Anti-C5 antibodies and their use

Abstract

To provide a method for producing a monoclonal antibody that bonds to a complement factor 5 (C5) protein.SOLUTION: A method for producing an antibody or an antigen-binding fragment thereof which specifically binds to a complement factor 5(C5) protein, and includes a heavy chain variable region (HCVR) including a specific amino acid sequence which includes HCDR1, HCDR2 and HCDR3 of the HCVR, and a light chain variable region (LCVR) including a specific amino acid sequence which includes LCDR1, LCDR2 and LCDR3 of the LCVR, where the method includes introducing a polynucleotide coding the HCVR and the LCVR into a host cell, culturing the host cell under a condition capable of producing the antibody or an antigen-binding fragment thereof, then recovering the antibody or an antigen-binding fragment thereof produced in this manner.SELECTED DRAWING: None

Description

Technical Field

[0001] This application was filed as a PCT international patent application on June 13, 2017, and claims the benefit of priority to U.S. Provisional Application No. 62 / 349,705, filed on June 14, 2016; No. 62 / 405,561, filed on October 7, 2016; and No. 62 / 422,107, filed on November 15, 2016 (the disclosures of each are incorporated herein by reference in their entireties).

[0002] Field of the Invention The present invention relates to antibodies and antigen-binding fragments of antibodies that specifically bind to complement factor C5, and to therapeutic and diagnostic methods using such antibodies.

Background Art

[0003] Background of the Invention The complement system is a group of plasma proteins that, when activated, result in target cell lysis and promote phagocytosis through opsonization. Complement is activated by a series of proteolytic steps via three major pathways: the classical pathway, which is typically activated by immune complexes, the alternative pathway, which can be induced by unprotected cell surfaces, and the mannose-binding lectin pathway. All three pathways of the complement cascade converge on the proteolytic cleavage of complement component 5 (C5) protein. Cleavage of complement component 5 (C5) results in the generation of fragments C5a and C5b, which are critical processes during activation of the complement cascade. C5a can elicit pleiotropic physiological responses through its receptor (Non-Patent Document 1). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates oxidative burst, and induces the release of various inflammatory mediators such as histamine or cytokines. C5b mediates the formation of the membrane attack complex (MAC, or C5b-9), which results in cell lysis at the late stage of complement-dependent cytotoxicity (CDC). Furthermore, in nucleated cells that are resistant to cell lysis by C5b-9, a sublytic amount of C5b-9 can cause cell activation, which results in cell proliferation, production of pro-inflammatory mediators, and production of extracellular matrix.

[0004] Monoclonal antibodies against C5 are known in the art and are described, for example, in Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, Patent Document 7, Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11, Patent Document 12, Patent Document 13, Patent Document 14, Patent Document 15, Patent Document 16, Patent Document 17, Patent Document 18, Patent Document 19, Patent Document 20, Patent Document 21, as well as Patent Document 22, Patent Document 23, Patent Document 24, Patent Document 25, Patent Document 26, and Patent Document 27.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Patent Document 15

Patent Document 16

Patent Document 17

Patent Document 18

Patent Document 19

Patent Document 20

Patent Document 21

Patent Document 22

[0006] [Non-Patent Document 1] Monk et al 2007, Br.J.Pharmacol.152:429-448 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] Fully human antibodies that specifically bind to C5 protein with high affinity and have improved pharmacokinetic properties can be important in the prevention and treatment of various C5-related diseases (e.g., atypical hemolytic uremic syndrome). [Means for Solving the Problems]

[0008] [Brief Summary of the Invention] The present invention provides an antibody that specifically binds to complement factor 5 (C5) protein and antigen-binding fragments thereof. The antibodies of the present invention are particularly useful for inhibiting or neutralizing the activity of C5 protein. In certain embodiments, the antibodies are useful in preventing, treating, or alleviating at least one symptom or sign of a C5-related disease or disorder in a subject. In certain embodiments, the antibodies can be administered prophylactically or therapeutically to a subject having or at risk of having a C5-related disease or disorder. In certain embodiments, the anti-C5 antibody is a fully human antibody that binds to C5 with high affinity and has improved pharmacokinetic (PK) and pharmacodynamic (PD) properties. Such high-affinity antibodies with improved PK / PD can be used to provide excellent efficacy with less frequent dosing in subjects having a C5-related disease or disorder.

[0009] The antibodies of the present invention can be full-length (e.g., IgG1 or IgG4 antibodies) or can contain only the antigen-binding portion (e.g., Fab, F(ab’) 2 or scFv fragment) and may be modified to affect function, e.g., to increase persistence in a host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.

[0010] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody that specifically binds to C5 protein or an antigen-binding fragment thereof. In some embodiments, the antibody is a fully human monoclonal antibody.

[0011] Exemplary anti-C5 antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 shows the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of the exemplary anti-C5 antibodies. Table 2 shows the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-C5 antibodies.

[0012] The present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0014] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any one of the HCVR amino acid sequences listed in Table 1 paired with any one of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 98 / 114, 122 / 106, 98 / 130, 138 / 106, 146 / 106, 122 / 130, 146 / 114, 146 / 130, 138 / 130, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 258, 274 / 282, 290 / 298, 306 / 314, 322 / 330, and 338 / 346. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NO: 50 / 58 (e.g., H4H12161P), 98 / 106 (e.g., H4H12166P), 138 / 106 (e.g., H4H12166P5), or 202 / 210 (e.g., H4H12170P). In certain embodiments, the present invention provides an anti-C5 antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 having 5 or fewer amino acid substitutions, and the LCVR comprises an amino acid sequence listed in Table 1 having 2 or fewer amino acid substitutions. For example, the present invention provides an anti-C5 antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 having 5 or fewer amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO: 106 having 2 or fewer amino acid substitutions. In another embodiment, the present invention provides an anti-C5 antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 having at least 1 amino acid substitution, and the LCVR comprises the amino acid sequence of SEQ ID NO: 106 having 1 amino acid substitution.

[0015] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0016] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0017] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0018] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acids listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0019] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0020] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising any one of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0021] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) and an LCDR3 amino acid pair (HCDR3 / LCDR3) comprising any one of the HCDR3 amino acid sequences listed in Table 1 paired with any one of the LCDR3 amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-C5 antibodies listed in Table 1. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 56 / 64 (e.g., H4H12161P), 104 / 112 (e.g., H4H12166P), 144 / 112 (e.g., H4H12166P5), and 208 / 216 (e.g., H4H12170P).

[0022] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid, an HCDR2 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid, and an HCDR3 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid. In certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid, an LCDR2 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid, and an LCDR3 comprising an amino acid sequence that differs from the amino acid sequence listed in Table 1 by 1 amino acid. For example, the present invention provides an anti-C5 antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that differs from SEQ ID NO: 100 by 1 amino acid, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that differs from SEQ ID NO: 102 by 1 amino acid, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that differs from SEQ ID NO: 104 by 1 amino acid. In another exemplary embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that differs from SEQ ID NO: 108 by 1 amino acid, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that differs from SEQ ID NO: 110 by 1 amino acid, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that differs from SEQ ID NO: 112 by 1 amino acid.

[0023] The present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 353, or a substantially similar sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0024] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 354, or a substantially similar sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0025] In certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 353, or a substantially similar sequence having at least 80% or at least 90% sequence identity thereto; and a light chain comprising the amino acid sequence of SEQ ID NO: 354, or a substantially similar sequence having at least 80% or at least 90% sequence identity thereto.

[0026] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 52-54-56-60-62-64 (e.g., H4H12161P), 100-102-104-108-110-112 (e.g., H4H12166P), 140-142-144-108-110-112 (e.g., H4H12166P5), and 204-206-208-212-214-216 (e.g., H4H12170P).

[0027] In related embodiments, the invention provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-C5 antibodies listed in Table 1. For example, the invention includes an antibody or an antigen-binding fragment thereof comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50 / 58 (e.g., H4H12161P), 98 / 106 (e.g., H4H12166P), 138 / 106 (e.g., H4H12166P5), or 202 / 210 (e.g., H4H12170P). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify the CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0028] In certain embodiments, the invention includes an antibody or antigen-binding fragment thereof that specifically binds to C5, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), wherein the HCVR comprises: (i) the amino acid sequence of SEQ ID NO: 98, (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 98, (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 98; or (iv) an amino acid sequence of SEQ ID NO: 98 having 5 or fewer amino acid substitutions; and the LCVR comprises: (i) the amino acid sequence of SEQ ID NO: 106, (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 106, (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 106; or (iv) an amino acid sequence of SEQ ID NO: 106 having 5 or fewer amino acid substitutions.

[0029] The invention includes anti-C5 antibodies having an altered glycosylation pattern. In some embodiments, modifications that remove undesirable glycosylation sites, e.g., antibodies lacking a fucose moiety present on the oligosaccharide chain to increase antibody-dependent cell-mediated cytotoxicity (ADCC) function, may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation may be made to modify complement-dependent cytotoxicity (CDC).

[0030] In certain embodiments, the invention provides an antibody and antigen-binding fragment thereof that exhibits pH-dependent binding to C5. For example, the invention includes an antibody and antigen-binding fragment thereof that binds to C5 with a higher affinity at neutral pH than at acidic pH (i.e., decreased binding at acidic pH).

[0031] In certain embodiments, the present invention provides antibodies and antigen-binding fragments that exhibit improved pharmacokinetic and pharmacodynamic properties. For example, the present invention provides anti-C5 antibodies having an extended serum half-life. In certain embodiments, the anti-C5 antibodies of the present invention have serum concentrations higher than 10 μg / mL in C5 humanized mice up to day 40. In certain embodiments, the anti-C5 antibodies of the present invention block CP hemolysis and AP hemolysis up to day 35 upon administration to C5 humanized mice.

[0032] The present invention also provides antibodies and antigen-binding fragments thereof that compete with an antibody or antigen-binding fragment thereof comprising the CDRs of HCVR and CDRs of LCVR for specific binding to C5, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0033] The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete with a reference antibody or antigen-binding fragment thereof comprising the CDRs of HCVR and CDRs of LCVR for binding to C5, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0034] The present invention also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof comprising the CDRs of HCVR and CDRs of LCVR, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof comprising the CDRs of HCVR and CDRs of LCVR, wherein the HCVR / LCVR amino acid sequence pair has SEQ ID NO: 98 / 106.

[0035] The present invention also includes anti-C5 antibodies and antigen-binding fragments thereof that bind to one or more amino acid residues contained in the alpha and / or beta chains of C5. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to one or more amino acids in the alpha chain of C5 and one or more amino acids in the beta chain of C5. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to one or more amino acids in the alpha and beta chains of C5, wherein the antibody does not bind to the C5a anaphylatoxin domain. In certain embodiments, the present invention provides anti-C5 antibodies that interact with one or more amino acids contained within human C5 (SEQ ID NO: 359). In certain embodiments, the present invention provides anti-C5 antibodies that interact with one or more amino acids contained within human C5 (SEQ ID NO: 359), wherein the antibody does not bind to the C5a anaphylatoxin domain of C5. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with an amino acid sequence selected from the group consisting of: (a) amino acids 591-599 of SEQ ID NO: 359; (b) amino acids 593-599 of SEQ ID NO: 359; (c) amino acids 775-787 of SEQ ID NO: 359; (d) amino acids 775-794 of SEQ ID NO: 359; and (e) amino acids 779-787 of SEQ ID NO: 359. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained within SEQ ID NO: 359, for example, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5, at least 10, or at least 15 amino acids contained within SEQ ID NO: 361. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained within SEQ ID NO: 359, for example, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids contained within SEQ ID NO: 360.In certain embodiments, the present invention provides an anti-C5 antibody and antigen-binding fragments thereof that interact with at least 5 amino acids contained within SEQ ID NOs: 360 and 361. In certain embodiments, the present invention provides an anti-C5 antibody and antigen-binding fragments thereof that interact with the amino acid sequence of SEQ ID NO: 360 (corresponding to amino acids 591-599 of SEQ ID NO: 359) and the amino acid sequence of SEQ ID NO: 361 (corresponding to amino acids 775-794 of SEQ ID NO: 359).

[0036] In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to C5 in an agonist-like manner, i.e., can enhance or stimulate C5 binding and / or activity; in other embodiments, the antibody can specifically bind to C5 in an antagonist-like manner, i.e., can block C5 binding and / or activity.

[0037] The present invention also provides an isolated antibody and antigen-binding fragments thereof that block the binding of C5 to C5 convertase. In some embodiments, the antibody or antigen-binding fragment thereof that blocks the binding of C5 to C5 convertase can bind to an epitope on C5 that is the same as that on the C5 convertase or can bind to an epitope on C5 that is different from that on the C5 convertase. In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that blocks the binding of C5 to a monkey C5 convertase.

[0038] In certain embodiments, the antibody or antigen-binding fragment of the present invention is bispecific, comprising a first binding specificity for a first epitope of the C5 protein and a second specificity for a second epitope of the C5 protein, wherein the first and second epitopes are different and do not overlap.

[0039] In certain embodiments, the antibody and antigen-binding fragment of the present invention have an IC 50It binds with. In certain embodiments, the antibody comprises an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 306, 322, and 338. In certain embodiments, the antibody comprises an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298, 314, 330, and 346.

[0040] In certain embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof having one or more of the following: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (K D ) of less than 0.9 nM at 25°C as measured by surface plasmon resonance; (c) binds to human C5 with a K D of less than 0.3 nM at 37°C as measured by surface plasmon resonance assay; (d) binds to cynomolgus C5 with a K D of less than 65 nM as measured by surface plasmon resonance assay; (e) binds to human C5 variant R885H (SEQ ID NO: 356) with a K D of less than 0.5 nM as measured by surface plasmon resonance assay; (f) binds to human C5 variant R885C (SEQ ID NO: 357) with a K D of less than 0.5 nM as measured by surface plasmon resonance assay; (g) blocks more than 95% of human C5-mediated classical pathway (CP) hemolysis with an IC 50 of less than 6 nM as measured by CP hemolysis assay; (h) blocks more than 70% of human C5-mediated alternative pathway (AP) hemolysis with an IC 50 of less than 165 nM as measured by AP hemolysis assay; (i) inhibits African green monkey C5-mediated CP hemolysis with an IC 50 of less than 185 nM as measured by CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with an IC 50 of less than 235 nM as measured by AP hemolysis assay; (k) inhibits cynomolgus C5-mediated CP hemolysis with an IC 50 of less than 145 nM as measured by CP hemolysis assay; and (l) inhibits cynomolgus C5-mediated AP hemolysis with an IC 50 of less than 30 nM as measured by AP hemolysis assay.

[0041] In certain embodiments, the invention provides an isolated recombinant monoclonal anti-C5 antibody or antigen-binding fragment thereof having one or more of the following characteristics: (a) comprising a set of six CDRs comprising the amino acid sequences of SEQ ID NOs: 100-102-104-108-110-112; (b) binding to human C5 with a dissociation constant (K D ) of less than 0.2 nM at 25° C. as measured by surface plasmon resonance assay; (c) binding to human C5 with a K D of less than 0.3 nM at 37° C. as measured by surface plasmon resonance assay; (d) binding to human C5 variant (R885H) with a K D of less than 0.4 nM at 37° C. as measured by surface plasmon resonance assay; (e) inhibiting classical pathway (CP)-mediated hemolysis of human serum with an IC 50 of less than 3 nM; (f) inhibiting alternative pathway (AP)-mediated hemolysis of human serum with an IC 50 of less than 27 nM; (g) inhibiting CP-mediated hemolysis of monkey serum with an IC 50 of less than 21 nM; (g) inhibiting AP-mediated hemolysis of monkey serum with an IC 50 of less than 10 nM; (h) having a serum half-life (t 1 / 2 ) of greater than 10 days in C5 humanized mice; (i) having a serum concentration of greater than 10 μg / mL up to day 40 when administered to C5 humanized mice; (j) blocking CP-mediated hemolysis up to day 50 in C5 humanized mice; and (k) binding to one or more amino acids contained in the alpha chain and / or beta chain of SEQ ID NO: 359, wherein the antibody does not bind to the C5a anaphylatoxin domain of C5.

[0042] In a second aspect, the invention provides a nucleic acid molecule encoding an anti-C5 antibody or portion thereof. For example, the invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the HCVR nucleic acid sequences listed in Table 2, or a polynucleotide sequence having substantially similar sequences thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0043] The present invention also provides a nucleic acid molecule encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from the LCVR nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0044] The present invention also provides a nucleic acid molecule encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from the HCDR1 nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0045] The present invention also provides a nucleic acid molecule encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from the HCDR2 nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0046] The present invention also provides a nucleic acid molecule encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from the HCDR3 nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0047] The present invention also provides a nucleic acid molecule encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and comprises a polynucleotide sequence.

[0048] The present invention also provides a nucleic acid molecule encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and comprises a polynucleotide sequence.

[0049] The present invention also provides a nucleic acid molecule encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and comprises a polynucleotide sequence.

[0050] The present invention also provides a nucleic acid molecule encoding HCVR, wherein HCVR comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), and wherein the HCDR1 - HCDR2 - HCDR3 amino acid sequence set is as defined by any of the exemplary anti - C5 antibodies listed in Table 1.

[0051] The present invention also provides a nucleic acid molecule encoding LCVR, wherein LCVR comprises a set of three CDRs (i.e., LCDR1 - LCDR2 - LCDR3), and wherein the LCDR1 - LCDR2 - LCDR3 amino acid sequence set is as defined by any of the exemplary anti - C5

[0052] The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule is selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-C5 antibody listed in Table 1.

[0053] In a related aspect, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-C5 antibody. For example, the present invention includes a recombinant expression vector comprising any of the above-described nucleic acid molecules, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 2. Also within the scope of the present invention are a host cell into which such a vector has been introduced, culturing the host cell under conditions that allow production of an antibody or antibody fragment, and recovering the antibody and antibody fragment thus produced to produce an antibody or a portion thereof.

[0054] In a third aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody that specifically binds to C5 or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-C5 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-C5 antibody. Examples of agents that can be advantageously combined with the anti-C5 antibody include, without limitation, other agents that bind to and / or inhibit C5 activity (including other antibodies or antigen-binding fragments thereof) and / or agents that do not bind directly to C5 but nevertheless treat or alleviate at least one symptom or sign of a C5-related disease or disorder. Further combination therapies and co-formulations comprising the anti-C5 antibodies of the invention are disclosed elsewhere herein.

[0055] In a fourth aspect, the present invention provides a method of treating a C5-related disease or disorder in a subject using an anti-C5 antibody or an antigen-binding portion of an antibody of the present invention, wherein the method of treatment comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of the antibody or an antigen-binding fragment of the antibody of the present invention. The disorder to be treated is any disease or condition that is ameliorated, remitted, inhibited or prevented by inhibition of C5 activity. In certain embodiments, the present invention provides a method of preventing, treating or remitting at least one symptom of atypical hemolytic uremic syndrome (aHUS), the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-C5 antibody or an antigen-binding fragment thereof of the present invention. In some embodiments, the present invention provides a method of remitting or reducing the severity of at least one symptom or sign of paroxysmal nocturnal hemoglobinuria (PNH) in a subject by administering an anti-C5 antibody of the present invention. In some embodiments, the antibody or an antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having or at risk of having a C5-related disease or disorder. In certain embodiments, the antibody or an antigen-binding fragment thereof of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent can be selected from the group consisting of anti-inflammatory agents (e.g., corticosteroids and non-steroidal anti-inflammatory agents), different antibodies against C5, nutritional supplements such as antioxidants, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent can be an agent that serves to counteract or reduce any possible side effects associated with the antibody or an antigen-binding fragment thereof of the present invention, if such side effects occur. The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally or intramuscularly. The antibody or fragment thereof can be administered at a dose of from about 0.1 mg / kg body weight to about 100 mg / kg body weight of the subject. In certain embodiments, the antibody of the present invention can be administered in one or more doses comprising between 50 mg and 600 mg.

