BR122022016112B1 - USES OF AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF WHICH SPECIFICALLY BINDS C5 - Google Patents

Abstract

The present invention provides monoclonal antibodies that bind to complement factor 5 (C5) protein, and methods of using them. In various embodiments of the invention, the antibodies are fully human antibodies that bind to the C5 protein. In some embodiments, antibodies of the invention are useful for inhibiting or neutralizing C5 activity, thereby providing a means of treating or preventing a C5-related disease or disorder in humans. In some embodiments, the invention provides an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, for example, a half-life of greater than 10 days.

Description

[001] This application is being filed on June 13, 2017 as a PCT International Patent Application and claims priority benefit for provisional applications Nos. 62/349,705, filed on June 14, 2016; 62/405,561, filed October 7, 2016; and 62/422,107, filed November 15, 2016, the descriptions of each incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] 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 of using these antibodies.

BACKGROUND OF THE INVENTION

[003] The complement system is a group of plasma proteins that, when activated, lead to target cell lysis and facilitate phagocytosis through opsonization. Complement is activated through a series of proteolytic steps by three main pathways: the classical pathway, which is typically activated by immune complexes, the alternative pathway that can be induced by unprotected cell surfaces, and the mannose-binding lectin pathway. All three pathways of the complement cascade converge in the proteolytic cleavage of complement component 5 (C5) protein. Cleavage of complement component 5 (C5) results in the production of C5a and C5b fragments, a critical process during activation of the complement cascade. C5a can generate pleiotropic physiological responses through binding to its receptors ( Monk et al., 2007, Br. J. Pharmacol. 152: 429-448 ). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates oxidative burst, and induces the release of various inflammatory mediators such as histamine or cytokines. C5b mediates the formation of the membrane attack complex (MAC or C5b-9) leading to cell lysis in the late stages of complement-dependent cytotoxicity (CDC). Furthermore, in nucleated cells resistant to cytolysis by C5b-9, sublytic amounts of C5b-9 can cause cell activation that results in cell proliferation, generation of pro-inflammatory mediators, and production of extracellular matrix.

[004] Monoclonal antibodies to C5 are known in the art and have been described, for example, in US Patent/Publication No. 9206251, 9107861, 9079949, 9051365, 8999340, 8883158, 8241628, 7999081, 7432356, 7361 339, 7279158, 6534058., 6355245, 6074642, 20160299305, 20160051673, 20160031975, 20150158936, 20140056888, 20130022615, 20120308559, and in WO2015198243 , WO2015134894, WO2015120130, EP2563813B1, EP2328616B1 and EP2061810B1.

[005] 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-associated diseases (eg, atypical hemolytic uremic syndrome).

BRIEF SUMMARY OF THE INVENTION

[006] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to complement factor 5 (C5) protein. Antibodies of the present invention are useful, inter alia, for inhibiting or neutralizing C5 protein activity. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom or indication of a C5-associated disease or disorder in an individual. In certain embodiments, the antibodies can be administered prophylactically or therapeutically to an individual with or at risk of having a C5-associated disease or disorder. In certain embodiments, anti-C5 antibodies are fully human antibodies that bind C5 with high affinity and have improved pharmacokinetic (PK) and pharmacodynamic (PD) properties. These PK/PD-enhanced high affinity antibodies can be used to provide superior efficacy along with less frequent dosing in an individual with a C5-associated disease or disorder.

[007] Antibodies of the invention may be full-length (e.g., an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and can be modified to affect functionality, for example to increase persistence in the host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925-1933). In certain embodiments, the antibodies can be bispecific.

[008] In a first aspect, the present invention provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the C5 protein. In some embodiments, the antibodies are fully human monoclonal antibodies.

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

[010] 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 a substantially similar sequence having at least 90 %, at least 95%, at least 98% or at least 99% sequence identity.

[011] 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 a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[012] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any one of the LCVR amino acid sequences listed in Table 1. In accordance with certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained in any one 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 present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, said HCVR comprising an amino acid sequence listed in Table 1 having no more than five amino acid substitutions, and the LCVR comprising an amino acid sequence listed in Table 1, having no more than two amino acid substitutions. For example, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, said HCVR comprising an amino acid sequence of SEQ ID NO: 98 having n more than five amino acid substitutions, and the said LCVR comprising an amino acid sequence of SEQ ID NO: 106 having no more than two amino acid substitutions. In another embodiment, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, said HCVR comprising an amino acid sequence of SEQ ID NO: 98 having at least one amino acid substitution, and the said LCVR comprising an amino acid sequence of SEQ ID NO: 106 having an amino acid substitution.

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

[014] The present invention also provides antibodies, or antigen-binding fragments 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 sequence substantially similar to the same containing at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[015] The present invention also provides antibodies, or antigen-binding fragments 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 sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

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

[017] The present invention also provides antibodies, or antigen-binding fragments 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 sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[018] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR3 light chain (LCDR3) comprising an amino acid sequence selected from 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.

[019] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any one of the LCDR3 amino acid sequences listed in Table 1. In accordance with certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained in any one of the anti-C5 antibodies examples are 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).

[020] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, mentioning HCVR comprising HCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, HCDR2 comprising an amino acid sequence that differs from an amino acid sequence listed in Table 1 by 1 amino acid, and HCDR3 comprising an amino acid sequence that differs from an amino acid sequence listed in Table 1 by 1 amino acid. In certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, LCDR2 comprising an amino acid sequence that differs from an amino acid sequence listed in Table 1 by 1 amino acid, and LCDR3 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid. For example, the present invention provides anti-C5 antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 100 or an amino acid sequence different from SEQ ID NO: 100 per 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO: 102 or an amino acid sequence other than SEQ ID NO: 102 per 1 amino acid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that differs from SEQ ID NO: 104 by 1 amino acid. In another exemplary embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 108 or an amino acid sequence other than SEQ ID NO: 108 by 1 amino acid, LCDR2 comprising an amino acid sequence of SEQ ID NO: 110 or an amino acid sequence other than SEQ ID NO: 110 by 1 amino acid, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 112 or a amino acid sequence that differs from SEQ ID NO: 112 by 1 amino acid.

[021] The present invention provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 353, or a substantially similar sequence thereof containing at least 80%, at least 85% , at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

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

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

[024] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any one of exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NOs: 52-54-5660-62-64 (for 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).

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

[026] 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 in a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR), which the HCVR comprises : (i) the amino acid sequence of SEQ ID NO: 98, (ii) an amino acid sequence with at least 90% identity with SEQ ID NO: 98, (iii) an amino acid sequence with at least 95% identity identity with SEQ ID NO: 98; or (iv) the amino acid sequence of SEQ ID NO: 98 having no more than 5 amino acid substitutions; and the LCVR comprises: (i) the amino acid sequence of SEQ ID NO: 106, (ii) an amino acid sequence with at least 90% identity to SEQ ID NO: 106, (iii) an amino acid sequence with at least less than 95% identity with SEQ ID NO: 106; or (iv) the amino acid sequence of SEQ ID NO: 106 having no more than 5 amino acid substitutions.

[027] The present invention includes anti-C5 antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present in the oligosaccharide chain, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see, Shield et al (2002) JBC 277: 26733 ). In other applications, modification of galactosylation can be done in order to modify complement-dependent cytotoxicity (CDC).

[028] 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 C5 with greater affinity at neutral pH than at acidic pH (i.e., reduced binding at acidic pH).

[029] In certain embodiments, the present invention provides antibodies and antigen-binding fragments that exhibit improved pharmacokinetic and pharmacodynamic properties, for example, the present invention provides anti-C5 antibodies that have extended serum half-lives. In certain embodiments, the anti-C5 antibodies of the present invention have a serum concentration of greater than 10 µg/ml by day 40 in C5 humanized mice. In certain embodiments, the anti-C5 antibodies of the present invention block CP and AP hemolysis by day 35 after administration to C5-humanized mice.

[030] 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 the LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[031] 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 , where the HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[032] 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 each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to the same epitope as an 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 NOs: 98/106.

[033] The present invention also includes anti-C5 antibodies and their antigen-binding fragments that bind to one or more amino acid residues comprised in the alpha chain and/or 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 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 C5 alpha and beta chains, where 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 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. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained in SEQ ID NO: 359, by 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 in SEQ ID NO: 361. In certain embodiments, the present invention provides anti-C5 antibodies and their antigen-binding fragments that interact with one or more amino acids contained in SEQ ID NO: 359, for example, the present invention provides anti-C5 antibodies and their antigen-binding fragments that interact with at least 5 amino acids contained in SEQ ID NO: 360. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids contained in 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 to 599 of SEQ ID NO: 359) and with the amino acid sequence of SEQ ID NO: 361 (corresponding to amino acids 775 to 794 of SEQ ID NO: 359).

[034] In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to C5 in an agonist manner, that is, it 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., it can block C5 binding and/or activity.

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

[036] In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first binding specificity for a first epitope of the C5 protein and a second binding specificity for a second epitope of the C5 protein, wherein the first and second epitopes are distinct and non-overlapping.

[037] In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to C5a with an IC50 of less than 0.5 nM. In certain embodiments, the antibodies comprise 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 antibodies comprise an LCVR comprising an amino acid sequence selected from from the group consisting of SEQ ID NOs: 298, 314, 330 and 346.

[038] In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that has one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25°C, as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37°C, as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM as measured in a surface plasmon resonance assay; (e) binds to the human C5 variant R885H (SEQ ID NO: 356) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (f) binds to the human C5 variant R885C (SEQ ID NO: 357) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (PC) hemolysis by greater than 95% and with an IC50 of less than 6 nM as measured in a CP hemolysis assay; (h) blocks human C5-mediated (AP) alternative pathway hemolysis by more than 70% and with an IC50 of less than 165 nM, as measured in an AP hemolysis assay; (i) inhibits African green monkey C5-mediated PC hemolysis with IC50 less than 185 nM as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with IC50 less than 235 nM as measured in a PA hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM as measured in a CP hemolysis assay; and (l) inhibits cynomolgus monkey C5-mediated PA hemolysis with an IC50 of less than 30 nM as measured in a PA hemolysis assay.

[039] In certain embodiments, the present invention provides an isolated recombinant anti-C5 monoclonal antibody or antigen-binding fragment thereof that has one or more of the following characteristics: (a) comprises a set of six CDRs comprising the amino acid sequences of SEQ ID NOs: 100102-104-108-110-112; (b) binds to human C5 with a dissociation constant (KD) of less than 0.2 nM at 25°C, as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37°C, as measured in a surface plasmon resonance assay; (d) binds to a human C5 variant (R885H) with a KD of less than 0.4 nM at 37°C, as measured in a surface plasmon resonance assay; (e) inhibits classical pathway (CP)-mediated hemolysis of human serum with an IC50 of less than 3 nM; (f) inhibits alternative pathway (AP)-mediated hemolysis of human serum with an IC50 of less than 27 nM; (g) inhibits CP-mediated hemolysis of monkey serum with an IC50 of less than 21 nM; (g) inhibits AP-mediated hemolysis of monkey serum with an IC50 of less than 10 nM; (h) has a serum half-life (t1/2) of greater than 10 days in C5 humanized mice; (i) has a serum concentration of more than 10 µg/mL by day 40 after administration to humanized C5 mice; (j) blocks CP-mediated hemolysis up to day 50 in C5 humanized mice; and (k) binds to one or more amino acids comprised in the alpha chain and/or the beta chain of SEQ ID NO: 359, wherein the antibody does not bind to the anaphylatoxin C5a domain of C5.

[040] 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.

[041] 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.

[042] 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.

[043] 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.

[044] 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 at least 98% or at least 99% sequence identity.

[045] 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.

[046] 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.

[047] 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.

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

[049] The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2- LCDR3 is as defined by any of the exemplary anti-C5 antibodies listed in Table 1.

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

[051] In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-C5 antibody. For example, the present invention includes recombinant expression vectors comprising any of the above mentioned nucleic acid molecules, i.e. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences as set out 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 of producing the antibodies or portions thereof by culturing the host cells under conditions that allow production of the antibodies or antibody fragments and recovery of the antibodies and antibody fragments. produced antibodies.

[052] In a third aspect, the 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 invention features a composition that is a combination of an anti-C5 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-C5 antibody. Exemplary agents that may be advantageously combined with an anti-C5 antibody include, without limitation, other agents that bind and/or inhibit C5 activity (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents which do not directly bind to C5 but nevertheless treat or ameliorate at least one symptom or indication of a disease or disorder associated with C5. Additional combination therapies and coformulations involving the anti-C5 antibodies of the present invention are described elsewhere herein.

[053] In a fourth aspect, the invention provides therapeutic methods for treating a C5-associated disease or disorder in an individual using an anti-C5 antibody or an antigen-binding portion of an antibody of the invention, wherein the methods Therapeutic treatments comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention to the subject in need thereof. The disorder treated is any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by inhibiting C5 activity. In certain embodiments, the invention provides methods for preventing, treating or ameliorating at least one symptom of atypical hemolytic uremic syndrome (aHUS), the method comprising administering a therapeutically effective amount of an anti-C5 antibody or antigen-binding fragment thereof of the invention to an individual in need thereof. In some embodiments, the present invention provides methods for ameliorating or reducing the severity of at least one symptom or indication of paroxysmal nocturnal hemoglobinuria (PNH) in a subject by administering an anti-C5 antibody of the invention. In some embodiments, the antibody or antigen-binding fragment thereof can be administered prophylactically or therapeutically to an individual with or at risk of having a C5-associated disease or disorder. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is administered in combination with a second therapeutic agent to the subject in need thereof. The second therapeutic agent may be selected from the group consisting of an anti-inflammatory drug (such as corticosteroids and non-steroidal anti-inflammatory drugs), an antibody other than C5 protein, a dietary supplement such as antioxidants, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent can be an agent that helps to neutralize or reduce any possible side effects associated with an antibody or antigen-binding fragment thereof of the invention, if such side effect(s) occur. (in). The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally or intramuscularly. The antibody or fragment thereof can be administered at a dose of from about 0.1 mg/kg body weight to about 100 mg/kg body weight of the subject. In certain embodiments, an antibody of the present invention can be administered in one or more doses comprising between 50 mg to 600 mg.

[054] The present 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 C5 binding and/or activity .

[055] Other embodiments will become apparent from a review of the detailed description that follows.

BRIEF DESCRIPTION OF THE FIGURES

[056] Figure 1 shows the inhibition of C5a levels by the anti-C5 antibody H4H12166P in a dose-dependent manner, as determined by ELISA (described in Example 9).

[057] Figure 2 shows the total serum concentration as a function of time after a single intravenous injection of 15 mg/kg of H4H12166P, H4H12161P or Comparator 2 in male cynomolgus monkeys (described in Example 10). Concentration-time profiles were plotted across the first dose result after dose below the limit of quantification (BLQ), if applicable, which was imputed as LLOQ/2. Each data point represents the mean (± SD) (n = 4 animals/group). Concentrations considered to be affected by ADA were excluded from 1 animal in the H4H12166P group and 1 animal in the H4H12161P group, starting on day 36 and day 29, respectively. LLOQ = Lower limit of quantification

[058] Figure 3 shows the percentage of hemolysis vs. time in Red Blood Cell (A) Classical Route (A) and Alternative Route (B) assays after a single intravenous injection of H4H12166P, H4H12161P or Comparator 2 in male cynomolgus monkeys. The % hemolysis, calculated as a ratio of experimental vs. maximum with % lysis base subtracted from both values, is related to the amount of C5 inhibited by the particular anti-C5 antibody present in the serum at a given time. Each data point represents the mean (± SD).

[059] Figure 4 shows total serum concentration versus time profiles of selected anti-C5 antibodies in C5 humanized mice (described in Example 11). Humanized C5 mice were given a single 15 mg/kg subcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each data point represents the mean s.e.p.m. (n = 4-5 each). Serum antibody concentrations were monitored 1, 10, 20, 30, and 40 days after injection using a sandwich ELISA.

[060] Figure 5 shows the percentage of hemolysis x time in a classical hemolysis assay of the ex vivo complement of selected anti-C5 antibodies in mice humanized for C5. Humanized C5 mice were given a single 15 mg/kg subcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each data point represents the mean s.e.p.m. (n = 45 each). Percentage of serum hemolysis was monitored pre-dose, 10, 20, 30, 40 and 50 days after injection. % hemolysis, calculated as a ratio of experimental vs maximal lysis with baseline % lysis subtracted from both values is related to the amount of C5 inhibited by the particular anti-C5 antibody present in the serum at a given time.

[061] Figure 6 shows total serum concentration versus time profiles of selected anti-C5 antibodies in C5 humanized mice (described in Example 11 here). Mice were given a subcutaneous dose of 15 mg/kg of H4H12166P, H4H12161P, Comparator 1, or IgG4P isotype control. Each data point represents the mean ± s.e.m. (n = 5 each). Serum antibody levels were monitored 6 hours, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45 and 59 days after injection using a sandwich ELISA.

[062] Figure 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) . **** p<0.0001, two-way ANOVA treatment with anti-C5 antibody at 50 mg/kg vs. no treatment or treatment with isotype control.

[063] Figure 8 shows the inhibition of hemolysis by the classical route by the 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).

[064] Figure 9 shows the cell cluster count in humanized C5 mice treated with isotype control or with 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

[065] Figure 10 is a graph showing the results of OCT in humanized C5 mice treated with isotype control or with 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

[066] Figure 11 is a graph showing OCT scores in humanized C5 mice treated with isotype control, anti-human C4 antibody H5H12166P at 3 mg/kg or 10 mg/kg, or Comparator 2 at 10 mg/kg. n = 6-12 eyes for each group (described in Example 15).

[067] Figure 12 shows the cell cluster count in humanized C5 mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg/kg or 10 mg/kg, or Comparator 2 at 10 mg/kg. n = 6-12 eyes for each group (described in Example 15).

[068] Figure 13 is a survival curve of NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described herein in Example 17).

[069] Figure 14 shows the levels of (A) urinary albumin and (B) urinary albumin normalized to urinary creatinine in NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17).

[070] Figure 15 shows blood urea nitrogen levels in NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17).

[071] Figure 16 is a graph showing the inhibition of antibody-dependent cytotoxicity of astrocytes by anti-C5 antibodies H4H12166P, H4H12170P, Comparator 1 and Comparator 2, as described in Example 18.

DETAILED DESCRIPTION

[072] Before the present methods are described, it should be understood that this invention is not limited to the particular methods and experimental conditions described, since such methods and conditions 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, as the scope of the present invention will be limited only by the appended claims.

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

Definitions

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

[075] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., molecules of antibody"), as well as their multimers (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (comprised of the CH1, CH2 and CH3 domains) Each light chain is composed of a light chain variable region ("LCVR or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be modified naturally or artificially. A consensus amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs.

