Anti-C5 antibodies and their use

Abstract

To provide monoclonal antibodies that bind to the complement factor 5 (C5) protein, and methods of use thereof.SOLUTION: In various embodiments of the present invention, the antibodies are fully human antibodies that bind to C5 protein. In some embodiments, the antibodies of the present invention are useful for inhibiting or neutralizing C5 activity, thus providing a means of treating or preventing a C5-related disease or disorder in humans. In some embodiments, the present invention provides an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, e.g., a half-life of more than 10 days.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application was filed on June 13, 2017, as a PCT international patent application, and claims priority over U.S. Provisional Applications No. 62 / 349,705 (filed June 14, 2016); No. 62 / 405,561 (October 7, 2016); and No. 62 / 422,107 (November 15, 2016) (each disclosure being incorporated herein by reference as a whole).

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

[0003] Background of the Invention The complement system is a group of plasma proteins that, when activated, lead to target cell lysis and promote phagocytosis through opsonization. Complement is activated by a series of proteolytic processes via three main pathways: the classical pathway, typically activated by immune complexes; an alternative pathway, which can be induced by unprotected cell surfaces; and the mannose-binding lectin pathway. All three pathways of the complement cascade converge on the proteolytic cleavage of the complement component 5 (C5) protein. Cleavage of complement component 5 (C5) results in the generation of fragments C5a and C5b, a critical process during the activation of the complement cascade. C5a can produce multifaceted physiological responses through its receptor (Non-Patent Literature 1). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates oxidative bursts, and induces the release of various inflammatory mediators such as histamine or cytokines. C5b mediates the formation of membrane invasion complexes (MACs, or C5b-9), leading to cell lysis in the later stages of complement-dependent cell damage (CDC). Furthermore, in nucleated cells resistant to C5b-9 cell lysis, sublytic amounts of C5b-9 can induce cell activation, resulting in cell proliferation, the generation of pro-inflammatory mediators, and the production of extracellular matrix.

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

[0005] [Patent Document 1] U.S. Patent No. 9206251 [Patent Document 2] U.S. Patent No. 9107861 [Patent Document 3] U.S. Patent No. 9079949 [Patent Document 4] U.S. Patent No. 9051365 [Patent Document 5] U.S. Patent No. 8999340 [Patent Document 6] U.S. Patent No. 8883158 [Patent Document 7] U.S. Patent No. 8241628 [Patent Document 8] U.S. Patent No. 7999081 [Patent Document 9] U.S. Patent No. 7432356 [Patent Document 10] U.S. Patent No. 7361339 [Patent Document 11] U.S. Patent No. 7279158 [Patent Document 12] U.S. Patent No. 6534058 [Patent Document 13] U.S. Patent No. 6355245 [Patent Document 14] U.S. Patent No. 6074642 [Patent Document 15] U.S. Patent Publication No. 20160299305 [Patent Document 16] U.S. Patent Publication No. 20160051673 [Patent Document 17] U.S. Patent Publication No. 20160031975 [Patent Document 18] U.S. Patent Publication No. 20150158936 [Patent Document 19] U.S. Patent Publication No. 20140056888 [Patent Document 20] U.S. Patent Publication No. 20130022615 [Patent Document 21] U.S. Patent Publication No. 20120308559 [Patent Document 22] WO2015198243 [Patent Document 23] WO2015134894 [Patent Document 24] WO2015120130 [Patent Document 25] EP2563813B1 [Patent Document 26] EP2328616B1 [Patent Document 27] EP2061810B1 [Non-patent literature]

[0006] [Non-Patent Document 1] Monk et al 2007,Br.J.Pharmacol.152:429-448 [Overview of the project] [Problems that the invention aims to solve]

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

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

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

[0010] In the first phase, the present invention relates to isolated recombinant proteins that specifically bind to C5 proteins. The present invention provides a monoclonal antibody or its antigen-binding fragment. In some embodiments, the antibody is a fully human monoclonal antibody.

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

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

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

[0014] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) which includes one of the HCVR amino acid sequences listed in Table 1 paired with one of the LCVR amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained within one of the example anti-C5 antibodies listed in Table 1. In a particular embodiment, 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 an anti-C5 antibody or antigen-binding fragment comprising HCVR and LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 having 5 or fewer amino acid substitutions, and the LCVR comprises an amino acid sequence listed in Table 1 having 2 or fewer amino acid substitutions. For example, the present invention provides an anti-C5 antibody or antigen-binding fragment comprising HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO. 98 having 5 or fewer amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO. 106 having 2 or fewer amino acid substitutions. In another embodiment, the present invention provides an anti-C5 antibody or antigen-binding fragment thereof comprising HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 98 having at least one amino acid substitution, and the LCVR comprises the amino acid sequence of SEQ ID NO: 106 having one amino acid substitution.

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

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

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

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

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

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

[0021] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid pair (HCDR3 / LCDR3) containing any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to a particular embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the example anti-C5 antibodies listed in Table 1. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 56 / 64 (e.g., H4H12161P), 104 / 112 (e.g., H4H12166P), 144 / 112 (e.g., H4H12166P5), and 208 / 216 (e.g., H4H12170P).

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

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

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

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

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

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

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

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

[0030] In certain embodiments, the present invention provides antibodies and antigen-binding fragments exhibiting pH-dependent binding to C5. For example, the present invention includes antibodies and antigen-binding fragments that bind to C5 with higher affinity at neutral pH than at acidic pH (i.e., reduced binding at acidic pH).

[0031] In certain embodiments, the present invention provides antibodies and antigen-binding fragments exhibiting improved pharmacokinetic and pharmacodynamic properties, for example, the present invention provides an anti-C5 antibody having an extended serum half-life. In certain embodiments, the anti-C5 antibody of the present invention has a serum concentration higher than 10 μg / mL up to day 40 in C5 humanized mice. In certain embodiments, the anti-C5 antibody of the present invention blocks CP hemolysis and AP hemolysis up to day 35 when administered to C5 humanized mice.

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

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

[0034] The present invention also provides an antibody and its antigen-binding fragment that bind to the same epitope as a reference antibody or its antigen-binding fragment, where HCVR and LCVR are the HCVR and LCVR sequences listed in Table 1, respectively. The present invention provides an antibody and its antigen-binding fragment that bind to the same epitope as a reference antibody or its antigen-binding fragment, the HCVR / LCVR amino acid sequence pair having SEQ ID NO: 98 / 106.

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

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

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

[0038] In certain embodiments, the antibody or antigen-binding fragment of the present invention has dual specificity, comprising a first binding specificity to a first epitope of the C5 protein and a second specificity to a second epitope of the C5 protein, where the first and second epitopes are different. , and they do not overlap.

[0039] In certain embodiments, the antibody and antigen-binding fragment of the present invention have an IC50 ratio of less than 0.5 nM relative to C5a. 50It binds with. In certain embodiments, the antibody comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 306, 322, and 338. In certain embodiments, the antibody comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298, 314, 330, and 346.

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

[0041] In certain embodiments, the present invention provides an isolated recombinant monoclonal anti-C5 antibody or its antigen-binding fragment having one or more of the following features: (a) comprising a set of six CDRs containing the amino acid sequence of SEQ ID NOs: 100-102-104-108-110-112; (b) having a dissociation constant (K) less than 0.2 nM at 25°C as measured by surface plasmon resonance assay. D (c) Binds to human C5; (c) K0.3 nM as measured by surface plasmon resonance assay at 37°C. D (d) Binds to human C5; (d) K is measured by surface plasmon resonance assay and is less than 0.4 nM at 37°C. D (e) IC2 is less than 3nM and binds to the human C5 mutant (R885H); 50 (f) inhibits classical pathway (CP)-mediated hemolysis in human serum; (f) IC < 27 nM 50 It inhibits alternative pathway (AP)-mediated hemolysis in human serum; (g) IC < 21 nM 50 It inhibits CP-mediated hemolysis in monkey serum; (g) IC < 10 nM 50 (h) Inhibits AP-mediated hemolysis in monkey serum; (h) In C5 humanized mice, serum half-life longer than 10 days (t 1 / 2 (i) having a serum concentration higher than 10 μg / mL up to day 40 when administered to C5 humanized mice; (j) blocking CP-mediated hemolysis up to day 50 in C5 humanized mice; and (k) binding to one or more amino acids in the alpha and / or beta chains of SEQ ID NO: 359, where the antibody does not bind to the C5a anaphylatoxin domain of C5.

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

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

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

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

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

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

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

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

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

[0051] The present invention also provides nucleic acid molecules encoding LCVR, where LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), where the LCDR1-LCDR2-LCDR3 amino acid sequence set is an example of anti-C5, as listed in Table 1. This is defined by one of the antibodies.

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

[0053] In a relevant context, the present invention provides recombinant expression vectors capable of expressing polypeptides comprising the heavy chain or light chain variable region of an anti-C5 antibody. For example, the present invention includes recombinant expression vectors comprising nucleic acid molecules encoding any of the above-mentioned nucleic acid molecules, i.e., any of the HCVR, LCVR, and / or CDR sequences shown in Table 2. Methods for producing antibodies or parts thereof by culturing host cells into which such vectors have been introduced, as well as by culturing host cells under conditions that enable the production of antibodies or antibody fragments, and by recovering the antibodies and antibody fragments thus produced, are also included within the scope of the present invention.

[0054] In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or its antigen-binding fragment that specifically binds to C5, and a pharmaceutically acceptable carrier. In a related aspect, the present invention features a composition that is a combination of an anti-C5 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that can be advantageously combined with the anti-C5 antibody. Examples of agents that can be advantageously combined with the anti-C5 antibody include, but are not limited to, other agents that bind to and / or inhibit C5 activity (including other antibodies or their antigen-binding fragments) and / or agents that do not directly bind to C5 but nevertheless treat or alleviate at least one symptom or sign of a C5-related disease or disorder. Further combination therapies and co-formulations comprising the anti-C5 antibody of the present invention are disclosed elsewhere herein.

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

[0056] The present invention also includes the use of the anti-C5 antibody or its antigen-binding fragment in the manufacture of a drug for treating a disease or disorder in which the blockage of C5 binding and / or activity would be beneficial.

