Anti-c5 antibodies and uses thereof

Abstract

To provide monoclonal antibodies that bind to the complement factor 5 (C5) protein, and methods of use thereof.SOLUTION: In various embodiments of the present invention, the antibodies are fully human antibodies that bind to C5 protein. In some embodiments, the antibodies of the present invention are useful for inhibiting or neutralizing C5 activity, thus providing a means of treating or preventing a C5-related disease or disorder in humans. In some embodiments, the present invention provides an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, e.g., a half-life of more than 10 days.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 Nos. 62 / 349,705 (filed June 14, 2016); 62 / 405,561 (filed October 7, 2016); and 62 / 422,107 (filed November 15, 2016), the disclosures of each of which are incorporated herein by reference in their entirety.

[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 these antibodies. [Background technology]

[0003] Background of the Invention The complement system is a group of plasma proteins that, when activated, leads to target cell lysis and promotes phagocytosis through opsonization. Complement is activated through 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 the complement component 5 (C5) protein. C5c results in the generation of the fragments C5a and C5b, which are crucial processes during activation of the complement cascade. C5a can produce pleiotropic physiological responses through its receptors (Non-Patent Document 1). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates the 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 leads to cell lysis at the late stage of complement-dependent cytotoxicity (CDC). Furthermore, in nucleated cells that are resistant to cytolysis by C5b-9, sublytic amounts of C5b-9 can cause cell activation, which results in cell proliferation, production of pro-inflammatory mediators, and extracellular matrix production.

[0004] Monoclonal antibodies against C5 are known in the art and are described, for example, in U.S. Patent No. 5,623,666, ... [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 9,206,251 [Patent Document 2] U.S. Patent No. 9,107,861 [Patent Document 3] U.S. Patent No. 9079949 [Patent Document 4] U.S. Patent No. 9,051,365 [Patent Document 5] U.S. Patent No. 8,999,340 [Patent Document 6] U.S. Patent No. 8,883,158 [Patent Document 7] U.S. Patent No. 8,241,628 [Patent Document 8] U.S. Patent No. 7,999,081 [Patent Document 9] U.S. Patent No. 7,432,356 [Patent Document 10] U.S. Patent No. 7,361,339 [Patent Document 11] U.S. Patent No. 7,279,158 [Patent Document 12] U.S. Patent No. 6,534,058 [Patent Document 13] U.S. Patent No. 6,355,245 [Patent Document 14] U.S. Patent No. 6,074,642 [Patent Document 15] U.S. Patent Publication No. 20160299305 [Patent Document 16] U.S. Patent Publication No. 20160051673 [Patent Document 17] U.S. Patent Publication No. 20160031975 [Patent Document 18] U.S. Patent Publication No. 20150158936 [Patent Document 19] U.S. Patent Publication No. 20140056888 [Patent Document 20] U.S. Patent Publication No. 20130022615 [Patent Document 21] U.S. Patent Publication No. 20120308559 [Patent Document 22] WO2015198243 [Patent Document 23] WO2015134894 [Patent Document 24] WO2015120130 [Patent Document 25] EP2563813B1 [Patent Document 26] EP2328616B1 [Patent Document 27] EP2061810B1 [Non-patent literature]

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

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

[0008] Brief summary of the invention The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to complement factor 5 (C5) protein. The antibodies of the present invention are useful, inter alia, for inhibiting or neutralizing the activity of C5 protein. In certain embodiments, the antibodies are useful for preventing, treating, or ameliorating 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 subjects with or at risk of having a C5-related disease or disorder. In certain embodiments, the anti-C5 antibodies are fully human antibodies that bind to C5 with high affinity and have improved pharmacokinetic (PK) and pharmacodynamic (PD) properties. Such high-affinity antibodies with improved PK / PD can be used to provide superior efficacy with less frequent dosing in subjects with C5-related diseases or disorders.

[0009] The antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect function, for example, to increase persistence in the host or to eliminate residual effector function (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 antibody that specifically binds to the C5 protein. 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 exemplary anti-C5 antibodies. Table 2 shows the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-C5 antibodies.

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

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

[0014] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments 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 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. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one 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). In certain embodiments, the invention provides an anti-C5 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 with five or fewer amino acid substitutions, and the LCVR comprises an amino acid sequence listed in Table 1 with two or fewer amino acid substitutions. For example, the invention provides an anti-C5 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 with five or fewer amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO: 106 with two or fewer amino acid substitutions. In another embodiment, the invention provides an anti-C5 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 with at least one amino acid substitution, and the LCVR comprises the amino acid sequence of SEQ ID NO: 106 with one amino acid substitution.

[0015] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acids listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

[0021] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and LCDR3 amino acid pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 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 antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an HCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an HCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. In certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an LCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an LCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. For example, the invention provides an anti-C5 antibody or 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 by one amino acid from SEQ ID NO: 100, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 102, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 104. provides an antibody or 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 which differs by one amino acid from SEQ ID NO: 108, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence which differs by one amino acid from SEQ ID NO: 110, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence which differs by one amino acid from SEQ ID NO: 112.

[0023] The present invention provides an antibody or 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 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 invention provides an antibody or 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 antibodies or antigen-binding fragments thereof comprising the 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 a related embodiment, the invention provides antibodies or antigen-binding fragments thereof that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-C5 antibodies listed in Table 1. For example, the invention includes antibodies or antigen-binding fragments thereof that comprise a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the 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 CDRs within the particular 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); See 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 present 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 a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a 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) the amino acid sequence of SEQ ID NO: 98 with five 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) the amino acid sequence of SEQ ID NO: 106 with five or fewer amino acid substitutions.

[0029] The present invention includes anti-C5 antibodies with altered glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites, such as antibodies lacking fucose moieties present on the oligosaccharide chains, can be useful to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0030] In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to C5. For example, the present invention includes antibodies and antigen-binding fragments thereof that bind to C5 with greater 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, e.g., the present invention provides anti-C5 antibodies with extended serum half-lives. In certain embodiments, the anti-C5 antibodies of the present invention have serum concentrations of greater 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 for specific binding to C5 with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

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

[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 an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR are selected from the HCVR and LCVR sequences listed in Table 1, respectively. 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 an HCVR and the CDRs of an 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 antibodies do 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 in 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 in human C5 (SEQ ID NO: 359), wherein the antibodies do 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 amino acids, at least 10 amino acids, 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, e.g., the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least five amino acids contained within SEQ ID NO: 360.In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least five amino acids contained within SEQ ID NOs: 360 and 361. In certain embodiments, the present invention provides anti-C5 antibodies 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 agonistic manner, i.e., can enhance or stimulate C5 binding and / or activity; in other embodiments, the antibody can specifically bind to C5 in an antagonistic manner, i.e., can block C5 binding and / or activity.

[0037] The present invention also provides isolated antibodies and antigen-binding fragments thereof that block the binding of C5 to C5 convertase. In some embodiments, the antibodies or antigen-binding fragments thereof that block the binding of C5 to C5 convertase may bind to the same epitope on C5 as the C5 convertase, or may bind to a different epitope on C5 than the C5 convertase. In some embodiments, the present invention provides antibodies or antigen-binding fragments thereof that block the binding of C5 to monkey C5 convertase.

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

[0039] In certain embodiments, the antibodies and antigen-binding fragments of the invention have an IC of less than 0.5 nM for C5a. 50In 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 present invention provides isolated antibodies or antigen-binding fragments thereof having one or more of the following: (a) being fully human monoclonal antibodies; (b) having a dissociation constant (K) of less than 0.9 nM at 25° C., as measured by surface plasmon resonance. D ) binds to human C5; (c) a K of less than 0.3 nM at 37°C as measured by surface plasmon resonance assay D (d) binds to human C5 with a K of less than 65 nM as measured by surface plasmon resonance assay; D (e) binds to monkey C5 with a K of less than 0.5 nM as measured by surface plasmon resonance assay; D (f) binds to human C5 mutant R885H (SEQ ID NO: 356) with a K of less than 0.5 nM as measured by surface plasmon resonance assay; D (g) binds to human C5 mutant R885C (SEQ ID NO: 357) with an IC of less than 6 nM as measured in a CP hemolysis assay; 50 (h) Blocks human C5-mediated classical pathway (CP) hemolysis by more than 95%; (h) IC<165 nM as measured by AP hemolysis assay 50 Blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70%; (i) an IC of less than 185 nM as measured by the CP hemolysis assay 50 (j) inhibits African green monkey C5-mediated CP hemolysis with an IC of less than 235 nM as measured by the AP hemolysis assay; 50 (k) inhibits African green monkey C5-mediated AP hemolysis with an IC of less than 145 nM as measured by the CP hemolysis assay; 50 inhibits cynomolgus monkey C5-mediated CP hemolysis at an IC of less than 30 nM as measured by the AP hemolysis assay; and 50 inhibits cynomolgus monkey C5-mediated AP hemolysis.

[0041] In certain embodiments, the present 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) a dissociation constant (K) of less than 0.2 nM at 25° C. as measured by a surface plasmon resonance assay. D ) binds to human C5; (c) a K of less than 0.3 nM at 37°C as measured by surface plasmon resonance assay D (d) binds to human C5 with a K of less than 0.4 nM at 37°C as measured by surface plasmon resonance assay D (e) binds to human C5 mutant (R885H) with an IC of less than 3 nM 50 Inhibits classical pathway (CP)-mediated hemolysis in human serum with an IC of less than 27 nM 50 inhibits alternative pathway (AP)-mediated hemolysis in human serum; (g) IC<21 nM 50 inhibits CP-mediated hemolysis of monkey serum at (g) IC<10 nM 50 inhibits AP-mediated hemolysis of monkey serum; (h) has a serum half-life (t) longer than 10 days in C5-humanized mice 1 / 2 (i) has a serum concentration of greater than 10 μg / mL for up to 40 days when administered to C5-humanized mice; (j) blocks CP-mediated hemolysis for up to 50 days in C5-humanized mice; and (k) binds to one or more amino acids contained in the alpha 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 present invention provides nucleic acid molecules encoding anti-C5 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR 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.

[0043] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR 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.

[0044] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 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.

[0045] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 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.

[0046] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 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.

[0047] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence 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.

[0048] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence 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.

[0049] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence 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.

[0050] The present invention also provides nucleic acid molecules encoding HCVRs, wherein the HCVRs comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), 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 nucleic acid molecules encoding LCVRs, wherein the LCVRs comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set corresponds to an exemplary anti-C5 as defined by any of the antibodies.

[0052] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR 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 a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof 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 an HCVR and an LCVR, wherein the HCVR and LCVR are both derived from the same anti-C5 antibody listed in Table 1.

[0053] In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising the heavy or light chain variable region of an anti-C5 antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 2. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibody or antibody fragment, and recovering the antibody and antibody fragment thus produced.

