural sequence. The synthetic heavy and kappa chain sequences may differ from the natural sequence in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; the optimal translation initiation site is incorporated according to Kozak's rule (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and, the Hind III site is manipulated upstream of the translation initiation site.

[0148] For both heavy and light chain variable regions, the optimized coding and corresponding non-coding strand sequences are decomposed into 30-50 nucleotides at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into a set of overlapping double strands spanning a segment of 150-400 nucleotides. The pool is then used as a template to produce a PCR amplification product of 150-400 nucleotides. Typically, a set of single variable region oligonucleotides will be decomposed into two pools that are amplified individually to produce two overlapping PCR products. These overlapping products are then combined via PCR amplification to form a complete variable region. Additionally, to generate a fragment that can be easily amplified into the expression vector construct, it may be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the κ light chain or the AgeI site in the case of the gamma heavy chain) in the PCR amplification.

[0149] The reconstructed heavy and light chain variable regions are combined in the order of a cloned promoter, leader sequence, translation initiation, leader sequence, constant region, 3' non-translation, polyadenylation, and transcription termination to form an expression vector construct. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, sequentially transfected, or individually transfected into host cells, and subsequently fused to form a host cell expressing both chains.

[0150] The plasmid for use in the production of expression vectors is configured such that PCR-amplified V heavy chain and V kappa light chain cDNA sequences can be used to reconstruct complete heavy chain and light chain minigenes. This plasmid can be used to express complete human IgG1κ or IgG4κ antibodies. The complete human and chimeric antibodies of the present invention also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be prepared for the expression of other heavy chain isotypes or for the expression of antibodies containing the lambda light chain.

[0151] Accordingly, in another aspect of the present invention, the structural features of the anti-C6 antibody of the present invention are used to produce a structurally related anti-C6 antibody having one or more functional characteristics of the antibody of the present invention, for example,

[0152] (a) Binding to the same epitope as the anti-C6 antibody of the present invention, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 or 7F02;

[0153] (b) IC50 in hemolysis analysis of 0.5 µg / ml or less 50 Having;

[0154] (c) K measured by surface plasmon resonance D 1×10 -8 M or less (or alternatively, 5 x 10 -8 M or less, 1 x 10 -9 M or less, 5 x 10 -9 M or less, or 5 x 10 -10 M or less);

[0155] (d) antibody-C6 binding half-life measured by surface plasmon resonance is 40 hours or more; or

[0156] (e) Cross-reacts with C6 of cynomolgus monkeys.

[0157] In one embodiment, one or more CDR regions of the antibody of the present invention may be recombinantly combined with known framework regions and CDRs to generate additional recombinantly engineered anti-C6 antibodies of the present invention. Heavy chain and light chain variable framework regions may be derived from the same or different antibody sequences. The antibody sequence may be the sequence of a naturally occurring antibody or a common sequence of several antibodies. Kettleborough et al. Protein Engineering 4 : 773 (1991); Kolbinger et al., Protein Engineering See 6:971 (1993) and Carter et al., WO 92 / 22653.

[0158] Accordingly, in another aspect, the present invention provides a method for producing an anti-C6 antibody: a step of producing an antibody comprising: (1) a heavy chain framework region and a heavy chain CDR comprising an amino acid sequence selected from the amino acid sequences of at least the CDRs shown in SEQ ID NOs 6, 7 and 8 among the heavy chain CDRs; and (2) a light chain framework region and a light chain CDR comprising an amino acid sequence selected from the amino acid sequences of the CDRs shown in SEQ ID NOs 11, 12 and 13 among one or more light chain CDRs, wherein the antibody possesses the ability to bind to C6. The ability of the antibody to bind to C6 can be determined using standard binding and / or functional analysis such as that described in the Examples. Preferably, the antibody exhibits one or more, two or more, three or more, four or five of the functional characteristics listed in (a) to (e) above. Examples of such antibodies disclosed herein include antibodies 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 (as described in Example 6).

[0159] It is known in the art that the CDR3 domains of antibody heavy and light chains play a particularly important role in the binding specificity / affinity of antibodies to antigens (Hall et al., J. Imunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol., 152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995); Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., See 116:2161-2162 (1994); Ditzel et al., J. Immunol., 157:739-749 (1996). Accordingly, the recombinant antibody of the present invention prepared as described above preferably comprises the heavy chain and / or light chain CDR3 of the 7E5 antibody as described in SEQ ID NOs 8 and 13, respectively. Examples of such antibodies disclosed herein include the antibodies 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 (as described in Example 6).

[0160] Additionally, in another aspect, the present invention further provides an anti-C6 antibody that binds to C6 comprising: (1) a heavy chain framework region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region comprising the sequence of SEQ ID NO. 8; and (2) a light chain framework region, a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region comprising the sequence of SEQ ID NO. 13. The antibody may further comprise the heavy chain CDR2 and / or light chain CDR2 of the 7E5 antibody described in SEQ ID NOs. 7 and 12, respectively. The antibody may further comprise the heavy chain CDR1 and / or light chain CDR1 of the 7E5 antibody described in SEQ ID NOs. 6 and 11, respectively. Examples of such antibodies disclosed herein include antibodies 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 (as described in Example 6).

[0161] Production of antibodies with modified sequences

[0162] In another aspect, the variable region sequence of the anti-C6 antibody of the present invention or a part thereof is modified to produce a structurally related anti-C6 antibody that retains the binding (i.e., the same epitope as the unmodified antibody), and thus is functionally identical. Methods for identifying residues that can be altered without removing antigen binding are widely known in the art (e.g., Marks et al. (Biotechnology (1992) 10 (7): 779-83 (diversification of monoclonal antibodies by shuffling light chain variable regions and heavy chain variable regions with fixed CDR3 sequence changes), Jespers et al. (1994) Biotechnology 12 (9): 899-903 (selection of human antibodies to a single epitoproto of an antigen from a phage display repertoire), Sharon et al. (1986) PNAS USA 83 (8): 2628-31 (site-specific mutagenesis of constant amino acid residues at the variable-diversity segment junction of antibodies); Casson et al. (1995) J. Immunol. 155 (12): 5647-54 (loss of evolution and change in specificity due to random mutagenesis of antibody heavy chain variable regions).

[0163] Accordingly, in one aspect of the invention, the CDR1, 2 and / or 3 regions of the antibody described above may comprise the exact amino acid sequence(s) shown in SEQ ID NOs. 6-7 and 11-13 (7E5 CDRs) described herein. However, in another aspect of the invention, the antibody comprises a derivative from the exact CDR sequence of 7E5 but still retains the ability to effectively bind to C6. Such sequence modifications may include one or more amino acid additions, deletions, or substitutions, for example, the conservative sequence modifications described above. Sequence modifications may also be based on the common sequences described above for specific CDR1, CDR2, and CDR3 sequences of the 7E5 antibody.

[0164] Accordingly, in another embodiment, the engineered antibody may consist of one or more CDRs of identical 7E5 antibodies, for example, 90%, 95%, 98%, or 99.5% (as shown in sequence numbers 6-8 and 11-13). Intermediate ranges of the aforementioned values, such as identical identity CDRs of 90-95%, 95-98%, or 98-100% for one or more of the sequences, are also intended to be included in the present invention.

[0165] In another aspect, the present invention provides an isolated antibody that binds to human C6, comprising the following.

[0166] (a) a heavy chain variable region comprising an amino acid sequence that is at least 90% (or 90-95%, 95%-98%, 98%-100%, 95%, 96%, 97%, 98%, or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44, and 46; and

[0167] (b) A light chain variable region comprising an amino acid sequence that is at least 90% (or 90-95%, 95%-98%, 98%-100%, 95%, 96%, 97%, 98%, or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45, and 47.

[0168] In another aspect, the isolated antibody is

[0169] (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46; and

[0170] (b) The above-mentioned light chain variable region is an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45 and 47.

[0171] Example 7 describes in detail a "mix and match" experiment in which each of these heavy chain and light chain variable regions paired with each other in all 81 possible combinations, and the functional activity of all 81 combinations that inhibit C6 activity was demonstrated.

[0172] Additionally, in another embodiment, one or more residues of the CDR may be modified to modify the bond to achieve a more desirable on-rate of the bond, a more desirable off-rate of the bond, or both, so that an ideal bond constant is achieved. Using this strategy, for example, 10 10 M -1 Antibodies having very high binding affinities or higher can be obtained. Affinity maturation techniques widely known in the art and the techniques described herein can be used to modify the CDR region(s) and then screen the resulting binding molecules for desired changes in binding. Thus, as the CDR(s) are modified, changes in binding affinity and immunogenicity are monitored, and antibodies optimized for optimal combined binding and low immunogenicity can be obtained.

[0173] Accordingly, for variable region modifications within the VH and / or VL CDR1, CDR2 and / or CDR3 regions, site-specific mutagenesis or PCR-mediated mutagenesis may be performed to introduce mutation(s) and effects on antibody binding, or other functional properties of interest, and may be evaluated by in vivo or in vitro assays as described herein and provided in the examples. Preferably, conservative modifications (as discussed herein) are introduced. Mutations may be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically one, two, three, four, or five residues within the CDR region are not altered.

[0174] Accordingly, in another aspect, the present invention provides an isolated anti-C6 monoclonal antibody or an antigen-binding portion thereof comprising:

[0175] (a) a VH CDR1 region containing the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 6;

[0176] (b) a VH CDR2 region containing the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 7;

[0177] (c) a VH CDR3 region containing the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 8;

[0178] (d) a VL CDR1 region containing the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 11;

[0179] (e) a VL CDR2 region containing the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 12;

[0180] (f) A VL CDR3 region containing the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO. 12.

[0181] In another aspect, the present invention provides an isolated anti-C6 monoclonal antibody or an antigen-binding portion thereof comprising:

[0182] (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46; and

[0183] (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45 and 47, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions compared to SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45 and 47.

[0184] In addition to or instead of modifications within the CDR, modifications may be made within one or more of the framework regions of FR1, FR2, FR3, and FR4 of the heavy chain and / or light chain variable region of the antibody. This is because such modifications do not eliminate the binding affinity of the antibody. For example, one or more non-germous lineage amino acid residues within the framework region of the heavy chain variable region and / or light chain variable region of the antibody of the present invention are replaced with germline lineage amino acid residues, i.e., amino acid residues corresponding to the human germline sequence for the heavy chain or light chain variable region, which have significant sequence identity with said antibody. For example, the antibody chain may be aligned with a germline antibody chain that shares significant sequence identity with it, and amino acid residues that do not match between the antibody framework sequence and the germline chain framework may be replaced with corresponding residues from the germline sequence. If there is a difference in amino acids between the antibody variable framework region and the equivalent human germline sequence variable framework region, the antibody framework amino acid should generally be replaced with an equivalent human germline amino acid where it is reasonably expected that the amino acid falls into one of the following categories:

[0185] (1) Amino acid residues that directly non-covalently bind to antigens,

[0186] (2) Amino acid residues adjacent to the CDR region,

[0187] (3) Amino acid residues interacting with the CDR region (e.g., located within approximately 3-6 Å of the CDR region determined by computer modeling), or

[0188] (4) Amino acid residues participating in the VL-VH interface.

[0189] The residue that "directly non-covalently binds to the antigen" comprises an amino acid of the framework region that has a good probability of directly interacting with an amino acid on the antigen through established chemical forces, such as hydrogen bonds, van der Waals forces, hydrophobic interactions, etc. Accordingly, in one embodiment, the amino acid residue of the framework region of the antibody of the present invention is substituted with a corresponding germline amino acid residue that directly non-covalently binds to the antigen.

[0190] The residue "adjacent to the CDR region" comprises a sequence immediately adjacent to one or more CDRs in the primary sequence of the antibody, for example, an amino acid residue at a position immediately adjacent to the CDR as defined by Kabat, or a CDR as defined by Chothia (see Chothia and Lesk J. Mol. Biol., 196:901 (1987)). Accordingly, in one embodiment, an amino acid residue within the framework region of the antibody of the present invention is substituted with a corresponding germline amino acid residue adjacent to the CDR region.

[0191] The residues “otherwise interacting with the CDR region” include residues determined by secondary structural analysis to be spatially oriented sufficient to influence the CDR region. These amino acids generally have side chain atoms within about 3 angstrom units (A) of some atoms within the CDR and must contain atoms capable of interacting with the CDR atoms according to established chemical forces as listed above. Accordingly, in one embodiment, amino acid residues within the framework region of the antibody of the present invention are substituted with corresponding germline amino acid residues that otherwise interact with the CDR region.

[0192] Amino acids at certain positions within the framework are known to be important in determining CDR identification (e.g., may interact with the CDR) in many antibodies (Chothia and Lesk, supra, Chothia et al., supra and Tramontano et al., J. Mol. Biol. 215:175 (1990); all of these are incorporated herein by reference). The authors analyzed the structures of several known antibodies to identify conserved framework residues important to the CDR structure. The analyzed antibodies were classified into a limited number of structural or "standard" classes based on the morphology of the CDR. Conserved framework residues within the standard class are referred to as "standard" residues. Standard residues include light chain residues 2, 25, 29, 30, 33, 48, 64, 71, 90, 94, and 95, and heavy chain residues 24, 26, 29, 34, 54, 55, 71, and 94. Additional residues (e.g., CDR structure-determining residues) can be identified according to the methodology of Martin and Thorton (1996) J. Mol. Biol. 263:800. In particular, amino acids at positions 2, 48, 64, and 71 of the light chain and amino acids at positions 26–30, 71, and 94 of the heavy chain (numbering according to Kabat) are known to interact with the CDR in many antibodies. Amino acids at position 35 of the light chain and positions 93 and 103 of the heavy chain are also likely to interact with the CDR. Additional residues that may result in the conformation of the CDR can be identified according to the methodology of Foote and Winter (1992) J. Mol. Biol. 224:487. These residues are referred to as "vernier" residues and are residues in the framework region that is closely fundamental (i.e., forms the "platform") of the CDR.

[0193] "Residues participating in the VL-VH interface" or "packing residues" include residues at the interface between VL and VH, as defined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al., supra.

[0194] Sometimes there is ambiguity as to whether a specific amino acid falls into one or more of the categories mentioned above. In such cases, alternative variant antibodies are produced, one having a specific substitution and the other not. These other variant antibodies produced in this way can be tested for desired activity using any assay described herein and a selected preferred antibody.

