EA039858B1 - Anti-c5 antibodies and uses thereof - Google Patents

Abstract

The invention provides monoclonal antibodies that bind to the complement factor 5 (C5) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to C5 protein. In some embodiments, the antibodies of the 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 invention provides for an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, e.g., a half-life of more than 10 days.

Description

The application for this invention, filed June 13, 2017 as a PCT International Patent Application, claims the benefit of priority of U.S. Provisional Application No. 62/349,705, filed June 2016; 62/405,561, filed October 7, 2016; and 62/422.10, filed November 15, 2016, the disclosures of each are incorporated herein by reference in their entirety.

Field of invention

The present invention relates to antibodies and antigen-binding antibody fragments that specifically bind to complement factor C5, and to therapeutic and diagnostic methods for using these antibodies.

Background of the invention

The complement system is a group of plasma proteins that, when activated, lead to lysis of the target cell and promote phagocytosis through opsonization. Complement is activated through a series of proteolytic steps in three main pathways: the classical pathway, which is normally 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 to proteolytic cleavage of the protein component 5 (C5) of complement. Cleavage of component 5 of complement (C5) leads to the formation of fragments C5a and C5b, this process is critical during the activation of the complement cascade. C5a can elicit pleiotropic physiological responses through binding to its receptors (Monk et al., 2007, Br. J. Pharmacol. 152:429-448). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates oxidative burst, and induces the release of various inflammatory mediators such as histamine or cytokines. C5b mediates the formation of a membrane attack complex (MAC or C5b-9), which leads to cell lysis in the late phases of complement-dependent cytotoxicity (CDC). Furthermore, in nucleated cells that are resistant to cytolysis by C5b-9, sublytic amounts of C5b-9 can induce cell activation resulting in cell proliferation, production of pro-inflammatory mediators, and extracellular matrix production.

Моноклональные антитела к С5 известны в данной области и описаны, например, в патентах США/публикациях № 9206251, 9107861, 9079949, 9051365, 8999340, 8883158, 8241628, 7999081, 7432356, 7361339, 7279158, 6534058, 6355245, 6074642, 20160299305, 20160051673 , 20160031975, 20158936, 20140056888, 20130022615, 20120308559 and in Wo 2015/198243, Wo 2015/134894, Wo 2015/120130, EP 2563813V1, EP 2328616V1 and EP 2061810V1.

Fully human antibodies that specifically bind to the C5 protein with high affinity and have improved pharmacokinetic properties may be important in the prevention and treatment of various C5-associated diseases (eg, atypical hemolytic uremic syndrome).

Brief summary of the invention

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind complement factor 5 (C5) protein. The antibodies of the present invention can be used, inter alia, to inhibit or neutralize the activity of the C5 protein. In certain embodiments, antibodies can be used in preventing, treating, or improving at least one symptom or indication of a C5-associated disease or disorder in a subject. In certain embodiments, antibodies can be administered prophylactically or therapeutically to a subject who is at risk of having or has a C5-associated disease or disorder. In certain embodiments, anti-C5 antibodies are fully human antibodies that bind to C5 with high affinity and have improved pharmacokinetic (PK) and pharmacodynamic (PD) properties. Such high affinity PK/PD improved antibodies can be used to provide a superior effect along with less frequent dosing in a subject with a C5 associated disease or disorder.

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

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

Exemplary antibodies against C5 of the present invention are listed in table. 1 and 2 in the present description. In table. 1 shows the amino acid sequence identifiers for the heavy chain variable regions (HCVR), the light chain variable regions (LCVR), the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of exemplary anti-C5 antibodies. . In table. 2 shows the nucleic acid sequence identifiers for HCVR, LCVR, HCDR1, HCDR2

HCDR3, LCDR1, LCDR2 and LCDR3 exemplary antibodies against C5.

The present invention provides antibodies or antigennegative fragments thereof, which contain HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to antibodies or antigennegative fragments thereof, which contain an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to antibodies or antigen-binding fragments thereof, which contain a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences of HCVR listed in table. 1, paired with any of the LCVR amino acid sequences listed in Table. 1. In accordance with certain variants of implementation of the present invention provides antibodies or antigennegative fragments thereof, which contain a pair of amino acid sequences HCVR/LCVR contained in any exemplary anti-C5 antibodies listed in table. 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 98/114 122/106 98/130 138/106 146/106 122/130 146/114 146/130 138/130 154/162 170/178 186/194 202/ 210, 218/226, 234/242, 250/258, 266/258, 274/282, 290/298, 306/314, 322/330 and 338/346. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 50/58 (e.g. H4H12161P), 98/106 (e.g. H4H12166P), 138/106 (e.g. H4H12166P5) or 202/210 (e.g. , H4H12170P). In certain embodiments, the implementation of the present invention provides antibodies against C5 or antigennegative fragments containing HCVR and LCVR, the specified HCVR contains the amino acid sequence listed in table. 1, having no more than five amino acid substitutions, and the specified LCVR contains the amino acid sequence listed in table. 1 having no more than two amino acid substitutions. For example, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, said HCVR comprising the amino acid sequence of SEQ ID NO: 98 having no more than five amino acid substitutions, and said LCVR comprising the amino acid sequence of SEQ ID NO: 106 having no more than two amino acid substitutions. In another embodiment, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, said HCVR comprising the amino acid sequence of SEQ ID NO: 98 having at least one amino acid substitution, and said LCVR comprising the amino acid sequence of SEQ ID NO: 106 having one amino acid substitution.

The present invention also relates to antibodies or antigennegative fragments thereof, which contain a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigennegative fragments thereof, which contain a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigennegative fragments thereof, which contain a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

The present invention also relates to antibodies, or antigen-binding fragments thereof, that comprise a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1. 1, or an essentially similar sequence having at least 90%, at least 95%,

- 2 039858 at least 98% or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof, which contain a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigennegative fragments thereof, which contain a pair of amino acid sequences HCDR3 and LCDR3 (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. 1. In accordance with certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise the HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary anti-C5 antibodies listed in Table 1. 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 56/64 (e.g., H4H12161P), 104/112 (e.g., H4H12166P), 144/112 (e.g., H4H12166P5), and 208 /216 (for example, H4H12170P).

The present invention also relates to antibodies or antigennegative fragments thereof, which contain HCVR and LCVR, the specified HCVR contains HCDR1 containing an amino acid sequence that differs from the amino acid sequence listed in table. 1, one amino acid, HCDR2, containing an amino acid sequence that differs from the amino acid sequence listed in table. 1, one amino acid, and HCDR3 containing an amino acid sequence that differs from the amino acid sequence listed in table. 1, per amino acid. In certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise HCVR and LCVR, said LCVR comprising an LCDR1 containing an amino acid sequence different from the amino acid sequence listed in Table 1. 1, one amino acid, LCDR2 containing an amino acid sequence that differs from the amino acid sequence listed in table. 1, one amino acid, and LCDR3 containing an amino acid sequence that differs from the amino acid sequence listed in table. 1, per amino acid. For example, the present invention provides antibodies against C5 or antigen-binding fragments thereof, which contain HCVR and LCVR, said HCVR contains HCDR1 containing the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that differs from SEQ ID NO: 100 by one amino acid, HCDR2 containing the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence different from SEQ ID NO: 102 by one amino acid, and HCDR3 containing the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence different from SEQ ID NO: 104 by one amino acid. In another exemplary embodiment, the present invention provides antibodies or antigen-binding fragments thereof that comprise HCVR and LCVR, said LCVR comprising an LCDR1 containing the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that differs from SEQ ID NO: 108 by one amino acid, LCDR2, containing the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that differs from SEQ ID NO: 110 by one amino acid, and LCDR3 containing the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that differs from SEQ ID NO: 112 by one amino acid.

The present invention provides antibodies or antigen-binding fragments thereof, which contain a heavy chain containing the amino acid sequence of SEQ ID NO: 353 or a sequence essentially similar to it, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

The present invention also relates to antibodies, or antigen-binding fragments thereof, which contain a light chain containing the amino acid sequence of SEQ ID NO: 354 or a sequence essentially similar to it, having at least 80%, at least 85%, at least 90 %, at least 95%, at least 98% or at least 99% sequence identity with it.

In certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 353, or a sequence substantially similar thereto, having at least 80% or at least 90% sequence identity with her;

and a light chain containing the amino acid sequence of SEQ ID NO: 354 or a sequence substantially similar to it, having at least 80% or at least 90% sequence identity with it.

The present invention also relates to antibodies, or antigen-binding fragments thereof,

- 3 039858 which contain a set of six CDRs (ie HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-C5 antibodies listed in table. one.

In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NO: 52-54-56-60-62-64 (e.g., H4H12161P), 100-102-104 -108-110-112 (for example, H4H12166P), 140-142-144-108-110-112 (for example, H4H12166P5) and 204-206-208-212-214-216 (for example, H4H12170P).

In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR/LCVR amino acid sequence pair as determined by any exemplary antibodies against C5 listed in table. 1. For example, the present invention includes antibodies, or antigen-binding fragments thereof, which comprise a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 50 /58 (eg H4H12161P), 98/106 (eg H4H12166P), 138/106 (eg H4H12166P5) or 202/210 (eg H4H12170P). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in specific HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the definition of Kabat, the definition of Chothia, and the definition of AbM. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences in an antibody.

In certain embodiments, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to C5, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region ( HCVR), and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in 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 no more than 5 amino acid substitutions;

and LCVR contains (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 no more than 5 amino acid substitutions.

The present invention includes anti-C5 antibodies having a modified glycosylation pattern. In some embodiments, a modification may be used to remove unwanted glycosylation sites, or an antibody lacking a fucose moiety present on the oligosaccharide chain, e.g., to increase the function of antibody-dependent cell-mediated cytotoxicity (ADCC) (see Shield et al. (2002) , JBC, 277:26733). In other applications, modification of galactosylation can be performed in order to modify complement-dependent cytotoxicity (CDC).

In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that inhibit pH-dependent binding to C5. For example, the present invention includes antibodies and antigen-binding fragments thereof that bind C5 with higher affinity at neutral pH than at acidic pH (ie, reduced binding at acidic pH).

In certain embodiments, the present invention provides antibodies and antigen binding fragments that exhibit improved pharmacokinetic and pharmacodynamic properties, for example, the present invention provides anti-C5 antibodies that have an increased serum half-life. In certain embodiments, the anti-C5 antibodies of the present invention have a serum concentration of greater than µg/mL up to day 40 in C5 humanized mice. In certain embodiments, the anti-C5 antibodies of the present invention block CP and AP hemolysis up to day 35 when administered to C5 humanized mice.

The present invention also relates to antibodies and antigen-binding fragments thereof that compete for specific binding to C5 with an antibody or antigen-binding fragment thereof containing a CDR from HCVR and a CDR from LCVR, where each HCVR and LCVR has an amino acid sequence selected from the sequences of HCVR and LCVR, listed in Table. one.

The present invention also relates to antibodies and antigen-binding fragments thereof that cross-compete for binding to C5 with a reference antibody or antigen-binding fragment thereof containing a CDR from HCVR and a CDR from LCVR, where each HCVR and LCVR has an amino acid sequence selected from the sequences of HCVR and LCVR listed in Table. one.

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

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

In some embodiments, the antibody, or antigen-binding fragment thereof, can bind specifically to C5 in an agonistic manner, ie. they can enhance or stimulate C5 binding and/or activity; in other embodiments, the antibody may bind specifically to C5 in an antagonistic manner, ie. it may block the binding and/or activity of C5.

The present invention also relates to isolated antibodies and antigen-binding fragments thereof which block the binding of C5 to C5 convertase. In some embodiments, an antibody or antigen-binding fragment thereof that blocks C5 binding to

C5 convertase may bind to the same epitope on C5 as C5 convertase, or may bind to a different epitope on C5 than C5 convertase. In some embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that block C5 binding to monkey C5 convertase.

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

In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to C5a with an IC _{50 of} less than 0.5 nM. In certain embodiments, the antibodies comprise an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 306, 322, and 338. In certain embodiments, the antibodies comprise an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298, 314, 330 and 346.

In certain embodiments, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that has one or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C. as measured in a surface plasmon resonance assay;

(c) binds to human C5 with a KD of less than 0.3 nM at 37° C. as measured in a surface plasmon resonance assay;

(d) binds to monkey C5 with a KD of less than 65 nM as measured in a surface plasmon resonance assay;

(e) binds to human C5 variant R885H (SEQ ID NO: 356) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(f) binds to human C5 variant R885C (SEQ ID NO: 357) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(g) blocks the human C5-mediated classical pathway (CP) of hemolysis by greater than 95% and with an IC _{50 of} less than 6 nM as measured in the hemolysis CP assay;

(h) blocks the human C5 mediated alternative pathway (AR) of hemolysis by more than 70% and with an IC _{50 of} less than 165 nM as measured by the hemolysis AR assay;

(i) inhibits African green monkey C5 mediated CP hemolysis with an IC _{50 of} less than 185 nM as measured in the Hemolysis CP assay;

(j) inhibits African green monkey C5-mediated AR hemolysis with an IC _{50 of} less than 235 nM as measured by the AR hemolysis assay;

(k) inhibits cynomolgus C5 mediated CP hemolysis with an IC _{50 of} less than 145 nM as measured in the CP hemolysis assay;

(l) inhibits cynomolgus C5 mediated AR hemolysis with an IC _{50 of} less than 30 nM as measured by the AR hemolysis assay.

In certain embodiments, the present invention provides an isolated recombinant anti-C5 monoclonal antibody, or antigen-binding fragment thereof, that has one or more of the following characteristics:

(a) contains a set of six CDRs containing the amino acid sequences of SEQ ID NO: 100-102-104-108-110-112;

(b) binds to human C5 with a dissociation constant (KD) of less than 0.2 nM at 25° C. as measured in a surface plasmon resonance assay;

(c) binds to human C5 with a KD of less than 0.3 nM at 37° C. as measured in a surface plasmon resonance assay;

(d) binds to human C5 variant (R885H) with a KD of less than 0.4 nM at 37° C. as measured in a surface plasmon resonance assay;

(e) inhibits classically mediated (CP) hemolysis in human serum with an IC _{50 of} less than 3 nM;

(f) inhibits alternative pathway (AP) mediated hemolysis in human serum with an IC _{50 of} less than 27 nM;

(g) inhibits CP-mediated hemolysis in monkey serum with an IC ₅₀ less than 21 nM;

(g) inhibits AP-mediated hemolysis in monkey serum with an IC50 of less than 10 nM;

(h) has a serum half-life (t _1/2 ) greater than 10 days in C5 humanized mice;

(i) has a serum concentration greater than 10 μg/ml up to day 40 when administered to C5 humanized mice;

- 6 039858 (j) blocks CP-mediated hemolysis up to day 50 in C5 humanized mice;

(k) binds to one or more amino acids contained in the α chain and/or β chain of SEQ ID NO: 359, wherein the antibody does not bind to the C5a anaphylatoxin domain of C5.

In a second aspect, the present invention provides nucleic acid molecules encoding anti-C5 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the nucleic acid sequences HCDR1 listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the nucleic acid sequences HCDR2 listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the nucleic acid sequences HCDR3 listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table. one; in certain embodiments, the implementation of the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in table. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table. one; in certain embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 1. 2, or a sequence that is substantially similar to them, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding HCVR, where HCVR contains a set of three CDRs (i.e. HCDR1-HCDR2-HCDR3), where the set of amino acid sequences HCDR1-HCDR2-HCDR3 is that determined using any exemplary antibodies against C5 listed in table. one.

The present invention also relates to nucleic acid molecules encoding LCVR, where the LCVR contains a set of three CDRs (i.e. LCDR1-LCDR2-LCDR3), where the set of amino acid sequences LCDR1-LCDR2-LCDR3 is that determined using any exemplary antibodies against C5 listed in table. one.

The present invention also relates to nucleic acid molecules encoding both

- 7 039858

HCVR and LCVR, where HCVR contains an amino acid sequence from any of the HCVR amino acid sequences listed in table. 1, and where LCVR contains an amino acid sequence from any of the LCVR amino acid sequences listed in table. one.

In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1. 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from the same anti-C5 antibody listed in Table 1. one.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide containing the heavy or light chain variable region of an anti-C5 antibody. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, ie. nucleic acid molecules encoding any sequence of HCVR, LCVR and/or CDR, as set forth in table. 2. Also included within the scope of the present invention are host cells into which such vectors are introduced, as well as methods for producing antibodies or parts thereof by culturing the host cells under conditions capable of producing antibodies or antibody fragments and recovering the antibodies and antibody fragments thus obtained. .

In a third aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds C5 and a pharmaceutically acceptable carrier. In a related aspect, the invention is characterized by 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 combines favorably with an anti-C5 antibody. Exemplary agents that may be usefully combined with an anti-C5 antibody include, without limitation, other agents that bind and/or inhibit C5 activity (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents that do not directly bind C5, but nevertheless treat or improve at least one symptom or indication of a C5-associated disease or disorder. Additional combination therapy and combination formulations involving antibodies against C5 of the present invention are disclosed elsewhere in the present description.

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

- 8 039858 body weight of the subject. In certain embodiments, an antibody of the present invention may be administered in one or more doses between 50 and 600 mg.

The present invention also relates to the use of an anti-C5 antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blockade of C5 binding and/or activity.

Other embodiments will become apparent upon review of the following detailed description.

Brief description of the figures

In FIG. 1 shows dose dependent inhibition of C5a levels by anti-C5 antibody H4H12166P as determined by ELISA (described in Example 9 herein).

In FIG. 2 shows the total serum concentration versus time after a single 15 mg/kg intravenous injection of H4H12166P, H4H12161P or reference 2 male cynomolgus monkeys (described in Example 10 herein). The concentration-time profiles were plotted from the first post-dose to the limit of quantitation (BLQ) result, if applicable, which was calculated as LLOQ/2. Each data point represents the mean (±SD) (n=4 animals/group); concentrations that were expected to be influenced by ADA were excluded in one animal in the H4H12166P group and one animal in the H4H12161P group, starting on day 36 and day 29, respectively. LLOQ = lower limit of quantification.

In FIG. 3 shows the percentage of hemolysis versus time ex vivo in red blood cells for (A) classical pathway and (B) alternative pathway assays after a single intravenous injection of H4H12166P, H4H12161P or reference 2 male cynomolgus monkeys. The % hemolysis, calculated as the ratio of experimental and maximum lysis minus % background lysis from both values, is related to the amount of C5 inhibited by a particular anti-C5 antibody present in serum at a given time. Each data point represents the mean (±SD).

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

In FIG. 5 shows the percent hemolysis versus time ex vivo in the classical complement pathway hemolysis assay for selected anti-C5 antibodies in C5 humanized mice. Humanized C5 mice were administered a single 15 mg/kg subcutaneous dose of H4H12166P, Comparator 1, or Comparator 2. Each data point represents mean±SEM (n=4-5 each). Carried out monitoring of the percentage of hemolysis in serum before the dose, 10, 20, 30, 40 and 50 days after injection. The % hemolysis, which was calculated as the ratio of experimental and maximum lysis minus % background lysis from both values, is related to the amount of C5 inhibited by a particular anti-C5 antibody present in serum at a given time.

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

In FIG. 7 is a graph showing optical coherence tomography (OCT) scores in mice treated with the isotype control or anti-C5 antibody M1M17628N at 10 or 50 mg/kg (described in Example 14 herein). ****p<0.0001, two-way ANOVA of anti-C5 antibody treatment at 50 mg/kg versus no treatment or isotype control treatment.

In FIG. 8 shows the classical pathway inhibition of hemolysis by the anti-C5 antibody M1M17628N in the absence of C3 (A) and in the presence of 80 μg/ml human C3 (B) (described in Example 14 herein).

In FIG. 9 shows the number of cell clusters in C5 humanized mice treated with isotype control or anti-human C5 antibody H4H12170P at 10 or 50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group.

In FIG. 10 is a graph showing OCT scores in C5 humanized mice treated with the isotype control or anti-human C5 antibody H4H12170P at 10 or 50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group.

In FIG. 11 is a graph showing OCT scores in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 or 10 mg/kg, or comparator 2 at 10 mg/kg. n=6-12 eyes for each group (described in Example 15 herein).

- 9 039858

In FIG. 12 shows the number of cell clusters in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 or 10 mg/kg, or comparator 2 at 10 mg/kg. n=6-12 eyes for each group (described in Example 15 herein).

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

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

In FIG. 15 shows blood urea nitrogen levels in NZBWF1 mice treated with isotype control or anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein).

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

Detailed description

Before describing these methods, it should be understood that this invention is not limited to the particular methods and experimental conditions described, as such, the methods and conditions may vary. It should also be understood that the terminology used in this specification is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is only limited by the appended claims.

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning as they are commonly understood by a person skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned in the present description are incorporated into the present description by reference in their entirety.

Definitions

The term C5, also called complement component 5 or complement factor 5, refers to a serum protein from the complement cascade. The C5 protein is a 1676 amino acid protein that contains two chains, α and β. The protein is the point of convergence of three pathways of complement activation: the classical pathway, the alternative pathway, and the mannose-binding lectin pathway. The amino acid sequence of the full-length C5 protein is exemplified in the amino acid sequence provided by GenBank Accession Number NP_001726.2 (SEQ ID NO: 355). The term C5 includes recombinant C5 protein or a fragment thereof. The term also encompasses a C5 protein or a fragment thereof conjugated to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1. For example, the term includes sequences, exemplified by those shown in SEQ ID NO: 356 or 357, which contain at the C-terminus a histidine tag conjugated to amino acid residues 19-1676 of the full-length C5 protein. The term also includes protein variants that contain a C-terminal histidine tag conjugated to amino acid residues 19-1676 of the full-length C5 protein with an R885H substitution or an R885C substitution.