[0056] The invention also encompasses the use of an anti-C5 antibody of the invention or an antigen-binding fragment thereof in the manufacture of a medicament for treating a disease or disorder that would benefit from blocking C5 binding and / or activity.

[0057] Other embodiments will become apparent upon consideration of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0058]

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Mode for Carrying Out the Invention

[0059] Detailed Description Before describing the method of the present invention, it is a matter of course that since the methods and experimental conditions described may vary, the present invention is not limited to the specific methods and experimental conditions described. Also, as a matter of course, since the scope of the present invention is limited only by the appended claims, the terms used in this specification are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0060] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described in this specification may be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All publications mentioned in this specification are incorporated herein by reference in their entirety.

[0061] Definitions The term "C5", also referred to as "complement component 5" or "complement factor 5", refers to a serum protein of the complement cascade. The C5 protein is a 1676 amino acid protein containing two chains, alpha and beta. The protein represents the convergence point of the three complement activation pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin pathway. The amino acid sequence of the full-length C5 protein is exemplified by the amino acid sequence provided in GenBank as accession number NP_001726.2 (SEQ ID NO: 355). The term "C5" includes recombinant C5 protein or fragments thereof. This term also includes, for example, a C5 protein or fragment thereof coupled to a signal sequence such as a histidine tag, mouse or human Fc, or ROR1. For example, this term includes a sequence exemplified by the sequence shown in SEQ ID NO: 356 or 357 containing a histidine tag at the C-terminus coupled to amino acid residues 19-1676 of the full-length C5 protein. This term also includes a protein variant having an R885H or R885C change and containing a histidine tag at the C-terminus coupled to amino acid residues 19-1676 of the full-length C5 protein.

[0062] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "complete antibody molecule") composed of four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "V H ") and a heavy chain constant region (domains C H 1, C H 2 and C H 3). Each light chain consists of a light chain variable region ("LCVR" or "V L ") and a light chain constant region (C L ). V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) incorporated into regions called more conserved framework regions (FRs). Each V H and V L consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of an antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be modified naturally or artificially. Amino acid consensus sequences can be defined based on a side-by-side analysis of two or more CDRs.

[0063] Substitution of one or more CDR residues or deletion of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be omitted for binding. Padlan et al. (1995 FASEB J. 9:133-139) have published Based on the crystal structures obtained, the contact regions between antibodies and their antigens were analyzed, and it was concluded that only approximately one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies that do not have amino acids in one or two CDRs that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0064] CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from regions of the Kabat CDR outside the Chothia CDR based on previous studies (e.g., residues H60-H65 in CDRH2 are often not required). When a CDR or its residue is deleted, it is usually replaced with the amino acid occupying the corresponding position in another human antibody sequence or the consensus of such sequences. The positions of substitutions within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be either conservative or non-conservative substitutions.

[0065] The fully human anti-C5 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to, for example, germline sequences available from public antibody sequence databases. The invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residue of the germline sequence from which the antibody was derived, or to the corresponding residue of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily produce a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only specific residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Further, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, where specific individual residues are mutated to the corresponding residues of a specific germline sequence while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (where appropriate), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the invention.

[0066] The invention also includes fully human anti-C5 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes anti-C5 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having conservative amino acid substitutions such as, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0067] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutation in vivo), for example, in the CDRs, and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in a cell of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[0068] As used herein, the term "recombinant" refers to an antibody of the invention or an antigen-binding fragment thereof that is produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transgenic expression. The term refers to an antibody expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0069] The term "specifically binds" or "binds specifically to" or the like means that an antibody or an antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding is at least about 1 x 10 -8 M or less (e.g., a smaller K Dmay be characterized by an equilibrium dissociation constant that indicates a tighter binding. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. The antibodies described herein have been identified by surface plasmon resonance, for example, BIACORE TM and specifically bind to C5. Further, multi-specific antibodies that bind to one domain in C5 and one or more additional antigens, or bispecific antibodies that bind to two different regions of C5, are nonetheless considered "specifically binding" antibodies as used herein.

[0070] The term "high affinity" antibody, as measured by surface plasmon resonance, for example, BIACORE TM or solution affinity ELISA, has a binding affinity for C5 of at least 10 -8 M; preferably 10 -9 M; more preferably 10 -10 M, even more preferably 10 -11 M, even more preferably 10 -12 M of K D and refers to an mAb having a binding affinity for C5 as represented.

[0071] The term "slow dissociation rate", "Koff" or "kd", as determined by surface plasmon resonance, for example, BIACORE TM means an antibody that dissociates from C5 with a rate constant of 1x10 -3 s -1 or less, preferably 1x10 -4 s -1 or less.

[0072] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to C5 protein.

[0073] In certain embodiments, the antibodies or antibody fragments of the invention can be conjugated to a moiety such as a ligand or a therapeutic moiety ("immunoconjugate"), a second anti-C5 antibody, or any other therapeutic moiety useful for treating a C5-related disease or disorder.

[0074] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to C5 is substantially free of Abs that specifically bind to antigens other than C5).

[0075] As used herein, the term "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes C5 activity" or "antagonist antibody") is intended to refer to an antibody whose binding to C5 results in inhibition of at least one biological activity of C5. For example, the antibodies of the invention can prevent or block complement-mediated hemolysis via the classical or alternative pathway.

[0076] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE TM system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0077] As used herein, the term "KD is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0078] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. This also refers to the region of an antigen that is bound by an antibody. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can also be structural, i.e., composed of non-linear amino acids. In certain embodiments, an epitope can include determinants that are a chemically active surface population of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural features, and / or specific charge features.

[0079] As used herein, the term "cross-competing" means that an antibody or its antigen-binding fragment binds to an antigen and inhibits or blocks the binding of another antibody or its antigen-binding fragment. This term also includes competition between two antibodies in both directions, i.e., a first antibody that binds and blocks the binding of a second antibody and vice versa. In certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that the binding of one inhibits or blocks the binding of the second antibody, e.g., by steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, e.g., by real-time label-free biolayer interferometry assays. Cross-competition between two antibodies can be expressed as the binding of the second antibody being lower than the background signal resulting from self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, e.g., as the percentage of binding of the second antibody being lower than the baseline self-self background binding (where the first and second antibodies are the same antibody).

[0080] When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" means that when optimally aligned with another nucleic acid (or its complementary strand) including appropriate nucleotide insertions or deletions, as considered below, by any of the well-known algorithms for sequence identity such as FASTA, BLAST or GAP it shows nucleotide sequence identity at at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0081] The terms "substantial similarity" or "substantially similar" when applied to polypeptides mean that when optimally aligned by a program such as GAP or BESTFIT using the default gap weights, the two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98% or 99% sequence identity. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are those in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent similarity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are changes having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. "Moderately conservative" substitutions are changes having a non-negative value in the PAM250 log-likelihood matrix.

[0082] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein, using default parameters. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; programs in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the region of maximum overlap between a query sequence and a search sequence and the percent sequence identity (Pearson (2000) supra). Another algorithm that is preferred when comparing the sequences of the present invention to databases containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402 (which are incorporated herein by reference, respectively).

[0083] The phrase "therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The exact amount depends on the purpose of the treatment and can be ascertained by using techniques known to those of skill in the art (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0084] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of remission, prevention and / or treatment of a C5-related disease or disorder such as atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH). This term includes human subjects having or at risk of having such a disease or disorder.

[0085] As used herein, the terms "treat", "treating" or "treatment" refer to a reduction or remission in the severity of at least one symptom or sign of a C5-related disease or disorder resulting from administration of a therapeutic agent, such as an antibody of the invention, to a subject in need thereof. These terms include inhibition of disease progression or worsening of symptoms / signs. These terms also include a positive prognosis for the disease, i.e., the subject may be disease-free or have a reduced disease upon administration of a therapeutic agent such as an antibody of the invention. The therapeutic agent may be administered at a therapeutic dose to the subject.

[0086] The terms "prevent", "preventing" or "prevention" refer to inhibiting the occurrence of a C5-related disease or disorder or symptoms of such a disease or disorder upon administration of an antibody of the invention.

[0087] Antigen-binding fragment of an antibody Unless specifically indicated otherwise, as used herein, the term "antibody" is understood to include antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains, i.e., "complete antibody molecules"), as well as antigen-binding fragments thereof. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen and form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to the C5 protein. Antibody fragments include Fab fragments, F(ab’) 2Examples include fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic manipulation including manipulation and expression of DNA encoding antibody variable domains and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0088] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) diabodies The minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies (diabodies), trispecific antibodies (triabodies), tetraspecific antibodies (tetrabodies), minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIP), and other engineered molecules such as shark variable IgNAR domains are also included within the expression "antigen-binding fragment" as used herein.

[0089] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR, which is adjacent to or in-frame with one or more framework sequences. V L domain associated with the V H In an antigen-binding fragment having a V H and V L domains, the V H -V H and V H -V L or V L -V L can be in any suitable arrangement relative to each other. For example, the variable regions can be dimeric, and the V H or V L domains can be monomeric.

[0090] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting examples of configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C Linclude. In any of the variable and constant domain configurations, including any of the configurations exemplified above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a mobile or semi-mobile linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of the present invention are non-covalently bound to each other and / or (e.g., by disulfide bonds) to one or more monomeric V H or V L domains and may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above.

[0091] Like full antibody molecules, the antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). The multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, where each variable domain can specifically bind to a different antigen or a different epitope on the same antigen. Any multispecific antibody format, including the bispecific antibody formats exemplified herein, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0092] Production of Human Antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to produce human antibodies that specifically bind to C5 protein.

[0093] An antibody against C5 protein can be generated using an immunogen comprising any one of the following. In certain embodiments, the antibodies of the invention are obtained from mice immunized with full-length native C5 protein (see, e.g., GenBank accession number NP_001726.2) (SEQ ID NO: 355), or with DNA encoding said protein or fragment thereof. Alternatively, the protein or fragment thereof can be produced, modified, and used as an immunogen using standard biochemical techniques. In certain embodiments of the invention, the immunogen is a fragment of the C5 protein spanning amino acid residues approximately 19 to 1676 of SEQ ID NO: 355.

[0094] In some embodiments, the immunogen can be a recombinant C5 protein or fragment thereof expressed in E. coli or any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.

[0095] VELOCIMMUNE (R) technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE (R) for reference) or any other known method for generating monoclonal antibodies to first isolate a high-affinity chimeric antibody to C5 having human variable regions and mouse constant regions. VELOCIMMUNE (R) technology involves generating transgenic mice having a genome comprising human heavy and light chain variable regions operably linked to the endogenous mouse constant region locus such that the mice produce antibodies comprising human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0096] Generally, VELOCIMMUNE (R)Load a mouse with the antigen of interest and recover lymphocytes (e.g., B cells) from a mouse expressing the antibody. The lymphocytes can be fused with a myeloma cell line to produce immortal hybridoma cell lines, and such hybridoma cells are screened and selected to identify a hybridoma cell line that produces an antibody specific for the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0097] First, isolate a high-affinity chimeric antibody having a human variable region and a mouse constant region. Characterize the antibody as described below and select for the desired characteristics including affinity, selectivity, epitope, etc. Replace the mouse constant region with the desired human constant region to generate a fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. The selected constant region can vary depending on the particular application, but the high-affinity antigen-binding and target-specificity characteristics reside in the variable region.

[0098] Biological equivalence The anti-C5 antibodies and antibody fragments of the present invention include proteins that have an amino acid sequence different from the described antibodies but retain the ability to bind to the C5 protein. Such variant antibodies and antibody fragments have one or more additions, deletions, or substitutions of amino acids when compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, the DNA sequences encoding the antibodies of the present invention include one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention. sequences that do.

[0099] Two antigen-binding proteins, or antibodies, are considered biologically equivalent if, for example, they show no significant differences in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either as a single dose or multiple doses. Some antibodies are considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in the rate of absorption, and furthermore, such differences in the rate of absorption are not intentional and are reflected in the labeling, for example, not essential for achieving the effective in vivo drug concentration in chronic use and not medically important for the particular drug being studied, and thus can be considered biologically equivalent.

[0100] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, or potency.

[0101] In one embodiment, two antigen-binding proteins are biologically equivalent if a patient can switch one or more times between a reference formulation and a biological formulation without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or a decrease in efficacy, compared to continued treatment without switching.

[0102] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by a common mechanism of action for the conditions of use, to the extent that such a mechanism is known.

[0103] Biological equivalence can be demonstrated by in vivo and / or in vitro methods. Examples of biological equivalence measurements include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and are reasonably predictable from human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, bioavailability, or biological permeability of an antibody.

[0104] Biologically equivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences, or by removing terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges during regeneration. In other situations, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0105] Anti-C5 antibody comprising an Fc variant According to certain embodiments of the invention, there is provided, for example, an anti-C5 antibody comprising an Fc domain that contains one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH as compared to neutral pH. For example, the invention relates to the C of the Fc domain H 2 or C HAn anti-C5 antibody containing a mutation in three regions, where the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T) modifications; or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0106] For example, the present invention includes an anti-C5 antibody comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the present invention.

[0107] The present invention also includes an anti-C5 antibody comprising a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region comprises segments derived from the C H regions of more than one immunoglobulin isotype. For example, the antibodies of the present invention may comprise a C H 3 domain, a portion or all of which is combined with a portion or all of a C H 2 domain, the chimeric C H region derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies of the present invention have a chimeric C Hincludes a region. For example, a chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region may include amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C H Antibodies that include a region may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety).

[0108] Biological characteristics of the antibody Generally, the antibodies of the invention function by binding to C5 protein and preventing its cleavage into C5a and C5b. For example, the invention provides antibodies that bind to C5 protein (e.g., at 25°C or 37°C) with a K D of less than 9 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein, and antigen-binding fragments of the antibody. In certain embodiments, the antibody or its antigen-binding fragment binds to C5 with a K of less than about 9 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, about 500 pM, less than 250 pM, or less than 100 pM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein, or an assay substantially similar thereto. D to C5.

[0109] The present invention also provides an antibody that binds to human C5 protein with a dissociation half-life (t 1 / 2 ) of longer than about 2 minutes, as measured by surface plasmon resonance at 25°C using an assay format defined, for example, in Example 4 of this specification or a substantially similar assay, and antigen-binding fragments thereof. In certain embodiments, the antibody or antigen-binding fragment of the present invention has a t 1 / 2 of longer than about 5 minutes, longer than about 10 minutes, longer than about 30 minutes, longer than about 50 minutes, longer than about 100 minutes, longer than about 150 minutes, longer than about 200 minutes, or longer than about 250 minutes when binding to C5 protein, as measured by surface plasmon resonance at 25°C using an assay format (e.g., mAb capture or antigen capture format) defined, for example, in Example 3 of this specification or a substantially similar assay.

[0110] The present invention also provides an antibody that binds to human C5 protein with a dissociation half-life of longer than about 1.5 minutes, as measured by surface plasmon resonance at 37°C using an assay format defined, for example, in Example 4 of this specification or a substantially similar assay, and antigen-binding fragments thereof. In certain embodiments, the antibody or antigen-binding fragment of the present invention has a t 1 / 2 of longer than about 2 minutes, longer than about 5 minutes, longer than about 10 minutes, longer than about 25 minutes, longer than about 50 minutes, longer than about 100 minutes, longer than about 150 minutes, or longer than about 200 minutes when binding to C5 protein, as measured by surface plasmon resonance at 37°C using an assay format (e.g., mAb capture or antigen capture format) defined, for example, in the examples of this specification or a substantially similar assay.

[0111] The present invention also provides an antibody that binds to human C5 protein with a K DIt includes antibodies and antigen-binding fragments of antibodies that bind to simian C5 protein (e.g., at 25 °C or 37 °C). In certain embodiments, the antibody or its antigen-binding fragment binds to simian C5 with a K of less than about 120 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, about 500 pM, or less than 250 pM, as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. D It binds to simian C5.

[0112] The present invention also provides antibodies and antigen-binding fragments of antibodies that bind to a modified human C5 protein having an R885H change (exemplified by SEQ ID NO: 356) with a K of less than 70 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein. D It includes antibodies and antigen-binding fragments of antibodies that bind to a modified human C5 protein having an R885H change (exemplified by SEQ ID NO: 356). The C5 variant showed an insufficient response to previously disclosed anti-C5 antibodies in the art (e.g., Nishimura et al 2014, New Engl. J. Med. 370:632 - 639). In certain embodiments, the antibody or its antigen-binding fragment binds to the modified human C5 with a K of less than about 65 nM, less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than 2 nM, as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or an assay substantially similar thereto. D It binds to the modified human C5.

[0113] The present invention also provides antibodies and antigen-binding fragments of antibodies that bind to a modified human C5 protein having an R885C change (exemplified by SEQ ID NO: 357) with a K of less than 160 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein. D It includes antibodies and antigen-binding fragments of antibodies that bind to the modified human C5 protein having an R885C change. The C5 variant showed an insufficient response to previously disclosed anti-C5 antibodies in the art (e.g., Nishimura et al 2014, New Engl. J. Med. 370:632 - 639). In certain embodiments The antibody or antigen-binding fragment thereof binds to human C5 modified with, for example, a K of less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than 2 nM as measured by surface plasmon resonance using the assay format defined in Example 3 herein or a substantially similar assay. D

[0114] The present invention also provides an antibody and antigen-binding fragment thereof that inhibits complement-dependent cytotoxicity (CDC) with an IC of less than 10 nM as measured by a luminescence assay using the assay format defined in Example 6 herein. In certain embodiments, the antibody or antigen-binding fragment thereof inhibits CDC with an IC of less than about 5 nM, less than about 3.5 nM, or less than about 2 nM as measured by a B cell luminescence assay using the assay format defined in Example 6 herein or a substantially similar assay. 50 50

[0115] The present invention also provides an antibody and antigen-binding fragment thereof that blocks more than 94% of human C5-mediated classical pathway (CP) hemolysis with an IC of less than 6 nM as measured by a CP hemolysis assay using the assay format defined in Example 8 herein. In certain embodiments, the antibody or antigen-binding fragment thereof blocks CP hemolysis with an IC of less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, or less than about 2 nM as measured by a CP hemolysis assay using the assay format defined in Example 8 herein or a substantially similar assay. 50 50

[0116] The present invention also provides an antibody and antigen-binding fragment thereof that blocks AP hemolysis with an IC of less than 165 nM as measured by an AP hemolysis assay using the assay format defined in Example 8 herein. 50It includes antibodies and antigen-binding fragments that block more than 70% of human C5-mediated alternative pathway (AP) hemolysis. In certain embodiments, the antibody or its antigen-binding fragment has an IC, as measured by an AP hemolysis assay, of less than about 160 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less than about 20 nM, using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto. 50 blocks AP hemolysis.