[0195] Additional candidate substances for substitution within the framework region are amino acids that are abnormal or rare in the antibody at the corresponding position. These amino acids may be substituted with amino acids from equivalent positions in human germline sequences or from equivalent positions in more typical antibodies. For example, substitution may be desirable when an amino acid in the framework region of the antibody is rare at that position and the corresponding amino acid in the germline sequence is common for that position in the immunoglobulin sequence; or when an amino acid in the antibody is rare at that position and the corresponding amino acid in the germline sequence is rare in relation to other sequences. It is considered that by replacing abnormal amino acids with amino acids from germline sequences that typically occur in antibodies, the antibody may be made less immunogenic.

[0196] As used herein, the term “rare” refers to an amino acid occurring at that position in less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 3%, more preferably less than about 2%, and much more preferably less than about 1% of a representative sample of the sequence, and the term “common” as used herein refers to an amino acid occurring in more than about 25%, usually more than about 50% of the sequence in a representative sample. For example, all light chain and heavy chain variable region sequences are classified into “subgroups” with sequences that are particularly homologous to one another and have the same amino acid at a specific critical position (Kabat et al., supra). When determining whether an anti-sequence sequence is “rare” or “common,” it is often desirable to consider only the sequences of the same subgroup as the antibody sequence.

[0197] Generally, the framework regions of antibodies are generally substantially identical, and more generally identical to the framework regions of the human germline sequences from which they originated. Of course, many amino acids in the framework regions do not directly contribute to the specificity or affinity of the antibody. Therefore, many individual conservative substitutions of framework residues may be allowed without significant changes in the specificity or affinity of the produced immunoglobulin. Thus, in one embodiment, the variable framework region of the antibody shares at least 85% sequence identity with the human germline variable framework region sequence or a match of such sequence. In another embodiment, the variable framework region of the antibody shares at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the human germline variable framework region sequence or a match of such sequence.

[0198] As described in Carr et al. US2003 / 0153043, a framework modification may also be made to reduce the immunogenicity of an antibody or to reduce or remove T cell epitopes present therein.

[0199] The engineered antibodies of the present invention include, for example, those in which modifications have been made to framework residues within VH and / or VL to enhance the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline. More specifically, an antibody that has undergone somatic mutation may contain framework residues different from the germline sequence from which the antibody originated. These residues can be identified by comparing the germline sequence from which the antibody originated with the antibody framework sequence.

[0200] Another type of framework modification involves mutating one or more residues within a framework region or even within one or more CDR regions to reduce the potential immunogenicity of the antibody by removing T cell epitopes. This approach is also called "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043.

[0201] In addition to simply binding to C6, the antibody may be selected to possess other functional characteristics of the antibody of the present invention, for example:

[0202] (a) Binding to the same epitope as the anti-C6 antibody of the present invention, such as 7E5, 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 or 7F02;

[0203] (b) IC50 in hemolysis analysis of 0.5 µg / ml or less50 Having;

[0204] (c) K measured by surface plasmon resonance D 5 x 10 -10 M or less;

[0205] (d) antibody-C6 binding half-life measured by surface plasmon resonance is 40 hours or more; or

[0206] (e) Cross-reacts with C6 of cynomolgus monkeys.

[0207] Additional antibody modifications

[0208] The antibodies of the present invention may contain one or more glycosylation sites in one or more of the light chain or heavy chain variable regions. These glycosylation sites may result in increased immunogenicity of antigen binding or changes in the antibody's pK due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glyco-biology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing NXS / T sequences. In some cases, it is desirable to have an anti-C6 antibody that does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by modifying residues within the glycosylation region.

[0209] For example, in one embodiment, the glycosylation of the antibody is modified, for example, the variable region is modified to remove one or more glycosylation sites residing in the variable region. In particular, it is desirable to remove sites prone to glycosylation in the sequence of the antibody. This can be achieved by altering the occurrence of one or more NX-(S / T) sequences occurring in the parent variable region (where X is any amino acid residue), particularly N residues and / or S or T residues. In one embodiment, T95 is mutated to K95. In another embodiment, N47 is mutated to R47.

[0210] For example, a glycoslated antibody may be prepared (i.e., lacking glycosylation). Glycosylation may be modified, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications may be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made to remove glycosylation at one or more variable region framework glycosylation sites by removing them. Such aglycosylation may increase the affinity of the antibody for an antigen. Reference is made to U.S. Patents No. 5,714,350 and No. 6,350,861, for example.

[0211] Additionally or alternatively, antibodies may have modified types of glycosylation, such as hypofucosylated antibodies having a reduced amount of fucosyl residues or antibodies having an increased bisected GlcNac structure. These modified glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells having a modified glycosylation mechanism. Cells having a modified glycosylation mechanism are described in the art and can be used as host cells to produce antibodies having modified glycosylation by expressing the recombinant antibody of the present invention. For example, since Ms704, Ms705, and Ms709 cell lines lack the fucosyltransferase gene FUT8 (α (1,6) -fucosyltransferase), antibodies expressed in Ms704, Ms705, and Ms709 cell lines do not contain fucose in their carbohydrates. Ms704, Ms705, and Ms709 FUT8 - / - cell lines were generated by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two alternative vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87: 614-22). As another example, European Patent No. 1,176,195 describes a cell line having a functionally disrupted FUT8 gene encoding fucosyltransferase so that an antibody is expressed in the cell line exhibiting hypofucosylation by reducing or removing the α-1,6 binding-related enzyme. European Patent No. 1,176,195 also describes a cell line having low enzymatic activity for binding to the Fc region of an antibody or adding fucose to N-acetylglucosamine, or a cell line having low enzymatic activity, e.g., rat myeloma cell line YB2 / 0 (ATCC CRL 1662).PCT Publication WO 03 / 035835 describes a variant CHO cell line, Luc13 cells, in which the ability to attach fucose to Asn-binding carbohydrates is reduced and also results in hypofucosylation of antibodies expressed in the host cells (Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with a modified glycosylation profile can also be produced in eggs as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells such as rhamna. A method for producing antibodies in a plant system is disclosed in the U.S. patent application corresponding to Alston & Bird LLP attorney docket No. 040989 / 314911, filed August 11, 2006. PCT Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modified glycosyl transferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit an increased bisecting GlcNac structure, which increases the antibody's ADCC activity (see Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, fucose residues of the antibody can be cleaved using fucosidase enzymes; for example, fucosidase α-L-fucosidase removes fucosyl residues from the antibody (Tarentino et al. (1975) Biochem., 14:5516-23).

[0212] The antibody of the present invention may be modified in a variable region to remove one or more glycosylation sites and / or to improve the physical stability of the antibody. For example, in one embodiment, the physical stability of the antibody is improved by substituting a serine at position 228 of the variable region with a proline residue (i.e., the antibody has a variable region containing an S228P mutation). The S228P modification significantly stabilizes the antibody structure against the formation of disulfide bonds within the chain. In one embodiment, the full-length antibody of the present invention is an IgG4 isotype (IgG4 S228P) having the S228P modification.

[0213] In another embodiment, the variable region is modified to remove one or more glycosylation sites residing in the variable region. In particular, it is desirable to remove sites prone to glycosylation in the sequence of the antibody. As previously mentioned, this can be achieved by altering one or more NX-(S / T) sequences occurring in the parent variable region (where X is any amino acid residue), particularly the occurrence of N residues and / or S or T residues. In one embodiment, T95 is mutated to K95. In another embodiment, N47 is mutated to R47.

[0214] In addition to or alternative to modifications made within the framework or CDR region, the antibody of the present invention may include modifications within the Fc region, typically comprising one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or cytotoxicity of antigen-dependent cells. The antibody may also be chemically modified to alter one or more functional properties of the antibody (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation. Each of these examples is described in more detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0215] In certain embodiments, the present invention considers antibody variants that possess some, but not all, effector functions, where the half-life of the in vivo antibody is important but specific effector functions (e.g., complement and ADCC) are unnecessary or harmful, and these become desirable candidates for use. In vitro and / or in vivo cytotoxicity assays may be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to maintain FcRn binding ability, as the antibody does not possess FcgR binding (and thus likely lacks ADCC activity). NK cells, the major cells mediating ADCC, express only FcgRIII, while monocytes express FcgRI, FcgRII, and FcgRIII. FcR expression in hematopoietic cells is Ravetch, JV and Kinet, JP, Annu Rev. This is summarized in Table 3 on page 464 of Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,002,303; U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063 and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive analysis methods may be used (e.g., ACTI.™ for flow cytometry; non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96.RTM; non-radioactive cytotoxicity assay (Promega, Madison, Wis.Useful effector cells for such analysis include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK). Optionally or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in the animal models disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay can also be performed to determine whether the antibody cannot bind to C1q and thus lacks CDC activity. Refer to C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, CDC analysis may be performed (see, e.g., ano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, M. S and MJ Glennie, Blood 103 (2004) 2738-2743). Determination of FcRn binding and in vivo clearance / half-life may be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006) 1759-1769).

[0216] In certain embodiments, the antibody includes a mutated variable region to enhance the physical stability of the antibody. In one embodiment, the antibody is an IgG4 isotype antibody containing a mutation from serine to proline at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy-chain constant region. This mutation has been reported to eliminate heterogeneity of the inter-heavy chain disulfide hybrid in the hinge region (Angal et al., supra; position 241 is based on the Kavat numbering system). For example, in various embodiments, the anti-C6 antibody of the present invention may include a heavy-chain variable region of any of the antibodies described herein linked to a human IgG4 constant region, in which the serine corresponding to position 241 in this region is mutated to proline as described in Angal et al., supra. Thus, in the case of a heavy-chain variable region linked to a human IgG4 constant region, this mutation corresponds to the S228P mutation by the EU index.

[0217] In another embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues within the hinge region is, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is modified to, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

[0218] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, which impairs the Staphylococcus protein A (SpA) binding compared to the original Fc-hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745.

[0219] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations, such as T252L, T254S, and T256F as described in U.S. Patent No. 6,277,375, may be introduced. Alternatively, to increase the biological half-life, the antibody may be modified to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG within the CH1 or CL region, as described in U.S. Patents No. 5,869,046 and No. 6,121,022. In another approach, the antibody is modified to increase its biological half-life by introducing two mutations, one at serine at position 434, and a second mutation selected from the group consisting of isoleucine at position 311, valine at position 311, isoleucine at position 436, and valine at position 436. This approach is described in U.S. Patent Publication No. 2012 / 6128663.

[0220] In another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced with other amino acid residues so that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered may be, for example, an Fc receptor or a C1 component of complement. This approach is described in more detail in U.S. Patents No. 5,624,821 and No. 5,648,260.

[0221] In another embodiment, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with different amino acid residues so that the antibody alters the C1q bond and / or reduces or disrupts complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551.

[0222] In another embodiment, one or more amino acid residues within amino acid positions 231 and 239 are altered to change the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94 / 29351.

[0223] In another embodiment, the Fc region is modified to increase the ability of an antibody to modulate antibody-dependent cell cytotoxicity (ADCC) and / or increase the affinity of the antibody for the Fcγ receptor by modifying one or more amino acids from the following: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication WO 00 / 42072. Additionally, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn were mapped, and variants having improved binding were described (Shields et al. (2001) J.Biol.Chem. 276(See : 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve binding to FcγRIII: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0224] In another embodiment, the Fc region reduces the ability of the antibody to mediate antibody-dependent cell cytotoxicity (ADCC) and / or reduces the affinity of the antibody for the Fcγ receptor by introducing an amino acid substitution at the Pro329 position, preferably comprising one or more additional amino acid substitutions selected from S228P, E233P, L234A, L235A, L235E, N297A, N297D, and P331S. This approach is described in U.S. Patent Publication No. 2012 / 0251531.

[0225] Antibodies having reduced effector function include substituents of one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). These Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, and so-called “DANA” Fc mutants having residues 265 and 297 substituted with alanine (U.S. Patent No. 7,332,581).

[0226] Specific antibody variants with improved or reduced binding to FcR have been described (see, e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312 and Shields, RL et al., J.Biol.Chem. 276 (2001) 6591-6604).

[0227] In a specific embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0228] In some embodiments, changes (i.e., improved or reduced) are made in the Fc region that causes C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0229] Antibodies having a half-life and improved binding to the neonatal Fc receptor (FcRn) that cause pregnant women to transfer IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593, Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) are described in US 2005 / 0014934. These antibodies comprise an Fc region having one or more substituents that improve the binding of the Fc region to FcRn. These Fc variants comprise one or more Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See Duncan, AR, and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0230] Additionally, antibodies may be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG), for example, a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" includes any form of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryl oxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and may be applied to the antibodies of the present invention. For example, refer to EP 0 154 316 and EP 0 401 384.

[0231] Description of the characteristics of monoclonal antibodies against C6

[0232] The monoclonal antibodies of the present invention may be characterized by binding to C6 and / or functional inhibition of C6 using various known techniques. Typically, the binding of the antibody to the target antigen is initially characterized by ELISA. Briefly, a microtiter plate is coated with PBS containing purified C6 and then blocked with an unrelated protein, such as bovine serum albumin (BSA) diluted with PBS. Plasma dilutions from C6-immunized mice were added to each well and incubated at 37°C for 1 to 2 hours. After washing the plates with PBS / Tween 20, they were incubated at 37°C for 1 hour with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to alkaline phosphatase. After washing, the plates were developed with ABTS substrate and analyzed at OD 405. Preferably, mice that develop the highest titer of antibody exhibiting the highest binding affinity and / or functional inhibitory activity are used for fusion.

[0233] Using the ELISA assay described above, hybridomas that produce antibodies exhibiting positive reactivity with antibodies and C6 immunogens can be screened. Subsequently, hybridomas that preferably bind to C6 with high affinity can be subcloned and further characterized. By ELISA, one clone can be selected from each hybridoma that retains the reactivity of the parent cell to create a cell bank and purify the antibody.

[0234] Additionally or alternatively, functional analyses determining the ability of antibodies to inhibit or block C6 activity can be used for screening and selecting antibodies of interest. Suitable in vitro functional analyses include hemolysis assays and MAC ELISA assays, as described in detail in Example 1. Suitable in vivo analyses for determining functional activity are described in detail in Example 4.