The term antibody, as used herein, is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains linked by disulfide bonds (i.e., complete antibody molecules), as well as their multimers (eg IgM) or their antigen-binding fragments. Each heavy chain is composed of a heavy chain variable region (HCVR or V _H ) and a heavy chain constant region (composed of C _H 1 , C _H 2 and C _H 3 domains). Each light chain is composed of a light chain variable region (LCVR or V _L ) and a light chain constant region (C _L ). The V _H and V _L regions can be further subdivided into regions of hypervariability, referred to as complementarity-determining regions (CDRs), which alternate with regions that are more conserved and are referred to as framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FR of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified.

The consensus amino acid sequence can be determined based on a comparative analysis of two or more CDRs.

It is also possible to replace one or more CDR residues or skip one or more CDRs. The scientific literature describes antibodies in which one or two CDRs can be distributed for binding. Padlan et al. (1995, FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one fifth to one third of the CDRs actually contact the antigen.

Padlan has also found many antibodies in which one or two CDRs lack amino acids in contact with the antigen (see also Vajdos et al. 2002, J. Mol. Biol. 320:415-428).

Non-antigen contacting CDR residues can be identified based on previous studies (eg, H60-H65 residues in CDRH2 are often not required), in Kabat CDR regions outside the no Chothia CDR, by molecular modeling and/or empirically. If a CDR or its residue(s) is omitted, it is usually replaced with an amino acid at the appropriate position in another human antibody sequence or consensus of such sequences. CDR substitution positions and amino acid substitutions can also be empirically selected. Empirical substitutions may be conservative or non-conservative substitutions.

The fully human anti-C5 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the heavy and light chain variable domain framework and/or CDR regions compared to the corresponding germline sequences. Such mutations are readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and/or CDR regions are mutated into the corresponding germline sequence residue(s) from which an antibody is generated, either to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence substitutions are collectively referred to herein as generative mutations). One skilled in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily generate a variety of antibodies and antigen-binding fragments that contain one or more individual generative mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues in the V _H and/or V _L domains are mutated back to residues occurring in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues occurring within the first 8 amino acids in FR1 or within the last 8 amino acids in FR4, or only mutated residues occurring within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to corresponding residues of a different germline sequence (ie, a germline sequence that differs from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention may contain any combination of two or more generative mutations in the framework and/or CDR regions, for example, where certain individual residues mutate into the corresponding residue of a particular germline sequence, while certain other residues that differ from the original sequence germline, retain or mutate into the corresponding residue of another germline sequence. Once antibodies and antigen-binding fragments have been obtained that contain one or more generative mutations, one or more desired properties can be easily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity, etc.

Antibodies and antigen-binding fragments obtained in this general way are included in the present invention.

The present invention also provides fully human anti-C5 monoclonal antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes anti-C5 antibodies having the amino acid sequences HCVR, LCVR and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), e.g., in CDRs and, in particular, in CDR3 . However, the term human antibody, as used herein, is not intended to include mAbs in which CDR sequences derived from a mammalian germline of other species (eg, mice) are grafted into human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

The term recombinant, as used herein, refers to antibodies or antigen-binding fragments thereof of the invention that are made, expressed, isolated, or produced by technologies or methods known in the art, such as recombinant DNA technology, which includes, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or in a cellular expression system (e.g., CHO cells) or isolated from a combinatorial library of recombinant human antibodies.

The term specifically binds or specifically binds to or the like means that an antibody, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-8 M or less (eg, lower K _D means stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As disclosed herein, using surface plasmon resonance, for example BIACORE™, antibodies have been identified that bind specifically to C5. In addition, polyspecific antibodies that bind to one domain at C5 and one or more additional antigens, or bispecific antibodies that bind to two different regions at C5, are nonetheless considered antibodies that specifically bind as used herein. .

The term high affinity antibody refers to those mAbs that have a C5 binding affinity, expressed as a KD, of at least 10 ^-8 M; preferably 10 ^-9 M; more preferably 10 ^-10 M, even more preferably 10 ^-11 M, even more preferably 10 ^-12 M, as measured by surface plasmon resonance, for example BIACORE™ or ELISA affinity in solution.

By the term low dissociation rate, K _off or kd is meant that the antibody dissociates from C5 with a rate constant of 1 x 10 ^-3 s ^-1 or less, preferably 1 x 10' ⁴ s ^-1 or less, as determined by surface plasmon resonance, for example BIACORE™.

The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen in order to form a complex. The terms antigen-binding antibody fragment or antibody fragment, as used herein, refers to one or more antibody fragments that retain the ability to bind to the C5 protein.

In specific embodiments, an antibody or antibody fragments of the invention can be conjugated to a fragment such as a ligand or therapeutic fragment (immunoconjugate), a second anti-C5 antibody, or any other therapeutic fragment that can be used to treat a C5 associated disease or disorder.

An isolated antibody, as used herein, is intended to mean an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds C5, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than C5.

A blocking antibody or neutralizing antibody as used herein (or an antibody that neutralizes C5 activity or an antagonist antibody) is intended to mean an antibody in which binding to C5 leads to inhibition of at least one C5 biological activity. For example, an antibody of the invention may prevent or block complement-mediated hemolysis via the classical pathway or an alternative pathway.

The term surface plasmon resonance, as used herein, refers to an optical phenomenon that makes it possible to analyze biomolecular interactions in real time by detecting changes in protein concentrations on a biosensor array, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term K _D , as used herein, is intended to mean the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a particular antigen-binding site in the variable region of an antibody molecule, known as a paratope. One

- 12 039858 an antigen may have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. The term epitope also refers to a location on an antigen to which B and/or T cells respond. It also refers to the region of an antigen that an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are reactive surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural and/or specific charge characteristics.

The term cross-compete, as used herein, means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, ie. the first antibody that binds and blocks the binding of the second antibody, and vice versa. In certain embodiments, the first antibody and the second antibody can bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that binding of one inhibits or blocks binding of the second antibody, eg, through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example by real-time analysis of unlabeled biolayer interferometry. Cross-competition between two antibodies can be expressed in the binding of the second antibody, which is less than the background signal due to binding to itself (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as % binding of the second antibody, which is less than the base level of background binding to itself (where the first and second antibodies are the same antibody).

The term substantial identity, or substantially identical with respect to a nucleic acid or fragment thereof, indicates that, when optimally aligned with suitable nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is a nucleotide sequence identity of at least about 90 % and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide bases, as measured by any commonly known algorithm for sequence identity such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule that has substantial identity with a reference nucleic acid molecule may, in certain instances, encode a polypeptide that has the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

When applied to polypeptides, the term significant similarity or substantially similar means that two peptide sequences, when aligned optimally, for example, using the GAP or BESTFIT programs using the default gap weights, have at least 90% sequence identity, even more preferably at least 95, 98 or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions.

A conservative amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution does not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to allow for the conservative nature of the substitution. The means of making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994), Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids that have side chains with similar chemical properties include:

1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;

2) aliphatic hydroxyl side chains: serine and threonine;

3) amide-containing side chains: asparagine and glutamine;

4) aromatic side chains: phenylalanine, tyrosine and tryptophan;

5) main side chains: lysine, arginine and histidine;

6) acidic side chains: aspartate and glutamate and

7) sulfur-containing side chains: cysteine and methionine.

Preferred groups of conservative amino acid substitutions are valineleucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log probability matrix disclosed in Gonnet et al.

(1992), Science 256:1443 45, incorporated herein by reference. A moderately conservative substitution is any substitution that has a non-negative value in the PAM250 log probability matrix.

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 conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides, from different species, or between a wild-type protein and his mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity for regions with the best overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm for comparing a sequence of the invention with a database containing a large number of sequences from different organisms is implemented in the BLAST computer program, in particular BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410 and (1997), Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. The exact amount depends on the purpose of the treatment and will be determined by those skilled in the art using known methods (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term subject refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention, and/or treatment of a C5-associated 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.

As used herein, the terms treat, treating, or treating refer to reducing or improving the severity of at least one symptom or indication of a C5-associated disease or disorder by administering a therapeutic agent, such as an antibody of the present invention, to a subject in need thereof. The terms include inhibition of disease progression or worsening of a symptom/indication. The terms also include a positive prognosis of the disease, ie. the subject may be freed from disease or may have reduced disease upon administration of a therapeutic agent, such as an antibody of the present invention. The therapeutic agent may be administered to a subject at a therapeutic dose.

The terms prevent, preventing, or prevention refer to the inhibition of the manifestation of a C5-associated disease or disorder, or any symptoms or indications of such a disease or disorder, upon administration of an antibody of the present invention.

Antigen-binding fragments of antibodies.

Unless specifically stated otherwise, the term antibody, as used herein, should be understood to encompass antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., complete antibody molecules), as well as antigen-binding fragments thereof. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms antigen-binding antibody fragment or antibody fragment, as used herein, refers to one or more antibody fragments that retain the ability to specifically bind to the C5 protein. An antibody fragment contains a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term antigen-binding fragment refers to a polypeptide fragment of a polyspecific antigen-binding molecule.

Antigen-binding fragments of an antibody can be extracted, for example, from whole antibody molecules using any suitable standard methods such as proteolytic cleavage or recombinant genetic engineering techniques using the manipulation and expression of DNA that encodes the variable and (optionally) constant domains of an antibody. Such DNA is known and/or readily available from, for example, commercial sources, DNA libraries (including, for example, antibody phage libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in an appropriate configuration or introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen-binding fragments include:

(i) Fab fragments;

(ii) F(ab')2 fragments;

(iii) Fd fragments;

(iv) Fv fragments;

(v) single chain Fv molecules (scFv);

(vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide.

Other engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.) .), small modular immunopharmaceuticals (SMIPs), and shark IgNAR variable domains are also encompassed by the term antigen binding fragment as used herein.

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

In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that can be found in an antigen-binding fragment of an antibody 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 -Ch1-C _h 2-C _h 3; (vi) V _H -C _H 2 -C _H 3; (vii) V _H -C _L ; (viii)

V _{L -CH} 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 2C _H 3; (xiii) V _L -C _H 2 -C _H 3; and (xiv) V _L -C _L .

In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be linked directly to each other, or linked via a complete or partial hinge or linker region. The hinge region may be at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible bond between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more V _H or V _L monomeric domains. (for example, via disulfide bond(s)).

As with full antibody molecules, antigen binding fragments can be monospecific or polyspecific (eg, bispecific).

A polyspecific antigen-binding fragment of an antibody typically contains at least two distinct variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope on the same antigen. A polyspecific antibody in any format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using standard techniques available in the art.

Obtaining human antibodies.

Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention in order to

- 15 039858 part of a human antibody that specifically binds to the C5 protein.

An immunogen containing any one of the following can be used to generate antibodies to the C5 protein. In certain embodiments, antibodies of the invention are obtained from mice immunized with full-length native C5 protein (see, for example, Genbank accession number NP_001726.2) (SEQ ID NO: 355) or DNA encoding the protein or fragment thereof. Alternatively, the protein or fragment thereof can be prepared using standard biochemical methods and modified and used as an immunogen. In certain embodiments, the immunogen is a C5 protein fragment that is within amino acid residues 19-1676 of SEQ ID NO: 355.

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

Using VELOCIMMUNE® technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for creating monoclonal antibodies, high affinity chimeric anti-C5 antibodies having a human variable region and a mouse constant region are initially isolated. The VELOCIMMUNE® technology involves generating a transgenic mouse having a genome that contains human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigen challenge . The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

In general, a VELOCIMMUNE® mouse is stimulated with an antigen of interest and lymphatic cells (such as B cells) are obtained from the mice that express antibodies. The lymphatic cells can be fused with a myeloma cell line to obtain immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies with specificity for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotypic heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, antibodies will be characterized and selected for desired characteristics, including affinity, selectivity, epitope, and so on. The mouse constant regions are replaced with the desired human constant region in order to generate a fully human antibody of the invention, eg wild-type or modified IgG1 or IgG4. Although the constant region chosen may vary according to the particular use, the characteristics of high affinity antigen binding and target specificity are present in the variable region.

Bioequivalents.

The anti-C5 antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies but retain the ability to bind the C5 protein. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions relative to the parent sequence to exhibit biological activity that is substantially equivalent to that of the disclosed antibodies. Similarly, DNA sequences encoding antibodies of the present invention encompass sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequence, but which encode an antibody or antibody fragment that is substantially bioequivalents of the antibody or antibody fragment according to invention.

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives in which the rate and extent of absorption does not show significant differences under similar experimental conditions when administered at the same molar dose, or a single dose, or multiple doses. Some antibodies should be considered equivalents or pharmaceutical alternatives if they are equivalent in their absorption but not in their absorption rate, and may still be considered bioequivalent because such differences in absorption rates are intentional and labeled, not essential to achieving effective concentrations drug in the body, such as chronic use, and are considered medically irrelevant for the particular investigational medicinal product.

- 16 039858

In one embodiment, two antigen-binding proteins are bioequivalent unless there are clinically significant differences in their safety, purity, or potency.

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

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

Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, for example:

(a) an in vivo test in humans or other mammals, in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluid as a function of time;

(b) an in vitro test that is correlated with and reasonably predictive of human in vivo bioavailability data;

(c) an in vivo test in humans or other mammals, in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a strictly controlled clinical trial that establishes the safety, effect, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention can be engineered, for example by creating various residue or sequence substitutions, or by deleting terminal or internal residues or sequences not necessary for biological activity. For example, cysteine residues that are not important for biological activity can be removed or replaced with other amino acids in order to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies may include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or abolish glycosylation.

Anti-C5 antibodies containing Fc variants.

In accordance with certain embodiments of the present invention, anti-C5 antibodies are provided that contain an Fc domain containing one or more mutations that increase or decrease the binding of the antibody to the FcRn receptor, for example, at acidic pH versus neutral pH. For example, the present invention includes anti-C5 antibodies containing a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome, where the pH is in the range of approximately 5.5 to about 6.0). Such mutations can lead to an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example:

modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or a modification at position 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 a modification at position 250 and/or 428; or modification at position 307 or 308 (eg 308F, V308F) and 434.

In one embodiment, the modification comprises modification 428L (eg M428L) and 434S (eg N434S);

modification 428L, 259I (for example, V259I) and 308F (for example, V308F);

modification 433K (for example, H433K) and 434 (for example, 434Y);

modification 252, 254 and 256 (for example, 252Y, 254T, and 256E);

modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or 308P).

In yet another embodiment, the modification comprises modification 265A (eg, D265A) and/or 297A (eg, N297A).

For example, the present invention includes anti-C5 antibodies containing an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of:

250Q and 248L (for example, T250Q and M248L);

252Y, 254T and 256E (eg M252Y, S254T and T256E);

428L and 434S (eg M428L and N434S);

257I and 311I (for example, P257I and Q311I);

2571 and 434H (eg P257I and N434H);

376V and 434H (for example, D376V and N434H);

- 17 039858

307A, 380A and 434A (for example, T307A, E380A and N434A) and

433K and 434F (for example, H433K and N434F).

All possible combinations of mutations of the above Fc domain and other mutations in the variable domains of antibodies disclosed in the present description are provided within the scope of the present invention.

The present invention also provides anti-C5 antibodies comprising a heavy chain chimeric constant region (C _n ), wherein the chimeric C _n region contains segments derived from C _n regions of more than one immunoglobulin isotype. For example, antibodies of the invention may comprise a C _H chimeric _region comprising a partial or complete CH 2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule, in combination with a partial or complete _CH 3 domain derived from a human IgG1 molecule, human IgG2 or human IgG4. In accordance with certain embodiments, the antibodies of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4, in combination with a lower hinge sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. An antibody containing a C _n chimeric region as disclosed herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, US Patent Application Publication 2014/0243504, the disclosure of which is hereby incorporated by reference in its entirety.)

Biological characteristics of antibodies.

In general, the antibodies of the present invention function by binding to the C5 protein and preventing it from being cleaved into C5a and C5b. For example, the present invention includes antibodies and antigen-binding antibody fragments that bind the C5 protein (e.g., at 25 or 37°C) with a KD of less than 9 nM as measured by surface plasmon resonance, for example, using the assay format as defined in example 3 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind C5 with a K _D of less than about 9 nM, about less than 5 nM, about less than 2 nM, about less than 1 nM, about less than 500 pM, less than 250 pM, or less than 100 pM as measured by surface plasmon resonance, for example, using the assay format as defined in Example 3 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind human C5 protein with a dissociative half-life (t _1/2 ) of greater than about 2 minutes, as measured by surface plasmon resonance at 25°C, for example, using the assay format, as defined in Example 4 herein, or a substantially similar analysis. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind the C5 protein with a t _1/2 greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 100 minutes, more greater than about 150 minutes, greater than about 200 minutes, or greater than about 250 minutes, as measured by surface plasmon resonance at 25°C, e.g., using the assay format as defined in Example 3 herein (e.g., mAb capture format or with antigen capture), or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind human C5 protein with a dissociation half-life (t _1/2 ) greater than about 1.5 min, as measured by surface plasmon resonance at 37°C, for example, using the format assay as defined in Example 4 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind the C5 protein with a t _1/2 greater than about 2 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 25 minutes, greater than about 50 minutes, more greater than about 100 minutes, greater than about 150 minutes, or greater than about 200 minutes, as measured by surface plasmon resonance at 37°C, e.g., using the assay format as defined in Example 3 herein (e.g., mAb capture format or with antigen capture), or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments of antibodies,

- 18 039858 which bind monkey C5 protein (for example at 25° C. or at 37° C.) with a K _D of less than 120 nM as measured by surface plasmon resonance, for example using the assay format as defined in Example 3 herein description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind monkey C5 with a K _D of less than about 120 nM, about less than 100 nM, about less than 50 nM, about less than 25 nM, about less than 10 nM, about less than 5 nM , less than about 1 nM, less than about 500 pM, or less than 250 pM, as measured by surface plasmon resonance, for example, using the assay format as defined in Example 3 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding antibody fragments that bind a modified human C5 protein with R885H substitution (an example is SEQ ID NO: 356) with a K _D of less than 70 nM, as measured by surface plasmon resonance, for example, using the assay format as defined in Example 3 herein. C5 variants showed a poor response to anti-C5 antibodies previously disclosed in the art (eg, Nishimura et al. 2014, New Engl. J. Med. 370:632-639). In certain embodiments, the antibodies, or antigen-binding fragments thereof, bind modified human C5 with a K _D of less than about 65 nM, about less than 50 nM, about less than 20 nM, about less than 10 nM, about less than 5 nM, about less than 3 nM or less than 2 nM as measured by surface plasmon resonance, for example using the assay format as defined in Example 3 herein, or a substantially similar assay.

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

The present invention also relates to antibodies and antigen-binding antibody fragments that inhibit complement dependent cytotoxicity (CDC) with an IC _{50 of} less than 10 nM as measured by a luminescence assay, e.g. using the assay format as defined in Example 6 herein. In certain embodiments, the antibodies, or antigen-binding fragments thereof, inhibit CDC with an IC _{50 of} less than about 5 nM, about less than 3.5 nM, or about less than 2 nM, as measured by a B cell luminescence assay, e.g., using the assay format, as defined in Example 6 herein, or a substantially similar analysis.

The present invention also relates to antibodies and antigen-binding fragments that block human C5-mediated classical pathway (CP) hemolysis by greater than 94% and with an IC _{50 of} less than 6 nM as measured by a hemolysis CP assay, e.g. using an assay format as defined in example 8 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block CP hemolysis with an IC _{50 of} less than about 6 nM, about less than 5 nM, about less than 4 nM, about less than 3 nM, or about less than 2 nM, as measured by CP assay. hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments that block human C5-mediated alternative pathway (AR) hemolysis by greater than 70% and with an IC _{50 of} less than 165 nM as measured by a hemolysis AR assay, e.g. using an assay format as defined in example 8 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block AP hemolysis with an IC _{50 of} less than about 160 nM, about less than 150 nM, about less than 100 nM, about less than 50 nM, or about less than 20 nM, as measured by AP assay. hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments that block the African green monkey C5 mediated classical pathway (CP) of hemolysis by greater than 40% and with an IC _{50 of} less than 185 nM as measured by a hemolysis CP assay, e.g.

- 19 039858 using the analysis format as defined in example 8 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block CP hemolysis with an IC _{50 of} less than about 180 nM, about less than 150 nM, about less than 100 nM, about less than 75 nM, or about less than 50 nM, as measured by CP assay hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments that block the African green monkey C5-mediated alternative pathway (AP) of hemolysis with an IC _{50 of} less than 235 nM as measured by a hemolysis AP assay, e.g. using the assay format as defined in Example 8 in this description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block AP hemolysis with an IC _{50 of} less than about 200 nM, about less than 150 nM, about less than 100 nM, about less than 50 nM, or about less than 20 nM, as measured by AP assay. hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen binding fragments that block greater than 90% of C5-mediated cynomolgus monkey classical pathway (CP) hemolysis with an IC _{50 of} less than 145 nM as measured by a hemolysis CP assay, e.g. using the assay format as defined in example 8 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block CP hemolysis with an IC _{50 of} less than about 140 nM, about less than 120 nM, about less than 100 nM, about less than 75 nM, or about less than 50 nM, as measured by CP assay. hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments that block the cynomolgus monkey C5-mediated alternative pathway (AP) of hemolysis with an IC _{50 of} less than 30 nM as measured by a hemolysis AP assay, e.g. using the assay format as defined in Example 8 in the present description. In certain embodiments, the antibodies, or antigen-binding fragments thereof, block AP hemolysis with an IC _{50 of} less than about 25 nM, about less than 20 nM, about less than 10 nM, about less than 5 nM, or about less than 2 nM, as measured by AP assay. hemolysis, for example, using the assay format as defined in Example 8 herein, or a substantially similar assay.