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

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

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

[0057] Other embodiments will become apparent from consideration of the following detailed description. [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 shows the dose-dependent inhibition of C5a levels by anti-C5 antibody H4H12166P as determined by ELISA (described in Example 9 herein). [Figure 2] Figure 2 shows total serum concentrations over time following a single 15 mg / kg intravenous injection of H4H12166P, H4H12161P, or Control Drug 2 to male cynomolgus monkeys (as described in Example 10 herein). Concentration-time profiles were plotted up to the first dose after a below-limit-of-quantitation (BLQ) result, where applicable (which was interpolated as LLOQ / 2). Each data point represents the mean (±SD) (n=4 animals per group); concentrations considered affected by ADA were excluded from one animal in the H4H12166P group and one animal in the H4H12161P group, starting on days 36 and 29, respectively. LLOQ = lower limit of quantitation. [Figure 3A] Figure 3 shows percent hemolysis over time in ex vivo erythrocyte (A) classical pathway and (B) alternative pathway assays following a single intravenous injection of H4H12166P, H4H12161P, or control drug 2 into male cynomolgus monkeys. % hemolysis, calculated as the ratio of experimental lysis to maximum lysis, with % background lysis subtracted from both values, relates to the amount of C5 inhibited by a specific anti-C5 antibody present in the serum at a given time point. Each data point represents the mean (±SD). [Figure 3B]Figure 3 shows percent hemolysis over time in ex vivo erythrocyte (A) classical pathway and (B) alternative pathway assays following a single intravenous injection of H4H12166P, H4H12161P, or control drug 2 into male cynomolgus monkeys. % hemolysis, calculated as the ratio of experimental lysis to maximum lysis, with % background lysis subtracted from both values, relates to the amount of C5 inhibited by a specific anti-C5 antibody present in the serum at a given time point. Each data point represents the mean (±SD). [Figure 4] Figure 4 shows the total serum concentration versus time profiles of selected anti-C5 antibodies in mice humanized for C5 (described in Example 11 herein). Humanized C5 mice were administered a single 15 mg / kg subcutaneous dose of H4H12166P, Control 1, or Control 2. Each data point represents the mean ± sem (n = 4-5 each). Antibody concentrations in serum were monitored 1, 10, 20, 30, and 40 days after injection using a sandwich ELISA. [Figure 5] Figure 5 shows percent hemolysis versus time in an ex vivo complement classical pathway hemolysis assay for selected anti-C5 antibodies in mice humanized for C5. Humanized C5 mice were administered a single 15 mg / kg subcutaneous dose of H4H12166P, Control 1, or Control 2. Each data point represents the mean ± sem (n = 4-5 each). Percent hemolysis in serum was monitored pre-dose and 10, 20, 30, 40, and 50 days after injection. Percent hemolysis, calculated as the ratio of experimental lysis to maximum lysis, with the background lysis percentage subtracted from both values, relates to the amount of C5 inhibited by a particular anti-C5 antibody present in serum at a given time point. [Figure 6]Figure 6 shows the total serum concentration versus time profiles of selected anti-C5 antibodies in mice humanized for C5 (described in Example 11 herein). Mice were administered a single 15 mg / kg subcutaneous dose of H4H12166P, H4H12161P, Control Drug 1, or IgG4P isotype control. Each data point represents the mean ± sem (n = 5 each). Antibody levels in serum were monitored 6 hours, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45, and 59 days after injection using a sandwich ELISA. [Figure 7] 7 is a graph showing optical coherence tomography (OCT) scores in mice treated with isotype control or anti-C5 antibody M1M17628N at 10 mg / kg or 50 mg / kg (described in Example 14 herein). ****p<0.0001, two-tailed ANOVA treatment with anti-C5 antibody at 50 mg / kg vs. untreated or isotype control treatment. [Figure 8] FIG. 8 shows inhibition of classical pathway hemolysis by anti-C5 antibody M1M17628N in the absence of C3 (A); and in the presence of 80 μg / mL human C3 (B) (described in Example 14 herein). [Figure 9] Figure 9 shows cell population counts in C5-humanized mice treated with isotype control or anti-human C5 antibody H4H12170P at 10 mg / kg or 50 mg / kg (described in Example 15 herein). n = 8-12 eyes for each group. [Figure 10] 10 is a graph showing OCT scores in C5-humanized mice treated with isotype control or anti-human C5 antibody H4H12170P at 10 mg / kg or 50 mg / kg (described in Example 15 herein). n=8-12 eyes for each group. [Figure 11] 11 is a graph showing OCT scores in C5-humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg / kg or 10 mg / kg, or control drug 2 at 10 mg / kg. n=6-12 eyes for each group (described in Example 15 herein). [Figure 12] Figure 12 shows cell population counts in C5-humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg / kg or 10 mg / kg, or control drug 2 at 10 mg / kg. n = 6-12 eyes for each group (described in Example 15 herein). [Figure 13] FIG. 13 is a survival curve of NZBWF1 mice treated with isotype control or anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein). [Figure 14A] FIG. 14 shows (A) urinary albumin and (B) urinary albumin levels normalized to urinary creatinine in NZBWF1 mice treated with isotype control or anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein). [Figure 14B] FIG. 14 shows (A) urinary albumin and (B) urinary albumin levels normalized to urinary creatinine in NZBWF1 mice treated with isotype control or anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein). [Figure 15] FIG. 15 shows blood urea nitrogen levels in NZBWF1 mice treated with isotype control or anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein). [Figure 16] FIG. 16 is a graph showing inhibition of antibody-dependent cellular cytotoxicity of astrocytes by anti-C5 antibodies H4H12166P, H4H12170P, Control 1 and Control 2, as described in Example 18. DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed Description Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

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

[0061] definition The term "C5," also known as "complement component 5" or "complement factor 5," refers to a serum protein of the complement cascade. The C5 protein is a 1,676-amino acid protein containing two chains, alpha and beta. The protein represents the convergence point of 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 under accession number NP_001726.2 (SEQ ID NO: 355). The term "C5" includes recombinant C5 proteins or fragments thereof. The term also encompasses C5 proteins or fragments thereof coupled to a signal sequence, such as, for example, a histidine tag, mouse or human Fc, or ROR1. For example, the term includes sequences exemplified by the sequences set forth in SEQ ID NOs: 356 or 357, which contain a C-terminal histidine tag coupled to amino acid residues 19 to 1,676 of the full-length C5 protein. The term also includes protein variants having an R885H or R885C change and containing a C-terminal histidine tag 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., an "intact antibody molecule") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (domain C H 1. C H 2 and C H Each light chain is composed of a light chain variable region ("LCVR" or "V L ) and light chain constant region (C L ) V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may 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 in which one or two CDRs can be omitted for binding have been described in the scientific literature. Padlan et al. (1995 FASEB J.9:133-139) report on published Based on the crystal structures obtained, they analyzed the contact regions between antibodies and their antigens and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs do not have amino acids 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 empirically based on previous studies, using molecular modeling and / or from regions of the Kabat CDRs outside the Chothia CDRs (e.g., residues H60-H65 in CDRH2 are often unnecessary). When a CDR or its residues are deleted, they are typically replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[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 and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in 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"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are mutated back to the residue 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, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, antibodies of the present invention can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in 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 (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed within the present invention.

[0066] The present 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 with one or more conservative substitutions. For example, the present invention includes anti-C5 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions 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. Human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, and particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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. This term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. This 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 or antigen-binding fragment thereof of the invention that is produced, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to antibodies that are expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse) or cell (e.g., a CHO cell) expression system, or that are isolated from a recombinant combinatorial human antibody library.

[0069] The terms "specifically bind" or "specifically binds to," or the like, mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding to an antigen with a specific binding affinity of at least about 1 x 10 -8 M or less (e.g., a smaller K DThe antibody may be characterized by an equilibrium dissociation constant (where a higher binding affinity indicates 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, and the like. The antibodies described herein may be characterized by surface plasmon resonance, e.g., BIACORE™. TM and specifically binds to C5. Furthermore, 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 nevertheless considered to be "specifically binding" antibodies as used herein.

[0070] The term "high affinity" antibody refers to antibodies that are highly avid, as determined by surface plasmon resonance, e.g., BIACORE TM or at least 10 as measured by solution affinity ELISA -8 M; preferably 10 -9 M; more preferably 10 -10 M, and even more preferably 10 -11 M, and even more preferably 10 -12 K of M D refers to mAbs with binding affinity for C5 expressed as

[0071] The terms "slow off rate", "Koff" or "kd" refer to the rate at which a given molecule is dissociated using surface plasmon resonance, e.g., BIACORE TM Determined by 1x10 -3 s -1 or less, preferably 1x10 -4 s -1 It means an antibody that dissociates from C5 with a rate constant equal to or less than that.

[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" of an antibody or "antibody fragment" 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 are used in combination with a moiety such as a ligand or therapeutic moiety (an "immunoconjugate"), a second anti-C5 antibody, or a C5-associated disease or may be conjugated to any other therapeutic moiety useful for treating the disorder.

[0074] As used herein, an "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, a "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, an antibody of the invention may prevent or block complement-mediated hemolysis by the classical or alternative pathway.

[0076] The term "surface plasmon resonance" as used herein refers to, for example, BIACORE TM It refers to an optical phenomenon that allows the analysis of real-time biomolecular interactions through the detection of changes in protein concentration within a biosensor matrix using systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

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

[0079] As used herein, the term "cross-compete" means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. This term also includes competition between two antibodies in both directions, i.e., a first antibody binding and blocking the binding of a second antibody, and vice versa. In certain embodiments, a first antibody and a 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, such as real-time label-free biolayer interference assays. Cross-competition between two antibodies can be expressed as the binding of the second antibody below the background signal due to self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as the % binding of the second antibody below baseline self-self background binding (where the first and second antibodies are the same antibody).

[0080] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand), including appropriate nucleotide insertions or deletions, as discussed below, can be used in a variety of applications, such as FASTA, BLAST, or GAP. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or a substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0081] The terms "substantial similarity" or "substantially similar," as applied to polypeptides, mean that two peptide sequences, when optimally aligned using programs such as GAP or BESTFIT with default gap weights, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When 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, e.g., 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 conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is one that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, incorporated herein by reference. A "moderately conservative" substitution is one that has 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 with default parameters to determine sequence homology or sequence identity between closely related polypeptides, for example, between homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters; a program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of maximum overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0083] The phrase "therapeutically effective amount" means the amount for which it is administered that produces the desired effect. The precise amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., 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 amelioration, 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 in the severity or amelioration 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 of the disease, i.e., the subject may be disease-free or have 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 appearance of a C5-related disease or disorder or a symptom of such a disease or disorder upon administration of an antibody of the invention.

[0087] Antigen-binding fragment of an antibody Unless otherwise specifically indicated, the term "antibody" as used herein is understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules"), as well as antigen-binding fragments thereof. As used herein, the terms "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 to 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 C5 protein. Antibody fragments include Fab fragments, F(ab')2 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 technique, such as proteolytic digestion or recombinant genetic engineering, including the manipulation and expression of DNA encoding antibody variable 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 molecular biology techniques can be used, for example, to place one or more variable and / or constant domains in the appropriate 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) antibodies. Examples of antigen-binding fragments include minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an IgNAR (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, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0089] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L Domain and associated V H In an antigen-binding fragment having a domain, V H and V L The domains can be positioned in any suitable orientation relative to each other. For example, the variable region can be a dimer, and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may be a dimer. H or V L It may contain domains.

[0090] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the 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 LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which creates a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be non-covalently linked to each other and / or to one or more monomeric V H Or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, with the domains (e.g., via disulfide bonds).

[0091] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain 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 exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0092] Human antibody production 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 the C5 protein.

[0093] Antibodies to the C5 protein can be generated using an immunogen comprising any one of the following: In certain embodiments, antibodies of the invention are obtained from mice immunized with the full-length native C5 protein (see, e.g., GenBank Accession No. NP_001726.2) (SEQ ID NO: 355) or with DNA encoding the protein or a fragment thereof. Alternatively, the protein or a 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 about 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 cell such as a Chinese hamster ovary (CHO) cell.

[0095] Velocimmune (R) technology (e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE (R) ) or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody to C5 having human variable regions and mouse constant regions is first isolated. (R) The technique involves generating transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions 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] In general, VELOCIMMUNE (R)Mice are challenged with the antigen of interest, and lymphocytes (e.g., B cells) are collected from the mice that express the antibody. The lymphocytes may be fused with a myeloma cell line to produce an immortal hybridoma cell line, and such hybridoma cells are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and linked to constant regions of the desired heavy and light chain isotype. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains may be isolated directly from the antigen-specific lymphocytes.

[0097] First, high-affinity chimeric antibodies having human variable regions and murine constant regions are isolated. As in the experimental section below, the antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to generate fully human antibodies of the invention, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the particular application, high-affinity antigen binding and target specificity characteristics reside in the variable regions.

[0098] bioequivalence The anti-C5 antibodies and antibody fragments of the present invention include proteins that have amino acid sequences that differ from the described antibodies but retain the ability to bind to C5 protein. Such variant antibodies and antibody fragments have one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially equivalent biological activity to the described antibodies. Similarly, DNA sequences encoding the antibodies of the present invention may contain 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. It contains the sequence to be loaded.

[0099] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in their rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antibodies may be considered equivalents or pharmaceutical substitutes if they are equivalent in their extent but not in their rate of absorption, and furthermore, such differences in absorption rate are unintentional and reflected in the label, are not essential, for example, to achieving effective body drug concentrations with chronic use, and are considered medically insignificant for the particular drug being studied.

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

[0101] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference and biologic products one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuing treatment without switching.

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

[0103] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which concentrations of an antibody or its metabolites are measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in well-controlled clinical trials that establish the safety, efficacy, or bioavailability or biological permeability of the 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 unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0105] Anti-C5 antibodies containing Fc variants Certain embodiments of the present invention provide anti-C5 antibodies comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present invention provides anti-C5 antibodies comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HAnti-C5 antibodies include those containing mutations in three regions, where the mutations increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, mutations at positions 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); or 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 at positions 250 and / or 428; or 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 invention includes anti-C5 antibodies comprising an Fc domain containing 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 aforementioned Fc domain mutations disclosed herein and other mutations in antibody variable domains are considered within the scope of the present invention.

[0107] The present invention also provides chimeric heavy chain constant (C H ) region, wherein the chimeric C5 antibody H The region may contain C domains of more than one immunoglobulin isotype. H For example, the antibodies of the present invention may comprise a segment derived from a C region derived from a human IgG1, human IgG2, or human IgG4 molecule. H C derived from a human IgG1, human IgG2 or human IgG4 molecule combined with some or all of the three domains. H Chimeric C containing part or all of the 2 domains H In certain embodiments, the antibodies of the present invention may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 216 to 227 according to EU numbering) combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 228 to 236 according to EU numbering). According to a particular embodiment, the chimeric hinge region comprises 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 described herein H Antibodies comprising the region may, in certain embodiments, exhibit altered Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety).