[076] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature where one or two CDRs can be dispensed for binding. Padlan et al. (1995, FASEB J. 9: 133-139) analyzed the regions of contact between antibodies and their antigens, based on published crystal structures, and concluded that only about one-fifth to one-third of CDR residues contact in any way. indeed the antigen. Padlan also found many antibodies in which one or two CDRs lacked amino acids in contact with an antigen (see also Vajdos et al. 2002 J Mol Biol 320: 415-428).

[077] CDR residues that do not contact antigen can be identified based on previous studies (for example, residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs outside of Chothia CDRs, for molecular and/or empirical modeling. If a CDR or residue(s) thereof is omitted, it is usually replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for replacement within CDRs and amino acids to replace can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[078] The fully human anti-C5 monoclonal antibodies described herein may comprise 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 germline sequences correspondents. Such mutations can be easily determined by comparing the amino acid sequences described herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences described herein, wherein one or more amino acids within one or more framework regions and/or CDRs are mutated to the corresponding residues. of the germline sequence(s) from which the antibody was derived, or the corresponding residue(s) of another human germline sequence, or a conservative amino acid substitution of the residue(s) (s) of the corresponding germline (such sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antibodies and antigen-binding fragments that comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR residues within the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework residues and/or CDRs are mutated into the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from the which the antibody was originally derived). Furthermore, antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a given germline sequence while certain other residues that differ from the original germline sequence are retained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired properties, such as better binding specificity, higher binding affinity, improved or improved antagonistic or agonistic biological properties ( as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[079] 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 described herein having 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 less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences described herein.

[080] The term "human antibody", when used herein, 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., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g. in the CDRs and in particular CDR3. However, the term "human antibody", when used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated or generated in a human subject.

[081] The term "recombinant", when used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, for example, DNA splicing and transgene expression. The term refers to antibodies expressed in an NO:human mammal (including NO:human transgenic mammals, e.g., transgenic mice), or in a cell expression system (e.g., CHO cells), or isolated from a library of recombinant human combinatorial antibodies.

[082] The term "specifically binds", or "specifically binds to", or similar, means that an antibody, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions . Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 -8 M or less (eg, lower KD 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. As described herein, antibodies have been identified by surface plasmon resonance, for example, BIACORE™, which specifically bind to C5. Furthermore, multispecific antibodies that bind to one domain in C5 and one or more additional antigens or a bispecific that binds to two different regions of C5 are, however, considered "specifically binding" antibodies when used herein.

[083] The term "high affinity" antibody refers to those mAbs with a C5 binding affinity, expressed as KD, of at least 10 -8 M; preferably 10-9 M; more preferably 10-10 M, even more preferably 10-11 M, even more preferably 10-12 M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution affinity ELISA.

[084] By the term "deceleration rate", "Koff" or "kd" means an antibody that dissociates from C5, with a rate constant of 1 x 10-3 s-1 or less, preferably 1 x 10-4 s-1 or less, as determined by surface plasmon resonance, eg, BIACORE™.

[085] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody and the like, as used herein, include any naturally occurring polypeptide or glycoprotein obtained enzymatically, synthetically or genetically modified that specifically binds to an antigen to form a complex. The terms "antigen-binding fragment" of an antibody, or "antibody fragment" when used herein, refer to one or more fragments of an antibody that retain the ability to bind the C5 protein.

[086] In specific embodiments, antibody or antibody fragments of the invention can be conjugated to a moiety of such a linker or a therapeutic moiety ("immunoconjugate"), a second anti-C5 antibody, or any other therapeutic moiety useful for treating a disease or disorder associated with C5.

[087] An "isolated antibody", when used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds to C5, or an additional fragment, is substantially free of Abs that specifically bind to antigens other than C5.

[088] A "blocking antibody" or a "neutralizing antibody", as used herein (or an "antibody that neutralizes C5 activity" or "antagonistic antibody"), refers to an antibody whose binding to C5 results in the inhibition of at least one biological activity of C5. For example, an antibody of the invention can prevent or block complement-mediated hemolysis either classically or alternatively.

[089] The term "surface plasmon resonance", when used herein, refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

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

[091] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site on the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas of an antigen and can have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. Also refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. composed of non-linear 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 charge characteristics specific.

[092] The term "cross-competition", when used herein, means 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. The term also includes competition between two antibodies in both orientations, i.e. a first antibody that binds and blocks the binding of the second antibody and vice versa. In certain embodiments, the first antibody and the second antibody can bind the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that binding of one inhibits or blocks binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, by a real-time unlabeled biolayer interferometry assay. Cross-competition between two antibodies can be expressed as binding of the second antibody that is less than the background signal due to self-self binding (wherein the first and second antibodies are the same antibody). Cross-competition between 2 antibodies can be expressed, for example, as % binding of the second antibody that is less than background binding of the background self-substance (where the first and second antibodies are the same antibody).

[093] The term "substantial identity" or "substantially identical", when referring to a nucleic acid or fragment thereof, indicates that, when ideally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand) , there is nucleotide sequence identity to at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well known sequence identity algorithm , such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[094] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by GAP or BESTFIT programs using standard range weights, share at least 90% identity sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, 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). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that have 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: aspartate and glutamate, 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, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log probability matrix described in Gonnet et al. (1992) Science 256: 1443-45, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log probability matrix.

[095] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software combines similar sequences using measures of similarity attributed to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein. and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with predefined or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identity of regions of best overlap between query and query sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention against a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403410 and (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

[096] The expression "therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be determinable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[097] When used herein, the term "individual" refers to an animal, preferably a mammal, most preferably a human, in need of amelioration, prevention and/or treatment of a disease or disorder associated with C5 such as uremic syndrome atypical hemolytic (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH). The term includes humans who have or are at risk of having such a disease or disorder.

[098] When used herein, the terms "treat", "treating", or "treatment" refer to reducing or ameliorating the severity of at least one symptom or indication of a C5-associated disease or disorder due to the administration of an agent therapeutic, such as an antibody of the present invention, to an individual in need thereof. Terms include inhibition of disease progression or worsening of a symptom/indication. The terms also include positive disease prognosis, i.e., the individual may be free of disease or may have reduced disease upon administration of a therapeutic agent, such as an antibody of the present invention. The therapeutic agent can be administered at a therapeutic dose to the subject.

[099] The terms "prevent", "preventing" or "prevention" refer to inhibiting the manifestation of a C5-associated disease or disorder or any symptoms or indications of such disease or disorder on administration of an antibody of the present invention.

Fragments of Antigen Binding Antibodies

[0100] Unless specifically noted otherwise, the term "antibody" when used herein shall comprise antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "complete antibody molecules") as well as their antigen-binding fragments. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtained, synthesized, or genetically engineered polypeptide or glycoprotein that binds specifically to an antigen to form a complex. The term "antigen-binding fragment" of an antibody, or "antibody fragment" when used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to the C5 protein. An antibody fragment can include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a CDR-containing fragment, or an isolated CDR. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a specific antigen-binding molecule. Antigen-binding fragments of an antibody may be derived, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally ) constant domains. Such DNA is known and/or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids , etc.

[0101] 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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a FR3-CDR3-FR4 peptide restricted. Other engineered molecules such as 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 shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" when used herein.

[0102] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent or in-frame with one or more framework sequences. In antigen-binding fragments with a VH domain associated with a VL domain, the VH and VL domains may be situated relative to each other in any suitable arrangement. For example, the variable region can be dimeric and contain VH - VH, VH - VL, or VL - VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a VH or VL monomeric domain.

[0103] 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. Exemplary non-limiting, exemplary variable and constant domain configurations that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VLCL. In 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. A hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variables and/or constant domains in a single molecule polypeptide. Furthermore, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more more VH or VL monomeric domains (e.g., by disulfide bond(s)).

[0104] As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, where each variable domain is capable of specifically binding a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

Preparation of Human Antibodies

[0105] Methods for generating human antibodies in transgenic mice are known in the art. Any of these known methods can be used in the context of the present invention to produce human antibodies that specifically bind to the C5 protein.

[0106] An immunogen comprising any of the following can be used to generate antibodies to the C5 protein. In certain embodiments, antibodies of the invention are obtained from mice immunized with a full-length native C5 protein (see, for example, GenBank accession number NP_001726.2) (SEQ ID N 355), or with DNA encoding the protein or fragment thereof. Alternatively, the protein or fragment thereof can be produced using conventional and modified biochemical techniques and used as an immunogen. In certain embodiments of the invention, the immunogen is a fragment of the C5 protein ranging from about amino acid residues 19 - 1676 of SEQ ID NO: 355.

[0107] In some embodiments, the immunogen can be a recombinant C5 protein or fragment thereof expressed in E. coli or in any other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0108] Using VELOCIMMUNE® technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to C5 are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE technology involves generating a transgenic mouse with a genome comprising human heavy and light chain variable regions operatively linked to endogenous mouse constant region sites, such that the mouse produces an antibody comprising a human variable region and a human variable region. mouse constant in response to antigenic stimulation. The DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

[0109] Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest and lymphatic cells (such as B cells) are recovered from mice expressing antibodies. Lymph cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and these hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and ligated to desirable isotypic heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chemical antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0110] Initially, high affinity chimeric antibodies are isolated with a human variable region and a mouse constant region. As in the experimental section below, antibodies are characterized and screened for desirable characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, e.g., wild-type or modified IgG1 or IgG4. Although the constant region selected may vary according to the specific use, the characteristics of high affinity antigen binding and target specificity reside in the variable region.

Bioequivalents

[0111] The anti-C5 antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that vary from those of the disclosed antibodies, but which retain the ability to bind to the C5 protein. Such variant antibodies and antibody fragments comprise one or more amino acid additions, deletions or substitutions as compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the disclosed antibodies. Likewise, DNA sequences encoding antibodies of the present invention encompass sequences that comprise one or more nucleotide additions, deletions, or substitutions as compared to the disclosed sequence, but which encode an antibody or antibody fragment that is essentially bioequivalent to an antibody. antibody or antibody fragment of the invention.

[0112] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, dose single or multiple doses. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet they can be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, they are not essential for the obtaining effective body medications the above concentrations, for example, chronic use, and are considered clinically insignificant for the particular drug being studied.

[0113] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

[0114] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biologic without an expected increase in risk of adverse effects, including a clinically significant change in immunogenicity or decreased efficacy compared with continuing therapy without such a switch.

[0115] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent such mechanisms are known.

[0116] Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals, where the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictable from in vivo bioavailability data in humans; c) An in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0117] Bioequivalent variants of the antibodies of the invention can be constructed, for example, by making various residue or sequence substitutions or deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine residues not essential for biological activity can be eliminated or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds by renaturation. In other contexts, bioequivalent antibodies can include antibody variants comprising amino acid changes that modify the glycosylation characteristics of the antibodies, for example, mutations that eliminate or remove glycosylation.

Anti-C5 Antibodies Comprising Fc Variants

[0118] In accordance with certain embodiments of the present invention, there are provided anti-C5 antibodies comprising an Fc domain comprising one or more mutations that increase or decrease binding of the antibody to the FcRn receptor, for example, acidic pH compared to pH neutral. For example, the present invention includes anti-C5 antibodies comprising a mutation in the CH2 or CH3 region of the Fc domain, wherein the mutation(s) increase(s) the affinity of the Fc domain for FcRn in an acidic environment (e.g. example, in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, Are or T), and 256 (for example, Are/R/Q/E/D or T); or a modification at heading 428 and/or 433 (e.g. H/L/R/are/P/Q or K) and/or 434 (e.g. A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification comprises a modification of 428L (eg, M428L) and 434S (eg, N434S); a modification of 428L, 259I (eg V259I) and 308F (eg V308F); a 433K modification (eg H433K) and 434 (eg 434Y); a modification 252, 254 and 256 (for example, 252Y, 254T and 256E); a modification of 250Q and 428L (for example, T250Q and M428L); and a modification 307 and/or 308 (e.g., 308F or 308P). In yet another embodiment, the modification comprises a modification 265A (e.g., D265A) and/or a 297A (e.g., N297A).

[0119] For example, the present invention includes anti-C5 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.G., T250Q and M248L); 252Y, 254T and 256E (e.g. M252Y, S254T and T256E); 428L and 434S (e.g. M428L and N434S); 257I and 311I (e.g. P257I and Q311I); 257I and 434H (e.g. P257I and N434H); 376V and 434H (for example, D376V and N434H); 307A, 380A and 434A (e.g. T307A, E380A and N434A); and 433K and 434F (for example, H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains described herein are contemplated within the scope of the present invention.

[0120] The present invention also includes anti-C5 antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, antibodies of the invention may comprise a chimeric CH region comprising part or all of the CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of the CH3 domain derived from a human IgG1 molecule, IgG2 human or human IgG4. According to certain embodiments, antibodies of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge can comprise a "higher hinge" amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region, combined with sequence of "lower hinge" (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1 hinge region, a human IgG2 or a human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chemical CH region as described herein can, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Patent Application Publication 2014/0243504, the disclosure of which is incorporated herein by reference in its entirety).

Biological Characteristics of Antibodies

[0121] In general, the antibodies of the present invention function by binding to the C5 protein and preventing its cleavage to C5a and C5b. For example, the present invention includes antibodies and antigen-binding fragments of antibodies that bind the C5 protein (e.g., at 25°C or 37°C) with a KD of less than 9 nM as measured by surface plasmon resonance, for example, using the essay format as defined in Example 3 here. In certain embodiments, antibodies or antigen-binding fragments thereof bind C5 with a K D 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, for example, using the assay format as defined in Example 3 herein, or a substantially similar assay.

[0122] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human C5 protein with a dissociative half-life (t7) greater than about 2 minutes, as measured by surface plasmon resonance at 25 °C , for example, using an assay format as defined in Example 4, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind the C5 protein with a t7 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 by surface plasmon resonance at 25°C, for example, using an assay format such as defined in Example 3 (eg, mAb capture or antigen capture format), or a substantially similar assay.

[0123] The present invention also includes antibodies and antigen-binding fragments thereof that bind to human C5 protein with a dissociative half-life (t7) greater than about 1.5 minutes, as measured by surface plasmon resonance at 37 °C, for example, using an assay format as defined in Example 4, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind the C5 protein with a t14 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 by surface plasmon resonance at 37°C, for example, using an assay format such as defined in Example 3 (e.g., mAb capture or antigen capture format), or a substantially similar assay.

[0124] The present invention also includes antibodies and antigen-binding fragments of antibodies that bind monkey C5 protein (e.g., at 25°C or 37°C) with a KD of less than 120 nM as measured by plasmon resonance surface, for example, using the test format as defined in Example 3 here. In certain embodiments, antibodies or antigen-binding fragments thereof bind monkey C5 with a K D of less than about 120 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about to about 10 nM, less than about 5 nM to about 1 nM, less than about 500 pM, or less than 250 pM, as measured by surface plasmon resonance, for example, using the assay format as herein defined in Example 3, or a substantially similar assay.

[0125] The present invention also includes antibodies and antigen-binding fragments of antibodies that bind modified human C5 protein with alteration R885H (exemplified by SEQ ID NO: 356) with a KD of less than 70 nM measured by surface plasmon resonance, for example, using the essay format as defined in Example 3 here. C5 variants showed a poor response to anti-C5 antibodies previously described in the art (eg, Nishimura et al., 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, antibodies or antigen-binding fragments thereof bind modified human C5 with a K D 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 3nM, or less than 2 nM, as measured by surface plasmon resonance, for example, using the assay format as defined herein in Example 3, or a substantially similar assay.

[0126] The present invention also includes antibodies and antigen-binding fragments of antibodies that bind modified human C5 protein with alteration R885C (exemplified by SEQ ID NO: 357) with a KD of less than 160 nM as measured by surface plasmon resonance, for example, using the essay format as defined in Example 3 here. C5 variants showed a poor response to anti-C5 antibodies previously described in the art (eg, Nishimura et al., 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, antibodies or antigen-binding fragments thereof bind modified human C5 with a K D of less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 20 nM, less at about 10 nM, less than about 5 nM, or less than 2 nM, as measured by surface plasmon resonance, for example, using the assay format as defined herein in Example 3, or a substantially similar assay.

[0127] The present invention also includes antibodies and antigen-binding fragments of antibodies that inhibit complement-dependent cytotoxicity (CDC) with IC50 less than 10 nM as measured by a luminescence assay, for example, using the assay format as defined in Example 6. In certain embodiments, the antibodies or antigen-binding fragments thereof inhibit CDC with 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, for example, using the assay format defined herein in Example 6, or a substantially similar assay.

[0128] The present invention also includes antibodies and antigen-binding fragments that block C5-mediated classical pathway hemolysis (CP) by more than 94% and with an IC50 of less than 6nM as measured by a CP hemolysis assay , for example, using the essay format as defined in Example 8 here. In certain embodiments, antibodies or antigen-binding fragments thereof block CP hemolysis with IC50s of less than about 6nM, less than about 5nM, less than about 4nM, less than about 3nM, or less than about 2nM , as measured by the CP hemolysis assay, for example, using the assay format as defined in Example 8, or a substantially similar assay.

[0129] The present invention also includes antibodies and antigen-binding fragments that block human C5-mediated (AP) alternative pathway hemolysis by more than 70% and with an IC50 of less than 165 nM, as measured by a human C5 hemolysis assay. AP, for example, using the essay format as defined in Example 8 here. In certain embodiments, antibodies or antigen-binding fragments thereof block AP hemolysis with IC50s of less than about 160 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less at about 20 nM, as measured by the AP hemolysis assay, for example, using the assay format as defined in Example 8, or a substantially similar assay.

[0130] The present invention also includes antibodies and antigen-binding fragments that block African green monkey C5-mediated classical pathway hemolysis (CP) by more than 40% and with an IC50 of less than 185 nM as measured by a CP hemolysis assay, for example, using the assay format as defined in Example 8 herein. In certain embodiments, antibodies or antigen-binding fragments thereof block CP hemolysis with IC50s of less than about 180 nM, less than about 150 nM, less than about 100 nM, less than about 75 nM, or less at about 50 nM, as measured by the CP hemolysis assay, for example, using the assay format as defined in Example 8, or a substantially similar assay.

[0131] The present invention also includes antibodies and antigen-binding fragments that block African green monkey C5-mediated alternative pathway hemolysis with an IC50 less than 235 nM, as measured by an AP hemolysis assay, for example, using the essay format as defined in Example 8 here. In certain embodiments, antibodies or antigen-binding fragments block AP hemolysis with IC50s 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 50 nM. of 20 nM, as measured by the AP hemolysis assay, for example, using the assay format as defined in Example 8, or a substantially similar assay.