The present invention also relates to antibodies and antigen-binding fragments that exhibit improved pharmacokinetic (PK) and pharmacodynamic (PD) properties compared to anti-C5 antibodies in the art. Anti-C5 antibodies of the present invention show less susceptibility to target-mediated clearance upon administration, as shown in Examples 9 and 10 herein. In certain embodiments, the present invention includes anti-C5 antibodies and antigen-binding fragments thereof that exhibit serum concentrations over extended periods, e.g., greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days. , greater than 45 days, greater than 50 days, greater than 55 days, or greater than 60 days, as described in examples 9 and 10 herein. In certain embodiments, the anti-C5 antibodies of the present invention exhibit an increased serum half-life of greater than 10 days compared to anti-C5 antibodies in the art.

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

In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C. as measured in a surface plasmon resonance assay;

(c) binds to human C5 with a KD of less than 0.3 nM at 37° C. as measured in a surface plasmon resonance assay;

(d) has a serum concentration greater than 10 µg/mL up to day 70 when administered to Javanese

- 20 039858 macaque;

(e) blocks SR and AR hemolysis up to day 35 when administered to cynomolgus monkey as measured in an ex vivo hemolysis assay;

(f) has a serum half-life greater than 10 days in the cynomolgus monkey;

(g) has a serum concentration greater than 10 μg/ml up to day 40 when administered to C5 humanized mice;

(h) blocks CP hemolysis up to day 30 when administered to C5 humanized mice as measured in an ex vivo hemolysis assay; and (i) has a serum half-life greater than 10 days in C5 humanized mice.

In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to human C5 with a dissociation constant (K _D ) of less than 0.9 nM at 25° C. as measured in a surface plasmon resonance assay;

(c) binds to human C5 with a K _D of less than 0.3 nM at 37° C. as measured in a surface plasmon resonance assay;

(d) binds to monkey C5 with a K _D of less than 65 nM as measured in a surface plasmon resonance assay;

(e) binds to human C5 variant R885H (SEQ ID NO: 356) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(f) binds to human C5 variant R885C (SEQ ID NO: 357) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(g) blocks the human C5-mediated classical pathway (CP) of hemolysis by greater than 95% and with an IC _{50 of} less than 6 nM as measured in the hemolysis CP assay;

(h) blocks the human C5 mediated alternative pathway (AR) of hemolysis by more than 70% and with an IC _{50 of} less than 165 nM as measured by the hemolysis AR assay;

(i) inhibits African green monkey C5 mediated CP hemolysis with an IC _{50 of} less than 185 nM as measured in the Hemolysis CP assay;

(j) inhibits African green monkey C5-mediated AR hemolysis with an IC _{50 of} less than 235 nM as measured by the AR hemolysis assay;

(k) inhibits cynomolgus C5 mediated CP hemolysis with an IC _{50 of} less than 145 nM as measured in the CP hemolysis assay; and (l) inhibits cynomolgus C5 mediated AR hemolysis with an IC _{50 of} less than 30 nM as measured by the AR hemolysis assay.

The antibodies of the present invention may have one or more of the above biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the present invention will be apparent to those skilled in the art upon review of the present disclosure, including the working examples in the present specification.

Epitope mapping and related technologies.

The present invention includes anti-C5 antibodies that interact with one or more amino acids occurring in one or more regions of the C5 protein molecule, including an a polypeptide and a β polypeptide. The epitope to which antibodies bind may be one contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in any of the above domains of the C5 protein molecule (eg, a linear epitope in the domain). Alternatively, an epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) that are located in any or both of the above domains of a protein molecule (eg, a conformational epitope).

Various methods known to those skilled in the art can be used to determine if an antibody interacts with one or more amino acids in a polypeptide or protein. Exemplary methods include, for example, standard cross-competitive assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine-scanning mutagenesis assay, peptide blot assay (Reineke (2004), Methods Mol. Biol. 248:443-63), crystallographic studies in peptide digestion assay, and NMR assay. In addition, methods such as excision of epitopes, extraction of epitopes and chemical modification of antigens can be used (Tomer (2000), Prot. Sci. 9:487496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen-deuterium exchange, detected by mass spectrometry. Generally speaking, the hydrogen-deuterium exchange method involves labeling the protein of interest with deuterium, followed by antibody binding.

- 21 039858 with deuterium labeled protein. The protein/antibody complex is then transferred to water and the protons to be exchanged in amino acids that are protected by the antibody complex are deuterium-to-hydrogen back exchanged at a slower rate than the protons to be exchanged in amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999), Analytical Biochemistry, 267:252-259; Engen and Smith (2001), Anal. Chem. 73:256A-265A.

The term epitope refers to a location on an antigen to which B and/or T cells respond. B-cell epitopes can be formed from continuous amino acids or non-contiguous amino acids that are brought together by tertiary folding of the protein. Epitopes formed from continuous amino acids are usually retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. An epitope usually contains at least 3 and more, usually at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method of classifying a large number of monoclonal antibodies (mAbs) found for the same antigen according to the similarities in the binding profile of each antibody to surfaces of chemically or enzymatically modified antigens (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope that is either distinctly different from, or partially overlaps with, the epitope represented by the other category. This technology allows rapid filtering of genetically identical antibodies so that characterization can be focused on genetically different antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP can be used to sort the antibodies of the invention into groups of antibodies that bind different epitopes.

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

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

In certain embodiments, the present invention includes anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more epitopes occurring in the α and/or β chain of C5 (SEQ ID NO: 359). The epitope(s) may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more) amino acids located in the α chain and/or β chain of C5. Alternatively, an epitope may be composed of a plurality of non-contiguous amino acids (or amino acid sequences) located at C5. As shown in Example 11 herein, the C5 epitope with which the exemplary antibody of the invention H4H12166P interacts is determined by:

(i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) which corresponds to amino acids 591 to 599 contained in the β chain of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361) which corresponds to amino acids 775 to 794 contained in a chain of SEQ ID NO: 359.

Accordingly, the present invention includes anti-C5 antibodies that interact with one or more amino acids contained in a region consisting of:

(i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) which corresponds to amino acids 591 to 599 of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361) which corresponds to amino acids 775 to 794 of SEQ ID NO: 359.

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

Whether an antibody binds to the same epitope or competes for binding with a reference anti-C5 antibody can be readily determined using standard methods known in the art. For example, in order to determine if a test antibody binds to the same epitope as a reference anti-C5 antibody of the invention, the reference antibody is allowed to bind to the C5 protein or peptide under saturating conditions. The ability of the test antibody to bind to the C5 protein molecule is then evaluated. If the test antibody is able to bind to C5 after saturation binding to the reference anti-C5 antibody, then it can be concluded that the test antibody binds to a different epitope than the reference anti-C5 antibody. On the other hand, if the test antibody is unable to bind to the C5 protein after saturation binding to the reference anti-C5 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-C5 antibody of the invention.

In order to determine if an antibody competes for binding with a reference anti-C5 antibody, the binding method described above is carried out in two orientations: in the first orientation, the reference antibody is allowed to bind to the C5 protein under saturating conditions, followed by evaluation of the binding of the test antibody to the C5 molecule. In the second orientation, the test antibody is allowed to bind to the C5 molecule under saturating conditions, followed by evaluation of the binding of the reference antibody to the C5 molecule. If in both orientations only the first (saturating) antibody is able to bind to the C5 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to C5. One skilled in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block reference antibody binding by binding to an overlapping or contiguous epitope.

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

Additional routine experiments (e.g., peptide mutations and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or if steric blocking (or other phenomenon) responsible for the lack of observable binding. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

Immunoconjugates.

The present invention relates to a human anti-C5 monoclonal antibody conjugated to a therapeutic moiety (immunoconjugate) to treat a C5-associated disease or disorder (eg, atypical hemolytic uremic syndrome). As used herein, the term immunoconjugate refers to an antibody that is chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter fragment, enzyme, peptide or protein, or therapeutic agent. Antibodies can be bound to a radioactive agent, cytokine, interferon, target or reporter fragment, enzyme, peptide, or therapeutic agent at any location along the molecule, provided they are capable of binding to their target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second distinct antibody to the C5 protein. The type of therapeutic moiety that can be conjugated to an anti-C5 antibody should take into account the condition being 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.

polyspecific antibodies.

The antibodies of the present invention may be monospecific, bispecific or polyspecific. Multispecific antibodies may have specificity for different epitopes of the same target polypeptide or may contain antigen-binding domains with specificity for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69;

- 23 039858

Kufer et al., 2004, Trends Biotechnol. 22:238-244.

Any polyspecific antigen-binding molecules of the invention, or variants thereof, can be constructed using standard molecular biology techniques (eg, recombinant DNA and protein expression technology) as is known to those skilled in the art.

In some embodiments, C5-specific antibodies are created in a bispecific format (bispecific) in which variable regions that bind to different domains of the C5 protein are linked together to confer dual domain specificity on a single binding molecule. Properly designed bispecific agents can enhance the overall inhibitory C5 protein effect by increasing both specificity and avidity of binding. Variable regions with specificity for individual domains (eg, segments of the N-terminal domain) or that can bind to different regions in the same domain are paired on a structural backbone that allows each region to bind simultaneously to separate epitopes or to different regions in the same domain. In one example, for a bispecific agent, heavy chain variable regions (VH) from a binding agent with specificity for one domain are recombined with variable light chain regions (V _L ) from a series of binding agents with specificity for a second domain in order to identify non-cognate V _L partners that can pair with the original VH without violating the original specificity for that V _H . Thus, one V _L segment (eg, V _L 1) can be combined with two different VH domains (eg, V _H 1 and V _H 2) to create a bispecific agent consisting of two binding arms (VH1-VL1 and VH2-VL1). The use of a single V _L segment reduces system complexity and thereby simplifies and increases efficiency in the cloning, expression and purification processes used to create a bispecific agent (see, for example, USSN13/022759 and US 2010/0331527).

Alternatively, antibodies that bind more than one domain and a second target, such as, but not limited to, for example, a second distinct anti-C5 antibody, can be generated in a bispecific format using the methods described herein or other methods known to those skilled in the art. in this area. Antibody variable regions that bind to distinct regions can be linked together with variable regions that bind to relevant sites, eg on the C5 extracellular domain, to confer dual antigenic specificity on a single binding molecule. Properly designed bispecific agents with these properties perform a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for the outer extracellular domain and paired on a structural backbone that allows each variable region to bind to separate antigens.

An exemplary bispecific antibody format that can be used in the context of the present invention includes the use of a first immunoglobulin (Ig) C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid. and wherein the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody having no amino acid difference. In one embodiment, the first Ig C _H 3 domain binds protein A and the second Ig C _H 3 domain contains a mutation that reduces or abolishes protein A binding, such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C _H 3 may additionally contain modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that can be found in the second CH3 include

D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies;

N44S, K52N and V82I (IMGT; N384S, K392N and V422I in EU) for IgG2 antibodies; and

Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies.

Variations in the bispecific antibody format as described above are contemplated within the scope of the present invention.

Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, Ig double variable domain (DVD), quadroma, ridge-in-trough , ordinary light chain (e.g., ordinary light chain with ridges and valleys, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, IgG with Dual Action Fab (DAF) and bispecific formats Mab2 (for a review of the above formats see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where non-natural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence, and geometry. . (Cm.,

- 24 039858 e.g. Kazane et al., J. Am. Chem. soc. [Epub: Dec. 4, 2012]).

Therapeutic administration and formulations.

The invention relates to therapeutic compositions containing antibodies against C5 or their antigen-binding fragments of the present invention. Therapeutic compositions in accordance with the invention should be administered with suitable carriers, excipients and other means, which are included in the formulations to provide improved transfer, delivery, tolerability, and the like. A variety of suitable formulations can be found in the reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences , Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, fats, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. Compendium of excipients for parenteral formulations, PDA (1998), J. Pharm. sci. Technol. 52:238-311.

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

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

The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail in Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat., vol. 2009, article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles can be designed and conjugated with antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery are also described, for example, in US 8257740 or US 8246995, each is included in this description in full.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, the controlled release system can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, and the like. These injectable preparations can be obtained using well-known methods. For example, injectable preparations can be obtained, for example, by dissolving, suspending

- 25 039858 or emulsifying an antibody or salt thereof as described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline, isotonic solution containing glucose and other adjuvants, etc., which can be used in combination with a suitable solubilizing agent such as an alcohol (for example, ethanol), polyhydric alcohol (eg, propylene glycol, polyethylene glycol), non-ionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, the pen-type delivery device easily finds applications for delivering the pharmaceutical composition of the present invention. Such a pen-type delivery device may be reusable or disposable. The reusable pen type delivery device generally uses a replaceable cartridge that contains a pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been introduced and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen type delivery device can then be reused. There is no replaceable cartridge in the pen-type disposable delivery device. Rather, the pen-type disposable delivery device comes pre-filled with a pharmaceutical composition contained in a reservoir in the device. When the pharmaceutical composition in the tank is over, discard the entire device.

Many reusable pen and auto-injector delivery devices find use for subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are definitely not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ handle (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ handle, HUMALOG™ handle, HUMALIN handle 70/30™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ handle (Becton Dickinson , Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), among others. Examples of disposable pen-type delivery devices having applications for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), among others.

Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared in unit dosage forms adjusted to match the dosage of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of antibody contained is generally from about 5 mg to about 500 mg per unit dosage form; in particular in the form of an injection, preferably the antibody ranged from about 5 to about 300 mg and from about 10 to about 300 mg for other dosage forms.

Therapeutic use of antibodies.

The antibodies of the present invention can be used to treat and/or prevent a disease, or disorder, or condition associated with C5, and/or to improve at least one symptom associated with such a disease, disorder, or condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention may be administered at a therapeutic dose to a patient with a disease or disorder or condition associated with C5.

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

In certain embodiments, the antibodies of the present invention can be used in the treatment or prevention of a symptom or indication of paroxysmal nocturnal hemoglobinuria (PNH). Symptoms and indications for PNH include, but are not limited to, red blood cell destruction, thrombosis (including deep vein thrombosis, pulmonary embolism), intravascular hemolytic anemia, red urine, symptoms of anemia such as fatigue, shortness of breath and palpitations, pain in the abdomen and difficulty swallowing.

In certain embodiments, the antibodies of the present invention can be used to treat or prevent at least one symptom or indication of a C5 associated disease or disorder selected from the group consisting of neurological disorders, renal disorders, multiple sclerosis, stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, inappropriate or unwanted complement activation disorders, complications of hemodialysis, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammatory disorders, inflammation in autoimmune diseases, Crohn's disease, respiratory distress - adult syndrome, thermal injury, including burns or frostbite, postischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, ep ilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, balloon angioplasty, posthemodialysis syndrome with cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease, or sepsis and autoimmune diseases, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, reperfusion injury in renal ischemia, lupus nephritis, glomerulopathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, membranoproliferative nephritis, hemolytic anemia, optomyelitis, kidney transplant, hereditary CD59 deficiency, psoriasis and myasthenia gravis. In certain other embodiments, the antibodies of the present invention can be used to treat or prevent at least one symptom or indication of a C5 associated 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, fibrogenic pneumoconiosis, inert dust and mineral damage (eg, silica, coal dust, beryllium, and asbestos), pulmonary fibrosis, organic pneumoconiosis, chemical damage (from - irritating gases and chemicals, e.g. chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia and hydrochloric acid), smoke damage, thermal damage (e.g. burns, frostbite), asthma, allergies, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, inherited native Quincke's edema and immune complex-associated inflammation.

In certain embodiments, the antibodies of the invention can be used to treat subjects suffering from an ocular disease such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, ocular angiogenesis (ocular neovascularization affecting choroid, cornea, or retina), geographic atrophy (GA), uveitis and myelitis optica. The antibodies of the present invention can be used to treat or improve at least one symptom or indication of dry AMD or wet AMD. In some embodiments, the antibodies of the invention can be used to prevent or reduce the rate of vision loss. In one embodiment, the antibodies of the present invention can be used to reduce drusen in the eye of a subject with AMD. In one embodiment, the antibodies of the present invention can be used to prevent or reduce/slow vision loss in a subject with AMD.

One or more antibodies of the present invention can be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions/indications of an ocular disease or disorder. Antibodies can be used to improve or reduce the severity of at least one symptom, including, but not limited to, loss of vision, visual distortion, difficulty adapting to low levels of illumination, distorted central vision, increased obscuration of central/general vision, the presence of drusen (small accumulations of extracellular material that form on the retina), pigmentary changes, distorted vision in the form of metamorphopsia, in which a grid of straight lines looks wavy and parts of the grid may look empty, exudative changes (hemorrhages in the eye, profuse exudate, subretinal / under the retinal pigment epithelium /intraretinal fluid), slow recovery of visual function after exposure to bright light (photostress test), initial and geographic atrophy, significant reduction in visual acuity (two levels or more), for example, from 20/20 to 20/80, predominant perimetric changes increased acute blurred vision (for wet AMD), blurred vision, gradual loss of central vision (in those with non-exudative macular degeneration, rapid onset of vision loss (often due to leakage and bleeding in abnormal blood vessels in a subject with exudative macular degeneration) yellow spots, central scotomas (shadows or loss of visual fields), difficulty in distinguishing colors, in particular between dark and dark and between light and light, loss of contrast sensitivity, straight lines look curved on the Amsler grid.

Also contemplated herein is the use of one or more antibodies of the present invention prophylactically in subjects at risk for developing macular degeneration, such as subjects over the age of 50, subjects with a family history of macular degeneration, smokers, and subjects with obesity, high cholesterol, heart vascular disease or an unhealthy diet.

In a further embodiment of the invention, these antibodies are used to prepare a pharmaceutical composition or medicament for the treatment of patients suffering from a disease or disorder associated with C5. In another embodiment, these antibodies are used as an adjuvant therapy with any other agent or therapy known to those skilled in the art that can be used to treat or ameliorate a C5 related disease or disorder.

Combined therapy.

Combination therapy may include an anti-C5 antibody of the invention and any additional therapeutic agent that may be useful in combination with an antibody of the invention or a biologically active fragment of an antibody of the invention. The antibodies of the present invention can be combined synergistically with one or more drugs or therapies that are used to treat a disease or disorder associated with C5. In some embodiments, the antibodies of the invention can be combined with a second therapeutic agent to improve one or more symptoms of said disease.

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

In certain embodiments, the second therapeutic agent is another antibody to the C5 protein. The present description contemplates the use of a combination (cocktail) of antibodies with broad neutralizing or inhibitory activity against C5. In some embodiments, non-competing antibodies can be combined and administered to a subject in need. In some embodiments, the antibodies contained in the combination bind to separate, non-overlapping epitopes on a protein. The antibodies contained in the combination may block the binding of C5 to C5 convertase and/or may prevent/inhibit the cleavage of C5 to C5a and C5b. In certain embodiments, the implementation of the second antibody may have a longer half-life in human serum.

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

The additional therapeutically active ingredient(s) may be administered to the subject prior to administration of the anti-C5 antibody of the present invention. For example, the first component can be considered to be administered before the second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 h before, 4 h before, 3 h before, 2 h before, 1 h before, 30 min before, 15 min before, 10 min before, 5 min before, or less than 1 min before the administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject following administration of an anti-C5 antibody of the present invention. For example, the first component may be considered to be administered after the second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 h after, 5 h after, 6 h after, 12 h after, 24 h after, 36 h after, 48 h after, 60 h after, 72 h after administration of the second component. In other embodiments, the implementation of additional therapeutically active component(s) can be administered to the subject simultaneously with the introduction of antibodies against C5 of the present invention. Simultaneous administration, for purposes of the present invention, includes, for example, administration of an anti-C5 antibody and additional

- 28 039858 therapeutically active component to the subject in a single dosage form or in separate dosage forms administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (eg, both anti-C5 antibodies and the additional therapeutically active moiety may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route (eg, the anti-C5 antibody may be administered intravenously and the additional therapeutically active ingredient may be administered orally). In any case, administration of the components in the same dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes, are all considered to be simultaneous administration for the purposes of this disclosure. For purposes of this disclosure, administration of an anti-C5 antibody prior to, simultaneously with, or after (as these terms are defined hereinbefore) administration of an additional therapeutically active ingredient is considered to be administration of an anti-C5 antibody in combination with an additional therapeutically active ingredient.

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

Introduction schemes.

In certain embodiments, a single dose of an anti-C5 antibody of the invention (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents described herein) may be administered to a subject in need thereof. In accordance with certain embodiments of the present invention, multiple doses of an anti-C5 antibody (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents described herein) may be administered to a subject over a period of time. Methods in accordance with this aspect of the invention include sequentially administering to a subject several doses of an anti-C5 antibody of the invention. As used herein, sequential administration means that each dose of anti-C5 antibody is administered to a subject at a different point in time, eg, on a different day separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of anti-C5 antibody, followed by one or more secondary doses of anti-C5 antibody, and optionally followed by one or more tertiary doses of anti-C5 antibody.