[0108] Biological characteristics of antibodies 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 of less than 9 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein. D In certain embodiments, the antibodies or antigen-binding fragments thereof are used in an assay format, such as those set forth in Example 3 herein, or a substantially similar assay. a K of less than about 9 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM as measured by surface plasmon resonance using a D binds to C5.

[0109] The present invention also provides compounds having a dissociation half-life (t) of greater than about 2 minutes, as measured by surface plasmon resonance at 25° C. using, for example, the assay format defined in Example 4 herein, or a substantially similar assay. 1 / 2 In certain embodiments, the antibodies or antigen-binding fragments of the invention have a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, greater than about 200 minutes, or greater than about 250 minutes, as measured at 25° C. by surface plasmon resonance using, for example, an assay format defined in Example 3 herein (e.g., mAb capture or antigen capture format), or a substantially similar assay. 1 / 2 It binds to the C5 protein.

[0110] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human C5 protein with a dissociation half-life of greater than about 1.5 minutes, as measured at 37°C by surface plasmon resonance using, for example, the assay format defined in Example 4 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention have a dissociation half-life of greater than about 2 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 25 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, or greater than about 200 minutes, as measured at 37°C by surface plasmon resonance using, for example, the assay format defined in the Examples herein (e.g., mAb capture or antigen capture format), or a substantially similar assay. 1 / 2 It binds to the C5 protein.

[0111] The present invention also provides compounds having a K of less than 120 nM, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein. DThe present invention also includes antibodies and antigen-binding fragments of antibodies that bind to monkey C5 protein at 25° C. (e.g., at 25° C. or 37° C.). In certain embodiments, the antibodies or antigen-binding fragments thereof have 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, less than about 500 pM, or less than 250 pM, as measured by surface plasmon resonance using, for example, the assay format set forth in Example 3 herein, or a substantially similar assay. D It binds to monkey C5.

[0112] The present invention also provides compounds having 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 and an R885H alteration (exemplified by SEQ ID NO: 356). C5 variants have shown poor responses to anti-C5 antibodies previously disclosed 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 has 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 set forth in Example 3 herein, or a substantially similar assay. D It binds to human C5 modified with

[0113] The present invention also provides a modified human C5 protein (exemplified by SEQ ID NO: 357) having an R885C change, which has 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 C5 variants include antibodies and antigen-binding fragments of antibodies that bind to C5. C5 variants have shown poor responses to anti-C5 antibodies previously disclosed in the art (e.g., Nishimura et al. 2014, New Engl. J. Med. 370:632-639). In certain embodiments, Thus, the antibody or antigen-binding fragment thereof may have 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, for example, the assay format set forth in Example 3 herein, or a substantially similar assay. D It binds to human C5 modified with

[0114] The present invention also provides compounds having an IC of less than 10 nM, as measured by a luminescence assay, for example, using the assay format defined in Example 6 herein. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an IC50 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, for example, the assay format set forth in Example 6 herein or a substantially similar assay. 50 inhibits CDC.

[0115] The present invention also provides human C5-mediated classical pathway (CP) hemolysis with an IC5 of less than 6 nM as measured by a CP hemolysis assay, for example, using the assay format defined in Example 8 herein. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has 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 hemolytic assay, e.g., using the assay format set forth in Example 8 herein or a substantially similar assay. 50 Blocks CP hemolysis.

[0116] The present invention also provides compounds having an IC of less than 165 nM as measured by an AP hemolytic assay, for example using the assay format defined in Example 8 herein. 50The present invention includes antibodies and antigen-binding fragments thereof that block human C5-mediated alternative pathway (AP) hemolysis by more than 70% at 1000 ng / mL. In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC5 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 as measured by an AP hemolysis assay, e.g., using the assay format set forth in Example 8 herein or a substantially similar assay. 50 Blocks AP hemolysis.

[0117] The present invention also provides compounds having an IC of less than 185 nM as measured by the CP hemolysis assay, for example using the assay format defined in Example 8 herein. 50 In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC5 of less than about 180 nM, less than about 150 nM, less than about 100 nM, less than about 75 nM, or less than about 50 nM as measured by a CP hemolysis assay, e.g., using the assay format set forth in Example 8 herein or a substantially similar assay. 50 Blocks CP hemolysis.

[0118] The present invention also provides compounds having an IC of less than 235 nM, as measured by an AP hemolytic assay, for example, using the assay format defined in Example 8 herein. 50 In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC5 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, as measured by an AP hemolytic assay, e.g., using the assay format set forth in Example 8 herein or a substantially similar assay. 50 Blocks AP hemolysis.

[0119] The present invention also provides compounds having an IC of less than 145 nM as measured by the CP hemolytic assay, for example, using the assay format defined in Example 8 herein. 50In certain embodiments, the antibodies or antigen-binding fragments thereof block C5-mediated classical pathway (CP) hemolysis in cynomolgus monkeys by more than 90%. In certain embodiments, the antibodies or antigen-binding fragments thereof block C5-mediated classical pathway (CP) hemolysis in cynomolgus monkeys by more than 90% using, for example, the assay format set forth in Example 8 herein or a substantially similar assay. 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 P hemolysis assay 50 Blocks CP hemolysis.

[0120] The present invention also provides compounds having an IC of less than 30 nM as measured by an AP hemolytic assay, for example, using the assay format defined in Example 8 herein. 50 The present invention includes antibodies and antigen-binding fragments thereof that block C5-mediated alternative pathway (AP) hemolysis in situ. In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC5 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 an AP hemolysis assay, e.g., using the assay format set forth in Example 8 herein or a substantially similar assay. 50 Blocks AP hemolysis.

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

[0122] In certain embodiments, the present invention provides antibodies with high affinity for human C5 (e.g., K<0.3 nM).D The present invention provides anti-C5 antibodies and antigen-binding fragments thereof that have increased serum half-life and reduced clearance (e.g., prolonged serum half-life, improved pharmacodynamic activity over a longer period of time than previously known anti-C5 antibodies). Such antibodies of the present invention can be advantageously used with less frequent dosing in subjects with a C5-related disease or disorder.

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

[0124] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) has a dissociation constant (K) of less than 0.9 nM at 25° C. as measured in a surface plasmon resonance assay. D ) binds to human C5; (c) a K of less than 0.3 nM at 37°C as measured in a surface plasmon resonance assay D(d) binds to human C5 with a K of less than 65 nM as measured in a surface plasmon resonance assay; D (e) binds to monkey C5 with a K of less than 0.5 nM as measured in a surface plasmon resonance assay; D (f) binds to human C5 mutant R885H (SEQ ID NO: 356) at 0.5n as measured in a surface plasmon resonance assay; K less than M D (g) binds to human C5 mutant R885C (SEQ ID NO: 357) with an IC of less than 6 nM as measured in a CP hemolysis assay; 50 (h) Blocks human C5-mediated classical pathway (CP) hemolysis by more than 95%; (h) IC<165 nM as measured in the AP hemolysis assay 50 Blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70%; (i) an IC of less than 185 nM as measured in the CP hemolysis assay 50 (j) inhibits African green monkey C5-mediated CP hemolysis with an IC of less than 235 nM as measured in an AP hemolysis assay; 50 (k) inhibits African green monkey C5-mediated AP hemolysis with an IC of less than 145 nM as measured in a CP hemolysis assay; 50 and (l) an IC5 of less than 30 nM as measured in an AP hemolysis assay. 50 inhibits cynomolgus monkey C5-mediated AP hemolysis.

[0125] The antibodies of the invention may have one or more of the aforementioned biological characteristics, or a combination thereof. Other biological characteristics of the antibodies of the invention will be apparent to those of skill in the art upon review of this disclosure, including the Examples herein.

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

[0127] Various 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 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 mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium methods involve deuterium labeling of the protein of interest followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface can retain deuterium and therefore exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0128] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. Epitopes can be formed both from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. 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 for classifying multiple monoclonal antibodies (mAbs) specific for the same antigen according to the similarity of each antibody's binding profile to chemically or enzymatically modified antigen surfaces (see US 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is highly distinct or partially overlapping with the epitope represented by another category. This technique allows for rapid filtering of genetically identical antibodies, so 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 or recombinantly produced C5 protein, or fragment thereof, exemplified in SEQ ID NO: 355. 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 to 1676 of the human C5 protein.

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

[0132] In certain embodiments, the present 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). An 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, an epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within C5. As shown in Example 11 herein, the epitope of C5 with which exemplary antibody H4H12166P of the invention interacts is defined by: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360), which corresponds to amino acids 591-599 in the beta chain of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775-794 in the alpha chain of SEQ ID NO: 359. Thus, the present 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), which corresponds to amino acids 591-599 in SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775-794 in SEQ ID NO: 359.

[0133] The present invention relates to antibodies that bind to the same epitope as or contain any of the specific exemplary antibodies listed in Table 1. The present invention includes anti-C5 antibodies that bind to a portion of the C5 protein. Similarly, the present invention also includes anti-C5 antibodies that compete for binding to the C5 protein or a fragment thereof with any of the specific exemplary antibodies listed in Table 1. For example, the present invention includes anti-C5 antibodies that cross-compete for binding to the C5 protein with one or more of the antibodies listed in Table 1.

[0134] Using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference anti-C5 antibody or competes for binding with the reference C5 antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-C5 antibody of the present invention, the reference antibody is allowed to bind to a C5 protein or peptide under saturating conditions. The ability of the test antibody to bind to a C5 molecule is then evaluated. If the test antibody is able to 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 than the reference anti-C5 antibody. On the other hand, if the test antibody is unable to 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 for binding with a reference anti-C5 antibody, the above binding methodology is performed in two directions: in the first direction, the reference antibody is allowed to bind to C5 protein under saturating conditions, followed by assessment of the binding of the test antibody to the C5 molecule. In the second direction, the test antibody is allowed to bind to C5 molecules under saturating conditions, followed by assessment of the binding of the reference antibody to the C5 molecule. In both directions, if only the first (saturating) antibody can bind to the C5 molecule, it is concluded that the test and reference antibodies compete for C5 binding. Those skilled in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0136] Two antibodies bind to the same or overlapping epitope 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 can be used in a competitive binding assay (e.g., as described by Junghans et al.). al., Cancer Res. 1990 50:1495-1502), but preferably inhibit binding of the other by at least 50%, but also by 75%, 90%, or 99%. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0137] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another reduction) is responsible for the observed lack of binding. These types of experiments 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 conjugated to a therapeutic moiety (immunoconjugates) 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, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. An antibody can be a molecule capable of binding to its target, as long as it is capable of binding to the target. The C5 antibody may be linked at any position along the C5 axis to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the drug may be a second, different antibody directed against the C5 protein. The type of therapeutic moiety that can be attached to an 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, e.g., WO 05 / 103081.

[0139] Multispecific antibodies The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide (see, e.g., 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 produced in a bispecific format ("bi-specific") in which variable regions that bind to different domains of the C5 protein are linked together, resulting in dual-domain specificity within a single binding molecule. Properly designed bispecifics can enhance overall C5 protein inhibitory efficacy by increasing both specificity and avidity. Variable regions that have specificity for individual domains (e.g., segments of the N-terminal domain) or can bind to different regions within a domain are paired on a structural scaffold, allowing each region to simultaneously bind to a separate epitope or different regions within a domain. In one example of bispecificity, a heavy chain variable region (V) from a binder with specificity for one domain is linked to a heavy chain variable region (V) from a binder with specificity for one domain. H ) is that V H without destroying the original singularity for the original V H Non-cognate V that can be paired with L To identify partners, we identify light chain variable regions (V) from a series of binders with specificity for the second domain. L ) and recombines with a single V L Segments (e.g., V L 1) is two different V H Domain (e.g., V H 1 and V H 2) and two connecting "arms" (V H 1-V L 1 and V H 2-V L 1) produces a bispecific antibody composed of a single V L The use of segments reduces the complexity of the system and thereby simplifies and increases the efficiency of the cloning, expression, and purification processes used to generate bispecifics (e.g., USSN 13 / 022759 and US2010 / 0331527).

[0142] Alternatively, more than one domain 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 skilled in the art. Antibody variable regions that bind to different regions can be linked together, for example, with variable regions that bind to related sites on the extracellular domain of C5, resulting in dual antigen specificity within a single binding molecule. Properly designed bispecifics of this nature lend themselves to dual functionality. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for the outside of the extracellular domain and paired on a structural scaffold that allows each variable region to bind to a separate antigen.

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

[0144] Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats (for a review of such formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al. al., J.Am.Chem.Soc.[Epub:Dec.4, 2012]).

[0145] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-C5 antibody of the present invention or an antigen-binding fragment thereof. The therapeutic compositions according to the present invention are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations can be found in formularies 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 Excipients for parenteral formulations include acetaminophen, DNA binders, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycol 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 dose of the antibody may 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 generally administered at 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 about 1 g. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. In certain embodiments, the antibody or antigen-binding fragment of the invention may 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 antigen-binding fragment thereof may be administered in an amount about 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] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and 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, epidermal, and oral routes. The compositions can be administered by any convenient route, such as infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0148] The use of nanoparticles to deliver 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 production and use are described in detail by Arruebo, M. et al., 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 can be developed for cell targeting and 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 in its entirety).