[0235] To purify the anti-C6 antibody, the selected hybridoma may be cultured in a roller bottle, a 2-liter spinner flask, or other culture system. The supernatant may be filtered and concentrated prior to affinity chromatography using Protein A-Sepharos (Pharmacia, Piscataway, NJ) to purify the protein. After exchanging the buffer with PBS, the concentration may be determined by an extinction coefficient of 1.43 or, preferably, by OD280 using nephelometric analysis. IgG may be tested by gel electrophoresis and antigen-specific methods.

[0236] To determine whether the selected anti-C6 monoclonal antibody binds to a unique epitope, each antibody can be biotinized using a commercially available reagent (Pierce, Rockford, IL). Conjugation of the biotinylated MAb can be detected with a streptavidin-labeled probe. To determine the isotype of the purified antibody, an isotype ELISA can be performed using techniques recognized in the art. For example, the wells of a microquantification plate can be coated with 10 µg / ml anti-Ig overnight at 4°C. After blocking with 5% BSA, the plate is reacted with 10 µg / ml monoclonal antibody or a purified isotype control at ambient temperature for 2 hours. Subsequently, the wells can be reacted with IgG1 or other isotype-specifically conjugated probes. The plate is developed and analyzed as described above.

[0237] A method for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-C6 antibodies includes Biacore™ surface plasmon resonance (SPR) using a Biacore™ 2000 SPR device (Biacore AB, Uppsala, Sweden), as described in Example 2, which is a standard analysis known in the art.

[0238] Preferably, the antibody of the present invention is 5×10 -8 Bond to C6 with M or less KD, and 2×10 -8 Bond to C6 with a KD of M or less, and 5×10 -9 It binds to C6 with a KD of M or less, and 4×10 -9 Bond to C6 with a KD of M or less, and 3×10 -9 Bond to C6 with M or less KD, and 2×10 -9 Bond to C6 with M's KD, and 1×10 -9 Bond to C6 with a KD of M or less, and 5×10 -10 Bond to C6 with a KD of M or less, or 2.5×10 -10 Combine with C6 using KD of M or less.

[0239] Preferably, the antibody of the present invention has a T of 24 hours or more, 30 hours or more, 36 hours or more, 40 hours or more, or 45 hours or more. 1 / 2 It has (measurements by surface plasmon resonance).

[0240] Physical properties of antibodies

[0241] The antibodies of the present invention can be characterized by their various physical properties to detect and / or distinguish their different classes.

[0242] In a preferred embodiment, the antibody of the present invention does not disclose an asparagine isomer site. Deamidation of asparagine may occur on the NG or DG sequence and result in the formation of isoaspartate residues that introduce a twist into the polypeptide chain and reduce its stability (isoaspartate effect).

[0243] Each antibody has a unique isoelectric point (pI), and the pH range is generally 6 to 9.5. The pI for IgG1 antibodies typically falls within the pH range of 7–9.5, while the pI for IgG4 antibodies typically falls within the pH range of 6–8. It is speculated that antibodies with pIs outside the normal range may be unstable and fail to fold under in vivo conditions. Therefore, it is desirable to have an anti-C6 antibody containing a pI value within the normal range. This can be achieved by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0244] In a preferred embodiment, an antibody that does not degrade rapidly is selected. The degradation of the antibody can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67: 3626-32).

[0245] In another preferred embodiment, an antibody having a minimal aggregation effect that may cause an unwanted immune response and / or altered or adverse pharmacokinetic properties is selected. Generally, the antibody is acceptable with aggregates of 25% or less, preferably 20% or less, more preferably 15% or less, much more preferably 10% or less, and much more preferably 5% or less. Aggregation can be measured by several techniques including size-exclusion column (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0246] Each antibody has a characteristic melting temperature with a high melting temperature, indicating higher overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3: 361-71). Generally, TM1 (temperature of initial development) is preferably greater than 60°C, preferably greater than 65°C, and more preferably greater than 70°C. The melting point of the antibody can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm Res 20: 1952-60; Ghirlando et al. (1999) Immunol Lett 68: 47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40: 343-9).

[0247] In one embodiment, the antibody of the present invention has a high melting temperature. In one embodiment, the antibody has a melting point of at least 65°C, more preferably at least 66°C, much more preferably at least 67°C, and much more preferably at least 68°C. Preferably, the antibody of the present invention has a melting point of 67°C to 72°C, more preferably 68°C to 72°C, or 69°C to 72°C, or 70°C to 72°C, or 69°C to 71.43°C.

[0248] II. Immunotoxins, Immunoconjugates, and Antibody Derivatives

[0249] In another embodiment, the antibody of the present invention is linked to a therapeutic moiety, such as a cytotoxin, drug, or radioisotope. When conjugated to a cytotoxin, these antibody conjugates are called "immunotoxins." Cytotoxins or cytotoxic agents comprise any agent that is harmful to (e.g., causes cell death) cells. For example, Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, It includes propranolol and puromycin and their analogs or homologs.Therapeutic agents include metabolic antagonists (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CBNU), cyclothosfamide, busulban, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), and anthracyclines. (e.g., daunorubicin (formerly daunomycin)) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (anthramycin, AMC)) and anti-mitotic inhibitors (e.g., vincristine and vinblastine). The antibodies of the present invention may be conjugated to a radioisotope, e.g., radioactive iodine, to produce a cytotoxic radiopharmaceutical.

[0250] The antibody conjugate of the present invention may be used to modify a given biological response, and the drug moiety should not be interpreted as being limited to classical chemotherapy agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins include, for example, enzymatically active toxins or fragments thereof, e.g., abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; and biological response modulators, e.g., lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors.

[0251] 이러한 치료 학적 모이어티를 항체에 접합시키는 기술은 잘 알려져 있다, 예를 들어, Arnon 외, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld 외 (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom 외, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson 외 (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera 외 (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin 외 (eds.), pp. 303-16 (Academic Press 1985), and Thorpe 외, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982) 참조.

[0252] Conjugates of antibodies and cytotoxics can be prepared using various bifunctional protein coupling agents, e.g., bifunctional derivatives of imide esters such as N-succinimidyl-3-(2-pyridyldithiol)propionate, iminothiolane, and dimethyladipimidate HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, diisocyanates such as bis-(p-diakonium benzoyl)-diethylenediamine, and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). C 14 - Labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a chelating agent suitable for conjugating antibodies against radionuclides.

[0253] The toxic components of an immunotoxin are, for example, toxins or fragments thereof, such as chemotherapy agents, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or small molecule toxins or radioisotopes, for example 212 Bi, 131 I, 131 In, 111 In, 90 Y and 186 It could be Re.

[0254] Chemotherapy agents useful for generating the above-mentioned immunoconjugate include maytansinoids including DM-1 and DM-4, ​​auristatins, adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel, and docetaxel, taxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, Includes etoposide, ifosamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related mustards. In addition, it includes hormone preparations such as tamoxifen or onapristone that regulate or inhibit hormone activity in tumors.Toxins and fragments thereof include yphtheria A chain, unbound active fragment of diphtheria toxin, cholera toxin, botulinum toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, phytolaca Americana protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor (Momordica charantia inhibitor), curcin, crotin, sapaonaria, officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin, and tricothcenes. Small molecule toxins include, for example, calicheamicins, maytansinoids, palytoxin, and CC1065.

[0255] Additional therapeutic agents that may be conjugated to the antibody to form an immunotoxin include metabolic antagonists, alkylating agents, DNA minor groove binders, DNA inserts, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In the conjugate, the antibody and the therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ No. 15), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The conjugate is U.S. Patents No. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038658; WO 07 / 051081; WO 07 / 059404; WO 08 / 083312; and WO 08 / 103693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295; the disclosures thereof are incorporated herein by reference.

[0256] The antibody of the present invention may also be used for diagnostic purposes, including sample testing and in vivo imaging, for which the antibody (or its binding fragment) may be conjugated to a suitable detectable agent to form an immunoconjugate. Suitable agents for diagnostic purposes are detectable labels, including radioisotopes for whole-body imaging, radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for sample testing.

[0257] For C6 detection, detectable labels may be any of the various types currently used in the field of in vitro diagnostics and may include metal sols such as colloidal gold, particulate labels containing isotopes such as I125 or Tc99 presented as peptide chelating reagents of type N2S2, N3S, or N4, chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker and polynucleotide tags revealed after amplification, such as those by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label may be an enzyme alkaline phosphatase detected by measuring the presence or formation of chemiluminescence after the conversion of a 1,2-dioxetane substrate, which may be adamantyl methoxyphosphoryloxyphenyldioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenyl phosphate (CSPD), as well as CDP and CDP-star® or other luminescent substrates known in the art, such as suitable lanthanide chelates like terbium(III) and europium(III). The detection means is determined by the selected label. The appearance of the label or its reaction product may be visually inspected if the label is particulate and accumulates to an appropriate level, or according to standard practice using instruments such as a spectrophotometer, illuminometer, fluorescence meter, etc. It can be confirmed.

[0258] In certain embodiments, the antibody provided herein may be further modified to contain additional non-protein moiety that is known in the art and readily available. Moiety suitable for derivatization of the antibody includes, but is not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved and whether the antibody derivative is used in treatment under defined conditions.

[0259] In another embodiment, a conjugate of a non-protein moiety and an antibody that can be selectively heated by radiation exposure is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature at which cells adjacent to the antibody-non-protein portion die.

[0260] Conjugation methods that induce substantially (or nearly) non-immunogenic bonds are particularly suitable. Thus, peptide (i.e., amide), sulfide, (sterically hindered), disulfide, hydrazone, or ether bonds are particularly suitable. These bonds are nearly non-immunogenic and exhibit reasonable stability in serum (see Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0261] Different conjugation strategies are involved depending on the biochemical properties of the moiety and the antibody. When the moiety is naturally occurring or recombinant between 50 and 500 amino acids, there are standard procedures in textbooks describing the chemistry for the synthesis of protein conjugates that can be easily followed by a skilled technician (e.g., Hackenberger, CPR and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of a maleimido moiety and a cysteine ​​moiety within the antibody or moiety is used. This is particularly suitable for coupling chemistry, for example, when the Fab or Fab'- fragment of the antibody is used. Alternatively, in one embodiment, coupling is performed at the C-terminus of the antibody or moiety. C-terminal modification of a protein, e.g., Fab-fragmentation, can be performed as described in (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0262] In general, site-specific reactions and covalent bonding are based on modifying natural amino acids into amino acids that have orthogonal reactivity to the reactivity of other functional groups. For example, specific cysteines within rare sequences can be enzymatically converted from aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be obtained by utilizing the specific enzymatic reactivity of a specific enzyme and natural amino acids within a given sequence context (e.g., Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and the formation of protease-catalyzed C-N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0263] Site-specific reactions and covalent bonds can also be achieved by the selective reaction of terminal amino acids with suitable modifiers. The reactivity of N-terminal cysteine ​​and benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent bonds.

[0264] Native chemical ligation can also rely on C-terminal cysteine ​​residues (Taylor, E. Vogel, Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0265] EP 1 074 563 describes a conjugation method based on the faster reaction of cysteine ​​within a negatively charged amino acid segment and cysteine ​​located in a positively charged amino acid segment.

[0266] The moiety can also be a synthetic peptide or a peptide mimic. When polypeptides are chemically synthesized, orthogonal chemically reactive amino acids may be mixed during such synthesis (see de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a wide variety of orthogonal functional groups are involved and can be introduced into synthetic peptides, the conjugation of such peptides with linkers is standard chemistry.

[0267] To obtain mono-labeled polypeptides, conjugates with 1:1 stoichiometry can be separated from other conjugate byproducts by chromatography. This procedure can be facilitated by using dye-labeled bond pairs and charged linkers. By using this type of labeled and highly negatively charged bond pair, mono-conjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides having one or more linkers. This is because differences in charge and molecular weight can be utilized for separation. Fluorescent dyes can be useful for purifying complexes from unbound components, such as labeled monovalent binders.

[0268] In one embodiment, the effector moiety is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0269] III. Composition

[0270] In another embodiment, the present invention provides a composition containing one or a combination of the monoclonal antibodies of the present invention, formulated with a composition, for example, a carrier (e.g., a pharmaceutically acceptable carrier). A composition containing a bispecific molecule comprising the antibody of the present invention is also provided. In one embodiment, the composition comprises a combination of a plurality (e.g., two or more) isolated antibodies of the present invention. Preferably, each antibody of the composition binds to a distinct, pre-selected C6 epitope.

[0271] The pharmaceutical composition of the present invention may also be administered as a combination therapy, that is, in combination with other agents. For example, the combination therapy may include the composition of the present invention having at least one additional therapeutic agent, such as an anti-inflammatory agent, a DMARD (disease-modifying anti-rheumatic drug), an immunosuppressant, and a chemotherapy agent. The pharmaceutical composition of the present invention may also be administered with radiotherapy. Administration with other antibodies is also included in the present invention.

[0272] As used herein, the terms “carrier” and “pharmaceutically acceptable carrier” include any and all physiologically compatible solvents, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic solutions, absorption retardants, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecules, may be coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

[0273] Adjuvants that may be used with the antibodies and components of the present invention include: Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines such as GM-CSF, interleukin-2, -7, -12, and other similar factors; 3D-MPL; CpG oligonucleotides; and monophosphoryl lipid A, e.g., 3-di-o-acylated monophosphoryl lipid A.

[0274] MPL adjuvants are available from Corixa Corporation (Wash, Seattle, see U.S. Patents No. 4,436,727, 4,877,611, 4,866,034 and 4,912,094). CpG-containing oligonucleotides (CpG dinucleotides that have been demethylated) are well known and are described, for example, in WO 96 / 02555, WO 99 / 33488 and U.S. Patents No. 6,008,200 and 5,856,462. Immunostimulating DNA sequences are also described, for example, in Sato et al., Science 273:352, 1996.