The terms initial dose, secondary doses, and tertiary doses refer to the chronological sequence of administration of the anti-C5 antibody of the invention. Thus, the starting dose is the dose that is administered at the start of a treatment regimen (also referred to as the baseline dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. All initial, secondary and tertiary doses may contain the same amount of anti-C5 antibody, but in general may differ from each other with respect to the frequency of administration. However, in certain embodiments, the amount of anti-C5 antibody contained in the initial, secondary, and/or tertiary doses varies from one to the other (eg, adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered on a less frequent basis (eg, maintenance doses).

In certain exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered 1-48 hours apart (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5 .5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 , 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 , 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or more) after the immediately preceding dose. The phrase immediately preceding dose as used herein means, in a multi-dose sequence, the dose of anti-C5 antibody that is administered to a patient prior to the next next dose in the sequence with no intermediate doses.

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

In certain embodiments, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of a treatment regimen. The frequency of administration can also be adjusted during the course of treatment by the physician, depending on the needs of the particular patient, after clinical examination.

- 29 039858

Diagnostic use of antibodies.

Anti-C5 antibodies of the present invention can be used to detect and/or measure C5 in a sample, for example for diagnostic purposes. Some embodiments provide for the use of one or more antibodies of the present invention in assays to detect a C5 associated disease or disorder. Exemplary diagnostic assays for C5 may include, for example, contacting a sample obtained from a patient with an anti-C5 antibody of the invention, where the anti-C5 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate C5 from patient samples.

Alternatively, an unlabeled anti-C5 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure C5 in a sample include ELISA, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in C5 diagnostic assays in accordance with the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of either C5 protein or fragments thereof, under normal or pathological conditions. In general, C5 protein levels in a particular sample from a healthy patient (eg, a patient not affected by a C5 related disease) are measured to initially establish a baseline or standard C5 level. This baseline C5 level can then be compared with C5 levels measured in samples obtained from individuals suspected of having a C5-associated condition or symptoms associated with such a condition.

Antibodies with specificity for the C5 protein may not contain additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the label or fragment is biotin. In a binding assay, the location of the label (if appropriate) may determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface.

Selected Embodiments.

Selected embodiments of the present disclosure include the following.

In Embodiment 1, the present invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to a complement factor 5 (C5) protein, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained in C5 (SEQ ID NO: 359), as determined by hydrogen-deuterium exchange.

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

In embodiment 3, the present invention includes the isolated antibody or antigen-binding fragment of embodiments 1 or 2, wherein the antibody or antigen-binding fragment thereof does not interact with an amino acid from the C5a anaphylatoxin region of C5, as determined by hydrogen-deuterium exchange.

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

In Embodiment 5, the present invention includes an isolated antibody or antigen-binding fragment according to any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of:

(a) amino acids 591 to 599 of SEQ ID NO: 359;

(b) amino acids 593 to 599 of SEQ ID NO: 359;

(c) amino acids 775 to 787 of SEQ ID NO: 359;

(d) amino acids 775 to 794 of SEQ ID NO: 359; and (e) amino acids 779 to 787 of SEQ ID NO: 359.

In Embodiment 6, the present invention includes an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with at least five amino acids contained in an amino acid sequence selected from the group, consisting of SEQ ID NO: 360 and 361.

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

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

In Embodiment 9, the present invention includes the isolated antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 8, wherein the antibody has one or more of the following:

(a) has a serum concentration of greater than 10 μg/ml up to day 70 when administered to the cynomolgus monkey;

(b) blocks the classical pathway (CP) of hemolysis up to day 35 when administered to cynomolgus monkey as measured in an ex vivo hemolysis assay;

(c) blocks the alternative pathway (AR) of hemolysis up to day 35 when administered to cynomolgus monkey as measured in an ex vivo hemolysis assay;

(d) has a serum half-life greater than 10 days in the cynomolgus monkey;

(e) has a serum concentration greater than 10 μg/ml up to day 40 when administered to C5 humanized mice;

(f) blocks CP hemolysis up to day 30 when administered to C5 humanized mice as measured in an ex vivo hemolysis assay; and (g) has a serum half-life greater than 10 days in C5 humanized mice.

In embodiment 10, the present invention includes the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the antibody has an additional characteristic selected from the group consisting of:

(a) is a fully human monoclonal antibody;

(b) binds to human C5 with a dissociation constant (K _D ) of less than 0.9 nM at 25° C. as measured in a surface plasmon resonance assay;

(c) binds to human C5 with a KD of less than 0.3 nM at 37° C. as measured in a surface plasmon resonance assay;

(d) binds to monkey C5 with a KD of less than 65 nM as measured in a surface plasmon resonance assay;

(e) binds to human C5 variant R885H (SEQ ID NO: 356) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(f) binds to human C5 variant R885C (SEQ ID NO: 357) with a K _D of less than 0.5 nM as measured in a surface plasmon resonance assay;

(g) blocks the human C5-mediated classical pathway (CP) of hemolysis by greater than 95% and with an IC _{50 of} less than 6 nM as measured in the hemolysis CP assay;

(h) blocks the human C5 mediated alternative pathway (AR) of hemolysis by more than 70% and with an IC _{50 of} less than 165 nM as measured by the hemolysis AR assay;

(i) inhibits African green monkey C5 mediated CP hemolysis with an IC _{50 of} less than 185 nM as measured in the Hemolysis CP assay;

(j) inhibits African green monkey C5-mediated AR hemolysis with an IC _{50 of} less than 235 nM as measured by the AR hemolysis assay;

(k) inhibits cynomolgus C5 mediated CP hemolysis with an IC _{50 of} less than 145 nM as measured in the CP hemolysis assay; and (l) inhibits cynomolgus C5 mediated AR hemolysis with an IC _{50 of} less than 30 nM as measured by the AR hemolysis assay.

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

In Embodiment 12, the present invention includes an isolated antibody or anti-gene-binding fragment thereof according to any one of Embodiments 1 to 11, which comprises:

(a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292,

308, 324 and 340;

(b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 126, 142, 150, 158, 174, 190, 206, 222, 238, 254, 270, 278, 294, 310, 326 and 342;

(c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 128, 144, 152, 160, 176, 192, 208, 224, 240, 256, 272, 280, 296, 312, 328 and 344;

(d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332 and 348;

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 118, 134, 166, 182, 198, 214, 230, 246, 262, 286, 302, 318, 334 and 350; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 120, 136, 168, 184, 200, 216, 232, 248 , 264, 288, 304, 320, 336 and 352.

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

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

In Embodiment 15, the present invention includes an isolated antibody or antigen-binding fragment according to any one of Embodiments 11 to 14, comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/ 42, 50/58, 66/74, 82/90, 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 embodiment 16, the present invention includes an isolated antibody or antigen-binding fragment thereof according to any one 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.

In Embodiment 17, the present invention includes the isolated antibody or antigen-binding fragment thereof of Embodiment 16, comprising CDRs selected from the group consisting of:

(a) SEQ ID NOs: 52, 54, 56, 60, 62 and 64;

(b) SEQ ID NOs: 1000, 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.

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

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

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

In embodiment 21, the present invention includes the antibody or antigen-binding fragment of embodiment 9 or 10 comprising a heavy chain variable region comprising the amino acid sequence listed in Table 1. 1 having no more than 5 amino acid substitutions.

In Embodiment 22, the present invention includes the antibody or antigen-binding fragment thereof of Embodiment 21, which comprise a light chain variable region comprising the amino acid sequence listed in Table 1. 1 having no more than 5 amino acid substitutions.

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

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

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

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

In Embodiment 27, the present invention includes an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes for an HCVR antibody as set forth in any one of Embodiments 1 to 25.

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

In embodiment 29, the present invention includes a vector containing the polynucleotide sequence of embodiment 27 or 28.

In Embodiment 30, the present invention includes a cell expressing the vector of Embodiment 29.

In Embodiment 31, the present invention includes a method for preventing, treating, or improving at least one symptom or indication of a C5-associated disease or disorder, the method comprising administering an antibody or antigen-binding fragment according to any one of Embodiments 1 to 25 to a subject in need thereof.

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

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

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

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

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

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

In Embodiment 38, the present invention includes the method of any one of Embodiments 31 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or into the skull.

Examples

The following examples are provided to provide those of ordinary skill in the art with a full disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure.

Example 1 Creation of Human Antibodies to Complement Factor 5 (C5) Protein.

Human antibodies to the C5 protein were generated in a VELOCIMMUNE® mouse containing DNA encoding heavy and light chain variable regions for the human immunoglobulin chain. Mice were immunized with purified human serum C5 protein (Calbiochem cat# 20-4888).

Monitoring of the antibody immune response was carried out by C5-specific immunoassay. When the desired immune response was achieved, the splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Carried out screening and selection of hybridoma cell lines in order to identify cell lines that produce C5-specific antibodies. The cell lines were used to generate several chimeric anti-C5 antibodies (ie, antibodies containing human variable domains and mouse constant domains); exemplary antibodies thus generated were designated as H2M11683N and H2M11686N.

Anti-C5 antibodies were also isolated directly from mouse antigen-positive B cells without fusion with myeloma cells, as described in US Pat. No. 7,582,298, incorporated herein by reference in its entirety. Using this method, several fully human anti-C5 antibodies (ie, antibodies containing human variable domains and human constant domains) were generated; образцовые антитела, созданные таким образом, обозначали как Н4Н12159Р, Н4Н12161Р, Н4Н12163Р, Н4Н12164Р, Н4Н12166Р, Н4Н12167Р, Н4Н12168Р, Н4Н12169Р, Н4Н12170Р, Н4Н12171Р, Н4Н12175Р, Н4Н12176Р2, Н4Н12177Р2 и Н4Н12183Р2.

The biological properties of exemplary antibodies created in accordance with the methods of this example are described in detail in the examples below.

Example 2. Amino acid and nucleotide sequences of the variable regions of the heavy and light chains.

In table. 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-C5 antibodies of the invention.

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Table 1

Amino acid sequence identifiers_______

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H2M11683N 2 4 6 eight ten 12 fourteen sixteen H2M11686N eighteen 20 22 24 26 28 thirty 32 H4N12159R 34 36 38 40 42 44 46 48 H4N12161R fifty 52 54 56 58 60 62 64 H4N12163R 66 68 70 72 74 76 78 80 H4H12164R 82 84 86 88 90 92 94 96 H4N12166R 98 100 102 104 106 108 110 112 H4H12166R2 98 100 102 104 114 116 118 120 H4N12166RZ 122 124 126 128 106 108 110 112 Н4Н12166Р4 98 100 102 104 130 132 134 136 H4H12166R5 138 140 142 144 106 108 110 112 H4H12166R6 146 148 150 152 106 108 110 112 Н4Н12166Р7 122 124 126 128 130 132 134 136 H4H12166R8 146 148 150 152 114 116 118 120 H4H12166R9 146 148 150 152 130 132 134 136 Н4Н12166Р1 0 138 140 142 144 130 132 134 136 H4N12167R 154 156 158 160 162 164 166 168 H4N12168R 170 172 174 176 178 180 182 184 H4N12169R 186 188 190 192 194 196 198 200 H4H12170R 202 204 206 208 210 212 214 216 H4N12171R 218 220 222 224 226 228 230 232 H4N12175R 234 236 238 240 242 244 246 248 H4H12176R2 250 252 254 256 258 260 262 264 H4H12177R2 266 268 270 272 258 260 262 264 H4H12183R2 274 276 278 280 282 284 286 288 H2M11682N 290 292 294 296 298 300 302 304 H2M11684N 306 308 310 312 314 316 318 320 H2M11694N 322 324 326 328 330 332 334 336 H2M11695N 338 340 342 344 346 348 350 352

The corresponding nucleic acid sequence identifiers are shown in Table 1. 2.

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table 2

Nucleic acid sequence identifiers

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H2M11683N one 3 5 7 nine eleven thirteen fifteen H2M11686N H4N12159R H4N12161R H4N12163R H4H12164R H4N12166R H4H12166R2 H4N12166RZ Н4Н12166Р4 H4H12166R5 H4H12166R6 Н4Н12166Р7 H4H12166R8 H4H12166R9 Н4Н12166Р1 0 H4N12167R H4N12168R H4N12169R H4H12170R H4N12171R 17 33 49 65 81 97 97 121 97 137 145 121 145 145 137 153 169 185 201 217 nineteen 35 51 67 83 99 99 123 99 139 147 123 147 147 139 155 171 187 203 219 21 37 53 69 85 101 101 125 101 141 149 125 149 149 141 157 173 189 205 221 23 39 55 71 87 103 103 127 103 143 151 127 151 151 143 159 175 191 207 223 25 41 57 73 89 105 113 105 129 105 105 129 113 129 129 161 177 193 209 225 27 43 59 75 91 107 115 107 131 107 107 131 115 131 131 163 179 195 211 227 29 45 61 77 93 109 117 109 133 109 109 133 117 133 133 165 181 197 213 229 31 47 63 79 95 111 119 111 135 111 111 135 119 135 135 167 183 199 215 231 H4N12175R 233 235 237 239 241 243 245 247 H4H12176R2 249 251 253 255 257 259 261 263 H4H12177R2 265 267 269 271 257 259 261 263 H4H12183R2 273 275 277 279 281 283 285 287 H2M11682N 289 291 293 295 297 299 301 303 H2M11684N 305 307 309 311 313 315 317 319 H2M11694N 321 323 325 327 329 331 333 335 H2M11695N 337 339 341 343 345 347 349 351

Antibodies are usually designated in the present description in accordance with the following nomenclature:

an Fc prefix (e.g. H4H, H2M, etc.) followed by a numeric identifier (e.g. 11686, 12166, 12183, etc., as shown in Table 2) followed by a P, P2, or N suffix. Thus, in accordance with this nomenclature, an antibody can be referred to herein, for example, as H2M1168 6N, H4H12183P2, H4H12168P, etc. The prefixes H4H and H2M in the designations of antibodies used in the present description indicate the specific isotype of the Fc region of the antibody. For example, the H4H antibody has a human IgG4 Fc containing a serine to proline mutation in the hinge region (S108P) to help stabilize the dimer, and the H2M antibody has a mouse IgG2 Fc (isotype a or b) (all variable regions are fully human, as indicated with using the first H in the designation of the antibody). One skilled in the art will appreciate that an antibody containing a particular Fc isotype can be converted into an antibody with another Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted into an antibody with a human IgG4, etc.), but in any case variable domains (including CDR), which are designated using digital identifiers, which are presented in table. 2 will remain the same, and antigen binding properties are expected to be identical or substantially similar, regardless of the nature of the Fc domain.

In certain embodiments, selected mouse IgG1 Fc antibodies are converted into human IgG4 Fc antibodies. In one embodiment, an IgG4 Fc domain contains two or more amino acid substitutions as disclosed in US 20100331527.

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

Control constructs used in the following examples.

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

Example 3 Antibody binding to C5 as determined by surface plasmon resonance.

Equilibrium dissociation constants (KD values) for C5 binding to purified anti-C5 antibodies were determined using real-time surface plasmon resonance biosensor analysis on a Biacore T200 instrument. The Biacore sensor surface was derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody (GE Healthcare, no. BR-1008-39) to capture anti-C5 antibodies expressed with human Fc constant regions. Biacore binding studies were performed in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v surfactant P20). Human C5 was obtained from a commercial source (EMD). Other C5 reagents were expressed with a C-terminal myc-myc-hexahistidine tag (hereinafter referred to as C5-mmh). Human C5-mmh reagents containing histidine and cysteine point mutations at arginine 885 (hereinafter referred to as C5 R885H-mmh and C5 R885C-mmh, respectively) were also expressed. Different concentrations of human C5, human C5 R885H-mmh (SEQ ID NO: 356), human C5 R885C-mmh (SEQ ID NO: 357), and monkey C5-mmh (SEQ ID NO: 358) (range 100 to 1, 23 nM, 3-fold dilutions) prepared in HBST running buffer was injected onto the surface with captured anti-C5 antibody at a flow rate of 30 μl/min. The association of all C5 reagents with each of the captured monoclonal antibodies was monitored for 3 min and their dissociation was monitored in HBST running buffer for 8 min. All experiments on the kinetics of binding was carried out at 25 or 37°C. The kinetic rate constants of association (k _a ) and dissociation (kd) were determined by fitting real time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding equilibrium dissociation constants (KD) and dissociation half-life (t _1/2 ) were calculated from the kinetic rate constants: K _D (M) = kd/k _a and t _1/2 (min) = ln ₂ / ( 60xkd).

Binding kinetic parameters for human C5 binding to anti-C5 antibodies at 25 and 37° C. are shown in Table 1. 3 and 4.

Table 3

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 at 25°C

Antibody Amount of antibody captured (RU) 100 nM C5 person is connected. (RU) to _a k _{d KD} t _1/2 (1/ms) (1/s) (M) (min) H4H11683N 231 439 4.17E+05 5.14Е-05 1.23E-10 225 H4N12171R 51 64 1.49E+05 8.16Е-05 5.49E-10 142 H4N12161R 38 64 2.58E+05 4.37Е-05 1.70E-10 264 H4H12176R2 fifty 96 3.36E+05 6.75Е-05 2.01E-10 171 H4N12163R 51 108 6.43E+05 2.08Е-04 3.24Е-10 55 H4N12167R 52 116 1.09E+06 1.31Е-04 1.21E-10 88

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H4N12175R 51 100 2.16E+05 1.96E-04 9.10E-10 59 H4N12159R 53 118 9.75E+05 7.13E-05 7.31E-11 162 H4H12164R 52 103 2.92E+05 8.84Е-05 3.02E-10 131 H4N12168R fifty 113 4.23E+05 4.75Е-05 1.12E-10 243 H4N12169R 51 eighteen 2.24E+05 4.40E-04 1.96E-09 26 H4H11686N 200 341 2.20E+05 3.31E-05 1.50E-10 349 H4H12170R 51 119 5.25E+05 6.79Е-05 1.29E-10 170 H4H12177R2 47 60 6.56E+04 6.29Е-05 9.59Е-10 184 H4H12183R2 46 fifty 1.66E+05 2.70E-05 1.63Е-10 427 H4N12166R 53 105 6.42E+05 1.10E-04 1.71Е-10 105 H4H12166R2 53 95 8.26E+05 3.61E-04 4.38E-10 32 H4N12166RZ 59 124 4.03E+05 4.65Е-04 1.15E-09 25 Н4Н12166Р4 49 92 4.46E+05 1.76E-04 3.95E-10 66 H4H12166R5 59 110 2.85E+05 3.28E-04 1.15E-09 35 H4H12166R6 64 131 4.89E+05 1.84Е-04 3.75E-10 63 Н4Н12166Р7 fifty 92 2.74E+05 1.01E-03 3.67E-09 eleven H4H12166R8 fifty 91 4.84E+05 6.86Е-04 1.42E-09 17 H4H12166R9 52 100 3.32E+05 2.64Е-04 7.94Е-10 44 Н4Н12166Р1 0 49 69 1.57E+05 1.32E-03 8.38Е-09 nine an object comparisons one 232 250 9.69E+04 1.46E-04 1.51Е-09 79 an object comparisons 2 117 170 2.62E+05 2.39Е-04 9.12E-10 48

Table 4

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 at 37°C

Antibody Amount of antibody captured (RU) 100 nM C5 person is connected. (RU) to _a k _{d KD} tl/2 (1/ms) (1/s) (M) (min) H4H11683N 257 492 4.54E+05 2.41Е-04 5.32E-10 48 H4N12171R 59 58 1.22E+05 7.62E-04 6.27Е-09 fifteen H4N12161R 40 66 1.16E+05 1.15E-04 9.90E-10 101 H4H12176R2 38 71 1.47E+05 2.34Е-04 1.59Е-09 49 H4N12163R 65 139 9.11E+05 6.65Е-04 7.29E-10 17 H4N12167R 75 153 1.29E+06 3.81E-04 2.95E-10 thirty H4N12175R 74 132 2.96E+05 6.37Е-04 2.15Е-09 eighteen H4N12159R 70 145 1.04E+06 1.07Е-04 1.03Е-10 108

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H4H12164R 66 140 3.96E+05 1.28E-04 3.23E-10 90 H4N12168R 34 12 2.50E+04 4.64Е-04 1.85Е-08 25 H4N12169R 59 65 1.15E+05 3.52E-04 3.06E-09 33 H4H11686N 206 406 3,3E+05 1.56Е-04 4.69Е-10 74 H4H12170R 34 55 2.97E+05 4.15Е-04 1.40E-09 28 H4H12177R2 41 37 4.42E+04 5.78Е-04 1.31Е-08 20 H4H12183R2 29 thirty 4.30E+04 2.50E-04 5.81E-09 46 H4N12166R 69 127 8.80E+05 2,ZOE-04 2.62E-10 fifty H4H12166R2 68 110 9.50E+05 1.23E-03 1.29Е-09 nine H4N12166RZ 86 147 6.12E+05 1.27E-03 2.07Е-09 nine Н4Н12166Р4 63 108 5.05E+05 4.69Е-04 9.30E-10 25 H4H12166R5 76 129 4.40E+05 1.22E-03 2.77Е-09 nine H4H12166R6 90 157 5.42E+05 4.74Е-04 8.75E-10 24 Н4Н12166Р7 64 105 3.49E+05 2.58Е-03 7.39Е-09 4 H4H12166R8 65 98 6.75E+05 2.09Е-03 3.10E-09 6 H4H12166R9 76 122 3.75E+05 6.39Е-04 1.70E-09 eighteen Н4Н12166Р1 0 64 82 2.27E+05 3.14Е-03 1.38Е-08 4 an object comparisons one 185 246 1.47E+05 5,ZOE-04 3.61E-09 22 an object comparisons 2 119 205 2.85E+05 b,57E-04 2.30E-10 eighteen

The binding of C5 monkey-mmh with antibodies against C5 at 25 and 37°C is presented in table. 5 and 6.