[0149] In certain situations, 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 proximity to the target of the composition, thus requiring only a fraction of the systemic dosage.

[0150] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, infusions, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like, which may be used in combination with appropriate solubilizers 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)], and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, and the like. The injectable formulations prepared in this manner are preferably filled into appropriate ampoules.

[0151] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, pen delivery devices are readily useful for delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge is all administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0152] Numerous reusable pen and auto-injector delivery devices have utility in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, the 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 Penn (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM , OPTIPEN PRO TM , OPTIPEN STARLET TM , and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany). Certainly non-limiting examples of disposable pen devices that have utility in the subcutaneous delivery of pharmaceutical compositions of the present invention include SOLOSTAR, to name a few. 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, LP) and HUMIRA TM Penn (Abbott Labs, Abbott Park, IL).

[0153] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses adapted to the dose of the active ingredient. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained per unit dose dosage form is generally about 5 to about 500 mg; particularly in the form of injections, the antibody is preferably contained in an amount of about 5 to about 300 mg, and for other dosage forms, the antibody is preferably contained in an amount of about 10 to about 300 mg.

[0154] Therapeutic Uses of Antibodies The antibodies of the present invention are useful for treating and / or preventing diseases, disorders, or conditions associated with C5, or for ameliorating at least one symptom associated with such diseases, disorders, or conditions. In certain embodiments, the antibodies or antigen-binding fragments thereof herein can be administered in therapeutic doses to patients with diseases, disorders, or conditions associated with C5.

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

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

[0157] In certain embodiments, the antibodies of the invention are used to treat neurological disorders, nephropathy, 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 in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, burns including 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 nephropathy (C3 glomerulopathy, membranoproliferative glomerulonephritis, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal artery bypass surgery, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disease and autoimmune disease, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, renal ischemia-reperfusion injury, lupus nephritis, glomerulopathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, membranoproliferative nephritis, hemolytic anemia, neuromyelitis optica, kidney transplant, inherited 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, fibrosing dust diseases, injury caused by inert dusts and minerals (e.g., silicon, coal dust, beryllium, and asbestos), pulmonary fibrosis, organic dust diseases, chemical injury (caused by irritant gases and chemicals, e.g., chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia, and hydrochloric acid), smoke injury, thermal injury (e.g., burns, frostbite), asthma, allergies, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, hereditary angioedema, and immune complex-associated inflammation.

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

[0159] One or more antibodies of the invention may be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions / signs of an ocular disease or injury, such as loss of vision, distorted vision, difficulty adapting to low light, crooked central vision, increased haziness in central / panoramic vision, the presence of drusen (extracellular deposits that accumulate on the retina), and / or the presence of ocular ocular disorders. Small accumulations of material), pigmentary changes, distorted vision in the form of metamorphopsia (where straight grids appear wavy and parts of the grid appear blank), exudative changes (ocular hemorrhages, hard exudates, subretinal / sub-RPE / intraretinal fluid), slow recovery of visual function after exposure to bright light (light stress test), initial and geographic atrophy, sudden decrease in visual acuity (2 levels or more), e.g., from 20 / 20 to 20 / 80, preferential hyperacuity perimetry The present invention may be used to ameliorate or reduce the severity of at least one symptom including, but not limited to, blurred vision (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 missing area in visual field), difficulty distinguishing colors, especially dark from dark and light from light, loss of contrast sensitivity, and lines that appear bent in an Amsler grid.

[0160] Also contemplated herein is the prophylactic use of one or more antibodies of the invention in subjects at risk of developing macular degeneration, such as subjects over the age of 50, 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 medicament 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 adjunctive therapy with any other drug or therapy known to those skilled in the art to be useful for treating or ameliorating a disease or disorder associated with C5.

[0162] Combination therapy Combination therapy can include an anti-C5 antibody of the present invention and any additional therapeutic agent that can be advantageously combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention. The antibody of the present invention can be synergistically combined with one or more drugs or therapies to treat a disease or disorder associated with C5. In some embodiments, the antibody of the present invention can be combined with a second therapeutic agent to ameliorate one or more symptoms of the disease.

[0163] Depending on the C5-related disease or disorder, the antibodies of the invention may 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 nonsteroidal anti-inflammatory drugs), antihypertensive drugs (e.g., angiotensin II inhibitors), anticoagulants ... converting enzyme inhibitors), immunosuppressants (e.g., vincristine, cyclosporin A, or methotrexate), fibrinolytic agents (e.g., ancrod, ε-aminocaproic acid, antiplasmin-a1, prostacyclin, and defibrotide), lipid-lowering agents such as inhibitors of hydroxymethylglutaryl CoA reductase, anti-CD20 agents such as rituximab, anti-TNF agents such as infliximab, anticonvulsants (e.g., magnesium sulfate), C3 inhibitors, or antithrombotic agents.

[0164] In certain embodiments, the second therapeutic agent is another antibody against the C5 protein. The use of a combination ("cocktail") of antibodies with 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 constituting the combination bind to different, non-overlapping epitopes on the protein. The antibodies constituting the combination can block the binding of C5 to the C5 convertase and / or inhibit the C5 activity of C5. It may prevent / inhibit cleavage into C5a and C5b. In certain embodiments, the second antibody may have a longer half-life in human serum.

[0165] The term "in combination with" as used herein means that an additional therapeutically active ingredient 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] An additional therapeutically active ingredient may be administered to a subject prior to administration of an anti-C5 antibody of the present invention. For example, a first component may be considered to be administered "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, an additional therapeutically active ingredient may be administered to a subject after administration of an anti-C5 antibody of the present invention. For example, a first component can be considered to be administered "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 yet other embodiments, an additional therapeutically active component can be administered to a subject simultaneously with administration of an anti-C5 antibody of the invention. "Concurrent" administration for purposes of this invention includes, for example, administration to a subject where the anti-C5 antibody and the additional therapeutically active component are administered in a single dosage form or in separate dosage forms that are administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form can be administered via the same route (e.g., both the anti-C5 antibody and the additional therapeutically active ingredient can be administered intravenously, etc.); or each dosage form can be administered by a different route (e.g., the anti-C5 antibody can be administered intravenously and the additional therapeutically active ingredient can be administered orally). In any event, a single dosage form, separate dosage forms by the same route, or separate dosage forms by different routes are all considered "co-administered" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-C5 antibody "before," "concurrently with," or "after" (as those terms are defined herein above) the administration of an additional therapeutically active ingredient is considered administration of the anti-C5 antibody "in combination with" the additional therapeutically active ingredient.

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

[0168] Dosing regimen According to certain embodiments, a single dose of an anti-C5 antibody of the present invention (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of an anti-C5 antibody (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. A method according to this aspect of the present invention comprises sequentially administering multiple doses of an anti-C5 antibody of the present invention to a subject. As used herein, "sequentially administering" means that each dose of an anti-C5 antibody is administered to a subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods comprising sequentially 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 administration of an anti-C5 antibody of the present invention. The term "initial dose" refers to a chronological order. 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 in terms of administration 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 administered less frequently (e.g., "maintenance doses").

[0170] According to an exemplary embodiment of the invention, each secondary and / or tertiary dose is administered within 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 the dose of anti-C5 antibody that, in a multiple dose sequence, is administered to a patient prior to the administration of the immediately next dose in the sequence, with no intervening doses.

[0171] The method according to this aspect of the invention can include administering any number of secondary and / or tertiary doses of anti-C5 antibody to the patient. 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 with which the secondary and / or tertiary doses are administered to a patient may vary during the treatment regimen. The frequency of administration may also be adjusted during treatment by the physician according to the needs of the individual patient after clinical testing.

[0173] Diagnostic Uses of Antibodies The anti-C5 antibodies of the present invention can be used to detect and / or measure C5 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present invention in assays for detecting a C5-related disease or disorder. An exemplary diagnostic assay for C5, for example, involves contacting a sample obtained from a patient with an anti-C5 antibody of the present invention, where the anti-C5 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate C5 from the patient sample. Alternatively, an unlabeled anti-C5 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The C5 may be a 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 exemplary 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 patients. The term "C5" includes any tissue or fluid sample that can be obtained from a patient, which contains a detectable amount of either C5 protein or a fragment thereof under normal or pathological conditions. Generally, the level of C5 protein in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a C5-related disease) is measured to first 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 C5-related condition or symptoms associated with such a condition.

[0175] Antibodies specific for 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 binding assays, the position of the label (if any) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface.

[0176] Selected Implementations Selected embodiments of the present disclosure include: In embodiment 1, 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 one or more amino acids contained within C5 (SEQ ID NO: 359), as determined by hydrogen / deuterium exchange.

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

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

[0179] In embodiment 4, the invention comprises an isolated antibody of the antigen-binding fragment of any one of embodiments 1 to 3, wherein the antibody or 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 invention comprises the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 591 to 599 of SEQ ID NO: 359; (b) amino acids 593 to 599 of SEQ ID NO: 359; (c) amino acids 775 to 787 of SEQ ID NO: 359; (d) amino acids 775 to 794 of SEQ ID NO: 359; and (e) amino acids 779 to 787 of SEQ ID NO: 359.

[0181] In embodiment 6, the present invention comprises the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with at least five 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 invention relates to the isolated antibody of 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. The present invention includes an antibody or antigen-binding fragment thereof.

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

[0185]

[0022] In embodiment 10, the invention comprises the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 9, wherein the antibody has additional characteristics selected from the group consisting of: (a) a fully human monoclonal antibody; (b) a dissociation constant (K) of less than 0.9 nM at 25°C, as measured in a surface plasmon resonance assay. D ) binds to human C5; (c) a K of less than 0.3 nM at 37°C as measured in a surface plasmon resonance assay D(d) binds to human C5 with a K of less than 65 nM as measured in a surface plasmon resonance assay; D (e) binds to monkey C5 with a K of less than 0.5 nM as measured in a surface plasmon resonance assay; D (f) binds to human C5 mutant R885H (SEQ ID NO: 356) with a K of less than 0.5 nM as measured in a surface plasmon resonance assay; D (g) binds to human C5 mutant R885C (SEQ ID NO: 357) with an IC of less than 6 nM as measured in a CP hemolysis assay; 50 (h) Blocks human C5-mediated classical pathway (CP) hemolysis by more than 95%; (h) IC<165 nM as measured in the AP hemolysis assay 50 Blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70%; (i) an IC of less than 185 nM as measured in the CP hemolysis assay 50 (j) inhibits African green monkey C5-mediated CP hemolysis with an IC of less than 235 nM as measured in an AP hemolysis assay; 50 (k) inhibits African green monkey C5-mediated AP hemolysis with an IC of less than 145 nM as measured in a CP hemolysis assay; 50 and (l) an IC5 of less than 30 nM as measured in an AP hemolysis assay. 50 inhibits cynomolgus monkey C5-mediated AP hemolysis.

[0186] In embodiment 11, the invention includes 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 provides: (a) SEQ ID NOs: 4, 20, 36, 52, 68, 84 , 100, 124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292, 308, 324, and 340; (b) an HCDR1 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 (c) an HCDR2 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 HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, (e) an LCDR1 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 LCDR2 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.

[0188] In embodiment 13, the invention comprises an isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 12, 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 comprises the isolated antibody or antigen-binding fragment thereof of embodiment 13, comprising 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 provides 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 / 19 15. The isolated antibody or antigen-binding fragment of any one of embodiments 11 to 14, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of: 4, 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 comprises an isolated antibody or antigen-binding fragment thereof of any one of embodiments 11 to 15, comprising 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 invention includes the isolated antibody or antigen-binding fragment thereof of embodiment 16, comprising 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 a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50 / 58, 98 / 106, 138 / 106, and 202 / 210. 18. The isolated antibody or antigen-binding fragment thereof of embodiment 17, comprising:

[0194] In embodiment 19, the invention includes an antibody or 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 present invention comprises an antibody or antigen-binding fragment that binds to the same epitope as the antibody or antigen-binding fragment of embodiment 17.

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

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

[0198] In embodiment 23, the invention comprises the antibody or 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 the antibody or 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 invention comprises an isolated monoclonal antibody or antigen-binding fragment thereof that blocks C5 cleavage into C5a and C5b, comprising three CDRs of an HCVR, wherein the 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 an LCVR, wherein the 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.

[0201] In embodiment 26, the present invention includes a pharmaceutical composition comprising an isolated antibody or 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 comprises an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of an antibody shown in any one of embodiments 1 to 25.

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

[0204] In embodiment 29, the present invention comprises a vector comprising the polynucleotide sequence of embodiment 27 or 28.

[0205] In embodiment 30, the invention includes a cell expressing the vector of embodiment 29.