[0117] The present invention also includes antibodies and antigen-binding fragments that block more than 40% of African green monkey C5-mediated classical pathway (CP) hemolysis, with an IC, as measured by a CP hemolysis assay, of less than 185 nM, using, for example, the assay format defined in Example 8 herein. 50 In certain embodiments, the antibody or its antigen-binding fragment has an IC, as measured by a CP hemolysis assay, of less than about 180 nM, less than about 150 nM, less than about 100 nM, about 75 nM, or less than about 50 nM, using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto. 50 blocks CP hemolysis.

[0118] The present invention also includes antibodies and antigen-binding fragments that block the African green monkey C5-mediated alternative pathway (AP) with an IC, as measured by an AP hemolysis assay, of less than 235 nM, using, for example, the assay format defined in Example 8 herein. 50 In certain embodiments, the antibody or its antigen-binding fragment has an IC, as measured by an AP hemolysis assay, of less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less than about 20 nM, using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto. 50 blocks AP hemolysis.

[0119] The present invention also includes antibodies and antigen-binding fragments that block the CP with an IC, as measured by a CP hemolysis assay, of less than 145 nM, using, for example, the assay format defined in Example 8 herein. 50It includes antibodies and antigen-binding fragments that block more than 90% of cynomolgus monkey C5-mediated classical pathway (CP) hemolysis. In certain embodiments, the antibody or its antigen-binding fragment blocks CP hemolysis with an IC of less than about 140 nM, less than about 120 nM, less than about 100 nM, less than about 75 nM, or less than about 50 nM as measured by the CP hemolysis assay, for example, using the assay format defined in Example 8 herein or an assay substantially similar thereto. 50 for CP hemolysis.

[0120] The present invention also includes antibodies and antigen-binding fragments that block cynomolgus monkey C5-mediated alternative pathway (AP) hemolysis with an IC 50 of less than 30 nM as measured by the AP hemolysis assay, for example, using the assay format defined in Example 8 herein. In certain embodiments, the antibody or its antigen-binding fragment blocks AP hemolysis with an IC 50 of less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than about 2 nM as measured by the AP hemolysis assay, for example, using the assay format defined in Example 8 herein or an assay substantially similar thereto.

[0121] The present invention also includes antibodies and antigen-binding fragments that exhibit improved pharmacokinetic (PK) and pharmacodynamic (PD) properties compared to anti-C5 antibodies in the art. The anti-C5 antibodies of the present invention exhibit lower sensitivity to target-mediated clearance upon administration, as shown in Examples 9 and 10 herein. In certain embodiments, the present invention includes anti-C5 antibodies and their antigen-binding fragments that exhibit serum concentrations for an extended period, for example, longer than 20 days, longer than 25 days, longer than 30 days, longer than 35 days, longer than 40 days, longer than 45 days, longer than 50 days, longer than 55 days, or longer than 60 days. In certain embodiments, the anti-C5 antibodies of the present invention exhibit an extended serum half-life of longer than 10 days compared to anti-C5 antibodies in the art.

[0122] In certain embodiments, the present invention has a high affinity for human C5 (e.g., KD ) and anti-C5 antibodies and antigen-binding fragments thereof having a lower clearance (e.g., an extended serum half-life, improved pharmacodynamic activity over a longer number of days than previously known anti-C5 antibodies). Such antibodies of the invention can be advantageously used at a lower dosing frequency in a subject having a C5-related disease or disorder.

[0123] In one embodiment, the present invention provides an isolated recombinant antibody or an antigen-binding fragment thereof that specifically binds to C5 protein, wherein the antibody or its fragment exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (K D ) of less than 0.9 nM at 25°C as measured in a surface plasmon resonance assay; (c) binds to human C5 with a K D of less than 0.3 nM at 37°C as measured in a surface plasmon resonance assay; (d) has a serum concentration higher than 10 μg / mL over 70 days upon administration to cynomolgus monkeys; (e) blocks CP hemolysis and AP hemolysis over 35 days upon administration to cynomolgus monkeys as measured in an ex vivo hemolysis assay; (f) has a serum half-life longer than 10 days in cynomolgus monkeys; (g) has a serum concentration higher than 10 μg / mL over 40 days upon administration to C5 humanized mice; (h) blocks CP hemolysis over 30 days upon administration to C5 humanized mice as measured in an ex vivo hemolysis assay; and (i) has a serum half-life longer than 10 days in C5 humanized mice.

[0124] In one embodiment, the present invention provides an isolated recombinant antibody or an antigen-binding fragment thereof that specifically binds to C5 protein, wherein the antibody or its fragment exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (K D ) of less than 0.9 nM at 25°C as measured in a surface plasmon resonance assay; (c) binds to human C5 with a K Dthat binds to human C5; (d) having a K of less than 65 nM as measured in a surface plasmon resonance assay D that binds to cynomolgus monkey C5; (e) having a K of less than 0.5 nM as measured in a surface plasmon resonance assay D that binds to human C5 variant R885H (SEQ ID NO: 356); (f) having a K of less than 0.5 nM as measured in a surface plasmon resonance assay D that binds to human C5 variant R885C (SEQ ID NO: 357); (g) having an IC of less than 6 nM as measured in a CP hemolysis assay 50 that blocks human C5-mediated classical pathway (CP) hemolysis by more than 95%; (h) having an IC of less than 165 nM as measured in an AP hemolysis assay 50 that blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70%; (i) having an IC of less than 185 nM as measured in a CP hemolysis assay 50 that inhibits African green monkey C5-mediated CP hemolysis; (j) having an IC of less than 235 nM as measured in an AP hemolysis assay 50 that inhibits African green monkey C5-mediated AP hemolysis; (k) having an IC of less than 145 nM as measured in a CP hemolysis assay 50 that inhibits cynomolgus monkey C5-mediated CP hemolysis; and (l) having an IC of less than 30 nM as measured in an AP hemolysis assay 50 that inhibits cynomolgus monkey C5-mediated AP hemolysis.

[0125] The antibodies of the present invention may have one or more of the foregoing biological characteristics, or combinations thereof. Other biological characteristics of the antibodies of the present invention will be apparent to those skilled in the art upon review of the present disclosure, including the examples herein.

[0126] Epitope mapping and related techniques The present invention includes an anti-C5 antibody that interacts with one or more amino acids found within one or more regions of a C5 protein molecule that includes an alpha polypeptide and a beta polypeptide. The epitope to which the antibody binds can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the C5 protein molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within any or both of the aforementioned domains of the protein molecule (e.g., a structural epitope).

[0127] A variety of techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface may retain deuterium and thus exhibit a relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry to reveal the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0128] The term "epitope" refers to the site on an antigen to which B and / or T cells respond. B cell epi A loop can be formed from both contiguous or non - contiguous amino acids juxtaposed by the three - dimensional folding of a protein. Epitopes formed from contiguous amino acids are typically exposed and retained in a denaturing solvent, while epitopes formed by three - dimensional folding are typically lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, and more usually at least 5 or 8 - 10 amino acids in a unique spatial arrangement.

[0129] Modification - Assisted Profiling (MAP), also known as Antigen - Structure - based Antibody Profiling (ASAP), is a method of classifying multiple monoclonal antibodies (mAbs) specific for the same antigen according to the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, which is specifically incorporated herein by reference in its entirety). Each category can reflect a distinct epitope that is very different from or partially overlaps with an epitope represented by another category. This technique enables rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to classify the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0130] In certain embodiments, the anti - C5 antibody or antigen - binding fragment thereof binds to an epitope within any one or more of the regions exemplified in the native form as set forth in SEQ ID NO: 355, or in a recombinantly produced C5 protein, or a fragment thereof. In some embodiments, the antibodies of the invention bind to a region comprising one or more amino acids selected from the group consisting of amino acid residues 19 - 1676 of the human C5 protein.

[0131] In certain embodiments, the antibodies of the invention interact with at least one amino acid sequence selected from the group consisting of amino acid residues spanning from approximately position 19 to approximately position 750 of SEQ ID NO: 355; or amino acid residues spanning from approximately position 751 to approximately position 1676.

[0132] In certain embodiments, the invention includes anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the alpha and / or beta chains of C5 (SEQ ID NO: 359). The epitope can consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the alpha and / or beta chains of C5. Alternatively, the epitope can consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within C5. As shown in Example 11 herein, the epitopes of C5 with which the exemplary antibody H4H12166P of the invention interacts are: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) corresponding to amino acids 591-599 contained in the beta chain of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361) corresponding to amino acids 775-794 contained in the alpha chain of SEQ ID NO: 359. Accordingly, the invention includes anti-C5 antibodies that interact with one or more amino acids contained within the region consisting of: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) corresponding to amino acids 591-599 of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361) corresponding to amino acids 775-794 of SEQ ID NO: 359.

[0133] The invention is directed to anti-C5 antibodies that interact with the same epitope as any of the specific exemplary antibodies listed in Table 1 or an epitope It includes an anti-C5 antibody that binds to a part of the epitope. Similarly, the present invention also includes any of the antibodies that are specific examples listed in Table 1, and anti-C5 antibodies that compete with respect to binding to C5 protein or its fragments. For example, the present invention includes an anti-C5 antibody that cross-competes with one or more antibodies listed in Table 1 with respect to binding to C5 protein.

[0134] By using conventional methods known in the art, it is possible to easily determine whether an antibody binds to the same epitope as a reference anti-C5 antibody or competes with respect to binding to the reference C5 antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-C5 antibody of the present invention, the reference antibody is bound to C5 protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the C5 molecule is evaluated. If the test antibody can bind to C5 after saturation binding with the reference anti-C5 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-C5 antibody. On the other hand, if the test antibody cannot bind to C5 after saturation binding with the reference anti-C5 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-C5 antibody of the present invention.

[0135] To determine whether an antibody competes with respect to binding to the reference anti-C5 antibody, the above binding methodology is performed in two directions: In the first direction, the reference antibody is bound to C5 protein under saturation conditions, and subsequently the binding of the test antibody to the C5 molecule is evaluated. In the second direction, the test antibody is bound to the C5 molecule under saturation conditions, and subsequently the binding of the reference antibody to the C5 molecule is evaluated. In both directions, if only the first (saturated) antibody can bind to the C5 molecule, it is concluded that the test antibody and the reference antibody compete with respect to C5 binding. It will be obvious to those skilled in the art that antibodies that compete with respect to binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0136] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, preferably 75%, 90%, or 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502), or all substantially amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have overlapping epitopes.

[0137] Subsequent conventional experiments (e.g., peptide mutagenesis and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric blocking (or another reduction) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0138] Immunoconjugate The present invention encompasses human anti-C5 monoclonal antibodies (immunoconjugates) conjugated to a therapeutic moiety for treating C5-related diseases or disorders (e.g., atypical hemolytic uremic syndrome). As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a radiopharmaceutical, cytokine, interferon, targeting or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody can be any molecule as long as it can bind to its target. It can be conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any position along. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second different antibody to the C5 protein. The type of therapeutic moiety that can be conjugated to the anti-C5 antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0139] Multispecific antibody The antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide, see, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0140] Any of the multispecific antigen-binding molecules of the present invention or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) as known to those skilled in the art.

[0141] In some embodiments, C5-specific antibodies are generated in a bispecific format (a “bi-specific”) where variable regions that bind to different domains of the C5 protein are linked together to provide dual domain specificity within a single binding molecule. Appropriately designed bispecifics can enhance overall C5 protein inhibition efficacy by increasing both specificity and binding affinity. Variable regions that are specific for individual domains (e.g., segments of the N-terminal domain) or that can bind to different regions within one domain pair up on a structural scaffold, enabling each region to simultaneously bind to a separate epitope or different regions within one domain. In one example of a bispecific, the heavy chain variable region (V H ) from a binder specific for one domain can pair with a non-cognate V H partner that can pair with the original V H without disrupting its original specificity for its V L . To identify the non-cognate V L partner, it is recombined with light chain variable regions (V L ) from a series of binders specific for a second domain. In this way, a single V L segment (e.g., V H 1) binds to two different V H domains (e.g., V H 1 and V H 2) to generate a bispecificity composed of two binding “arms” (V L 1-V H 1 and V L 2-V L 1). The use of a single V

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[0142] <000Alternatively, one or more domains, and an antibody that binds to a second target, such as, but not limited to, a second different anti-C5 antibody, can be produced in a bispecific format using the techniques described herein or other techniques known to those of skill in the art. Antibody variable regions that bind to different regions can be linked together, for example, with a variable region that binds to a relevant site on the extracellular domain of C5, to confer bispecific antigenicity within a single binding molecule. Appropriately designed bispecificities of this nature serve a dual function. The variable region having specificity for the extracellular domain is paired on a structural scaffold that binds to a variable region having specificity for the outside of the extracellular domain and allows each variable region to bind to a separate antigen.

[0143] An example of a bispecific antibody format that can be used in the context of the present invention is a first immunoglobulin (Ig)C H 3 domain and a second Ig C H 3 domain, where the first and the second Ig C H 3 domains differ by at least 1 amino acid from each other, and where at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without an amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (by IMGT exon numbering; H435R in EU numbering). The second C H 3 may further contain the Y96F modification (by IMGT; Y436F in EU). The second C HAdditional modifications that may be seen within 3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; in EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (IMGT in EU; N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; in EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations in the bispecific antibodies above are considered within the scope of the present invention.

[0144] Other exemplary bispecific formats that can be used in the context of the present invention include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (for an overview of the formats mentioned above, see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates, for example, here using non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence, and arrangement. (For example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0145] Therapeutic Administration and Formulations The present invention provides a therapeutic composition comprising an anti-C5 antibody of the present invention or an antigen-binding fragment thereof. The therapeutic composition according to the present invention is administered together with other agents incorporated into the formulation to provide a suitable carrier, excipient, and improved transport, delivery, resistance, etc. A number of suitable formulations can be found in the pharmacopoeias known to all pharmacists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN TM ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (emulsions carbowax) (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0146] The dosage of the antibody can vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient, the antibody of the present invention is usually administered in a single dose of about 0.1 to about 100 mg / kg body weight, more preferably about 5 to about 80, about 10 to about 70, or about 20 to about 50 mg / kg body weight It is advantageous to administer a single dose of g. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment of the invention can be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, following the initial dose, a second or multiple subsequent doses of the antibody or its antigen-binding fragment can be administered in an amount approximately the same as or less than the initial dose, where the subsequent doses are separated by at least 1 day to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0147] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epithelial, and oral routes. The composition can be administered by absorption through the epithelium or mucosal lining (e.g., oral mucosa, rectal, and intestinal mucosa) by any convenient route, such as injection or bolus injection, and can be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0148] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of manufacture and use are described in detail by Arruebo, M. et al. in 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389) (incorporated herein by reference). Nanoparticles are developed to target cells and can be conjugated to antibodies contained in pharmaceutical compositions. Nanoparticles for drug delivery are also described, for example, in US 8257740 or US 8246995 (each incorporated herein by reference in its entirety).

[0149] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in the vicinity of the target of the composition and thus only a small portion of systemic dosing is required.

[0150] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, infusions, etc. These injectable preparations can be manufactured by known methods. For example, an injectable preparation can be manufactured by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the aqueous medium for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and these can be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and these can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. The injectable thus manufactured is preferably filled into appropriate ampoules.

[0151] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Further, with respect to subcutaneous delivery, pen-type delivery devices are readily useful in the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Thereafter, the pen-type delivery device can be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition has emptied from the reservoir, the entire device is discarded.

[0152] A number of reusable pen-type and auto-injector delivery devices are useful in the subcutaneous delivery of the pharmaceutical compositions of the present invention. By way of example, but certainly not limited to, a few examples include AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM , OPTIPEN PRO TM , OPTIPEN STARLET TM , and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type devices useful in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are certainly not limited to, a few examples include SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Auto-injector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA TM Pen (Abbott Labs, Abbott Park, IL).

[0153] Advantageously, the pharmaceutical composition for oral or parenteral use as described above is manufactured in unit doses adapted to the dose of the active ingredient to form a dosage form. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of the antibody contained is generally about 5 to about 500 mg per unit dose dosage form; particularly in the form of an injection, it is preferably contained at about 5 to about 300 mg of the antibody, and for other dosage forms, it is preferably contained at about 10 to about 300 mg.

[0154] Therapeutic use of the antibody The antibody of the present invention is useful for treating and / or preventing a disease, disorder or condition associated with C5, or for alleviating at least one symptom associated with such a disease, disorder or condition. In certain embodiments, the antibody or antigen-binding fragment thereof herein can be administered in a therapeutic dose to a patient having a disease, disorder or condition associated with C5.

[0155] In certain embodiments, the antibody of the present invention is useful in treating or preventing the symptoms or signs of atypical hemolytic uremic syndrome (aHUS). Symptoms and signs of aHUS include, but are not limited to, platelet activation, hemolysis, stroke, heart attack, kidney failure and / or all systemic thrombotic microangiopathy (formation of blood clots in small blood vessels throughout the body), end-stage renal disease, persistent kidney injury, abdominal pain, confusion, edema, fatigue, nausea / vomiting, diarrhea, and microangiopathic anemia.

[0156] In certain embodiments, the antibody of the present invention is useful in treating or preventing the symptoms or signs of paroxysmal nocturnal hemoglobinuria (PNH). Symptoms and signs of PNH include, but are not limited to, red blood cell destruction, thrombosis (including deep vein thrombosis, pulmonary embolism), intravascular hemolytic anemia, red urine, fatigue, shortness of breath, and symptoms of anemia such as palpitations, abdominal pain and dysphagia.

[0157] In certain embodiments, the antibodies of the invention are useful for treating or preventing at least one symptom or sign of a C5-related disease or disorder selected from the group consisting of neurological disorders, kidney disorders, multiple sclerosis, stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, disorders of inappropriate or unwanted complement activation, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammatory disorders, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, burns or frostbite including thermal injury, post-ischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, post-pump syndrome in balloon angioplasty, cardiopulmonary bypass or renal artery bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious diseases or sepsis, immune complex diseases and autoimmune diseases, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, renal ischemia-reperfusion injury, lupus nephritis, glomerulitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, membranoproliferative nephritis, hemolytic anemia, neuromyelitis optica, kidney transplantation, hereditary CD59 deficiency, psoriasis, and myasthenia gravis. In certain other embodiments, the antibodies of the invention are useful for treating or preventing at least one symptom or sign of a C5-related disease or disorder selected from the group consisting of pulmonary diseases and disorders such as dyspnea, hemoptysis, ARDS, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, fibrotic dust diseases, injuries due to inert dusts and minerals (e.g., silica, carbon dust, beryllium, and asbestos), pulmonary fibrosis, organic dust diseases, chemical injuries (due to irritant gases and chemicals such as chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia, and hydrochloric acid), smoke injury, thermal injury (e.g., burns, frostbite), asthma, allergy, bronchoconstriction, hypersensitivity pneumonia, parasitosis, Goodpasture syndrome, pulmonary vasculitis, hereditary angioedema, and immune complex-related inflammation.