[0275] Other additional adjuvants include, for example, saponins such as Quil A or its derivatives including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); escin; digitonin; or Gypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, United States); ISCOMS (CSL), MF-59 (Chiron); SBAS-class adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium); Detox (Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.); and other aminoalkyl glucosaminid 4-phosphates (AGPs). Polyoxyethylene ether adjuvants such as those described in WO 99 / 52549 A1; synthetic imidazoquinoline such as [S-26308, R-837] (Harrison, et al., Vaccine 19: 1820-1826, 2001); and resiquimod [S-28463, R-848] (Vasilakos, et al., Cellular Immunology 204: 64-74, 2000); Schiff bases of carbonyl and amine persistently expressed on the surface of antigen-presenting cells and T cells such as Tucaresol (Rhodes, J.et al., Nature 377: 71-75, 1995); cytokines as proteins or peptides including pro-inflammatory cytokines such as interferon, IL-1α, IL-1β, TGF-α and TGF-β, and interferon gamma; chemokines and co-stimulatory molecules; Th1 inducers such as IL2, IL-12, IL-15, IL-18 and IL-21; Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13; and other chemokines; and co-stimulatory genes such as MCP-1, MIP-1α, MIP-1β, RNATES, TCA-3, CD80, CD86 and CD40L; immunostimulators targeting ligands such as CTLA-4 and L-selectin; apoptosis-promoting peptides and proteins such as Fas; vaxfectin, (Reyes et al., Vaccine 19: 3778-3786, 2001); synthetic lipid-based adjuvants such as squalene, alpha-tocopherol, polysorbate 80, DOPC, and cholesterol; endotoxin, [LPS], (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000); ligands that induce Toll receptors to produce Th1-induced cytokines such as mycobacterial protein p19, peptidoglycan, teicoic acid, and lipid A; and CT (cholera toxin, subunits A and B) and LT (heat labile enterotoxin from Escherichia coli, subunits A and B), LLO (listeriolysin O; WO 01 / 72329). These and various additional Toll-like receptor (TLR) agonists are described, for example, in Kanzler et al., Nature Medicine, May 2007, Vol 13, No 5.

[0276] "Pharmaceuticalally acceptable salts" refer to salts that retain the desirable biological activity of the parent compound and do not impart any unwanted toxic effects (see Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid-added salts and base-added salts. Acid-added salts include those derived from non-toxic organic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, bromic acid, hydroiodic acid, phosphorus, etc., as well as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base-added salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucarmine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0277] The compositions of the present invention may be administered by various methods known in the art. As will be known to those skilled in the art, the route and / or mode of administration will vary depending on the desired result. The active compound may be prepared as a carrier that protects the compound from rapid release, such as in controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for manufacturing such formulations are patented or are generally known to those skilled in the art. For example, see Continuous and Controlled-Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0278] To administer the compound of the present invention via a specific route of administration, it may be necessary to coat the compound with a substance to prevent its inactivation or to administer it together with the compound. For example, the compound may be administered to a target on a suitable carrier, e.g., a liposome or a diluent. Acceptable diluents include saline solution and aqueous buffer. Liposomes include conventional liposomes as well as water-in-oil-in-water CGF emulsions (Strejan et al., (1981) J. Neuroimmunol. 7:27).

[0279] The carrier comprises a sterile aqueous solution or dispersant and a sterile powder for the immediate preparation of a sterile injectable solution or dispersant. The uses of media and formulations for pharmaceutically active substances are known in the art. The use of any conventional media or formulation in the pharmaceutical composition of the present invention is considered, except where such media or formulations are incompatible with the active compound. A supplementary active compound may also be included in the composition.

[0280] The therapeutic composition must typically be sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, liposome, or other regular structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity may be maintained by the use of a coating, for example, lecithin, the maintenance of particle size required in the case of dispersion, and the use of surfactants. In many cases, it will be desirable to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol or sorbitol, or sodium chloride, in the composition. Long-term absorption of the injectable composition may be achieved by including an absorption-delaying agent, for example, monostearates and gelatin, in the composition.

[0281] Sterile injectable solutions can be prepared by mixing the required amount of the active compound with one or a combination of the components listed above in a suitable solvent as needed, followed by sterile microfiltration. Generally, dispersions are prepared by mixing the active compound with a basic dispersion medium and an excipient containing other necessary components among the components listed above. For sterile powders for the preparation of sterile injectable solutions, a preferred preparation method is vacuum drying and freeze-drying (freeze-drying) to produce any additional desired component from the powder of the active ingredient plus a previously sterile-filtered solution thereof.

[0282] The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For example, the antibody of the present invention may be administered once or twice a week by subcutaneous or intramuscular injection, or once or twice a month by subcutaneous or intramuscular injection.

[0283] It is particularly desirable to formulate parenteral compositions in the form of dosing units for ease of administration and uniformity of dosage. As used herein, the dosing unit form refers to a physically separated unit suitable as a single dose for a subject of treatment; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in relation to the required pharmaceutical carrier. Description of the dosing unit forms of the present invention is directed by and depends on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of formulating active compounds to treat such individual sensitivities.

[0284] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0285] In the case of therapeutic compositions, the formulations of the present invention include those suitable for intravenous, intraperitoneal, oral, nasal, topical (including oral and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may be conveniently provided as a single formulation and may be prepared by any method known in the pharmaceutical field. The amount of active ingredient that may be combined with a carrier material to prepare a single formulation will depend on the target being treated and the specific mode of administration. The amount of active ingredient that may be combined with a carrier material to prepare a single formulation will generally be the amount of the composition exhibiting a therapeutic effect. Generally, out of 100%, this amount will be about 0.001% to about 90% of the active ingredient, preferably about 0.005% to about 70%, most preferably about 0.01% to about 30%.

[0286] Formulations of the present invention suitable for vaginal administration include vaginal suppositories, tampons, creams, gels, pastes, foams, or spray formulations containing a carrier known in the art as suitable. Formulations of the compositions of the present invention for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservative, buffer, or propellant that may be necessary.

[0287] As used herein, the terms “parenteral administration” and “parenterally administered” generally refer to a mode of administration other than intestinal and local administration by injection, and may be administered without limitation by intravenous, intramuscular, intra-arterial, intravertebral, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intrabronchial, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion.

[0288] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, e.g. olive oil, and injectable organic esters, e.g. ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating material such as lecithin, by maintaining the required particle size in the case of a dispersant, and by the use of a surfactant.

[0289] These compositions may also contain auxiliary agents such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization procedure and the inclusion of various antimicrobial and antifungal agents such as parabens, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Additionally, long-term absorption in the form of an injectable medicine may be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0290] When the compounds of the present invention are administered to humans and animals as medicines, they may be provided alone or as pharmaceutical compositions containing, for example, 0.001 to 90% (more preferably 0.005 to 70%, for example 0.01 to 30%) of an active ingredient together with a pharmaceutically acceptable carrier.

[0291] Regardless of the selected route of administration, compounds of the present invention and / or pharmaceutical compositions of the present invention that can be used in an appropriate hydrated form are formulated into pharmaceutically acceptable formulations by conventional methods obvious to those skilled in the art.

[0292] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a specific patient, composition, and mode of administration, except that it is toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the specific composition of the present invention or its esters, salts, or amides used, the route of administration, the time of administration, the elimination rate of the specific compound used, the duration of treatment, other drugs, other compounds and / or substances used in combination with the specific compound used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and factors well known in the medical field. A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may start the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to achieve a therapeutic effect, in order to gradually increase the dosage until the desired effect is achieved. Generally, the appropriate daily dosage of the composition of the present invention will be the amount of the compound that is the lowest dosage effective in producing a therapeutic effect. Such an effective dosage generally depends on the factors described above. Administration is preferably performed intravenously, intramuscularly, intraperitoneally, or subcutaneously, preferably near the target site. If necessary, the effective daily dosage of the therapeutic composition may be administered individually in 2, 3, 4, 5, 6, or more subdoses at appropriate intervals over a day, optionally in a unit dosage form. The compounds of the present invention may be administered alone, but are preferably administered as a pharmaceutical preparation (composition).

[0293] The therapeutic composition may be administered by a medical device known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention may be administered by a needleless subcutaneous injection device such as the device disclosed in U.S. Patents No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603 disclosing an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering a drug through the skin; and U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering a drug at an accurate infusion rate. U.S. Patent No. 4,447,224 disclosing a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing an osmotic drug delivery system having a multi-chamber compartment; U.S. Patent No. 4,475,196 disclosing an osmotic drug delivery system. Many other implants, delivery systems, and modules are known to those skilled in the art.

[0294] In certain embodiments, the antibodies of the present invention may be formulated to ensure adequate distribution in vivo. For example, the blood-brain barrier (BBB) ​​rejects many hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if necessary), they may be formulated, for example, within liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents No. 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see V.V. Ranade (1989) J.Clin.Pharmacol.29:685). Exemplary target moieties include folic acid or biotin (for example, see Low et al. U.S. Patent No. 5,416,016); Mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153 : 1038); Antibodies (PG Bloeman et al. (1995) FEBS Lett. 335:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180; surfactants of different species that may include components of the molecule of the present invention as well as the formulations of the present invention (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J.Biol.Chem. 269: 9090); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273; also see. In one aspect of the present invention, the therapeutic compound of the present invention is formulated in a liposome; in a more preferred aspect, the liposome targets It contains moiety. The composition must be fluid enough to have soft syringability.It must be stable under manufacturing and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi.

[0295] The composition must be sterile and fluid enough to be delivered by a syringe. In addition to water, the carrier may be isotonic buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity may be maintained by the use of a coating, for example, lecithin, by maintaining the particle size required in the case of dispersion, and by the use of a surfactant. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable composition may be achieved by including an absorption-delaying agent in the composition, for example, aluminum monostearate or gelatin.

[0296] If the active compound is adequately protected, as described above, the compound may be administered orally, for example, with an inert diluent or an assimilated edible carrier.

[0297] IV. Use and Method of the Invention

[0298] The anti-C6 antibody of the present invention can functionally inhibit C6 in vivo and in vitro, thereby inhibiting the formation of membrane attack complexes that require C6. Accordingly, in another aspect, the present invention relates to a method for inhibiting the formation or activity of membrane attack complexes (MACs) in a subject, said method comprising administering the antibody of the present invention to the subject in an amount effective for inhibiting MAC formation or inhibiting activity in the subject. In another aspect, the present invention provides a method for treating, preventing, or reducing symptoms of a disorder mediated by unwanted activation of the complement system in a subject, said method comprising administering an effective amount of the antibody of the present invention to the subject. Examples of such disorders are further described below.

[0299] As described in detail in U.S. Patent No. 8,703,136 (the entire contents of which are incorporated herein by reference), it has been confirmed that axonal regeneration can be enhanced by inhibition of the complement system. Accordingly, the use of anti-C6 antibodies for inhibition of the complement system, particularly for inhibition of MAC formation, can be used to treat symptoms requiring axonal regeneration, for example, in mammals affected by damage or disease of the central or peripheral nervous system. Symptoms requiring axonal regeneration that can be treated according to the present invention include physical damage as well as neurodegenerative diseases of the peripheral or central nervous system.

[0300] In one embodiment, the antibody of the present invention promotes axonal regeneration. As used herein, the terms "promotion of axonal regeneration" or "promotion of nerve regeneration" are distinguished from reducing or preventing axonal or nerve degeneration. Promotion (or facilitation) of axonal or nerve regeneration is understood herein to mean that the regeneration of axons or nerves is improved in a treated subject compared to an untreated subject. Improved axonal regeneration is preferably regeneration occurring in a treated subject at an early stage (after axonal or nerve damage or after the start of treatment) compared to an untreated subject. Improved axonal regeneration may also include regeneration occurring at a higher rate and / or on a larger scale in a treated subject compared to an untreated subject. Accordingly, the antibody according to the present invention preferably produces the acquisition of sensory or motor function.

[0301] Accordingly, in one embodiment, the present invention provides a method for regenerating nerves in a patient, comprising administering a therapeutically effective amount of the antibody of the present invention to the patient. In another embodiment, the present invention provides a method for promoting the recovery of damaged or degenerated nerves in a patient, comprising administering a therapeutically effective amount of the antibody of the present invention to the patient. In yet another embodiment, the present invention provides a method for reducing or delaying nerve degeneration in a patient, comprising administering a therapeutically effective amount of the antibody of the present invention to the patient.

[0302] The patient may suffer from neurological trauma resulting from physical injury, e.g., damage to the peripheral nervous system (PNS) or central nervous system (CNS), e.g., neurological trauma from physical injury (discussed further below). Physical injury may be, e.g., trauma (e.g., accident), surgical injury, or non-traumatic injury (e.g., nerve compression). In one embodiment, the antibody is administered at or near the site of injury. Additionally, the patient may suffer from acquired and / or hereditary immune-mediated inflammatory diseases and / or neurodegenerative diseases, chronic demyelinating neuropathy, multiple sclerosis (MS), or other neurodegenerative diseases such as myasthenia gravis or amyotrophic lateral sclerosis (ALS) (discussed further below).

[0303] Improvement in axonal regeneration is preferably determined by functional tests that are relatively easy to perform in human patients; for example, the recovery of sensory or motor function is preferably determined by standardized tests available in the relevant art (see e.g., g. Wong, KH et al. (2006) Scand. J. Plast. Reconstr. Surg. Hand Surg. 40:219-224; Jerosch-Herold (2005) Hand Surg. 30:252-264). Suitable tests are preferably quantitative, standardized, and more preferably, in which their psychometric characteristics are evaluated and quantified. Such tests include the Weinstein Enhanced Sensory Test (WEST) or the Semmes-Weinstein Monofilament Test (SWMT) and the shape-texture identification (STI) for tactile perception testing. Improved axonal regeneration is [see] Hare, GMT et al. (1992) Plastic and Reconstr. Surg. As described by De Koning, P. et al. (1986) J. Neurol. Sci. 74:237-246, the recovery of sensory or motor function can be experimentally determined in test animals by functional tests. The antibody, preferably, produces the acquisition of sensory or motor function as can be determined in the aforementioned tests.

[0304] Example 8 describes in detail an animal model that can be used to test the effect of the anti-C6 antibody of the present invention on sensory function. This nerve compression model (compression of the sciatic nerve (nervus ischiadicus)) is used to test the effect of the anti-human C6 monoclonal antibody on the recovery of sensory function in C6-knockout rats (PVC) supplemented with human C6. Nerve compression is a model of peripheral nerve injury. See WO 2010 / 005310 (PCT / NL2009 / 050418); and de Jonge et al. (2004) Hum Mol Genet. 13 (3): 295-302.

[0305] Improved axonal regeneration can also be experimentally determined in test animals by histological examination; for example, improved remyelination can be determined by comparing measurements of myelin sheaths around axons in treated animals versus untreated animals, where thicker myelin sheaths indicate improved remyelination. More efficient axonal regeneration can be determined by the generation of large-diameter single axon sprouts in treated animals compared to small axon clusters in untreated animals.

[0306] An appropriate dose of antibody is an effective amount for promoting axonal regeneration, as can be seen from the improvement of sensory or motor function described above. "Effective dose," "therapeutic dose," or "effective dosage" refers to an amount sufficient to induce the desired pharmacological or therapeutic effect, thereby causing effective treatment of the injury or disorder.