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

[0207] In embodiment 32, the present invention provides a method for treating a disease or disorder comprising administering to a patient in need thereof a therapeutically effective amount of ... 32. The method of embodiment 31, wherein the disease is selected from the group consisting of cancer, atherosclerosis, vasculitis, bullous pemphigoid, C3 nephropathy, 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 surgery, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disease and autoimmune disease, nephropathy, 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 invention includes the method of embodiment 31, wherein the disease or disorder is aHUS.

[0209] In embodiment 34, the invention comprises the method of embodiment 31, wherein the disease or disorder is PNH.

[0210] In embodiment 35, the invention comprises the method of any one of embodiments 31 to 34, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof.

[0211] In embodiment 36, the invention comprises the method of any one of embodiments 31 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0212] Embodiment 37: The invention includes the method of embodiment 36, wherein the therapeutic agent is selected from the group consisting of an anticoagulant, an anti-inflammatory, an antihypertensive, an immunosuppressant, a lipid-lowering agent, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an antiepileptic agent, a C3 inhibitor, a second anti-C5 antibody, and an antithrombotic agent.

[0213] In embodiment 38, the invention comprises the method of any one 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 presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

[0215] Example 1: Generation of human antibodies against complement factor 5 (C5) protein Human antibodies against the C5 protein are produced using VELOCIMMUNE, which contains DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions. (R) Mice were immunized with serum-purified human C5 protein (Calbiochem catalog number 20-4888) produced in mice.

[0216] Antibody immune responses were monitored by C5-specific immunoassays. 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 producing C5-specific antibodies. These cell lines were used to obtain several anti-C5 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains); exemplary antibodies generated in this manner were designated H2M11683N and H2M11686N.

[0217] Anti-C5 antibodies were also isolated directly from antigen-positive mouse B cells without fusion to 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 with human variable and human constant domains) were obtained; exemplary antibodies generated in this manner 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 methods of this example are described in detail in the Examples provided 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 numeric 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, antibodies may be referred to herein as, for example, "H2M11686N," "H4H12183P2," "H4H12168P," etc. The H4H and H2M prefixes in antibody designations used herein indicate the identity of the antibody. A particular Fc region isotype is designated, for example, an "H4H" antibody has a human IgG4 with a serine to proline mutation (S108P) in the hinge region to promote dimer stabilization, and an "H2M" antibody has a murine IgG2 Fc (a or b isotype) (all variable regions are fully human, indicated by an initial "H" in the antibody designation). Those skilled in the art will appreciate that antibodies with a particular Fc isotype can be converted to a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted to an antibody with human IgG4, etc.), but in any case, the variable domains (including CDRs) - these are indicated by the numerical identifiers shown in Table 2 - remain the same, and the binding characteristics 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 with a murine IgG1 Fc was converted to an antibody with a human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains two or more amino acid changes disclosed in US20100331527.

[0225] To generate the 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 target antigens, resulting in improved pharmacokinetics (Igawa et al. 2010, Nat. Biotechnol. 28:1203-1207).

[0226] Control constructs used in the following examples The following control construct (anti-C5 antibody) was included in the experiments disclosed herein for comparison purposes: Control drug 1 , V of the antibody "h5G1.1" according to U.S. Patent No. 6,355,245 (Alexion Pharmaceuticals, Inc.) H / V L a monoclonal antibody against human C5 having the sequence Control drug 2 ", V of antibody "8109" according to US Patent Application Publication No. 2013 / 0022615 (Novartis) H / V L A human monoclonal antibody against human C5 having the sequence

[0227] Example 3: Antibody Binding to C5 Determined by Surface Plasmon Resonance The equilibrium dissociation constant (K) for C5 binding to purified anti-C5 antibody DThe binding activity (RI) of C5 was determined using a real-time surface plasmon resonance biosensor assay on a Biacore T200 instrument. To capture expressed anti-C5 antibodies with a human Fc constant region, the Biacore sensor surface was derivatized with a monoclonal mouse anti-human Fc antibody (GE Healthcare, no. BR-1008-39) via amine coupling. Biacore binding studies were 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). Another C5 reagent was expressed with a C-terminal myc-myc-hexahistidine tag (hereafter referred to as C5-mmh). Human C5-mmh reagents containing histidine and cysteine ​​point mutations at arginine 885 were also expressed (hereafter 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) (ranging from 100 nM to 1.23 nM, 3-fold dilutions) prepared in HBST running buffer were injected over the anti-C5 antibody capture surface at a flow rate of 30 μL / min. Binding of the total C5 reagent to each of the captured monoclonal antibodies was monitored for 3 min, and their dissociation in HBST running buffer was monitored for 8 min. All binding kinetic experiments were performed at either 25°C or 37°C. Kinetic binding (k a ) and dissociation (k d ) Rate constants were calculated by Scrub real-time sensorgrams against a 1:1 binding model. The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) from the kinetic rate constant: K D (M)=k d / k a and t 1 / 2 (minutes)=ln2 / (60xk d ) was calculated as

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

[0229] [Table 3]

[0230] [Table 4]

[0231] Monkey C5-mmh binding to anti-C5 antibodies 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 antibodies 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 antibodies 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 have K values ​​for human C5 ranging from 73 pM to 8.4 nM, as shown in Table 3. D At 37°C, the anti-C5 antibodies of the invention bound with K values ​​ranging from 103 pM to 18.5 nM, as shown in Table 4. D At 25°C, 25 of the 25 anti-C5 antibodies of the invention tested bound to human C5 with K values ​​ranging from 133 pM to 64 nM, as shown in Table 5. D At 37°C, 25 of the 25 anti-C5 antibodies of the invention tested bound to monkey C5-mmh with K values ​​ranging from 133 pM to 118 nM, as shown in Table 6. D Sixteen of the anti-C5 antibodies of the invention tested at 25°C bound to monkey C5-mmh with K values ​​ranging from 147 pM to 10.9 nM, as shown in Table 7. D Sixteen of the 16 anti-C5 antibodies of the invention tested at 25°C bound to human C5 R885H-mmh with K values ​​ranging from 251 pM to 190 nM, as shown in Table 8. D Sixteen of the 16 anti-C5 antibodies of the invention tested at 37°C bound to human C5 R885C-mmh with K values ​​ranging from 1.49 nM to 69.4 nM, as shown in Table 9. D Sixteen of the 16 anti-C5 antibodies of the invention tested at 25°C bound to human C5 R885H-mmh with K values ​​ranging from 1.74 nM to 159 nM, as shown in Table 10. D It bound to human C5 R885C-mmh at 1000kJ / mL.

[0241] Example 4: Antibodies that bind to C5 at different pH The effect of pH on the dissociation rate of recombinant human C5 bound to purified anti-C5 monoclonal antibody was determined using a Biacore T200 real-time surface plasmon resonance biosensor. The Biacore sensor surface was first derivatized with a monoclonal mouse anti-human Fc antibody (GE, no. BR-1008-39) by amine coupling to capture the expressed anti-C5 monoclonal antibody bearing the human IgG4 Fc. All Biacore binding experiments were performed using two running buffers: PBS-T, pH 7.4 (0.01 M NaHPO / NaHPO, 0.15 M NaCl, 0.05% v / v Tween-20, adjusted to pH 7.4) and PBS-T, pH 6.0 (0.01 M NaHPO / NaHPO, 0.15 M NaCl, 0.05% v / v Tween-20, adjusted to pH 6.0). Various concentrations of human C5 (EMD, Cat. No. 204888) or monkey C5.mmh (prepared in PBS-T, pH 7.4, 3-fold dilutions ranging from 100 nM to 11.11 nM) were injected over the anti-C5 monoclonal antibody-captured surface at a flow rate of 50 μL / min for 3 min, and their dissociation in two running buffers, PBS-T, pH 7.4 and PBS-T, pH 6.0, was monitored for 6 min. All binding kinetic experiments were performed at 25°C and 37°C. The kinetic dissociation constant (k d ) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. 1 / 2 ) to k d from:

number

[0242] The ratios of half-lives for 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, are shown in Tables 11 and 12.

[0243] [Table 11]

[0244] [Table 12]

[0245] The ratios of half-lives for 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, are shown in Tables 13 and 14.

[0246] [Table 13]

[0247] [Table 14]

[0248] As shown in Tables 11 to 14, the selected anti-C5 antibodies 1 / 2 The ratios showed pH dependent binding.

[0249] Example 5: Octet cross-competition between anti-C5 antibodies Binding competition between anti-C5 monoclonal antibodies (mAbs) was determined using a real-time, label-free biolayer interferometry assay with an Octet RED384 biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v Surfactant Tween-20, 0.1 mg / mL BSA (Octet HBS-P buffer) with plate shaking at 1000 rpm. To assess whether the two antibodies can compete with each other for binding to their respective epitopes on human C5 (hC5 purified from plasma, EMD), approximately 1.5 nm of anti-human C5 mAb was first captured onto an anti-hFc antibody-coated Octet biosensor chip (Pall ForteBio Corp., No. 18-5060) by submerging the chip in a well containing a 50 μg / mL solution of anti-human C5 mAb (hereafter referred to as mAb1) for 3 minutes. The blocking H4H isotype control mAb (hereafter referred to as blocking mAb) was then saturated by immersing the captured biosensor chip in a well containing a 200 μg / mL solution of the blocking mAb for 4 minutes. This was followed by a complex of 50 nM hC5 and 1 μM of a second anti-human C5 mAb (hereafter referred to as mAb2), which had been pre-incubated for 2 hours. The biosensor chip was immersed in the well containing the co-complexed solution 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 recorded at the end of each step. The binding of mAb2 pre-complexed with human C5 to mAb1 was corrected for background binding and compared to determine the competitive / non-competitive behavior of various anti-C5 monoclonal antibodies.

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

[0251] [Table 15]

[0252] Example 6: Inhibition of C5-mediated complement-dependent cytotoxicity in a B-cell bioassay This example describes a bioassay using an anti-CD20 antibody to test the role of C5 in the classical complement pathway. Therapeutic anti-CD20 antibodies against the B cell-specific cell surface antigen CD20 have been shown to result in CDC of B cells (Glennie et al. 2007, Mol. Immunol. 44:3823-3837), and a CDC assay using a CD20-expressing cell line has been previously described (Flieger et al. 2000, Cell. Immunol. 204:55-63). Daudi cells, a human B cell line expressing CD20, complement-preserved serum or C5-depleted serum for exogenous C5 variants, and an anti-CD20 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 cultured at 10,000 cells / well in 96-well assay plates in 10% FBS, penicillin / streptomycin, L Cells were seeded in either RPMI containing -glutamine, sodium pyruvate, and non-essential amino acids (RPMI complete) or RPMI containing 1% BSA, penicillin / streptomycin, and L-glutamine (RPMI / BSA). All assays testing mutated anti-hC5 antibodies, along with tests of non-mutated antibodies using C5-containing human serum, were run in RPMI complete medium, whereas assays testing non-mutated antibodies used African green monkey serum, and human C5 mutants were tested in RPMI / BSA medium. To measure CDC using human or monkey serum, anti-CD20 antibodies were diluted 1:3 from 100 nM to 2 pM (including a no-antibody control sample) and incubated with the cells for 10 min at 25°C, followed by the addition of 1.66% serum or 1.66% C5-depleted serum and 6.6 nM C5 mutant protein. The amount of C5 protein to be added to the C5-depleted serum was based on the reported C5 concentration in human serum of 0.37 μM (Rawal et al. 2008, J. Biol. Chem. 283:7853-7863). To test C5 antibody inhibition of CDC, C5 antibody was diluted 1:3 from 100 nM to 2 pM (including a control sample containing no antibody) and incubated with 1.66% serum or 1.66% C5-depleted serum and 6.6 nM C5 mutant protein for 30 minutes. Ten minutes before adding the antibody and serum to the cells, 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. Upon completion of the incubation with anti-CD20 antibody, the antibody / serum mixture was added to the cells. Cytotoxicity was assessed by CytoTox-Glo after 3.5 hours of incubation at 37°C and 5% CO2. TM Measurements were taken after adding the reagent (Promega, no. G9292). TM CytoTox-Glo is a luminescence-based reagent that measures cell killing; increased luminescence is observed as increased cytotoxicity increases (measured in relative light units, RLU). Untreated cells in control wells were incubated with CytoTox-Glo. TM Immediately after adding the reagent, the cells were rinsed with digitonin to determine maximum cell killing. TMLuminescence was read on a Victor X instrument (Perkin Elmer) 15 minutes after addition. Where calculated, the percentage of cytotoxicity was calculated with the RLU values ​​by using the following equation:

number

[0254] In this equation, "background cytolysis" is the luminescence from cells treated with medium and serum alone without any CD20 antibody, and "maximum cytolysis" is the luminescence from cells treated with digitonin. Results, expressed as % cytotoxicity or RLU, were analyzed using nonlinear regression (four-parameter logistics) with Prism 5 software (GraphPad) to calculate EC 50 Value and IC 50 Values ​​were obtained. Antibody inhibition was calculated such that 0-100% inhibition was the range of inhibition from the concentration of anti-CD20 antibody used in the assay without inhibitor to 0 nM anti-CD20 antibody.