[0158] In certain embodiments, the antibodies of the invention are useful for treating subjects suffering from eye diseases such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, intraocular angiogenesis (intraocular neovascularization affecting choroidal, corneal or retinal tissue), geographic atrophy (GA), uveitis, and neuromyelitis optica. The antibodies of the invention can be used to treat or alleviate at least one symptom or sign of dry or wet AMD. In some embodiments, the antibodies of the invention are useful in preventing or slowing vision loss. In one embodiment, the antibodies of the invention are useful in reducing drusen in the eyes of subjects with dry AMD. In one embodiment, the antibodies of the invention are useful in preventing or reducing / delaying vision loss in subjects with AMD.

[0159] One or more antibodies of the invention can be administered to reduce, prevent or decrease the severity of one or more symptoms or conditions / signs of an eye disease or injury. The antibodies can be used to treat vision loss, visual distortion, difficulty adapting to low light levels, distorted central vision, increased haziness of the central / overall visual field, the presence of drusen (extracellular Small accumulations of substances, pigment changes, distorted vision in the form of metamorphopsia (where a straight grid appears wavy and parts of the grid appear blank), exudative changes (eye bleeding, hard exudates, subretinal / sub-RPE / intraretinal fluid), slow recovery of visual function after exposure to bright light (light stress test), early and geographic atrophy, rapid decrease in visual acuity (2 levels or more), for example, from 20 / 20 to 20 / 80, preferential hyperacuity perimetry changes (for exudative AMD), blurred vision, gradual loss of central vision (for those with non-exudative macular degeneration), rapid onset of visual field loss (often caused by leakage and bleeding of abnormal blood vessels in subjects with exudative macular degeneration), central scotoma (shadow or area of missing vision), difficulty in distinguishing colors, especially dark colors from dark colors and light colors from light colors, loss of contrast sensitivity, and can be used to relieve or reduce the severity of at least one symptom including but not limited to straight lines that appear bent in the Amsler grid.

[0160] The prophylactic use of one or more antibodies of the present invention is also contemplated herein in subjects at risk of developing macular degeneration, such as subjects over 50 years of age, subjects with a family history of macular degeneration, smokers, and subjects with obesity, high cholesterol, cardiovascular disease, or an unhealthy diet.

[0161] In a further embodiment of the invention, the antibody is used for the manufacture of a pharmaceutical composition or agent for treating a patient suffering from a disease or disorder associated with C5. In another embodiment of the invention, the antibody is used as an adjuvant therapy together with any other agent or any other treatment known to those skilled in the art that is useful for treating or alleviating a disease or disorder associated with C5.

[0162] Combination therapy Combination therapy may include the anti-C5 antibody of the invention, and any additional therapeutic agent that can be advantageously combined with the antibody of the invention and / or a biologically active fragment of the antibody of the invention. The antibody of the invention can be synergistically combined with one or more drugs or therapies for treating C5-related diseases or disorders. In some embodiments, the antibody of the invention can be combined with a second therapeutic agent to relieve one or more symptoms of the disease.

[0163] Depending on the C5-related disease or disorder, the antibody of the invention can be used in combination with one or more additional therapeutic agents, including but not limited to anticoagulants (e.g., warfarin, aspirin, heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran)), anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), antihypertensive drugs (e.g., angiotensin-converting enzyme inhibitors), immunosuppressive agents (e.g., vincristine, cyclosporine A, or methotrexate), fibrinolytic agents (e.g., ancrod, ε-aminocaproic acid, anti-plasmin-a 1 , prostacyclin, and defibrotide), lipid-lowering agents such as inhibitors of hydroxymethylglutaryl CoA reductase, anti-CD20 drugs such as rituximab, anti-TNF drugs such as infliximab, anti-seizure drugs (e.g., magnesium sulfate), C3 inhibitors, or antithrombotic drugs.

[0164] In certain embodiments, the second therapeutic agent is another antibody against the C5 protein. The use of combinations ( "cocktails") of antibodies having broad neutralizing or inhibitory activity against C5 is contemplated herein. In some embodiments, non-competing antibodies can be administered in combination to a subject in need thereof. In some embodiments, the antibodies that make up the combination bind to different non-overlapping epitopes of the protein. The antibodies that make up the combination can block the binding of C5 to the C5 convertase, and / or the C It can prevent / inhibit cleavage into C5a and C5b. In certain embodiments, the second antibody may have a longer half-life in human serum.

[0165] As used herein, the term "in combination with" means that additional therapeutic active ingredients can be administered before, simultaneously with, or after administration of the anti-C5 antibody of the present invention. The term "in combination with" also includes sequential or simultaneous administration of the anti-C5 antibody and a second therapeutic agent.

[0166] Additional therapeutic active ingredients may be administered to a subject prior to administration of the anti-C5 antibody of the present invention. For example, a first component may be considered to be "before" a second component if the first component is administered 1 week, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second component. In other embodiments, additional therapeutic active ingredients may be administered to a subject after administration of the anti-C5 antibody of the present invention. For example, a first component may be considered to be "after" a second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours after administration of the second component. In still other embodiments, additional therapeutic active ingredients may be administered to a subject simultaneously with administration of the anti-C5 antibody of the present invention. "Simultaneous" administration for the purposes of the present invention includes, for example, administration of the anti-C5 antibody and the additional therapeutic active ingredient to the subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-C5 antibody and the additional therapeutic active ingredient may be administered intravenously); or each dosage form may be administered via a different route (e.g., the anti-C5 antibody may be administered intravenously and the additional therapeutic active ingredient may be administered orally). In any case, a single dosage form, separate dosage forms by the same route, or separate dosage forms by different routes are all considered "simultaneous administration" for the purposes of this disclosure. For the purposes of this disclosure, administration of the anti-C5 antibody "before," "simultaneously," or "after" (as defined by the terms above in this specification) administration of an additional therapeutic active ingredient is considered administration of the anti-C5 antibody "in combination with" the additional therapeutic active ingredient.

[0167] The present invention includes pharmaceutical compositions in which the anti-C5 antibody of the present invention is co-formulated with one or more additional therapeutic active ingredients as described elsewhere herein.

[0168] Dosing regimen According to certain embodiments, a single dose of an anti-C5 antibody of the invention (or a pharmaceutical composition comprising an anti-C5 antibody or a combination of an anti-C5 antibody and any additional therapeutically active agent mentioned herein) can be administered to a subject in need thereof. According to certain embodiments of the invention, multiple doses of an anti-C5 antibody (or a pharmaceutical composition comprising an anti-C5 antibody or a combination of an anti-C5 antibody and any additional therapeutically active agent mentioned herein) can be administered to a subject over a defined period of time. Methods according to this aspect of the invention include continuously administering multiple doses of the anti-C5 antibody of the invention to a subject. As used herein, "administering continuously" means that each dose of the anti-C5 antibody is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising continuously administering to a patient a single initial dose of an anti-C5 antibody, followed by one or more secondary doses of an anti-C5 antibody, and optionally followed by one or more tertiary doses of an anti-C5 antibody.

[0169] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of the anti-C5 antibody of the invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-C5 antibody, but generally may differ from each other with respect to dosing frequency. However, in certain embodiments, the amount of anti-C5 antibody contained in the initial, secondary, and / or tertiary doses differs from each other during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as "loading doses," followed by subsequent doses being administered less frequently (e.g., "maintenance doses").

[0170] According to an exemplary embodiment of the present invention, each secondary and / or tertiary dose is administered 1 to 48 (e.g., 1, 1 1 / 2 、2、2 1 / 2 、3、3 1 / 2 、4、4 1 / 2 、5、5 1 / 2 、6、6 1 / 2 、7、7 1 / 2 、8、8 1 / 2 、9、9 1 / 2 、10、10 1 / 2 、11、11 1 / 2 、12、12 1 / 2 、13、13 1 / 2 、14、14 1 / 2 、15、15 1 / 2 、16、16 1 / 2 、17、17 1 / 2 、18、18 1 / 2 、19、19 1 / 2 、20、20 1 / 2 、21、21 1 / 2 、22、22 1 / 2 、23、23 1 / 2 、24、24 1 / 2 、25、25 1 / 2 、26、26 1 / 2 、 or more) hours later. As used herein, the phrase "immediately preceding dose" means, in the order of multiple administrations, the dose of the anti-C5 antibody administered to the patient immediately prior to the administration of the next dose in that order without intervening administrations.

[0171] Methods according to this aspect of the invention may include administering to a patient any number of secondary and / or tertiary doses of an anti-C5 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0172] In certain embodiments of the invention, the frequency at which secondary and / or tertiary doses are administered to a patient can vary during the treatment regimen. The dosing frequency can also be adjusted by a physician during treatment according to the needs of an individual patient after clinical examination.

[0173] Diagnostic uses of the antibody The anti-C5 antibodies of the invention can be used, for example, for diagnostic purposes, to detect and / or measure C5 in a sample. Some embodiments contemplate the use of one or more of the antibodies of the invention in an assay for detecting a C5-related disease or disorder. Example diagnostic assays for C5 include, for example, contacting a sample obtained from a patient with an anti-C5 antibody of the invention, where the anti-C5 antibody is labeled with a detectable label or reporter molecule, or used as a capture ligand for selectively isolating C5 from the patient's sample. Alternatively, an unlabeled anti-C5 antibody can be used in a diagnostic application in combination with a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules are 3 H, 14 C, 32 P, 35 S, or 125A radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific examples of assays that can be used to detect or measure C5 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0174] Samples that can be used in the C5 diagnostic assay according to the present invention can be obtained from a patient and include any tissue or fluid sample that can be obtained, which contains either a detectable amount of C5 protein or a fragment thereof under normal or pathological conditions. Generally, the level of C5 protein in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease associated with C5) is measured to initially establish a baseline or standard level of C5. This baseline level of C5 can then be compared to the C5 level measured in a sample obtained from an individual suspected of having a condition associated with C5 or symptoms related to such a condition.

[0175] Antibodies specific for the C5 protein may not contain additional labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if any) can determine the orientation of the peptide with respect to the surface to which the peptide binds. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin is oriented such that the C-terminal portion of the peptide is distal to the surface.

[0176] Selected embodiments Selected embodiments of the present disclosure include the following: In Embodiment 1, the present invention includes an isolated antibody or an antigen-binding fragment thereof that specifically binds to complement factor 5 (C5) protein, wherein the antibody or the antigen-binding fragment thereof interacts with one or more amino acids contained within C5 (SEQ ID NO: 359) as determined by hydrogen / deuterium exchange.

[0177] In Embodiment 2, the present invention includes an isolated antibody of the antigen-binding fragment of Embodiment 1, wherein the antibody or the antigen-binding fragment thereof interacts with one or more amino acids contained within the alpha and / or beta chain of C5 as determined by hydrogen / deuterium exchange.

[0178] In Embodiment 3, the present invention includes an isolated antibody of the antigen-binding fragment of Embodiment 1 or 2, wherein the antibody or the antigen-binding fragment thereof does not interact with the amino acids of the C5a anaphylatoxin region of C5 as determined by hydrogen / deuterium exchange.

[0179] In Embodiment 4, the present invention includes an isolated antibody of the antigen-binding fragment of any one of Embodiments 1 to 3, wherein the antibody or the antigen-binding fragment thereof interacts with one or more amino acids contained within SEQ ID NO: 360 and / or SEQ ID NO: 361 as determined by hydrogen / deuterium exchange.

[0180] In Embodiment 5, the present invention includes an isolated antibody or an antigen-binding fragment thereof of any one of Embodiments 1 to 4, wherein the antibody or the antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 591-599 of SEQ ID NO: 359; (b) amino acids 593-599 of SEQ ID NO: 359; (c) amino acids 775-787 of SEQ ID NO: 359; (d) amino acids 775-794 of SEQ ID NO: 359; and (e) amino acids 779-787 of SEQ ID NO: 359.

[0181] In Embodiment 6, the present invention includes an isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with at least 5 amino acids contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 360 and 361.

[0182] In Embodiment 7, the present invention includes an isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with the amino acid sequences of SEQ ID NOs: 360 and 361. and is isolated.

[0183] In Embodiment 8, the present invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to complement factor 5 (C5) protein, wherein the antibody or antigen-binding fragment thereof interacts with at least one of the following amino acid residues: N591, M592, A593, T594, G595, M596, D597, S598, W599, W775, E776, V777, H778, L779, V780, P781, R782, R783, K784, Q785, L786, Q787, F788, A789, L790, P791, D792, S793, or L794 of SEQ ID NO: 359.

[0184] In Embodiment 9, the invention includes an isolated antibody or an antigen-binding fragment thereof according to any one of Embodiments 1-8, wherein the antibody has one or more of the following characteristics: (a) having a serum concentration higher than 10 μg / mL by day 70 upon administration to cynomolgus monkeys; (b) blocking classical pathway (CP) hemolysis by day 35 upon administration to cynomolgus monkeys as measured in an ex vivo hemolysis assay; (c) blocking alternative pathway (AP) hemolysis by day 35 upon administration to cynomolgus monkeys as measured in an ex vivo hemolysis assay; (d) having a serum half-life longer than 10 days in cynomolgus monkeys; (e) having a serum concentration higher than 10 μg / mL by day 40 upon administration to C5 humanized mice; (f) blocking CP hemolysis by day 30 upon administration to C5 humanized mice as measured in an ex vivo hemolysis assay; and (g) having a serum half-life longer than 10 days in C5 humanized mice.

[0185] In Embodiment 10, the invention includes an isolated antibody or an antigen-binding fragment thereof according to any one of Embodiments 1-9, wherein the antibody has a further characteristic selected from the group consisting of: (a) being a fully human monoclonal antibody; (b) binding to human C5 with a dissociation constant (K D ) of less than 0.9 nM at 25°C as measured in a surface plasmon resonance assay; (c) binding to human C5 with a K D of less than 0.3 nM at 37°C as measured in a surface plasmon resonance assay; (d) binding to monkey C5 with a K D of less than 65 nM as measured in a surface plasmon resonance assay; (e) binding to human C5 mutant R885H (SEQ ID NO: 356) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (f) binding to human C5 mutant R885C (SEQ ID NO: 357) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (g) blocking more than 95% of human C5-mediated classical pathway (CP) hemolysis with an IC 50 of less than 6 nM as measured in a CP hemolysis assay; (h) blocking human C5-mediated alternative pathway (AP) hemolysis with an IC 50blocks more than 70% of human C5-mediated alternative pathway (AP) hemolysis; (i) as measured in a CP hemolysis assay, an IC of less than 185 nM 50 inhibits African green monkey C5-mediated CP hemolysis; (j) as measured in an AP hemolysis assay, an IC of less than 235 nM 50 inhibits African green monkey C5-mediated AP hemolysis; (k) as measured in a CP hemolysis assay, an IC of less than 145 nM 50 inhibits cynomolgus monkey C5-mediated CP hemolysis; and (l) as measured in an AP hemolysis assay, an IC of less than 30 nM 50 inhibits cynomolgus monkey C5-mediated AP hemolysis.

[0186] In Embodiment 11, the present invention comprises an isolated antibody or antigen-binding fragment of any one of Embodiments 1 to 10, wherein the antibody or antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy-chain variable region (HCVR) sequences listed in Table 1; and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light-chain variable region (LCVR) sequences listed in Table 1.

[0187] In Embodiment 12, the present invention is: (a) SEQ ID NOs: 4, 20, 36, 52, 68, 84 An HCDR1 domain having an amino acid sequence selected from the group consisting of 100, 124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292, 308, 324, and 340; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 126, 142, 150, 158, 174, 190, 206, 222, 238, 254, 270, 278, 294, 310, 326, and 342; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 128, 144, 152, 160, 176, 192, 208, 224, 240, 256, 272, 280, 296, 312, 328, and 344; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332, and 348; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 118, 134, 166, 182, 198, 214, 230, 246, 262, 286, 302, 318, 334, and 350; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 120, 136, 168, 184, 200, 216, 232, 248, 264, 288, 304, 320, 336, and 352, comprising an isolated antibody of any one of Examples 1 to 11 or an antigen-binding fragment thereof.

[0188] In Embodiment 13, the present invention comprises an isolated antibody of any one of Embodiments 1 to 12 or an antigen-binding fragment thereof, comprising an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1.

[0189] In Embodiment 14, the present invention includes the isolated antibody of Embodiment 13 or an antigen-binding fragment thereof, which comprises an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0190] In Embodiment 15, the present invention includes the isolated antibody or antigen-binding fragment of any one of Embodiments 11 to 14, which comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 98 / 114, 122 / 106, 98 / 130, 138 / 106, 146 / 106, 122 / 130, 146 / 114, 146 / 130, 138 / 130, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 258, 274 / 282, 290 / 298, 306 / 314, 322 / 330, and 338 / 346.

[0191] In Embodiment 16, the present invention includes the isolated antibody of any one of Embodiments 11 to 15 or an antigen-binding fragment thereof, which comprises three CDRs contained within an HCVR selected from the group consisting of SEQ ID NOs: 50, 98, 138, and 202; and three CDRs contained within an LCVR selected from the group consisting of SEQ ID NOs: 58, 106, and 210.

[0192] In Embodiment 17, the present invention includes the isolated antibody of Embodiment 16 or an antigen-binding fragment thereof, which comprises CDRs selected from the group consisting of: (a) SEQ ID NOs: 52, 54, 56, 60, 62, and 64; (b) SEQ ID NOs: 100, 102, 104, 108, 110, and 112; (c) SEQ ID NOs: 140, 142, 144, 108, 110, and 112; and (d) SEQ ID NOs: 204, 206, 208, 212, 214, and 216.

[0193] In Embodiment 18, the present invention provides an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50 / 58, 98 / 106, 138 / 106, and 202 / 210 Comprising an isolated antibody of embodiment 17 or an antigen-binding fragment thereof.

[0194] In embodiment 19, the invention comprises an antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiment 17 for binding to C5.

[0195] In embodiment 20, the invention comprises an antibody or an antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment of embodiment 17.

[0196] In embodiment 21, the invention comprises an antibody or an antigen-binding fragment thereof of embodiment 9 or 10, comprising a heavy-chain variable region comprising an amino acid sequence listed in Table 1 having 5 or fewer amino acid substitutions.

[0197] In embodiment 22, the invention comprises an antibody or an antigen-binding fragment thereof of embodiment 21, comprising a light-chain variable region comprising an amino acid sequence listed in Table 1 having 5 or fewer amino acid substitutions.

[0198] In embodiment 23, the invention comprises an antibody or an antigen-binding fragment thereof of embodiment 9 or 10, comprising a heavy-chain variable region having at least 90% sequence identity to SEQ ID NO: 98.

[0199] In embodiment 24, the invention comprises an antibody or an antigen-binding fragment thereof of embodiment 23, comprising a light-chain variable region having at least 90% sequence identity to SEQ ID NO: 106.

[0200] In Embodiment 25, the present invention relates to three CDRs of HCVR [where HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 122, 138, 146, 154, 170, 186, 202, 218, 234, 250, 266, 274, 290, 306, 322, and 338]; and three CDRs of LCVR, [where LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 114, 130, 162, 178, 194, 210, 226, 242, 258, 282, 298, 314, 330, and 346], and comprises an isolated monoclonal antibody or an antigen-binding fragment thereof that blocks C5 cleavage into C5a and C5b.

[0201] In Embodiment 26, the present invention relates to a pharmaceutical composition comprising an isolated antibody or an antigen-binding fragment thereof that binds to C5 according to any one of Embodiments 1 to 25, and a pharmaceutically acceptable carrier or diluent.