[0132] The present invention also includes antibodies and antigen-binding fragments that block greater than 90% of cynomolgus monkey C5-mediated classical pathway (CP) hemolysis with an IC50 of less than 145 nM as measured by a hemolysis assay by CP, for example, using the essay format as defined in Example 8 here. In certain embodiments, antibodies or antigen-binding fragments thereof block CP hemolysis with IC50s of less than about 140 nM, less than about 120 nM, less than about 100 nM, less than about 75 nM, or less than about 50 nM, as measured by the CP hemolysis assay, for example, using the assay format as defined in Example 8 herein or a substantially similar assay.

[0133] The present invention also includes antibodies and antigen-binding fragments that block cynomolgus monkey (AP) C5-mediated alternative pathway hemolysis with an IC50 of less than 30 nM, as measured by an AP hemolysis assay, by example, using the essay format as defined in Example 8 here. In certain embodiments, antibodies or antigen-binding fragments thereof block AP hemolysis with IC50s of less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less. at about 2 nM, as measured by the AP hemolysis assay, for example, using the assay format as defined in Example 8, or a substantially similar assay.

[0134] The present invention also includes antibodies and antigen-binding fragments that exhibit improved pharmacokinetic (PK) and pharmacodynamic (PD) properties compared to anti-C5 antibodies in the art. The anti-C5 antibodies of the present invention are less susceptible to target-mediated clearance after administration, as shown in Examples 9 and 10 of the present invention. In certain embodiments, the present invention includes anti-C5 antibodies and antigen-binding fragments thereof that show serum concentrations for prolonged periods, e.g., greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days. days, greater than 45 days, greater than 50 days, greater than 55 days, or greater than 60 days, as described in Examples 9 and 10 of the present invention. In certain embodiments, anti-C5 antibodies of the present invention show a prolonged serum half-life of greater than 10 days compared to anti-C5 antibodies in the art.

[0135] In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that have high affinity for human C5 (e.g., KD less than 0.3nM) and a lower clearance (e.g., half -extended serum life, improved pharmacodynamic activity over more days than previously known anti-C5 antibodies). Such antibodies of the present invention can be advantageously used with less frequent dosing in an individual with a C5-associated disease or disorder.

[0136] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25°C, as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37°C, as measured in a surface plasmon resonance assay; (d) has a serum concentration of greater than 10 µg/mL by day 70 after administration to the cynomolgus monkey; (e) blocks CP and AP hemolysis at day 35 after administration to the cynomolgus monkey, as measured in an ex vivo hemolysis assay; (f) has a serum half-life of more than 10 days in cynomolgus monkeys; (g) has a serum concentration of more than 10 µg/mL by day 40 after administration to humanized C5 mice; (h) blocks CP hemolysis up to day 30 after administration to C5 humanized mice, as measured in an ex vivo hemolysis assay; and (i) it has a serum half-life of more than 10 days in humanized C5 mice.

[0137] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the C5 protein, wherein the antibody or fragment thereof has one or more of the following characteristics: (a) an antibody fully human monoclonal; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25°C, as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37°C, as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM as measured in a surface plasmon resonance assay; (e) binds to the human C5 variant R885H (SEQ ID NO: 356) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (f) binds to the human C5 variant R885C (SEQ ID NO: 357) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (PC) hemolysis by greater than 95% and with an IC50 of less than 6 nM as measured in a CP hemolysis assay; (h) blocks human C5-mediated (AP) alternative pathway hemolysis by more than 70% and with an IC50 of less than 165 nM, as measured in an AP hemolysis assay; (i) inhibits African green monkey C5-mediated PC hemolysis with IC50 less than 185 nM as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with IC50 less than 235 nM as measured in a PA hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM as measured in a CP hemolysis assay; and (l) inhibits cynomolgus monkey C5-mediated PA hemolysis with an IC50 of less than 30 nM as measured in a PA hemolysis assay.

[0138] The antibodies of the present invention may possess one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the present invention will be apparent to a person of ordinary skill in the art from a review of the present disclosure including the Working Examples presented herein.

Epitope Mapping and Related Technologies

[0139] 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 the beta polypeptide. The epitope to which antibodies bind can consist of a single contiguous sequence of 3 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 in any of the above-mentioned domains of the C5 protein molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within either or both of the above-mentioned domains of the protein molecule (e.g., a conformational epitope).

[0140] Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scan mutational analysis, peptide transfer analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves labeling the protein of interest with deuterium, followed by binding of the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and the exchangeable protons within amino acids that are protected by the antibody complex undergo reverse hydrogen-to-hydrogen exchange at a slower rate than exchangeable protons within amino acids that do not. part of the interface. As a result, amino acids that are part of the protein/antibody interface can retain deuterium and therefore exhibit a relatively greater mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage analysis and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. chem. 73: 256A-265A.

[0141] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B cell epitopes can be formed from either contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0142] Modification-Assisted Profiling (MAP), also known as Antigen Structure-Based Antibody Profiling (ASAP), is a method that categorizes a large number of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see, US 2004/0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a single epitope that is distinctly different from or partially overlaps with the epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, so 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 the desired characteristics. MAP can be used to classify antibodies of the invention into groups of antibodies that bind to different epitopes.

[0143] In certain embodiments, anti-C5 antibodies or antigen-binding fragments thereof bind to an epitope within any one or more of the regions exemplified in the C5 protein, either in the natural form, as exemplified in SEQ ID NO : 355, or produced recombinantly, or a fragment thereof. In some embodiments, antibodies of the invention bind to a region comprising one or more amino acids selected from the group consisting of amino acid residues 19 - 1676 of the human C5 protein.

[0144] In certain embodiments, antibodies of the 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; or amino acid residues ranging from about position 751 to about position 1676 of SEQ ID NO: 355.

[0145] 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 chain of C5 (SEQ ID NO: 359). The epitope(s) may(s) consist of one or more contiguous sequences of 3 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 in the alpha and/or beta chain of C5. Alternatively, the epitope can consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within C5. As shown in Example 11, the C5 epitope with which the exemplary antibody of the invention H4H12166P interacts is defined by: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) which corresponds to amino acids 591 to 599 comprised in the chain beta of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775 to 794 comprised in the alpha chain of SEQ ID NO: 359. Accordingly, the present invention includes anti-C5 antibodies that interact with one or more further amino acids contained in the region consisting of (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360), which corresponds to amino acids 591 to 599 of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775 to 794 of SEQ ID NO: 359.

[0146] The present invention includes anti-C5 antibodies that bind the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in Table 1. Likewise, the present invention also includes anti-C5 antibodies that compete for binding to the C5 protein or 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 antibodies listed in Table 1.

[0147] One can readily determine whether an antibody binds the same epitope as, or competes for binding with, a reference anti-C5 antibody using routine methods known in the art. For example, to determine whether a test antibody binds the same epitope as a reference anti-C5 antibody of the invention, the reference antibody is allowed to bind a C5 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the C5 protein molecule is 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 the C5 protein after saturation binding with the reference anti-C5 antibody, then the test antibody may bind to the same epitope as the epitope bound by the antibody. reference anti-C5 of the invention.

[0148] To determine whether an antibody competes for binding with a reference anti-C5 antibody, the binding methodology described above is performed in two orientations: in a first orientation, the reference antibody is allowed to bind to a C5 protein under saturation conditions, followed by evaluation of test antibody binding to the C5 molecule. In a second orientation, the test antibody is allowed to bind to a C5 molecule under saturation conditions, followed by evaluation of reference antibody binding to the C5 molecule. If, in both orientations, only the first (saturating) antibody is able to bind to the C5 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to C5. As will be appreciated by one of ordinary skill in the art, an antibody which competes for binding to a reference antibody may not necessarily bind the identical epitope on the reference antibody, but may sterically block binding of the reference antibody by binding an overlay. or adjacent epitope.

[0149] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to antigen. That is, a one-, 1-, 5-, 10-, 20-, or 100-fold excess inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay ( see, for example, Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding to one antibody reduce or eliminate binding to the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding to one antibody reduce or eliminate binding to the other.

[0150] Additional routine experimentation (e.g., peptide mutation and binding analyses) can be performed to confirm whether the observed lack of test antibody binding is indeed due to binding to the same epitope as the reference antibody or whether steric blockade (or another phenomenon) is responsible for the observed lack of binding. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

Immunoconjugates

[0151] The invention encompasses a human anti-C5 monoclonal antibody conjugated with a therapeutic moiety ("immunoconjugate"), to treat a disease or disorder associated with C5 (eg, atypical hemolytic uremic syndrome). When used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein, or a therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule, provided it is capable of binding its target. Examples of immunoconjugates include antibody and antibody conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a different second antibody to the C5 protein. The type of therapeutic moiety that can be conjugated to the anti-C5 antibody will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05/103081.

Multispecific Antibodies

[0152] The antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244.

[0153] Any of the multispecific antigen-binding molecules of the invention, or variants thereof, may be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of experience ordinary in technique.

[0154] In some embodiments, C5-specific antibodies are generated in a bispecific format (a "bispecific") in which variable regions that bind to distinct domains of the C5 protein are linked to confer dual-domain specificity within a single binding molecule. Appropriately designed bispecifics can increase the overall inhibitory efficacy of the C5 protein by increasing specificity and binding avidity. Variable regions that have specificity for individual domains (e.g., segments of the N-terminal domain) or that can bind to different regions within a domain are paired in a structural framework that allows each region to simultaneously bind to separate epitopes or to different epitopes. regions within a domain. In an example for a bispecific, heavy chain (VH) variable regions from a linker with specificity for one domain are recombined with light chain variable regions (VL) from a series of linkers with specificity for a second domain to identify non-VL partners. cognates that can be paired with an original VH without interrupting the original specificity for that VH. In this way, a single VL segment (e.g. VL1) can be combined with two different VH domains (e.g. VH1 and VH2) to generate a bispecific composite of two linkage "subdivisions" (VH1-VL1 and VH2-VL1) . The use of a single VL segment reduces system complexity and therefore simplifies and increases efficiency in the cloning, expression and purification processes used to generate the bispecific (see, for example, USSN13/022759 and US2010/0331527).

[0155] Alternatively, antibodies that bind to more than one domain and a second target, such as, but not limited to, for example, a different second anti-C5 antibody, can be prepared in a bispecific format using techniques herein described, or other techniques known to those skilled in the art. Antibody variable regions that bind to distinct regions can be linked together with variable regions that bind to relevant sites, for example, in the extracellular domain of C5, to confer dual antigen specificity within a single binding molecule. Appropriately designed bispecifics of this nature serve a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity outside the extracellular domain and are paired in a structural framework that allows each variable region to bind separate antigens.

[0156] An exemplary bispecific antibody format that may be used in the context of the present invention involves the use of a first immunoglobulin (Ig) domain CH3 and a second Ig3 domain of CH3, wherein the first and second Ig3 domains of CH3 differ each other by at least one amino acid, and wherein 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. In one embodiment, the first CH3 Ig domain binds Protein A and the second CH3 Ig domain contains a mutation that reduces or eliminates Protein A binding such as a modification of H95R (by IMGT exon numbering; H435R by EU numbering ). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Additional modifications that can be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (for IMGT; D356E, L358M, N384S, K392N, V397M and V422I for EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I per EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies. Variations on the bispecific antibody format described above are contemplated within the scope of the present invention.

[0157] Other exemplary bispecific formats that may be used in the context of the present invention include, without limitation, for example, bispecific or scFv-based formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, knobs -into-holes, common light chain (eg, common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1/IgG2, dual-action Fab ( DAF) - bispecific formats of IgG and Mab2 (see, for example, Klein et al. 2012, mAbs 4: 6, 1-11 and the references cited therein, for a review of previous formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, in which unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with composition, valence, and geometry. defined. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

Therapeutic Administration and Formulations

[0158] The invention provides therapeutic compositions comprising the anti-C5 antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions according to the invention will be administered with suitable vehicles, excipients and other agents which are incorporated into formulations to provide improved delivery, delivery, tolerance and the like. A multitude of suitable formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions and water-in-oil, carbocera emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbocera. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0159] The dose of antibody may vary depending on the age and size of an individual to be administered, target disease, conditions, route of administration and the like. When an antibody of the present invention is used to treat a disease or disorder in an adult patient, or to prevent such a disease, it is advantageous to administer the antibody of the present invention usually at a single dose of from about 0.1 to about 100 mg/kg of body weight, more preferably about 5 to about 80, about 10 to about 70, or about 20 to about 50 mg/kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can be administered as 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 to about 400 mg. In certain embodiments, the initial dose can be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than the initial dose, where doses subsequent days are separated by at least 1 day to 3 days; at least one 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.

[0160] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see e.g. , Wu et al., 1987) J. Biol, Chem 262: 4429-4432 ). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be administered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249: 1527-1533).

[0161] The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation 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 and conjugated with antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995, each incorporated herein in its entirety.

[0162] In certain situations, the pharmaceutical composition can be released in a controlled release system. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in close proximity to the target of the composition, thereby requiring only a fraction of the systemic dose.

[0163] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or in an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (for example, ethanol) , a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adducted from hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

[0164] A pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a conventional needle and syringe. Furthermore, as far as subcutaneous delivery is concerned, a pen delivery device easily has applications in delivering a pharmaceutical composition of the present invention. Such a pen release device may be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can easily be discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen release device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In contrast, the disposable pen delivery device comes 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.

[0165] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are certainly not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ Pen, HUMALOG™ Pen, HUMALIN 70 /30™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™,PTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are certainly not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk) and the KWIKPEN™ ( Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, LP) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0166] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally from about 5 to about 500 mg per dosage form in a unit dose; especially in the injection form, it is preferred that the antibody is contained in about 5 to about 300 mg and in about 10 to about 300 mg for the other dosage forms.

Therapeutic Uses of Antibodies

[0167] The antibodies of the present invention are useful for treating and/or preventing a disease or disorder or condition associated with C5 and/or for ameliorating at least one symptom associated with such disease, disorder or condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention can be administered at a therapeutic dose to a patient having a disease or disorder or condition associated with C5.

[0168] In certain embodiments, antibodies of the present invention are useful in treating or preventing a symptom or indication of atypical hemolytic uremic syndrome (aHUS). Symptoms and indications of aHUS include, but are not limited to, platelet activation, hemolysis, systemic thrombotic microangiopathy (formation of blood clots in small blood vessels throughout the body) leading to stroke, heart attack, kidney failure and/or or death, end-stage renal disease, permanent kidney damage, abdominal pain, confusion, edema, fatigue, nausea/vomiting, diarrhea, and microangiopathic anemia.

[0169] In certain embodiments, antibodies of the present invention are useful in treating or preventing a symptom or indication of paroxysmal nocturnal hemoglobinuria (PNH). PNH symptoms and indications include, but are not limited to, red blood cell destruction, thrombosis (including deep vein thrombosis, pulmonary embolism), intravascular hemolytic anemia, red discoloration of the urine, symptoms of anemia such as tiredness, shortness of breath, and palpitations, abdominal pain and difficulty swallowing.

[0170] In certain embodiments, antibodies of the present invention are useful for treating or preventing at least one symptom or indication of a C5-associated disease or disorder selected from the group consisting of neurological disorders, kidney disorders, multiple sclerosis, stroke stroke, Guillain Barre 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 from autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal injury including burns or frostbite, reperfusion conditions following ischemia, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia , stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, balloon angioplasty, syndrome after pump on cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disorders and autoimmune diseases, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney diseases, renal ischemia-reperfusion injury, lupus nephritis, glomerulopathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis , membranoproliferative nephritis, hemolytic anemia, neuromyelitis optica, kidney transplantation, hereditary CD59 deficiency, psoriasis and myasthenia gravis. In certain other embodiments, antibodies of the present invention are useful for treating or preventing at least one symptom or indication of a C5-associated disease or disorder selected from the group consisting of lung disease and disorders such as dyspnoea, hemoptysis, ARDS, asthma , chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolisms and infarctions, pneumonia, fibrogenic dust diseases, injuries due to dust and inert minerals (eg silicon, coal dust, beryllium and asbestos), lung fibrosis, organic diseases from dust, chemical injury (due to irritating gases and chemicals, eg chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia and hydrochloric acid), smoke injury, thermal injury (e.g. burn, frostbite), asthma, allergy, bronchoconstriction, hypersensitivity pneumonitis, parasites, Goodpasture Syndrome, pulmonary vasculitis, hereditary angioedema, and inflammation associated with the immune complex.

[0171] In certain embodiments, antibodies of the invention are useful for treating subjects suffering from an ocular disease such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, ocular angiogenesis (ocular neovascularization that affects the choroid, cornea or retinal tissue), geographic atrophy (GA), uveitis and neuromyelitis optica. Antibodies of the present invention can be used to treat or ameliorate at least one symptom or indication of dry AMD or wet AMD. In some embodiments, antibodies of the invention are useful in preventing or reducing the rate of vision loss. In one embodiment, antibodies of the present invention are useful in reducing drusen in the eye of a subject with dry AMD. In one embodiment, antibodies of the present invention are useful in preventing or reducing vision loss in an individual with AMD.

[0172] One or more antibodies of the present invention can be administered to alleviate or prevent or lessen the severity of one or more of the symptoms or conditions/indications of the ocular disease or disorder. The antibodies can be used to improve or reduce the severity of at least one symptom including, but not limited to, vision loss, visual distortion, difficulty adapting to low light levels, skewed central vision, increased cloudiness of central/global vision , presence of drusen (tiny accumulations of extracellular material that accumulate in the retina), pigmentary changes, distorted vision in the form of metamorphopsia, in which a grid of straight lines appears wavy and parts of the grid may appear white, exudative changes (bleeding in eye, hard exudates, subretinal/sub-RPE/intraretinal), slow recovery of visual function after exposure to bright light (photosynthesis test), incipient and geographic atrophy, drastically decreasing visual acuity (two levels or more), e.g. 20 /20 to 20/80, changes in perimetry of preferred hyperacuity (for wet AMD), blurred vision, gradual loss of central vision (for those with non-exudative macular degeneration, rapid onset of vision loss ten caused by leaking and bleeding vessels abnormal blood cells in individuals with exudative macular degeneration, central scotomas (missing shadows or areas of vision), difficulty discerning colors, specifically dark from dark and light from dark, loss of contrast sensitivity, straight lines appearing curved on a grid of Amsler.

[0173] It is also contemplated here the prophylactic use of one or more antibodies of the present invention to individuals at risk of developing macular degeneration, such as individuals over 50 years of age, individuals with a family history of macular degeneration, smokers and individuals with obesity , high cholesterol, cardiovascular disease or unhealthy diet.