[0057] Other embodiments will become apparent from the examination of the detailed description below. [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 shows the dose-dependent inhibition of C5a levels by the anti-C5 antibody H4H12166P, as determined by ELISA (as described in Example 9 of this specification). [Figure 2] Figure 2 shows the total serum concentration over time after a single 15 mg / kg intravenous injection of H4H12166P, H4H12161P, or control drug 2 in male cynomolgus monkeys (as described in Example 10 herein). Concentration-time profiles were plotted up to the first dose after a limit of quantification (BLQ) result, where applicable (this was imputed as LLOQ / 2). Each data point represents the mean (±SD) (n=4 animal groups); concentrations considered affected by ADA were excluded from one animal in the H4H12166P group and one animal in the H4H12161P group, initiated on days 36 and 29, respectively. LLOQ = limit of quantification. [Figure 3A] Figure 3 shows the percentage of hemolysis over time in ex vivo erythrocyte assays (A) classical and (B) alternative pathways after a single intravenous injection of H4H12166P, H4H12161P, or control drug 2 into male cynomolgus monkeys. The percentage of hemolysis, calculated as the ratio of experimental lysis to maximum lysis by subtracting the background lysis percentage from both values, is related to the amount of C5 inhibited by the specific anti-C5 antibody present in the serum at a given time. Each data point represents the mean (±SD). [Figure 3B]Figure 3 shows the percentage of hemolysis over time in ex vivo erythrocyte assays (A) classical and (B) alternative pathways after a single intravenous injection of H4H12166P, H4H12161P, or control drug 2 into male cynomolgus monkeys. The percentage of hemolysis, calculated as the ratio of experimental lysis to maximum lysis by subtracting the background lysis percentage from both values, is related to the amount of C5 inhibited by the specific anti-C5 antibody present in the serum at a given time. Each data point represents the mean (±SD). [Figure 4] Figure 4 shows the total serum concentration versus time profile of selected anti-C5 antibodies in humanized mice for C5 (as described in Example 11 of this specification). Humanized C5 mice were administered a single subcutaneous dose of 15 mg / kg of H4H12166P, control 1, or control 2. Each data point represents the mean ± sem (n=4-5, respectively). Serum antibody concentrations were monitored 1, 10, 20, 30, and 40 days after injection using a sandwich ELISA. [Figure 5] Figure 5 shows the percentage of hemolysis versus time in an ex vivo complement classical pathway hemolysis assay for selected anti-C5 antibodies in humanized mice for C5. Humanized C5 mice were administered a single 15 mg / kg subcutaneous dose of H4H12166P, control 1, or control 2. Each data point represents mean ± sem (n=4-5, respectively). Serum hemolysis percentage was monitored before administration and at 10, 20, 30, 40, and 50 days after injection. The hemolysis percentage, calculated as the ratio of experimental lysis to maximum lysis (both values ​​minus background lysis percentage), is related to the amount of C5 inhibited by the specific anti-C5 antibody present in the serum at a given time point. [Figure 6]Figure 6 shows the total serum concentration versus time profile of selected anti-C5 antibodies in humanized mice for C5 (as described in Example 11 of this specification). Mice were administered a single 15 mg / kg subcutaneous dose of H4H12166P, H4H12161P, control 1, or IgG4P isotype control. Each data point represents the mean ± sem (n=5 for each). Serum antibody levels were monitored using sandwich ELISA at 6 hours, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45, and 59 days after injection. [Figure 7] 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 (as described in Example 14 of this specification). ****p<0.0001, bilateral ANOVA: treatment with anti-C5 antibody at 50 mg / kg vs. untreated or isotype control. [Figure 8] Figure 8 shows the inhibition of classical pathway hemolysis by the anti-C5 antibody M1M17628N in the absence of C3 (A) and in the presence of 80 μg / mL of human C3 (B) (as described in Example 14 of this specification). [Figure 9] Figure 9 shows the cell population size in C5 humanized mice treated with isotype control or anti-human C5 antibody H4H12170P 10 mg / kg or 50 mg / kg (as described in Example 15 of this specification). n=8-12 eyes for each group. [Figure 10] Figure 10 is a graph showing OCT scores in C5 humanized mice treated with isotype control or anti-human C5 antibody H4H12170P 10 mg / kg or 50 mg / kg (as described in Example 15 of this specification). n=8-12 eyes for each group. [Figure 11] Figure 11 is a graph showing OCT scores in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg / kg or 10 mg / kg, or control drug 2 at 10 mg / kg. Each group had n=6–12 eyes (as described in Example 15 of this specification). [Figure 12] Figure 12 shows the cell population size in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P 3 mg / kg or 10 mg / kg, or control drug 2 10 mg / kg. Each group had n=6 to 12 eyes (as described in Example 15 of this specification). [Figure 13] Figure 13 shows the survival curves of NZBWF1 mice treated with isotype control or anti-C5 antibody M1M17628N or M1M17627N (as described in Example 17 of this specification). [Figure 14A] Figure 14 shows (A) urinary albumin levels and (B) urinary albumin levels normalized to urinary creatinine in NZBWF1 mice treated with isotype control or anti-C5 antibody M1M17628N or M1M17627N (as described in Example 17 of this specification). [Figure 14B] Figure 14 shows (A) urinary albumin levels and (B) urinary albumin levels normalized to urinary creatinine in NZBWF1 mice treated with isotype control or anti-C5 antibody M1M17628N or M1M17627N (as described in Example 17 of this specification). [Figure 15] Figure 15 shows the levels of blood urea nitrogen in NZBWF1 mice treated with an isotype control or the anti-C5 antibody M1M17628N or M1M17627N (as described in Example 17 of this specification). [Figure 16] Figure 16 is a graph showing the inhibition of antibody-dependent cell-mediated cytotoxicity of astrocytes by anti-C5 antibodies H4H12166P, H4H12170P, control drug 1, and control drug 2, as described in Example 18. [Modes for carrying out the invention]

[0059] Detailed explanation Before describing the methods of the present invention, it should be noted that, naturally, the methods and experimental conditions described are subject to change, and therefore this invention is not limited to the specific methods and experimental conditions described. Also, naturally, the scope of the present invention is limited solely by the appended claims, and therefore, the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference as a whole.

[0061] definition The term "C5," also called "complement component 5" or "complement factor 5," refers to a serum protein in the complement cascade. The C5 protein is a 1676-amino acid protein comprising two chains, alpha and beta. The protein represents the convergence point of three complement activation pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin pathway. The amino acid sequence of the full-length C5 protein is exemplified by the amino acid sequence provided in GenBank as acceptance number NP_001726.2 (SEQ ID NO: 355). The term "C5" includes recombinant C5 proteins or fragments thereof. This term also encompasses C5 proteins or fragments thereof coupled to signal sequences such as, for example, a histidine tag, mouse or human Fc, or ROR1. For example, this term includes sequences exemplified by the sequence shown in SEQ ID NO: 356 or 357, which contains a histidine tag at the C-terminus coupled to amino acid residues 19–1676 of the full-length C5 protein. This term also includes protein variants that have either an R885H or R885C mutation and contain a histidine tag at the C-terminus coupled to amino acid residues 19–1676 of the full-length C5 protein.

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

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

[0064] CDR residues not in contact with the antigen can be identified by molecular modeling and / or empirically from the region of the Kabat CDR outside the Chothia CDR, based on previous studies (e.g., residues H60-H65 in CDRH2 are often unnecessary). If a CDR or its residues are deleted, it is usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such a sequence. The position of the substitution within the CDR and the amino acid to be substituted can also be selected empirically. The empirical substitution may be either a conserved or non-conserved substitution.

[0065] The fully human anti-C5 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention comprises antibodies and antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in the framework and / or CDR region are mutated to the corresponding residues of the germline sequence from which the antibody was derived, or to the corresponding residues of another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody was induced. In other embodiments, only specific residues are mutated back to the original germline sequence, for example, only mutated residues found in the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally induced). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, where specific individual residues are mutated to corresponding residues in a specific germline sequence, while other specific residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. When antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (if applicable), or reduced immunogenicity. Antibodies and antigen-binding fragments obtained by this general method are included within the scope of the present invention.

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

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

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

[0069] The terms "specifically bind" or "specifically bind to," or similar, mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least approximately 1 x 10⁻¹⁶. -8 M or less (for example, a smaller K) DThe equilibrium dissociation constant may be characterized by (showing a tighter binding). Methods for determining whether two molecules bind specifically are well known in the art, and these include, for example, equilibrium dialysis and surface plasmon resonance. The antibodies described herein may be characterized by surface plasmon resonance, for example, BIACORE TM It has been identified by and specifically binds to C5. Furthermore, a multi-specific antibody that binds to one domain of C5 and one or more further antigens, or a bispecific antibody that binds to two different regions of C5, is nevertheless considered a “specifically binding” antibody as used herein.

[0070] The term "high affinity" refers to antibodies that exhibit surface plasmon resonance, such as BIACORE. TM Alternatively, it can be measured by solution affinity ELISA, and at least 10 -8 M; preferably 10 -9 M;comfort10 -10 M, more preferably 10 -11 M, more preferably 10 -12 M's K D This refers to an mAb that has binding affinity to C5, which is represented as follows.

[0071] The terms "slow dissociation rate," "Koff," or "kd" refer to surface plasmon resonance, e.g., BIACORE TM Determined by, 1x10 -3 s -1 Or less, preferably 1x10 -4 s -1 This refers to an antibody that dissociates from C5 at a rate constant of or less than that.

[0072] As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically available, synthetically, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” refer to one or more fragments of an antibody that retain the ability to bind to the C5 protein.

[0073] In certain embodiments, the antibody or antibody fragment of the present invention comprises a ligand or therapeutic portion ("immunoconjugate"), a second anti-C5 antibody, or a C5-related disease. It can be combined with any other therapeutic component that is useful for treating the disorder.

[0074] As used herein, “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies (Abs) with different antigen specificities (for example, an isolated antibody or fragment thereof that specifically binds to C5 substantially does not contain Abs that specifically bind to antigens other than C5).

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

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

[0077] The term "K" used in this specificationD This term is intended to refer to the equilibrium dissociation constant of a specific antibody-antigen interaction.

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

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

[0080] When referring to nucleic acids or their fragments, the term "substantial identity" or "substantially identical" means that they are optimally aligned with another nucleic acid (or its complementary strand) including appropriate nucleotide insertions or deletions, as considered below, such as FASTA, BLAST, or GAP. The sequence identity is measured by any known algorithm for sequence identity, demonstrating that nucleotide sequence identity exists in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity with respect to a reference nucleic acid molecule may, in certain examples, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0081] The term “substantial similarity” or “substantially identical” as applied to polypeptides means that two peptide sequences share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99%, when optimally aligned by a program such as GAP or BESTFIT using default gap weights. Preferably, non-identical residue positions are distinguished by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the similarity percentage or similarity can be adjusted up to compensate for the conservative nature of the substitutions. 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 having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is a change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443 45 (incorporated herein by reference). A "moderately conservative" permutation is a change that has a non-negative value in the PAM250 log-likelihood matrix.

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

[0083] The phrase “therapeutic dose” refers to the amount administered to produce the desired effect. The exact amount depends on the purpose of the treatment and can be determined using techniques known to those skilled in the art (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

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

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

[0086] The terms "prevent," "preventing," or "prevention" refer to inhibiting the appearance of C5-related disease or disorder, or signs of such disease or disorder, upon administration of the antibody of the present invention.