[0307] To minimize nerve damage and / or promote axonal regeneration as quickly as possible, the antibody may be administered preferably immediately after the occurrence of nerve damage, i.e., within 24, 12, 6, 3, 2, or 1 hour, more preferably within 45 minutes, 30 minutes, 20 minutes, or 10 minutes. In one embodiment of the present invention, the antibody may be administered prior to surgery at risk of nerve damage (see below) to minimize nerve damage and / or promote axonal regeneration immediately upon surgical damage to the nerve (e.g., as a precautionary measure).

[0308] Various diseases requiring axonal regeneration can be treated with the antibodies of the present invention. Such conditions include damage to the CNS as well as damage to the PNS. Such diseases include neurological trauma resulting from physical injury. Such diseases include immune-mediated inflammatory diseases or progressive neurodegenerative diseases of injury and / or acquired and / or hereditary origin.

[0309] Physical damage to the PNS and CNS may be traumatic damage, including surgical damage or non-traumatic damage. Traumatic PNS and CNS damage that can be treated with the method and / or agent of the present invention includes spinal cord injury as well as trauma to peripheral nerves, including collisions, automobile accidents, gunshot wounds, fractures, dislocations, lacerations, or any other form of penetrating trauma. Peripheral nerves damaged by trauma that can be treated include finger nerves, median nerve, ulnar nerve, radial nerve, facial nerve, spinal accessory nerve, and brachial plexus nerve.

[0310] Surgical PNS injury is understood as damage to peripheral nerves that occurs when it is clinically necessary to remove or dissect the nerve during a surgical procedure. This occurs in thousands of surgical procedures annually. One example of a surgically damaged peripheral nerve that can be treated with the method and / or agent of the present invention is, for example, the cavernous nerve that supports erectile function and bladder control; such nerves are often damaged during the surgical removal of prostate tumors and surrounding tissues. Another example of a surgically damaged peripheral nerve that can be treated according to the present invention is the phrenic nerve after coronary artery bypass graft (CABG).

[0311] Non-traumatic physical PNS injuries that can be treated with the antibody of the present invention include compression and / or attachment of peripheral nerves, also known as nerve entrapment syndromes. The most common nerve entrapment syndrome is carpal tunnel syndrome.

[0312] Additionally, immune-mediated inflammatory diseases or injuries can be treated with the antibodies of the present invention. These include demyelinating diseases of the central and peripheral nervous systems that are thought to have an autoimmune basis and cause neuronal demyelination as a result of damage directly caused to oligodendrocytes or myelin. Such demyelinating diseases include, for example, Guillain-Barré syndrome (GBS; also known as inflammatory demyelinating polyneuropathy, acute idiopathic polyradiculoneuritis, acute idiopathic polyneuritis, French polio, and Landry's ascending paralysis). Preferably, the antibodies of the present invention are applied to promote axonal regeneration after the acute phase in GBS. Similarly, chronic inflammatory demyelinating polyneuropathy (CIDP), considered the chronic counterpart of GBS, can be treated with the antibodies of the present invention. Multiple sclerosis (MS) is another demyelinating disease that can be treated with the antibody of the present invention.

[0313] Neurodegenerative CNS and / or PNS diseases having genetic components that can be treated with the antibodies of the present invention include amyotrophic lateral sclerosis (ALS, sometimes referred to as Lou Gehrig's disease), Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy, HMSN), and Huntington's disease (HD).

[0314] The present invention is further explained by the following examples, which should not be interpreted as being more limiting. The sequence lists, numbers, and all references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

[0315] Examples

[0316] Example 1: Production of rat anti-human C6 monoclonal antibody

[0317] Rat anti-human C6 monoclonal antibodies were generated by immunizing five rats of the PVG C6 - / - strain with human C6 protein. C6-deficient rats were selected because, according to current understanding in this field, it is extremely difficult to generate functional C6 antibodies in normal rodents. It was hypothesized that immunization against C6 in wild-type animals is inefficient due to the high homology of C6 proteins between humans and rodents. The antibody response in C6-deficient animals is more active because C6 is considered completely "foreign" since there is no functional C6 protein in the circulatory system. Human C6 was purified from whole human serum by affinity chromatography using the 23D1 mouse monoclonal antibody 23D1 coupled to Cepharose (GE Healthcare Cat No. 17-0717-01) (described in L. Clayton (2005) Ph.D. Thesis, Cardiff University).

[0318] Antigens and vaccinationsOne week prior to vaccination, 100 µl of blood was collected from the tail vein and pre-vaccination was administered to rats. On day 1 of vaccination, 100 µg of C6 antigen in Complete Freund's Adjuvant (CFA) was injected subcutaneously (sc) into four sites in rats at a volume of 250 µl per injection. Booster injections were administered on days 14 and 21, again at four sc sites, with 50 µg of C6 antigen in Incomplete Freund's Adjuvant (IFA) at a volume of 250 µl per injection. Test hemorrhage was performed on day 36 by collecting 100 µl of blood from the tail vein for in vitro testing. These test hemorrhages, in which all five rats showed a positive immune response to C6, were analyzed using C6 ELISA, C6 Western blot, and hemolysis assay (described below): all five rats possessed antibodies preventing hemolysis, and all five rats possessed antibodies recognizing purified C6 in the hemolysis assay and Western blot (denaturation conditions). Preliminary fusion boosters were administered on day 62 by intraperitoneal injection of 100 µg of antigen in 250 µl of PBS. Finally, preliminary fusion boosters were administered intravenously (tail vein) in 250 µl of PBS It was performed on day 64 by injecting 100 µg of antigen. The spleens of two rats were harvested on day 66 (the other three were left as backups), and the isolated spleen cells used for hybridoma production were harvested.

[0319] Hybridoma manufacturingHybridomas were prepared by fusing splenocytes from human C6-immunized rats with Y3-Ag1.2.3 fusion partner cells using a standard polyethylene glycol (PEG)-mediated fusion, as described in Luk, JM et al. (1990) J. Immunol. Methods 129:243-250. The supernatant was harvested and used for initial screening for anti-human C6 antibodies via ELISA using 96-well plates coated with human C6 antigen. Positive clones were selected and subcloned. Thirty-eight positive clones were selected for further analysis.

[0320] Hemolysis analysis These 38 supernatants and control supernatants were further tested in a 1:50 dilution hemolysis assay using human serum as a supplement. In this assay, red blood cells coated with complement-activating antigens are incubated in the presence of serum. Serum contains components of the complement system and is activated via a typical pathway when coated red blood cells are detected. The Membrane Attack Complex (MAC) is formed as part of the terminal complement system, and the MAC initiates the lysis of red blood cells. Red blood cell lysis can be quantified by measuring the OD at 405 or 415 nm in the supernatant, which directly measures the activity of the MAC. Since complement inhibitors can quantitatively prevent red blood cell lysis if effective, complement inhibitors can be tested in this system.

[0321] To perform the analysis, a ready-to-use hemolytic system was commercially obtained with CFT buffer (Virion / Serion GmbH, Wurzburg, Germany). The CFT buffer was prepared according to the manufacturer's instructions. The hemolytic system was placed in a roller bank in a cold room to thoroughly mix the red blood cells. To prepare the CFT serum cocktail, 100 µl of human serum was added to 5 ml of CFT buffer. A 50 µl dilution of the test inhibitor was added to a round-bottom 96-well plate, 50 µl of the CFT serum cocktail was added to each well, mixed carefully by pipetting, and the plate was incubated at 37°C for 30 minutes. The positive control was EDTA. The negative control was serum-free or C6-deficient serum. After incubation, the plate was spun at 2000 rpm for 5 minutes (Hettich tabletop centrifuge), and 80 µl of the supernatant was transferred to a flat bottom plate for measurement at 405 or 415 nm. OD was measured within 10 minutes of transfer.

[0322] To determine whether it prevents erythrocyte lysis, the test supernatant was diluted and added to the hemolysis assay. Exemplary results demonstrating that certain supernatants exhibit stronger inhibitory activity than others are shown in Figure 1a. In particular, supernatants #6-12 showed stronger inhibition than other supernatants, and supernatants #11 and #12 showed the strongest inhibition. The supernatant (1:50 dilution) was also tested in a hemolysis assay using rat serum as a supplement, no inhibitory effect was observed, and the inhibitory activity of the antibody was demonstrated to be specific to human C6.

[0323] MAC ELISA analysisA second assay was used to determine whether the supernatant could block MAC formation. In this assay, the ELISA wells of the plate are coated with Mannan or IgG to trigger the typical lectin or complement pathways, respectively, in the presence of serum. Serum contains components of the complement system and is activated via either pathway upon exposure to the coated plate. Membrane Attack Complexes (MACs) are formed as part of the terminal complement system, and MACs precipitate on the ELISA plate. MAC precipitation on the plate can be detected by HRP-binding antibodies and visualized by enzymatic reactions in the presence of chromogens and substrates. This reaction produces a color that can be quantified by measuring the OD at 450 or 655 nm. OD is a direct measure of the amount of MAC formed. Since complement inhibitors can prevent or inhibit MAC precipitation on the plate if effective, they can be tested in this system.

[0324] In the second assay used to test the hybridoma supernatant, a mannan-activated complement ELISA assay was performed. Briefly, ELISA plates were diluted with mannan, coated with the diluted hybridoma supernatant, and human serum was added. Complement components forming complexes on mannan-coated plates can be detected using antibodies. In this specific test, C9 was detected as an indicator of MAC formation. If less C9 is detected in the presence of the supernatant than in its absence, this indicates MAC inhibition. The positive control used was EDTA (since the reaction is calcium-dependent).

[0325] To perform the analysis, coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 15 mM NaN3, pH 9.6), blocking buffer (1 mg / ml BSA / HAS, 10 mM Tris / HCl, pH 7.4, 145 mM NaCl, 15 nM NaN3, pH 7.4), wash buffer (1×TBS, 0.05% Tween 20, 5 mM CaCl2), and dilution buffer (4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 0.3% BSA, 0.02% Tween 20) were prepared. Wells of a flat-bottomed, highly bound 96-well plate were coated with 100 µl of coating buffer containing 10 µg / ml mannan (Sigma, catalog number M7504) and incubated overnight at 4°C. The plate was blocked with 200 µl of blocking buffer for 1 hour at room temperature. Human serum in dilution buffer (1:100) was diluted to the supernatant (1:50) in a round-bottom plate and 50 µl was added per well to a flat-bottom high-binding plate. The plate was incubated at 37°C for 1 hour, then washed 3 times with wash buffer. Anti-C5b-9neo (clone aE11, DAKO, catalog number M0777) was diluted to 1:100 in dilution buffer, 50 µl was added per well, and the plate was incubated at room temperature for 1 hour, then washed 3 times with wash buffer. Anti-mouse HRP (DAKO, Cat. No. P0447) was diluted to 1:2000 in dilution buffer, 50 µl was added per well, and the plate was incubated at room temperature for 30 minutes, then washed 3 times with wash buffer. For development, 50 µl of TMB chromosome (TMB: Sigma T2885, stock solution prepared at 10 mg / ml TMB in DMSO) and 10 µl of 3% H2O2 were added to 5 ml of NaAc buffer (8.2 gm sodium acetate, 21 gm citric acid monohydrate in 1 liter of H2O) and distributed into a 96-well plate.The reaction was stopped with 25 µl of 1M H2SO4, and the OD was measured at 450 nm / 655 nm using a spectrophotometer.

[0326] An exemplary result of this analysis is shown in Figure 1b, demonstrating that two supernatants, #11 and #17, have significantly superior inhibitory activity compared to the other 36 supernatants, along with the best inhibitory activity among all analyzed clones, supernatant #11.

[0327] Since supernatant #11 showed the strongest inhibition in both the hemolysis assay and the MAC ELISA assay, this hybridoma was selected for further characterization. The monoclonal antibody produced by this hybridoma is referred to herein as 7E5.

[0328] Example 2: Characterization of 7E5 Monoclonal Antibody

[0329] In this example, additional experiments were performed to further investigate the binding and functional characteristics of rat anti-human C6 monoclonal antibody 7E5.

[0330] Cross-reactivity Western blotting was performed using cynomolgus monkey (Cyno) serum and human serum. Human and Cyno serums were used for PAGE (10% gel) and standard Western blotting. Antibodies were diluted 1:500 and incubated for 1 hour. Detection was performed using anti-rat horseradish peroxidase (HRP) (DAKO, 1:1000) and Lumilight (Roche) on a LAS3000 (Fuji) darkbox imaging system. The results indicated that 7E5 could recognize both human and cynomolgus monkey C6.

[0331] coupling dynamicsTo investigate the kinetics of 7E5 binding to C6, surface plasmon resonance measurements were performed on a BIACORE 2000 (GE Healthcare) equipped with a research CM5 sensor chip. The ligand (C6, 113 kDa) was immobilized using amine-coupling chemistry. The surface of flow cell 2 was activated for 7 minutes at a flow rate of 5 µl / min with a 1:1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.4 M EDC (3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide). The ligand was immobilized at a density of 955 RU in 10 mM sodium acetate at pH 5.0 at a concentration of 10 µg / ml. The surface was blocked by injecting 1 M ethanolamine (pH 8.0) for 7 minutes.

[0332] Flow cell 1 was fixed with the antibody of the initial experiment (αvWWF; 987 RU) and used as a reference surface.

[0333] To collect kinetic binding data, the analyte (anti-C6 antibody, 150 kDa) was injected into two flow cells at a temperature of 25°C at a flow rate of 30 µl / min in 10 mM HEPES, 150 mM NaCl, 0.005% P2O, and pH 7.4. The injected concentration varied for each antibody. Data were collected at a rate of 1 Hz. The complexes were able to bind and dissociate for 90 seconds and 300 seconds, respectively. The surfaces were regenerated by injecting 0.1 M HCl for 10 seconds. Each sample and buffer blank were injected equally (in a random order) onto both surfaces.

[0334] This data was fitted to a simple 1:1 interaction model using the global data analysis options available in BiaEvaluation 4.1 software. The Biacore dynamics results are shown in Figure 2. Representative experimental results are also summarized in Table 1-4 below.