Table 5

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Monkey C5-mmh at 25°C

Antibody Quantity captured about antibodies (RU) 100 nM monkey C5mmh bound. (RU) to _a (1/Ms) k _d (1/s) K _D (M) tl/2 (min) H4H11683N 228 403 3.86E+05 2.47Е-04 6.40E-10 47 H4N12171R 51 17 4.60E+04 2.26E-04 4.92E-09 51 H4N12161R 38 45 6,33E+04 2.48Е-05 3.92E-10 465 H4H12176R2 fifty 69 1.82E+05 5.88E-05 3.22E-10 196 H4N12163R fifty 98 3.11E+05 7.75Е-04 2.49Е-09 fifteen

- 39 039858

H4N12167R 52 111 4.19E+05 1.32E-04 3.15E-10 88 H4N12175R 51 59 6.42E+04 1.65Е-03 2.57Е-08 7 H4N12159R 53 116 3.54E+05 4.69Е-05 1,ZZE-10 246 H4H12164R 51 66 1.27E+05 1.53Е-03 1.20E-08 eight H4N12168R fifty 86 1.73E+05 1.14Е-04 6.60E-10 101 H4N12169R 51 22 1.64E+05 4.55Е-03 2.78Е-08 3 H4H11686N 196 247 1.57E+05 4.89E-04 3.11E-09 24 H4H12170R 51 92 2.62E+05 5.21E-05 1.99E-10 222 H4H12177R2 47 32 4.62E+04 9.92E-04 2.15E-08 12 H4H12183R2 47 23 4.88E+04 4.94Е-04 1.01Е-08 23 H4N12166R 52 90 2.05E+05 1.06E-03 5.15E-09 eleven H4H12166R2 53 71 3.00E+05 3.16E-03 1.05Е-08 4 H4N12166RZ 59 72 1.68E+05 4.47Е-03 2.66Е-08 3 Н4Н12166Р4 49 69 2.10E+05 1.78E-03 8.50Е-09 6 H4H12166R5 59 56 1.44E+05 3.46E-03 2.40E-08 3 H4H12166R6 64 94 2.39E+05 2.66Е-03 1.11E-08 4 Н4Н12166Р7 fifty 36 1.36E+05 6,ZZE-OZ 4.65Е-08 2 H4H12166R8 fifty 47 2.31E+05 4.99E-03 2.16E-08 2 H4H12166R9 52 55 1.70E+05 3.18E-03 1.87Е-08 4 Н4Н12166Р1 0 49 fifteen 9.56E+04 6.16E-03 6.44Е-08 2 an object comparisons one* 228 eleven N/A N/A 3.11E-07 N/A

N/A - not available;

*SS - steady state analysis.

Table 6

Binding kinetic parameters for anti-C5 monoclonal antibody binding to monkey C5-mmh at 37°C

Antibody Amount of antibody captured (RU) 100 nM monkey C5mmh bound. (RU) to _a k _{d KD} t _1/2 (1/ms) (1/s) (M) (min) H4H11683N 192 303 5.35E+05 1.15E-03 2.16E-09 ten H4N12171R 59 78 5.56E+05 1.03Е-03 1.85Е-09 eleven H4N12161R 41 53 1.34E+05 7.45Е-04 5.56Е-09 sixteen H4H12176R2 36 47 1.35E+05 1.29Е-03 9.60E-09 nine H4N12163R 64 129 3.90E+05 1.25E-03 3.20E-09 nine H4N12167R 74 146 5.37E+05 2.89Е-04 5.39E-10 40 H4N12175R 74 74 1.77E+05 2.76Е-03 1.56Е-08 4 H4N12159R 70 137 4.12E+05 5.50E-05 1,ZZE-10 210 H4H12164R 65 99 1.86E+05 1.17E-03 6,ZOE-09 ten

- 40 039858

H4N12168R 34 29 5,3E+04 6.76Е-04 1.27Е-08 17 H4N12169R 59 64 2.51E+05 3.61E-03 1.43Е-08 3 H4H11686N 145 195 2,33E+05 2.07Е-03 8.88Е-09 6 H4N12170R 34 60 5.21E+05 8.71Е-04 1.67Е-09 thirteen H4H12177R2 41 27 1.50E+05 7.17E-03 4.77Е-08 2 H4H12183R2 28 thirteen 5.40E+04 6.37Е-03 1.18E-07 2 H4N12166R 68 110 2.19E+05 1.87Е-03 8.55Е-09 6 H4H12166R2 68 83 3.93E+05 2.97Е-03 7.56Е-09 4 H4N12166RZ 85 92 2.23E+05 2.92E-03 1.31Е-08 4 Н4Н12166Р4 62 80 2.23E+05 1.83Е-03 8.20E-09 6 H4H12166R5 75 70 1.50E+05 3.13E-03 2.09Е-08 4 H4H12166R6 90 112 2.53E+05 2.32E-03 9.18E-09 5 Н4Н12166Р7 63 48 1.25E+05 2.41Е-03 1.93Е-08 5 Н4Н12166Р8 64 53 2.03E+05 2.69Е-03 1,ZZE-08 4 H4H12166R9 75 69 1.81E+05 2.61E-03 1.44Е-08 4 Н4Н12166Р1 0 63 24 6.60E+04 2.79Е-03 4.22E-08 4 an object comparisons one 132 4 NB NB NB NB

The binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibodies at 25° C. is shown in Table 1. 7 and 8, respectively.

Table 7

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 R885H-mmh at 25°C

Antibody Amount of antibody captured (RU) 100 nM human C5 R885H-mmh bound. (RU) to _a k _{d KD} %/2 (1/ms) (1/s) (M) (min) H4H11683N 183 118 5.26E+05 2.46Е-04 4.68E-10 47 H4N12171R 119 51 6.59E+05 1.42E-04 2.16E-10 81 H4N12161R 105 199 8.36E+04 8.32E-05 9.96E-10 139 H4N12176R 2 170 65 1.78E+05 2.17E-04 1.22E-09 53 H4N12163R 111 214 6.72E+05 4.34Е-04 6.46Е-10 27 H4N12167R 93 187 6.89E+05 2.98E-04 4,ZZE-10 39 H4N12175R 104 207 1.81E+05 1.98E-03 1.09Е-08 6 H4N12159R 101 177 7.06E+05 1.76E-04 2.50E-10 66 H4H12164R 143 295 1.58E+05 1.87Е-04 1.19E-09 62 H4N12168R 138 197 5.29E+04 2.14Е-04 4.05Е-09 54 H4N12169R 116 173 4.84E+05 7.09Е-05 1.47Е-10 163 H4H11686N 145 259 2.16E+05 1.06E-04 4.91Е-10 109 H4H12170R 244 442 4.09E+05 1.61E-04 3.94Е-10 72 H4N12177R 137 232 1.48E+05 5.92E-04 4.01Е-09 20 H4N12183R 158 99 3.77E+04 4.37Е-05 1.16E-09 264 H4N12166R 188 366 5.28E+05 2.12E-04 4.02E-10 54 an object comparisons one 87 eleven NB NB NB NB an object comparisons 2 118 249 1.08E+06 6.53Е-04 6.06Е-10 eighteen

Table 8

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Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 R885C-mmh at 25°C

Antibody Quantity captured about antibodies (RU) 100 nM human C5 R885C-mmh bound. (RU) to _a k _d K _D t _1/2 (1/ms) (1/s) (M) (min) H4H11683N 174 116 4.99E+05 2.39Е-04 4.79E-10 48 H4N12171R 109 51 3.79E+05 1.39Е-04 3.66E-10 83 H4N12161R 103 147 1.30E+05 8.71Е-05 6.72E-10 133 H4N12176R 2 164 63 1.07E+05 2.18E-04 2.03Е-09 53 H4N12163R 110 211 5.04E+05 4.32E-04 8.58E-10 27 H4N12167R 85 163 7.11E+05 2.94Е-04 4.13E-10 39 H4N12175R 99 128 8.18E+04 1.55Е-02 1.90E-07 one H4N12159R 93 168 5.86E+05 1.69Е-04 2.89Е-10 68 H4H12164R 139 249 1.53E+05 1.82E-04 1.19E-09 63 H4N12168R 128 144 6.09E+04 1.99E-04 3.27E-09 58 H4N12169R 108 168 2.78E+05 6.99E-05 2.51Е-10 165 H4H11686N 143 253 1.78E+05 9.49Е-05 5.34Е-10 122 H4H12170R 244 427 3.57E+05 1.60E-04 4.47E-10 72 H4N12177R 2 138 177 1.00E+05 1.32E-03 1.32E-08 nine H4N12183R 2 158 80 2.99E+04 2.20E-05 7.37E-10 525 H4N12166R 188 356 4.26E+05 2.07Е-04 4.87E-10 56 an object comparisons one 87 nine NB NB NB NB an object comparisons 2 117 241 1.17E+06 6.19E-04 5,ZOE-10 nineteen

The binding of human C5 R885H-mmh and human C5 R885C-mmh to anti-C5 antibodies at 37° C. is shown in Table 1. 9 and 10 respectively.

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Table 9

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 R885H-mmh at 37°C

Antibody Amount of antibody captured (RU) 100 nM C5 human R885H-mmh to _a k _d K _D tl/2 Related (RU) (1/ms) (1/s) (M) (min) H4H11683N 49 81 5.48E+05 1.47Е-03 2.69Е-09 eight H4N12171R 59 80 5.92E+05 9.63Е-04 1.63Е-09 12 H4N12161R 41 54 1.18E+05 9.25Е-04 7.84Е-09 12 H4N12176R 2 45 69 2.57E+05 9.58Е-04 3.73E-09 12 H4N12163R 60 85 7.24E+05 2.90E-03 4.00E-09 4 H4N12167R 38 65 8.81E+05 2.57Е-03 2.91Е-09 5 H4N12175R 25 thirty 1.37E+05 9.50Е-03 6.94Е-08 one H4N12159R 51 82 6.38E+05 9.48Е-04 1.49Е-09 12 H4H12164R 59 68 1.95E+05 1.06E-03 5.46Е-09 eleven H4N12168R 34 29 2.43E+04 1.23E-03 5.04Е-08 nine H4N12169R 61 79 4.29E+05 7.39E-04 1.72E-09 sixteen H4H11686N 40 74 4.19E+05 8.00Е-04 1.91Е-09 fourteen H4H12170R 36 64 5.59E+05 8.39Е-04 1.50E-09 fourteen H4N12177R 2 45 51 2.76E+05 2.76Е-03 1.00Е-08 4 H4N12183R 2 33 36 9.58E+04 7.12E-04 7.43Е-09 sixteen H4N12166R 71 58 6.24E+05 1.31Е-03 2.09Е-09 nine an object comparisons one 41 5 NB NB NB NB an object comparisons 23 47 8.39E+05 1.05Е-03 1.25E-09 eleven 2

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Table 10

Kinetic Binding Parameters for Anti-C5 Monoclonal Antibodies Binding to Human C5 R885C-mmh at 37°C

Antibody Amount of antibody captured (RU) 100 nM human C5 R8 8 5C-mmh bound. (RU) to _a k _d K _D tl/2 (1/ms) (1/s) (M) (min) H4H11683N 48 78 4.38E+05 1.43Е-03 3.25E-09 eight H4N12171R 59 78 4.77E+05 9.57Е-04 2.01Е-09 12 H4N12161R 41 49 1.10E+05 9.01Е-04 8.17E-09 thirteen H4N12176R 2 44 55 1.41E+05 1.03Е-03 7.32E-09 eleven H4N12163R 59 83 5.66E+05 2.81E-03 4.97Е-09 4 H4N12167R 38 64 6.84E+05 2.49Е-03 3.64Е-09 5 H4N12175R 25 4 1.12E+05 1.79Е-02 1.59Е-07 one H4N12159R 51 68 5.61E+05 9.75Е-04 1.74Е-09 12 H4H12164R 59 64 1.77E+05 1.04Е-03 5.85Е-09 eleven H4N12168R 34 21 6.38E+04 5.69E-04 8.90E-09 20 H4N12169R 61 75 3.29E+05 7.37Е-04 2.24Е-09 sixteen H4H11686N 39 69 2.84E+05 7.91E-04 2.78Е-09 fifteen H4H12170R 36 61 4.24E+05 8.70E-04 2.05Е-09 thirteen H4N12177R 2 43 31 1.07E+05 5.07Е-03 4.76Е-08 2 H4N12183R 2 31 25 5.12E+04 9.97Е-04 1.95Е-08 12 H4N12166R 72 54 4.91E+05 1.26E-03 2.56Е-09 nine an object comparisons one 41 2 NB NB NB NB an object comparisons 2 23 42 7.34E+05 1.07Е-03 1.45Е-09 eleven

At 25° C., all 25 anti-C5 antibodies of the invention bound human C5 with KD values ranging from 73 pM to 8.4 nM, as shown in Table 1. 3. At 37° C., the anti-C5 antibodies of the invention bound human C5 with K _D values ranging from 103 pM to 18.5 nM, as shown in Table 3. 4. At 25° C., 25 of the 25 tested anti-C5 antibodies of the invention bound monkey-mmh C5 with KD values ranging from 133 pM to 64 nM, as shown in Table 5. At 37° C., 25 of the 25 tested anti-C5 antibodies of the invention bound monkey-mmh C5 with KD values ranging from 133 pM to 118 nM, as shown in Table 6. At 25° C., 16 of the 16 tested anti-C5 antibodies of the invention bound to human C5 R885H-mmh with KD values ranging from 147 pM to 10.9 nM, as shown in Table 1. 7. At 25° C., 16 out of 16 tested anti-C5 antibodies of the invention bound to human C5 R885C-mmh with KD values ranging from 251 pM to 190 nM, as shown in Table 7. 8. At 37° C., 16 of the 16 anti-C5 antibodies tested according to the invention bound to human C5 R885Hmmh with KD values ranging from 1.49 to 69.4 nM, as shown in Table 8. 9. At 25° C., 16 out of 16 anti-C5 antibodies tested according to the invention bound to human C5 R885C-mmh with KD values ranging from 1.74 to 159 nM, as shown in Table 9. ten.

Example 4 Antibody binding to C5 at different pH.

The effect of pH on the dissociation rate of recombinant human C5 bound to purified anti-C5 monoclonal antibodies was determined using a real-time surface plasmon resonance biosensor on a Biacore T200 instrument. The surface of the Biacore sensor was first derivatized by amine coupling with a mouse anti-human Fc monoclonal antibody (GE, no. BR-1008-39) in order to capture anti-C5 monoclonal antibodies expressed with human IgG4 Fc. All Biacore binding studies were performed using two PBS-T pH 7.4 running buffers (0.01 M Na ₂ HPO ₄ /NaH ₂ PO ₄ , 0.15 M NaCl, 0.05% v/v Tween-20 , pH adjusted to 7.4) and PBS-T pH 6.0 (0.01 M Na2HPO4/NaH2PO4, 0.15 M NaCl, 0.05% v/v Tween-20, pH adjusted to 6.0 ). Various concentrations of human C5 (EMD, cat. no. 204888) or monkey C5.mmh (prepared in PBS-T pH 7.4 running buffer) in diapa

- 44 039858 zone from 100 to 11.11 nM, 3-fold dilutions were injected onto the surface with captured anti-C5 monoclonal antibody for 3 min at a flow rate of 50 μl/min and their dissociation was monitored in two moving buffers PBS-T pH 7 .4 and PBS-T pH 6.0 for 6 min. All experiments on the kinetics of binding was carried out at 25 and 37°C. The kinetic dissociation constant (kd) was determined by fitting real time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. The dissociative half-life (t _1/2 ) upon binding was calculated from kd as:

t1/2(MHH) = ln(2)/60xkd.

Half-life ratios for binding of human C5 to various anti-C5 monoclonal antibodies at 25 and 37° C. in two running buffers PBS-T pH 7.4 and PBS-T pH 6.0 are shown in Table 1. 11 and 12.

Table 11

Half-life ratios of selected anti-C5 antibodies for human C5 at 25°C

Captured Ratio ti _/2 mAb pH 7.4/pH 6.0 H4N12169R IC H4H12176R2 IC H4N12161R IC H4N12159R < 0.3 H4H12170R < 0.5 H4N12166R 4.5 H4H12183R2 IC H4N12167R 0.6 H4H12164R 0.3 H4N12163R 1.2 H4N12175R 0.9 H4H12177R2 < 0.5 H4N12171R 0.6 H4N12168R 1.5 H4H12166R2 9, 3 H4N12166RZ 7.9 Н4Н12166Р4 7.8 H4H12166R5 8.3 H4H12166R6 7, 8 Н4Н12166Р7 35 H4H12166R8 47 H4H12166R9 31 Н4Н12166Р10 33 H4H11683N 2 H4H11686N 2

IC - unconvincing.

- 45 039858

Table 12 Half-life ratios of selected anti-C5 antibodies for human C5 at 37°C

Captured mAb Ratio ti _/2 pH 7.4/pH 6.0 H4N12169R IC H4H12176R2 < 0.4 H4N12161R < 0.7 H4N12159R < 0.2 H4H12170R < 0.2 H4N12166R 3, 8 H4H12183R2 IC H4N12167R 0.2 H4H12164R <0.1 H4N12163R 0.8 H4N12175R 0.9 H4H12177R2 1.3 H4N12171R 3.7 H4N12168R one H4H12166R2 7.3 H4N12166RZ 6, 6 H4H12166R4 7, 6 H4H12166R5 7, 6 H4H12166R6 8.2 Н4Н12166Р7 21 Н4Н12166Р8 36 H4H12166R9 28 Н4Н12166Р10 nineteen H4H11683N 1.4 H4H11686N 0.8

IC - unconvincing.

The half-life ratios for monkey C5 binding to various anti-C5 monoclonal antibodies at 25 and 37° C. in two running buffers PBS-T pH 7.4 and PBS-T pH 6.0 are shown in Table 1. 13 and 14.

- 46 039858

Table 13

Half-life ratios of selected anti-C5 antibodies for C5 monkey at 25°C______

Captured mAb Ratio ti/ ₂ pH 7.4/pH 6.0 H4N12169R 3.4 H4H12176R2 > 9, 1 H4N12161R IC H4N12159R 1.2 H4H12170R > 1.7 H4N12166R 18.5 H4H12183R2 5, 8 H4N12167R 9, 2 H4H12164R 2.9 H4N12163R 9, 7 H4N12175R 3, 6 H4H12177R2 3.7 H4N12171R 2.1 H4N12168R 3, 8 H4H11683N 0.34 H4H11686N 0.37

IC - unconvincing.

Table 14

Half-life ratios of selected anti-C5 antibodies for C5 monkey at 37°C

Captured Ratio ti/ ₂ mAb pH 7.4/pH 6.0 H4N12169R 2 H4H12176R2 2.8 H4N12161R 10, 7 H4N12159R 6, 3 H4H12170R 4.7 H4N12166R 7.1 H4H12183R2 2.4 H4N12167R 4.4 H4H12164R 1.1 H4N12163R 3.3 H4N12175R 0.4 H4H12177R2 1.5 H4N12171R 4.7 H4N12168R 4 H4H11683N 0.7 H4H11686N 0.5

IC - unconvincing.

As shown in Table. 11-14, the selected anti-C5 antibodies exhibited pH dependent binding as seen from the t _1/2 ratios.

- 47 039858

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

Binding competition between anti-C5 monoclonal antibodies (mAb) was determined using real-time labelless biolayer interferometry analysis on an Octet RED384 biosensor (Pall ForteBio Corp.). The entire experiment was carried out at 25° C. in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v/v. Tween-20 surfactant, 0.1 mg/ml BSA (Octet HBS-P buffer) with plate shaking at 1000 rpm. In order to assess whether two antibodies were able to compete with each other for binding to their respective epitopes on human C5 (plasma-purified hC5, EMD), approximately 1.5 nM anti-human C5 mAb was first captured on antibody-coated Octet biosensor tips. against hFc (Pall ForteBio Corp., no. 18-5060), by immersing the tips for 3 minutes in wells containing a solution of anti-human C5 mAb 50 μg/ml (hereinafter referred to as mAb1). The captured antibody biosensor tips were then saturated with the H4H blocking isotype control mAb (hereinafter referred to as the blocking mAb) by immersion in wells containing a 200 μg/mL blocking mAb solution for 4 min. Then, the biosensor tips were immersed in wells containing a co-complex solution of 50 nM hC5 and 1 μM of the second anti-human C5 mAb (hereinafter referred to as mAb2), which was preincubated for 2 h, for 4 min. The biosensor tips were washed in Octet HBS-P buffer between all stages of the experiment. Real-time monitoring of the binding response was carried out during the course of the experiment and the binding response was recorded at the end of each stage. The binding response of human C5 previously complexed with mAb2 to mAb1 was corrected for background binding, compared, and the competitive/non-competitive behavior of various anti-C5 monoclonal antibodies was determined.