[0087] Antibody antigen-binding fragments Unless otherwise specifically indicated, the term “antibody” as used herein is understood to include antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “complete antibody molecules”), and furthermore, their antigen-binding fragments. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc., as used herein include naturally occurring, enzymatically obtained, synthetically, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. The terms “antigen-binding fragment” or “antibody fragment” as used herein refer to one or more fragments of an antibody that retain the ability to specifically bind to the C5 protein. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multiselective antigen-binding molecule. Antibody antigen-binding fragments can be derived from a complete antibody molecule using any suitable standard technique, such as protein lysis digestion or recombinant genetic engineering, which includes manipulation and expression of DNA encoding the antibody variable domain and (optionally) the constant domain. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or, by the use of molecular biology techniques, for example, to position one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

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

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

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

[0091] Like complete antibody molecules, antigen-binding fragments can be monospecific or multiselective (e.g., bispecific). A multiselective antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multiselective antibody form, including the example bispecific antibody forms disclosed herein, can be adapted for use in the context of the antibody antigen-binding fragment of the present invention using conventional techniques available in the art.

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

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

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

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

[0096] Generally, VELOCIMMUNE (R)Mice are loaded with the target antigen, and lymphocytes (e.g., B cells) are recovered from the mice expressing the antibody. The lymphocytes may be fused with myeloma cell lines to produce immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains may be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0097] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired features, including affinity, selectivity, and epitope, as described in the experimental section below. The mouse constant region is replaced with the desired human constant region to generate the fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. The selected constant region may vary depending on the specific application, but high-affinity antigen binding and target specificity features reside in the variable region.

[0098] bioequivalence The anti-C5 antibodies and antibody fragments of the present invention include proteins that have a different amino acid sequence from the described antibodies but retain the ability to bind to the C5 protein. Such mutant antibodies and antibody fragments have one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially equivalent biological activity to the described antibodies. Similarly, the DNA sequences encoding the antibodies of the present invention include one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequence, but are essentially biologically equivalent to the antibodies or antibody fragments of the present invention. It contains an array that does not contain the specified elements.

[0099] Two antigen-binding proteins or antibodies are considered bioequivalent or pharmaceutically equivalent if, for example, they are administered in the same molar dose under similar experimental conditions, whether in single or multiple doses, and their absorption rates and extents do not show significant differences. Some antibodies are considered equivalent or pharmaceutically equivalent if they are equivalent in their degree of absorption but not in their absorption rate, and furthermore, such differences in absorption rate are unintentional, reflected in labeling, and are not essential, for example, to achieving effective drug concentrations in the body in chronic use, and are not considered medically important to the particular drug being studied, and therefore may be considered bioequivalent.

[0100] In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference between them in terms of safety, purity, or efficacy.

[0101] In one embodiment, the two antigen-binding proteins are bioequivalent if the patient can switch between the reference formulation and the biological formulation once or more times, without any anticipated increase in the risk of adverse effects, including clinically significant changes in immunogenicity, or any decrease in efficacy, compared to continuous treatment without switching.

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

[0103] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measures include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antibody or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with human in vivo bioavailability data and are reasonably predictable; (c) in vivo studies 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) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or biological permeability of the antibody.

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

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

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

[0107] The present invention also relates to the chimeric heavy chain steady state (C H Contains an anti-C5 antibody that includes the ) region, where chimeric C H The region is C, which contains one or more immunoglobulin isotypes. H Includes segments derived from the region. For example, the antibody of the present invention is derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from human IgG1, human IgG2, or human IgG4 molecules, combined with some or all of the three domains. H Chimera C containing part or all of two domains H It may include a region. According to a particular embodiment, the antibody of the present invention is a chimeric C having a chimeric hinge region. HThe region includes. For example, the chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from the human IgG1, human IgG2, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from the human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. Chimeric C described herein H Antibodies containing the region may, in certain embodiments, exhibit modified Fc effector functionality 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 in its entirety by reference).

[0108] Biological characteristics of antibodies Generally, the antibodies of the present invention function by binding to the C5 protein and preventing its cleavage into C5a and C5b. For example, the present invention binds to the C5 protein (e.g., at 25°C or 37°C) and measures a K of less than 9 nM by surface plasmon resonance using the assay format specified in Example 3 of this specification. D The present invention includes an antibody and an antigen-binding fragment of the antibody that are bound by an assay. In certain embodiments, the antibody or its antigen-binding fragment is used in an assay format, for example, as defined in Example 3 of this specification, or a substantially similar assay. Using a ssey, surface plasmon resonance was measured to determine K levels below approximately 9 nM, below approximately 5 nM, below approximately 2 nM, below approximately 1 nM, below approximately 500 pM, below 250 pM, or below 100 pM. D Then merge it into C5.

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

[0110] The present invention also includes antibodies that bind to human C5 protein and their antigen-binding fragments, which, when measured by surface plasmon resonance at 37°C using, for example, the assay format specified in Example 4 of this specification or substantially similar assays, have a dissociation half-life longer than about 1.5 minutes (longer than about 2 minutes, longer than about 5 minutes, longer than about 10 minutes, longer than about 25 minutes, longer than about 50 minutes, longer than about 100 minutes, longer than about 150 minutes, or longer than about 200 minutes). 1 / 2 It then binds to the C5 protein.

[0111] The present invention also measures, for example, by surface plasmon resonance using the assay format specified in Example 3 of this specification, to a K of less than 120 nM. DThe present invention comprises an antibody and an antigen-binding fragment of the antibody that bind to the monkey C5 protein (for example, at 25°C or 37°C). In certain embodiments, the antibody or its antigen-binding fragment is measured by surface plasmon resonance using, for example, the assay format specified in Example 3 of this specification, or substantially similar assays, to a K content of less than about 120 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, about 500 pM, or less than 250 pM. D It then binds to monkey C5.

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

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

[0114] The present invention also allows for measurement by luminescence assay using, for example, the assay format specified in Example 6 of this specification, to achieve an IC of less than 10 nM. 50 The present invention includes an antibody that inhibits complement-dependent cell-mediated cytotoxicity (CDC) and an antigen-binding fragment of the antibody. In certain embodiments, the antibody or its antigen-binding fragment is measured by a B-cell luminescence assay using, for example, the assay format specified in Example 6 of this specification or a substantially similar assay, to an IC of less than about 5 nM, less than about 3.5 nM, or less than about 2 nM. 50 This inhibits CDC.

[0115] The present invention also relates to human C5-mediated classical pathway (CP) hemolysis, measured by a CP hemolysis assay using, for example, the assay format specified in Example 8 herein, with an IC5 concentration of less than 6 nM. 50 The present invention includes antibodies and antigen-binding fragments that block more than 94%. In certain embodiments, the antibody or its antigen-binding fragment is measured by a CP hemolysis assay using, for example, the assay format specified in Example 8 herein or a substantially similar assay, to have an IC of less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, or less than about 2 nM. 50 This blocks CP hemolysis.

[0116] The present invention also includes measuring an IC of less than 165 nM by an AP hemolysis assay using, for example, the assay format specified in Example 8 herein. 50The present invention comprises an antibody and antigen-binding fragment that block more than 70% of human C5-mediated alternative pathway (AP) hemolysis. In certain embodiments, the antibody or its antigen-binding fragment has an IC50 of less than approximately 160 nM, less than approximately 150 nM, less than approximately 100 nM, less than approximately 50 nM, or less than approximately 20 nM, as measured by an AP hemolysis assay using, for example, the assay format specified in Example 8 herein or a substantially similar assay. 50 This blocks AP hemolysis.

[0117] The present invention also includes measuring IC25 nM less than 185 nM by a CP hemolysis assay using, for example, the assay format specified in Example 8 of this specification. 50 The present invention comprises an antibody and an antigen-binding fragment that block more than 40% of African green monkey C5-mediated classical pathway (CP) hemolysis. In certain embodiments, the antibody or its antigen-binding fragment is measured by a CP hemolysis assay using, for example, the assay format specified in Example 8 herein or a substantially similar assay, to an IC of less than approximately 180 nM, less than approximately 150 nM, less than approximately 100 nM, about 75 nM, or less than approximately 50 nM. 50 This blocks CP hemolysis.

[0118] The present invention also measures, for example, by an AP hemolysis assay using the assay format specified in Example 8 herein, an IC of less than 235 nM. 50 The present invention comprises an antibody and an antigen-binding fragment that block the African green monkey C5-mediated alternative pathway (AP). In certain embodiments, the antibody or its antigen-binding fragment is measured by an AP hemolysis assay using, for example, the assay format specified in Example 8 herein or a substantially similar assay, to have an IC of less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less than about 20 nM. 50 This blocks AP hemolysis.

[0119] The present invention also measures, for example, by a CP hemolysis assay using the assay format specified in Example 8 herein, that an IC of less than 145 nM is obtained. 50The present invention comprises an antibody and an antigen-binding fragment that block more than 90% of C5-mediated classical pathway (CP) hemolysis in cynomolgus monkeys. In certain embodiments, the antibody or its antigen-binding fragment is used, for example, in the assay format specified in Example 8 of this specification or a substantially similar assay, to block C IC50 is measured by a P-hemolysis assay and is less than approximately 140 nM, less than approximately 120 nM, less than approximately 100 nM, less than approximately 75 nM, or less than approximately 50 nM. 50 This blocks CP hemolysis.

[0120] The present invention also measures, for example, by an AP hemolysis assay using the assay format specified in Example 8 herein, that an IC of less than 30 nM is obtained. 50 The present invention comprises an antibody and an antigen-binding fragment that block C5-mediated alternative pathway (AP) hemolysis in pulp-bearing monkeys. In certain embodiments, the antibody or its antigen-binding fragment is measured by an AP hemolysis assay using, for example, the assay format specified in Example 8 herein or a substantially similar assay, to have an IC of less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than about 2 nM. 50 This blocks AP hemolysis.

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

[0122] In certain embodiments, the present invention provides a high affinity for human C5 (for example, less than 0.3 nM of K).D The present invention provides anti-C5 antibodies and their antigen-binding fragments having lower clearance (e.g., extended serum half-life, improved pharmacodynamic activity over longer periods than previously known anti-C5 antibodies). Such antibodies of the present invention can be advantageously used with less frequent dosing in subjects with C5-related diseases or disorders.