[0174] In another embodiment of the invention, the present antibodies are used for the preparation of a pharmaceutical composition or medicament for the treatment of patients suffering from a disease or disorder associated with C5. In another embodiment of the invention, the present antibodies are used as adjunctive therapy with any other agent or any other therapy known to those of ordinary skill in the art useful for treating or ameliorating a disease or disorder associated with C5.

Combination Therapies

[0175] Combination therapies can include an anti-C5 antibody of the invention and any additional therapeutic agent that can advantageously be combined with an antibody of the invention or a biologically active fragment of an antibody of the invention. Antibodies of the present invention can be synergistically combined with one or more drugs or therapy used to treat a C5-associated disease or disorder. In some embodiments, antibodies of the invention can be combined with a second therapeutic agent to ameliorate one or more symptoms of said disease.

[0176] Depending on the disease or disorder associated with C5, the antibodies of the present invention can be used in combination with one or more additional therapeutic agents including, but not limited to, an anticoagulant (e.g., warfarin, aspirin, heparin), phenindione , fondaparinux, idraparinux, and thrombin inhibitors such as argatroban, lepirudin, bivalirudin, or dabigatran) an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs), an antihypertensive (eg, an angiotensin-converting enzyme inhibitor) ), an immunosuppressive agent (e.g., vincristine, cyclosporine A, or methotrexate), a fibrinolytic agent (e.g., ancrode, ε-aminocaproic acid, a1-antiplasmin, prostacyclin, and defibrotide), a lipid-lowering agent such as an inhibitor of hydroxymethylglutaryl CoA reductase, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an anticonvulsant agent (eg, magnesium sulfate), a C3 inhibitor, or an antithrombotic agent.

[0177] In certain embodiments, the second therapeutic agent is another antibody to C5 protein. It is contemplated herein to use a combination ("cocktail") of antibodies with broad neutralizing or inhibitory activity against C5. In some embodiments, non-competing antibodies can be combined and administered to an individual in need thereof. In some embodiments, the antibodies comprising the combination bind distinct non-overlapping epitopes on the protein. Antibodies comprising the combination can block the binding of C5 to C5 convertase and/or can prevent/inhibit the cleavage of C5 into C5a and C5b. In certain embodiments, the second antibody may have a longer half-life in human serum.

[0178] When used herein, the term "in combination with" means that the additional therapeutically active component(s)(s) may(s) be administered before, concomitant(s), or after administration of the anti-C5 antibody of the present invention. The term "in combination with" also includes the sequential or concurrent administration of an anti-C5 antibody and a second therapeutic agent.

[0179] The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-C5 antibody of the present invention. For example, it can be considered that a first component is administered "before" a second component if the first component is administered one week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject following 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 later, 5 minutes later, 10 minutes later, 15 minutes later, 30 minutes later, 1 hour later, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with the administration of an anti-C5 antibody of the present invention. "Concurrent" administration, for purposes of the present invention, includes, for example, administering an anti-C5 antibody and an additional therapeutically active component to an individual in a single dosage form, or in separate dosage forms administered to the individual. within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form can be administered by the same route (eg, both the anti-C5 antibody and the additional therapeutically active component can be administered intravenously, etc.); alternatively, each dosage form can be administered via a different route (for example, the anti-C5 antibody can be administered intravenously and the additional therapeutically active component can be administered orally). In any event, administration of the components in a single dosage form from separate dosage forms via the same route, or from separate dosage forms via different routes, is considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-C5 antibody "prior to", "concurrent with" or "after" (as the terms are defined hereinabove) administration of an additional therapeutically active component is considered administration of an anti-C5 antibody. C5 antibody "in combination with" an additional therapeutically active component.

[0180] The present invention includes pharmaceutical compositions in which an anti-C5 antibody of the present invention is co-formulated with one or more of additional therapeutically active components, as described elsewhere herein.

Administration Regimes

[0181] According to certain embodiments, a single dose of an anti-C5 antibody of the invention (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a individual in need. In accordance with 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) can be administered to an individual over the course of a definite time course. Methods according to this aspect of the invention comprise sequentially administering multiple doses of an anti-C5 antibody of the invention to a subject. As used herein, "administrate sequentially" means that each dose of anti-C5 antibody is administered to the individual at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months ). The present invention includes methods comprising sequentially administering to the patient a single initial dose of an anti-C5 antibody, followed by one or more further doses of the anti-C5 antibody, and optionally followed by one or more tertiary doses of the anti-C5 antibody. .

[0182] The terms "initial dose", "secondary doses" and "tertiary doses" refer to the temporal sequence of administration of the anti-C5 antibody of the invention. Thus, the "initial dose" is the dose that is administered at the start of the treatment regimen (also referred to as the "baseline dose"); "secondary doses" are doses given after the initial dose; and the "tertiary doses" are the doses given after the secondary doses. Initial, secondary, and tertiary doses may all contain the same amount of anti-C5 antibody, but they may generally differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-C5 antibody contained in the initial, secondary, and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[0183] In certain exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered from 1 to 48 hours (e.g., 1.1%, 2.2%, 3.3%, 4.4%, 5 , 5%, 6.6%, 7.7%, 8.8%, 9.9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15 , 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25 , 25%, 26, 26%, or more) after the immediately preceding dose. The phrase "the immediately preceding dose", when used herein, means, in a multiple administration sequence, the dose of anti-C5 antibody that is administered to a patient prior to the administration of the next dose in the sequence without intervening doses.

[0184] Methods according to this aspect of the invention may comprise administering any number of minor and/or tertiary doses of an anti-C5 antibody to a patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more secondary doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) are administered to the patient. Also, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more tertiary (e.g., 2, 3, 4, 5, 6, 7, 8 or more) doses are administered to the patient.

[0185] In certain embodiments of the invention, the frequency with which minor and/or tertiary doses are administered to a patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the individual patient's needs after the clinical examination.

Diagnostic Uses of Antibodies

[0186] The anti-C5 antibodies of the present invention can be used to detect and/or measure C5 in a sample, for example, for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present invention in assays to detect a C5-associated disease or disorder. Exemplary diagnostic assays for C5 may comprise, for example, contacting a sample, obtained from a patient, with an anti-C5 antibody of the invention, wherein the anti-C5 antibody is labeled with a detectable marker or reporter molecule or used as a capture ligand to selectively isolate C5 from patient samples. Alternatively, an unlabeled anti-C5 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable marker or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I; a fluorescent or chemiluminescent moiety such as rhodamine or fluorescein isothiocyanate; 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).

[0187] Samples that can be used in C5 diagnostic assays according to the present invention include any sample of tissue or fluid obtainable from a patient that contains detectable amounts of C5 protein, or fragments thereof, under normal freezing or pathological conditions. Generally, C5 protein levels in a particular sample obtained from a healthy patient (for example, a patient not afflicted with a C5-associated disease) will be measured to initially establish a baseline or standard level of C5. This baseline C5 level can then be compared to C5 levels measured in samples obtained from individuals suspected of having a C5-associated condition, or symptoms associated with that condition.

[0188] Antibodies specific for the C5 protein may not contain additional tags or moieties, or may contain an N-terminal or C-terminal tag or portion. In one embodiment, the marker or moiety is biotin. In a binding assay, the location of a marker (if any) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

Selected Modalities

[0189] Selected embodiments of this description include the following:

[0190] 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 a or more amino acids contained within C5 (SEQ ID NO: 359), as determined by hydrogen/deuterium exchange.

[0191] In Embodiment 2, the present invention includes the antibody isolated from the antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained in the alpha chain and/or in the alpha chain. C5 beta as determined by hydrogen/deuterium exchange.

[0192] In Embodiment 3, the present invention includes the antibody isolated from the antigen-binding fragment of Embodiments 1 or 2, wherein the antibody or antigen-binding fragment thereof does not interact with an amino acid from the C5 anaphylatoxin region of C5, as determined by hydrogen/deuterium exchange.

[0193] In Embodiment 4, the present invention includes the antibody isolated from 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 in SEQ ID NO: 360 and/or SEQ ID NO: 361, as determined by hydrogen/deuterium exchange.

[0194] In Embodiment 5, the present invention includes the isolated antibody or antigen-binding fragment of any 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.

[0195] In Embodiment 6, the present invention includes the isolated antibody or antigen-binding fragment thereof of any of Embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with at least five amino acids contained in a amino acid sequence selected from the group consisting of SEQ ID NOs: 360 and 361.

[0196] In Embodiment 7, the present invention includes the isolated antibody or antigen-binding fragment thereof of any 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.

[0197] 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 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.

[0198] In Embodiment 9, the present invention includes the isolated antibody or antigen-binding fragment thereof of any of Embodiments 1 to 8, wherein the antibody has one or more of the following characteristics: (a) has a serum concentration of more 10 µg/mL until day 70 after administration to the cynomolgus monkey; (b) blocks classical hemolysis (PC) up to day 35 after administration to the cynomolgus monkey, as measured in an ex vivo hemolysis assay; (c) blocks alternative route (AP) hemolysis up to day 35 after administration to the cynomolgus monkey, as measured in an ex vivo hemolysis assay; (d) it has a serum half-life of more than 10 days in the cynomolgus monkey; (e) has a serum concentration of more than 10 µg/mL by day 40 after administration to humanized C5 mice; (f) blocks CP hemolysis up to day 30 after administration to C5 humanized mice, as measured in an ex vivo hemolysis assay; and (g) it has a serum half-life of more than 10 days in humanized C5 mice.

[0199] In embodiment 10, the present invention includes the isolated antibody or antigen-binding fragment thereof of any of embodiments 1 to 9, wherein the antibody has an additional feature selected from the group consisting of: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25°C, as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37°C, as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM as measured in a surface plasmon resonance assay; (e) binds to the human C5 variant R885H (SEQ ID NO: 356) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (f) binds to the human C5 variant R885C (SEQ ID NO: 357) with a K D of less than 0.5 nM as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (PC) hemolysis by greater than 95% and with an IC50 of less than 6 nM as measured in a CP hemolysis assay; (h) blocks human C5-mediated (AP) alternative pathway hemolysis by more than 70% and with an IC50 of less than 165 nM, as measured in an AP hemolysis assay; (i) inhibits African green monkey C5-mediated PC hemolysis with IC50 less than 185 nM as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated after hemolysis with an IC50 of less than 235 nM as measured in a PA hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM as measured in a CP hemolysis assay; and (l) inhibits cynomolgus monkey C5-mediated PA hemolysis with an IC50 of less than 30 nM as measured in a PA hemolysis assay.

[0200] In Embodiment 11, the present invention includes the 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 in any of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the light chain variable region (LCVR) sequences listed in Table 1.

[0201] In Embodiment Form 12, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 11, comprising: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of 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 HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 126, 142, 150, 158, 174, 190, 206 , 222, 238, 254, 270, 278, 294, 310, 326 and 342; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 128, 144, 152, 160, 176, 192, 208 , 224, 240, 256, 272, 280, 296, 312, 328 and 344; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332 and 348; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 118, 134, 166, 182, 198, 214, 230 , 246, 262, 286, 302, 318, 334 and 350; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 120, 136, 168, 184, 200, 216, 232, 248, 264, 288, 304, 320, 336 and 352.

[0202] In Embodiment 13, the present invention includes the 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 HCVR sequences listed in Table 1 .

[0203] In Embodiment 14, the present invention includes an isolated antibody or antigen-binding fragment thereof of Embodiment 13 comprising an LCVR having an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.

[0204] In Embodiment 15, the present invention includes the isolated antibody or antigen-binding fragment of any of Embodiments 11 to 14 comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/ 10, 18/26, 34/42, 50/58, 66/74, 82/9 0, 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.

[0205] In embodiment 16, the present invention includes the isolated antibody or antigen-binding fragment thereof of any of embodiments 11 to 15 comprising three CDRs contained in 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.

[0206] In Embodiment 17, the present 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.

[0207] In Embodiment 18, the present invention includes the isolated antibody or antigen-binding fragment thereof of Embodiment 17 comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50/58, 98 /106, 138/106 and 202/210.

[0208] In embodiment 19, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding to C5 with the antibody or antigen-binding fragment thereof of embodiment 17.

[0209] In embodiment 20, the present invention includes an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof of embodiment 17.

[0210] In embodiment 21, the present invention includes 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 having no more than 5 amino acid substitutions .

[0211] In embodiment 22, the present invention includes 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 having no more than 5 amino acid substitutions.

[0212] In Embodiment 23, the present invention includes the antibody or antigen-binding fragment thereof of Embodiment 9 or 10 comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 98.

[0213] In Embodiment 24, the present invention includes an antibody or antigen-binding fragment thereof of Embodiment 23 comprising a light chain variable region with at least 90% sequence identity to SEQ ID NO: 106.

[0214] In embodiment 25, the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that blocks cleavage from C5 to C5a and C5b comprising three CDRs of an HCVR, wherein the HCVR has an amino acid sequence selected from 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.

[0215] 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.

[0216] In embodiment 27, the present invention includes an isolated polynucleotide molecule comprising a polynucleotide sequence encoding an HCVR of an antibody, as set forth in any one of embodiments 1 to 25.

[0217] In Embodiment 28, the present invention includes an isolated polynucleotide molecule comprising a polynucleotide sequence encoding an LCVR of an antibody as set forth in any one of Embodiments 1 to 25.

[0218] In Embodiment 29, the present invention includes a vector comprising the polynucleotide sequence of Embodiment 27 or 28.

[0219] In Embodiment 30, the present invention includes a cell that expresses the Embodiment 29 vector.

[0220] In Embodiment 31, the present invention includes a method of preventing, treating, or ameliorating at least one symptom or indication of a disease or disorder associated with C5, the method comprising administering an antibody or antigen-binding fragment of any one of modalities 1 to 25 to an individual in need thereof.

[0221] In embodiment 32, the present invention includes the method of embodiment 31, wherein the disease or disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), related macular degeneration age, geographic atrophy, uveitis, neuromyelitis optica, multiple sclerosis, stroke, Guillain Barré syndrome, traumatic brain injury, Parkinson's disease, disorders of inappropriate or undesirable complement activation, hemodialysis complications, hyperacute allogeneic rejection, xenograft rejection , interleukin-2-induced toxicity during IL-IL. 2 therapy, inflammatory disorders, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal injury including burns or burns, reperfusion conditions after ischemic stroke, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, gl C3 omerulopathy, membramproliferative glomerulonephritis, diabetic nephropathy, Alport's syndrome, progressive renal failure, proteinuric renal diseases, renal ischemia-reperfusion injury, lupus nephritis, angioplasty balloon pump syndrome, post-pump syndrome on cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disorders and autoimmune diseases, renal disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE ), SLE nephritis, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and pulmonary infarcts, pneumonia and myasthenia gravis.

[0222] In embodiment 33, the present invention includes the method of embodiment 31, wherein the disease or disorder is aHUS.

[0223] In embodiment 34, the present invention includes the method of embodiment 31, wherein the disease or disorder is PNH.

[0224] In embodiment 35, the present invention includes the method of any one of embodiments 31 to 34, wherein the pharmaceutical composition is administered prophylactically or therapeutically to the subject in need thereof.

[0225] In embodiment 36, the present invention includes the method of any one of embodiments 31 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0226] In embodiment 37, the present invention includes the method of embodiment 36, in which the second therapeutic agent is selected from the group consisting of an anticoagulant, an anti-inflammatory drug, an antihypertensive, an immunosuppressive agent, a lipid-lowering agent, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an anticonvulsant agent, a C3 inhibitor, a second anti-C5 antibody, and an antithrombotic agent.

[0227] In embodiment 38, the present invention includes the method of any one of embodiments 31 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.

EXAMPLES

[0228] The following examples are presented in order to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the 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 regard to the numbers used (eg, amounts, temperature, etc.), however, some experimental errors and biases must be considered. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, ambient temperature is about 25°C, and pressure is close to atmospheric.

Example 1: Generation of Human Antibodies to Complement Factor (C5) protein

[0229] Human antibodies to the C5 protein were generated in a mouse from VELOCIMMUNE® comprising DNA encoding heavy and light chain variable regions of human Immunoglobulin coat. Mice were immunized with purified human C5 protein in serum (Calbiochem Cat # 20-4888).

[0230] The antibody immune response was monitored by a C5-specific immunoassay. When a desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were screened and screened to identify cell lines that produce C5-specific antibodies. Cell lines were used to obtain various chimeric anti-C5 antibodies (ie, antibodies having human variable domains and mouse constant domains); Exemplary antibodies generated in this manner have been designated as H2M11683N and H2M11686N.

[0231] Anti-C5 antibodies were also isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Pat. 7582298, incorporated herein by reference in its entirety. Using this method, several fully human anti-C5 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; Exemplary antibodies generated in this manner have been designated as H4H12159P, H4H12161P, H4H12163P, H4H12164P, H4H12166P, H4H12167P, H4H12168P, H4H12169P, H4H12170P, H4H12171P, H4H12175P, H 4H12176P2, H4H12177P2 and H4H12183P2.

[0232] The biological properties of exemplary antibodies generated according to the methods of this Example are described in detail in the Examples presented below.

Example 2: Heavy and Light Chain Variable Region Nucleotide and Amino Acid Sequences

[0233] Table 1 presents the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-C5 antibodies of the invention. Table 1: Amino Acid Sequence Identifiers

[0234] The corresponding nucleic acid sequence identifiers are shown in Table 2. Table 2: Nucleic Acid Sequence Identifiers

[0235] Antibodies are typically referred to herein according to the following nomenclature: 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, in accordance with this nomenclature, an antibody may be referred to herein as, for example, "H2M11686N," "H4H12183P2," "H4H12168P," etc. The prefixes H4H and H2M on antibody designations used herein indicate the particular Fc region isotype of the antibody. For example, an "H4H" antibody has a human IgG4 Fc comprising a serine to proline mutation in the hinge region (S108P) to promote dimer stabilization, and an "H2M" antibody has a mouse IgG2Fc (isotype a or b) (all variable regions are fully human as denoted by the first 'H' in the antibody designation). As will be appreciated by one of ordinary skill in the art, an antibody having a particular Fc isotype can be converted into an antibody with a different Fc isotype (for example, an antibody with a mouse IgG1 Fc can be converted into a antibody with a human IgG4, etc.) however, in any case, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 2 - will remain the same, and it is expected that the antigen-binding properties are identical or substantially similar, regardless of the nature of the Fc domain.

[0236] In certain embodiments, antibodies selected with a mouse IgG1 Fc have been converted to antibodies with human IgG1 Fc. In one embodiment, the IgG4 Fc domain comprises 2 or more amino acid changes, as described in US20100331527.

[0237] To generate mutated antibodies, several residues in the complementary determining regions (CDRs) of H4H12166P were mutated to histidine to generate 9 mutated antibodies, identified as H4H12166P2 to H4H12166P10. Histidine mutations in CDRs have been shown to confer pH dependence on target antigen binding, leading to improved pharmacokinetics ( Igawa et al., 2010, Nat. Biotechnol. 28: 1203-1207 ).