In table. 15 explicitly defines dependencies for antibodies competing in both directions, regardless of the binding order.

Table 15

Cross-competition between pairs of selected anti-C5 antibodies

First mAb (mAbl), using ANS Octet captured biosensors mAb2 antibodies shown to compete with mAbl H4H12183R2 H4H12167R; H4N12163R H4H12166R; H4N12167R H4H12183R2; H4N12163R H4H12166R; H4N12166R H4H12183R2; H4N12163R H4H12167R; H4N12163R H4H12183R2; H4N12166R H4H12167R; H4N12159R H4H12169R; H4H11683N; H4H12170R H4N12169R H4H12159R; H4H12170R H4H11683N; H4H11683N H4H12159R; H4H12170R H4H12169R; H4H12170R H4H12159R; H4H11683N H4H12169R; H4N12175R H4H12177R2 H4H12177R2 H4N12175R H4H12176R2 H4H12164R H4H12164R H4H12176R2 H4N12168R No H4N12161R No H4H11686N No

Example 6 Inhibition of C5-Mediated Complement-Dependent Cytotoxicity in a B Cell Bioassay.

This example describes a bioassay to test the role of C5 using an anti-CD20 antibody in the classical complement pathway. It has been shown that therapeutic antibodies against CD20 to

- 48 039858 cell surface specific antigen CD20 of B cells leads to CDC in B cells (Glennie et al. 2007, Mol. Immunol. 44:3823-3837), and CDC analysis using cell lines expressing CD20 has been previously described (Flieger et al 2000, Cell Immunol 204:55-63). Daudi cells, a human B cell line expressing CD20, complement conserved or C5-depleted serum with exogenous C5 variants, and an anti-CD20 antibody (antibody containing VH/VL from 2F2 from US Pat. No. 8,529,902) were used to evaluate the role of C5 activity in CDC.

In the CDC bioassay for C5, Daudi cells were seeded in 96-well assay plates at 10,000 cells/well, or in RPMI containing 10% FBS, penicillin/streptomycin, L-glutamine, sodium pyruvate, and non-essential amino acids (RPMI complete medium), or in RPMI containing 1% BSA, penicillin/streptomycin and L-glutamine (RPMI/BSA). All assays testing for mutated antibodies against hC5, along with testing for non-mutated antibodies with C5-containing human sera, were performed in complete RPMI medium, while assays testing for non-mutated antibodies with African green monkey sera and human C5 variants were performed in RPMI/ B.S.A. In order to measure CDC using human or monkey sera, anti-CD20 antibody was diluted 1:3 from 100 nM to 2 pM (including control sample containing no antibody) and incubated with cells for 10 min at 25°C, followed by addition of 1.66% serum or 1.66% C5-depleted serum and 6.6 nM C5 protein variants. The amount of C5 protein added to C5-depleted serum is based on a reported human serum C5 concentration of 0.37 μM (Rawal et al. 2008, J. Biol. Chem. 283:7853-7863). In order to test CDC inhibition with anti-C5 antibody, anti-C5 antibodies were diluted 1:3 from 100 nM to 2 pM (including a blank control) and incubated with 1.66% serum or 1.66% C5-depleted serum and 6.6 nM protein C5 variants for 30 min. 10 min before adding serum antibodies to cells, anti-CD20 antibody was added to cells at 1, 2, 3, 3.5, 7, 10, or 30 nM. At the end of the incubation with the anti-CD20 antibody, the antibody/serum mixture was added to the cells. Cytotoxicity was measured after 3.5 hours of incubation at 37° C. and 5% CO ₂ , followed by 15 minutes of incubation at 25° C. and addition of CytoTox-Glo™ reagent (Promega, no. G9292). CytoTox-Glo™ is a luminescent reagent that measures cell death such that increased luminescence is observed with increased cytotoxicity (measured in relative light units, RLU). Untreated cells in control wells were lysed by digitonin treatment immediately after the addition of CytoTox-Glo™ reagent in order to determine maximum cell death. Luminescence in the plates was read using a Victor X instrument (Perkin Elmer) 15 min after the addition of CytoTox-Glo™. The percentage of cytotoxicity was calculated using the RLU values using the following equation:

% cytotoxicity=100 x (experimental cell lysis background cell lysis)/(maximum cell lysis - background cell lysis)

In this equation, background cell lysis is the luminescence of cells that were treated with media and serum alone without any anti-CD20 antibody, and maximum cell lysis is the luminescence of cells that were treated with digitonin. The results, expressed as % cytotoxicity or RLU, were analyzed using non-linear regression (4-parameter logistic) and Prism 5 software (GraphPad) to obtain EC ₅₀ and IC ₅₀ values. Inhibition for antibodies was calculated such that 0 to 100% inhibition is the range of inhibition from the concentration of anti-CD20 antibody used in the assay without inhibitor to 0 nM of anti-CD20 antibody.

Results.

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

- 49 039858

Table 16

Inhibition of CDC with non-mutated anti-hC5 antibody using 1.66% serum and anti-CD20 antibody in Daudi cells

Serum Man Man African green monkey emaciated on C5 human and emaciated on C5 the age and EC ₅₀ [M] anti-CD20 antibody (with 1.66% serum) Permanent anti-CD20 antibody (with 1.66% serum) 1.0E-09 1 nM 1C ₅₀ [M] Not tested Not tested 3.2E-09 2.4E-09 3.4E-09 2.4E-09 2.6E-09 2.5E-09 1.4Е-09 1.2E-09 1.5E-09 Not tested Not tested Not tested Not tested Not tested Not tested 2.4E-09 3 nM 1C ₅₀ [M] (max. % inhibition) * 4.0E-09 4.4E-09 3.4E-09 2.6E-09 3.7E-09 5.8E-09 4.5E-09 3.5E-09 6, 6 nM variant C5 R885H 1.9E-09 3.5 nM 1C ₅₀ [M] 1.3E-09 1.1E-09 1.4E-09 1.8E-09 2.1E-09 1.8E- 09 1,ZE-09 1,9E-09 6, 6 nM variant C5 R885C 2.7E-09 3 0 nM 1C ₅₀ [M] (max. % inhibition) * 9.0E-10 4.5E-10 1.0E-09 1.0E-09 1, 1E-09 8.2E-10 4.6E-10 1.0E-09 H4N12168R 1.5E-09 Not tested 2.0E-09 2,ZE-09 8.6E-10 H4N12169R 1.7E-09 Not tested 2.9E-09 1,ZE-09 6.7E-10 H4H12170R 2.0E-09 Not 3.7E-09 4.8E-10 4,ZE-10

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tested H4N12171R 1.9E-09 Not tested 3,ZE-09 1.6E-09 6.5E-10 H4N12175R 2.2E-09 Not tested 5.2E-09 4.2E-09 >2.0E-08 (67%) H4H12176R2 2.7E-09 Not tested 3.5E-09 2.1E-09 1,ZE-09 H4H12177R2 2.2E-09 Not tested 6.1E-09 2.4Е-09 1.6E-09 H4H12183R2 1.7E-09 Not tested 1.4E-08 1.2E-09 4.5E-10 an object comparisons one 2,ZE-09 1.8E-09 >9.0E-08 (49%) Without inhibited and I Without inhibited and I control e mAb 1 Without inhibited AND I Without inhibited AND I Not tested Not tested Not tested control e mAb 2 Not tested Not tested Without inhibited AND I Without inhibited and I Without inhibited and I

*Unless otherwise noted, inhibition is ~100% everywhere.

As shown in Table. 16 and 17, all 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by C5 present in 1.66% human serum. IC ₅₀ for non-mutated antibodies ranged from 1.2 to 3.4 nm. The IC _{50 of the} mutated antibodies ranged from 3.0 to 12 nM. The parental non-mutated H4H12166P antibody gave complete inhibition with IC _{50s of} 2.6 and 2.9 nM.

Table 17

Inhibition of CDC by mutated anti-hC5 antibody using 1.66% serum and anti-CD20 antibody in Daudi cells

Serum Man African green monkey Depleted at human C5 and 6.6 nM variant C5 R885H Depleted at human C5 and 6.6 nM variant C5 R885C EC ₅₀ [M] anti-CD20 antibody (with 1.66% 1.9E-09 2.6E-09 6,ZE-09 9.5E-09

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serum) Permanent anti-CD20 antibody (from 1.66% serum) 2 nM 10 nM 7 nM 30 nM Antibody 1C ₅₀ [M] 1C ₅₀ [M] (max. % inhibition) * 1C ₅₀ [M] 1C ₅₀ [M] H4N12166R 2.9E-09 5.6E-09 1,ZE-09 7.6E-10 H4H12166R2 3.7E-09 9.7E-09 1.7E-09 1.2E-09 H4N12166RZ 7.8E-09 >3.0E-08 (64%) 2.9E-09 1.7E-09 Н4Н12166Р4 3.5E-09 7.9E-09 1.5E-09 9.6E-10 H4H12166R5 4.9E-09 >3.0E-08 (75%) 2.1E-09 1.4Е-09 H4H12166R6 3.0E-09 9.9E-09 1,ZE-09 7.9E-10 Н4Н12166Р7 7,ZE-09 >6.0E-08 (61%) 4.2E-09 2,ZE-09 H4H12166R8 4.1E-09 >2.0E-08 (79%) 2.1E-09 1.2E-09 H4H12166R9 3.9E-09 >1.0E-08 (85%) 1.7E-09 7.7E-10 Н4Н12166Р10 1.2E-08 >1.0E-07 (34%) 7.0E-09 3.5E-09 an object comparisons 1 2.7E-09 >1.0E-07 (35%) Without inhibition Without inhibition Control mAb 2 Without inhibition Without inhibition Without inhibition Without inhibition

*Unless otherwise noted, inhibition is ~100% everywhere.

unmutated anti-hC5 antibodies showed complete inhibition of African green monkey C5-mediated CDC with an IC ₅₀ ranging from 2.0 to 14 nM.

of the 9 mutated antibodies showed complete inhibition of African green monkey C5 mediated CDC with an IC ₅₀ ranging from 7.1 to 9.9 nM. The remaining 6 mutated antibodies are blockers with IC ₅₀ greater than 10 nM and maximum inhibition (at 100 nM antibody) ranging from 34 to 85%. The parental non-mutated H4H12166P antibody produced complete inhibition with IC _{50s of} 4.5 and 5.6 nM.

In order to test whether anti-hC5 antibodies inhibit human C5 variants, R885H and R885C, C5-depleted human serum was tested with 6.6 nM of each C5 variant. All 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by R885H variant C5 with IC ₅₀ for unmutated antibodies ranging from 0.48 to 4.2 nM, while IC ₅₀ for mutated antibodies ranged from 1.3 to 7 .0 nM. The parental unmutated H4H12166P antibody gave complete inhibition with an IC _{50 of} 1/3 and 1.3 nM.

of 16 non-mutated hC5 antibodies showed complete inhibition of CDC mediated by the C5 R885C variant with an IC ₅₀ ranging from 0.43 to 1.6 nM. One non-mutated antibody showed weak CDC inhibition with a maximum inhibition of 67% (at 100 nM antibody) and an IC ₅₀ >20 nM. All 9 mutated antibodies showed complete inhibition of CDC mediated by the C5 R885C variant, with an IC ₅₀ ranging from 0.77 to 3.5 nM. The parental non-mutated H4H12166P antibody gave complete inhibition with IC _{50s of} 0.46 and 0.76 nM.

Anti-CD20 antibody showed CDC of Daudi cells with 1.66% serum with EC _{50 of} 1.0, 1.4 and 1.9 nM for human serum, 2.4 and 2.6 nM for African green monkey serum, 1.9 and 6.3 nM for hC5 depleted serum with hC5 R885H variant and 2.7 and 9.5 nM for hC5 depleted serum with hC5 R885C variant. None of the irrelevant control IgG antibodies, control mAb1 and control mAb2, showed any inhibition of CDC.

Example 7 Inhibition of C5a activity as determined by luciferase assay.

This example describes an assay for testing C5a activation through one of its receptors, C5aR1. C5aR1 is a G protein coupled receptor (GPCR) and can initiate various GPCR coupled signaling pathways (Monk et al. 2007, Br. J. Pharmacol. 152:429-448).

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A bioassay was generated using HEK293 cells stably transfected with human C5aR1 (accession no. NP 001727.1) and human G16 (accession no. NP_002059.3) along with a luciferase reporter [NFAT response element (4X)-luciferase]. Ga 16 is a relatively promiscuous G protein that can bind to various types of GPCRs, leading to PLC-β activation and a subsequent increase in Ca ⁺⁺ , which in turn activates NFAT translocation and reporter gene transcription (Kostenis et al. 2005 , Trends Pharmacol Sci 26:595-602). The resulting HEK293/hGa16/hC5aR1/NFAT-luc cell line was isolated and maintained in 10% DMEM containing 10% FBS, NEAA, penicillin/streptomycin, 500 μg/ml G418, 100 μg/ml hygromycin B, and 7 μg/ml blasticidin.

For C5a luciferase bioassay, HEK293/hGa16/hC5aR1/NFAT-luc cells were seeded in 96-well assay plates at 20,000 cells/well in OPTIMEM (Invitrogen, no. 31985-070) supplemented with 0.5% BSA, penicillin/streptomycin and L- glutamine and then incubated at 37°C and 5% CO ₂ overnight. BSA was used instead of FBS as serum has been shown to cleave and inactivate hC5a (Klos et al., 2013, Pharmacol. Rev. 65:500-543). The next morning, hC5a was titrated from 100 nM to 2 pM (including a control containing no hC5a) and added to the cells in order to determine a dose-dependent titration curve for the cell line. In order to test hC5a inhibition with an anti-hC5a antibody, 500 pM hC5a was added to the cells. Immediately thereafter, antibodies diluted 1:3 from 100 nM to 2 pM (including a control containing no antibody) were added to the cells. Cells were incubated for 5.5 h at 37°C in the presence of 5% CO ₂ . Luciferase activity was detected after incubation with OneGlo™ reagent (Promega, no. E6051). OneGlo™ is a luminescent reagent that measures the amount of luciferase present in cells. In this assay, increased hC5a activation leads to increased luciferase production and luminescence (measured in relative light units, RLU). Luminescence was measured using a Victor X instrument (Perkin Elmer). The results were analyzed using non-linear regression (4-parameter logistic) and Prism 5 software (GraphPad) to obtain EC ₅₀ and IC ₅₀ values. Antibody inhibition was calculated such that 0 to 100% inhibition represents a range of inhibition from 500 pM hC5a without inhibitor to 0 nM hC5a.

Four anti-hC5 antibodies were tested for their ability to inhibit hC5a activation of its receptor, hC5aR1, by measuring the degree of inhibition for 500 pM hC5a activation of HEK293/hGal6/hC5aR1/NFAT-luc cells.

Table 18

Inhibition by anti-hC5 antibody for 500 pM hC5a in HEK293/Ga16/hC5aR1/NFAT-luc cells

EU ₅₀ [M] hC5a 3.9E-10 mAb PID or REGN No. Inhibition for 500 pM hC5a _IC50 [M] H2aM11682N 4.6E-10 H2aM11684N 3.5E-11 H2aM11694N 1.4E-10 H2aM11695N 4.2E-11 Control mAb Without inhibition

As shown in Table. 18, all 4 antibodies of the invention showed complete inhibition for 500 pM hC5a with an IC ₅₀ ranging from 0.035 to 0.46 nM. The irrelevant control IgG antibody, control mAb3, did not show any inhibition of hC5a. hC5a activated HEK293/Ga16/hC5aR1/NFAT-luc cells with an EC _{50 of} 0.39 nM.

Example 8 Hemolysis Bioassay.

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

SN is a screening assay for classical complement pathway activation that is sensitive to the reduction, absence and/or inactivity of any component of the pathway. In CH, the functional ability of serum components of the classical pathway to complement lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (hemolysin) is tested. When antibody-coated SRBCs are incubated with test serum, the classical complement pathway is activated and the result is hemolysis. If the complement component is absent, the CH level will be zero; if one or more components of the classical pathway are reduced, SN will be reduced. (Nilsson et al. 1984, J. Immunol. Meth. 72:49-59). This assay is used to characterize and screen for high affinity anti-human C5 antibodies.

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Ways.

(A) Analysis of hemolysis by the classical complement pathway.

The desired number of sheep red blood cells (SRBC) was washed in GVB++ buffer and resuspended at 1 x 10 ⁹ cells/ml. For sensitization, SRBC was mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/ml) at 37° C. for 20 minutes. Sensitized SRBC cells were diluted to 2±10 ⁸ cells/ml in GVB++ prior to use in the hemolysis assay. Normal human serum or cynomolgus monkey serum was diluted to 2% or 10% in GVB++ buffer. In order to test inhibition of C5-mediated hemolytic activity, test antibodies were pre-incubated for 20 min at 4°C at concentrations ranging from 0.6 to 800 nM in 2-10% normal human serum or 10% cynomolgus or African monkey serum. green monkey. Round bottom 96 well plates were used to measure hemolytic activity. A total of 100 µl of sensitized ovine RBCs (2×10 ⁸ cells/ml) were seeded into a 96-well plate, followed by the addition of 100 µl of the appropriate serum samples, which had been pre-incubated with test antibodies. Cells were mixed gently and incubated at 37°C for 60 min. After the incubation time, the cells were pelleted by centrifugation at 1250xg and 4°C. A total of 100 μl of the supernatant was transferred to a fresh 96-flat bottom plate and read at 412 nm on a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 1-5% for the treatments.

The percentage of hemolysis was calculated as follows:

% hemolysis = 100 x (experimental cell lysis - background cell lysis)/(maximum cell lysis - background cell lysis).

In this equation, background cell lysis is the OD at A412 nm from cells incubated in GVB++ buffer only containing no serum. Maximum cell lysis is the OD at A412 nm from cells that were treated with water. The results, expressed as % hemolysis, were analyzed using non-linear regression (4-parameter logistic) and Prism 5 software (GraphPad) to obtain IC ₅₀ values - Data were presented as mean ± standard error of the mean.

(B) Alternative complement analysis.

The desired number of rabbit red blood cells (RbRBC) was washed in GVB-Mg ²⁺ /EGTA buffer and resuspended at 2x10 ⁸ cells/ml. Normal human serum or cynomolgus monkey serum was diluted to 10% in GVB-Mg ²⁺ /EGTA buffer. In order to test inhibition of C5 mediated hemolytic activity, antibodies at concentrations ranging from 3 to 800 nM were pre-incubated for 20 minutes at 4° C. in 5-10% normal human serum or cynomolgus monkey serum. Round bottom 96 well plates were used to measure hemolytic activity. A total of 100 µl of RbRBC (2 x 10 ⁸ cells/ml) was plated in a 96-well plate followed by the addition of 100 µl of 10% normal human serum or cynomolgus monkey serum or African green monkey serum, which had been preincubated with anti-C5 antibodies. Cells were gently mixed and incubated at 37°C for 60 min. After the incubation time, the cells were pelleted by centrifugation at 1250xg at 4°C. A total of 100 µl of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 5% serum.

The percentage of hemolysis was calculated as follows:

% hemolysis = 100 x (experimental cell lysis - background cell lysis)/(maximum cell lysis - background cell lysis).

In this equation, background cell lysis is the OD at A412 nm from cells that were incubated in GVB-Mg/EGTA buffer containing no serum alone or without any anti-C5 antibody. Maximum cell lysis is the OD at A412 nm from cells that were treated with water. Inhibition by anti-C5 antibodies IC ₅₀ values were calculated using non-linear regression (4-parameter logistic) and Prism 6 software (GraphPad).

Results.

(A) Inhibition of human C5 mediated hemolysis.

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

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Table 19

Inhibition of CP and AR activity by anti-hC5 antibody in 1% or 5% normal human serum (NHS)

mAb PID human SR Human AR human SR Human AR 1C ₅₀ [M] 1C ₅₀ [M] % max inhibition % max inhibition H4H12183R2 5.88Е-09 1.60E-07 99.9% 78.9% H4H12176R2 4.58Е-09 1.65Е-08 94.1% 69.9% H4N12168R 3,ZZE-09 2.85Е-08 97.5% 66.2% H4H11686N 3.09Е-09 1,ZOE-08 97.4% 76.2% H4N12167R 3.68E-09 1.55Е-08 99.9% 64.8% H4N12161R 2.56Е-09 2.55Е-08 93.7% 56.1% H4N12163R 2.72Е-09 2.05Е-08 96.1% 66.0% H4N12166R 2.80E-09 2.60E-08 95.0% 70.9% H4H11683N 2.54Е-09 3.40E-08 98.1% 73.2% H4N12159R 2.50E-09 1.75Е-08 97.9% 73.4% H4H12177R2 2.34Е-09 1.70E-08 97.5% 71.0% H4H12170R 2.39Е-09 1.80E-08 98.2% 81.1% H4N12175R 2.36E-09 2.00Е-08 98.0% 80.2% H4N12171R 2,ZZE-09 1.55Е-08 94.9% 42.0% H4H12164R 2.10E-09 1.45Е-08 95.9% 69.7% H4N12169R 2.36E-09 2.00Е-08 98.3% 44.5% isotypic Not Not Not Not the control activity activity activity activity

As shown in Table. 19, 16 anti-hC5 antibodies of the invention exhibited greater than 94% inhibition of classical pathway (CP) hemolysis mediated by C5 present in 1% human serum. IC ₅₀ for antibodies were in the range from 2.1 to 5.9 nm, and the percentage of inhibition was in the range of 95-99%. All 16 anti-C5 antibodies showed more than 60% inhibition (with the exception of H4H12169P) in an alternative pathway (AP) hemolysis assay that is mediated by C5 present in 5% NHS. IC ₅₀ for antibodies were in the range from 13 to 160 nm, and the percentage of inhibitory activity was in the range from 44 to 81%.