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

[0124] In one embodiment, the present invention provides an isolated recombinant antibody or its antigen-binding fragment that specifically binds to the C5 protein, wherein the antibody or its fragment exhibits one or more of the following characteristics: (a) it is a fully human monoclonal antibody; (b) it has a dissociation constant (K) less than 0.9 nM at 25°C, as measured by a surface plasmon resonance assay. D (c) Binds to human C5; (c) Measured in surface plasmon resonance assay, with a K content of less than 0.3 nM at 37°C. D(d) Binds to human C5; (d) Measured in a surface plasmon resonance assay, K is less than 65 nM D (e) Binds to monkey C5; (e) Measured in surface plasmon resonance assay, K < 0.5 nM D (f) 0.5n is measured in a surface plasmon resonance assay and binds to the human C5 mutant R885H (SEQ ID NO: 356); (f) 0.5n Less than M K D It binds to the human C5 mutant R885C (SEQ ID NO: 357); (g) measured in a CP hemolysis assay, IC2 is less than 6 nM. 50 (h) Blocks more than 95% of human C5-mediated classical pathway (CP) hemolysis; (h) As measured in the AP hemolysis assay, IC < 165 nM 50 (i) Blocks more than 70% of human C5-mediated alternative pathway (AP) hemolysis; (i) As measured in a CP hemolysis assay, IC < 185 nM 50 (j) It inhibits C5-mediated CP hemolysis in African green monkeys; measured in the AP hemolysis assay, IC < 235 nM 50 It inhibits C5-mediated AP hemolysis in African green monkeys; measured in a (k)CP hemolysis assay, IC2 is less than 145 nM. 50 It inhibits C5-mediated CP hemolysis in cynomolgus monkeys; and (1) measured in the AP hemolysis assay, IC <30 nM. 50 This inhibits C5-mediated AP hemolysis in cynomolgus monkeys.

[0125] The antibodies of the present invention may have one or more of the aforementioned biological characteristics, or a combination thereof. Other biological characteristics of the antibodies of the present invention will become apparent to those skilled in the art by examining this disclosure, including the examples herein.

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

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

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

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

[0130] In certain embodiments, an anti-C5 antibody or its antigen-binding fragment binds to an epitope in one or more regions of the native form or recombinant C5 protein exemplified in SEQ ID NO: 355, or the fragment thereof. In some embodiments, the antibody of the present invention binds to a region of the human C5 protein containing one or more amino acids selected from the group consisting of amino acid residues 19 to 1676.

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

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

[0133] The present invention relates to any of the antibodies listed in Table 1 as specific examples, or to the same epitope or said antibody. This includes an anti-C5 antibody that binds to a portion of a pitope. Similarly, the present invention also includes an anti-C5 antibody that competes for binding to the C5 protein or a fragment thereof with any of the antibodies listed in Table 1, which are specific examples. For example, the present invention includes an anti-C5 antibody that cross-competes for binding to the C5 protein with one or more of the antibodies listed in Table 1.

[0134] By using conventional methods known in the art, it is possible to easily determine whether an antibody binds to the same epitope as the reference anti-C5 antibody, or whether it competes for binding with the reference anti-C5 antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-C5 antibody of the present invention, the reference antibody is conjugated to a C5 protein or peptide under saturated conditions. The ability of the test antibody to bind to the C5 molecule is then evaluated. If the test antibody can bind to C5 after saturated 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 cannot bind to C5 after saturated binding with the reference anti-C5 antibody, the test antibody may bind to the same epitope as the epitope conjugated by the reference anti-C5 antibody of the present invention.

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

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

[0137] Next, further conventional experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether stereoblocking (or other reduction) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the field.

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

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

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

[0141] In some embodiments, C5-specific antibodies are produced in a bi-specific form ("bi-specific"), where variable regions that bind to different domains of the C5 protein are linked together, resulting in bi-domain specificity within a single binding molecule. A well-designed bi-specificity can enhance the overall C5 protein inhibitory efficacy by increasing both specificity and binding affinity. Variable regions that are specific to individual domains (e.g., segments of the N-terminal domain) or that can bind to different regions within a single domain pair on a structural scaffold, allowing each region to bind simultaneously to separate epitopes or different regions within a single domain. In one example of bi-specificity, a heavy chain variable region (V) derived from a binder that is specific to one domain is used. H ) is that V H Without destroying the original singularity with respect to the original V H Non-homogeneous V that can be paired with it L To identify the partner, a series of binder-derived light chain variable regions (V) have specificity for the second domain. L ) is recombined with. In this way, a single V L segment (for example, V L 1) is two different V H Domain (for example, V H 1 and V H 2) is combined with the two joints "arms" (V H 1-V L 1 and V H 2-V L 1) results in a double singularity. Single V L The use of segments reduces the complexity of the system and thereby simplifies the cloning, expression, and purification processes used to generate bispecificity, increasing their effectiveness (e.g., USSN13 / 022759 and US2010 / 0331527).

[0142] Alternatively, two or more domains, and antibodies that bind to a second target, such as, without limitation, a second different anti-C5 antibody, can be produced in a bispecific format using the techniques described herein or other techniques known to those of skill in the art. Antibody variable regions that bind to different regions can be linked together, for example, with a variable region that binds to a relevant site on the extracellular domain of C5, to confer bispecific antigenicity within a single binding molecule. Appropriately designed bispecifics of this nature serve a dual function. Variable regions having specificity for the extracellular domain are paired on a structural scaffold that binds to a variable region having specificity for outside of the extracellular domain and that allows each variable region to bind to a separate antigen.

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

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

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

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

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

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

[0149] In certain circumstances, pharmaceutical compositions may be delivered by a controlled-release system. In one embodiment, a pump may be used. In another embodiment, a polymer material may be used. In yet another embodiment, the controlled-release system may be placed near the target of the composition, thus requiring only a small portion of systemic delivery.

[0150] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection, as well as intravenous infusion. These injectable formulations may be manufactured by known methods. For example, an injectable formulation may be manufactured by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable formulation thus manufactured is preferably filled into a suitable ampoule.

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

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

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

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

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

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

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

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

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

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

[0161] In a further embodiment of the present invention, the antibody is used to manufacture a pharmaceutical composition or agent for treating patients suffering from a C5-related disease or disorder. In another embodiment of the present invention, the antibody is used as an adjunct therapy in conjunction with any other agent or treatment known to those skilled in the art that is useful for treating or relieving a C5-related disease or disorder.

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

[0163] Depending on the C5-related disease or disorder, the antibody of the present invention can be used in combination with one or more additional therapeutic agents, including but not limited to anticoagulants (e.g., warfarin, aspirin, heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran)), anti-inflammatory drugs (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), antihypertensive drugs (e.g., angiotensin-converting enzyme inhibitors), immunosuppressive agents (e.g., vincristine, cyclosporin A, or methotrexate), fibrinolytic agents (e.g., ancrod, ε-aminocaproic acid, antiplasmin-a1, prostacyclin, and defibrotide), lipid-lowering agents such as inhibitors of hydroxymethylglutaryl CoA reductase, anti-CD20 drugs such as rituximab, anti-TNF drugs such as infliximab, antiepileptic drugs (e.g., magnesium sulfate), C3 inhibitors, or antithrombotic drugs.

[0164] In certain embodiments, the second therapeutic agent is another antibody against the C5 protein. The use of combinations ("cocktails") of antibodies having broad neutralizing or inhibitory activity against C5 is contemplated herein. In some embodiments, non-competing antibodies can be administered in combination to a subject that requires them. In some embodiments, the antibodies that make up the combination bind to different non-overlapping epitopes of the protein. The antibodies that make up the combination can block the binding of C5 to the C5 convertase and / or the C of C5 It may prevent / inhibit cleavage to 5a and C5b. In certain embodiments, the second antibody may have a longer half-life in human serum.

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

[0166] Further therapeutic active ingredients may be administered to the subject prior to the administration of the anti-C5 antibody of the present invention. For example, the first component may be considered administered "before" the second component if the first component is administered one week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before the administration of the second component. In other embodiments, further therapeutic active ingredients may be administered to the subject after the administration of the anti-C5 antibody of the present invention. For example, the first component may be considered to be administered "after" the second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after the administration of the second component. In yet another embodiment, further therapeutic active ingredients may be administered to the subject simultaneously with the administration of the anti-C5 antibody of the present invention. "Simultaneous" administration for the purposes of the present invention includes, for example, administration to the subject of the anti-C5 antibody and further therapeutic active ingredients in a single dosing form, or in separate dosing forms administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (for example, both the anti-C5 antibody and the further therapeutic active ingredient may be administered intravenously); or each dosage form may be administered via different routes (for example, the anti-C5 antibody may be administered intravenously, and the further therapeutic active ingredient may be administered orally). In any case, a single dosage form, separate dosage forms via the same route, or separate dosage forms via different routes are all considered “concurrent administration” for the purposes of this disclosure. For the purposes of this disclosure, administration of the anti-C5 antibody “before,” “at the same time as” or “after” administration of the further therapeutic active ingredient (as defined herein) is considered administration of the anti-C5 antibody “in combination with” the further therapeutic active ingredient.

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

[0168] Administration regimen According to certain embodiments, a single dose of the anti-C5 antibody of the present invention (or a pharmaceutical composition comprising a combination of the anti-C5 antibody and any further therapeutic active agent referred to herein) may be administered to a subject in need. According to certain embodiments of the present invention, multiple doses of the anti-C5 antibody (or a pharmaceutical composition comprising a combination of the anti-C5 antibody and any further therapeutic active agent referred to herein) may be administered to a subject over a specified period of time. A method according to this aspect of the present invention involves administering multiple doses of the anti-C5 antibody of the present invention to a subject in succession. As used herein, “administering in succession” means that each dose of the anti-C5 antibody is administered to the subject at different points in time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising administering a single initial dose of the anti-C5 antibody, followed by one or more secondary doses of the anti-C5 antibody, and optionally followed by one or more tertiary doses of the anti-C5 antibody, to a patient in succession.

[0169] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the administration of the anti-C5 antibody of the present invention. This refers to the chronological order. Therefore, the “initial dose” is the dose administered at the beginning of the treatment regimen (also called the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-C5 antibody, but may generally differ from one another in terms of administration frequency. However, in certain embodiments, the amounts of anti-C5 antibody contained in the initial, secondary, and / or tertiary doses may differ from one another throughout the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more doses (e.g., two, three, four, or five) may be administered at the beginning of the treatment regimen as a “loading dose,” followed by subsequent doses administered at a lower frequency (e.g., “maintenance doses”).

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

[0171] A method following this aspect of the present invention may involve administering any number of secondary and / or tertiary doses of anti-C5 antibody to a patient. For example, in a particular embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in a particular embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0172] In certain embodiments of the present invention, the frequency with which secondary and / or tertiary doses are administered to a patient may vary during the treatment regimen. The frequency of administration may also be adjusted by the physician between treatments according to the individual patient's needs after clinical examinations.

[0173] Diagnostic use of antibodies The anti-C5 antibody of the present invention may be used, for example, to detect and / or measure C5 in a sample for diagnostic purposes. Several embodiments explore the use of one or more antibodies of the present invention in assays for detecting C5-related diseases or disorders. An example diagnostic assay for C5 includes, for example, contacting a sample obtained from a patient with the anti-C5 antibody of the present invention, where the anti-C5 antibody is labeled with a detectable label or reporter molecule, or used as a capture ligand for selectively isolating C5 from the patient sample. Alternatively, an unlabeled anti-C5 antibody may be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is, 3 H, 14 C, 32 P, 35 S, or 125 This could be a radioactive isotope such as C1; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific examples of assays that can be used to detect or measure C5 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent cell sequencing (FACS).