[0202] In Embodiment 27, the present invention relates to an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody shown in any one of Embodiments 1 to 25.

[0203] In Embodiment 28, the present invention relates to an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody shown in any one of Embodiments 1 to 25.

[0204] In Embodiment 29, the present invention relates to a vector comprising the polynucleotide sequence of Embodiment 27 or 28.

[0205] In Embodiment 30, the present invention relates to a cell expressing the vector of Embodiment 29.

[0206] In Embodiment 31, the present invention includes a method of preventing, treating, or causing at least one symptom or sign of a C5-related disease or disorder, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment of any one of Embodiments 1-25.

[0207] In Embodiment 32, the present invention includes the method of Embodiment 31, wherein the disease or disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, geographic atrophy, uveitis, neuromyelitis optica, multiple sclerosis, stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, disorders of inappropriate or undesirable complement activation, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammatory disorders, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal burns such as burns or frostbite, post-ischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, renal ischemia-reperfusion injury, lupus nephritis, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal artery bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious diseases or sepsis, immune complex diseases and autoimmune diseases, kidney disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, and myasthenia gravis.

[0208] In Embodiment 33, the present invention includes the method of Embodiment 31, wherein the disease or disorder is aHUS.

[0209] In Embodiment 34, the present invention includes the method of Embodiment 31, wherein the disease or disorder is PNH.

[0210] In Embodiment 35, the present invention includes any one of the methods of Embodiments 31 to 34, wherein the pharmaceutical composition is prophylactically or therapeutically administered to a subject in need thereof.

[0211] In Embodiment 36, the present invention includes any one of the methods of Embodiments 31 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0212] In Embodiment 37, the present invention includes the method of Embodiment 36, which is selected from the group consisting of anticoagulants, anti-inflammatory drugs, antihypertensive drugs, immunosuppressants, lipid-lowering agents, anti-CD20 drugs such as rituximab, anti-TNF drugs such as infliximab, antiepileptic drugs, C3 inhibitors, second anti-C5 antibodies, and antithrombotic agents.

[0213] In Embodiment 38, the present invention includes any one of the methods of Embodiments 31 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

Example

[0214] The following examples are provided to fully disclose and describe to those skilled in the art the methods of making and using the methods and compositions of the present invention, and are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be expected. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25 °C, and pressures are at or near atmospheric pressure.

[0215] Example 1: Generation of Human Antibodies Against Complement Factor 5 (C5) Protein Human antibodies against C5 protein were generated in VELOCIMMUNE mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions. (R) Mice were immunized with serum-purified human C5 protein (Calbiochem catalog number 20-4888).

[0216] The antibody immune response was monitored by a C5-specific immune assay. When the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce C5-specific antibodies. Using these cell lines, several anti-C5 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this way were named H2M11683N and H2M11686N.

[0217] Also, anti-C5 antibodies were isolated directly from antigen-positive mouse B cells without fusing with myeloma cells as described in U.S. Patent No. 7,582,298 (specifically incorporated herein by reference in its entirety). Using this method, several fully human anti-C5 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies generated in this way were designated H4H12159P, H4H12161P, H4H12163P, H4H12164P, H4H12166P, H4H12167P, H4H12168P, H4H12169P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2, H4H12177P2, and H4H12183P2.

[0218] The biological properties of exemplary antibodies generated according to the method of this example are described in detail in the examples shown below.

[0219] Example 2: Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-C5 antibodies of the present invention.

[0220] [Table 1]

[0221] The corresponding nucleic acid sequence identifiers are shown in Table 2.

[0222]

Table 2

[0223] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", "H2M", etc.), followed by a numerical identifier (e.g., "11686", "12166", "12183", etc. as shown in Table 2), followed by a "P", "P2", or "N" suffix. Thus, according to this nomenclature, an antibody may be referred to herein, for example, as "H2M11686N", "H4H12183P2", "H4H12168P", etc. The H4H and H2M prefixes in the antibody symbol designations used herein indicate a particular Fc region isotype. For example, an "H4H" antibody has a human IgG4 that contains a serine-to-proline mutation (S108P) in the hinge region to promote dimer stabilization, and an "H2M" antibody has a mouse IgG2 Fc (a or b isotype) (all variable regions are fully human and are first indicated by "H" in the antibody symbol designation). It will be understood by those skilled in the art that an antibody having a particular Fc isotype can be converted to a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.), but in any case, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 2 - remain the same, and the binding properties to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0224] In certain embodiments, a selected antibody having a mouse IgG1 Fc was converted to an antibody having a human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains 2 or more amino acid changes disclosed in US20100331527.

[0225] To generate mutant antibodies, various residues in the complementarity determining regions (CDRs) of H4H12166P were mutated to histidine, generating nine mutant antibodies identified as H4H12166P2 to H4H12166P10. Histidine mutations in the CDRs have been shown to confer pH-dependence of binding to the target antigen, resulting in improved pharmacokinetics (Igawa et al. 2010, Nat. Biotechnol. 28:1203-1207).

[0226] Control constructs used in the following examples The following control constructs (anti-C5 antibodies) were included in the experiments disclosed herein for comparison purposes: " Control drug 1 , the V H / V L sequence of the antibody "h5G1.1" against human C5 according to U.S. Patent No. 6,355,245 (Alexion Pharmaceuticals, Inc.); and " Control drug 2 ", the V H / V L sequence of the human monoclonal antibody against human C5 having the sequence according to U.S. Patent Application Publication No. 2013 / 0022615 (Novartis).

[0227] Example 3: Binding of antibodies to C5 determined by surface plasmon resonance Equilibrium dissociation constant (K DThe value) was determined using a real-time surface plasmon resonance biosensor assay on a Biacore T200 instrument. To capture the expressed anti-C5 antibody having a human Fc constant region, the Biacore sensor surface was derivatized with a monoclonal mouse anti-human Fc antibody (GE Healthcare, number BR-1008-39) by amine coupling. The Biacore binding assay was performed in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% volume / volume Surfactant P20). Human C5 was obtained from a commercial source (EMD). Other C5 reagents having a C-terminal myc-myc-hexahistidine tag were expressed (hereinafter referred to as C5-mmh). Human C5-mmh reagents containing histidine and cysteine point mutations at arginine 885 were also expressed (hereinafter referred to as C5 R885H-mmh and C5 R885C-mmh, respectively). Various concentrations of human C5, human C5 R885H-mmh (SEQ ID NO: 356), human C5 R885C-mmh (SEQ ID NO: 357) and monkey C5-mmh (SEQ ID NO: 358) (3-fold dilutions ranging from 100 nM to 1.23 nM) prepared in HBST running buffer were injected onto the anti-C5 antibody capture surface at a flow rate of 30 μL / min. The binding of all C5 reagents to each of the captured monoclonal antibodies was monitored for 3 minutes, and their dissociation in HBST running buffer was monitored for 8 minutes. All binding kinetics experiments were performed at either 25 °C or 37 °C. The kinetic binding (k a ) and dissociation (k d ) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrub ber 2.0c curve fitting software. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) were calculated from the kinetic rate constants: K D (M) = k d / k a and t 1 / 2 (min) = ln2 / (60 x k d ).

[0228] The binding kinetic parameters for human C5 binding to anti-C5 antibody at 25°C and 37°C are shown in Tables 3 and 4.

[0229] [Table 3]

[0230] [Table 4]

[0231] The monkey C5-mmh binding to anti-C5 antibody at 25°C and 37°C is shown in Tables 5 and 6.

[0232] [Table 5]

[0233] [Table 6]

[0234] The binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibody at 25°C is shown in Tables 7 and 8, respectively.

[0235] [Table 7]

[0236] [Table 8]

[0237] The binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibody at 37°C is shown in Tables 9 and 10, respectively.

[0238] [Table 9]

[0239]

Table 10

[0240] At 25°C, all 25 anti-C5 antibodies of the present invention bound to human C5 with K values ranging from 73 pM to 8.4 nM as shown in Table 3. D At 37°C, the anti-C5 antibodies of the present invention bound to human C5 with K values ranging from 103 pM to 18.5 nM as shown in Table 4. D At 25°C, 25 out of the 25 anti-C5 antibodies of the present invention tested bound to monkey C5-mmh with K values ranging from 133 pM to 64 nM as shown in Table 5. D At 37°C, 25 out of the 25 anti-C5 antibodies of the present invention tested bound to monkey C5-mmh with K values ranging from 133 pM to 118 nM as shown in Table 6. D At 25°C, 16 of the anti-C5 antibodies of the present invention tested bound to human C5 R885H-mmh with K values ranging from 147 pM to 10.9 nM as shown in Table 7. D At 25°C, 16 of the 16 anti-C5 antibodies of the present invention tested bound to human C5 R885C-mmh with K values ranging from 251 pM to 190 nM as shown in Table 8. D At 37°C, 16 of the 16 anti-C5 antibodies of the present invention tested bound to human C5 R885H-mmh with K values ranging from 1.49 nM to 69.4 nM as shown in Table 9. D At 25°C, 16 of the 16 anti-C5 antibodies of the present invention tested bound to human C5 R885C-mmh with K values ranging from 1.74 nM to 159 nM as shown in Table 10. D At 25°C, 16 of the 16 anti-C5 antibodies of the present invention tested bound to human C5 R885C-mmh with K values ranging from 1.74 nM to 159 nM as shown in Table 10.

[0241] Example 4: Antibodies That Bind to C5 at Different pHs The effect of pH on the dissociation rate of recombinant human C5 bound to a purified anti-C5 monoclonal antibody was determined using a Biacore T200 with real-time surface plasmon resonance biosensor. The Biacore sensor surface was first derivatized with a monoclonal mouse anti-human Fc antibody (GE, number BR-1008-39) by amine coupling to capture the expressed anti-C5 monoclonal antibody having human IgG4 Fc. All Biacore binding tests were performed using two running buffers PBS-T, pH 7.4 (0.01M Na 2 HPO 4 / NaH 2 PO 4 , 0.15M NaCl,, 0.05% volume / volume Tween-20, adjusted to pH 7.4) and PBS-T, pH 6.0 (0.01M Na 2 HPO 4 / NaH 2 PO 4 , 0.15M NaCl, 0.05% volume / volume Tween-20, adjusted to pH 6.0). Various concentrations of human C5 (EMD, catalog number 204888) or cyno C5.mmh (prepared in PBS-T, ranging from 100 nM to 11.11 nM, pH 7.4, 3-fold dilution) were injected onto the surface capturing the anti-C5 monoclonal antibody at a flow rate of 50 μL / min for 3 minutes, and their dissociation in two running buffers PBS-T, pH 7.4 and PBS-T, pH 6.0 was monitored for 6 minutes. All binding kinetics experiments were performed at 25 °C and 37 °C. The kinetic dissociation constant (k d ) was determined by fitting the real-time sensorgram to a 1:1 binding model using Scrubber 2.0c curve fitting software. The binding dissociation half-life (t 1 / 2 ) was calculated from k d as:

Equation

[0242] Table 11 and 12 show the half-life ratios of human C5 binding to various anti-C5 monoclonal antibodies at 25 °C and 37 °C in two running buffers, PBS-T, pH 7.4 and PBS-T, pH 6.0.

[0243]

Table 11

[0244]

Table 12

[0245] Table 13 and 14 show the half-life ratios of monkey C5 binding to various anti-C5 monoclonal antibodies at 25 °C and 37 °C in two running buffers, PBS-T, pH 7.4 and PBS-T, pH 6.0.

[0246]

Table 13

[0247]

Table 14

[0248] As shown in Tables 11 - 14, the selected anti-C5 antibodies showed pH-dependent binding as indicated by the t 1 / 2 ratio.

[0249] Example 5: Octet Cross-Competition between Anti-C5 Antibodies The binding competition between anti-C5 monoclonal antibodies (mAbs) was determined using a real-time label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25 °C with the plate shaken at 1000 rpm in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% volume / volume Surfactant Tween-20, 0.1 mg / mL BSA (Octet HBS-P buffer). To evaluate whether two antibodies can compete with each other for binding to their respective epitopes on human C5 (hC5 purified from plasma, EMD), an anti-human Fc antibody-coated Octet biosensor chip (Pall ForteBio Corp., number 18-5060) was first loaded with approximately 1.5 nm of anti-human C5 mAb (hereinafter referred to as mAb1) by submerging the chip into a well containing a 50 μg / mL solution of anti-human C5 mAb for 3 minutes. Subsequently, the antibody-loaded biosensor chip was saturated with a blocking H4H isotype control mAb (hereinafter referred to as the blocking mAb) by immersing it into a well containing a 200 μg / mL solution of the blocking mAb for 4 minutes. Subsequently, the biosensor chip was immersed into a well containing a co-complexed solution of 50 nM hC5 and 1 μM of a second anti-human C5 mAb (hereinafter referred to as mAb2) that had been pre-incubated for 2 hours for 4 minutes. The biosensor chip was washed with Octet HBS-P buffer between each step of the experiment. The real-time binding response was monitored during the course of the experiment, and the binding response was recorded at the end of each step. The binding of pre-complexed mAb2 to mAb1 in the presence of human C5 was corrected against the background binding, compared, and the competitive / non-competitive behavior of various anti-C5 monoclonal antibodies was determined. The co-complexed solution was prepared by mixing 50 nM hC5 and 1 μM of a second anti-human C5 mAb (hereinafter referred to as mAb2) that had been pre-incubated for 2 hours.

[0250] Table 15 clearly defines the relationship of antibodies that compete in both directions, independent of the order of binding.

[0251]

Table 15

[0252] Example 6: Inhibition of C5-mediated complement-dependent cytotoxicity in B cell bioassay This example describes a bioassay that tests the role of C5 using an anti-CD20 antibody in the classical complement pathway. Therapeutic anti-CD20 antibodies against the B cell-specific cell surface antigen CD20 have been shown to effect CDC of B cells (Glennie et al. 2007, Mol. Immunol. 44:3823-3837), and a CDC assay using cell lines expressing CD20 has been previously described (Flieger et al. 2000, Cell. Immunol. 204:55-63). The human B cell line Daudi cells expressing CD20, complement-depleted serum or C5-deficient serum with exogenous C5 variants, and an anti-CD20 antibody (antibody containing the VH / VL of "2F2" from U.S. Patent No. 8,529,902) were used to evaluate the role of C5 in CDC.

[0253] For the C5 CDC bioassay, Daudi cells were seeded at 10,000 cells / well on a 96-well assay plate in 10% FBS, penicillin / streptomycin, L -Glutamine, sodium pyruvate, and non-essential amino acids (RPMI complete medium) or RPMI containing 1% BSA, penicillin / streptomycin, and L-glutamine (RPMI / BSA). All assays testing the mutated anti-hC5 antibody were tested in RPMI complete medium along with the test of the non-mutated antibody using C5-containing human serum, while the assays testing the non-mutated antibody used African green monkey serum, and the human C5 variant was tested in RPMI / BSA medium. To measure CDC using human or monkey serum, the anti-CD20 antibody was diluted 1:3 from 100 nM to 2 pM (including control samples without antibody), and incubated with cells at 25 °C for 10 minutes, followed by the addition of 1.66% serum or 1.66% C5-deficient serum and 6.6 nM C5 variant protein. The amount of C5 protein to be added to the C5-deficient serum was based on the reported value of 0.37 μM for the C5 concentration in human serum (Rawal et al 2008, J. Biol. Chem. 283:7853-7863). To test the C5 antibody inhibition of CDC, the C5 antibody was diluted 1:3 from 100 nM to 2 pM (including control samples without antibody), and incubated with 1.66% serum or 1.66% C5-deficient serum and 6.6 nM C5 variant protein for 30 minutes. 10 minutes before adding the antibody to the cells with serum, the anti-CD20 antibody was added to the cells at 1 nM, 2 nM, 3 nM, 3.5 nM, 7 nM, 10 nM, or 30 nM. At the end of the incubation with the anti-CD20 antibody, the antibody / serum mixture was added to the cells. Cytotoxicity was measured after a 3.5-hour incubation at 37 °C 5% CO 2 After incubation for 3.5 hours at TM and adding the CytoTox-Glo TM reagent (Promega, number G9292). CytoTox-Glo TM is a luminescence-based reagent for measuring cell death, and increased luminescence is observed as increased cytotoxicity increases (measured in relative light units, RLU). Untreated cells in control wells were rinsed by treating with digitonin immediately after adding the CytoTox-Glo TM reagent to determine the maximum killing of cells. The plates were incubated with CytoTox-GloTM Luminescence was read using a Victor X instrument (Perkin Elmer) 15 minutes after addition. When calculated, the percentage of cytotoxicity was calculated using the RLU values by using the following equation: [Number]

[0254] In this equation, "background cell lysis" is luminescence from cells treated with only medium and serum without any CD20 antibody, and "maximum cell lysis" is luminescence from cells treated with digitonin. Results expressed as % cytotoxicity or RLU were analyzed using Prism 5 software (GraphPad) using non-linear regression (4-parameter logistics) to obtain EC 50 values and IC 50 values. Antibody inhibition was calculated such that 0 - 100% inhibition was in the range of inhibition from the concentration of anti-CD20 antibody used in the assay without inhibitor to 0 nM anti-CD20 antibody.

[0255] Results A total of 25 anti-human C5 antibodies (16 non-mutated and 9 mutated) were tested for their ability to inhibit C5 in a CDC assay using Daudi cells with anti-CD20 antibody and human serum (including normal hC5 or C5 variants) or African green monkey serum. Various residues in the complementarity determining region (CDR) of H4H12166P were mutated to histidine to generate 9 mutated antibodies H4H12166P2 - H4H12166P10. Histidine mutations in the CDR have been shown to confer pH-dependence of binding to the target antigen and result in improved pharmacokinetics (Igawa et al. 2010, Nat. Biotechnol. 28:1203 - 1207).

[0256] [Table 16]

[0257] As shown in Tables 16 and 17, all 25 anti-hC5 antibodies showed complete inhibition of C5-mediated CDC present at 1.66% of human serum. The IC50 of the non-mutated antibodies ranged from 1.2 to 3.4 nM. The IC50 of the mutated antibodies ranged from 3.0 nM to 12 nM. The parental non-mutated antibody H4H12166P produced complete inhibition at IC50s of 2.6 nM and 2.9 nM.

[0258] [Table 17]

[0259] Sixteen non-mutated anti-hC5 antibodies showed complete inhibition of C5-mediated CDC by African green monkey C5 with IC50s ranging from 2.0 nM to 14 nM.

[0260] Four out of nine mutated antibodies showed complete inhibition of C5-mediated CDC by African green monkey C5 with IC50s ranging from 7.1 nM to 9.9 nM. The remaining six mutated antibodies were blockers with IC50s greater than 10 nM, and the maximum inhibition (with 100 nM antibody) ranged from 34% to 85%. The parental non-mutated antibody H4H12166P produced complete inhibition at IC50s of 4.5 nM and 5.6 nM.

[0261] To test whether the anti-hC5 antibodies inhibit the human C5 variants, R885H and R885C, C5-deficient human serum was tested with each C5 variant at 6.6 nM. All 25 anti-hC5 antibodies showed complete inhibition of C5 variant R885H-mediated CDC with IC50s of non-mutated antibodies ranging from 0.48 nM to 4.2 nM, while the IC50s of the mutated antibodies ranged from 1.3 nM to 7.0 nM. The parental non-mutated antibody H4H12166P produced complete inhibition at IC50s of 1.3 nM and 1.3 nM.