Table 20

Inhibition of CP and AR activity by anti-hC5 antibody in 5% normal human serum (NHS)

rnAb PID human SR Human AR human SR Human AR 1C ₅₀ [M] 1C ₅₀ [M] % max inhibition % max inhibition H4N12166R 1.09Е-08 2.09Е-08 99.4% 86.9% H4H12166R2 1.59Е-08 4.78Е-08 98.2% 81.3% H4N12166RZ 1.34Е-08 6.00Е-08 95.9% 78.3% Н4Н12166Р4 1.32E-08 3.17E-08 98.6% 77.0% H4H12166R5 1.49Е-08 6.55Е-08 97.1% 77.7% H4H12166R6 1.03Е-08 2.84Е-08 98.1% 82.4% Н4Н12166Р7 2.43Е-08 1.56E-07 93.7% 83.2% H4H12166R8 1.41Е-08 7,ZOE-08 95.7% 73.3% H4H12166R9 1.16E-08 5.35Е-08 93.7% 72.2% Н4Н12166Р10 4.44Е-08 Not activity 74.0% Not activity isotypic the control Not activity Not activity Not activity Not activity

As shown in Table. 20, all 9 mutated hC5 antibodies showed inhibition of CP and AR hemolytic activity mediated by C5 present in 5% human serum. In the hemolysis CP assay, the parental unmutated H4H12166P antibody showed greater than 98% inhibition with an IC _{50 of} 10.9 nM. The 8 mutated hC5 antibodies showed greater than 90% inhibition with an IC ₅₀ ranging from 10.3 to 24.3 nM. The mutated anti-C5 antibody 12166P10 showed a partial inhibition of 74%. In the hemolysis AP assay, the parental non-mutated H4H12166P antibody showed greater than 85% inhibition with an IC _{50 of} 20.9 nM.

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Mutated antibodies against hC5 showed inhibition in the range of 72-83%, and IC ₅₀ antibodies ranged from 28 nm to 0.15 μm.

(B) Inhibition of monkey C5-mediated hemolysis.

A total of 25 anti-human C5 (hC5) antibodies, 16 unmutated and 9 mutated, were tested for their ability to inhibit cynomolgus monkey and African green monkey C5 in CH50 assay using sensitized sheep red blood cells (SRBC) and AH50 assay using red blood cells rabbit (RRBC).

Table 21

Inhibition of CP and AR activity by anti-hC5 antibody in 5% normal African green monkey (AGM) sera

SR with AGM serum AP with AGM serum AGM SR AGM AR rnAb PID 1C _5O [M] 1C _5O [M] % max inhibition % max inhibition H4H12183R2 Not activity Not activity Not activity Not activity H4H12176R2 3.04Е-08 4.77Е-08 91.0% 83.2% H4N12168R 2.80E-08 2.25Е-08 90.8% 88.2% H4H11686N 4.82E-08 1.63E-07 49.3% 50.4% H4N12167R 6.95Е-08 6.95Е-08 90.5% 53.4% H4N12161R 3.19E-08 4.75Е-08 78.9% 35.7% H4N12163R 6.90E-08 2.16E-07 83.2% 58.5% H4N12166R 1,ZOE-07 2,ZZE-07 81.0% 44.2% H4H11683N 2.92E-08 4.08Е-08 81.1% 88.1% H4N12159R 2.58Е-08 2.70E-08 93.4% 93.5% H4H12177R2 1.80E-07 1.01E-07 80.6% 8.80% H4H12170R 2.54Е-08 2.69Е-08 94.9% 90.9% H4N12175R 1.18E-07 9.85Е-08 84.5% 17.4% H4N12171R Not activity 2,ZZE-08 17.8% 69.70% H4H12164R 2.47Е-07 1.78E-07 85.8% 15.60% H4N12169R 3.44Е-08 9.15Е-08 43.3% 45.70%

As shown in Table. 21, anti-hC5 antibodies showed varying levels of inhibition of CP or AR hemolytic activity in 5% African green monkey sera. In the SR analysis, 2 of 16 anti-hC5 antibodies did not show inhibition of hemolytic activity. The 14 antibodies showed inhibition in the range of 43-94% with an IC ₅₀ ranging from 25 to 180 nM. In the hemolysis AP assay, 13 of 17 antibodies showed inhibitory activity ranging from 17-93% with an IC ₅₀ ranging from 22.5 to 233 nM.

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Table 22

Inhibition of CP and AR activity by anti-hC5 antibody in 5% normal sera from Cynomolgus monkeys (Cyno)

Wed with serum Supo AR with Serum Supo Supo SR Supo AR mAb PID H4H12183R2 H4N12171R H4H12170R H4N12159R H4N12168R H4H11683N H4H12176R2 H4N12161R H4N12169R H4H11686N H4N12163R H4N12167R H4N12175R H4N12166R H4H12177R2 H4H12164R 1C ₅₀ [M] 1.42E-07 8.70E-09 8.20E-09 7.75E-09 1.03E-08 9.00E-09 1.47E-08 2.79E-08 1.99E-08 1.41E-08 1.65E-08 2.13E-08 1.09E-08 2.01E-08 1.71E-08 1.96E-08 1C ₅₀ [M] 2.96E-08 7.20E-09 7.00E-09 7.05E-09 5.45E-09 7.15E-09 7.70E-09 7.80E-09 7.95E-09 9.00E-09 1.02E-08 7.60E-09 8.05E-09 8.85E-09 8.80E-09 9.10E-09 % max, inhibition 64.6% 92.9% 99.0% 99.3% 99.0% 98.8% 97.5% 100.0% 92.8% 94.5% 96.4% 100.0% 96.7% 94.2% 94.90% 94.7% % max inhibition I 100.0% 98.8% 99.2% 99.6% 99.7% 98.9% 99.3% 98.4% 96.30% 98.7% 98.5% 98.30% 98.10% 98.60% 98.10% 98.70% Isotype control No activity No activity Not activity Not activity

As shown in Table. 22, anti-hC5 antibodies (with the exception of H4H12183P2, which showed 64% inhibition of CP) showed greater than 90% inhibition in the CP or AP assay of hemolysis in 5% cynomolgus serum. In the hemolysis CP assay, the IC _{50s of} the antibodies ranged from 7.15 to 142 nM. In the analysis of AP hemolysis IC ₅₀ antibodies ranged from 5.4 to 29.6 nm.

(C) Inhibition of hemolysis mediated by human variant C5.

Selected anti-C5 antibodies were tested for their ability to inhibit the human C5 variant (see Example 3 herein) from C5-depleted human serum in a CH50 assay. In C5-depleted human serum supplemented with exogenous C5 variant R885H, H4H12166P and Comparator 2 blocked CP hemolysis with IC ₅₀ values of 6.0 and 4.4 nM, respectively, and IC ₈₀ values of 7.6 and 5.5 nM, respectively. For the R885C variant, H4H12166P and Comparator 2 blocked CP hemolysis in C5 depleted human serum with exogenous C5 variants with IC _{50s of} 9.3 and 6.8 nM, respectively, and IC ₈₀ values of 11 and 8.2 nM, respectively. As expected, Comparator 1 did not block the hemolytic activity of human C5 variants.

(D) Inhibition of the human C5b-6 complex.

Selected anti-C5 antibodies were tested for their ability to inhibit the human C5b-6 complex from C5-depleted human serum in the CH50 assay. H4H12166P strongly blocked CP hemolysis in C5-depleted human serum supplemented with exogenous huC5b-6 complex with an IC _{50 of} 3.8 nM and an IC ₈₀ value of 5.8 nM. In contrast, Comparator 1 blocked C5b-6 complex-mediated hemolysis with less force, with IC ₅₀ values of 5.0 nM and IC ₈₀ values of 46 nM, respectively. Comparator 1 only inhibited 70% of total hemolysis at the highest concentrations tested. Comparison 2 did not block the hemolytic activity of the human C5b-6 complex.

Example 9 Antibodies against C5 block the formation of C5a in the hemolysis CP assay.

To assess whether anti-C5 antibodies inhibit C5a production, supernatants from the classical pathway (CP) hemolysis assay were analyzed for C5a levels by ELISA.

C5a, resulting from the cleavage of C5, is a protein fragment of 74 amino acids. C5a is rapidly metabolized by serum carboxypeptidases to a more stable, less active 73 amino acid form of C5a des-Arg via removal of the C-terminal arginine. Quantification of C5a des-Arg therefore provides a reliable measurement for monitoring C5a formation in vivo and in vitro. According to the information provided by the manufacturer, the MicroVue C5a ELISA kit used here is capable of detecting C5a des-Arg. Preliminary experiments (data not shown) show that they also detect the primary form C5a from 74

- 57039858 amino acids. For the purposes of this example, both forms are collectively referred to as C5a.

C5a protein levels were determined in supernatants from the hemolysis CP assay using normal human serum (NHS) with complement conserved that had been preincubated with H4H12166P or an isotype control antibody as described in Example 8. C5a protein levels were measured using the MicroVue C5a ELISA kit as instructed manufacturer. Briefly, samples were diluted and incubated in pre-coated plates with capture antibody (mouse anti-C5a with specificity for human C5 neoepitope). Human C5a protein provided by the manufacturer was used as a calibration standard. C5a in the supernatants was detected with an HRP-conjugated identifying antibody (mouse monoclonal antibody to the C5a region of C5). The chromogenic HRP substrate 3-, 3'-, 5-, 5'-tetramethylbenzidine (TMB) was added in order to detect HRP activity. Used a solution of 1N. hydrochloric acid in order to stop the reaction, and measured the optical density at 450 nm (OD ₄₅₀ ) on a plate reader SpectraMax. Data was analyzed using non-linear regression (4-parameter logistic) in GraphPad Prism. The concentration of C5a was expressed in ng/ml of the supernatant.

In an assay using 5% NHS, H4H12166P strongly blocked the increase in C5a protein levels in a dose dependent manner with an IC _{50 of} 8.5 nM, while the isotype control antibody had no effect on C5a levels (FIG. 1). Maximum blocking at the highest concentration of H4H12166P tested (267 nM) resulted in ~10-fold reduction in C5a levels to 3.8 ng/mL (0.3 nM) compared to 34 ng/mL (2.8 nM) seen with lowest tested concentration of H4H12166P (1 nM) in 5% serum. The C5a concentration observed for maximum blocking is close to the C5a baseline of 2.3 ng/mL (0.2 nM) in untreated 5% NHS.

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

This example describes the characterization of pharmacokinetics (PK) and pharmacodynamics (PD) of selected antibodies against C5, which was performed in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody dosing and used to stratify animal groups by dose.

Total circulating levels of C5 in the cynomolgus monkey were determined using the Human Complement C5 ELISA (Abeam, cat# ab125963) performed according to the manufacturer's recommendations. The mean C5 protein concentrations in monkeys were determined to be 90.85±19.17 µg/ml.

For each anti-C5 antibody, each of 4 cynomolgus monkeys received one intravenous (IV) injection at a dose of 15 mg/kg. Blood samples were taken from each animal pre-dose and up to 1680 hours (70 days), serum was obtained and frozen at -80°C until analysis for PK and PD.

Analysis of the total level of IgG antibodies using immunoassay ELISA.

Total antibody concentrations in monkey serum samples were measured using a non-validated direct ELISA. The ELISA procedure used a microtiter plate coated with a mouse monoclonal antibody against human Fc IgG1/IgG4. Various anti-C5 antibodies were added to the plate and anti-C5 antibodies captured on the plate were detected using a biotinylated monoclonal mouse anti-human Fc IgG4 antibody, followed by horseradish peroxidase conjugated neutravidin (Neutravidin-HRP). A peroxidase specific luminol substrate was then added to obtain a signal intensity that is proportional to the concentration of total captured anti-C5 antibody. Measurements of the calibration standards in relative light units (RLU) and their corresponding nominal concentrations were approximated using a weighted 4-parameter logistic equation in order to create a calibration equation that describes the dependence of the concentration of antibodies against C5 and the response in the analysis. The lower limit of quantification (LLOQ) was 1.56 ng/ml in the assay (2% monkey serum) and 78 ng/ml in pure monkey serum.

Determination of PK parameters.

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

All PK parameters were derived from the respective mean concentrations, including observed maximum serum concentration (C _max ), time of observed peak concentration (t _max ) and observed estimated half-life (T _1/2 ). For each antibody, the area under the concentration-time curve up to the last measured concentration (AUC _last ) and extrapolated from time zero to infinity (AUC _inf ) was determined using the linear trapezoidal rule with linear interpolation and uniform weights.

PD analysis by ex vivo hemolysis assay.

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

Analysis of the classical pathway of hemolysis: Sheep red blood cells (SRBC) were washed in buffer

GVB++ (gelatin-veronal buffer with CaCl2 and MgCl2) (Boston BioProducts) and resuspended in

1x10 ⁹ cells/ml. To sensitize SRBC, a total of 1 x 10 ⁹ SRBC/ml was mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/ml) at 37° C. for 20 minutes.

Sensitized SRBCs were diluted to 2x10 ⁸ cells/ml in GVB++ buffer prior to use in the hemolysis assay. Blood was taken from cynomolgus monkeys before dosing and 5 min, 4 and 8 h and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56, 63 and 70 days after dosing. doses for PD analysis. Serum was obtained and frozen until further use. On the day of analysis, Cynomolgus sera from the respective time points were diluted to 10% in GVB++ buffer. Round bottom 96 well plates were used to measure hemolytic activity. A total of 100 μl of sensitized SRBCs (2x10 ⁸ cells/ml) were plated in a 96-well plate at 37°C followed by the addition of 100 μl of 10% cynomolgus monkey serum from the appropriate time points. SRBC was gently mixed and incubated at 37°C for 10 min. After the incubation time, the cells were centrifuged at 1250xg at 4°C. A total of 100 µl of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on a Spectramax microplate reader. Hemolytic activity was calculated at a final serum concentration of 5%. Percent hemolysis was calculated using absorbance values and the following equation:

% hemolysis = 100 x (experimental cell lysis - background cell lysis)/(maximum cell lysis - background cell lysis).

In this equation, background cell lysis is the OD at A412 nm for SRBCs that were incubated in GVB++ buffer containing no serum. Maximum cell lysis is the OD at A412 nm for SRBCs that were treated with water. The results, expressed as % hemolysis, were analyzed using non-linear regression (4-parameter logistic) and Prism 5 software (GraphPad) to obtain IC ₅₀ values. Data are presented as mean ± standard error of the mean.

Alternate hemolysis pathway assay: Desired number of rabbit red blood cells (RbRBC) were washed in GVB-Mg ²⁺ /EGTA buffer and resuspended at 2x10 ⁸ cells/ml. Blood was taken from cynomolgus monkeys before dosing and 5 min, 4 and 8 h and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42 and 49 days post-dose for PD analysis. Serum was obtained and frozen until further use. Round bottom 96 well plates were used to measure hemolytic activity. A total of 100 μl of RbRBC (2x10 ⁸ cells/ml) were plated in a 96-well plate at 37° C. followed by the addition of 100 μl of 10% cynomolgus serum from the respective time points listed above. RbRBC was gently mixed and incubated at 37°C for 60 min. After the incubation time, the cells were centrifuged at 1250χg at 4°C. A total of 100 μl of the supernatant was transferred to a fresh 96-flat bottom plate, which was read at 412 nm on a Spectramax microplate reader. Hemolytic activity was calculated for a final serum concentration of 5% and expressed as a percentage of total RBC hemolysis using water. The percentage of hemolysis was calculated as described above.

Results.

Selected anti-C5 antibodies (listed in Table 1) were tested in initial experiments for prolonged pharmacokinetic profiles in cynomolgus monkey and C5 humanized mice (described in Example 10). H4H12166P and H4H12161P were selected as having high affinity in combination with prolonged PK and used in subsequent experiments in the present description with the object of comparison 1 and the object of comparison 2.

Cynomolgus monkeys were administered a single 15 mg/kg IV bolus dose of H4H12166P, H4H12161P, or Comparator 2. Serum antibody concentrations of all and percent classical pathway (CP) hemolysis activity were determined at 19 time points over a 70-day lifetime. The alternative pathway (AR) of hemolysis was determined at 17 time points during a 50-day life span. In table. 23 summarizes the mean antibody concentrations for all three antibodies. Profiles of mean total antibody concentrations versus time are shown in FIG. 2. The average PK parameters are described in Table. 24.

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Table 23

Mean serum total IgG concentrations after a single 15 mg/kg intravenous injection of selected anti-C5 antibodies in male cynomolgus monkeys

dose time) (clock after No. Concentration mean ± SD Serum AB (mcg/ml) H4N12166R H4N12161R an object comparisons 2 0 0.083 4 4 BLQ 445 ± 30 BLQ 456 + 26.2 BLQ 459+55.2 4 4 328 ± 27 360 + 28.2 363+43.8 eight 4 353 ± 29.9 316 + 21.5 357 + 16.1 24 4 282 ± 43.6 276 + 32.4 248 + 19.6 48 4 225 ± 15.2 221±21.5 212 ± 19.3 72 4 180±15.0 181 + 17.2 196 + 36.2 120 4 194 ± 20.9 162+10.7 179 + 23.3 168 4 171 ± 29.9 132 + 17.0 157 + 18.4 240 4 157 ± 12.7 96.1+17.3 114 + 13.0 336 4 120 + 10.2 49.3 + 18.7 67.9 + 25.8 432 4 105 ± 13.9 24.6 ± 11.8 42.6 ± 15.9 504 4 92.2 ± 10.6 13.8 + 9.95 28.6+13.1 672 4 75.1+15.8 6.16 + 2.40 10.9 + 6.25 840 3 59.6+4.79 2.44 + 0.85 4.45 + 2.63 1008 3 43.3 + 2.89 1.16 + 0.52 2.17 + 1.58 1176 3 30.6 + 1.42 0.57 + 0.25 1.29 + 1.16 1344 3 25.9 + 3.74 0.315+ 0.16 0.492 + 0.49 1512 3 18.2 + 2.41 0.17 + 0.08 0.270 + 0.27 1680 3 11.5 + 1.51 0.079+ 0.07 0.123 + 0.15 Time - time in hours after injection of a single dose;

SD - standard deviation;

BLQ - below the limit of quantitation.

Following IV bolus administration, total IgG concentration versus time profiles for H4H12166P, H4H12161P and Comparator 2 were characterized by an initial short distribution phase followed by a single elimination phase throughout the lifetime. The peak concentrations of H4H12166P, H4H12161P and Comparator 2 were well comparable as the respective Cmax/dose values among all antibodies were within 1.1 times (29.7, 30.4 and 30.6 [(µg/mL)/(mg /kg)] respectively) (Table 24).

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Table 24

Mean pharmacokinetic parameters for serum total IgG concentrations following a single intravenous injection of selected anti-C5 antibodies 15 mg/kg in male cynomolgus monkeys

Parameter H4N12166R H4N12161R Comparison object 2 15 mg/kg IV (n=4) The average SD The average SD The average SD _Cmax (µg/ml) 445 30.0 456 26.2 459 55.2 Stax/dose (mcg/ml)/(mg/kg) 29.7 2.00 30.4 1.75 30.6 3.68 C _o (µg/ml) 448 30.5 458 26, 6 461 55.6 t _max (hours) 0.083 0 0.083 0 0.083 0 AUC _last day (mcg/ml) 5080 1040 2350 357 2810 470 AUC _last / _H o ₃ a (mcg / ml) / (mg / kg) per day 339 69.3 157 23.8 187 31.3 AUC _inf days (µg/ml) 5550 671 2350 356 2810 470 AUC _inf /flosa (µg/ml)/(mg/kg) per day 370 44.7 157 23.7 188 31.4 CL (ml/h/kg) 0.114 0.0143 0.270 0.0411 0.228 0.0425 _Vss (ml/kg) 60.4 4.85 44.0 4.34 45.3 3.06 t _1/2 (day) 15.6 1.43 5.50 2.45 5.91 1.13

IV - intravenously;

n is the number of animals;

C _max - peak concentration;

Co - initial concentration, determined by extrapolation;

tmax ^- time ^to Cmax;

AUC - area under the concentration-time curve;

AUC _last - AUC calculated from time zero to the time of the last positive concentration;

AUCinf - AUC from time zero and extrapolated to infinity;

CL - total body clearance;

V _ss is the volume of distribution at steady state;

t _1/2 - half-life;

SD - standard deviation.

Note - t _max is expressed in nominal hours.

Evaluation of concentration-time profiles showed that H4H12166P showed the slowest elimination with final antibody concentrations >10 μg/ml up to day 71 of the study. The kinetics of H4H12161P and Comparator 2 were similar; both showed faster elimination than H4H12166P, with mAb concentrations >10 µg/mL by days 22 and 29, respectively.

Therefore, dose-normalized exposures (AUQast/dose) indicated that H4H12166P had the highest exposure of 339 days(µg/mL)/(mg/kg), while H4H12161P and comparator 2 had approximately 2-fold lower exposure, 157 and 187 days (µg/ml)/(mg/kg), respectively, than H4H12166P.