[0255] result A total of 25 anti-human C5 antibodies (16 unmutated and 9 mutated) were tested for their ability to inhibit C5 in a CDC assay using Daudi cells with anti-CD20 antibodies and human serum (containing normal hC5 or C5 mutants) or African green monkey serum. Various residues in the complementarity-determining regions (CDRs) of H4H12166P were mutated to histidines to generate nine mutated antibodies, H4H12166P2 to H4H12166P10. Histidine mutations in the CDRs have been shown to confer pH-dependence of binding to target antigens 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 CDC mediated by C5, which is present in 1.66% of human serum. The IC50s of the non-mutated antibodies ranged from 1.2 to 3.4 nM. The IC50s of the mutated antibodies ranged from 3.0 nM to 12 nM. The parent non-mutated antibody H4H12166P produced complete inhibition with IC50s of 2.6 nM and 2.9 nM.

[0258] [Table 17]

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

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

[0261] To test whether anti-hC5 antibodies inhibit the human C5 mutants, R885H and R885C, C5-depleted human serum was tested with 6.6 nM of each C5 mutant. All 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by the C5 mutant R885H, with IC50s for the non-mutated antibodies ranging from 0.48 nM to 4.2 nM, whereas the IC50s for the mutant antibodies ranged from 1.3 nM to 7.0 nM. The parent non-mutated antibody H4H12166P inhibited CDC with IC50s of 1.3 nM and 4.2 nM. and produced complete inhibition with an IC50 of 1.3 nM.

[0262] Fifteen of the 16 non-mutated hC5 antibodies showed complete inhibition of CDC mediated by the C5 mutant R885C with IC50s 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 an IC50 > 20 nM. All nine mutant antibodies showed complete inhibition of CDC mediated by the C5 mutant R885C with IC50s ranging from 0.77 nM to 3.5 nM. The parent non-mutated antibody H4H12166P produced complete inhibition with IC50s of 0.46 nM and 0.76 nM.

[0263] The anti-CD20 antibodies exhibited CDC of Daudi cells with 1.66% serum with EC50s 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 the hC5 variant R885H, and 2.7 nM and 9.5 nM for hC5-deficient serum using the hC5 variant R885C. None of the irrelevant IgG control antibodies, control mAb1, or control mAb2, exhibited 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 the 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 that couples to various types of GPCRs to activate PLC-β and subsequently upregulate Ca. ++This can result in an increase in NFAT translocation and reporter gene transcription, 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 at 20,000 cells / well in 96-well assay plates in OPTIMEM (Invitrogen, product number 31985-070) supplemented with 0.5% BSA, penicillin / streptomycin, and L-glutamine, followed by overnight incubation at 37°C and 5% CO. BSA was used instead of FBS because serum has been shown to cleave and activate hC5a (Klos et al., 2013, Pharmacol. Rev. 65:500-543). The following morning, hC5a was titrated from 100 nM to 2 pM (including a control sample containing no hC5a) and a dose-response titration curve was determined for the cell line in addition to the cells. To test hC5a antibody inhibition of hC5a, 500 pM hC5a was added to cells. Immediately after, antibody diluted 1:3 from 100 nM to 2 pM was added to the cells (including a control sample containing no antibody). Cells were incubated for 5.5 hours at 37°C in the presence of 5% CO2. TM Luciferase activity was detected after incubation with the reagent (Promega, no. E6051). TM is a luminescence-based reagent that measures the amount of luciferase present in cells. In this assay, increased hC5a activation leads to increased luciferase production and luminescence (measured in relative light units, RLU). Luminescence measurements were performed using a Victor X instrument (Perkin Elmer). Prism 5 The results were analyzed using nonlinear regression (four-parameter logistics) with software (GraphPad) to determine EC 50 Value and IC 50 The inhibition of the antibodies was calculated such that 0-100% inhibition was the range of inhibition from 500 pM hC5a in the absence of 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 extent of inhibition of 500 pM hC5a activation of HEK293 / hGα16 / hC5aR1 / NFAT-luc cells.

[0267] [Table 18]

[0268] As shown in Table 18, all four antibodies of the invention showed complete inhibition of 500 pM hC5a with IC50s ranging from 0.035 nM to 0.46 nM. An irrelevant IgG control antibody, control mAb3, did not show any inhibition of hC5a. hC5a activated HEK293 / Gα16 / hC5aR1 / NFAT-luc cells with an EC50 of 0.39 nM.

[0269] Example 8: Hemolytic 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 classical complement pathway activation, which is sensitive to the reduction, absence, and / or inactivity of any component of the pathway. CH tests the functional ability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBCs) precoated with rabbit anti-sheep red blood cell antibodies (hemolysins). When antibody-coated SRBCs are incubated with test serum, the classical pathway of complement is activated and hemolysis occurs. In the absence of complement components, CH levels are zero; if one or more components of the classical pathway are reduced, CH decreases. (Nilsson et al. 1984, J. Immunol. Meth. 72:49-59) This assay is used to characterize and screen high-affinity anti-human C5 antibodies.

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

[0272] Percent hemolysis was calculated as follows:

number

[0273] In this equation, "background cell lysis" is the OD at A412nm from cells incubated with serum-free GVB++ buffer alone. "Maximum cell lysis" is the OD at A412nm from cells treated with water. Results, expressed as % hemolysis, were analyzed using nonlinear regression (four-parameter logistics) with Prism 5 software (GraphPad) to obtain IC50 values. Data are presented as mean ± standard error of the mean.

[0274] (B) Alternative complement assay The desired number of rabbit red blood cells (RbRBCs) was added to GVB-Mg 2+The cells were washed with EGTA / EGTA buffer and resuspended at 2x10^8 cells / ml. Normal human or cynomolgus monkey serum was added to GVB-Mg 2+ Antibodies were diluted to 10% in EGTA / EGTA buffer. To test for inhibition of C5-mediated hemolytic activity, antibodies at concentrations ranging from 3 nM to 800 nM were preincubated in 5% to 10% normal human serum or cynomolgus monkey serum for 20 minutes at 4°C. Hemolytic activity was measured using a round-bottom 96-well plate. A total of 100 μl of RbRBCs (2 x 10^8 cells / ml) was plated into a 96-well plate, followed by the addition of 100 μl of 10% normal human serum, cynomolgus monkey serum, or African green monkey serum preincubated with anti-C5 antibody. The cells were gently mixed and incubated at 37°C for 60 minutes. After the incubation period, the cells were pelleted by centrifugation 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 on a Spectramax microplate reader. Hemolytic activity was calculated based on a final serum concentration of 5% serum.

[0275] Percent hemolysis was calculated as follows:

number

[0276] In this equation, "background cell lysis" refers to the amount of lysis that occurs without or with serum. "Maximum cell lysis" is the OD at A412 nm from cells incubated with GVB-Mg / EGTA buffer alone without any anti-C5 antibody. "Maximum cell lysis" is the OD at A412 nm from cells treated with water. Inhibition by anti-C5 antibody, IC 50 Values ​​were calculated using nonlinear regression (four-parameter logistics) with Prism 6 software (GraphPad).

[0277] result (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 sensitized sheep red blood cells (SRBCs) and in the AH50 assay using rabbit red blood cells (RRBCs).

[0278] [Table 19]

[0279] As shown in Table 19, 16 anti-hC5 antibodies of the present invention demonstrated greater than 94% inhibition of hemolysis in the classical pathway (CP) mediated by C5 present in 1% human serum. The antibody IC50s ranged from 2.1 to 5.9 nM, and the percent inhibition ranged from 95% to 99%. All 16 anti-C5 antibodies demonstrated greater than 60% inhibition (except for H4H12169P) in the alternative pathway (AP) hemolysis assay mediated by C5 present in 5% NHS. The antibody IC50s ranged from 13 to 160 nM, and the percent inhibitory activity ranged from 44% to 81%.

[0280] [Table 20]

[0281] As shown in Table 20, all nine mutated anti-hC5 antibodies demonstrated inhibition of CP and AP hemolytic activity mediated by C5 present in 5% human serum. In the CP hemolysis assay, the parent non-mutated antibody H4H12166P demonstrated greater than 98% inhibition with an IC50 of 10.9 nM. The eight mutant anti-hC5 antibodies demonstrated greater than 90% inhibition with IC50s ranging from 10.3 nM to 24.3 nM. The mutant anti-C5 antibody 12166P10 demonstrated partial inhibition of 74%. In the AP hemolysis assay, the parent non-mutated antibody H4H12166P demonstrated greater than 85% inhibition with an IC50 of 20.9 nM. The mutant anti-hC5 antibodies demonstrated a range of inhibition of 72-83%, with IC50s ranging 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 the CH50 assay using sensitized sheep red blood cells (SRBCs) and the AH50 assay using rabbit red blood cells (RRBCs).

[0283] [Table 21]

[0284] As shown in Table 21, anti-hC5 antibodies showed varying levels of inhibition of CP or AP hemolytic activity in 5% African green monkey serum. In the CP assay, two of the 16 anti-hC5 antibodies showed no inhibition of hemolytic activity. Fourteen antibodies showed inhibition ranging from 43 to 94% with IC50s ranging from 25 nM to 180 nM. In the AP hemolytic assay, 13 of the 17 antibodies showed inhibitory activity ranging from 17% to 93% with IC50s ranging from 22.5 nM to 233 nM.

[0285] [Table 22]

[0286] As shown in Table 22, anti-hC5 antibodies (except H4H12183P2, which showed 64% CP inhibition) showed greater than 90% inhibition in either the CP or AP hemolytic assays in 5% cynomolgus serum. In the CP hemolytic assay, the IC50s of the antibodies ranged from 7.15 nM to 142 nM. In the AP hemolytic assay, the IC50s of the antibodies ranged from 5.4 to 29.6 nM.

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

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

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

[0290] C5a, generated as a result of C5 cleavage, is a 74 amino acid protein fragment. C5a is metabolized by serum carboxypeptidases to a more stable and less active 73 amino acid form, C5a des-Arg, by removal of the C-terminal arginine. Quantitation of C5a des-Arg therefore provides a reliable measure for monitoring C5a generation in vivo and in vitro. MicroVue as used herein The 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 supernatants from CP hemolytic assays using complement-preserved normal human serum (NHS) preincubated with H4H12166P or an isotype control antibody as described in Example 8. C5a protein levels were measured using a MicroVue C5a ELISA kit according to the manufacturer's instructions. Briefly, samples were diluted and incubated on plates precoated with a capture antibody (mouse anti-C5a specific for a neo-epitope on human C5a). Human C5a protein provided by the manufacturer was used as a standard for calibration. C5a in the supernatants was detected with an HRP-conjugated detection antibody (mouse monoclonal antibody against the C5a region of C5). A 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 using a SpectraMax plate reader. Data were analyzed using nonlinear regression (four-parameter logistics) in GraphPad Prism. C5a concentrations were analyzed as ng / mL of supernatant.

[0292] In assays using 5% NHS, H4H12166P potently blocked increases in C5a protein levels in a dose-dependent manner with an IC50 of 8.5 nM, whereas an isotype control antibody had no effect on C5a levels (Figure 1). 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: Pharmacokinetic and pharmacodynamic characterization of anti-C5 antibodies in cynomolgus monkeys This example describes the pharmacokinetic (PK) and pharmacodynamic (PD) characterization of selected anti-C5 antibodies performed in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody dosing and used to stratify animal dosing groups.

[0294] Total circulating C5 levels in cynomolgus monkeys were determined using a human complement C5 ELISA (Abcam, catalog no. ab125963) 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 Total antibody concentrations in monkey serum samples were measured using an unvalidated direct ELISA. The ELISA procedure used a microtiter plate coated with a mouse anti-human IgG1 / IgG4 Fc monoclonal antibody. Various anti-C5 antibodies were added to the plate, and the anti-C5 antibodies captured on the plate were detected using a biotinylated mouse anti-human IgG4 Fc monoclonal antibody followed by NeutrAvidin conjugated with horseradish peroxidase (NeutrAvidin HRP). A luminol-based substrate specific for the peroxidase was then added to achieve a signal intensity proportional to the total concentration of captured anti-C5 antibodies. The relative light unit (RLU) measurements of the calibration standards and their respective nominal concentrations were fitted using a weighted four-parameter logistic equation to generate a calibration equation describing the relationship between anti-C5 antibody concentration and assay response. The lower limit of quantitation (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, Phoenix (R) WinNonlin (R) The doses were determined by non-compartmental analysis (NCA) using software (version 6.4, Certara, LP) and an IV bolus dosing model.

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

[0299] PD analysis by ex vivo hemolysis assay The pharmacodynamics of selected anti-C5 antibodies were analyzed using ex vivo classical and proximal pathway hemolysis assays.

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

number

[0301] In this equation, "background cell lysis" is the OD at A412nm from SRBCs incubated with serum-free GVB++ buffer alone. "Maximum cell lysis" is the OD at A412nm from SRBCs treated with water. Results, expressed as % hemolysis, were analyzed using nonlinear regression (four-parameter logistics) with Prism 5 software (GraphPad) to determine IC 50 Values ​​were obtained. Data are expressed as the mean ± standard error of the mean.