[0335]

[0336] Dynamics of 7E5 coupling determined by surface plasmon resonance

[0337]

[0338] 7E5 bond complex half-life

[0339]

[0340] Time to 5% dissociation of the 7E5 bond

[0341]

[0342] Time until 95% dissociation of the 7E5 bond

[0343] 7E5's K D is 2.5×10 -10 It is calculated as M. This high affinity is mainly caused by the high antibody-antigen complex half-life (45 hours). Therefore, 7E5 binding is very stable, and the half-life of the 7E5-C6 complex is estimated to be more than 40 hours.

[0344] To determine whether the antigen-antibody complex could be released from endosomes and lysosomes following binding by the Fc gamma receptor and cellular uptake, the sensitivity of the 7E5-C6 complex was tested at low pH. Since the pH in lysosomes is approximately 4.8, the stability of the complex was tested up to pH = 4. In this BIACORE experiment, the 7E5-C6 complex on the chip was washed with a pH-reducing buffer. Hepes buffered saline (HBS) was used at pH 7.4, 7.0, and 6.5, and 10 mM sodium acetate was used at pH 6.0, 5.5, 5.0, 4.5, and 4.0. It was observed that the stability of the complex was not sensitive to low pH.

[0345] Effect of pre-culture on hemolysis analysis The BIACORE experiment showed that the slow emission of 7E5 from C6 is the K of 7E5 DSince it was revealed that it is a primary determinant, we investigated whether pre-incubation of 7E5 with complement source (human serum) before the addition of red blood cells increased the inhibitory efficacy in the hemolysis assay. After pre-incubating 7E5 with human serum at room temperature (20°C) for 30, 90, or 180 minutes, red blood cells were added and the reaction was started at 37°C. As a result, the hemolytic inhibitory effect did not improve further even without increasing the pre-incubation time up to 3 hours. This implies that the kinetics of C6 binding by 7E5 in this reaction are equivalent to C6 being effectively fully complexed and neutralized within minutes.

[0346] Example 3: Epitope mapping of 7E5 monoclonal antibody

[0347] A peptide array was used to determine the 7E5 epitope from human C6. A continuously overlapping 16mer peptide (peptide length 16 amino acids, overlapping 14 amino acids) was synthesized from the C6 protein sequence and spotted in a grid pattern on a membrane. Subsequently, the membrane was incubated with a 7E5 antibody to detect which peptide was recognized by the antibody. The primary peptide sequence recognized by 7E5 was GSCQDGRQLEWGLERT (peptide 418) (sequence number 1).

[0348] Subsequently, an alanine scan (using alanine to replace amino acids one by one) was performed on the selected peptides to accurately reveal the epitopes. In this study, in addition to the modification of peptide 418, peptide 420 (DGRQLEWGLERTRLSS) (SEQN 2), in which 4 amino acids are shifted compared to 418, and several of its alanine modifications exhibited the binding of 7E5. Therefore, the amino acid forming part of the main epitope of 7E5 is expected to be within the peptide sequence combining peptides 418 and 420: GSCQDGRQLEWGLERTRLSS (SEQN 3).

[0349] Figure 4a shows the sequences of peptide 418 and the surrounding region of human (SEQ No. 50) and rat C6 (SEQ No. 51). As schematically illustrated in Figure 4b, peptide 418 is partially located at the end of the first FIM domain of C6.

[0350] To determine whether other antibodies bind to the same epitope as the 7E5 antibody, Biacore cross-blocking experiments were performed by binding the C6 antigen to the chip and then using a single anti-C6 antibody (control) or the first anti-C6 antibody (antibody 1), followed by the second anti-C6 antibody (antibody 2). The results of the cross-blocking experiments to determine whether mouse mAb 27B1 binds to the same epitope as the rat monoclonal antibody 7E5 are shown in Figures 3A-D, where Figure 3A shows the results using 27B1 as antibody 1 and 7E5 as antibody 2, Figure 3B shows the results using 7E5 as antibody 1 and 27B1 as antibody 2, Figure 3C shows the results for 27B1 alone, and Figure 3D shows the results for 7E5 alone.

[0351] Example 4: In vivo effects of 7E5 monoclonal antibody

[0352] To test whether 7E5 can block C6 in living animals, C6-deficient PGR rats supplemented with human C6 were used. This approach was used because 7E5 is specific to human C6 and cannot block rat C6. In C6-deficient rats, human C6 can be injected to restore full complement system functionality and MAC activity, and the effects of 7E5 can be measured without the confounding effects of rat C6.

[0353] First, this approach was tested by measuring hemolytic activity in two rats injected with human C6. By collecting multiple blood samples over time following C6 injection, the half-life of C6 in rats was estimated to be approximately 48 hours. Two C6-deficient rats were injected intravenously with 4 mg / kg of human C6. Blood samples were collected 10 minutes, 24 hours, and 48 hours after C6 injection. After all blood samples coagulated, the clots were centrifuged at 13,000 rpm in an Eppendorf table centrifuge for 10 minutes at room temperature to separate the serum. The serum was used in the hemolytic assay described in Example 1 to measure MAC activity. Serum from wild-type PVG rats and untreated C6-deficient rats were used as references for maximum and minimum hemolytic activity. Using the hemolytic assay, the half-life of human C6 in rats was estimated to be approximately 48 hours.

[0354] In a pilot experiment, a female C6-deficient PVG rat (body weight 220 g) was injected intraperitoneally with a high dose of 12 mg of 7E5 and supplemented with 2 mg of human C6 (intravenous injection). Human C6 was isolated from human serum using an affinity purifier on a column coated with C6 antibodies. C6 was administered at 1 mg doses 24 hours prior to the 7E5 bolus injection and 5 minutes later. Control rats (same body weight as the 7E5-treated rats) received only the C6 injection. Blood samples for hemolytic analysis were collected 60 minutes after the 7E5 injection. The results are shown in Figure 5. The results demonstrated that hemolytic activity was blocked by 7E5 60 minutes after administration, proving that 7E5 can block MAC formation in vivo.

[0355] In a follow-up experiment, 1 mg of C6 was injected into two C6-deficient female rats (PVG strain). Blood samples were collected before and after C6 injection (1 mg IV) to confirm normal and supplemented hemolytic activity. Ten minutes after C6 injection, 7E5 was administered as 8 mg IP or 2 mg IV. Blood samples were collected 60 minutes after 7E5 administration to evaluate the effect of 7E5 on hemolytic activity. Both dosing strategies blocked MAC activity in the blood. Then, another 1 mg of C6 was injected IV into the same rats. Blood samples taken 15 minutes after the new C6 supplementation showed only a gradual increase in hemolytic activity, but it was presumed that hemolytic activity was inhibited in both rats by free-circulating 7E5.

[0356] The experiment described above shows that 7E5 can block C6 in living animals.

[0357] Example 5: Sequencing and Recombinant Expression of 7E5 Monoclonal Antibody

[0358] The nucleotide and amino acid sequences of the heavy and light chain variable regions of the 7E5 monoclonal antibody were determined by standard procedures.

[0359] The nucleotide sequence of the VH region is as follows:

[0360] gaggtgcagctggtggagtctgatggaggcttagtgcagcctggagggtccctgaaactctcctgtgtagcctcaggattctctttcagtgactattacatggcctgggtccgccagggtccaacgaaggggctggagtgggtcgcaaccattaattatgatggtagtagtacttac tatcgagagtccgtgaagggccgattcactatctccagagataatgcgaaacgcaccctatacctgcaaatggacagtctgaggtctgaggacacggccacttattactgttcaagaccttctacggaggccctgtttgcttactggggccacggcactctggtcactgtctcctca (SEQ ID NO: 4)

[0361] The amino acid sequence of the VH region is as follows:

[0362] EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAYWGHGTLVTVSS

[0363] (Sequence No. 5)

[0364] The amino acid sequences of VH CDR1, CDR, and CDR 3 are as follows:

[0365] CDR1: DYYMA (Sequence No. 6)

[0366] CDR2: TINYDGSSTYYRESVKG (Sequence No. 7)

[0367] CDR3: PSTEALFAY (Sequence No. 8)

[0368] The nucleotide sequence of the VL region is as follows:

[0369] gatgttgtgctgacccagactccatccacattatcggctaccatggacaatcggtctccatctcttgcaggtcaagtcagagtctcttaaatgatgttggaaacacctatttatattggtatctacagaggcctggccaatctccacagcttctaatttatttggtc tccgacctgggatctggggtccccaacaggttcagtggcagtgggtcaggaacagatttcacactcaaaatcagtggagtggaggctgaggatttgggaatttattactgcatgcaagctagtcatgctccgtacacgtttggagctgggaccaacctggaactgaaa (SEQ ID NO: 9)

[0370] The amino acid sequence of the VL region is as follows:

[0371] DVVLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNLELK (SEQ ID NO: 10)

[0372] The amino acid sequences of VL CDR1, CDR, and CDR3 are as follows:

[0373] CDR1: RSSQSLLNDVGNTYLY (Sequence No. 11)

[0374] CDR2: LVSDLGS (Sequence No. 12)

[0375] CDR3: MQASHAPYT (Sequence No. 13)

[0376] Following the optimization of the coding sequence for expression in production cell lines (Hek-293 cells) and the introduction of appropriate restriction sites for cloning, an expression cassette was prepared. Human-rat chimeric recombinant antibodies were generated by cloning the heavy and light chain variable regions of the synthesized 7E5 into a set of pMQR eukaryotic expression vectors (pMQR-hIgG1 and pMQR-hIgK). Sequence analysis of the generated clones confirmed that both sequences were correctly cloned. Hek-293 cells were transfected with the pMQR eukaryotic expression vectors containing both 7E5 variable regions, and these cells were allowed to produce recombinant antibodies. After production, hIgG1 / hIgK antibodies were detected in the pulmonary supernatant via a capture ELISA. The transfection supernatant was found to contain 0.019 mg / ml of recombinant 7E5.

[0377] Example 6: Humanization of 7E5 Monoclonal Antibody

[0378] Instead of antibody humanization methods based on site-specific mutagenesis processes, rat 7E5 monoclonal antibodies were humanized using a humanization approach based on CDR-homology between human and murine antibodies, as described by Hwang and his colleagues (Methods. 2005, 36:35-42). This method is based on the principle that if non-human and human antibodies have similarly structured CDRs, the human framework also supports the non-human CDR while maintaining good affinity. In this method, human framework sequences are selected from a set of human germline genes based on the structural similarity of the human CDR to the antibody to be humanized (identical Chothian canonical structure). A phage display library of Fab variant sequences containing out-of-FR residues is generated. After selection by affinity, individual clones are screened for binding and off-rate, and sequence human identity and homology are determined.

[0379] The process of humanizing the 7E5 rat antibody applied in this work consists of the following steps:

[0380] 1 - Design of Humanized Library: Identification of Closest Human Germ Lineages and Identification of Rat VH and VK FR Residues Deviating from These Human Germ Lineages.

[0381] 2 - Assembly of 7E5 gene libraries (using nested oligonucleotides to synthetically generate variable heavy chain (VH) and light chain (VL) encoding genes via PCR).

[0382] 3 - These gene libraries are cloned into a phagemid (pCB13-CK1 / 3) containing human constant heavy chain (CH1) and light chain (Cκ) (library composition).

[0383] 4 - Selection of a functional Fab using phasing display and affinity selection.

[0384] 5 - Screening for off-rate (Biacore) and sequencing.

[0385] Selection of Fab having the highest human identity and homology without losing the binding to 6 - hC6.

[0386] 7 - Production and purification of 8 humanized leads used for additional affinity measurements and functional analysis.

[0387] Design of a Humanized Library Using the nucleotide and amino acid sequences of the variable region of rat 7E5 antibody and the public database Ant tool, it was confirmed that 7E5 uses IGHV5S45*01, IGHD1-6*01, IGHJ3*01 and IGKV2S27*01, IGKJ2-3*01 as germline segments. The canonical fold combinations for CDR H1 and CDR H2 of 7E5 are 1-3, and the canonical fold combinations for CDR L1 and CDR L2 of 7E5 are 4-1.

[0388] Comparison of the 7E5 VH sequence with the human germline having the same canonical fold combinations 1-3 for CDR1 and CDR2 revealed human germline VH3 family 1 as the closest match. The closest human JH germline is IGHJ4. Alignment for these germline segments is shown in Fig. 6a. The 7E5 heavy chain amino acid sequence is also shown in SEQ ID NO. 5. The human germline VH3_1 amino acid sequence is also shown in SEQ ID NO. 48. FR and CDR are indicated to enable the identification of FR residues deviating from the human germline.

[0389] Using similar analysis, it was confirmed that the closest human germline for the 7E5 Vκ sequence is human VK2 family 5. The closest human JH germlines are IGKJ2 and IGKJ5. The alignment for these germline segments is shown in Fig. 6b. The 7E5 light chain amino acid sequence is also shown in SEQ ID NO. 10. The human germline VK2_5 amino acid sequence is also shown in SEQ ID NO. 49. FR and CDR are indicated to enable the identification of FR residues deviating from the human germline.

[0390] As shown in Figures 6a and 6b, for the humanized libraries, there were 13 positions for the 7E5 VH sequence containing human residues and 16 positions for the 7E5 V sequence, respectively, but rat residues were also included where the change was detrimental to antigen binding. Considering the number of mutagenic positions and the number of variants per position, the library sizes capable of accommodating the introduced diversity are 8.2×10³ and 9.8×10⁴ for the VH and Vκ libraries, respectively.

[0391] Building the Humanized 7E5 Fab Library To build the final humanized 7E5 Fab phage display library, two different sub-libraries were initially built:

[0392] 1 - Fab sublibrary in which the VH humanized 7E5 VH gene is cloned along with WT 7E5Vκ into pCB13-CK3 phagemid containing the gene encoding the human invariant region CH1 and Cκ.

[0393] 2 - Humanized Fab sub-libraries in which the VL humanized 7E5 Vκ gene is cloned into WT 7E5VH and phagemid vectors pCB13-CK1 and pCB13-CK3 containing genes encoding the human invariant regions CH1 and Cκ.

[0394] Due to the cloning strategy and the sequences of the two different phagemids used, the residues at positions 104 to 107 of the light chain V region of the clone produced from pCB13-CK1 will correspond to LEIK (humanized 7E5 sequence), but the light chain of the V region of the clone produced from pCB13-CK3 will show amino acid LELK (7E5 WT Vκ sequence) at the same position.

[0395] To proceed with the construction of the final Fab library in which both heavy and light chains were humanized, two final sub-libraries were panned against human C6, and the combined clone was recovered.