Control Constructs Used in the Following Examples

[0238] The following control constructs (anti-C5 antibodies) were included in the experiments described herein, for comparison purposes: "Comparator 1", a monoclonal antibody against human C5 having VH/VL sequences from the antibody "h5G1.1" according to US Patent No. 6,355,245 (Alexion Pharmaceuticals, Inc.); and "Comparator 2", a human monoclonal antibody against human C5 having VH/VL sequences from the "8109" antibody according to US Patent Application Publication No. 2013/0022615 (Novartis).

Example 3: Antibody binding to C5 as determined by Surface Plasmon MRI

[0239] Equilibrium dissociation constants (KD values) for C5 binding to purified anti-C5 antibodies were determined using a real-time surface plasmon resonance biosensor assay on a Biacore T200 instrument. The Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc monoclonal antibody (GE Healthcare, #BR-1008-39) to capture anti-C5 antibodies expressed with human Fc constant regions. Biacore binding studies were performed in flowing HBST buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20). Human C5 was obtained from a commercial source (EMD). Other C5 reagents were expressed with a C-terminal myc-myc-hexahistidine tag (subsequently referred to as C5-mmh). Human C5-mmh reagents were also expressed containing histidine and cysteine point mutations in arginine 885 (subsequently referred to as C5 R885H-mmh and C5 R885C-mmh, respectively). Different 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 flowing buffer were injected onto the surface captured with anti-C5 antibody at a flow rate of 30 μL/min. The association of all C5 reagents with each of the captured monoclonal antibodies was monitored for 3 minutes and their dissociation in HBST flowing buffer was monitored for 8 minutes. All binding kinetics experiments were performed at 25oC or 37oC. On-rate (ka) and off-rate (kd) kinetic constants were determined by fitting real-time sensors to a 1:1 binding model using Scrubber 2.0c curve-fitting software. Equilibrium dissociation binding constants (KD) and dissociative half-lives (t7) were calculated from the kinetic rate constants as: KD (M) = kd/ka and t1/2 (min) = ln2/( 60xkd)

[0240] The binding kinetic parameters for the binding of human C5 to anti-C5 antibodies at 25°C and 37°C are shown in Tables 3 and 4. Table 3: Binding kinetic parameters of anti-C5 monoclonal antibodies ligand- if to human C5 at 25°C Table 4: Binding kinetic parameters of anti-C5 monoclonal antibodies binding to human C5 at 37°C

[0241] Monkey C5-mmh binding to anti-C5 antibodies at 25°C and 37°C is shown in Tables 5 and 6. Table 5: Kinetic Binding Parameters of anti-C5 monoclonal antibodies binding to C5 -mmh from monkey at 25°C N/A = not available; * SS = steady-state analysis Table 6: Binding Kinetic Parameters of anti-C5 monoclonal antibodies binding to monkey C5-mmh at 37°C

[0242] Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodies at 25°C are shown in Tables 7 and 8, respectively. Table 7: Kinetic Binding Parameters of anti-C5 monoclonal antibodies binding to human C5 R885H-mmh at 25°C Table 8: Kinetic Binding Parameters of anti-C5 monoclonal antibodies binding to human C5 R885C-mmh at 25°C

[0243] Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodies at 37°C are shown in Tables 9 and 10, respectively. Table 9: Binding Kinetic Parameters of anti-C5 monoclonal antibodies binding to human C5 R885H-mmh at 37oC Table 10: Binding Kinetic Parameters of anti-C5 monoclonal antibodies binding to human C5 R885C-mmh at 37°C

[0244] At 25°C, all 25 anti-C5 antibodies of the invention bound to human C5 with KD values ranging from 73 pM to 8.4 nM, as shown in Table 3. At 37°C, anti-C5 antibodies C5 of the invention bound to human C5 with KD values ranging from 103 pM to 18.5 nM, as shown in Table 4. At 25, 25 of the 25 anti-C5 antibodies of the invention tested bound to C5-mmh of monkey with KD values ranging from 133 pM to 64 nM as shown in Table 5. At 37°C, 25 of the 25 anti-C5 antibodies of the invention tested bound to monkey C5-mmh with KD values ranging from 133 pM at 118 nM, as shown in Table 6. At 25°C, 16 of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885H-mmh with KD values ranging from 147 pM to 10.9 nM, as shown in Table 7. At 25°C, 16 of the 16 anti-C5 antibodies of the invention tested bound human C5 R885C-mmh with KD values ranging from 251pM to 190nM, as shown in Table 8. At 37°C, 16 of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885H-mmh with KD values ranging from 1.49 nM to 69.4 nM as shown in Table 9. At 25°C, 16 of the 16 anti-C5 antibodies of the invention C5 of the invention tested bound human C5 R885C-mmh with K D values ranging from 1.74 nM to 159 nM, as shown in Table 10.

Example 4: Antibody binding to C5 due to different pH

[0245] The effect of pH on the dissociation rate of recombinant human C5 bound to purified anti-C5 monoclonal antibodies was determined using a real-time surface plasmon resonance biosensor using a Biacore T200 instrument. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, # BR-1008-39) to capture anti-C5 monoclonal antibodies expressed with human IgG1 Fc. All Biacore binding studies were performed using two PBS-T flow buffers, pH 7.4 (0.001M Na2HPO4/0.001M NaH2PO4, 0.15M NaCl, 0.05% v/v Tween-20, adjusted to pH 7.4) and PBS-T, pH6.0, (0.01 M Na2HPO4/NaH2PO4, 0.15 M NaCl, 0.05% v/v Tween-20, adjusted to pH 6.0) . Different concentrations of human C5 (EMD, Catalog No. # 204888) or monkey C5.mmh (prepared in PBS-T, flow buffer at pH 7.4 (ranging from 100 nM to 11.11 nM, 3 times) were injected onto the captured surface of anti-C5 monoclonal antibody for 3 minutes at a flow rate of 50 μL/minute and their dissociation in two fluent buffers PBS-T, pH 7.4 and PBS-T, pH 6 ,0 was monitored for 6 minutes. All kinetic binding experiments were performed at 25°C and 37°C. Kinetic dissociation constant (kd) was determined by fitting the sensors in real time in a 1:1 binding model using Scrubber 2.0c curve fitting software. Dissociative binding half-lives (t^) were calculated from kd as:

[0246] The half-life relationships for the binding of human C5 to different anti-C5 monoclonal antibodies at 25°C and 37°C in two fluent buffers PBS-T, pH7.4 and PBS-T, pH6.0 are shown in Tables 11 and 12. Table 11: Half-life relationships of selected anti-C5 antibodies to human C5 at 25°C Table 12: Half-life relationships of selected anti-C5 antibodies in human C5 at 37°C

[0247] Half-life relationships for binding of monkey C5 to different anti-C5 monoclonal antibodies at 25°C and 37°C in two fluent buffers PBS-T, pH7.4 and PBS-T, pH6.0 are shown in Tables 13 and 14. Table 13: Half-life relationships of selected anti-C5 antibodies in monkey C5 at 25°C Table 14: Half-life relationships of selected anti-C5 antibodies in monkey C5 at 37°C

[0248] As shown in Tables 11-14, the selected anti-C5 antibodies showed pH-dependent binding, as seen by the t1A ratios

Example 5: Octet cross-competition between anti-C5 antibodies

[0249] Competition binding between anti-C5 monoclonal antibodies (mAbs) was determined using an unlabeled, real-time biolayer interferometry assay on 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 Tween-20 Surfactant, 0.1 mg/mL BSA (buffer Octet HBS-P) with the plate agitated at a speed of 1000 rpm. To assess whether 2 antibodies were able to compete with each other for binding to their respective epitopes on human C5 (hC5 purified from plasma, EMD), about 1.5 nm of anti-human C5 mAb was captured first. onto anti-hFc antibody-coated Octet biosensor ends (Pall ForteBio Corp., # 18-5060) by submerging the ends for 3 minutes in wells containing a 50 µg/ml solution of anti-human C5 mAb (subsequently referred to as mAb1). The antibody-captured ends of the biosensor were then saturated with a blocking H4H isotype control mAb (subsequently referred to as the blocking mAb) by immersion in wells containing 200 µg/ml blocking mAb solution for 4 minutes. The ends of the biosensor were then subsequently immersed in wells containing a co-complexed solution of 50 nM hC5 and 1 µM of a second anti-human C5 mAb (subsequently referred to as mAb2), which had been pre-incubated for 2 hours, for 4 minutes. The ends of the biosensor were washed in Octet HBS-P buffer between each step of the experiment. The real-time binding response was monitored during the course of the experiment and the binding response at the end of each step was recorded. The response of human C5 precomplex mAb2 binding to mAb1 was corrected for antecedent binding, compared and the competitive/non-competitive behavior of different anti-C5 monoclonal antibodies was determined.

[0250] Table 15 explicitly defines the relationships of antibodies that compete in both directions, regardless of binding order. Table 15: Cross-competition between pairs of selected anti-C5 antibodies

Example 6: Inhibition of C5-mediated complement-dependent cytotoxicity in a B-cell bioassay

[0251] This example describes a bioassay to test the role of C5 using an anti-CD20 antibody in the classical complement pathway. Therapeutic anti-CD20 antibodies against the B cell specific cell surface antigen CD20 have been shown to lead to B cell CDC (Glennie et al. 2007, Mol. Immunol. 44: 3823-3837) and CDC assay using cell lines expressing CD20 have been described previously ( Flieger et al. 2000, Cell. Immunol. 204: 55-63 ). Daudi cells, a human B cell line expressing CD20, complementary preserved serum or depleted serum of C5 with exogenous C5 variants and an anti-CD20 antibody (antibody comprising "2F2" VH/VL of US Patent 8,529,902) were used to assess the role of C5 activity in CDC.

[0252] For the C5 CDC bioassay, Daudi cells were seeded in 96-well assay plates at 10,000 cells/well in RPMI containing 10% FBS, penicillin/streptomycin, L-glutamine, sodium pyruvate and Non-Essential Amino Acids (Complete RPMI media) or RPMI containing 1% BSA, penicillin/streptomycin and L-glutamine (RPMI/BSA). All assays testing mutated anti-hC5 antibodies, along with testing the non-mutated antibodies with human serum containing C5 were tested in Complete RPMI media, while assays testing the non-mutated antibodies with Afrian Green monkey serum and human C5 variants were tested on RPMI/BSA media. To measure CDC with human or monkey serum, anti-CD20 antibody was diluted 1:3 from 100 nM to 2 pM (including a control sample without antibody) and incubated with cells for 10 minutes at 25°C followed by addition of 1.66% serum or 1.66% C5 depleted serum and 6.6 nM C5 variant proteins. The amount of C5 protein to be added to C5 depleted serum was based on the reported value of C5 concentration in human serum of 0.37 uM ( Rawal et al., 2008, J. Biol. Chem. 283: 7853-7863 ) . To test CDC C5 antibody inhibition, C5 antibodies were diluted 1:3 from 100 nM to 2 pM (including a control sample without antibody) and incubated with either 1.66% or 1.66% serum. depleted serum of C5 and 6.6nM of C5 variant for 30 minutes. Ten minutes prior to addition of serum antibodies to cells, anti-CD20 antibody was added to cells at 1 nM, 2 nM, 3 nM, 3.5 nM, 7 nM, 10 nM, or 30 nM. At the end of the incubation with the anti-CD20 antibody, the antibody/serum mixture was added to the cells. Cytotoxicity was measured after 3.5 hours of incubation at 37°C and 5% CO2, followed by 15 minutes of incubation at 25°C and addition of CytoTox-Glo™ reagent (Promega, # G9292). CytoTox-Glo™ is a luminescence-based reagent that measures cell death in such a way that increased luminescence is observed with increased cytotoxicity (measured in relative light units, RLUs). Untreated cells in control wells were lysed by digitonin treatment immediately after addition of CytoTox-Glo™ reagent to determine maximum cell kill. Plates were read by luminescence by a Victor X instrument (Perkin Elmer) 15 minutes after addition of CytoTox-Glo™. When calculated, the percentage of cytotoxicity was calculated with the RLU values, using the following equation: % Cytotoxicity = 100 x (Experimental Cell Lysis - Baseline Cell Lysis) (Maximum Cell Lysis - Baseline Cell Lysis)

[0253] In this equation, "baseline cell lysis" is the luminescence of cells treated with media and serum alone without any anti-CD20 antibody and "maximal cell lysis" is the luminescence of digitonin-treated cells. Results, expressed as % cytotoxicity or RLUs, were analyzed using non-linear regression (4-parameter logistic) with Prism 5 software (GraphPad) to obtain EC50 and IC50 values. Antibody inhibition was calculated such that 0 - 100% inhibition is the inhibition range of the anti-CD20 antibody concentration used in the assay without 0nM anti-CD20 antibody inhibitor.

Results

[0254] A total of 25 anti-human C5 antibodies, 16 non-mutated and 9 mutated, were tested for their ability to inhibit C5 in the CDC assay using Daudi cells with an anti-CD20 antibody and human serum (with normal hC5 or C5 variants) or African Green monkey sera. Several residues in the Complementarity Determining Regions (CDRs) of H4H12166P were mutated to histidines to generate 9 mutated antibodies, H4H12166P2-H4H12166P10. Histidine mutations in CDRs have been shown to confer pH dependence on target antigen binding, leading to improved pharmacokinetics ( Igawa et al., 2010, Nat. Biotechnol. 28: 1203-1207 ). Table 16: Inhibition of non-mutated CDC anti-hC5 antibody with 1.66% serum and anti-CD20 antibody in Daudi cells *Unless otherwise noted, all inhibition is ~100%

[0255] As shown in Tables 16 and 17, all 25 anti-hC5 antibodies showed complete inhibition of C5-mediated CDC present in 1.66% of human serum. IC50's of non-mutated antibodies ranged from 1.2 to 3.4 nM. The IC50 of the mutated antibodies ranged from 3.0nM to 12nM. The parent, unmutated antibody H4H12166P produced complete inhibition with IC50s of 2.6 nM and 2.9 nM. Table 17: Inhibition of CDC mutated anti-hC5 antibody with 1.66% serum and anti-CD20 antibody in Daudi cells *Unless otherwise noted, all inhibition is ~100%

[0256] The 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.

[0257] Four of the 9 mutated antibodies showed complete African Green monkey C5-mediated inhibition of CDC with IC50s ranging from 7.1 nM to 9.9 nM. The remaining six mutated antibodies were blockers with IC50s greater than 10 nM and minimal inhibition (at 100 nM antibody) ranging from 34% to 85%. The parent, unmutated antibody H4H12166P produced complete inhibition with IC50s of 4.5 nM and 5.6 nM.

[0258] To test whether anti-hC5 antibodies inhibit the human C5 variants, R885H and R885C, C5 Depleted Human Serum was tested with 6.6 nM of each C5 variant. All 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by the C5 variant R885H, with IC50s of the non-mutated antibodies ranging from 0.48 nM to 4.2 nM, while IC50s of the mutated antibodies ranged from 1.3 nM to 7 ,0 nM. The parent, unmutated antibody H4H12166P produced complete inhibition with IC50s of 1.3 nM and 1.3 nM.

[0259] Fifteen of the 16 non-mutated anti-hC5 antibodies showed complete inhibition of CDC mediated by the C5 variant R8855C, with IC50s ranging from 0.43 nM to 1.6 nM. An unmutated antibody showed weak inhibition of CDC with maximum inhibition of 67% (at 100 nM antibody) and an IC50 > 20 nM. All nine mutated antibodies showed complete inhibition of CDC mediated by the C5 variant R885C with IC50s ranging from 0.77 nM to 3.5 nM. The parent, unmutated antibody H4H12166P produced complete inhibition with an IC50s of 0.46 nM and 0.76 nM.

[0260] The anti-CD20 antibody showed 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 depleted serum with hC5 variant R885H and 2.7 nM and 9.5 nM for hC5 depleted serum with hC5 variant R885C. None of the irrelevant IgG control antibodies, Control mAb1 and Control mAb2, demonstrated any inhibition of CDC.

Example 7: Inhibition of C5a activity as determined by the luciferase assay

[0261] This example describes an assay to test the activation of C5a through one of its receptors, C5aR1. C5aR1 is a G protein-coupled receptor (GPCR) and can initiate several GPCR-coupled signaling pathways ( Monk et al., 2007, Br. J. Pharmacol. 152: 429-448 ). A bioassay was established using HEK293 cells stably transfected with human C5aR1 (Accession No. NP_001727.1) and human Gα16 (Accession No. NP_002059.3) together with a luciferase reporter [NFAT (4X)-luciferase response element] . Gα16 is a relatively promiscuous G protein that can couple to different types of GPCRs leading to activation of PLC-β and subsequent elevation of Ca++, 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, 500μg/ml G418, 100μg/ml hygromycin B and 7μg/mL of blasticidin.

[0262] For the C5a luciferase bioassay, HEK293/hGD16/hC5aR1/NFAT-luc cells were seeded in 96-well assay plates at 20,000 cells/well in OPTIMEM (Invitrogen, # 31985-070) supplemented with 0 BSA .5%, penicillin/streptomycin and L-glutamine, and then incubated at 37oC and 5% CO2 overnight. BSA was used instead of FBS, as serum has been shown to cleave and inactivate hC5a ( Klos et al., 2013, Pharmacol. Rev. 65: 500-543 ). The next morning, hC5a was titrated from 100 nM to 2 pM (including a control sample containing no hC5a) and added to the cells to determine the dose-response titration curve for the cell line. To test hC5a antibody inhibition of hC5a, 500pM of hC5a was added to the cells. Immediately thereafter, 1:3 diluted antibodies from 100 nM to 2 pM (including a control sample containing no antibody) were added to the cells. Cells were incubated for 5.5 hours at 37°C in the presence of 5% CO 2 . Luciferase activity was detected after incubation with OneGlo™ reagent (Promega, # E6051). OneGlo™ 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, RLUs). Luminescence measurement was performed using a Victor X instrument (Perkin Elmer). Results were analyzed using non-linear regression (4-parameter logistic) with Prism 5 software (GraphPad) to obtain EC50 and IC50 values. Antibody inhibition was calculated such that 0 - 100% inhibition is the inhibition range from 500 pM hC5a without inhibitor to 0 nM hC5a.

[0263] Four anti-hC5 antibodies were tested for their ability to inhibit hC5a activation of its receptor, hC5aR1, by measuring the extent of inhibition of activation of 500pM hC5a from HEK293/hGα16/hC5aR1/NFAT-luc cells. Table 18: Anti-hC5 antibody inhibition of 500 pM hC5a in HEK293/GD16/hC5aR1/NFAT-luc cells

[0264] As shown in Table 18, all four antibodies of the invention showed complete inhibition of 500pM of hC5a with IC50s ranging from 0.035nM to 0.46nM. An Irrelevant IgG control antibody, Control mAb3, did not demonstrate any inhibition of hC5a. hC5a activated HEK293/Gα16/hC5aR1/NFAT-luc cells with an EC50 of 0.39 nM.