[0174] The sample that may be used in the C5 diagnostic assay according to the present invention is obtained from a patient. The sample includes any tissue or fluid sample that contains either a detectable amount of C5 protein or a fragment thereof under normal or pathological conditions. Generally, the level of C5 protein in a specific sample obtained from a healthy patient (e.g., a patient without a C5-related disease) is measured to initially establish a baseline or standard level of C5. This baseline level of C5 can then be compared to the C5 level measured in a sample obtained from an individual suspected of having a C5-related condition or symptoms associated with such a condition.

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

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

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

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

[0179] In Embodiment 4, the present invention includes an isolated antibody of any one antigen-binding fragment from Embodiments 1 to 3, wherein the antibody or its antigen-binding fragment 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.

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

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

[0182] In Embodiment 7, the present invention provides an isolated antibody or its antigen-binding fragment that interacts with the amino acid sequences of SEQ ID NOs. 360 and 361, one of the embodiments 1 to 5. It contains an antibody or its antigen-binding fragment.

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

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

[0185] In Embodiment 10, the present invention comprises one isolated antibody or antigen-binding fragment thereof from Embodiments 1 to 9, wherein the antibody has further features selected from the group consisting of: (a) being a fully human monoclonal antibody; and (b) having a dissociation constant (K) less than 0.9 nM at 25°C, as measured by surface plasmon resonance assay. D (c) Binds to human C5; (c) Measured in surface plasmon resonance assay, with a K content of less than 0.3 nM at 37°C. D(d) Binds to human C5; (d) Measured in a surface plasmon resonance assay, K is less than 65 nM D (e) Binds to monkey C5; (e) Measured in surface plasmon resonance assay, K < 0.5 nM D (f) Binds to human C5 mutant R885H (SEQ ID NO: 356); (f) Measured in surface plasmon resonance assay, K < 0.5 nM D It binds to the human C5 mutant R885C (SEQ ID NO: 357); (g) measured in a CP hemolysis assay, IC2 is less than 6 nM. 50 (h) Blocks more than 95% of human C5-mediated classical pathway (CP) hemolysis; (h) As measured in the AP hemolysis assay, IC < 165 nM 50 (i) Blocks more than 70% of human C5-mediated alternative pathway (AP) hemolysis; (i) As measured in a CP hemolysis assay, IC < 185 nM 50 (j) It inhibits C5-mediated CP hemolysis in African green monkeys; measured in the AP hemolysis assay, IC < 235 nM 50 It inhibits C5-mediated AP hemolysis in African green monkeys; measured in a (k)CP hemolysis assay, IC2 is less than 145 nM. 50 It inhibits C5-mediated CP hemolysis in cynomolgus monkeys; and (1) measured in the AP hemolysis assay, IC <30 nM. 50 This inhibits C5-mediated AP hemolysis in cynomolgus monkeys.

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

[0187] In Embodiment 12, the present invention includes: (a) Sequence IDs 4, 20, 36, 52, 68, 84 (b) HCDR1 domain having an amino acid sequence selected from the group consisting of 100, 124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292, 308, 324, and 340; (b) SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 126, 142, 150, 158, 174, 190, 206, 222, 238, 254, 270, 278, 294, 310, 326 (c) HCDR2 domain having an amino acid sequence selected from the group consisting of (a) domains 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) HCDR3 domain having an amino acid sequence selected from the group consisting of (a) domains 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) domains 12, 28, 44, 60, 76, LCDR1 domain having an amino acid sequence selected from the group consisting of 92, 108, 116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332, and 348; (e) 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 The antibody comprises one isolated antibody or antigen-binding fragment thereof from Examples 1 to 11, comprising (f) an LCDR2 domain having a selected amino acid sequence, and 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.

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

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

[0190] In Embodiment 15, the present invention relates to SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 98 / 114, 122 / 106, 98 / 130, 138 / 106, 146 / 106, 122 / 130, 146 / 114, 146 / 130, 138 / 130, 154 / 162, 170 / 178, 186 / 19 The embodiment comprises one isolated antibody or antigen-binding fragment from any of embodiments 11 to 14, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of 4, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 258, 274 / 282, 290 / 298, 306 / 314, 322 / 330, and 338 / 346.

[0191] In Embodiment 16, the present invention comprises one isolated antibody or antigen-binding fragment thereof from 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 in an LCVR selected from the group consisting of SEQ ID NOs: 58, 106, and 210.

[0192] In Embodiment 17, the present invention comprises an isolated antibody or antigen-binding fragment thereof from Embodiment 16, comprising: (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, selected from the group.

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

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

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

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

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

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

[0199] In Embodiment 24, the present invention includes the antibody of Embodiment 23 or its antigen-binding fragment, which includes a light chain variable region having at least 90% sequence identity with SEQ ID NO: 106.

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

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

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

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

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

[0205] In Embodiment 30, the present invention includes cells expressing the vector of Embodiment 29.

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

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

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

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

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

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

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

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

[0214] The following examples are provided to the art to provide a complete disclosure and description of the methods and compositions of the present invention and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation are to be expected. Unless otherwise indicated, parts are parts by mass, molecular weight is average molecular weight, temperature is degrees Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or near atmospheric pressure.

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

[0216] Antibody immune responses were monitored using C5-specific immunoassays. Once the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to preserve their viability, forming hybridoma cell lines. These hybridoma cell lines were screened and selected to identify those producing C5-specific antibodies. Using these cell lines, several anti-C5 chimeric antibodies (i.e., antibodies possessing both a human variable domain and a mouse constant domain) were obtained; example antibodies produced in this manner were named H2M11683N and H2M11686N.

[0217] Furthermore, anti-C5 antibodies were isolated directly from antigen-positive mouse B cells without fusing to myeloma cells, as described in U.S. Patent No. 7,582,298 (which is specifically added herein in whole by reference). Using this method, several fully human anti-C5 antibodies (i.e., antibodies having a human variable domain and a human constant domain) were obtained; example antibodies produced by this method were designated as H4H12159P, H4H12161P, H4H12163P, H4H12164P, H4H12166P, H4H12167P, H4H12168P, H4H12169P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2, H4H12177P2, and H4H12183P2.

[0218] The biological properties of an example antibody produced according to the method of this embodiment are described in detail in the following examples.

[0219] Example 2: Heavy chain and light chain variable region amino acids and nucleic acid sequences Table 1 shows the amino acid sequence identifiers of the heavy chain and light chain variable regions and CDRs of the selected anti-C5 antibodies of the present invention.

[0220] [Table 1]

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

[0222] [Table 2]

[0223] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", "H2M", etc.), followed by a numerical identifier (e.g., "11686", "12166", "12183", etc., as shown in Table 2), followed by a suffix of "P", "P2", or "N". Thus, according to this nomenclature, antibodies may be referred to herein, for example, "H2M11686N", "H4H12183P2", "H4H12168P", etc. The H4H and H2M prefixes in antibody symbolization used herein refer to the antibody For example, an antibody exhibiting a specific Fc region isotype, such as the "H4H" antibody, has human IgG4 containing a serine-to-proline mutation (S108P) in the hinge region to promote dimerization, and an "H2M" antibody has mouse IgG2 Fc (a or b isotype) (all variable regions are fully human, indicated by "H" first in the antibody symbolization). As is obvious to those skilled in the art, antibodies with a specific Fc isotype can be converted to a different Fc isotype (for example, an antibody with mouse IgG1 Fc can be converted to an antibody with human IgG4, etc.), but in any case, the variable domain (including the CDR) These remain the same, as indicated by the numerical identifiers shown in Table 2, and their binding properties to the antigen are expected to be identical or substantially the same regardless of the properties of the Fc domain.

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

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

[0226] Control structures used in the following examples The following control construct (anti-C5 antibody) was included in the experiments disclosed herein for comparative purposes: Control drug 1" The antibody "h5G1.1" in accordance with U.S. Patent No. 6,355,245 (Alexion Pharmaceuticals, Inc.) H / V L Monoclonal antibodies against human C5 having the sequence; and " Control drug 2 ", the antibody "8109" in accordance with U.S. Patent Application Publication No. 2013 / 0022615 (Novartis) H / V L A human monoclonal antibody against human C5 containing a specific sequence.

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

[0228] Tables 3 and 4 show the binding dynamics parameters for human C5 binding to anti-C5 antibodies at 25°C and 37°C.

[0229] [Table 3]

[0230] [Table 4]

[0231] Tables 5 and 6 show the binding of monkey C5-mmh to anti-C5 antibodies at 25°C and 37°C.

[0232] [Table 5]

[0233] [Table 6]

[0234] Tables 7 and 8 show the binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibodies at 25°C, respectively.

[0235] [Table 7]

[0236] [Table 8]

[0237] Tables 9 and 10 show the binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibodies at 37°C, respectively.

[0238] [Table 9]

[0239] [Table 10]

[0240] At 25°C, all 25 anti-C5 antibodies of the present invention exhibit a K2 level in human C5 ranging from 73 pM to 8.4 nM, as shown in Table 3. D The values ​​were bound. At 37°C, the anti-C5 antibody of the present invention has a K content ranging from 103 pM to 18.5 nM, as shown in Table 4. D The antibodies bound to human C5 at a specific value. At 25°C, 25 of the 25 anti-C5 antibodies of the present invention tested showed K levels ranging from 133 pM to 64 nM, as shown in Table 5. D The antibody bound to monkey C5-mmh at a specific value. At 37°C, 25 of the 25 anti-C5 antibodies of the present invention tested showed K levels ranging from 133 pM to 118 nM, as shown in Table 6. D The antibody bound to monkey C5-mmh was measured at a value. Of the anti-C5 antibodies of the present invention tested at 25°C, 16 had K levels ranging from 147 pM to 10.9 nM, as shown in Table 7. D The values ​​were found to bind to human C5 R885H-mmh. Of the 16 anti-C5 antibodies of the present invention tested at 25°C, 16 had K levels ranging from 251 pM to 190 nM, as shown in Table 8. D The values ​​were found to bind to human C5 R885C-mmh. Of the 16 anti-C5 antibodies of the present invention tested at 37°C, 16 had K levels ranging from 1.49 nM to 69.4 nM, as shown in Table 9. D The antibody bound to human C5 R885H-mmh was measured at a value. Of the 16 anti-C5 antibodies of the present invention tested at 25°C, 16 had K levels ranging from 1.74 nM to 159 nM, as shown in Table 10. D The value indicated binding to human C5 R885C-mmh.