[0262] Of the 16 non-mutated antibodies hC5 antibodies, 15 showed complete inhibition of CDC mediated by C5 variant R885C with IC50 values ranging from 0.43 nM to 1.6 nM. One non-mutated antibody showed weak inhibition of CDC with a maximum inhibition of 67% (at 100 nM antibody) and IC50 > 20 nM. All 9 mutated antibodies showed complete inhibition of CDC mediated by C5 variant R885C with IC50 values ranging from 0.77 nM to 3.5 nM. The parental non-mutated antibody H4H12166P produced complete inhibition with IC50 values of 0.46 nM and 0.76 nM.

[0263] The anti-CD20 antibody showed CDC of Daudi cells with EC50 values of 1.0 nM, 1.4 nM, and 1.9 nM for human serum, 2.4 nM and 2.6 nM for African green monkey serum, 1.9 nM and 6.3 nM for hC5-deficient serum using hC5 variant R885H, and 2.7 nM and 9.5 nM for hC5-deficient serum using hC5 variant R885C, using 1.66% serum. None of the irrelevant IgG control antibodies, control mAb1 and control mAb2 showed any inhibition of CDC.

[0264] Example 7: Inhibition of C5a activity determined by luciferase assay This example describes an assay for testing the activation of C5a by one of its receptors, C5aR1. C5aR1 is a G protein-coupled receptor (GPCR) and can initiate various GPCR-coupled signaling pathways (Monk et al. 2007, Br. J. Pharmacol. 152:429-448). A bioassay was established using HEK293 cells stably transfected with luciferase receptor [NFAT response element (4X)-luciferase] using human C5aR1 (accession number NP_001727.1) and human Gα16 (accession number NP_002059.3). Gα16 is a relatively promiscuous G protein, which can couple with various types of GPCRs to activate PLC-β and subsequent Ca ++can bring about an increase, which in turn activates NFAT translocation and reporter gene transcription (Kostenis et al. 2005, Trends Pharmacol. Sci. 26:595 - 602). The resulting cell line HEK293 / hGα16 / hC5aR1 / NFAT-luc was isolated and maintained in 10% DMEM containing 10% FBS, NEAA, penicillin / streptomycin, 500 μg / mL G418, 100 μg / mL hygromycin B, and 7 μg / mL blasticidin.

[0265] For the C5a luciferase bioassay, HEK293 / hG□16 / hC5aR1 / NFAT-luc cells were seeded in 96-well assay plates at 20,000 cells / well in OPTIMEM (Invitrogen, number 31985 - 070) supplemented with 0.5% BSA, penicillin / streptomycin, and L-glutamine, and then incubated overnight at 37 °C and 5% CO 2 . Since serum has been shown to cleave and activate hC5a, BSA was used instead of FBS (Klos et al., 2013, Pharmacol. Rev. 65:500 - 543). The next morning, hC5a was titrated from 100 nM to 2 pM (including control samples without hC5a), and added to the cells to determine the dose-response titration curve for the cell line. To examine hC5a antibody inhibition of hC5a, 500 pM of hC5a was added to the cells. Immediately thereafter, antibodies diluted 1:3 from 100 nM to 2 pM (including control samples without antibody) were added to the cells. The cells were incubated for 5.5 hours at 37 °C in 5% CO 2 . The cells were incubated in the presence of OneGlo TM reagent (Promega, number E6051), and luciferase activity was detected after incubation. OneGlo TMis a luminescence-based reagent that measures the amount of luciferase present in cells. In this assay, increased hC5a activation results in increased luciferase production and luminescence (measured in relative light units, RLU). Luminescence measurements were performed using a Victor X instrument (Perkin Elmer). Results were analyzed using Prism 5 software (GraphPad) using nonlinear regression (4-parameter logistics) to obtain EC 50 values and IC 50 values. Antibody inhibition was calculated such that 0 - 100% inhibition was in the inhibition range from 500 pM hC5a without inhibitor to 0 nM hC5a.

[0266] Four anti-hC5 antibodies were tested for their ability to inhibit hC5a activation of its receptor hC5aR1 by measuring the degree of inhibition of 500 pM hC5a activation in HEK293 / hGα16 / hC5aR1 / NFAT-luc cells.

[0267]

Table 18

[0268] As shown in Table 18, all four antibodies of the present invention showed complete inhibition of 500 pM hC5a with IC50 values ranging from 0.035 nM to 0.46 nM. The irrelevant IgG control antibody, control mAb3, showed no inhibition of hC5a. hC5a activated HEK293 / Gα16 / hC5aR1 / NFAT-luc cells with an EC50 of 0.39 nM.

[0269] Example 8: Hemolysis Bioassay Classical pathway hemolysis assay (CH) and alternative pathway hemolysis assay (AH) were developed to test antibody activity.

[0270] CH is a screening assay for the activation of the classical complement pathway, which is sensitive to a decrease, absence, and / or inactivation of any component of the pathway. CH tests the functional ability of the serum complement components of the classical pathway to lyse sheep red blood cells (SRBCs) pre-coated with rabbit anti-sheep red blood cell antibody (hemolysin). When SRBCs coated with antibody are incubated with test serum, the classical pathway of complement is activated and hemolysis occurs. In the absence of complement components, the CH level is zero; when one or more components of the classical pathway are decreased, CH is decreased. (Nilsson et al 1984, J. Immunol. Meth. 72:49-59). This assay is used for the characterization and screening of high affinity anti-human C5 antibodies.

[0271] Methods (A) Classical pathway complement hemolysis assay A desired number of sheep red blood cells (SRBCs) were washed with GVB++ buffer and resuspended at 1 x 10^9 cells / mL. To sensitize the SRBCs, an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg / mL) was mixed with them at 37°C for 20 minutes. The sensitized S RBC cells were diluted to 2x10^8 cells / ml in GVB++ before use in the hemolysis assay. Normal human serum or cynomolgus monkey serum was diluted to 2% or 10% with GVB++ buffer. To examine the inhibition of C5-mediated hemolytic activity, test antibodies were pre-incubated at 4°C for 20 minutes at concentrations ranging from 0.6 nM to 800 nM in 2% - 10% normal human or 10% cynomolgus monkey serum or African green monkey serum. A round-bottom 96-well plate was used to measure hemolytic activity. A total of 100 ul of sensitized rabbit RBC (2x10^8 cells / ml) was plated into the 96-well plate, followed by the addition of 100 ul of each serum sample pre-incubated with the test antibody. The cells were gently mixed and incubated at 37°C for 60 minutes. After the incubation time, the cells were sedimented by centrifugation at 1250 x g at 4°C. A total of 100 uL of the supernatant was transferred to a new 96 flat-bottom plate and read at 412 nm with a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 1 - 5% for the treatment.

[0272] The percent hemolysis was calculated as follows:

Equation

[0273] In this equation, "background cell lysis" is the OD at A412nm from cells incubated with GVB++ buffer alone without serum. "Maximum cell lysis" is the OD at A412nm from cells treated with water. The results expressed as % hemolysis were analyzed using non-linear regression (4-parameter logistics) with Prism 5 software (GraphPad) to obtain the IC50 value. The data are presented as mean ± standard error of the mean.

[0274] (B) Alternative complement assay The desired number of rabbit red blood cells (RbRBC) in GVB-Mg 2+Washed with / EGTA buffer and resuspended at 2x10^8 cells / ml. Normal human or cynomolgus monkey serum was diluted to 10% in GVB-Mg 2+ / EGTA buffer. To test for inhibition of C5-mediated hemolytic activity, antibodies at concentrations ranging from 3 nM to 800 nM were pre-incubated for 20 minutes at 4°C in 5 - 10% normal human serum or cynomolgus monkey serum. Hemolytic activity was measured using round-bottom 96-well plates. A total of 100 ul of RbRBC (2x10^8 cells / ml) was plated into 96-well plates, followed by addition of 100 ul of 10% normal human serum or cynomolgus or African green monkey serum that had been pre-incubated with anti-C5 antibody. The cells were gently mixed and incubated at 37°C for 60 minutes. After the incubation period, the cells were sedimented by centrifugation at 1250 x g at 4°C. A total of 100 uL of the supernatant was transferred to a new 96-well flat-bottom plate and read at 412 nm using a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 5% serum.

[0275] The percent hemolysis was calculated as follows: [Number]

[0276] In this equation, "background cell lysis" is the OD at A412nm from cells incubated with only GVB-Mg / EGTA buffer without serum or any anti-C5 antibody. "Maximum cell lysis" is the OD at A412nm from cells treated with water. Inhibition by anti-C5 antibody, IC values were calculated using non-linear regression (4-parameter logistics) with Prism 6 software (GraphPad). 50 values were calculated using non-linear regression (4-parameter logistics) with Prism 6 software (GraphPad).

[0277] Results (A) Inhibition of human C5 hemolysis A total of 25 anti-human C5 (hC5) antibodies (16 non-mutated and 9 mutated) were tested for their ability to inhibit C5 from normal human serum (NHS) in the CH50 assay using sheep red blood cells (SRBC) sensitized in the assay and in the AH50 assay using rabbit red blood cells (RRBC).

[0278]

Table 19

[0279] As shown in Table 19, the 16 anti-hC5 antibodies of the present invention showed more than 94% inhibition of hemolysis in the classical pathway (CP) mediated by C5 present at 1% of human serum. The IC50 of the antibodies ranged from 2.1 to 5.9 nM, and the percent inhibition ranged from 95% to 99%. All 16 anti-C5 antibodies showed more than 60% inhibition (except for H4H12169P) in the alternative pathway (AP) hemolysis assay mediated by C5 present in 5% NHS. The IC50 of the antibodies ranged from 13 to 160 nM, and the percent inhibition activity ranged from 44% to 81%.

[0280]

Table 20

[0281] As shown in Table 20, all 9 mutated anti-hC5 antibodies showed inhibition of CP and AP hemolytic activities mediated by C5 present in 5% human serum. In the CP hemolysis assay, the parental non-mutated antibody H4H12166P showed more than 98% inhibition with an IC50 of 10.9 nM. Eight mutated anti-hC5 antibodies showed more than 90% inhibition with IC50s ranging from 10.3 nM to 24.3 nM. The mutant anti-C5 antibody 12166P10 showed 74% partial inhibition. In the AP hemolysis assay, the parental non-mutated antibody H4H12166P showed more than 85% inhibition with an IC50 of 20.9 nM. The mutated anti-hC5 antibodies showed an inhibition range of 72 - 83%, and the IC50 antibodies ranged from 28 nM to 0.15 μM.

[0282] (B) Inhibition of monkey C5 hemolysis A total of 25 anti-human C5 (hC5) antibodies (16 non-mutated and 9 mutated) were tested for their ability to inhibit C5 from cynomolgus monkeys and African green monkeys in a CH50 assay using sensitized sheep red blood cells (SRBC) and an AH50 assay using rabbit red blood cells (RRBC).

[0283]

Table 21

[0284] As shown in Table 21, the anti-hC5 antibodies showed inhibition of CP or AP hemolytic activity at various levels in 5% African green monkey serum. In the CP assay, 2 out of 16 anti-hC5 antibodies showed no inhibition of hemolytic activity. Fourteen antibodies showed inhibition ranging from 43% to 94% with IC50 values ranging from 25 nM to 180 nM. In the AP hemolytic assay, 13 out of 17 antibodies showed inhibitory activity ranging from 17% to 93% with IC50 values ranging from 22.5 nM to 233 nM.

[0285]

Table 22

[0286] As shown in Table 22, the anti-hC5 antibodies (except for H4H12183P2 which showed 64% CP inhibition) showed inhibition higher than 90% of the CP or AP hemolytic assay in 5% cynomolgus monkey serum. In the CP hemolytic assay, the IC50 of the antibodies ranged from 7.15 nM to 142 nM. In the AP hemolytic assay, the IC50 of the antibodies ranged from 5.4 nM to 29.6 nM.

[0287] (C) Inhibition of mutant human C5 hemolysis The selected anti-C5 antibodies were tested for their ability to inhibit mutant human C5 from C5-deficient human serum in the CH50 assay (see Example 3 herein). In C5-deficient human serum supplemented with exogenous C5 variant R885H, H4H12166P, and control drug 2 blocked CP hemolysis with IC50 values of 6.0 nM and 4.4 nM, and IC80 values of 7.6 nM and 5.5 nM, respectively. For variant R885C, H4H12166P and control drug 2 blocked CP hemolysis in C5-deficient human serum containing the exogenous C5 variant with IC50 values of 9.3 nM and 6.8 nM, and IC80 values of 11 nM and 8.2 nM, respectively. As predicted, control drug 1 did not block the hemolytic activity of the human C5 variants.

[0288] (D) Inhibition of human C5b-6 complex The selected anti-C5 antibodies were tested for their ability to inhibit human C5 from C5-deficient human serum in the CH50 assay of the b-6 complex. H4H12166P strongly blocked CP hemolysis in C5-deficient human serum supplemented with exogenous huC5b-6 complex with an IC50 value of 3.8 nM and an IC80 value of 5.8 nM. In contrast, control drug 1 blocked C5b-6 complex-mediated hemolysis with lower potency, with IC50 and IC80 values of 5.0 nM and 46 nM, respectively. Control drug 1 did not inhibit 70% of total hemolysis at the highest concentration tested. Control drug 2 did not block the hemolytic activity of the human C5b-6 complex.

[0289] Example 9: Anti-C5 antibodies block C5a production in the CP hemolysis assay To evaluate whether anti-C5 antibodies inhibit C5a production, the supernatants from the assay for classical pathway (CP) hemolysis were analyzed for C5a levels by ELISA.

[0290] C5a, a protein fragment of 74 amino acids, is generated as a result of C5 cleavage. C5a is metabolized by serum carboxypeptidase to a more stable and less active 73 - amino - acid form, C5a des - Arg, by removal of the C - terminal arginine. Thus, quantification of C5a des - Arg provides a reliable measure for monitoring C5a production in vivo and in vitro. The MicroVue C5a ELISA kit detects C5a des - Arg according to the information provided by the manufacturer. Preliminary experiments (data not shown) indicate that the primary 74 - amino - acid form of C5a is also detected. For the purposes of this example, both forms are collectively referred to as "C5a".

[0291] C5a protein levels were determined in the supernatant from the CP hemolysis assay using normal human serum (NHS) in which complement pre - incubated with H4H12166P or isotype control antibody as described in Example 8 was preserved. C5a protein levels were measured using the MicroVue C5a ELISA kit according to the manufacturer's instructions. Briefly, samples were diluted and incubated in plates pre - coated with a capture antibody (mouse anti - C5a specific for a new epitope on human C5a). Human C5a protein provided by the manufacturer was used as a standard for calibration. C5a in the supernatant was detected with an HRP - conjugated detection antibody (mouse monoclonal antibody against the C5a region of C5). The chromogenic HRP - substrate, 3,3’,5,5’ - tetramethylbenzidine (TMB), was added to detect HRP activity. The reaction was stopped using 1N hydrochloric acid solution, and the optical density at 450 nm (OD450) was measured with a SpectraMax plate reader. Data were analyzed using non - linear regression (4 - parameter logistics) in GraphPad Prism. C5a concentration was analyzed as ng / supernatant mL.

[0292] In an assay using 5% NHS, H4H12166P strongly blocked the increase in C5a protein levels in a dose-dependent manner with an IC50 of 8.5 nM, while the isotype control antibody had no effect on C5a levels (Figure 1). The maximal blockade at the highest tested H4H12166P concentration (267 nM) resulted in an approximately 10-fold decrease in C5a levels to 3.8 ng / mL (0.3 nM) compared to 34 ng / mL (2.8 nM) observed at the lowest tested H4H12166P concentration (1 nM) in 5% serum. The C5a concentration observed for maximal blockade was close to the baseline C5a level of 2.3 ng / mL (0.2 nM) in untreated 5% NHS.

[0293] Example 10: Pharmacokinetics and Pharmacodynamics Characterization of Anti-C5 Antibodies in Cynomolgus Monkeys This example describes the pharmacokinetic (PK) and pharmacodynamic (PD) characterization of selected anti-C5 antibodies conducted in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody dosing and used to stratify the animal dosing groups.

[0294] Total circulating C5 levels in cynomolgus monkeys were determined using a human complement C5 ELISA (Abcam, catalog number ab125963), which was performed according to the manufacturer's recommendations. The mean concentration of C5 protein in the monkeys was determined to be 0.85 μg / mL ± 19.17 μg / mL.

[0295] For each anti-C5 antibody, four cynomolgus monkeys were each administered a single intravenous (IV) injection at a dose of 15 mg / kg. Blood samples were collected from each animal from pre-dose up to 1680 hours (70 days), processed to serum, and frozen at -80 °C until analyzed for PK and PD.

[0296] Total IgG antibody level analysis by ELISA immunoassay The total antibody concentration in the monkey serum samples was measured using an unvalidated direct ELISA. The ELISA procedure used microtiter plates coated with mouse anti-human IgG1 / IgG4 Fc monoclonal antibodies. Various anti-C5 antibodies were added to the plates, and the anti-C5 antibodies captured on the plates were detected using biotinylated mouse anti-human IgG4 Fc monoclonal antibody, followed by NeutrAvidin conjugated to horseradish peroxidase (NeutrAvidin HRP). A luminol-based substrate specific for peroxidase was then added to achieve a signal intensity proportional to the total concentration of the captured anti-C5 antibodies. Relative light unit (RLU) measurements of calibration standards and their respective nominal concentrations were fitted using a weighted four-parameter logistic equation to generate a calibration equation that described the concentration of the anti-C5 antibody and the response relationship of the assay. The lower limit of quantification (LLOQ) was 1.56 ng / mL in this assay (2% monkey serum) and 78 ng / mL in neat monkey serum.

[0297] Determination of PK parameters PK parameters were determined by non-compartmental analysis (NCA) using Phoenix (R) WinNonlin (R) software (version 6.4, Certara, L.P.) and an IV bolus dosing model.

[0298] All PK parameters were derived from the respective mean concentration values, including the maximum concentration observed in serum (C max ), the time to the observed peak concentration, t max , and the observed estimated half-life (T 1 / 2 ). For each antibody, the area under the concentration-time curve from the last measurable concentration to infinity (AUC last ) and the extrapolated value from time zero to infinity (AUC inf ) were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

[0299] PD analysis by ex vivo hemolysis assay The pharmacodynamics of the selected anti-C5 antibody was analyzed using ex vivo classical and alternative pathway hemolysis assays.