Example 8: Hemolysis Bioassay

[0265] The classical pathway hemolysis (CH) assay and the alternative pathway hemolysis (AH) assay were developed to test for antibody activity.

[0266] The CH is an evaluation assay for the activation of the classical complementary classical pathway, which is sensitive to the decrease, absence and/or inactivity of any component of the pathway. CH tests the functional ability of serum complementary components of the classical pathway to lyse sheep red blood cells (SRBC) precoated with rabbit anti-sheep antibody to red blood cells (hemolysin). When antibody-coated SRBC are incubated with test serum, the classical complement pathway is activated and hemolysis results. If a complementary component is missing, the CH level will be zero; if one or more components of the classic track are turned down, the CH will be turned down. (Nilsson et al. 1984, J. Immunol. Meth. 72: 49-59). This assay is used for the characterization and evaluation of high affinity anti-human C5 antibodies.

Methods (A) Classical pathway complement hemolysis assay

[0267] The desired number of sheep red blood cells (SRBCs) were washed in GVB++ buffer and resuspended in 1x10A9 cells/ml. To sensitize red blood cells, equal volumes of diluted 1:50 rabbit anti-sheep hemolysin (1.5mg/mL) were mixed at 37°C for 20 minutes. Sensitized SRBC cells were diluted to 2x10A8 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 test for inhibition of C5-mediated hemolysis activity, test antibodies were preincubated for 20 minutes at 4°C at concentrations ranging from 0.6 nM to 800 nM in 2%-10% normal human serum or serum. 10% cynomolgus monkey serum or African green monkey serum. Round bottom 96-well plates were used to measure hemolysis activity. A total of 100ul of sensitized sheep RBCs (2x10Acells/ml) were seeded in a 96-well plate followed by the addition of 100ul of respective serum samples that were pre-incubated with the test antibodies. Cells were gently mixed and incubated at 37°C for 60 minutes. After the incubation time, the cells were spun by centrifugation at 1250xg at 4°C. A total of 100 ul of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on the Spectramax microplate reader. Hemolytic activity was calculated at the final serum concentration of 1-5% for treatments.

[0268] The percentage of hemolysis was calculated as follows: % Hemolysis = 100 x (Experimental Cell Lysis - Baseline Cell Lysis) (Maximum Cell Lysis - Baseline Cell Lysis)

[0269] In this equation, "baseline cell lysis" is the OD at A412nm of cells incubated in GVB++ buffer only containing no serum. "Maximum cell lysis" is the OD at A412nm of water-treated cells. Results, expressed as % hemolysis, were analyzed using non-linear regression (4-parameter logistic) with Prism 5 software (GraphPad) to obtain IC50 values. Data represented as mean ± standard error of mean

(B) Alternative complementary test

[0270] The desired number of rabbit red blood cells (RbRBCs) were washed in GVB-Mg2+/EGTA buffer and resuspended at 2x10A8cells/ml. Cynomolgus monkey or normal human serum was diluted to 10% in GVB-Mg2+/EGTA buffer. To test inhibition of C5-mediated hemolysis activity, antibodies at concentrations ranging from 3 nM to 800 nM were pre-incubated for 20 minutes at 4°C in 5-10% normal human serum or cynomolgus monkey serum. Round bottom 96-well plates were used to measure hemolysis activity. A total of 100ul of RbRBCs (2x10A8 cells/ml) was seeded into a 96-well plate followed by the addition of 100ul of 10% normal human serum or cynomolgus monkey serum or African green monkey serum that was pre-incubated with the anti-C5 antibodies. Cells were gently mixed and incubated at 37°C for 60 minutes. After the incubation time, the cells were spun by centrifugation at 1250xg at 4°C. A total of 100 µL of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on the Spectramax microplate reader. Hemolytic activity was calculated at the final serum concentration of 5% serum.

[0271] The percentage of hemolysis was calculated as follows: % Hemolysis = 100 x (Experimental Cell Lysis - Baseline Cell Lysis) (Maximum Cell Lysis - Baseline Cell Lysis)

[0272] In this equation, "baseline cell lysis" is the OD at A412nm of cells incubated in GVB-Mg/EGTA buffer containing only no serum or no anti-C5 antibody. "Maximum cell lysis" is the OD at A412nm of water-treated cells. Inhibition by anti-C5 antibodies, IC50 values were calculated using non-linear regression (4-parameter logistic) with Prism 6 software (GraphPad).

Results (A) Inhibition of Human C5 Hemolysis

[0273] A total of 25 anti-human C5 antibodies (hC5), 16 non-mutated and 9 mutated, were tested for their ability to inhibit normal human serum (NHS) C5 in the CH50 assay using sheep red blood cells (SRBCs) and AH50 assay using rabbit red blood cells (RRBCs). Table 19: Anti-hC5 antibody inhibition of CP and AP activity in 1% or 5% normal human serum (NHS)

[0274] As shown in Table 19, sixteen anti-hC5 antibodies of this invention showed greater than 94% inhibition of hemolysis in the classical pathway (CP) mediated by C5 present in 1% human serum. Antibody IC50s ranged from 2.1 to 5.9 nM and percent inhibition ranged from 95% - 99%. All 16 anti-C5 antibodies showed greater than 60% inhibition (except H4H12169P) in the alternative pathway (AP) hemolysis assay mediated by C5 present in 5% NHS. Antibody IC50s ranged from 13 to 160 nM and percent inhibition activity ranged from 44% to 81%. Table 20: Anti-hC5 antibody inhibition of CP and AP activity in 5% normal human serum (NHS)

[0275] As shown in Table 20, all 9 mutated anti-hC5 antibodies showed inhibition of C5-mediated CP and AP hemolysis activity present in 5% of human serum. In the CP hemolysis assay, the parent, unmutated antibody H4H12166P showed greater than 98% inhibition with an IC50 of 10.9 nM. Eight mutated anti-hC5 antibodies showed greater than 90% inhibition with IC50s ranging from 10.3 nM to 24.3 nM. Mutant anti-C5 antibody 12166P10 showed 74% partial inhibition. In the AP hemolysis assay, the parent, unmutated antibody H4H12166P showed greater than 85% inhibition with IC50s of 20.9 nM. The mutated anti-hC5 antibodies showed an inhibition range of 72-83% and the IC50s antibodies ranged from 28 nM to 0.15 µM.

(B) Inhibition of monkey C5 hemolysis

[0276] A total of 25 anti-human C5 (hC5) antibodies, 16 non-mutated and 9 mutated, were tested for their ability to inhibit Cynomolgus monkey and African green monkey C5 in the CH50 assay using sensitized sheep red blood cells (SRBCs) and AH50 assay using rabbit red blood cells (RRBCs).

[0277] As shown in Table 21, anti-hC5 antibodies showed different levels of inhibition of CP or AP hemolysis activity in 5% African green monkey serum. In the CP assay, two of the 16 anti-hC5 antibodies showed no inhibition of hemolysis activity. Fourteen antibodies showed inhibition ranging from 43-94%, with IC50s ranging from 25 nM to 180 nM. In the AP hemolysis assay, thirteen of the 17 antibodies showed inhibition activity ranging from 17% - 93% with IC50s ranging from 22.5 nM to 233 nM. Table 22: Anti-hC5 antibody inhibition of CP and AP activity in 5% normal Cynomolgus (Cyno) monkey serum

[0278] As shown in Table 22, anti-hC5 antibodies (except H4H12183P2, which showed 64% CP inhibition) showed greater than 90% inhibition of CP or AP hemolysis assay in 5% Cynomolgus monkey serum . In the CP hemolysis assay, antibody IC50s ranged from 7.15 nM to 142 nM. In the AP hemolysis assay, antibody IC50s ranged from 5.4 to 29.6 nM.

(C) Inhibition of variant human C5 hemolysis

[0279] Selected anti-C5 antibodies were tested for their ability to inhibit variant human C5 (see, here Example 3) of C5 depleted human serum in the CH50 assay. In C5-depleted human serum supplemented with the exogenous C5 variant R885H, H4H12166P, and Comparator 2 blocked CP hemolysis with IC50 values of 6.0nM and 4.4nM, respectively, and IC80 values of 7.6nM and 5 .5nM, respectively. For the R885C variant, H4H12166P and Comparator 2 blocked CP hemolysis in C5-depleted human serum with exogenous C5 variants with an IC50 of 9.3 nM and 6.8 nM, respectively, and IC80 values of 11 nM and 8, 2 nM, respectively. As expected, Comparator 1 did not block the hemolytic activity of human C5 variants. (D) Inhibition of the human C5b-6 complex

[0280] Selected anti-C5 antibodies were tested for their ability to inhibit the human C5b-6 complex from C5 depleted human serum in the CH50 assay. H4H12166P potentially blocked CP hemolysis in C5-depleted human serum supplemented with exogenous huC5b-6 complex with an IC50 of 3.8 nM and IC80 value of 5.8 nM. In contrast, Comparator 1 blocked C5b-6 complex-mediated hemolysis to a lesser extent, with IC50 values of 5.0 nM and IC80 values of 46 nM, respectively. Comparator 1 inhibited only 70% of total hemolysis at the highest concentrations tested. Comparator 2 did not block the hemolytic activity of the human C5b-6 complex.

Example 9: Anti-C5 antibodies block generation of C5a in CP hemolysis assay

[0281] To assess whether anti-C5 antibodies inhibit C5a generation, classical pathway hemolysis (CP) assay supernatants were analyzed for C5a levels by ELISA.

[0282] C5a, generated as a result of cleavage of C5, is a protein fragment of 74 amino acids. C5a is rapidly metabolized by serum carboxypeptidases to a more stable, less active, 73-amino acid form, C5a des-Arg, by removal of the C-terminal arginine. Quantification of C5a des-Arg therefore provides a reliable measure to monitor C5a generation in vivo and in vitro. The MicroVue C5a ELISA kit used here detects C5a des-Arg according to 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 purpose of this example, both forms will be referred to collectively as "C5a".

[0283] C5a protein levels were determined in CP hemolysis assay supernatants using normal human serum (NHS) pre-incubated with complement pre-incubated with H4H12166P or isotype control antibody as described in Example 8. C5a protein were measured using the MicroVue C5a ELISA kit according to the manufacturer's instructions. Briefly, samples were diluted and incubated on plates pre-coated with capture antibody (mouse anti-C5a specific for a neo-epitope on human C5a). The human C5a protein provided by the manufacturer was used as a standard for calibration. C5a in the supernatants was detected by HRP-conjugated detection antibody (mouse monoclonal antibody to the C5a region of C5). The chromogenic HRP substrate, 3,3',5,5'-tetramethylbenzidine (TMB), was added to detect HRP activity. A 1N hydrochloric acid solution was used to stop the reaction and the optical density was measured at 450 nm (OD450) in a SpectraMax plate reader. Data were analyzed using non-linear regression (4-parameter logistic) in GraphPad Prism. C5a concentration was expressed as ng/ml of supernatant.

[0284] In the assay using 5% NHS, H4H12166P potentially blocked increases in C5a protein levels in a dose-dependent manner with an IC50 of 8.5nM, whereas the isotype control antibody had no effect on C5a levels ( Figure 1). Maximum blockade at the highest tested H4H12166P concentration (267nM) resulted in a ~10-fold decrease in C5a levels to 3.8 ng/mL (0.3nM) compared to 34 ng/mL (2.8nM) observed at the lowest concentration of H4H12166P tested (1 nM) in 5% serum. The observed C5a concentration for maximal blockade was close to the baseline C5a level of 2.3 ng/ml (0.2 nM) in 5% untreated NHS.

Example 10: Characterization of pharmacokinetics and pharmacodynamics of anti-C5 antibodies in cynomolgus monkey

[0285] This Example describes the characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of selected anti-C5 antibodies raised in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody dosing and used to stratify animal dose groups.

[0286] Total circulating C5 levels in cynomolgus monkeys were determined using a Human Complementary C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations. Mean C5 protein concentrations in monkeys were determined to be 90.85 μg/mL ± 19.17 μg/mL.

[0287] For each anti-C5 antibody, 4 cynomolgus monkeys each received 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 into serum and frozen at -80°C until analyzed for PK and PD.

Analysis of Total IgG Antibody Level by ELISA Immunoassay

[0288] Total antibody concentrations in monkey serum samples were measured using an unvalidated direct ELISA. The ELISA procedure employed utilized a microtiter plate coated with a mouse anti-human IgG1/IgG1 Fc monoclonal antibody. Different anti-C5 antibodies were added to the plate and 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 peroxidase-specific luminol-based substrate was then added to achieve a signal intensity that is proportional to the concentration of total captured anti-C5 antibody. The relative light unit (RLU) measurements of the calibration standards and their respective nominal concentrations were fitted using a weighted 4-parameter logistic equation to generate a calibration equation that described the concentration of anti-C5 antibodies and the response relationship of the rehearsal. The lower limit of quantitation (LLOQ) was 1.56 ng/ml in the assay (2% monkey serum) and 78 ng/ml in pure monkey serum.

Determination of PK parameters

[0289] PK parameters were determined by non-compartmental analysis (NCA) using Phoenix®WinNonlin® software (Version 6.4, Certara, L.P.) and an IV bolus dosing model.

[0290] All PK parameters were derived from the respective mean concentration values, including the maximum observed serum concentration (Cmax), the time of observed peak concentration, tmax, and the estimated observed half-life (T1/2). For each antibody, the area under the concentration versus time curve to the last measurable concentration (AUClast) and extrapolated from time zero to infinity (AUCinf) was determined using a linear trapezoidal rule with linear interpolation and uniform weighting.

PD analysis by ex vivo hemolysis assay

[0291] The pharmacodynamics of selected anti-C5 antibodies were analyzed using ex vivo classical pathway and alternative pathway hemolysis assays.

[0292] Classical Trail Hemolysis Assay: Sheep red blood cells (SRBCs) were washed in GVB++ buffer (Gelatin Veronal Buffer with CaCl2 and MgCl2) (Boston BioProducts) and resuspended in 1x10A9 cells/ml. To sensitize the SRBCs, a total of 1x10A9/ml of SRBCs was 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 2x10A8 cells/ml in GVB++ buffer before use in the hemolysis assay. Cynomolgus monkey blood was collected before dosing and at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56, 63 and 70 days post dose for PD analysis. The serum was prepared and frozen until later use. On the day of the assay, cynomolgus serum from the respective time points was diluted to 10% in GVB++ buffer. Round bottom 96-well plates were used to measure hemolysis activity. A total of 100μl of sensitized SRBCs (2x10A8 cells/mL) were seeded in 96-well plates at 37°C followed by the addition of 100μL of cynomolgus monkey serum at 10% of the respective time points. The SRBCs were gently mixed and incubated at 37°C for 10 minutes. After the incubation time, the cells were centrifuged at 1250xg at 4°C. A total of 100μL of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412nm on a Spectramax microplate reader. Hemolytic activity was calculated at the final serum concentration of 5%. The percentage of hemolysis was calculated from the absorbance values using the following equation: % Hemolysis = 100 x (Experimental Cell Lysis - Baseline Cell Lysis) (Maximum Cell Lysis - Baseline Cell Lysis)

[0293] In this equation, "baseline cell lysis" is the OD at A412nm of SRBCs incubated in GVB++ buffer alone containing no serum. "Maximum cell lysis" is the OD at A412nm of water-treated SRBCs. Results, expressed as % hemolysis, were analyzed using non-linear regression (4-parameter logistic) with Prism 5 software (GraphPad) to obtain IC50 values. Data are represented as mean + Standard Error of Mean.

[0294] Alternative Lane Hemolysis Assay: The desired number of rabbit red blood cells (RbRBCs) were washed in GVB-Mg2+/EGTA buffer and resuspended in 2x10A8 cells/ml. Cynomolgus monkeys blood was collected before dosing and at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42 and 49 days post dose for PD analysis. The serum was prepared and frozen until later use. Round bottom 96-well plates were used to measure hemolysis activity. A total of 100μl of RbRBCs (2x10A8 cells/ml) were seeded into 96-well plates at 37°C, followed by the addition of 100μl of cynomolgus monkey serum at 10% of the respective time points listed above. The RbRBCs were gently mixed and incubated at 37°C for 60 minutes. After the incubation time, the cells were centrifuged at 1250xg at 4°C. A total of 100μL of the supernatant was transferred to a fresh 96 flat bottom plate which was read at 412nm on 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 described above.

Results

[0295] Selected anti-C5 antibodies (listed in Table 1) were tested in early experiments for prolonged pharmacokinetic profiles in Cynomolgus monkeys and in C5-humanized mice (described in Example 10). H4H12166P and H4H12161P were selected as having high affinity along with prolonged PK and used in subsequent experiments with Comparator 1 and Comparator 2.

[0296] Cynomolgus monkeys received a single IV bolus dose of 15 mg/kg of H4H12166P, H4H12161P, or Comparator 2. Serum total antibody concentrations and percent classical pathway hemolysis (PC) activity were determined in 19 time points over a 70-day period in the lifetime. Alternate path (AP) hemolysis was determined at 17 time points over a 50-day lifetime. Table 23 summarizes the mean antibody concentrations for all 3 antibodies. Mean total antibody concentrations versus time profiles are shown in Figure 2. Mean PK parameters are described in Table 24. Table 23: Mean Total IgG Serum Concentrations After a Single Intravenous Injection of 15 mg/kg of C5 Selected for Male Cynomolgus Monkeys Time = time in hours after single-dose injection; SD = standard deviation; BLQ = Limit Below Quantification

[0297] Following IV bolus administration, the total IgG concentration-time profiles of H4H12166P, H4H12161P, and Comparator 2 were characterized by an initial brief distribution phase followed by single elimination phases throughout the lifetime. Peak H4H12166P, H4H12161P, and Comparator 2 concentrations were highly comparable, as the corresponding Cmax/Dose values among all antibodies were within 1.1 fold (29.7, 30.4, and 30.6 [(ug/ mL)/(mg/kg)], respectively) (Table 24). Table 24: Mean Pharmacokinetic Parameters of Total Serum IgG Concentrations After a Single Intravenous Injection of 15 mg/kg of Selected Anti-C5 Antibodies in Male Cynomolgus Monkeys IV = Intravenous; n = Number of animals; Cmax = Peak concentration; C0 = Initial concentration determined by extrapolation; tmax = Time to Cmax; AUC = Area under the concentration-time curve; AUClast = AUC calculated from time zero to the time of the last positive concentration; AUCinf = AUC from time zero extrapolated to infinity; CL = Total Body Clearance; Vss = Volume of distribution at steady state; t1/2 = Half-life; SD = Standard Deviation. Note: tmax is expressed in nominal hours

[0298] Evaluation of concentration-time profiles revealed that H4H12166P demonstrated the slowest clearance with terminal antibody concentrations > 10 µg/mL through day 71 of the study. The kinetics of H4H12161P and Comparator 2 were similar; both demonstrated faster clearance than H4H12166P, with mAb concentrations > 10 µg/mL at day 22 and 29, respectively.