[0396] Synthetic Gene Assembly : To construct different humanized heavy and light chain sublibraries, humanized 7E5 VH and Vκ genes were generated by gene assembly (Cherry, J. et al. (2008) J Biochem Biophys Methods, 70: 820-2, Stemmer, WP et al. (1995) Gene, 164: 49-53).

[0397] Building Humanized 7E5 VH and Vκ Sub-libraries To construct the 7E5 VH Fab sub-library, a synthetic VH gene of approximately 400 bp generated by gene assembly and a DNA fragment encoding 7E5 VWTW were cloned into pazimid pCB13-CK3 (containing human constant heavy chain and kappa light chain encoding genes).

[0398] To construct the 7E5 Vκ Fab sub-library, a DNA fragment encoding a synthetic Vκ gene of approximately 400 bp and 7E5 VH WT generated by gene assembly was cloned into an equimolar mixture of phagemids pCB13-CK1 and pCB13-CK3 (containing human constant heavy chain and kappa light chain encoding genes), respectively, via the ApaLI / XhoI site and NcoI / NheI site.

[0399] The novel vector generated during the cloning process was transformed into E. coli TG1 cells by electroporation. The library size was calculated from 5 µl spots of TG1 transformed cells in LBA Carbenicillin (100 µg / ml) and Glucose 2%, and the percentage of Fab insertions was determined by colony PCR. The sub-library sizes and insertion percentages are summarized in Table 5 below.

[0400] Size and insertion percentage obtained from the Humanization 7E5 sub-library sub-library Library size Insert % Final library size Maximum theoretical diversity Scope of diversity application (exceeding theoretical diversity) Humanization 7E5 VH 2.4 x 10 8 95% 2.3 x 10 8 8.2 x 10 3 ~28,000 folds Humanization 7E5 Vκ 1.7 x 10 8 91% 1.5 x 10 8 9.8 x 10 4 ~1,500 folds

[0401] The sub-libraries were also QCed by DNA sequencing of 48 clones per library. Amino acid sequences were extracted using CLC Main Workbench Software. As a result of analyzing the frequency of WT or mutated residues per position and valid VH and Vκ sequences, the sub-libraries were successfully designed and constructed with a WT / mutation ratio of approximately 50 / 50 (33 / 33 / 33 for the 103rd position of the V gene), and the average FR mutation number was obtained as designed.

[0402] Panning selection of humanized 7E5 VH and Vκ sublibraries : Phages were prepared from two sub-libraries and used for primary selection on coated human C6. The goal of this selection round was to clean sub-libraries from non-coupled Fabs, and therefore strict conditions were not applied.

[0403] For panning selection, 5 and 0 µg / ml of human C6 were coated onto 96-well Maxisorp plates (Nunc) and blocked with low-fat milk powder (Marvell 4% in PBS). After incubation for 2 hours with sub-library phage and subsequent washing, trypsin elution (10 mg / ml) was performed at room temperature. Protease activity was immediately neutralized by applying the 16 mM protease inhibitor ABSF.

[0404] All phage products were infected into logarithmically grown E. coli TG1 cells, and 5 µl of the infected bacteria were cultured on agar plates (LBAGluc 2% Carb 100 µg / ml) for output analysis and concentration measurements. Concentration was calculated as the ratio between the number of phages eluted from human C6 and the number of phages eluted from protein-free conditions. Excellent concentrations were observed for both humanized 7E5 Vκ and VH Fab sub-libraries compared to the background (PBS).

[0405] Creation of the Final Humanization 7E5 Fab Pag Display Library The final humanized 7E5 Fab library is constructed by combining humanized heavy chains (VHCH) recovered from clones selected from the 7E5 VH Fab sub-library with humanized light chains (VκCκ) recovered from the 7E5 VH Fab sub-library. The size of the generated library was calculated from 5 µl spots of TG1-transformed cells in LBA Carbenicillin (100 µg / ml) and Glucose 2%, and the percentage of Fab insertion was determined by colony PCR.

[0406] Humanization 7E5 Fab Library SelectionTo select humanized variants with no loss of affinity or improved affinity compared to rat WT 7E5 antibodies, in-solution phage display selection using a humanized 7E5 Fab library was performed using biotinylated hC6 antigens. To detect biotinylated C6 captured on nutravidin-coated plates, human C6 was biotinylated and QC was performed by SDS-PAGE, Western blot, and ELISA using the anti-human C6 antibody 7E5. Three consecutive rounds of affinity selection were performed in which antigen concentrations were reduced from round to round, and phage inputs were also reduced from round 1 to round 2. In the second and third selections, phages incubated with nutravidin-captured human C6 were incubated for 2 hours or overnight (off-rate selection) in the presence of excess non-biotinized C6 after several washes to remove high off-rate binding clones. As a control, in parallel, similar selection was performed where phages were cultured with nutravidin-captured human C6 and PBS instead of non-biotinized hC6 (no off-rate selection).

[0407] All selected phage products were infected into logarithmically grown E. coli TG1 cells, and 5 µl of the infected bacteria were plate-cultured on agar plates (LBAGluc 2% Carb 100 µg / ml) for output analysis and concentration measurement. Concentration was calculated as the ratio between the number of phages eluted from human C6 and the number of phages eluted from protein-free conditions. A very high concentration was obtained compared to the background (PBS).

[0408] Combined screening of selected clones from the Humanization 7E5 Fab library :

[0409] Individual colonies of E. coli TG1 infected with eluted phage pools obtained after the second and third rounds of off-rate selection were grown for 8 hours at 37°C in two 96-well plates (master plates) containing 100 µl of 2TYGlucose 2% Carbenicillin. They were stored in 20% glycerol at 100 µg / ml at -80°C and used for subsequent sequencing and cytoplasmic extract generation. A total of two master plates (MPs) were generated from the clones from the second round and the clones from the third round. Bacterial extracts containing soluble monoclonal Fab (cytoplasmic extract) were produced from these MPs. Small-scale cultures of monoclonal bacteria were induced to an OD600 of 0.8 by adding isopropyl-bD-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Cytoplasmic extracts (PEs) containing Fab were prepared by freeze-thawing of bacterial pellets in PBS and subsequent centrifugation to remove cell debris.

[0410] To determine the target binding ability of the selected clones, 1:5 diluted PEs were tested for binding to 10 nM biotinylated hC6 captured on neutravidin-coated Maxisorp plates. PEs prepared from rat 7E5 WT Fab were used as a positive control. Blank PEs (prepared from uninoculated wells in MP) were used as a negative control. Binding of the PEs to targets was detected using anti-c-myc mouse antibodies conjugated to HRP (Horseradish peroxidase). A binding hit rate of 40% was obtained for both MPs (OD 450 nm value), and the binding signal of the positive clones was similar to the signal obtained from the parent rat 7E5 Fab.

[0411] Off-rate screening of human identity of selected clones and homology of selected human C6 combined clonesFor positively coupled clones, the off-rate for hC6 was determined using the SPR method, and simultaneously, the DNA encoding the variable regions of the heavy and light chains was sequenced.

[0412] A Biacore 3000 (GE Healthcare) was used to determine the off-rate. For this purpose, 50 µg / ml of hC6 in acetate buffer pH 4.5 was fixed at approximately 2000 RU on a CM5 sensor chip (GE Healthcare BR-1000-12). Regeneration conditions were tested, and 2×10 µl of 10 mM NaOH and 1 M NaCl were used for regeneration between sample injections. 30 µl of PE prepared as previously described was diluted in 120 µl of HBS-EP buffer, and 60 µl of this was injected at a flow rate of 30 µl / min. Dissociation was measured for 400 seconds, and the off-rate was determined by applying a 1:1 Langmuir dissociation fitting model.

[0413] To analyze human identity and homology, DNA encoding the variable heavy and light chains of clones exhibiting specific binding to hC6 was sequenced. Amino acid sequences were extracted using CLC Main Workbench Software. Vκ and VH sequences were individually aligned to a reference sequence (7E5 WT). All sequences were analyzed to determine human identity (the proportion of framework residues found in the closest match germline) and human homology (the proportion of framework residues found in the closest match germline or other germlines of the same subclass) using Abligner software.

[0414] Overall, a good correlation was observed between the ELISA and Biacore data, and excellent human identity and homology percentage values ​​of 88-99% were observed.

[0415] A lead panel of eight clones with excellent binding, off-rate, and human identity and homology data, designated as 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02, was selected. The complete nucleotide and amino acid sequences of the variable regions of the heavy and light chains of the lead panels of the eight humanized clones are shown below:

[0416] 8G09 VH and VL nucleotide sequences:

[0417] 8G09 VH

[0418] GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTAC TATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAACGCACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCCTCA (SEQ ID NO: 14)

[0419] 8G09 Vκ

[0420] GATATTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA (서열번호 15)

[0421] 7E12 VH and VL nucleotide sequences :

[0422] 7E12 VH

[0423] GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 16)

[0424] 7E12 Vκ

[0425] GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAACCTCGAGATCAAA (서열번호 17)

[0426] 7G09 VH and VL nucleotide sequences :

[0427] 7G09 VH

[0428] GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 18)

[0429] 7G09 Vκ

[0430] GATGTTGTGCTGACCCAGACTCCATCGTCATTATCGGTTACCCCTGGACAATCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAAACTCGAGCTGAAA (서열번호 19)

[0431] 8F07 VH and VL nucleotide sequences :

[0432] 8F07 VH

[0433] GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCAGGATTCTCTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 20)

[0434] 8F07 Vκ

[0435] GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAAACTCGAGATCAAA (서열번호 21)

[0436] 7F06 VH and VL Nucleotide Sequence :

[0437] 7F06 VH

[0438] GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGGGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 22)

[0439] 7F06 Vκ

[0440] GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA (서열번호 23)

[0441] 7F11 VH and VL Nucleotide Sequences :

[0442] 7F11 VH

[0443] GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTTCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 24)

[0444] 7F11 Vκ

[0445] GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA (서열번호 25)

[0446] 7E11 VH and VL nucleotide sequences :

[0447] 7E11 VH

[0448] GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCCTCA (서열번호 26)

[0449] 7E11 Vκ

[0450] GATATTGTGCTGACCCAGACTCCATTGTCATTATCGGCTACCCCTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAGGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAACCTCGAGATCAAA (서열번호 27)

[0451] 7F02 VH and VL nucleotide sequences :

[0452] 7F02 VH

[0453] GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCGTTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTCACTGTCTCCTCA (서열번호 28)

[0454] 7F02 Vκ

[0455] GATGTTGTGATGACCCAGACTCCATCGACATTATCGGCTACCCCTGGACAATCGGCCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCCAATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTACTGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA (서열번호 29)

[0456] 8G09 VH and VL amino acid sequences :

[0457] 8G09 VH

[0458] EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAYWGQGTLVTVSS (서열번호 30)

[0459] 8G09 Vκ

[0460] DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLEIK (서열번호 31)

[0461] 7E12 VH and VL amino acid sequences :

[0462] 7E12 VH

[0463] EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAYWGHGTLVTVSS (서열번호 32)

[0464] 7E12 Vκ

[0465] DVVLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNLEIK (서열번호 33)

[0466] 7G09 VH and VL amino acid sequences :

[0467] 7G09 VH

[0468] EVQLVESDGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAYWGHGTLVTVSS (서열번호 34)

[0469] 7G09 Vκ

[0470] DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLEIK (서열번호 35)

[0471] 8F07 VH and VL amino acid sequences :

[0472] 8F07 VH

[0473] EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAYWGHGTLVTVSS (서열번호 36)

[0474] 8F07 Vκ

[0475] DVVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKLEIK (서열번호 37)

[0476] 7F06 VH and VL amino acid sequences :

[0477] 7F06 VH

[0478] EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAYWGHGTLVTVSS (서열번호 38)

[0479] 7F06 Vκ

[0480] DVVLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLELK (서열번호 39)

[0481] 7F11 VH and VL amino acid sequences :

[0482] 7F11 VH

[0483] EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPTKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAYWGHGTLVTVSS (서열번호 40)

[0484] 7F11 Vκ

[0485] DVVLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRLEIK (서열번호 41)

[0486] 7E11 VH and VL amino acid sequences:

[0487] 7E11 VH

[0488] EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAYWGQGTLVTVSS (서열번호 42)

[0489] 7E11 Vκ

[0490] DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNLEIK (서열번호 43)

[0491] 7F02 VH and VL amino acid sequences :

[0492] 7F02 VH

[0493] EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAYWGHGTLVTVSS (서열번호 44)

[0494] 7F02 Vκ

[0495] DVVMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRLELK (서열번호 45)

[0496] The alignment of the amino acid sequences of the rat 7E5 heavy chain variable region to the amino acid sequences of the heavy chain variable region of humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 is shown in Fig. 8a along with CDRs 1, 2, and 3. The alignment of the amino acid sequences of the rat 7E5 light chain variable region to the amino acid sequences of the light chain variable region of humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02 is shown in Fig. 8b along with CDRs 1, 2, and 3. The heavy chain CDR1, 2, and 3 sequences for the eight human variants of the rat 7E5 antibody are identical to the amino acid sequences of the rat 7E5 monoclonal antibody (their amino acid sequences appear in SEQ ID NOs 6, 7, and 8, respectively). Likewise, the light chain CDR1, 2, and 3 sequences for the eight human variants of the rat 7E5 antibody are identical to the amino acid sequences of the rat 7E5 monoclonal antibody (their amino acid sequences appear in SEQ ID NOs 11, 12, and 13, respectively).

[0497] Fab expression, purification, and QC : To characterize a portion of the 7E5 humanized variant in further analysis (i.e., complement-mediated lysis, affinity measurements, melting temperature, and agglutination behavior analysis of pre-sensitized erythrocytes), soluble Fab was prepared and purified from the lead panel of the eight clones described above. The Fab genes of all eight humanized clones and the 7E5 WT control were cloned into a pCB4 expression vector (very similar to pCB13 but without gene 3 standardization sequences) via SfiI / NotI digestion and transformed into TG1 E. coli strains via heat shock. Sequences were verified using CLC Main Workbench Software.

[0498] The production of PEs containing soluble Fab from pCB4-cloned 7E5 humanized variants and 7E5 WT was carried out in 800 ml of 2xYT with the addition of 0.1% glucose and 100 µg / ml of carbenicillin. After inducing an OD 600 of 0.5–0.8 with IPTG at a final concentration of 1 mM, the culture medium was incubated for at least 20 hours, with a 24-hour period. The soluble Fab was purified with a TALON metal-affinity resin.