[0300] Classical pathway hemolysis assay: Sheep red blood cells (SRBC) were washed with GVB++ buffer (gelatin-veronal buffer containing CaCl2 and MgCl2) (Boston BioProducts) and resuspended at 1x10^9 cells / mL. To sensitize the SRBC, a total of 1x10^9 / mL of SRBC was mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg / mL) at 37°C for 20 minutes. The sensitized SRBC were diluted to 2x10^8 cells / mL in GVB++ buffer prior to use in the hemolysis assay. Blood from cynomolgus monkeys was collected prior to dosing, as well as at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56, 63 and 70 days after dosing for PD analysis. Serum was prepared and frozen until further use. On the day of the assay, cynomolgus monkey serum from each time point was diluted to 10% with GVB++ buffer. A round-bottom 96-well plate was used to measure hemolytic activity. A total of 100 μl of sensitized SRBC (2x10^8 cells / mL) was plated in a 96-well plate at 37°C, followed by the addition of 100 μl of 10% cynomolgus monkey serum from each time point. The SRBC were gently mixed and incubated at 37°C for 10 minutes. After the incubation time, the cells were centrifuged at 1250 x g at 4°C. A total of 100 μL of the supernatant was transferred to a new 96-well flat-bottom plate and read at 412 nm using a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 5%. The percent hemolysis was calculated using the absorbance value by using the following formula:

Equation

[0301] In this formula, "background cell lysis" is the OD at A412nm from SRBC incubated with only GVB++ buffer without serum. "Maximum cell lysis" is the OD at A412nm from SRBC treated with water. The results expressed as % hemolysis were analyzed using non-linear regression (4-parameter logistics) with Prism 5 software (GraphPad) to obtain IC 50 values. The data are presented as mean ± standard error of the mean.

[0302] Alternative pathway hemolysis assay: A desired number of rabbit red blood cells (RbRBC) were washed with GVB-Mg 2+ / EGTA buffer and resuspended at 2x10^8 cells / mL. Blood from cynomolgus monkeys was collected before dosing and at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, and 49 days after dosing for PD analysis. Serum was prepared and frozen until further use. A round-bottom 96-well plate was used to measure hemolytic activity. A total of 100 μl of RbRBC (2x10^8 cells / mL) was plated in the 96-well plate at 37°C, followed by addition of 100 μl of 10% cynomolgus monkey serum from each of the time points listed above. The RbRBC were gently mixed and incubated at 37°C for 60 minutes. After the incubation time, the cells were centrifuged at 1250 x g at 4°C. A total of 100 μL of the supernatant was transferred to a new 96 flat-bottom plate and read at 412 nm using a Spectramax microplate reader. Hemolytic activity was calculated for a final serum concentration of 5% and expressed as a percentage of total hemolysis of RBC by water. The percent hemolysis was calculated as described above.

[0303] Results The selected anti-C5 antibodies (listed in Table 1) were tested in initial experiments for an extended pharmacokinetic profile in cynomolgus monkeys and C5 humanized mice (described in Example 10). H4H12166P and H4H12161P were selected as having high affinity conjugated with extended PK and were used in subsequent experiments herein together with Control Drug 1 and Control Drug 2.

[0304] Cynomolgus monkeys were administered a single 15 mg / kg IV bolus dose of H4H12166P, H4H12161P, or Control Drug 2. Total antibody serum concentration and percent classical pathway (CP) hemolytic activity were determined at 19 time points during a 70-day survival period. Alternative pathway (AP) hemolysis was determined at 17 time points during a 50-day survival period. Table 23 summarizes the mean antibody concentrations for all three antibodies. The mean total antibody concentration vs. time profile is shown in Figure 2. The mean PK parameters are described in Table 24.

[0305]

Table 23

[0306] Following IV bolus administration, the total IgG concentration-time profiles of H4H12166P, H4H12161P, and Control Drug 2 were characterized by an initial short-term distribution phase followed by a single elimination phase over the survival period. The peak H4H12166P, H4H12161P, and Control Drug 2 concentrations were very similar and the corresponding C max / dose values among all antibodies were within 1.1-fold (29.7, 30.4, and 30.6 [(ug / mL) / (mg / kg)], respectively) (Table 24).

[0307]

Table 24

[0308] Evaluation of the concentration-time profile revealed that the slowest elimination occurred with terminal antibody concentration ≥ 10 μg / mL up to Day 71 of the study. The kinetics of H4H12161P and comparator 2 were similar; both showed faster elimination than H4H12166P up to Days 22 and 29, respectively, at mAb concentration ≥ 10 μg / mL.

[0309] As a result, dose-normalized exposure (AUC last / dose) was highest for H4H12166P at 339 day*(μg / mL) / (mg / kg), while H4H12161P and comparator 2 had exposures approximately two-fold lower than H4H12166P, at 157 and 187 day*(μg / mL) / (mg / kg), respectively.

[0310] The calculated antibody half-life (t 1 / 2 ) during the elimination phase ranged from 5.5 to 15.6 days across dosing groups, and also correlated with exposure, with H4H12166P having the highest corresponding t 1 / 2 of 15.6 days, while H4H12161P and comparator 2 had t 1 / 2 values of 5.5 and 5.9 days, respectively.

[0311] The pharmacologic effects of anti-C5 antibody from cynomolgus monkey serum samples were determined ex vivo by complement classical pathway (CP) hemolysis of sensitized sheep red blood cells (SRBC) and alternative pathway (AP) hemolysis of rabbit red blood cells (RbRBC). Inhibition of hemolytic activity was calculated for a final serum concentration of 5%, and expressed as a percentage of total hemolysis of RBC by water. Table 25 summarizes the ex vivo activities of the three antibodies determined by mean percent hemolysis.

[0312]

Table 25

[0313] As shown in Table 25 and Figure 2, the PD effect was measured by complement CP (10-minute incubation) up to day 70. H4H12166P blocked more than 95% of the CP hemolytic activity up to day 35. The activity returned to the maximum hemolytic level before the test by day 70. Control drug 2 blocked approximately 95% of the CP hemolytic activity up to day 10, and the activity rapidly returned to the maximum hemolytic level before the test by day 18.

[0314] The PD effect was also measured by the complement AP pathway (60-minute incubation) hemolysis assay up to day 49. As shown in Table 25 and Figure 3, H4H12166P blocked 80% of the total AP hemolytic activity up to day 18, and the activity returned to the maximum hemolytic level before the test by day 59. H4H1216P and control drug 2 blocked 90% of the AP hemolytic activity up to day 7, and the act ivity returned to the maximum hemolytic level before the test by day 21.

[0315] Example 11: Characterization of PK / PD of an anti-C5 antibody in C5 humanized mice In this experimental set, the pharmacokinetics and pharmacodynamics of the selected anti-C5 antibody were evaluated in humanized mice engineered to express human C5 protein using the Velocigene (R) technology (Valenzuela et al 2003, Nat. Biotechnol. 21:652-659). The humanized mice were engineered to replace exons 2 to 41 of the mouse C5 gene with exons 2 - 42 of the human C5 gene (disclosed in US Patent Application Publication 2015 / 0313194, incorporated herein by reference in its entirety).

[0316] Total circulating human C5 levels were determined using a human complement C5 ELISA (Abcam, catalog number ab125963) performed according to the manufacturer's recommendations.

[0317] Determination of total drug level in serum by ELISA The concentrations of both circulating anti-C5 antibody, bound and unbound, were determined by ELISA using total human antibody analysis. Briefly, goat anti-human IgG polyclonal antibody was immobilized overnight at 1 μg / mL in PBS in 96-well plates; the plates were washed to remove unbound IgG and then blocked with 5% BSA. Serial dilutions (6 points) of anti-C5 antibody-containing serum samples and reference standards of each antibody (12 points) were transferred to the anti-human IgG-coated plates and incubated for 1 hour. The anti-C5 antibody bound to the plate was then detected using a goat anti-human IgG polyclonal antibody conjugated to horseradish peroxidase. The plates were developed with TMB substrate according to the manufacturer's recommended protocol, and the signal of optical density (OD) at 450 nm was recorded using a Perkin Elmer Victor X4 multimode plate reader. The anti-C5 antibody concentration in the serum was calculated based on a reference standard calibration curve generated using GraphPad Prism software.

[0318] Determination of PK parameters PK parameters were determined by non-compartmental analysis (NCA) using Phoenix (R) WinNonlin (R) software (version 6.3, Certara, L.P.) and an extravascular dosing model. All PK parameters, including the observed estimated half-life (t 1 / 2 ), and the area under the concentration-time curve (AUC last ) to the last measurable concentration, were determined using linear interpolation and uniform weighting, using the respective mean concentrations for each antibody.

[0319] PD analysis by hemolysis assay The pharmacodynamics of the selected anti-C5 antibody was determined using a classical pathway complement hemolysis assay. Sheep red blood cells (SRBC) (sheep blood in Alsever's solution) were washed with GVB++ buffer (gelatin veronal buffer containing CaCl2 and MgCl2) (Boston BioProducts) and resuspended at 1x10^9 cells / mL. For sensitization, 1x10^9 / mL of SRBC was mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg / mL) for 20 minutes at 37°C. The sensitized SRBC were diluted to 2x10^8 cells / mL in GVB++ prior to the hemolysis assay. Serum samples from humanized C5 mice administered with anti-C5 antibody collected on days 0 (pre-dose) or 10, 20, 30, 40, and 50 post-dose were diluted to 20% with GVB++ buffer. A total of 100 μL of sensitized SRBC (2x10^8 cells / mL) was plated in a 96-well round-bottom plate at 37°C, followed by the addition of 100 μL of 20% serum supplemented with 160 - 180 μg / mL human complement 3 (huC3) protein. The cells were gently mixed and incubated at 37°C for 1 hour. After incubation, the cells were centrifuged at 1250 x g for 4°C. A total of 1 00 μL of the supernatant was transferred to a new 96 flat-bottom plate and read at A412nm using a Spectramax microplate reader. The percent hemolysis was calculated using the following formula with the absorbance values:

Equation

[0320] In this formula, "background cell lysis" is the OD at A412nm from SRBC incubated with GVB++ buffer alone without serum. "Maximum cell lysis" is the OD at A412nm from SRBC treated with water. The results expressed as % hemolysis were analyzed using nonlinear regression (4-parameter logistics) with Prism 6 software (GraphPad) to obtain the IC 50 value. The data are presented as mean ± standard error of the mean.

[0321] Experiment 1 In this experiment, the pharmacokinetics and pharmacodynamics of the exemplary antibody H4H12166P were evaluated by comparison with control drug 1 and control drug 2 in humanized C5 mice. Total circulating human C5 levels were determined using a human complement C5 ELISA (Abcam, catalog number ab125963) performed according to the manufacturer's recommendations. The mean concentration of human C5 in mice was determined to be 39.73 μg / mL ± 17.82 μg / mL. There was a difference between male (55.4 ± 1.7 μg / ml, n = 47) and female (24.7 ± 0.6 μg / ml, n = 49) mice.

[0322] Prior to antibody dosing, male and female humanized C5 mice were stratified according to a human C5 level of an average of 40 μg / mL. For each anti-C5 antibody, a single 15 mg / kg dose of H4H12166P, control drug 1, or control drug 2 was administered by subcutaneous (s.c.) injection to a cohort of 22 mice. All mice had blood drawn before dosing and 1 day after injection for PK analysis. Further, 4 or 5 mice from each cohort were euthanized 10, 20, 30, 40, and 50 days after injection, and terminal blood was collected for PK and PD analysis. The serum samples on day 1 were the average of the entire cohort of 22 mice. Blood was processed to serum and frozen until analyzed at -80°C.

[0323] Over a 50-day survival period, total antibody concentrations were determined at 7 time points, and percent hemolytic activity was determined at 6 time points. The total anti-C5 antibody concentrations are summarized in Table 26. The mean total antibody concentration versus time profile is shown in Figure 4. The mean PK parameters are described in Table 27.

[0324]

Table 26

[0325]

Table 27

[0326] The mean concentration-time profiles on Day 1 showed that the three antibodies, H4H12166P, Control Drug 1, and Control Drug 2 had similar serum concentrations of 178, 229, and 164 μg / mL, respectively. Control Drug 1 had a similar elimination profile to H4H12166P until Day 30, but showed a rapid increase in clearance relative to H4H12166P on Days 40 and 50. On Day 50, H4H12166P had an average antibody serum concentration of approximately 9 μg / mL, while both Control Drug 1 and Control Drug 2 had an average antibody serum concentration 30-fold lower, at 0.3 μg / mL. Control Drug 2 had the lowest exposure among the three antibodies tested and had an AUC last (1408 day-μg / mL) that was approximately half (1408 day-μg / mL) compared to H4H12166P (2801 day-μg / mL) and Control Drug 1 (2708 day-μg / mL).

[0327] The pharmacological effects of the anti-C5 antibodies H4H12166P, Control Drug 1, and Control Drug 2 were measured up to Day 50 from humanized C5 mouse serum samples supplemented with human C3 and determined ex vivo by complement classical pathway (CP) hemolysis of sensitized SRBC. The mean percent hemolysis for each anti-C5 antibody is summarized in Table 28 and the mean percent hemolysis-time profiles are shown in Figure 5.

[0328]

Table 28

[0329] H4H12166P, Control Drug 1, and Control Drug 2 inhibited terminal complement hemolytic activity, which appeared to correlate with antibody exposure. H4H12166P blocked more than 85% of hemolytic activity until Day 30, and the activity returned to pre-dose baseline levels by Day 50. Control Drug 1 and Control Drug 2 blocked approximately 80% of hemolytic activity until Days 20 and 10, respectively, and the activity returned to baseline for both by Day 30.

[0330] Experiment 2 In this experiment, the pharmacokinetics and pharmacodynamics of anti-C5 antibodies H4H12166P, H4H12161P, control drug 1, and isotype control were evaluated in humanized C5 mice (mice homozygous for human C5 expression). Total circulating C5 levels were determined using a human complement C5 ELISA (Abcam, catalog number ab125963) performed according to the manufacturer's recommendations. The mean concentration of human C5 in the mice was determined to be 48.98 μg / mL ± 15.1 μg / mL.

[0331] Prior to antibody dosing, humanized male and female C5 mice were stratified according to human C5 levels, which were on average 50 μg / mL. A cohort of 5 mice for each anti-C5 mAb was administered a single 15 mg / kg subcutaneous (s.c.) injection of H4H12166P, H4H12161P, control drug 1, or isotype control. All mice were bled prior to dosing and at 6 hours, 1, 2, 3, 4, 7, 10, 13, 21, 30, and 45 days after injection for PK analysis. Additionally, on day 59, all mice from each cohort were euthanized and terminal blood was collected for PK and PD analysis. Blood was processed to serum and frozen at -80°C until analyzed.

[0332] Total antibody concentrations were determined at 12 time points over the 59-day survival period, and percent hemolytic activity was determined at one time point. The total serum antibody concentrations for each anti-C5 antibody are summarized in Table 29. The mean total antibody concentration vs. time profile is shown in Figure 6. The mean PK parameters are described in Table 30.

[0333]

Table 29

[0334]

Table 30

[0335] The average concentration-time profiles of H4H12166P, H4H12161P, control drug 1, and isotype control were similar C within 1.3-fold, max with maximum serum concentrations (C max ) reaching 178, 225, 183, and 221 μg / mL on days 1 to 3, respectively. H4H12166P and isotype control had similar elimination profiles, with residual drug levels of approximately 4 μg / mL on day 59. H4H12161P showed faster clearance than H4H12166P and isotype control but was removed more slowly than control drug 1. On day 59, H4H12161P had an average serum drug level of 0.6 μg / mL, while control drug 1 had a drug level of approximately undetectable level of 0.08 μg / mL.

[0336] The isotype control, H4H12166P, and H4H12161P showed similar exposure (AUC last ) values within 1.3-fold (4080, 3490, and 3040 day-μg / mL, respectively), while control drug 1 showed 1.6-fold lower exposure (2240 day-μg / mL) compared to H4H12166P.

[0337] Example 12: LC-MRM-MS-based assay for determining total human C5 concentration In this example, the serum concentration of total human C5 was determined using liquid chromatography coupled with multiple reaction monitoring mass spectrometry (LC-MRM-MS) in the pharmacokinetic / pharmacodynamic study of anti-C5 antibody H4H12166P.

[0338] The serum concentration of total human C5 was determined by measuring the concentration of the 10 - amino acid peptide LQGTLPVEAR (amino acids (aa) 1129 - 1138 of SEQ ID NO: 359) contained in the C5 sequence as a surrogate for C5. In theory, this method could also detect the C5 cleavage product C5b. However, due to the instability of free C5b, the concentration of C5b in serum is generally low, and most of the C5b is bound to the cell surface in the form of the MAC complex (Cooper & Muller - Eberhard 1970, J. Exp. Med. 132:775 - 93; Hadders et al 2012, Cell Rep. 1:200 - 7). Therefore, the processed serum samples analyzed here may contain only negligible amounts of C5b products, if any.

[0339] Methods For PK / PD studies, mice were administered a single 15 mg / kg dose of H4H12166P by subcutaneous (s.c.) injection. All mice were bled before dosing and on day 1 after injection for PK analysis. Additionally, at 10, 20, 30, 40, 50, and 60 days after injection, the mice were euthanized and terminal blood was collected for PK and PD analysis.

[0340] Human C5 was used as a reference standard for calibration; and the human C5 peptide produced with a C - terminal stable isotope - labeled arginine residue was used as an internal standard (LQGTLPVEAR - 13 C 6 15 N 4)。Reference standards were used at concentrations ranging from 3.9 to 250 μg / mL (1:2 dilution steps) in serum from C5 knockout mice generated in-house with the mouse C5 gene deleted (C5- / -). Serum from C5- / - mice was also used as a negative control (blank). Calibration standards, blanks, and test serum samples (10 μL each) were dried and then denatured at 37 °C for 1 h in 100 μL of 8 M urea / 20 mM Tris(2-carboxyethyl)phosphine (TCEP) buffer. Next, 10 μL of 25 nM internal standard was added to all samples. Samples were alkylated with 10 mM iodoacetamide for 30 min at room temperature and then diluted to a final volume of 500 μL using 50 mM ammonium bicarbonate. Samples were then digested overnight at 37 °C with trypsin (1:20 mass / mass). The C5-derived tryptic peptide LQGTLPVEAR was detected and quantified by LC-MRM-MS using a Waters Xevo TQ-S with an ACQUITY UPLC system. Each processed sample (10 μL) was injected onto a pre-equilibrated ACQUITY UPLC BEH C18 column. The flow rate was 0.6 mL / min (mobile phase A: water:formic acid / 100:0.1 [volume:volume] and mobile phase B: acetonitrile:formic acid / 100:0.1 [volume:volume]). Retention times and peak areas were determined using Masslynx Analyst data software (Waters). The concentration of the C5 analyte was calculated from a calibration curve constructed by plotting the peak area ratio of ( 12 C 6 14 N 4 ) against the nominal concentration of the C5 reference standard. Concentrations were calculated using linear regression . The lowest concentration of the C5 reference standard (3.9 μg / mL) was within the dynamic range of the assay and was defined as the LLOQ of the assay.

[0341] Results The total human C5 concentration in serum was evaluated for samples collected from the corresponding animals via tail bleeding (before dosing) and via terminal blood on days 10, 30, and 35 before dosing. The total hC5 concentration after dosing with H4H12166P on days 10, 30, and 35 of dosing was similar to the pre-dosing levels (within about 1 to 0.9-fold). The small differences observed were not statistically significant as evaluated by the Mann-Whitney test using GraphPad Prism software. Analysis of the C5 / H4H12166P molar ratio demonstrated that H4H12166P remained in molar excess of C5 until day 35 after dosing (Table 31).