[0299] Consequently, dose-normalized exposures (AUClast/dose) indicated that H4H12166P had the highest exposure at 339 days* (μg/mL)/(mg/kg), while H4H12161P and Comparator 2 had approximately 2 times shorter, 157 and 187 days* (μg/mL)/(mg/kg), respectively, than that of H4H12166P.

[0300] Antibody half-life (t1/2) calculated during the elimination phase ranged from 5.5 to 15.6 days between dose groups and also correlated with exposure, since H4H12166P had the t1/ 2 correspondingly higher of 15.6 days, while H4H12161P and Comparator 2 had t1/2 values of 5.5 and 5.9 days, respectively.

[0301] The pharmacological effects of anti-C5 antibodies from cynomolgus monkey serum samples were determined ex vivo by complementary classical pathway hemolysis (PC) of sensitized sheep red blood cells (SRBCs) and alternative pathway hemolysis (AP) of red blood cells rabbit red blood cells (RbRBCs). 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 3 antibodies as determined by the mean percent hemolysis. Table 25: Percent Hemolysis Activity of the Alternative and Classic Ex Vivo Track of Selected Anti-C5 Antibodies Time = time in hours after single-dose injection; SEM = standard error of the mean; BLQ = Limit Below Quantification; NC = Not calculated

[0302] As shown in Table 25 and Figure 2, the effects of PD were measured by complementary CP (10 minutes of incubation) through Day 70. H4H12166P blocked more than 95% of the hemolytic activity of CP through Day 35. The activity returned to pre-study maximum hemolysis levels on day 70. Comparator 2 blocked about 95% of CP hemolytic activity by day 10, and activity quickly returned to pre-study maximum hemolysis levels on day 18.

[0303] The effects of PD also page were measured using complementary AP trail hemolysis assays (60 minutes of incubation) through day 49. As shown in Table 25 and Figure 3, H4H12166P blocked 80% of the hemolytic activity of Total AP by day 18 and activity returned to pre-study maximum hemolysis level by day 50. H4H1216P and Comparator 2 blocked 90% of AP hemolytic activity by day 7, and activity returned to hemolysis levels pre-study maximum on the 21st day.

Example 11: Characterization of PK/PD of anti-C5 antibodies in C5-humanized mice

[0304] In this set of experiments, the pharmacokinetics and pharmacodynamics of selected anti-C5 antibodies were evaluated in mice humanized to express human C5 protein using Velocigene® technology (Valenzuela et al., 2003, Nat. Biotechnol. 21: 652- 659). Humanized mice were constructed to replace exon 2 to exon 41 of the murine C5 gene with exons 2 - 42 of the human C5 gene (described in US Patent Application Publication 2015/0313194, incorporated herein in its entirety).

[0305] Total circulating human C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations.

Determination of Total Drug Level in Serum by ELISA

[0306] Circulating anti-C5 antibody concentrations, both bound and unbound to C5, were determined by total human antibody analysis using ELISA. Briefly, a polyclonal goat anti-human IgG antibody at 1 µg/ml in PBS was immobilized in 96-well plates overnight; plates were washed to remove unbound IgG and then blocked with 5% BSA. Serial dilutions of anti-C5 antibody containing serum samples (6 points) and reference standards (12 points) of the respective antibodies were transferred to anti-human IgG coated plates and incubated for one hour. Plate-bound anti-C5 antibodies were then detected using a polyclonal goat anti-human IgG antibody conjugated with horseradish peroxidase. Plates were developed with TMB substrate according to the manufacturer's recommended protocol and optical density (OD) signals at 450 nm were recorded using a Perkin Elmer Victor X4 Multimodal Plate Reader. Serum anti-C5 antibody concentrations were calculated based on the standard reference calibration curve generated using GraphPad Prism software.

Determination of PK parameters

[0307] PK parameters were determined by non-compartmental analysis (NCA) using Phoenix®WinNonlin® software (Version 6.3, Certara, L.P.) and an extravascular dosing model. Using the respective mean concentration values for each antibody, all pharmacokinetic parameters, including estimated observed half-life (t1/2) and area under the concentration versus time curve to last measurable concentration (AUClast) were determined using a trapezoidal rule. linear with linear interpolation and uniform weighting.

PD Analysis by Hemolysis Assay

[0308] The pharmacodynamics of selected anti-C5 antibodies were determined using a classical pathway complementary hemolysis assay. Sheep red blood cells (SRBCs) (Sheep Blood in Alsevers solution) were washed in GVB++ buffer (Gelatin Veronal Buffer with CaCl2 and MgCl2) (Boston BioProducts) and resuspended in 1x10A9 cells/ml. To sensitize, 1x10A9/ml of 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 2x10A8 cells/ml in GVB++ before use in the hemolysis assay. Serum samples from pre-dosed animals or humanized C5 mice dosed with anti-C5 antibodies collected on days 10, 20, 30, 40 and 50 post-dose were diluted to 20% in GVB++ buffer. A total of 100μl of sensitized SRBCs (2x10Acells/mL) were seeded in 96-well round bottom plates at 37°C, followed by the addition of 100μl of 20% serum that was supplemented with 160-180μg/mL of complement protein 3 human (huC3). Cells were gently mixed and incubated at 37°C for 1 hour. After incubation, cells were centrifuged at 1250xg at 4°C. A total of 100μL of supernatant was transferred to a fresh 96 flat bottom plate and read at A412nm on a Spectramax microplate reader. The percentage of hemolysis was calculated from the absorbance values using the following equation: % Hemolysis = 100 x (Experimental Cell Lysis - Baseline Cell Lysis) (Maximum Cell Lysis - Baseline Cell Lysis)

[0309] In this equation, "baseline cell lysis" is the OD at A412nm of SRBCs incubated in GVB++ buffer alone containing no serum. "Maximum cell lysis" is the OD at A412nm of water-treated SRBCs. Results, expressed as % hemolysis, were analyzed using nonlinear regression (4-parameter logistic) with Prism 6 software (GraphPad) to obtain IC50 values. Data represented as Mean ± (Standard Error of Mean).

Experience 1

[0310] In this experiment, the pharmacokinetics and pharmacodynamics of the exemplary antibody H4H12166P were evaluated in comparison to Comparator 1 and Comparator 2 in humanized C5 mice. Total circulating human C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations. Mean concentrations of human C5 in mice were 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.

[0311] Prior to antibody measurement, male and female humanized C5 mice were stratified according to human C5 levels, with an average of 40 μg/mL. For each anti-C5 antibody, cohorts of twenty-two mice received a single 15 mg/kg dose of H4H12166P, Comparator 1 or Comparator 2 by subcutaneous (s.c.) injection. All mice were bled pre-dose and one day after injection for PK analysis. Furthermore, at 10, 20, 30, 40 and 50 days after injection, groups of 4 or 5 mice from each cohort were euthanized and terminal bleedings were collected for PK and PD analysis. Day 1 serum samples were averaged across the entire cohort of 22 mice. Blood was processed into serum and frozen at -80°C until analyzed.

[0312] Total antibody concentrations were determined at 7 time points and percent hemolysis activity was determined at 6 time points over the 50-day period of life. Total anti-C5 antibody concentrations are summarized in Table 26. Mean total antibody concentrations versus time profile are shown in Figure 4. Mean PK parameters are described in Table 27. Table 26: Mean Total IgG concentrations in Serum after a Single Subcutaneous Injection of 15 mg/kg of anti-C5 antibodies in Humanized C5 mice Time = time in hours after single-dose injection; Day = Study day; SD = Standard deviation; SEM = Standard error of the mean; NA = Not detected; NS = No sample. * For Comparator 2, day 50, n = three due to the inability of a sample to be analyzed due to technical issues. Table 27: PK Parameters Cmax = maximum concentration; AUC = Area under the concentration-time curve; AUClast= AUC calculated from time zero to the time of the last positive concentration; T1/2 = Observed Estimated Half-Life

[0313] Mean concentration versus time profiles on day 1 show that the three antibodies, H4H12166P, Comparator 1 and Comparator 2, had comparable serum concentrations of 178, 229, and 164 µg/mL, respectively. Comparator 1 had a similar clearance profile to H4H12166P through day 30, but on days 40 and 50 it exhibited a rapid increase in clearance versus H4H12166P. At day 50, H4H12166P had a mean serum antibody concentration of approximately 9 µg/mL, whereas Comparator 1 and Comparator 2 both had a 30-fold lower mean serum antibody concentration of 0.3 µg/mL. Comparator 2 exhibited the lowest exposure of the three antibodies tested, with an AUCultimate approximately 2-fold lower (1408 dia.μg/mL) compared to H4H12166P (2801 dia.μg/mL) and Comparator 1 (2708 dia.μg /mL).

[0314] The pharmacological effects of anti-C5 antibodies H4H12166P, Comparator 1 and Comparator 2 of humanized C5 mouse serum samples supplemented with human C3 were measured until day 50 and were determined ex vivo by hemolysis of the classical complementary pathway (CP) of sensitized SRBC. The mean percent hemolysis for each anti-C5 antibody is summarized in Table 28 and the mean percent hemolysis versus time is shown in Figure 5. Table 28: Percent Hemolysis Activity of the Ex Vivo Classic Track of Anti-Human C5 Antibodies Time = Time in hours after single-dose injection; Day = Study day; SEM = Standard error of the mean; NA = Not detected; NS = No sample. *For Comparator 2, day 50, n = three due to the inability of a sample to be analyzed due to technical issues

[0315] H4H12166P, Comparator 1 and Comparator 2 inhibited terminal complement hemolytic activity that appeared to correlate with antibody exposures. H4H12166P blocked greater than 85% of hemolytic activity through day 30, with activity returning to pre-dose baseline levels by day 50. Comparator 1 and Comparator 2 blocked approximately 80% of hemolytic activity through day 20 and day 10, respectively, with activity returning to baseline levels by day 30 for both.

Experience 2

[0316] In this experiment, the pharmacokinetics and pharmacodynamics of anti-C5 antibodies H4H12166P, H4H12161P, Comparator 1 and an isotype control were evaluated in humanized C5 mice (mice homozygous for expression of human C5). Total circulating C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations. Mean concentrations of human C5 in mice were determined to be 48.98 μg/mL ± 15.1 μg/mL.

[0317] Prior to antibody measurement, male and female C5 humanized mice were stratified according to human C5 levels, on average 50 μg/mL. For each anti-C5 mAb, cohorts of five mice received a single 15 mg/kg subcutaneous (s.c.) injection of H4H12166P, H4H12161P, Comparator 1, or an isotype control. All mice were bled pre-dose, 6 hours, 1, 2, 3, 4, 7, 10, 13, 21, 30 and 45 days after injection for PK analysis. Furthermore, on day 59, all rats from each cohort were euthanized and terminal bleeds were collected for PK and PD analysis. Blood was processed into serum and frozen at -80°C until analyzed.

[0318] Total antibody concentrations were determined at 12 time points and percent hemolysis activity was determined at 1 time point over a lifetime of 59 days. The total serum antibody concentrations for each anti-C5 antibody are summarized in Table 29. Mean levels of total antibody concentration versus time are shown in Figure 6. Mean PK parameters are described in Table 30. Table 29: Mean concentrations of Total serum IgG following a single subcutaneous injection of 15 mg/kg of selected anti-C5 antibodies into humanized C5 mice Time = time in hours after single-dose injection; Day = study day; SD = standard deviation; SEM = standard error of the mean; NA = Not detected; NS = no sample. Table 30: PK Parameters Cmax = maximum concentration; AUC = Area under the concentration-time curve; AUClast= AUC calculated from time zero to the moment of the last positive concentration; T1/2 = Observed estimated half-life.

[0319] Mean concentration versus time profiles show that H4H12166P, H4H12161P, Comparator 1 and isotype control reached a maximum serum concentration (Cmax) between days 1 to 3, with comparable Cmax values 1.3 times (178, 225, 183 and 221) µg/mL, respectively. H4H12166P and isotype control had similar clearance profiles, with levels remaining approximately 4μg/mL by day 59. H4H12161P exhibited faster clearance than H4H12166P and isotype control but cleared more slowly than Comparator 1. By day 59, H4H12161P had medium level serum drug level of 0.6 µg/mL, while Comparator 1 had an almost undetectable drug level of 0.08 µg/mL.

[0320] The isotype control, H4H12166P and H4H12161P exhibited comparable exposure values (AUClast) at 1.3-fold (4080, 3490 and 3040 day • μg/mL, respectively), while Comparator 1 exhibited 1.6-fold exposure lower (2240 days • μg/mL) compared to H4H12166P.

Example 12: LC-MRM-MS based assay to determine human total C5 concentration

[0321] In this Example, human total C5 serum concentrations were determined using liquid chromatography coupled to a multiple reaction monitoring mass spectrometry (LC-MRM-MS) method in a pharmacokinetic/pharmacodynamic study of anti-C5 antibody H4H12166P.

[0322] Human total C5 serum concentrations were determined by measuring the concentration of a 10 amino acid peptide contained in the C5 sequence LQGTLPVEAR (aa 1129-1138 of SEQ ID NO: 359) as a substitute for C5. Theoretically, this method can also detect the C5 division product, C5b. However, due to the instability of free C5b, serum C5b concentrations are generally low, with the majority of C5b 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 are likely to contain only negligible amounts of C5b product, if any.

Methods

[0323] For the PK/PD study, mice received a single dose of 15 mg/kg of H4H12166P by subcutaneous (s.c.) injection. All mice were bled pre-dose and one day after injection for PK analysis. Furthermore, at 10, 20, 30, 40, 50 and 60 days after injection, the mice were euthanized and the terminal bleeds were collected for analysis of PK and PD.

[0324] Human C5 was used as a reference standard for calibration; and a human C5 peptide produced with an arginine residue labeled with a stable C-terminal isotope was used as an internal standard (LQGTLPVEAR-13C615N4). The reference standard was used at concentrations ranging from 3.9 to 250 µg/mL (1:2 serial dilutions) in the serum of in-house generated C5 knockout mice in which the mouse C5 gene has been deleted (C5 -/-) . Serum from C5 -/- mice was also used as a negative control (blank). Calibration standards, samples and study serum (10 µL each) were dried and then denatured in 100 µL Tris (2-carboxyethyl) phosphine (TCEP) 8 M urea/20 mM buffer at 37°C for 1 hour. Then 10 µL of 25 nM internal standard was added to all samples. Samples were alkylated with 10 mM 2-iodoacetamide at room temperature for 30 minutes and diluted using 50 mM ammonium bicarbonate to a final volume of 500 μL. Samples were then digested by trypsin (1:20 w/w) overnight at 37°C. The C5-derived LQGTLPVEAR tryptic peptide was detected and quantified by LC-MRM-MS using a Waters Xevo TQ-S system with ACQUITY UPLC. Each processed sample (10 μL) was injected into 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 [V:V] and mobile phase B: acetonitrile: formic acid/100: 0.1 [V:V] ). Retention time and peak area were determined using Masslynx Analyst Data software (Waters). C5 analyte concentrations were calculated from the calibration curve that was constructed by plotting the peak area ratio of the C5 reference standard (C5 peptide NO: labeled LQGTLPVEAR-12C614N4 generated by tryptic digest of hC5) to internal standard (peptide stable isotope labeled C5) versus nominal concentration of C5 reference standard. Concentrations were calculated using linear regression. The lowest concentration of the C5 Reference Standard (3.9 μg/mL) was within the dynamic range of the assay and was defined as the LLOQ of the assay.

Results

[0325] Total human C5 serum concentrations were evaluated for samples collected and via tail bleeding before dosing (Pre-dose) and via terminal bleeding on days 10, 30 and 35, from the corresponding animals. Total hC5 concentrations after H4H12166P dosing were similar (within ~1 to 0.9 fold) to predict levels at days 10, 30, and 35 post dosing. Minor differences observed were not statistically significant as assessed by the Mann-Whitney test using GraphPad Prism software. Analysis of the C5/H4H12166P molar ratio demonstrated that H4H12166P remained in molar excess of C5 at day 35 after administration (Table 31). Table 31: Summary of H4H12166P PD characteristics aPercent inhibition of CP hemolytic activity was calculated from the mean values of % PC hemolysis at the indicated daytime dose versus the mean value of PC hemolysis on day 0. bDoubling Change = terminal (indicated day after dosing) C5: Pre-dose C5. SD = standard deviation; MAHA = mouse anti-human antibody; no/d = not determined

[0326] Animals with data impacted by MAHA were completely excluded from the calculations (2x day 30, 1x day 35, 2x day 40 and all 4 mice on day 50)

Example 13: Binding epitope mapping of H4H12166P to C5 by hydrogen/deuterium exchange

[0327] H/D exchange epitope mapping with mass spectrometry was performed to determine the hC5 amino acid residues [(amino acids M1-C1676 of SEQ ID No: 359) with which H4H12166P interacts. A general description of the H/D exchange method is provided in, for example, Ehring (1999) Analytical Biochemistry 267(2): 252-259; and Engen and Smith (2001) Anal. chem. 73: 256A-265A.

[0328] HDX-MS experiments were performed on an integrated Waters 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 (binary solvent manager) for the column analytical gradient, and a Synapt G2-Si mass spectrometer for peptide-peptide mass measurement.

[0329] The labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of C5 (6 pmol/μL) or C5 premixed with the antibody at 1 : 1 molar ratio was incubated with 56.2 μL of D2O labeling solution for various time points ( eg uninduced control = 0 sec, timed for 1 min and 20 min). Deuteration was quenched by transferring 50 μL of sample into 50 μL of pre-cooled buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0°C for two minutes. The quenched sample was then injected into a Waters HDX Manager for online pepsin/protease XIII digestion. Digested peptides were retained on an ACQUITY UPLC BEH C18 1.7 µm, 2.1 x 5 mm VanGuard pre-column at 0°C and eluted onto an ACQUITY UPLC BEH C18 1.7 µm, 1.0 x 50 analytical column mm over a 9 minute gradient separation of 5% -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, sweep time of 0.5 s and mass/charge range of 50-1700 Thomson units (Th).