[0241] Example 4: Antibodies that bind to C5 at different pH levels The effect of pH on the dissociation rate of recombinant human C5 bound to purified anti-C5 monoclonal antibody was determined using a Biacore T200 with a real-time surface plasmon resonance biosensor. The Biacore sensor surface was first derivatized with monoclonal mouse anti-human Fc antibody (GE, no. BR-1008-39) by amine coupling to capture expressed anti-C5 monoclonal antibody containing human IgG4 Fc. All Biacore binding tests were performed using two running buffers: PBS-T, pH 7.4 (0.01M Na2HPO4 / NaH2PO4, 0.15M NaCl, 0.05% vol / vol Tween-20, adjusted to pH 7.4) and PBS-T, pH 6.0 (0.01M Na2HPO4 / NaH2PO4, 0.15M NaCl, 0.05% vol / vol Tween-20, adjusted to pH 6.0). Human C5 (EMD, catalog number 204888) or monkey C5.mmh (prepared in PBS-T, pH 7.4, 3-fold dilution, ranging from 100 nM to 11.11 nM) at various concentrations were injected at a flow rate of 50 μL / min for 3 minutes onto a surface containing anti-C5 monoclonal antibody, and their dissociation in two running buffers, PBS-T, pH 7.4 and PBS-T, pH 6.0, was monitored for 6 minutes. All coupling dynamics experiments were performed at 25°C and 37°C. Dynamical dissociation constant (k d The ) was determined by fitting the real-time sensorgram to a 1:1 coupling model using Scrubber 2.0c curve fitting software. The coupling / dissociation half-life (t) was determined. 1 / 2 ) to k d from:

number

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

[0243] [Table 11]

[0244] [Table 12]

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

[0246] [Table 13]

[0247] [Table 14]

[0248] As shown in Tables 11-14, the selected anti-C5 antibodies are t 1 / 2 The ratio shows pH-dependent binding.

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

[0250] Table 15 clearly defines the relationships between antibodies that compete in both directions, independently of the binding order.

[0251] [Table 15]

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

[0253] For the C5 CDC bioassay, Daudi cells were administered at 10,000 cells / well on a 96-well assay plate with 10% FBS, penicillin / streptomycin, and L. Cells were seeded in either RPMI (RPMI complete medium) containing glutamine, sodium pyruvate, and non-essential amino acids, or RPMI containing 1% BSA, penicillin / streptomycin, and L-glutamine (RPMI / BSA). All assays testing mutated anti-hC5 antibodies, along with assays testing non-mutant antibodies using C5-containing human serum, were performed in RPMI complete medium. Assays testing non-mutant antibodies used African green monkey serum, and human C5 mutants were tested in RPMI / BSA medium. To measure CDC using human or monkey serum, anti-CD20 antibody was diluted 1:3 from 100 nM to 2 pM (including a control sample without antibody), incubated with cells at 25°C for 10 minutes, and then 1.66% serum or 1.66% C5-deficient serum and 6.6 nM C5 mutant protein were added. The amount of C5 protein to be added to C5-deficient serum was based on the reported value of 0.37 μM for C5 concentration in human serum (Rawal et al 2008, J. Biol. Chem. 283:7853-7863). To test C5 antibody inhibition of CDC, C5 antibody was diluted 1:3 from 100 nM to 2 pM (including a control sample without antibody) and incubated with 1.66% serum or 1.66% C5-deficient serum and 6.6 nM C5 mutant protein for 30 minutes. Anti-CD20 antibody was added to cells at 1 nM, 2 nM, 3 nM, 3.5 nM, 7 nM, 10 nM, or 30 nM concentrations 10 minutes before adding the antibody with serum to the cells. The antibody / serum mixture was added to the cells upon completion of incubation with the anti-CD20 antibody. After 3.5 hours of incubation at 37°C and 5% CO2, the cytotoxicity was treated with CytoTox-Glo TM Measurements were taken after adding the reagent (Promega, number G9292). CytoTox-Glo TM CytoTox-Glo is a luminescence-based reagent that measures cell death, where increased luminescence is observed as the amount of cytotoxicity increases (measured in relative luminous units, RLU). Untreated cells in a control well are subjected to CytoTox-Glo TM The maximum cell toxicity was determined by rinsing with digitonin immediately after adding the reagent. The plates were then treated with CytoTox-Glo TMFifteen minutes after the addition of the substance, luminescence was measured using a Victor X instrument (Perkin Elmer). If calculated, the percentage of cytotoxicity was calculated using the RLU value by the following equation:

number

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

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

[0256] [Table 16]

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

[0258] [Table 17]

[0259] Sixteen non-mutant anti-hC5 antibodies showed complete inhibition of African green monkey C5-mediated CDC with IC50 ranging from 2.0 nM to 14 nM.

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

[0261] To test whether anti-hC5 antibodies inhibit human C5 variants R885H and R885C, C5-deficient human serum was tested using each C5 variant at 6.6 nM. All 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by the C5 variant R885H, with non-mutant antibodies ranging from 0.48 nM to 4.2 nM IC50, while mutant antibodies ranged from 1.3 nM to 7.0 nM IC50. The parental non-mutant antibody H4H12166P showed an IC50 of 1.3 nM. Complete inhibition occurred at an IC50 of 1.3 nM.

[0262] Fifteen of the sixteen non-mutant hC5 antibodies showed complete inhibition of CDC mediated by the C5 mutant R885C with an IC50 ranging from 0.43 nM to 1.6 nM. One non-mutant antibody showed weak inhibition of CDC with a maximum inhibition of 67% (at 100 nM) and an IC50 > 20 nM. All nine mutant antibodies showed complete inhibition of CDC mediated by the C5 mutant R885C with an IC50 ranging from 0.77 nM to 3.5 nM. The parental non-mutant antibody H4H12166P produced complete inhibition with IC50s of 0.46 nM and 0.76 nM.

[0263] Anti-CD20 antibodies inhibited CDC in Daudi cells at EC50 of 1.0 nM, 1.4 nM, and 1.9 nM for human serum, 2.4 nM and 2.6 nM for African green monkey serum, 1.9 nM and 6.3 nM for hC5-deficient serum using the hC5 mutant R885H, and 2.7 nM and 9.5 nM for hC5-deficient serum using the hC5 mutant R885C. Neither the unrelated IgG control antibody, control mAb1, nor control mAb2 showed any inhibition of CDC.

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

[0265] For the C5a luciferase bioassay, HEK293 / hG□16 / hC5aR1 / NFAT-luc cells were seeded at 20,000 cells / well in OPTIMEM (Invitrogen, no. 31985-070) supplemented with 0.5% BSA, penicillin / streptomycin, and L-glutamine in 96-well assay plates, and then incubated overnight at 37°C and 5% CO2. BSA was used instead of FBS because serum has been shown to cleave and activate hC5a (Klos et al., 2013, Pharmacol. Rev. 65:500-543). The following morning, hC5a was titrated from 100 nM to 2 pM (including a control sample without hC5a), and dose-response titration curves were determined for cells and cell lines. To investigate the inhibition of hC5a by hC5a antibody, 500 pM hC5a was added to cells. Immediately afterward, an antibody diluted 1:3 from 100 nM to 2 pM (including a control sample without antibody) was added to the cells. The cells were incubated for 5.5 hours at 37°C in the presence of 5% CO2. OneGlo TM Luciferase activity was detected after incubation with reagent (Promega, number E6051). OneGlo TM This is a luminescence-based reagent for measuring the amount of luciferase present in cells. In this assay, increased hC5a activation results in increased luciferase production and luminescence (measured in relative luminescence units, RLU). Luminescence was measured using a Victor X instrument (Perkin Elmer). Prism 5 Using software (GraphPad), I analyzed the results using nonlinear regression (4-parameter logistics) and performed EC. 50 Value and IC 50 The values ​​were obtained. Antibody inhibition was calculated so that 0-100% inhibition was within the inhibition range from 500 pM hC5a (no inhibitor) to 0 nM hC5a.

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

[0267] [Table 18]

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

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

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

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

[0272] The percentage of hemolysis was calculated as follows:

number

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

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

[0275] The percentage of hemolysis was calculated as follows:

number

[0276] In this equation, "background cell lysis" means that it does not contain serum or either does not contain serum or does not contain serum. This is the OD at A412nm from cells incubated with GVB-Mg / EGTA buffer alone, without the use of anti-C5 antibody. "Maximum cell lysis" is the OD at A412nm from cells treated with water. Inhibition by anti-C5 antibody, IC 50 The values ​​were calculated using nonlinear regression (4-parameter logistics) with Prism 6 software (GraphPad).

[0277] result (A) Inhibition of human C5 hemolysis A total of 25 anti-human C5 (hC5) antibodies (16 non-mutant and 9 mutant) were tested for their ability to inhibit C5 from normal human serum (NHS) in the CH50 assay using sensitized sheep erythrocytes (SRBCs) and in the AH50 assay using rabbit erythrocytes (RRBCs).

[0278] [Table 19]

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

[0280] [Table 20]

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

[0282] (B) Inhibition of monkey C5 hemolysis A total of 25 anti-human C5 (hC5) antibodies (16 non-mutant and 9 mutant) were tested for their ability to inhibit C5 from cynomolgus monkeys and African green monkeys in the CH50 assay using sensitized sheep erythrocytes (SRBCs) and the AH50 assay using rabbit erythrocytes (RRBCs).

[0283] [Table 21]

[0284] As shown in Table 21, anti-hC5 antibodies showed inhibition of CP or AP hemolytic activity at varying levels in 5% African green monkey serum. In the CP assay, two of the 16 anti-hC5 antibodies did not show inhibition of hemolytic activity. Fourteen antibodies showed inhibition ranging from 43% to 94% with an IC50 ranging from 25 nM to 180 nM. In the AP hemolytic assay, 13 of the 17 antibodies showed inhibitory activity ranging from 17% to 93% with an IC50 ranging from 22.5 nM to 233 nM.

[0285] [Table 22]

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

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

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

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

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

[0291] C5a protein levels were determined using complement-conserved normal human serum (NHS) pre-incubated with H4H12166P or isotype control antibody as described in Example 8, in the supernatant from a CP hemolysis assay. C5a protein levels 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 a capture antibody (mouse anti-C5a specific to a neo-epitope on human C5a). Human C5a protein provided by the manufacturer was used as a calibration standard. C5a in the supernatant was detected with an HRP-binding detection antibody (mouse monoclonal antibody against the C5a region of C5). A chromogenic HRP substrate, 3,3',5,5'-tetramethylbenzidine (TMB), was added to detect HRP activity. The reaction was stopped using a 1N hydrochloric acid solution, and the optical density at 450 nm (OD450) was measured using a SpectraMax plate reader. The data were analyzed using nonlinear regression (4-parameter logistics) in GraphPad Prism. C5a concentration was analyzed as ng / supernatant mL.