[0302] Alternative pathway hemolysis assay: The desired number of rabbit red blood cells (RbRBCs) was added to GVB-Mg 2+ The RbRBCs were washed with EGTA buffer and resuspended at 2x10^8 cells / mL. Blood from the cynomolgus monkeys was collected for PD analysis 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. Serum was prepared and frozen until further use. Round-bottom 96-well plates were used to measure hemolytic activity. A total of 100 μl of RbRBCs (2x10^8 cells / mL) was plated into a 96-well plate at 37°C, followed by the addition of 100 μl of 10% cynomolgus serum from each time point listed above. The RbRBCs were gently mixed and incubated at 37°C for 60 minutes. After the incubation period, the cells were centrifuged at 1250xg at 4°C. A total of 100 μL of supernatant was transferred to a new 96-well flat-bottom plate and read at 412 nm in a Spectramax microplate reader. Hemolytic activity was calculated for a final serum concentration of 5% and expressed as a percentage of total hemolysis of RBCs by water. Percent hemolysis was calculated as above.

[0303] result Selected anti-C5 antibodies (listed in Table 1) were tested for extended pharmacokinetic profiles in cynomolgus monkeys and C5-humanized mice in initial experiments (described in Example 10). H4H12166P and H4H12161P were selected as having high affinity coupled with extended PK and were used along with Control Drug 1 and Control Drug 2 in subsequent experiments.

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

[0305] [Table 23]

[0306] After IV bolus administration, the total IgG concentration-time profiles of H4H12166P, H4H12161P, and Control 2 were characterized by an initial short-term distribution phase followed by a single elimination phase over the lifespan. Peak H4H12166P, H4H12161P, and Control 2 concentrations were very similar, with corresponding C values ​​between all antibodies. max The / dose values ​​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 profiles revealed that terminal antibody concentrations ≥ 10 μg / mL showed the slowest elimination through study day 71. The kinetics of H4H12161P and control drug 2 were similar; both showed faster elimination than H4H12166P through days 22 and 29, respectively, at mAb concentrations ≥ 10 μg / mL.

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

[0310] Calculated antibody half-life (t 1 / 2 ) ranged from 5.5 to 15.6 days across the dosing groups, and H4H12166P also had the highest corresponding t 1 / 2 15.6 days, while H4H12161P and Control 2 had a t of 5.5 days and 5.9 days, respectively. 1 / 2 values ​​and were therefore correlated with exposure.

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

[0312] [Table 25]

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

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

[0315] Example 11: PK / PD characterization of anti-C5 antibodies in C5-humanized mice In this set of experiments, the pharmacokinetics and pharmacodynamics of selected anti-C5 antibodies were evaluated using Velocigene (R) The efficacy of this method was evaluated in mice humanized to express the human C5 protein using techniques (Valenzuela et al. 2003, Nat. Biotechnol. 21:652-659). Humanized mice were engineered to replace exons 2 through 41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in U.S. Patent Application Publication No. 2015 / 0313194, incorporated herein in its entirety).

[0316] Total circulating human C5 levels were determined using a human complement C5 ELISA (Abcam, catalogue no. ab125963) performed according to the manufacturer's recommendations.

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

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

[0319] PD analysis by hemolysis assay The pharmacodynamics of selected anti-C5 antibodies were determined using a classical pathway complement hemolysis assay. Sheep red blood cells (SRBCs) (sheep blood in Orsever's solution) were washed with GVB++ buffer (gelatin veronal buffer containing CaCl2 and MgCl2) (Boston BioProducts) and resuspended at 1 x 10^9 cells / mL. To sensitize, 1 x 10^9 / mL SRBCs were mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg / mL) at 37°C for 20 minutes. Sensitized SRBCs were diluted to 2 x 10^8 cells / mL in GVB++ before the hemolysis assay. Serum samples from humanized C5 mice administered with anti-C5 antibodies, collected pre-dose or on days 10, 20, 30, 40, and 50 post-dose, were diluted to 20% with GVB++ buffer. A total of 100 μl of sensitized SRBC (2 x 10^8 cells / mL) was plated into 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 at 4°C. A total of 100 μl of supernatant was collected. 100 μL was transferred to a new 96-well flat-bottom plate and read on a Spectramax microplate reader at A412 nm. Percent hemolysis was calculated using the absorbance values ​​by using the following formula:

number

[0320] In this equation, "background cell lysis" is the OD at A412nm from SRBCs incubated with serum-free GVB++ buffer alone. "Maximum cell lysis" is the OD at A412nm from SRBCs treated with water. Results, expressed as % hemolysis, were analyzed using nonlinear regression (four-parameter logistics) with Prism 6 software (GraphPad) to determine IC 50 Values ​​were obtained. Data are expressed as the mean ± standard error of the mean.

[0321] Experiment 1 In this experiment, the pharmacokinetics and pharmacodynamics of the exemplary antibody H4H12166P were evaluated in comparison to Control 1 and Control 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 their human C5 levels, which averaged 40 μg / mL. For each anti-C5 antibody, cohorts of 22 mice were administered a single 15 mg / kg dose of H4H12166P, Control 1, or Control 2 via subcutaneous (sc) injection. All mice were bled pre-dose and 1 day post-injection for PK analysis. Additionally, at 10, 20, 30, 40, and 50 days post-injection, groups of 4 or 5 mice from each cohort were euthanized, and terminal blood was collected for PK and PD analysis. Day 1 serum samples were averaged across the entire 22-mouse cohort. Blood was processed to serum and frozen at -80°C until analysis.

[0323] Total antibody concentrations were determined at seven time points and percent hemolytic activity was determined at six time points over the 50-day survival period. Total anti-C5 antibody concentrations are summarized in Table 26. The mean total antibody concentration versus time profile is shown in Figure 4. Mean PK parameters are listed in Table 27.

[0324] [Table 26]

[0325] [Table 27]

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

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

[0328] [Table 28]

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

[0330] Experiment 2 In this study, the pharmacokinetics and pharmacodynamics of anti-C5 antibodies H4H12166P, H4H12161P, Control Drug 1, and an 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 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 their human C5 levels, which averaged 50 μg / mL. Cohorts of five mice for each anti-C5 mAb were administered a single 15 mg / kg subcutaneous (sc) injection of H4H12166P, H4H12161P, Control 1, or isotype control. All mice were bled for PK analysis at pre-dose, 6 hours, and 1, 2, 3, 4, 7, 10, 13, 21, 30, and 45 days post-injection. 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 analysis.

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

[0333] [Table 29]

[0334] [Table 30]

[0335] The mean concentration versus time profiles for H4H12166P, H4H12161P, Control 1, and the isotype control each had similar C values ​​within 1.3-fold. max The maximum serum concentrations (C max H4H12166P and the isotype control had similar elimination profiles, with residual drug levels of approximately 4 μg / mL at day 59. H4H12161P showed faster clearance than H4H12166P and the isotype control, but was eliminated more slowly than Control 1. At day 59, H4H12161P had a mean serum drug level of 0.6 μg / mL, while Control 1 had a mean serum drug level of approximately 0.08 μg / mL. There were undetectable drug levels.

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

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

[0338] Serum concentrations of total human C5 were determined by measuring the concentration of the 10-amino acid peptide LQGTLPVEAR (amino acids (aa) 1129-1138 of SEQ ID NO: 359), which is 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 C5b is bound to cell surfaces in the form of MAC complexes (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 likely contain negligible amounts, if any, of C5b products.

[0339] method For PK / PD studies, mice were administered a single 15 mg / kg dose of H4H12166P via subcutaneous (sc) injection. All mice were bled pre-dose and on day 1 post-injection for PK analysis. Additionally, at 10, 20, 30, 40, 50, and 60 days post-injection, mice were euthanized and terminal blood was collected for PK and PD analysis.

[0340] Human C5 was used as the reference standard for calibration; and a prepared human C5 peptide with a C-terminal stable isotope-labeled arginine residue was used as the internal standard (LQGTLPVEAR- 13 C6 15N4). Reference standards were used in serum from in-house generated C5 knockout mice in which the mouse C5 gene had been deleted (C5- / -), at concentrations ranging from 3.9 to 250 μg / mL (1:2 serial dilutions). 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 in 100 μL of 8 M urea / 20 mM Tris(2-carboxyethyl)phosphine (TCEP) buffer at 37°C for 1 hour. Next, 10 μL of 25 nM internal standard was added to all samples. Samples were alkylated with 10 mM 2-iodoacetamide for 30 minutes at room temperature and diluted to a final volume of 500 μL using 50 mM ammonium bicarbonate. Samples were then digested with trypsin (1:20 mass / mass) overnight at 37°C. 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 [vol:vol] and mobile phase B: acetonitrile:formic acid / 100:0.1 [vol:vol]). Retention times and peak areas were determined using Masslynx Analyst data software (Waters). The C5 reference standard (unlabeled C5 peptide LQGTLPVEAR- generated by tryptic digestion of hC5) was compared with the internal standard (stable isotope-labeled C5 peptide). 12 C6 14 The concentrations of C5 analytes were calculated from a calibration curve constructed by plotting the peak area ratios of N4 against the nominal concentration of the C5 reference standard. 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] result Serum total human C5 concentrations were assessed for samples collected from matched animals via tail bleeds prior to dosing (pre-dose) and via terminal bleeds on days 10, 30, and 35. Total hC5 concentrations after H4H12166P dosing at 10, 30, and 35 days post-dose were similar (within approximately 1- to 0.9-fold) to pre-dose levels. The small differences observed were analyzed using GraphPad Statistical evaluation was performed using the Mann-Whitney test using Prism software. Analysis of the C5 / H4H12166P molar ratio demonstrated that H4H12166P remained in molar excess over C5 up to 35 days post-dosing (Table 31).

[0342] [Table 31]

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

[0344] HDX-MS experiments were 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 gradients, and a Synapt G2-Si mass spectrometer for digested peptide mass measurement. It was.

[0345] For deuterium labeling, 3.8 μL of C5 (6 pmol / μL) or C5 premixed with antibody at a 1:1 molar ratio was incubated with 56.2 μL of DO labeling solution for various time points (e.g., non-deuterated control = 0 s, 1 min, and 20 min of labeling). For deuterium labeling, 50 μL of sample was added to 50 μL of pre-chilled quench buffer (0.2 M PBS). The peptides were quenched by transferring TCEP (6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5), and the combined sample was incubated at 1.0 °C for 2 min. The quenched sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were captured on an ACQUITY UPLC BEH C18 1.7-μm, 2.1 × 5 mm VanGuard precolumn at 0 °C and eluted on an ACQUITY UPLC BEH C18 1.7-μm, 1.0 × 50 mm analytical column with a 9-min gradient from 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set to a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50–1700 Thomson units (Th).

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

[0347] MSE 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 62% sequence coverage. Five peptides had significantly reduced deuterium uptake when bound to H4H12166P (centroid delta values ​​>0.9 Daltons, p-values ​​<0.05), as shown in Table 32.

[0348] [Table 32]

[0349] Recorded peptide masses are centroids MH from three replicates + These peptides, corresponding to amino acids 591-599 and 775-794, correspond to the average mass of H4H121. It had a slower deuteration rate upon binding to 66P. These identified residues also correspond 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 undertaken to evaluate the role of C5 in experimental autoimmune uveitis (EAU) using both genetic [C5 knockout (KO) and C3 / C5 double KO mice] and pharmacological (anti-C5 antibody) experimental approaches.

[0351] method Adult C57BL / 6J mice (n=25, Jackson labs), C5 KO (n=13), and C3 / C5 KO (n=8) mice (Regeneron Pharmaceuticals Inc.) were used. 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 NOs: 362 / 363. SPECTRALIS (R) HRA+OCT (Heidelberg Engineering, Inc.) was used to assess inflammation levels on days -1, 7, 14, 21, and 28. 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] result Compared with wild-type mice, inflammation incidence (30-50%) and the number of vitreous cell clusters were significantly reduced in C5 KO mice (p<0.01). Optical coherence tomography (OCT) scores in C5 KO mice were also significantly reduced by 50% at week 3 (p<0.0001). Interestingly, C3 / C5 double KO mice had significantly more vitreous cell clusters and higher disease scores at day 28 compared with wild-type mice (p<0.05). In animals administered anti-mC5 Ab (50 mg / kg), inflammation incidence and vitreous cell clusters were significantly lower at day 21 compared with either the untreated or isotype control groups (p<0.01). At weeks 3 and 4, OCT scores in the anti-C5 antibody-treated groups were significantly lower compared with the untreated or isotype control groups (p<0.0001). (FIG. 7) The inhibitory effect of anti-C5 antibody was confirmed at week 4 by hemolysis assay with or without human C3 (FIG. 8).