[0330] For the identification of human C5 peptides, the LC-MSE data from the non-deuterated sample were processed and searched against the database, including human C5, pepsin, and its random sequence through the Waters ProteinLynx Global Server software (PLGS ). The identified peptides were imported into the DynamX software and filtered by two criteria: (1) minimum products per amino acid = 0.3 and (2) replication file threshold = 3. The DynamX software automatically determined the deuterium uptake of each peptide based on retention time and High Mass Accuracy (<10 ppm) at multiple time points with 3 replicates at a time.

[0331] Using the online pepsin/protease XIII column coupled to MSE data acquisition, 189 human C5 peptides were identified in the absence or presence of antibody, representing 62% sequence coverage. Five peptides significantly reduced deuteration uptake (delta centroid values >0.9 daltons with p values <0.05) when bound to H4H12166P and are shown in Table 32. Table 32: Deuteration of human C5 peptides after binding to H4H12166P

[0332] The registered peptide mass corresponds to the average value of the MH+ centroid mass of three repetitions. These peptides, corresponding to amino acids 591-599 and 775-794, had a slower rate of deuteration upon binding to H4H12166P. 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).

Example 14: Effect of anti-C5 antibodies on ocular inflammation in Experimental Autoimmune Uveitis in mice

[0333] The present study was undertaken to assess the role of C5 in experimental autoimmune uveitis (UAE). Both genetic [C5 knockout (KO), C3/C5 double KO] and pharmacological (anti-C5 antibody) experimental approaches were used.

Methods

[0334] Adult C57BL/6J mice (n = 25, Jackson labs), C5 KO (n = 13) and C3/C5 KO (n = 8) (Regeneron Pharmaceuticals Inc.) mice were used. UAE was induced by subcutaneous injection of human retinoid interphotoreceptor 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 injections every 3 days from day 5 to 28. The C5 anti-mouse antibody used in this study (M1M17628N) included an HCVR/LCVR of SEQ ID NOs: 362/363. SPECTRALIS® 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 collection of eyes and blood. Hemolysis assay with/without human C3 was performed to validate complement inhibition. Data were analyzed by ANOVA.

Results

[0335] Compared with wild-type mice, the occurrence of inflammation (30-50%) and vitreous cell cluster counts were significantly decreased in C5 KO mice (p < 0.01). Optical coherence tomography (OCT) scores in C5 KO mice also significantly reduced by 50% at week 3 (p < 0.0001). Interestingly, in C2/C5 double KO mice, there were significantly more vitreous cell clusters and higher disease scores at day 28 compared to wild-type mice (p < 0.05). In animals receiving anti-mC5 Ab (50mg/kg), the incidence of inflammation and vitreous cell clusters were significantly lower compared to no treatment or isotypic control group on day 21 (p < 0.01). At Weeks 3 and 4, OCT scores in the anti-C5 antibody-treated group were significantly lower compared with no treatment or isotype control (p < 0.0001). (Figure 7) Hemolysis assays with/without human C3 confirmed the inhibition effect of the anti-C5 antibody at week 4 (Figure 8).

Conclusion

[0336] Ocular inflammation due to UAE was attenuated by inhibition of C5 activity, either by genetic deletion or pharmacological inhibition with a specific anti-C5 antibody. C5 depletion delayed the occurrence of UAE and reduced the OCT disease score. These results indicate that C5 is a potential therapeutic target for autoimmune uveitis. Anti-C5 antibody has protective effect on UAE disease in wild-type mice. Our results also suggest that C3 may be beneficial for UAE disease in mice.

Example 15: Effect of human anti-C5 antibodies on Experimental Autoimmune Uveitis

[0337] This Example describes the effects of anti-C5 antibodies against human C5 in a mouse model of experimental autoimmune uveitis (EAU). Mice used for this study were humanized to express human C5 protein using Velocigene® technology ( Valenzuela et al., 2003, Nat. Biotechnol. 21: 652-659 ). Humanized mice were engineered to replace exon 2 through exon 41 of the murine C5 gene with exons 2 - 42 of the human C5 gene (described in US Patent Application Publication 2015/0313194, incorporated herein in its entirety).

Methods

[0338] Adult male mice were immunized subcutaneously on each thigh with 150 µg of human retinoid binding interphotoreceptor peptide (IRBP) 1-20 (GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO: 364) (Avichezer et al 2000, Invest. Ophthalmol. Vis. Sci. 41: 127-131) in 0.2 ml of CFA emulsion, supplemented with the Mycobacterium tuberculosis strain H37RA up 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, promoting a Th1 polarization of the immune response (Thurau et al., 1997, Clin. Exp. Immunol. 109: 370-376, Silver et al., 1999, Invest.Ophthalmol.Vis.Sci.40:2898-2905). The animals' body weights were monitored twice a week.

[0339] Ophthalmic examinations were performed on day 1, before UAE induction, and on days 7, 14, 21, and 28. Mice were anesthetized with ketamine (120 mg/kg, IP) and xylazine (5 mg/kg, IP ). The pupils were dilated using a 0.5% Tropicamide ophthalmic solution, and the base of the eye was examined using a contact lens with a base camera on a Heidelberg Spectralis platform (HRA) + OCT system (Heidelberg Engineering, Carlsbad, CAUSE).

[0340] A series of 61 lateral optical sections were obtained for each eye using the OCT function on the Spectralis HRA + OCT system (Heidelberg Engineering, Carlsbad, CA, USA). The OCT image area was centered on the optic disc allowing for an equal image above and below the optic nerve head. Retinal thickness was measured as the distance between the base of the RPE layer and the inner limiting membrane of the eye. Measurements were taken at 1500 μm from the optic disc, and values from 4 different retinal quadrants (eg, superior, inferior, temporal, and nasal) were calculated for an average retinal thickness of the eye.

[0341] The severity of inflammatory cell infiltration into the vitreous was also graded on OCT images by assessing the mean cluster number of inflammatory cells in the vitreous in four lateral OCT scans that transected the optic nerve, per eye.

[0342] A 4-point scale was developed for assessing disease severity on OCT images (OCT scores) (Table 33). Table 33: In vivo UAE score using Optical Coherence Tomography (OCT)

Statistical analysis

[0343] Statistical analyzes for parametric data (body weight, cluster of inflammatory cells in the vitreous and retinal thickness) were performed by one-way ANOVA and Tukey's multiple comparison test. For non-parametric data (OCT scores and histological scores), analyzes were performed by Kruskal-Wallis test and Dunn test with GraphPad Prism software version 5.0d, relative to isotypy or untreated groups. Data show mean values ± SEM. A p-value of less than 0.05 was considered statistically significant.

Results

[0344] In a first study (Study A), mice were treated subcutaneously every 3 days from day 5 with antibody isotype control (50 mg/kg) or 10 mg/kg or 50 mg/kg of H4H12170P. Treatment with 10 mg/kg of H4H12170P resulted in a reduction in inflammation and retinal damage (Figure 9). Mice treated with 10 mg/kg of H4H12170P also showed a statistically significant reduction in OCT scores on day 21 and day 28 (Figure 10).

[0345] In a second study (study B), mice were treated subcutaneously every 3 days from the sixth day with isotype control (10 mg/kg), 3 mg/kg or 10 mg/kg of H4H12166P or with Comparator 2 (see Example 2 here, "Control Constructs Used in the Examples Below"). Treatment with H4H12166P at 3 mg/kg or 10 mg/kg produced a dose-related reduction in OCT scores that was statistically significant on days 14 through 28 (Figure 11). Treatment with 10 mg/kg of H4H12166P in C5 humanized mice starting 6 days after UAE induction resulted in a dose-related reduction in retinal inflammation and damage as determined by OCT obtained on days 14 to 28 (Figure 12) .

[0346] For both studies, non-invasive evaluation in life by OCT showed a progressive development of inflammation, increased retinal thickness and morphological abnormalities in control animals after immunization with IRBP.

Conclusion

[0347] These experiments provide additional pharmacological evidence that C5 plays a role in the pathogenesis of autoimmune uveitis. Pharmacological depletion of human C5 by fully human anti-C5 antibodies postponed the incidence of UAE and reduced disease severity, establishing the efficacy of these antibodies in autoimmune uveitis.

Example 16: Effect of anti-C5 antibodies on renal ischemia-reperfusion injury

[0348] The present study was undertaken to assess the role of C5 in renal ischemia-reperfusion injury. Both genetic (using C3 knockout and C5 knockout mice) and pharmacological (using anti-C5 antibodies) were used. The ischemia-reperfusion model was induced by bilateral renal pedicle clamping for 45 min followed by 48 h of reperfusion. Sham laparotomy served as controls. Anti-C5 antibody was administered intravenously at 50 mg/kg, in a single dose, immediately after ischemia (curative); or subcutaneously as two doses, day -1 and day 1 (preventive) surgery. The anti-C5 antibody used for this study was M1M17628N comprising HCVR/LCVR of SEQ ID NO: 362/363. Blood urea nitrogen (BUN) and serum creatinine markers were used to assess levels of disease and protection in mice. Table 34: Percent change in blood urea nitrogen levels in mice treated with anti-C5 antibody (M1M17628N) in preventive and therapeutic modes Table 35: Percent change in serum creatinine levels in mice treated with anti-C5 antibody (M1M17628N) in preventive and therapeutic modes

[0349] 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 reductions in blood urea nitrogen and serum creatinine levels. The anti-C5 antibody showed functional protection in the RIRI model in preventive and therapeutic modes (Tables 34-35).

Example 17: Effect of anti-C5 antibodies on Lupus Nephritis

[0350] This example describes the effectiveness of anti-C5 antibodies in the treatment of lupus nephritis in a mouse model.

[0351] Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, production of autoantibodies, and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and specific organs, with lupus nephritis being 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 antibodies in the treatment of lupus nephritis was studied in NZBWF1 mice, a spontaneous mouse model of lupus nephritis (Yang et al 1996, PNAS). Mice develop autoimmune disease similar to human SLE, autoantibodies to nuclear antigens and cell membrane proteins, hypergammaglobulinemia, albuminuria, proteinuria, early immune complex glomerulonephritis, and renal failure and end-stage renal disease between 35 and 50 weeks of age.

[0352] For this study, 25 week old NZBWF1 mice were treated subcutaneously with 30 mg/kg of isotype control, or anti-C5 antibodies twice a week for 8 weeks followed by three times a week for 10 weeks. The C5 anti-mouse antibodies used for this study were M1M17628N and M1M17627N, comprising HCVR/LCVR of SEQ ID NOs: 362/363 and 365/366, respectively.

[0353] Treatment with anti-C5 antibodies significantly improved the survival rate in mice (Figure 13). Both antibodies improved albuminuria at 8-14 weeks of treatment (Figure 14) and blood urea nitrogen levels at 12-16 weeks of treatment (Figure 15).

Example 18: Effect of anti-C5 antibodies against astrocyte cell death

[0354] Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS) that primarily affects the optic nerve and spinal cord. In NMO, anti-aquaporin-4 autoantibodies (AQP4-Ab) damage astrocytes by activating complement-dependent cytotoxicity (CDC). The objectives of this study were to evaluate the role of the complement system in the progression of NMO and the use of an antibody against a complement protein as a potential therapeutic treatment for NMO.

[0355] Rat primary cortical astrocytes were obtained from the cerebral cortex of rat pups after birth and were cultured with AQP4-Ab (antibody "rAb-53" from U.S. Patent Application Publication 2014/0170140; Bennett et al 2009, Ann. Neurol 66: 617-629) and complement proteins to demonstrate cell-mediated cytotoxicity. Then, the experiments were repeated with the addition of an anti-C5 antibody to demonstrate blocking astrocyte cell destruction.

[0356] To quantify cell death, a CytoTox-Glo™ luminescence cytotoxicity assay was performed. The assay used various concentrations of anti-C5 antibodies (0.001μg/mL, 0.01μg/mL, 0.1μg/mL, 1μg/mL, 10μg/mL, 100μg/mL, or 1000μg/mL) or an isotype control antibody .

[0357] In order to determine whether anti-C5 antibody could block AQP4-Ab-induced CDC, astrocytes were plated and the CytoTox-GloTM cytotoxicity assay was repeated to find an optimal dose of AQP4-Ab for this coating. The optimal concentration of AQP4-Ab was found to be 50 μg/mL, and in a subsequent experiment a constant dose of AQP4-Ab (50 μg/mL) was used while the dose of anti-C5 antibody was varied. As shown in Figure 16, a decrease in RLU (averaging 300k to averaging 100k) was observed with increasing amounts of anti-C5 antibody, demonstrating that the anti-C5 antibody blocked astrocyte cell death. For both experiments, the RLU did not vary with the isotype control antibody. As shown in Figure 16, anti-C5 antibodies inhibited AQP4 Ab-induced cytotoxicity in primary cortical astrocytes with an IC50 of 15-17 nM.

[0358] In a subsequent study, anti-AQP4 antibody and anti-C5 antibody will be injected into rat brains to assess therapeutic efficacy against astrocyte complement-mediated cytotoxicity in the CNS.

Example 19: Endothelial Assay

[0359] This example describes an in vitro glomerular endothelial assay to examine whether anti-C5 antibodies block C5b-9 and C3 deposition.

[0360] Reproducible methods to assess the inhibitory effects of drug candidates on complement activation are essential for preclinical development. Due to the complexity of complement activation pathways, an assay must use cells and parameters relevant to the given therapeutic indication. Here, using an immortalized human glomerular endothelial cell line (HGECs), a model of C3 and C5 complement deposition was validated for use in evaluating the blocking activity of anti-C3 or C5 mAbs.

Methods

[0361] Primary human renal glomerular endothelial cells (HGEC; Cell Biologics) were seeded overnight in complete medium in 96-well plates of clear black base coated with Collagen I. Cells were treated with PBS (control) or activated for 10 min with 10 µM ADP. After washing with PBS, 50% human serum (complement-preserved, C3-depleted, or C5-depleted) was added for 4 hours. Anti-C5 antibodies were added at 1 mg/ml to the serum before treatment. Cells were washed and fixed and probed with anti-C3b antibodies (Thermofisher) and/or anti-C5b-9 antibodies (Abcam), secondary antibodies and stained with DAPI. Images were captured in ImagExpress and high content image analysis was used to quantify the fluorescent coloration for each image and calculate the average per condition.

Results

[0362] C3 and C5b-9 deposition was observed in ADP-activated HGECs exposed to normal human serum, but not in non-activated HGECs (C3: 1.5x107 ± 1.0x107; C5: 7.9x106 ± 6.6x106 , P < 0.05 vs HGEC not activated by ADP). C3 and C5b-9 deposition was significantly reduced in ADP-activated HGEC exposed to serum depleted in C3 or C5 (C3: 3.3x105 ± 4.8x104; C5: 1.5x106 ± 6.0x105, P < 0.05 ). Addition of an anti-C5 blocking mAb significantly reduced deposition of C5b-9 derived from normal human serum on ADP-activated HGEC, deposition was comparable to C5-depleted serum (C5 mAb: 1.02x106 ± 6.0x105, mAb 3.7x106 ± 1.6x106 control, P < 0.05 vs. mAb control).

Conclusion

[0363] These data demonstrate the usefulness of an in vitro human glomerular endothelial assay to model C3 and C5 complement deposition. In addition to in vitro evaluation, this assay offers potential as a translational model to evaluate anticomplement strategies in kidney disease using patient-derived serum samples.

[0364] 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 (18)

1. Use of an antibody or antigen-binding fragment thereof that specifically binds to C5, characterized in that it is in the manufacture of a medicament and/or pharmaceutical composition for treating or ameliorating at least one symptom or indication of a disease or C5-associated disorder in a human subject in need thereof, wherein the antibody or antigen-binding fragment thereof that specifically binds to C5 comprises: a heavy chain variable region comprising: an HCDR1 consisting of a sequence of amino acids as set forth in SEQ ID NO: 100, an HCDR2 consisting of an amino acid sequence as set forth in SEQ ID NO: 102, and an HCDR3 consisting of an amino acid sequence as set forth in SEQ ID NO: 104; and a light chain variable region comprising an LCDR1 consisting of an amino acid sequence as set forth in SEQ ID NO: 108, an LCDR2 consisting of an amino acid sequence as set forth in SEQ ID NO: 110 and an LCDR3 consisting of a sequence of amino acids as set forth in SEQ ID NO: 112; and wherein the disease or disorder is selected from the group consisting of atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, geographic atrophy, uveitis, neuromyelitis optica, multiple sclerosis, stroke, Guillain Barré syndrome, injury traumatic brain injury, Parkinson's disease, a disorder of inappropriate or unwanted complement activation, a complication of hemodialysis, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, an inflammatory disorder, inflammation of an autoimmune disease, Crohn's disease, adult respiratory distress syndrome, thermal injury, burns, frostbite, a post-ischemic reperfusion condition, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia , epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, renal ischemia-reperfusion injury, lupus nephritis, post-pump syndrome on cardiopulmonary bypass syndrome post-pump cardiopulmonary bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease, sepsis, an immune complex disorder, an autoimmune disease, a kidney disorder, rheumatoid arthritis, systemic lupus erythematosus, lupus erythematosus nephritis systemic disease, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary embolism, pulmonary infarction, pneumonia and myasthenia gravis. 2. Use according to claim 1, characterized in that the disease or disorder is atypical hemolytic uremic syndrome. 3. Use according to claim 1, characterized in that the disease or disorder is paroxysmal nocturnal hemoglobinuria. 4. Use according to claim 1, characterized in that the pharmaceutical composition is formulated to be used in combination with a second therapeutic agent. 5. Use according to claim 4, characterized in that the second therapeutic agent is selected from the group consisting of an anticoagulant, an anti-inflammatory drug, an antihypertensive, an immunosuppressive agent, a hypolipidemic agent, a anti-CD20 agent, rituximab, an anti-TNF agent, infliximab, an anticonvulsant agent, a C3 inhibitor, a second anti-C5 antibody, and an antithrombotic agent. 6. Use according to claim 1, characterized in that the pharmaceutical composition is formulated to be used subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially. 7. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98. 8. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 106. 9. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98 and a variable region light chain comprising the amino acid sequence shown in SEQ ID NO: 106. 10. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 353. 11. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO: 354. 12. Use according to claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 353; and a light chain comprising an amino acid sequence of SEQ ID NO: 354. 13. Use according to claim 12, characterized in that the antibody or antigen-binding fragment thereof is an antibody. 14. Use according to claim 13, characterized in that the disease or disorder is paroxysmal nocturnal hemoglobinuria. 15. Use according to claim 13, characterized in that the disease or disorder is myasthenia gravis. 16. Use according to claim 13, characterized in that the disease or disorder is neuromyelitis optica. 17. Use according to any one of claims 14 to 16, characterized in that the antibody is formulated to be used by the individual intravenously and subcutaneously. 18. Use according to claim 17, characterized in that the antibody is formulated to be used in combination with a second therapeutic agent.