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

[0293] Example 10: Characterization of the pharmacokinetics and pharmacodynamics of anti-C5 antibodies in cynomolgus monkeys This example describes the pharmacokinetic (PK) and pharmacodynamic (PD) characterization of selected anti-C5 antibodies performed in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody administration and used to stratify the animal treatment groups.

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

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

[0296] Total IgG antibody level analysis by ELISA immunoassay The total antibody concentration in monkey serum samples was measured using an unvalidated direct ELISA. The ELISA procedure used microtiter plates coated with mouse anti-human IgG1 / IgG4 Fc monoclonal antibody. Various anti-C5 antibodies were added to the plates, and the anti-C5 antibodies captured on the plates were detected using biotinylated mouse anti-human IgG4 Fc monoclonal antibody, followed by NeutrAvidin conjugated with horseradish peroxidase (NeutrAvidin HRP). A peroxidase-specific luminol-based substrate was then added to achieve a signal intensity proportional to the total concentration of captured anti-C5 antibodies. The relative light unit (RLU) measurements of the calibration standards and their respective nominal concentrations were fitted using a weighted 4-parameter logistic equation to generate a calibration equation describing the relationship between anti-C5 antibody concentration and assay response. The limit of quantification (LLOQ) was 1.56 ng / mL in this assay (2% monkey serum), and 78 ng / mL in untreated monkey serum.

[0297] Determining PK parameters PK parameter, Phoenix (R) WinNonlin (R) The results were determined by non-compartmental analysis (NCA) using software (version 6.4, Certara, LP) and an IV bolus delivery model.

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

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

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

number

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

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

[0303] result The selected anti-C5 antibodies (listed in Table 1) were tested in initial experiments for their extended pharmacokinetic profiles in cynomolgus monkeys and C5 humanized mice (described in Example 10). H4H12166P and H4H12161P were selected for their high affinity coupled to the extended PK and were used in subsequent experiments in conjunction with control agents 1 and 2.

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

[0305] [Table 23]

[0306] Following IV bolus administration, the total IgG concentration-time profiles of H4H12166P, H4H12161P, and control 2 were characterized over survival by an initial short-term distribution phase followed by a single efflux phase. The peak concentrations of H4H12166P, H4H12161P, and control 2 were very similar, and the corresponding C2 levels across all antibodies were observed. max The dose values ​​were within 1.1 times (29.7, 30.4, and 30.6 [(ug / mL) / (mg / kg)], respectively) (Table 24).

[0307] [Table 24]

[0308] Evaluation of the concentration-time profile revealed that the slowest elimination was observed up to day 71 of the study when the terminal antibody concentration was ≥10 μg / mL. The dynamics of H4H12161P and control drug 2 were similar; both showed faster elimination than H4H12166P up to days 22 and 29, respectively, when the mAb concentration was ≥10 μg / mL.

[0309] As a result, dose-normalized exposure (AUC) last In terms of exposure (dose), H4H12166P had the highest exposure at 339 days*(μg / mL) / (mg / kg), while H4H12161P and control drug 2 had approximately twice as low exposure as H4H12166P, at 157 and 187 days*(μg / mL) / (mg / kg), respectively.

[0310] Calculated antibody half-life during the efflux phase (t 1 / 2 ) ranged from 5.5 to 15.6 days across the drug administration groups, and also H4H12166P corresponds to the highest t 1 / 2 While the average duration was 15.6 days, H4H12161P and control drug 2 had durations of 5.5 days and 5.9 days, respectively. 1 / 2 Since it had a value, it correlated with exposure.

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

[0312] [Table 25]

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

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

[0315] Example 11: Characterization of PK / PD of anti-C5 antibodies in C5 humanized mice In this experimental setup, the pharmacokinetics and pharmacodynamics of the selected anti-C5 antibody were determined by Velocigene. (R) We evaluated humanized mice expressing human C5 protein using the technique (Valenzuela et al 2003, Nat. Biotechnol. 21:652-659). The humanized mice were manipulated to replace exons 2 through 41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in U.S. Patent Application Publication 2015 / 0313194, which is joined herein in its entirety).

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

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

[0318] Determining PK parameters PK parameter, Phoenix (R) WinNonlin (R) Non-compartmental analysis (NCA) was performed using software (version 6.3, Certara, LP) and an extravascular drug delivery model. The observed estimated half-life (t) was determined using the mean concentration of each antibody. 1 / 2 This includes all PK parameters, as well as the area under the concentration-time curve (AUC) up to the last measurable concentration. last The values ​​were determined using linear interpolation and uniform weighting.

[0319] PD analysis using hemolysis assay The pharmacodynamics of selected anti-C5 antibodies were determined using a classical pathway complement hemolysis assay. Sheep erythrocytes (SRBCs) (sheep blood in Orsiever's solution) were washed with GVB++ buffer (gelatin veronal buffer containing CaCl2 and MgCl2) (Boston BioProducts) and resuspended at 1 x 10^9 cells / mL. To sensitize, 1 x 10^9 / mL 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 2 x 10^8 cells / mL in GVB++ before the hemolysis assay. Serum samples from pre-administration animals or from humanized C5 mice administered with anti-C5 antibodies, collected at 10, 20, 30, 40, and 50 days post-administration, were diluted to 20% in GVB++ buffer. A total of 100 μl (2 x 10^8 cells / mL) of sensitized SRBCs was plated in a 96-well round-bottom plate at 37°C, followed by the addition of 100 μl of 20% serum supplemented with 160-180 μg / mL human complement 3 (huC3) protein. The cells were gently mixed and incubated at 37°C for 1 hour. After incubation, the cells were centrifuged at 1250xg at 4°C. Total supernatant 1 00 μL was transferred to a new 96 flat-bottom plate and read using a Spectramax microplate reader at A4 12 nm. The hemolysis percentage was calculated using the absorbance value with the following formula:

number

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

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

[0322] Prior to antibody administration, male and female humanized C5 mice were stratified according to their human C5 levels, which averaged 40 μg / mL. For each anti-C5 antibody, a single 15 mg / kg dose of H4H12166P, control 1, or control 2 was administered subcutaneously (sc) to a cohort of 22 mice. All mice were exsanguined before administration and 1 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 blood was collected for PK and PD analysis. Serum samples on day 1 were average for the entire cohort of 22 mice. The blood was processed into serum and frozen at -80°C until analysis.

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

[0324] [Table 26]

[0325] [Table 27]

[0326] The mean concentration-versus-time profiles on day 1 show that the three antibodies, H4H12166P, control 1, and control 2, had similar serum concentrations of 178, 229, and 164 μg / mL, respectively. Control 1 had a similar efflux profile to H4H12166P up to day 30, but showed a rapid increase in clearance compared to H4H12166P on days 40 and 50. On day 50, H4H12166P had a mean serum antibody concentration of approximately 9 μg / mL, while both control 1 and control 2 had a mean serum antibody concentration 30 times lower at 0.3 μg / mL. Control 2 had the lowest exposure of the three antibodies tested, with an AUC approximately twice as low compared to H4H12166P (2801 days μg / mL) and control 1 (2708 days μg / mL). last (1408 days μg / mL)

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

[0328] [Table 28]

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

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

[0331] Prior to antibody administration, humanized male and female C5 mice were stratified according to their human C5 levels, which averaged 50 μg / mL. For each anti-C5 mAb, a cohort of five mice received a single 15 mg / kg subcutaneous (sc) injection of H4H12166P, H4H12161P, control 1, or isotype control. All mice underwent bleeding before administration, 6 hours after injection, and at 1, 2, 3, 4, 7, 10, 13, 21, 30, and 45 days for PK analysis. Furthermore, on day 59, all mice from each cohort were euthanized, and terminal blood was collected for PK and PD analysis. The blood was processed into serum and frozen at -80°C until analysis.

[0332] Total antibody concentrations were determined at 12 time points during the 59-day survival period, and hemolytic activity percentage was determined at one time point. Table 29 summarizes the total serum antibody concentrations for each anti-C5 antibody. Figure 6 shows the mean total antibody concentration versus time profile. Table 30 lists the mean PK parameters.

[0333] [Table 29]

[0334] [Table 30]

[0335] The mean concentration-time profile showed that H4H12166P, H4H12161P, control drug 1, and isotype control were all within 1.3 times the C value. max The maximum serum concentration (C) was reached on days 1-3 at values ​​(178, 225, 183, and 221) μg / mL. max This indicates that the drug level reached 0. H4H12166P and the isotype control had similar elimination profiles, with residual drug levels of approximately 4 μg / mL on day 59. H4H12161P showed faster clearance than H4H12166P and the isotype control, but was removed more slowly than control drug 1. On day 59, H4H12161P had a mean serum drug level of 0.6 μg / mL, while control drug 1 had a mean serum drug level of 0.08 μg / mL. The drug levels were undetectable.

[0336] Isotype controls, H4H12166P and H4H12161P, showed similar exposure (AUC) within 1.3 times. last The values ​​were 4080, 3490, and 3040 μg / mL per day, respectively, but control drug 1 showed a 1.6 times lower exposure (2240 ​​μg / mL per day) compared to H4H12166P.

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

[0338] The total human C5 serum concentration was determined by measuring the concentration of the 10-amino acid peptide LQGTLPVEAR (amino acids (aa) 1129-1138 of SEQ ID NO: 359), which is contained in the C5 sequence as a surrogate for C5. Theoretically, this method could also detect the C5 fission product C5b. However, due to the instability of free C5b, the concentration of C5b in serum is generally low, and the majority of C5b is bound to the cell surface 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 may contain only negligible amounts of C5b products, if any.

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

[0340] Human C5 was used as the reference standard for calibration; and a manufactured human C5 peptide with a C-terminal stable isotope-labeled arginine residue was used as the internal standard (LQGTLPVEAR- 13 C6 15N4). Reference standards were used in serum from in-house generated C5 knockout mice in which the mouse C5 gene had been deleted (C5- / -), at concentrations ranging from 3.9 to 250 μg / mL (1:2 serial dilutions). Serum from C5- / - mice was also used as a negative control (blank). Calibration standard, blank, and test serum samples (10 μL each) were dried and then denatured in 100 μL of 8 M urea / 20 mM Tris(2-carboxyethyl)phosphine (TCEP) buffer at 37°C for 1 hour. Next, 10 μL of 25 nM internal standard was added to all samples. The samples were alkylated with 10 mM 2-iodoacetamide at room temperature for 30 minutes and then diluted to a final volume of 500 μL using 50 mM ammonium bicarbonate. The samples were then digested with trypsin (1:20 mass / mass) overnight at 37°C. The C5-derived trypsin peptide LQGTLPVEAR was detected and quantified by LC-MRM-MS using Waters Xevo TQ-S with the ACQUITY UPLC system. Each processed sample (10 μL) was injected into a pre-equalized ACQUITY UPLC BEH C18 column. The flow rate was 0.6 mL / min (mobile phase A: water: formic acid / 100:0.1 [vol:vol] and mobile phase B: acetonitrile: formic acid / 100:0.1 [vol:vol]). Retention time and peak area were determined using Masslynx Analyst data software (Waters). The C5 reference standard (unlabeled C5 peptide LQGTLPVEAR- produced by trypsin digestion of hC5) was compared to the internal standard (stable isotope-labeled C5 peptide). 12 C6 14 The concentration of the C5 sample was calculated from a calibration curve constructed by plotting the peak area ratio of N4) against the nominal concentration of the C5 reference standard. The concentration was then linearly regressed. The calculations were performed using the following: The lowest concentration of the C5 reference standard (3.9 μg / mL) was within the dynamic range of the assay and was defined as the LLOQ of the assay.