[0499] When 500 ng of the generated purified product was subjected to SDS-PAGE, several extra bands were observed, excluding the Fab-specific band (50 kDa and approximately 25 kDa under non-reducing and reducing conditions, respectively). To further purify these samples, a Life Technologies resin containing VHH that specifically binds to the human CH1 domain (CaptureSelect™ affinity resin IgG-CH1, cat# 194320005) was used in accordance with the manufacturer's instructions. The concentration of the generated purified protein was estimated by measuring the OD280 nm using a microvolume spectrophotometer and assuming a molar extinction coefficient of ε = 1.53. SDS-PAGE analysis of the purified samples showed high purity. The function of the purified Fab was confirmed in ELISA, and the binding of serial dilutions of these Fabs to 10 nM biotinylated hC6 captured on neutravidin-coated Maxisorp plates was examined. All 8 purified Fabs showed effective binding to hC6.

[0500] Biacore AnalysisTo determine whether humanization of 7E5 altered the binding specificity or activity of the resulting humanized antibodies, Biacore affinity analysis was performed on eight selected humanized Fabs (7E12, 7E11, 7F2, 7F6, 7F11, 7G9, 8F7, and 8F9) compared to (parental) wild-type rat 7E5 mAb and mouse 27B1 mAb. The results are shown in Figure 9. The results indicate that humanization of 7E5 did not alter the specificity or activity of the antibodies.

[0501] Example 7: "Mix and Match" Characterization of Humanized Anti-C6 Antibodies

[0502] In this experiment, a panel of humanized VH and VL chains derived from selected humanized anti-C6 antibodies was expressed as full-length antibodies in mammalian cells in various combinations, and their functional activity was evaluated.

[0503] The humanized VH chains used were the eight VH chains (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02) described in Example 6, as well as chain 9, 7C02, whose amino acid sequence corresponds to the amino acid sequence of SEQ ID NO. 46. The alignment of these nine chains is shown in FIG. 8a.

[0504] The humanized VL chains used were the eight VH chains (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, and 7F02) described in Example 6, as well as chain 9, 7G08, which represents the amino acid sequence of SEQ ID NO. 47. The alignment of these nine chains is shown in FIG. 8b.

[0505] To generate a coding sequence for the full-length chain containing a stabilized IgG4 (S228P) constant region, heavy and light chain nucleotide sequences were cloned into an expression vector. In all possible combinations in CHO host cells, 9 heavy chains and 9 light chains were co-expressed in pairs. Thus, 81 possible "mix and match" combinations of 9 heavy chains and 9 light chains were evaluated. The 81 pairs were tested in a hemolysis assay and MAC ELISA, respectively. For each assay, 4 µg of humanized 7E5 mAb from the CHO supernatant was used. The results of the hemolysis assay are shown in Fig. 7A. The results for the MAC ELISA are shown in Fig. 7B. The results demonstrate that the 81 possible "mix and match" combinations of 9 VH and 9 VL chains exhibit potent inhibitory activity in both assays.

[0506] Example 8: Animal model for evaluating the effect of C6 antibodies on nerve regeneration

[0507] A nerve compression model (compression of the sciatic nerve) is used to test the effects on the recovery of sensory function in C6-knockout rats (PVC) supplemented with human C6 anti-human C6 monoclonal antibodies, such as rat 7E5 or humanized 7E5. Nerve compression is a model of peripheral nerve injury. See WO 2010 / 005310 (PCT / NL2009 / 050418); and de Jonge et al. (2004) Hum Mol Genet. 13(3):295-302.

[0508] For treatment, C6 - / - rats (PVG, 6–8 weeks) were supplemented with human C6 or a control (PBS). C6 was administered intravenously to C6 - / - rats at a dose of 4 mg / kg in PBS prior to compression injury (Day 1) and once daily from Day 0 to 6. C6-supplemented rats and the control group were treated with anti-human C6 mAb (4 mg / rat intraperitoneal injection) 10 minutes prior to compression (Day 0). PVG rats were treated again with anti-human C6 mAb (4 mg / rat IP) 5 minutes prior to nerve compression. Subsequent doses of anti-human C6 mAb were administered on Days 1 through 6 (4 mg / rat IP). Control animals were subjected to the same nerve compression but were not treated with antibodies. To study the histology of the nerves, a portion of the animals was sacrificed 72 hours after crushing. 72 hours was selected because Wallerian degeneration is at its maximum in WT animals, and this time point is very useful for evaluating therapeutic efficacy.

[0509] Nerve compression was performed as follows. All surgical procedures were performed aseptically under deep isoflurane anesthesia (isoflurene 2.5 vol%, 1 L / min O2 and 1 L / min N2O). The left thigh was constricted, and the sciatic nerve was exposed through an incision in the femur. The nerve was compressed three times for 10 seconds at the level of the sciatic notch using soft, curved forceps (No. 7), and the area where the nerve was compressed appeared completely translucent. The right leg was used as an internal control. The muscles and skin were sutured.

[0510] Table 6 below is the experimental setup for treatment with recombinant anti-human C6 mAb 7E5 (12 mg / kg):

[0511] Experimental setup for nerve compression experiments Rat number Temgesic Reconstituted (4 mg / kg) pre-bleeding Treatment (12 mg / Kg) Compression injury Post-hemorrhage 1 yes C6 yes 7 E 5 yes yes 2 yes C6 yes 7 E 5 yes yes 3 yes C6 yes 7 E 5 yes yes 4 yes C6 yes 7 E 5 yes yes 5 yes C6 yes 7 E 5 yes yes 6 yes C6 yes PBS yes yes 7 yes C6 yes PBS yes yes 8 yes C6 yes PBS yes yes 9 yes doesn't exist yes PBS yes yes 10 yes doesn't exist yes PBS yes yes

[0512] On the third day after injury, all animals were intracardiaclysed with 4% paraformaldehyde in piperazine-N-N'-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.6). The left and right sciatic nerves were removed from each animal, and a 5 mm long segment was collected from the compression site. Each segment was conventionally treated with paraffin wax for immunohistochemistry.

[0513] 7-micron thick paraffin sections were placed on Super Frost Plus slide glasses (Knittel Glass, Germany). The sections were deparaffinized and rehydrated. Epitopes were exposed by heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0). Non-specific binding of the antibody was blocked using 10% normal goat serum in PBS (DAKO, Glostrup, Denmark) for 20 minutes at room temperature. The primary antibody was diluted in normal antibody diluent (Immunologic, Duiven, Netherlands) and incubated at room temperature for 1 hour. Detection was performed by incubating the sections in goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugate or sheep anti-mouse Cy3-conjugated IgG from Sigma-Aldrich (St. Louis, MO) diluted 1:200 in 1% bovine serum albumin. When instructed, slides were counterstained with 4,6-diamidin-2-phenylindole (DAPI) (Sigma-Aldrich) and mounted on Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Images were taken using a digital camera (DP12, Olympus, Zoeterwoude, The Netherlands) attached to a fluorescence microscope (Vanox, AHBT3, Olympus, The Netherlands).

[0514] The results are shown in Fig. 10. Cells were stained with anti-C9 for MAC detection, anti-pan-neurofilament (SMI312) for axon detection, anti-myelin basic protein (MBP) for myelin detection, and phagocytes (macrophages) for lysosomal membrane detection (CD68). Panel A shows the results for an intact sciatic nerve, demonstrating the absence of MAC, strong axonal staining, annular myelin staining, and the absence of activated macrophages. Panel B shows MAC precipitation, loss of axons and myelin, and macrophage influx as the results following injury in rats with normal complement activity. Panel C shows the results after treatment of C6-reconstituted rats with anti-C6, demonstrating that the antibody completely blocks MAC formation, inhibits axonal and myelin destruction, and suppresses macrophage influx. Panel D shows the results for unreconstituted C6 - / - rats, indicating the absence of MAC deposition and rapid neurodegeneration.

[0515] Therefore, the results of the nerve compression experiment demonstrate the efficacy of the antibody in vivo in an animal model of peripheral nerve injury by successfully blocking MAC formation, destroying axons and myelin, and inhibiting macrophage influx.

[0516] Equivalents

[0517] A person skilled in the art will be able to recognize or identify a number of equivalents of the specific embodiments of the invention described herein by using only ordinary experiments. Such equivalents are intended to be included in the following claims.

[0518] Summary of sequence list

[0519]

[0520]

Claims

Claim 1 An antibody or an antigen-binding fragment thereof that binds to complement component C6 (C6), wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1, 2, and 3 sequences shown in SEQ ID NOs 6, 7, and 8, respectively, and light chain CDR1, 2, and 3 sequences shown in SEQ ID NOs 11, 12, and 13, respectively, wherein (a) the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44, and 46; and (b) the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45, and 47. Claim 2 In claim 1, the antibody or its antigen-binding fragment is selected from the group consisting of: (a) an antibody or its antigen-binding fragment comprising a heavy chain variable region having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 30 and a light chain variable region having at least 90% identical to the sequence shown in SEQ ID NO. 31; (b) an antibody or its antigen-binding fragment comprising a heavy chain variable region having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 32 and a light chain variable region having at least 90% identical to the sequence shown in SEQ ID NO. 33; (c) an antibody or its antigen-binding fragment comprising a heavy chain variable region having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 34 and a light chain variable region having at least 90% identical to the sequence shown in SEQ ID NO. 35; (d) an antibody or its antigen-binding fragment comprising a heavy chain variable region having an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 36 and a light chain variable region having at least 90% identical to the sequence shown in SEQ ID NO. 37 Antigen-binding fragment; (e) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 38 and a light chain variable region comprising at least 90% identical to the sequence shown in SEQ ID NO. 39; (f) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 40 and a light chain variable region comprising at least 90% identical to the sequence shown in SEQ ID NO. 41; (g) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to the sequence shown in SEQ ID NO. 42 and a light chain variable region comprising at least 90% identical to the sequence shown in SEQ ID NO. 43;and (h) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having at least 90% identical amino acid sequence to the sequence shown in SEQ ID NO. 44 and a light chain variable region having at least 90% identical to the sequence shown in SEQ ID NO. 45.; Claim 3 An antibody or an antigen-binding fragment thereof, wherein, in claim 1, (a) the heavy chain variable region comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 30, 32, 34, 36, 38, 40, 42, 44 and 46; and (b) the light chain variable region comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45 and 47. Claim 4 In paragraph 3, the antibody or its antigen-binding fragment is selected from the group consisting of: (a) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 30 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 31; (b) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 32 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 33; (c) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 34 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 35; (d) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 36 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 37 Antigen-binding fragment; (e) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 38 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 39; (f) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 40 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 41; (g) an antibody or its antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO. 42 and a light chain variable region at least 95% identical to the sequence shown in SEQ ID NO. 43;and (h) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having at least 95% identical amino acid sequence to the sequence shown in SEQ ID NO. 44 and a light chain variable region having at least 95% identical to the sequence shown in SEQ ID NO. 45.; Claim 5 In claim 1, the antibody or its antigen-binding fragment exhibits at least three of the following characteristics: (a) an IC of 0.5 μg / ml or less in a hemolytic assay. 50 (b) having; K measured by surface plasmon resonance D 1×10 -8 (c) antibody-C6 binding half-life measured by surface plasmon resonance is 40 hours or more; and (d) cross-reacts with C6 of cynomolgus monkeys. Claim 6 In claim 5, the antibody or its antigen-binding fragment has an IC of 0.5 μg / ml or less in the hemolytic analysis. 50 An antibody or its antigen-binding fragment that inhibits C6 activity. Claim 7 In claim 1, the antibody or its antigen-binding fragment is a humanized or chimeric antibody or its antigen-binding fragment. Claim 8 In claim 1, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment of IgG1 isotype, IgG2 isotype, or IgG4 isotype. Claim 9 An expression vector comprising a nucleotide sequence encoding an antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof. Claim 10 In claim 9, the expression vector comprises a nucleotide selected from the group consisting of sequences shown in SEQ ID NOs 14, 16, 18, 20, 22, 24, 26, and 28. Claim 11 In claim 9, the expression vector comprises a nucleotide selected from the group consisting of sequences shown in SEQ ID NOs 15, 17, 19, 21, 23, 25, 27, and 29. Claim 12 In claim 10, the expression vector comprises the nucleotide sequence shown in SEQ ID NO.

16. Claim 13 In claim 10, the expression vector comprises the nucleotide sequence shown in SEQ ID NO.

17. Claim 14 In claim 12, the expression vector further comprises the nucleotide sequence shown in SEQ ID NO.

17. Claim 15 A host cell transformed with the expression vector of claim 9, wherein the host cell is not present in the human body. Claim 16 A pharmaceutical composition for treating an immune-mediated inflammatory disease or a progressive neurodegenerative disease, wherein the pharmaceutical composition comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8; and a carrier, wherein the composition promotes nerve regeneration, promotes the recovery of damaged or degenerated nerves, or reduces or delays neurodegeneration. Claim 17 A pharmaceutical composition for treating physical damage to a nerve, wherein the pharmaceutical composition comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8; and a carrier, wherein the composition promotes nerve regeneration or promotes the recovery of a damaged or degenerated nerve, and wherein the damaged or degenerated nerve is caused by physical damage to the nerve. Claim 18 A pharmaceutical composition according to claim 17, wherein the physical injury is a traumatic injury, a surgical injury, or a non-traumatic injury. Claim 19 A pharmaceutical composition according to claim 17, wherein the physical damage is to the Peripheral Nervous System (PNS) or the Central Nervous System (CNS). Claim 20 A pharmaceutical composition in which the above disease is acquired, as described in Clause 16. Claim 21 In Clause 16, a pharmaceutical composition in which the above disease is hereditary. Claim 22 A pharmaceutical composition according to claim 16, wherein the disease is chronic demyelinating neuropathy. Claim 23 A pharmaceutical composition according to claim 16, wherein the disease is a neurodegenerative disease. Claim 24 A pharmaceutical composition according to claim 16, wherein the disease is entrapment syndromes, carpal tunnel syndrome, Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), multiple sclerosis (MS), Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy; HMSN) or Huntington's disease (HD). Claim 25 A pharmaceutical composition according to claim 16, wherein the disease is amyotrophic lateral sclerosis (ALS). Claim 26 A pharmaceutical composition according to claim 16, wherein the disease is myasthenia gravis. Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete

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