[0342]

Table 31

[0343] Example 13: Epitope Mapping of the Binding of H4H12166P to C5 by Hydrogen / Deuterium Exchange H / D exchange epitope mapping using mass spectrometry was performed to determine the amino acid residues of hC5 [(amino acids M1-C1676 of SEQ ID NO: 359)] with which H4H12166P interacts. A general description of the H / D exchange method is shown, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0344] The HDX-MS experiment was performed on an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-class (Auxiliary Solvent Manager) for sample digestion and loading, a Waters Acquity M-class (μBinary Solvent Manager) for analytical column gradient, and a Synapt G2-Si mass spectrometer for digested peptide mass measurement

[0345] ​​Labeled solution D 2 For the deuterium labeling prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6), 3.8 μL of C5 (6 pmol / μL) or C5 pre-mixed with the antibody at a 1:1 molar ratio was incubated with 56.2 μL of D 2 2O labeled solution at various time points (e.g., non-deuterated control = 0 seconds, 1 minute, and 20 minutes of labeling). Deuteration was quenched by transferring 50 μL of the sample to 50 μL of pre-cooled quench buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5), and the mixed sample was incubated at 1.0 °C for 2 minutes. Then, the quenched sample was injected onto a Waters HDX manager for on-line pepsin / protease XIII digestion. The digested peptides were trapped at 0 °C on an ACQUITY UPLC BEH C18 1.7-μm, 2.1 × 5 mm VanGuard pre-column and eluted onto an analytical column ACQUITY UPLC BEH C18 1.7-μm, 1.0×50 mm with a gradient separation from 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile) for 9 minutes. The mass spectrometer was set at a cone voltage of 37 V, a scan time of 0.5 seconds, and a mass / charge range of 50 - 1700 Thomson units (Th).

[0346] For the identification of peptides from human C5, LC-MS E data from the non-deuterated samples was processed and the identified peptides searched against a database containing human C5, pepsin, and their randomized sequences via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered on two criteria: (1) = minimum product per amino acid = 0.3 and (2) iterative file threshold = 3. Then DynamX software automatically determined the deuterium incorporation of each peptide based on retention time and high mass accuracy (<10 ppm) over a number of time points with three replicates at each time point.

[0347] MS E Using an online pepsin / protease XIII column coupled with data acquisition, a total of 189 peptides from human C5 were identified in the absence or presence of antibody, representing a 62% sequence coverage. Five peptides had significantly decreased deuterium incorporation when bound to H4H12166P (Centroid delta value > 0.9 Dalton, p-value < 0.05), as shown in Table 32.

[0348]

Table 32

[0349] The recorded peptide masses corresponded to the average value of the centroid MH masses from three replicates. These peptides corresponding to amino acids 591 - 599 and 775 - 794 had slower deuteration rates when bound to H4H121 + 66P. These identified residues also corresponded to residues 591 - 599 and 775 - 794 of human C5 as defined by Uniprot entry P01031 (CO5_HUMAN; SEQ ID NO: 359).

[0350] Example 14: Effect of anti-C5 antibody on ocular inflammation in experimental autoimmune uveitis in mice This study was initiated to evaluate the role of C5 in experimental autoimmune uveitis (EAU). Both genetic [C5 knockout (KO), C3 / C5 double KO mice] and pharmacological (anti-C5 antibody) experimental approaches were used.

[0351] Methods ​Adult C57BL / 6J mice (n = 25, Jackson labs), C5 KO (n = 13) and C3 / C5 KO (n = 8) mice (Regeneron Pharmaceuticals Inc.) were used. Experimental autoimmune uveitis (EAU) was induced by subcutaneous injection of human interphotoreceptor retinoid-binding protein peptide (IRBP, New England Peptide) in complete Freund's adjuvant and intraperitoneal injection of pertussis toxin. Anti-mouse C5 mAb or isotype control mAb was administered by subcutaneous injection every 3 days from day 5 to day 28. The anti-C5 antibody (M1M17628N) used in this study contained the HCVR / LCVR of SEQ ID NO: 362 / 363. SPECTRALIS (R) The inflammation levels on days -1, 7, 14, 21 and 28 were evaluated using SPECTRALIS HRA+OCT (Heidelberg Engineering, Inc.). All animals were euthanized on day 28 for eye and blood collection. Hemolysis assays with or without human C3 were performed to demonstrate complement inhibition. Data were analyzed by ANOVA.

[0352] Results Compared with wild-type mice, the incidence of inflammation (30 - 50%) and the number of vitreous cell clusters were significantly decreased in C5 KO mice (p < 0.01). The optical coherence tomography (OCT) score in C5 KO mice also significantly decreased by 50% at week 3 (p < 0.0001). Interestingly, in C3 / C5 double KO mice, there were significantly more vitreous cell clusters and a higher disease score on day 28 compared with wild-type mice (p < 0.05). In animals administered anti-mC5 Ab (50 mg / kg), the incidence of inflammation and the number of vitreous cell clusters were significantly lower on day 21 compared with either the untreated or isotype control group (p < 0.01). At weeks 3 and 4, the OCT scores in the anti-C5 antibody-treated group were significantly lower compared with the untreated or isotype control (p < 0.0001). (Figure 7) The inhibitory effect of the anti-C5 antibody was confirmed by hemolysis assays with or without human C3 at week 4 (Figure 8).

[0353] Conclusion Eye inflammation caused by EAU was reduced by inhibiting C5 activity either by genetic deficiency or pharmacological inhibition using specific anti-C5 antibodies. C5 deficiency delayed the development of EAU and decreased the OCT disease score. These results indicate that C5 is a potential therapeutic target for autoimmune uveitis. Anti-C5 antibodies have a protective effect against EAU disease in wild-type mice. Our findings also suggest that C3 may be beneficial for EAU disease in mice.

[0354] Example 15: Effect of anti-human C5 antibody on experimental autoimmune uveitis This example describes the effect of an anti-C5 antibody against human C5 in a mouse model of experimental autoimmune uveitis (EAU). The mice used in this study were humanized using the Velocigene (R) technology (Valenzuela et al 2003, Nat. Biotechnol. 21:652-659) to express human C5 protein. The humanized mice were engineered to replace exons 2-41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in US Patent Application Publication No. 2015 / 0313194, which is incorporated herein by reference in its entirety).

[0355] Method Adult male mice were immunized subcutaneously with 150 μg of human interphotoreceptor retinoid-binding protein (IRBP) peptides 1-20 (GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO: 364) (Avichezer et al 2000, Invest. Ophthalmol. Vis. Sci. 41:127-131) in 0.2 ml of an emulsion of complete Freund's adjuvant (CFA) supplemented with Mycobacterium tuberculosis strain H37RA up to 2.5 mg / ml. Then, to facilitate the induction of cell-mediated autoimmunity by promoting Th1 polarization of the immune response, mice were inoculated intraperitoneally with 1.0 μg of pertussis toxin (PTX) (Thurau et al 1997, Clin. Exp. Immunol. 109:370-376; Silver et al 1999, Invest. Ophthalmol. Vis. Sci. 40:2898-2905). The body weight of the animals was monitored twice a week.

[0356] Eye tests were performed on day -1, before EAU induction, and on days 7, 14, 21, and 28. Mice were anesthetized with ketamine (120 mg / kg, IP) and xylazine (5 mg / kg, IP). The pupils were dilated using 0.5% tropicamide eye drops, and the eyes were examined in a Spectralis Heidelberg retinal angiography platform (HRA)+OCT system (Heidelberg Engineering, Carlsbad, CA, USA) using a contact lens equipped with a fundus camera.

[0357] A series of 61 lateral optical sections were obtained for each eye using the OCT function of the Spectralis HRA+OCT system (Heidelberg Engineering, Carlsbad, CA, USA)). The OCT imaging region was centered on the optic nerve head to enable equal imaging above and below the optic nerve disc. The retinal thickness was measured as the distance between the lower part of the RPE layer and the inner limiting membrane of the eye. Measurements were made 1500 μm from the optic nerve disc, and values from four different retinal quadrants (e.g., superior, inferior, lateral, and nasal) were averaged for the mean retinal thickness of the eye.

[0358] The severity of inflammatory cell infiltration into the vitreous was also graded in OCT images by evaluating the average number of inflammatory cell clusters in the vitreous in four lateral OCT scans across the optic nerve for each eye.

[0359] A 4 - point scale (OCT score) was developed for the assessment of disease severity in OCT images (Table 33).

[0360]

Table 33

[0361] Statistical analysis Statistical analysis of parametric data (body weight, inflammatory cell clusters in the vitreous, and retinal thickness) was performed by one - way ANOVA test and Tukey's multiple comparison test. For non - parametric data (OCT score and histological score), the Kruskal - Wallis test and Dunn's test were performed using GraphPad Prism version 5.0d software and compared with the isotype control or untreated group. The data are shown as mean ± SEM. A p - value of less than 0.05 was considered statistically significant.

[0362] Results In the first study (Study A), mice were subcutaneously treated every 3 days from day 5 with an isotype control antibody (50 mg / kg), or H4H12170P at 10 mg / kg or 50 mg / kg. Treatment with 10 mg / kg H4H12170P resulted in a decrease in inflammation and retinal damage (Figure 9). Mice treated with 10 mg / kg H4H12170P also showed a statistically significant decrease in OCT score on days 21 and 28 (Figure 10).

[0363] In the second study (Study B), an isotype control (10 mg / kg), H4 Mice were subcutaneously treated every 3 days starting on day 6 with either H12166P at 3 mg / kg or 10 mg / kg, or control agent 2 (see Example 2 herein; “control construct used in the following examples”). Treatment with H4H12166P at either 3 mg / kg or 10 mg / kg resulted in a dose-related decrease in OCT scores that was statistically significant from day 14 to day 28 (Figure 11). Treatment with 10 mg / kg H4H12166P starting 6 days after EAU induction in C5 humanized mice resulted in a dose-related decrease in inflammation and retinal damage as determined by OCT obtained from day 14 to day 28 (Figure 12).

[0364] For both studies, non-invasive in-life evaluations by OCT showed progressive inflammation, increased retinal thickness, and morphological abnormalities in immunized control mice using IRBP.

[0365] Conclusion These experiments provide additional pharmacological evidence that C5 plays a role in the pathology of autoimmune uveitis. The pharmacological depletion of full human anti-human C5 antibodies delays the onset of EAU and reduces disease severity, establishing the efficacy of these antibodies in autoimmune uveitis.

[0366] Example 16: Effect of anti-C5 antibody on renal ischemia-reperfusion injury This study was conducted to evaluate the role of C5 in renal ischemia-reperfusion injury. Both genetic (using C3 knockout and C5 knockout mice) and pharmacological approaches (using anti-C5 antibody) were used. The ischemia-reperfusion model was induced by clamping both renal pedicles for 45 minutes followed by 48 hours of reperfusion. Sham laparotomy served as a control. The anti-C5 antibody was administered intravenously as a single dose at 50 mg / kg immediately after ischemia (therapeutic); or subcutaneously as two doses at the time of surgery on days -1 and 1 (prophylactic). The anti-C5 antibody used in this study was M1M17628N containing the HCVR / LCVR of SEQ ID NO: 362 / 363. Blood urea nitrogen (BUN) and serum creatinine markers were used to evaluate the level of disease and protection in mice.

[0367]

Table 34

[0368]

Table 35

[0369] Compared with wild-type mice, C3 and C5 knockout mice showed significant functional protection in the RIRI model of acute kidney injury, as demonstrated by decreased blood urea nitrogen and serum creatinine levels. Anti-C5 antibody showed functional protection in the RIRI model in both preventive and therapeutic modes (Tables 34 - 35).

[0370] Example 17: Effect of Anti-C5 Antibody on Lupus Nephritis This example describes the efficacy of anti-C5 antibody in treating lupus nephritis in a mouse model.

[0371] Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by the loss of tolerance to self-antigens, the production of autoantibodies, and the deposition of complement-binding immune complexes (ICs) in damaged tissues. SLE is characterized by a wide range of clinical symptoms and target organs, and lupus nephritis is one of the most serious complications. Complement activation in the kidneys of lupus nephritis patients contributes to inflammation and tissue damage. The efficacy of anti-C5 antibody in the treatment of lupus nephritis was investigated in NZBWF1 mice, a spontaneous mouse model of lupus nephritis (Yang et al 1996, PNAS). Mice develop an autoimmune disease similar to human SLE, autoantibodies against nuclear antigens and cell membrane proteins, hypergammaglobulinemia, albuminuria, proteinuria, develop immune complex glomerulonephritis, and die of renal failure and end-stage renal disease at 35 - 50 weeks of age.

[0372] For this study, 25-week-old NZBWF1 mice were subcutaneously treated twice weekly for 8 weeks and then three times weekly for 10 weeks with 30 mg / kg of isotype control or anti-C5 antibody. The anti-mouse C5 antibodies used for this study were M1M17628N and M1M17627N, which contain the HCVR / LCVR of SEQ ID NOs: 362 / 363 and 365 / 366, respectively.

[0373] Treatment with anti-C5 antibody significantly improved survival in mice (Figure 13). Both antibodies improved albuminuria (Figure 14) at weeks 8 - 14 of treatment and blood urea nitrogen (Figure 15) at weeks 12 - 16 of treatment.

[0374] Example 18: Effect of anti-C5 antibody on astrocyte cell death Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS) that mainly affects the optic nerve and spinal cord. In NMO, anti-aquaporin 4 autoantibodies (AQP4-Ab) cause damage to normal cells by activating complement-dependent cytotoxicity (CDC). The goal of this study was to evaluate the role of the complement system in NMO progression and the use of antibodies against complement as a potential therapeutic treatment for NMO.

[0375] Primary rat cortical astrocytes were obtained from the cerebral cortex of neonatal rat pups and cultured with AQP4-Ab (antibody "rAb-53" from US Patent Application Publication 2014 / 0170140; Bennett et al 2009, Ann. Neurol. 66:617 - 629) and complement proteins to demonstrate cell-mediated cytotoxicity. The experiment was then repeated with the addition of anti-C5 antibody to demonstrate blockade of astrocyte destruction.

[0376] To quantify cell death, a CytoTox-Glo TM luminescent cytotoxicity assay was performed. This assay used anti-C5 antibody at various concentrations (0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, or 1000 μg / ml) or isotype control antibody.

[0377] To determine whether anti-C5 antibody can block AQP4-Ab-induced CDC, astrocytes were plated and CytoTox-Glo was used to find the optimal dose of AQP4-Ab for that plating. TM The cytotoxicity assay was repeated. The optimal concentration of AQP4-Ab was found to be 50 μg / mL, and in the following experiments, the dose of anti-C5 antibody was varied while using a constant dose of AQP4-Ab (50 μg / mL). As shown in Figure 16, a decrease in RLU was seen as the amount of anti-C5 antibody was increased (from an average of 300k to an average of 100k), demonstrating that anti-C5 antibody blocks astrocyte cell death. For both experiments, RLU did not change with the isotype control antibody. As shown in Figure 16, anti-C5 antibody inhibited AQP4 Ab-induced cytotoxicity in primary cortical astrocytes with an IC50 of 15 - 17 nM.

[0378] In subsequent studies, anti-AQP4 antibody and anti-C5 antibody are injected into rat brains to evaluate the therapeutic efficacy against complement-mediated cytotoxicity of astrocytes in the CNS.

[0379] Example 19: Endothelial assay This example describes an in vitro glomerular endothelial assay to examine whether anti-C5 antibody blocks C5b-9 and C3 deposition.

[0380] A reproducible method for evaluating the inhibitory effect of drug candidates on complement activation is essential for all clinical development. Due to the complexity of the complement activation pathway, the assay should use appropriate cells and evaluation items for a given therapeutic indication. Here, the immortalized human glomerular endothelial cell line (HGEC) was used to demonstrate the use of a complement C3 and C5 deposition model to evaluate the blocking activity of anti-C3 or C5 mAb.

[0381] Method Human primary glomerular endothelial cells (HGEC; Cell Biologics) were plated overnight in a collagen I-coated black clear bottom 96-well plate in complete medium. Cells were treated with PBS (control) or activated with 10 uM ADP for 10 minutes. After PBS washing, 50% human serum (complement-preserved, C3-deficient, or C5-deficient) was added for 4 hours. Anti-C5 antibody was added to the serum at 1 mg / mL before treatment. Cells were washed, fixed, and examined using anti-C3b antibody (Thermofisher) and / or anti-C5b-9 antibody (Abcam), secondary antibodies, and counterstained with DAPI. Images were recorded with ImagExpress, and fluorescence staining was quantified for each image using high-content image analysis and averaged for each condition.

[0382] Results C3 and C5b-9 depositions were observed in ADP-activated HGEC exposed to normal human serum but not in non-activated HGEC (C3: 1.5x10 7 ±1.0x10 7 ; C5: 7.9x10 6 ±6.6x10 6 , P<0.05 vs non-ADP-activated HGEC). C3 and C5b-9 depositions were significantly decreased in ADP-activated HGEC exposed to C3- or C5-deficient serum (C3: 3.3x10 5 ±4.8x10 4 ; C5: 1.5x10 6 ±6.0x10 5 , P<0.05). By adding blocking anti-C5 mAb, C5b-9 deposition from normal human serum onto ADP-activated HGEC was significantly decreased, and the deposition was similar to that in C5-deficient serum (C5 mAb: 1.02x10 6 ±6.0x10 5 , vs control mAb 3.7x10 6 ±1.6x10 6 , P<0.05 vs control mAb).

[0383] Conclusions These data demonstrate the usefulness of in vitro human glomerular deposition for creating models of complement C3 and C5 deposition. In addition to in vitro screening, this assay shows potential as a translational model for evaluating anti-complement strategies in kidney disease using patient-derived serum samples.

[0384] The invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

**Claim 1** An antibody or antigen-binding fragment thereof that specifically binds to complement factor 5 (C5) protein and comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 100, HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, HCDR3 comprises the amino acid sequence of SEQ ID NO: 104, LCDR1 comprises the amino acid sequence of SEQ ID NO: 108, LCDR2 comprises the amino acid sequence of SEQ ID NO: 110, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 112, a method for producing the antibody or antigen-binding fragment thereof, comprising: introducing polynucleotides encoding the HCVR and the LCVR into a host cell; culturing the host cell under conditions that allow production of the antibody or antigen-binding fragment thereof; and recovering the antibody or antigen-binding fragment thereof thus produced. A method comprising the above. **Claim 2** wherein HCDR1 is encoded by the nucleotide sequence of SEQ ID NO: 99; HCDR2 is encoded by the nucleotide sequence of SEQ ID NO: 101; HCDR3 is encoded by the nucleotide sequence of SEQ ID NO: 103; LCDR1 is encoded by the nucleotide sequence of SEQ ID NO: 107; LCDR2 is encoded by the nucleotide sequence of SEQ ID NO: 109; and LCDR3 is encoded by the nucleotide sequence of SEQ ID NO: 111, The method according to claim 1. **Claim 3** The method according to claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 and / or the LCVR comprises the amino acid sequence of SEQ ID NO:

106. **Claim 4** The method according to claim 3, wherein the heavy chain variable region is encoded by the nucleotide sequence of SEQ ID NO:

97. **Claim 5** The method according to claim 3, wherein the light chain variable region is encoded by the nucleotide sequence of SEQ ID NO:

105. **Claim 6** The method according to claim 3, wherein the heavy chain variable region is encoded by the nucleotide sequence of SEQ ID NO: 97; and the light chain variable region is encoded by the nucleotide sequence of SEQ ID NO:

105. **Claim 7** The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

353. **Claim 8** The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:

354. **Claim 9** The method according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 353 and a light chain comprising the amino acid sequence of SEQ ID NO: 354.

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