[0353] conclusion Ocular inflammation caused by EAU was attenuated by inhibiting C5 activity, either through genetic deficiency or pharmacological inhibition using a specific anti-C5 antibody. C5 deficiency delayed the development of EAU and reduced OCT disease scores. 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 C5 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 anti-C5 antibodies against human C5 in a mouse model of experimental autoimmune uveitis (EAU). Mice used in this study were from Velocigene. (R) Human C5 protein was expressed using the technique (Valenzuela et al. 2003, Nat. Biotechnol. 21:652-659). Humanized mice were engineered to replace exons 2 to 41 of the mouse C5 gene with exons 2 to 42 of the human C5 gene (as disclosed in U.S. Patent Application Publication No. 2015 / 0313194, which is incorporated herein in its entirety).

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

[0356] Ocular examinations 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). Pupils were dilated using tropicamide 0.5% eye drops, and the eyes were examined using a Spectralis Heidelberg Retinal Angiography Platform (HRA) + OCT system (Heidelberg Engineering, Carlsbad, CA, USA) with a contact lens equipped fundus camera.

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

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

[0359] A 4-point scale was developed for the assessment of disease severity on OCT images (OCT score) (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 using one-way ANOVA and Tukey's multiple comparison test. Nonparametric data (OCT scores and histology scores) were compared with the isotype control or untreated group using the Kruskal-Wallis test and Dunn's test using GraphPad Prism version 5.0d software. Data are shown as mean ± SEM. A p value of less than 0.05 was considered statistically significant.

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

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

[0364] In both studies, in-life evaluation by non-invasive OCT showed the development of progressive inflammation, increased retinal thickness and morphological abnormalities in control mice after immunization with IRBP.

[0365] conclusion These experiments provide further pharmacological evidence that C5 plays a role in the pathogenesis of autoimmune uveitis. Pharmacological depletion of fully human anti-human C5 antibodies delayed the development of EAU and reduced 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 antibodies) were used. The ischemia-reperfusion model was induced by bilateral renal pedicle clamping for 45 minutes, followed by 48 hours of reperfusion. Sham laparotomy served as a control. Anti-C5 antibodies were administered intravenously at 50 mg / kg as a single dose immediately after ischemia (therapeutic); or as two doses subcutaneously at the time of surgery on days -1 and 1 (prophylactic). The anti-C5 antibody used in this study was M1M17628N, which contains the HCVR / LCVR of SEQ ID NOs: 362 / 363. Blood urea nitrogen (BUN) and serum creatinine markers were used to assess the level of disease and protection in mice.

[0367] [Table 34]

[0368] [Table 35]

[0369] Compared to wild-type mice, C3 and C5 knockout mice showed significant functional protection in the RIRI model of acute kidney injury, as evidenced by reduced blood urea nitrogen and serum creatinine levels. Anti-C5 antibodies demonstrated functional protection in the RIRI model in both prophylactic and therapeutic ways (Tables 34-35).

[0370] Example 17: Effect of anti-C5 antibody on lupus nephritis This example describes the effectiveness of anti-C5 antibodies in treating lupus nephritis in a mouse model.

[0371] Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to autoantigens, production of autoantibodies, and deposition of complement-fixing 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 patients with lupus nephritis contributes to inflammation and tissue damage. The efficacy of anti-C5 antibody in treating 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, including autoantibodies against nuclear antigens and cell membrane proteins, hypergammaglobulinemia, albuminuria, and proteinuria, 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 with 30 mg / kg of an isotype control or an anti-C5 antibody twice a week for 8 weeks, followed by three times a week for 10 weeks. 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 antibodies significantly improved survival in mice (Figure 13). Both antibodies improved albuminuria between 8 and 14 weeks of treatment (Figure 14) and blood urea nitrogen between 12 and 16 weeks of treatment (Figure 15).

[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 primarily affects the optic nerves 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 postnatal rat pups and cultured with AQP4-Ab (antibody "rAb-53" from US Patent Application Publication No. 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, CytoTox-Glo TM A luminescent cytotoxicity assay was performed using various concentrations of anti-C5 antibody (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 an isotype control antibody.

[0377] To determine whether anti-C5 antibodies can block AQP4-Ab-induced CDC, astrocytes were plated and the optimal AQP4-Ab dose was found using CytoTox-Glo. TM The cytotoxicity assay was repeated. The optimal AQP4-Ab concentration was found to be 50 μg / mL, and in the following experiments, a fixed dose of AQP4-Ab (50 μg / mL) was used while varying the dose of anti-C5 antibody. As shown in Figure 16, a decrease in RLU was observed with increasing amounts of anti-C5 antibody (from an average of 300k to an average of 100k), demonstrating that anti-C5 antibody blocks astrocyte cell death. For both experiments, RLU was unchanged with the isotype control antibody. As shown in Figure 16, anti-C5 antibody inhibited AQP4 Ab-induced cytotoxicity with an IC50 of 15-17 nM in primary cortical astrocytes.

[0378] In subsequent studies, anti-AQP4 and anti-C5 antibodies will be infused into rat brains to assess their 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 antibodies block C5b-9 and C3 deposition.

[0380] Reproducible methods for assessing the inhibitory effects of drug candidates on complement activation are essential for all clinical development. Due to the complexity of the complement activation pathway, assays should use cells and endpoints appropriate for a given therapeutic indication. Here, we demonstrate the use of a complement C3 and C5 deposition model using an immortalized human glomerular endothelial cell line (HGEC) to assess the blocking activity of anti-C3 or C5 mAbs.

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

[0382] result C3 and C5b-9 deposition was 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 deposition was significantly reduced 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). Addition of blocking anti-C5 mAb significantly reduced C5b-9 deposition from normal human serum onto ADP-activated HGEC, and deposition was similar to that of C5-deficient serum (C5 mAb: 1.02x10 6 ±6.0x10 5 , control mAb 3.7x10 6 ±1.6x10 6 , P<0.05 vs. control mAb).

[0383] conclusion These data demonstrate the utility of in vitro human glomerular deposition to model complement C3 and C5 deposition. In addition to in vitro screening, this assay demonstrates its potential as a translational model for evaluating anti-complement strategies in renal disease using patient-derived serum samples.

[0384] The present invention is not to 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 accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. An 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 one or more amino acids contained within SEQ ID NO: 360 or SEQ ID NO: 361, as determined by hydrogen / deuterium exchange.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment interacts with one or more amino acids contained within the alpha and / or beta chains of C5 as determined by hydrogen / deuterium exchange.

3. 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment does not interact with amino acids in the C5a anaphylatoxin region of C5 as determined by hydrogen / deuterium exchange.

4. 4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or 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.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof interacts with at least five amino acids contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 360 and 361.

6. 6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within SEQ ID NO: 360 and SEQ ID NO: 361 as determined by hydrogen / deuterium exchange.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof interacts with the amino acid sequences of SEQ ID NOs: 360 and 361 as determined by hydrogen / deuterium exchange.

8. The antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVR: (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) the amino acid sequence of SEQ ID NO: 98 with five or fewer amino acid substitutions; Including, And LCVR: (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) the amino acid sequence of SEQ ID NO: 106 with 5 or fewer amino acid substitutions Including, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The antibody has the following characteristics: (a) having a serum concentration of greater than 10 μg / mL by Day 70 upon administration to cynomolgus monkeys; (b) blocks classical pathway (CP) hemolysis upon administration to cynomolgus monkeys for up to 35 days as measured in an ex vivo hemolysis assay; (c) blocks alternative pathway (AP) hemolysis upon administration to cynomolgus monkeys for up to 35 days as measured in an ex vivo hemolysis assay; (d) has a serum half-life in cynomolgus monkeys of greater than 10 days; (e) has a serum concentration greater than 10 μg / mL by day 40 upon administration to C5-humanized mice; (f) blocks CP hemolysis upon administration to C5-humanized mice for up to 30 days, as measured in an ex vivo hemolysis assay; and (g) C5-humanized mice have a serum half-life of more than 10 days; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, having one or more of:

10. The antibody: (a) is a fully human monoclonal antibody; (b) a dissociation constant (K) of less than 0.9 nM at 25°C as measured by surface plasmon resonance assay; Ｄ ) binds to human C5; (c) a K of less than 0.3 nM at 37°C as measured by surface plasmon resonance assay Ｄ binds to human C5 at (d) a K of less than 65 nM as measured by surface plasmon resonance assay Ｄ binds to monkey C5 at (e) a K of less than 0.5 nM as measured by surface plasmon resonance assay Ｄ binds to human C5 mutant R885H (SEQ ID NO: 356) at (f) a K of less than 0.5 nM as measured by surface plasmon resonance assay Ｄ binds to human C5 mutant R885C (SEQ ID NO: 357) at (g) an IC of less than 6 nM as measured by CP hemolysis assay ５０ blocks human C5-mediated classical pathway (CP) hemolysis by more than 95%; (h) an IC of less than 165 nM as measured by AP hemolysis assay ５０ Blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70%; (i) an IC of less than 185 nM as measured by CP hemolysis assay ５０ inhibits African green monkey C5-mediated CP hemolysis; (j) an IC of less than 235 nM as measured by AP hemolysis assay ５０ inhibits African green monkey C5-mediated AP hemolysis; (k) an IC of less than 145 nM as measured by CP hemolysis assay ５０ inhibits cynomolgus monkey C5-mediated CP hemolysis; and (l) IC of less than 30 nM as measured by AP hemolysis assay ５０ inhibits cynomolgus monkey C5-mediated AP hemolysis 10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, having a further feature selected from the group consisting of:

11. The antibody or antigen-binding fragment thereof of any one of claims 8 to 10, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 98 with five or fewer amino acid substitutions.

12. The antibody or antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 106 with five or fewer amino acid substitutions. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 11, comprising an LCVR comprising the amino acid sequence:

13. The antibody or antigen-binding fragment thereof of claim 11 or 12, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 98 with not more than five amino acid substitutions and an LCVR comprising the amino acid sequence of SEQ ID NO: 106 with not more than five amino acid substitutions.

14. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 100; HCDR2 comprising the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 102; HCDR3 comprising the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 104; LCDR1 comprising the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 108; LCDR2 comprising the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 110; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that differs by one amino acid from SEQ ID NO:

112.

15. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 14, comprising an HCVR having the amino acid sequence of SEQ ID NO:

98.

16. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 14, comprising an LCVR having the amino acid sequence of SEQ ID NO:

106.

17. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising the amino acid sequence of SEQ ID NOs: 100-102-104-108-110-112.

18. 18. The antibody or antigen-binding fragment thereof of claim 17, wherein the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 98 / 106.

19. 19. The antibody or antigen-binding fragment thereof of claim 18, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

353.

20. 19. The antibody or antigen-binding fragment thereof of claim 18, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

354.

21. 19. The antibody or antigen-binding fragment of claim 18, comprising the heavy chain / light chain amino acid sequence pair of SEQ ID NOs: 353 / 354.

22. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of claim 8 for binding to C5.

23. An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof of claim 8.

24. The following amino acid residues: N591, M592, A593, T594, G595, M596, D597, S598, W599, W775, E776, V777, H of SEQ ID NO: 359 778, L779, V780, P781, R782, R783, K784, Q785, L786, Q787, F788, A789, L790, P791, D792, S793, or L794.

25. 25. The antibody or antigen-binding fragment thereof of claim 24, comprising HCVR and LCVR, wherein HCVR has at least 90% sequence identity to SEQ ID NO:

98.

26. 26. The antibody or antigen-binding fragment thereof of claim 24 or 25, comprising HCVR and LCVR, wherein LCVR has at least 90% sequence identity to SEQ ID NO:

106.

27. The antibody or antigen-binding fragment thereof according to any one of claims 24 to 26, comprising HCVR / LCVR of SEQ ID NO: 98 / 106.

28. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to C5 according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier or diluent.

29. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of claims 1 to 27.

30. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody of any one of claims 1 to 27.

31. A vector comprising the polynucleotide sequence of claim 29 or 30.

32. A cell expressing the vector of claim 31.

33. 28. A method for preventing, treating, or ameliorating at least one symptom or sign of a disease or disorder associated with C5, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 27.

34. The disease or disorder may be 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 unwanted complement activation, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 therapy, inflammatory disorders, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, burns, including burns or frostbite, post-ischemic reperfusion states, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, 34. The method of claim 33, wherein the disease is selected from the group consisting of arteriosclerosis, vasculitis, bullous pemphigoid, C3 nephropathy, 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 surgery, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disease and autoimmune disease, nephropathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, and myasthenia gravis.

35. 34. The method of claim 33, wherein the disease or disorder is aHUS.

36. 34. The method of claim 33, wherein the disease or disorder is PNH.

37. The method of any one of claims 33 to 36, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof.

38. The method of any one of claims 33 to 37, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

39. 39. The method of claim 38, wherein the second therapeutic agent is selected from the group consisting of an anticoagulant, an anti-inflammatory, an antihypertensive, an immunosuppressant, a lipid-lowering agent, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an anticonvulsant, a C3 inhibitor, a second anti-C5 antibody, and an antithrombotic agent.

40. 40. The method of any one of claims 33 to 39, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

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