[0341] result Total human C5 concentrations in serum were evaluated in samples collected from corresponding animals via tail hemorrhage before administration (pre-administration) and via terminal blood at days 10, 30, and 35. Total hC5 concentrations after H4H12166P administration at 10, 30, and 35 days post-administration were similar to pre-administration levels (within approximately 1 to 0.9 times). Small differences observed were noted using GraphPad. The statistical evaluation was performed using the Mann-Whitney test with Prism software. The results were not statistically significant. Analysis of the C5 / H4H12166P molar ratio demonstrated that H4H12166P remained in C5 molar excess until day 35 after administration (Table 31).

[0342] [Table 31]

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

[0344] HDX-MS experiments are performed using an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradients, and a Synapt G2-Si mass spectrometer for digested peptide mass measurement. .

[0345] For deuterium labeling, 3.8 μL (6 pmol / μL) of C5 (or C5 pre-mixed with antibody in a 1:1 molar ratio) was incubated with 56.2 μL of D2O-labeled solution at various time points (e.g., non-deuterated control = labeled for 0 seconds, 1 minute, and 20 minutes). 50 μL of the deuterated sample was then incubated with 50 μL (0.2 M) of pre-cooled quench buffer. The sample was quenched by transferring TCEP (6M guanidine chloride in 100 mM phosphate buffer, pH 2.5), and the mixed sample was incubated at 1.0°C for 2 minutes. The quenched sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptide was captured at 0°C on an ACQUITY UPLC BEH C18 1.7-μm, 2.1 × 5 mm VanGuard pre-column, and eluted for 9 minutes for gradient separation on an ACQUITY UPLC BEH C18 1.7-μm, 1.0 × 50 mm analytical column in 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 37V, a scan time of 0.5 seconds, and a mass / charge range of 50–1700 Thomson units (Th).

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

[0347] MSE Using an online pepsin / protease XIII column linked to data acquisition, a total of 189 peptides from human C5 were identified in the absence or presence of antibody, representing 62% sequence ranges. Five peptides exhibited significantly reduced deuterium uptake when bound to H4H12166P (centroid delta value > 0.9 Daltons, p value < 0.05), as shown in Table 32.

[0348] [Table 32]

[0349] The recorded peptide mass is the center of gravity MH from three copies. + Corresponds to the average mass. These peptides, corresponding to amino acids 591-599 and 775-794, are H4H121. When bound to 66P, it exhibited a slower deuteration rate. These identified residues also correspond to residues 591-599 and 775-794 of human C5 as defined by Uniprot entry P01031 (CO5_HUMAN; SEQ ID NO: 359).

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

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

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

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

[0354] Example 15: Effect of anti-human C5 antibody on experimental autoimmune uveitis This example describes the effect of an anti-C5 antibody against human C5 in a mouse model of experimental autoimmune uveitis (EAU). The mice used in this study were Velocigene (R) Using the technology (Valenzuela et al 2003, Nat. Biotechnol. 21:652-659), we have learned to express human C5 protein. Humanized mice were modified to replace exons 2-41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in U.S. Patent Application Publication 2015 / 0313194, which is incorporated herein in whole).

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

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

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

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

[0359] A four-point scale was developed to assess disease severity in OCT images (OCT score) (Table 33).

[0360] [Table 33]

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

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

[0363] In the second trial (Trial B), isotype control (10 mg / kg), H4 Mice were subcutaneously treated every 3 days starting from day 6 with either H12166P at 3 mg / kg or 10 mg / kg, or control drug 2 (see Example 2 of this specification; "Control construct used in the following examples"). Treatment with either 3 mg / kg or 10 mg / kg of H4H12166P resulted in a statistically significant dose-related reduction in OCT scores from day 14 to day 28 (Figure 11). In C5 humanized mice, treatment with 10 mg / kg of H4H12166P, initiated 6 days after EAU induction, resulted in a dose-related reduction in inflammation and retinal damage as determined by OCT obtained from day 14 to day 28 (Figure 12).

[0364] In both studies, non-invasive OCT survival assessments showed progressive inflammation, increased retinal thickness, and morphological abnormalities in control mice immunized with IRBP.

[0365] conclusion These experiments provide further pharmacological evidence that C5 plays a role in the pathology of autoimmune uveitis. Pharmacological deficiency of fully human anti-human C5 antibodies delays EAU development and reduces disease severity, establishing the efficacy of these antibodies in autoimmune uveitis.

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

[0367] [Table 34]

[0368] [Table 35]

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

[0370] Example 17: Efficacy of anti-C5 antibody against lupus nephritis This example describes the efficacy of an anti-C5 antibody in treating lupus nephritis in a mouse model.

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

[0372] For this study, 25-week-old NZBWF1 mice were subcutaneously treated with either an isotype control at 30 mg / kg or an anti-C5 antibody twice a week for 8 weeks, followed by treatment three times a week for 10 weeks. The anti-mouse C5 antibodies used for this study were M1M17628N and M1M17627N, containing HCVR / LCVR sequences 362 / 363 and 365 / 366, respectively.

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

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

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

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

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

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

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

[0380] Reproducible methods for evaluating the inhibitory effects of candidate drugs on complement activation are essential for all clinical development. Due to the complexity of the complement activation pathway, assays should use cells and endpoints appropriate to the specified therapeutic indication. Here, we demonstrate the use of immortalized human glomerular endothelial cell lines (HGECs) to evaluate the blocking activity of anti-C3 or C5 mAbs in complement C3 and C5 deposition models.

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

[0382] result C3 and C5b-9 deposition was observed in ADP-activated HGECs exposed to normal human serum, but not in inactivated HGECs (C3: 1.5x10 7 ±1.0x10 7 ;C5:7.9x10 6 ±6.6x10 6 (P<0.05 vs. non-ADP-activated HGEC). Deposition of C3 and C5b-9 was significantly reduced in ADP-activated HGEC exposed to C3 or C5-deficient serum (C3: 3.3x10). 5 ±4.8x10 4 ;C5:1.5x10 6 ±6.0x10 5 (P<0.05). Addition of a blocking anti-C5 mAb significantly reduced the deposition of C5b-9 from normal human serum on ADP-activated HGECs, and the deposition was similar to that of C5-deficient serum (C5 mAb: 1.02 x 10⁻¹⁰). 6 ±6.0x10 5 , control mAb 3.7x10 6 ±1.6x10 6 (P < 0.05 vs. control mAb).

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

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

Claims

1. A pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to the C5 protein, for use in a method of preventing, treating, or relieving at least one symptom or sign of a disease or disorder related to complement factor 5 (C5), The antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 100, HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, HCDR3 comprises the amino acid sequence of SEQ ID NO: 104, LCDR1 comprises the amino acid sequence of SEQ ID NO: 108, LCDR2 comprises the amino acid sequence of SEQ ID NO: 110, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 112; and, The subject has a C5 R885H or R885C variant, The above-mentioned pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment is an antibody.

3. The pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment is an antigen-binding fragment of an antibody.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO:

98.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or its antigen-binding fragment comprises an LCVR having the amino acid sequence of SEQ ID NO:

106.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO: 98 and an LCVR containing the amino acid sequence of SEQ ID NO:

106.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO:

353.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the light chain comprises the amino acid sequence of SEQ ID NO:

354.

9. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 353 and a light chain containing the amino acid sequence of SEQ ID NO:

354.

10. The aforementioned C5-related diseases or disorders include adult respiratory distress syndrome, age-related macular degeneration, Alport syndrome, Alzheimer's disease, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune diseases, balloon angioplasty, bullous pemphigoid, capillary leak syndrome, Crohn's disease, chronic obstructive pulmonary disease (COPD), C3 nephropathy, diabetes mellitus, diabetic nephropathy, impaired inappropriate or undesirable complement activation, emphysema, epilepsy, geographic atrophy, Guillain-Barré syndrome, hemodialysis complications, hemolytic anemia, hyperacute allograft rejection, immune complex disease, infectious diseases, inflammation of autoimmune diseases, inflammatory disorders, interleukin-2 induction toxicity during IL-2 treatment. A pharmaceutical composition according to any one of claims 1 to 9, selected from lupus nephritis, membranoproliferative glomerulonephritis, mesenteric artery reperfusion after aortic reconstruction, multiple sclerosis, myocardial infarction, myasthenia gravis, neuromyelitis optica, obesity, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pneumonia, post-ischemic reperfusion state, post-pump syndrome in cardiopulmonary bypass or renal bypass surgery, progressive renal failure, proliferative glomerulonephritis, proteinuric nephropathy, pulmonary embolism, pulmonary infarction, renal impairment, renal ischemia-reperfusion injury, rheumatoid arthritis, schizophrenia, sepsis, stroke, systemic lupus erythematosus, systemic lupus erythematosus nephritis, burns including burns and frostbite, traumatic brain injury, uveitis, vasculitis, and xenograft rejection.

11. The pharmaceutical composition according to claim 10, wherein the C5-related disease or disorder is atypical hemolytic uremic syndrome (aHUS).

12. The pharmaceutical composition according to claim 10, wherein the C5-related disease or disorder is paroxysmal nocturnal hemoglobinuria (PNH).

13. The pharmaceutical composition according to claim 10, wherein the C5-related disease or disorder is myasthenia gravis.

14. The pharmaceutical composition according to any one of claims 1 to 13, which is administered to the subject in combination with a second therapeutic agent.

15. The pharmaceutical composition according to claim 14, wherein the second therapeutic agent is selected from anti-CD20 drugs, anticoagulants, anti-inflammatory drugs, antihypertensive drugs, antiepileptic drugs, antithrombotic drugs, anti-TNF drugs, C3 inhibitors, immunosuppressants, infliximab, lipid-lowering agents, rituximab, and a second anti-C5 antibody.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the pharmaceutical composition is administered to a subject by intracranial administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, oral administration, or subcutaneous administration.

17. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is administered to a subject by intravenous or subcutaneous administration.

18. A reusable pen-type delivery device comprising the pharmaceutical composition according to any one of claims 1 to 17.

19. An automated syringe delivery device comprising the pharmaceutical composition according to any one of claims 1 to 17.

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