The half-life of the antibody (t _1/2 ) calculated during the elimination phase ranged from 5.5 to 15.6 days among dose groups and also correlated with exposure, where H4H12166P had the highest t _1/2 of 15.6, respectively. days, while H4H12161P and the object of comparison 2 had t _1/2 values of 5.5 and 5.9 days, respectively.

The pharmacological effects of anti-C5 antibodies from cynomolgus monkey serum samples were determined ex vivo by classical complement (CP) pathway hemolysis using sensitized sheep red blood cells (SRBC) and the alternative pathway (AR) rabbit red blood cell (RbRBC) hemolysis. Inhibition of hemolytic activity was calculated for a final serum concentration of 5% and expressed as a percentage of total RBC hemolysis using water. In table. 25 summarizes the activity of 3 antibodies ex vivo, as determined by the average percentage of hemolysis.

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Table 25

Percentage of classical and alternative pathway hemolysis activity ex vivo for selected anti-C5 antibodies

Time watch after dose) No. Cynomolgus Serum % Classical Hemolysis Pathway, 10 min, Mean ± SEM Cynomolgus serum % alternative hemolysis pathway, 60 min, mean ± SEM H4H1216 6R H4N12161R an object compared ia 2 H4N12166R H4N12161R an object comparisons 2 0 4 91.34± 7.6 Not tested and 84.36± 20.28 73.44± 17.26 64.90 ± 19.51 55.77± 10.82 0.083 4 3.5± 1.4 Not tested and 6, 6 ± 6, 5 5.53± 1.98 5.90 ± 3.92 3.83± 3.93 4 4 2.35± 1.06 Not tested and 3.16± 2.2 7.43± 2.54 6.53 ± 2.7 5.30 ± 2.80 eight 4 1.55 ± 0.21 Not tested and 1.25± 0.21 3.53± 0.91 4.70 ± 2.77 1.98± 0.83 24 4 7.7± 6, 08 Not tested and 4.55± 2.05 13.40 ± 2.77 4.68± 1.89 4.65± 2.35 48 4 2.85± 2.19 Not tested and 2.6± 0.7 5.53± 2.40 7.68± 5.22 2.28± 0.67 72 4 0.9 ± 0.42 Not tested and 1.3 ± 0.28 7.95± 3.36 5.95± 2.23 1.45 ± 0.33

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120 4 1.75 0.07 ± Not tested and 1.3 0.14 ± 16, 38 6, 91 ± 7.60 1.94 ± 1.68 0.22 ± 168 4 1.6 1.13 ± Not tested and 1.4 0.56 + 21.28 8.24 + 10.75 2.27 + 2.15 0.19 + 240 4 one 0.14 ± Not tested and 3.7 2.83 ± 19.18 10.20 ± 13.53 7.17 ± 14.20 16.73 ± 336 4 2.55 2.05 ± Not tested and 37.85 5.3 + 21.10 7.55 + 50.58 12.91 + 65.60 26, 04 + 432 4 1.35 0.91 ± Not tested and 105.25 ±3.3 15.20 10.86 + 59.75 12.65 + 54.55 19.11 + 504 4 3.55 2.05 ± Not tested and 107.1 4.38 ± 33.15 8.80 ± 88.55 24.63 ± 85.63 27.48 ± 672 4 2.2 0.56 ± Not tested and 88.9 23.05 ± 75.25 18.30 ± 88.55 8.53 ± 91.58 18.55 ± 840 3 3.075 2.70 ± Not tested and 105.37 ± 53.4 46, 65 5.30 + 92.33 5.16 + 91.85 2.33 + 1008 3 15.5 26, 6 ± Not tested and 108.85 ±2.35 58.60 9.48 + 92.45 6.27 + 92.30 2.69 + 1176 3 58.33 39.55 ± Not tested and 113.85 ±2.62 72.95 5.87 + 104.90 3.5 + 101.90 0.42 + 1344 3 71.55 43.02 ± Not tested and 110.3 1.98 + Not tested and Not tested and Not tested and 1512 3 91.375 ±29.7 Not tested and 110.6 0.85 ± Not tested and Not tested and Not tested and 1680 3 112.22 ±4.06 Not tested and 112.15 ±0.5 Not tested and Not tested and Not tested and

Time - time in hours after injection of a single dose;

SEM standard error of the mean;

BLQ - below the limit of quantification;

NC - not calculated.

As shown in Table. 25 and in FIG. 2, PD effects were measured with complement CP (10 min incubation) by day 70. H4H12166P blocked more than 95% of CP of hemolytic activity until day 35. Activity returned to pre-study maximum hemolysis levels by day 70. Comparator 2 blocked approximately 95% CP of hemolytic activity up to day 10, and the activity quickly returned to the maximum levels of hemolysis before the study by day 18.

PD effects were also measured by complement AR pathway hemolysis assay (60 min incubation) up to day 49. As shown in Table. 25 and in FIG. 3, H4H12166P blocked 80% of the total AR of hemolytic activity until day 18, and the activity returned to the maximum level of hemolysis before the study by day 50. H4H1216P and comparator 2 blocked 90% of the AR of hemolytic activity until day 7, and the activity returned to the maximum levels of hemolysis until research by day 21.

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

In this group of experiments, the pharmacokinetics and pharmacodynamics of the selected antibodies against C5

- 63 039858 was evaluated in mice humanized to express human C5 protein using Velocigene® technology (Valenzuela et al. 2003, Nat. Biotechnol. 21:652-659). Humanized mice were created with the replacement of exons 2-41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in US Patent Application Publication 2015/0313194, incorporated herein in its entirety).

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

Determination of the total drug level in serum using ELISA.

The concentrations of circulating anti-C5 antibodies, both associated with C5 and not, were determined by analysis of total human antibodies using ELISA. Briefly, goat anti-human IgG polyclonal antibody 1 μg/ml in PBS was immobilized in 96-well plates overnight; plates were washed to remove unbound IgG and then blocked with 5% BSA. Serial dilutions of serum samples containing anti-C5 antibodies (6 points) and reference standards (12 points) for the respective antibodies were transferred to anti-human IgG-coated plates and incubated for 1 hour. Then, anti-C5 antibodies bound to the plate were detected using goat anti-human IgG polyclonal antibody conjugated with horseradish peroxidase. The plates were developed using TMB substrate according to the manufacturer's recommended protocol, and optical density (OD) signals at 450 nm were recorded using a Perkin Elmer Victor X4 Multimode plate reader. Serum anti-C5 antibody concentrations were calculated from a calibration curve of reference standards generated using GraphPad Prism software.

Determination of PK parameters.

PK parameters were determined using non-compartmental analysis (NCA) and Phoenix®WinNonlin® software (version 6.3, Certara, LP) and an extravascular dosing model. Using the respective mean concentrations for each antibody, all PK parameters, including the observed estimated half-life (t _1/2 ) and the area under the concentration versus time curve up to the last measured concentration (AUC _last ), were determined using the linear trapezoid rule. with linear interpolation and uniform weights.

PD analysis by haemolysis assay.

Pharmacodynamics for selected anti-C5 antibodies were determined using a classical complement pathway hemolysis assay. Sheep red blood cells (SRBC) (sheep blood in Olswer's solution) were washed in GVB++ buffer (gelatin-veronal buffer with CaCl ₂ and MgCl ₂ ) (Boston BioProducts) and resuspended at 1x109 cells/ml. For sensitization, 1x109 SRBC/ml was mixed with an equal volume of 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/ml) at 37°C for 20 minutes. Sensitized SRBCs were diluted to 2x10 ⁸ cells/ml in GVB++ prior to use in the hemolysis assay. Serum samples from pre-dose animals or humanized C5 mice dosed with anti-C5 antibodies taken on days 10, 20, 30, 40 and 50 post-dose were diluted to 20% in GVB++ buffer. A total of 100 µl of sensitized SRBCs (2x10 ⁸ cells/ml) were plated in 96-well round bottom plates at 37°C followed by the addition of 100 µl of 20% serum supplemented with 160-180 µg/ml of human complement protein 3 (huC3) . The cells were gently mixed and incubated at 37°C for 1 hour. After incubation, the cells were centrifuged at 1250xg at 4°C. A total of 100 µl of the supernatant was transferred to a fresh 96-flat bottom plate and read at A412 nm in a Spectramax microplate reader. Percent hemolysis was calculated using absorbance values and the following equation:

% hemolysis = 100 x (experimental cell lysis - background cell lysis)/(maximum cell lysis - background cell lysis).

In this equation, the background lysis of cells is the OD at A412 nm from SRBCs that were incubated in GVB++ buffer only containing no serum. Maximum cell lysis is the OD at A412 nm from SRBCs that were treated with water. The results, expressed as % hemolysis, were analyzed using non-linear regression (4-parameter logistic) in Prism 6 software (GraphPad) to obtain IC ₅₀ values. Data are presented as mean ± standard error of the mean.

Experiment 1

In this experiment, the pharmacokinetics and pharmacodynamics of the exemplary H4H12166P antibody were compared with Comparator 1 and Comparator 2 in humanized C5 mice. Total human circulating C5 levels were determined using the Human Complement C5 ELISA (Abcam, cat# ab125963) performed according to the manufacturer's recommendations. The mean human C5 concentrations in mice were determined to be 39.73±17.82 μg/ml. There was a difference between male (55.4±1.7 µg/ml, n=47) and female (24.7±0.6 µg/ml, n=49) mice.

Prior to antibody dosing, male and female humanized C5 mice were stratified according to human C5 levels, which averaged 40 μg/ml. For each anti-C5 antibody, a cohort of 22 mice received a single dose of H4H12166P, Comparator 1 or Comparator 2 15 mg/kg via subcutaneous (s.c.) injection. All mice were bled pre-dose and day 1 post-injection for PK analysis. In addition, 10, 20, 30, 40 and 50 days after injection, groups of 4 or 5 mice from each cohort were sacrificed and terminal blood samples were collected for PK and PD analysis. Serum samples on day 1 were the average of the entire cohort of 22 mice. Serum was obtained from blood and frozen at -80°C until analysis.

Total antibody concentrations were determined at 7 time points and percent hemolysis activity was determined at 6 time points over a 50-day lifetime. All concentrations of antibodies against C5 are summarized in table. 26. Profiles of mean total antibody concentrations versus time are shown in FIG. 4. Average PK parameters are described in Table. 27.

Table 26

Mean serum total IgG concentrations after a single subcutaneous injection of 15 mg/kg anti-C5 antibody in humanized C5 mice

Time (day) No. Serum AB concentration (mcg/ml) mean + SD H4N12166R Comparison object 1 Comparison object 2 one 22 178 ± 22.7 229+40.7 164 + 24.1 ten 4 83.7 + 22.2 102 ± 22.9 44±24.1 20 5 57.1 ± 26.8 29 ± 31.3 11.4 ± 10.3 thirty 5 38.1±7.6 30.1 + 34.2 3.6 ± 3.2 40 4 11.9 + 5.0 0.4 + 0.4 0.5 + 0.4 fifty 4* 9.3 ± 12.2 0.3±0.3 0.3±0.2

Time - time in hours after injection of a single dose;

day-day of research;

SD - standard deviation;

SEM standard error of the mean;

ND - not detected;

NS - no samples.

*For object of comparison 2 per day 50 n=3 due to the impossibility of analyzing one sample due to technical problems.

Table 27

PC parameters

Parameter Units H4N12166R Comparison object 1 Comparison object 2 mAb concentration in day 1 mcg/ml 178 229 164 AUClast day hmkg/ml 2801 2708 1418 tl/2 d 11.3 4.7 7, 6

C _max - peak concentration;

AUC - area under the concentration-time curve;

AUC _last - AUC calculated from time zero to the time of the last positive concentration;

ti/2 is the observed estimated half-life.

Mean concentration versus time profiles on day 1 show that the three antibodies H4H12166P Comparator 1 and Comparator 2 had comparable serum concentrations of 178, 229 and 164 μg/mL, respectively. Comparator 1 had an elimination profile similar to H4H12166P up to day 30, but on days 40 and 50 showed a rapid increase in clearance compared to H4H12166P. On day 50, H4H12166P had a mean serum antibody concentration of approximately 9 μg/ml, while both Comparator 1 and Comparator 2 had a 30-fold reduced mean serum antibody concentration of 0.3 μg/ml. Comparator 2 showed the lowest exposure of the three antibodies tested, with approximately 2-fold lower AUC _last (1408 days xmcg/mL) compared to H4H12166P (2801 days xmcg/ml) and Comparator 1

- 65 039858 (2708 days hmcg/ml).

The pharmacological effects of anti-C5 antibodies H4H12166P, Comparator 1 and Comparator 2 from humanized C5 mouse sera samples supplemented with human C3 were measured up to day 50 and determined ex vivo by classical complement pathway (CP) sensitized SRBC hemolysis. The average percentage of hemolysis for each antibody against C5 is given in table. 28 and the profile of the mean percent hemolysis versus time is shown in FIG. 5.

Table 28

Percentage of classical pathway hemolysis activity for anti-human C5 antibodies ex vivo

Time (day) No. % classical pathway hemolysis in 10% mouse serum, 60 min, mean ± SEM H4N12166R comparison object one comparison object 2 one 22 NS NS NS ten 4 12.6 ± 7.79 10.39±2.88 12.06±9.12 20 5 18.8±8.1 21.59 ± 17.53 65.08 ± 52.87 thirty 5 13.76 ± 10.9 78.98 ± 40.3 91.67 ± 16.74 40 4 41.71 ± 40.7 101.09 ± 4.01 68.99 ± 42.47 fifty 4* 62.2±56.6 88.99 ± 17.51 105.14 ± 4.07

Time - time in hours after injection of a single dose;

day - day of research;

SEM - standard error of the mean;

ND - not detected;

NS - no samples.

*For object of comparison 2 per day 50 n=3 due to the impossibility of analyzing one sample due to technical problems.

H4H12166P, Comparator 1 and Comparator 2 inhibited terminal hemolytic complement activity, apparently correlated with antibody exposures. H4H12166P blocked more than 85% of hemolytic activity up to day 30 with activity returning to baseline levels before dose by day 50. Comparator 1 and Comparator 2 blocked approximately 80% of hemolytic activity up to day 20 and day 10, respectively, with activity returning to baseline levels by day 50. 30 days for both.

Experiment 2

In this experiment, the pharmacokinetics and pharmacodynamics of antibodies against C5 H4H12166P, H4H12161P, reference 1 and isotype control were evaluated in humanized C5 mice (mice homozygous for human C5 expression). Total circulating C5 levels were determined using the Human Complement C5 ELISA (Abcam, catalog # ab125963) performed according to the manufacturer's recommendations. The mean human C5 concentrations in mice were determined to be 48.98±15.1 µg/mL.

Prior to antibody dosing, male and female humanized C5 mice were stratified according to human C5 levels, which averaged 50 μg/ml. For each anti-C5 mAb, a cohort of 5 mice received one subcutaneous (s.c.) injection of 15 mg/kg H4H12166P, H4H12161P, Comparator 1, or isotype control. All mice were bled pre-dose, 6 hours, 1, 2, 3, 4, 7, 10, 13, 21, 30 and 45 days post-injection for PK analysis. In addition, on day 59, all mice from each cohort were sacrificed and terminal blood samples were taken for PK and PD analysis. Serum was obtained from the blood, which was frozen at -80°C until analysis.

Total antibody concentrations were determined at 12 time points and percent hemolysis activity was determined at 1 time point over a 59-day lifetime. Total serum antibody concentrations for each anti-C5 antibody are summarized in Table 1. 29. Profiles of mean total antibody concentrations versus time are shown in FIG. 6. Average PK parameters are described in Table. thirty.

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Table 29

Mean serum total IgG concentrations after a single subcutaneous injection of selected anti-C5 antibodies 15 mg/kg in humanized C5 mice

Time (day) Serum AB concentration (µg/mL) mean ± SD H4N12166R H4N12161R An object isotypic 0 0.25 one 2 3 7 ND 31.2 ± 4.2 149.9 ± 16.1 160.8±20 166.2 ± 12.4 158.6±8.5 ND 43.5 ± 16.3 193.8 ± 24.1 221 ± 26.5 210 ± 31.2 162.5 ± 34.8 comparisons 1 ND 59.2 ± 24.1 179.0 ± 9.8 166.6 ± 22.3 159.2 ± 33.2 136.1 ± 38.1 the control ND 61.5 ± 29.4 218.1 ± 17 188.8 ± 25.8 177.9 ± 26.2 184.9 ± 33.9 ten 123.5 ± 28.7 133.2 ± 20.2 107.2 ± 45.7 159.5 ± 28.8 thirteen 93.7 ± 23.6 97.2 ± 24.6 70.6 ± 38 117.2 ± 24.1 21 60.4 ± 14.9 42.4 ± 30.3 29.5 ± 20.6 80.0±17.5 thirty 37.8 ± 10.8 15.3 ± 19.7 4.2 ± 3.5 42.1 ± 6.7 45 20.7±5.2 3.5±5.2 0.4 ± 0.3 16.5 ± 13.9 59 4.1 ± 1.9 0.6 ± 1.0 0.08 ± 0.04 4.6 ± 4.5

Time - time in hours after injection of a single dose;

day - day of research;

SD - standard deviation;

SEM standard error of the mean;

ND - not detected;

NS - no samples.

Table 30

PC parameters

Parameter Units Test antibody (mean ± SD) H4N12166R H4N12161R Comparison object 1 isotypic the control Stakh mcg/ml 178±10 225 ± 22 183±18 221 ± 19 AUC _last day hmkg/ml 3490±590 3040±900 2240±780 4080 ± 480 tl/2 D 11±1 5.8±2 4.2±1 9.9±4

C _max - peak concentration;

AUC - area under the concentration-time curve;

AUClast - AUC calculated from time zero to the time of the last positive concentration;

T _1/2 - observed estimated half-life.

Mean concentration-time profiles show that H4H12166P, H4H12161P, reference 1, and isotype control reached maximum serum concentration ( _Cmax ) between days 1 and 3, with comparable _Cmax values within 1.3-fold (178, 225 , 183 and 221) µg/mL, respectively. H4H12166P and isotype control had similar elimination profiles, with residual drug levels of approximately 4 µg/mL on day 59. H4H12161P showed faster clearance than H4H12166P and isotype control, but was cleared more slowly than comparator 1. On day 59, H4H12161P had an average serum drug level of 0.6 μg/ml, while Comparator 1 had an almost undetectable drug level of 0.08 μg/ml.

The isotype control, H4H12166P and H4H12161P showed comparable exposure values (AUC _last ) within 1.3 times (4080, 3490 and 3040 days xmcg/ml, respectively), while the object of comparison 1 showed a 1.6 times lower exposure (2240 days xmcg/ml, respectively). ml) compared with H4H12166P.

Example 12 LC-MRM-MS based assay for determination of human total C5 concentration.

In this example, human total C5 serum concentrations were determined using liquid chromatography in combination with a multi-reaction mass spectrometric monitoring (LC-MRM-MS) method in a pharmacokinetic/pharmacodynamic study of the anti-C5 antibody H4H12166P.

Serum human total C5 concentrations were determined by measuring the concentration

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The 10-amino acid peptide LQGTLPVEAR (a.o. 1129-1138 in SEQ ID NO: 359) contained in the C5 sequence as a C5 mediator. Theoretically, this method also allows the detection of C5b, a C5 cleavage product. However, because of the instability of free C5b, serum C5b concentrations are generally low, and most C5b is bound to cell surfaces in the form of MAC complexes (Cooper & Muller-Eberhard, 1970, J. Exp. Med. 132:775-93; Hadders et al 2012, Cell Rep. 1:200-7). Therefore, the processed serum samples analyzed here probably contain only insignificant amounts of the C5b product, if at all.

Ways.

For the PK/PD study, mice received a single dose of H4H12166P 15 mg/kg via subcutaneous (s.c.) injection. All mice were bled pre-dose and day 1 post-injection for PK analysis. In addition, 10, 20, 30, 40, 50 and 60 days after injection, mice were sacrificed and terminal blood samples were taken for PK and PD analysis.

Human C5 was used as a reference standard for calibration, and human C5 peptide prepared using a stable isotope labeled C-terminal arginine residue was used as an internal standard (LQGTLPVEAR-13C615N4). The reference standard was used at concentrations ranging from 3.9 to 250 μg/ml (1:2 serial dilutions) in sera from self-generated C5 knockout mice that had the mouse C5 gene deleted (C5 ^-/- ). Serum from C5 ^−/− mice was also used as a negative control (blank). Calibration standards, blanks and test serum samples (10 µl each) were dried and then denatured in 100 µl of 8 M urea/20 mM tris-(2-carboxyethyl)phosphine (TCEP) buffer at 37°C for 1 hour. Then 10 μl of 25 nM internal standard was added to all samples. Samples were alkylated with 10 mM 2-iodoacetamide at room temperature for 30 min and diluted with 50 mM ammonium bicarbonate to a final volume of 500 µl. The samples were then digested with trypsin (1:20 w/w) overnight at 37°C. The C5-derived tryptic peptide LQGTLPVEAR was detected and quantified by LC-MRM-MS using a Waters Xevo TQ-S with an ACQUITY UPLC system. Each treated sample (10 μl) was injected into a pre-equilibrated ACQUITY UPLC BEN C18 column. The flow rate was 0.6 ml/min (mobile phase A: water:formic acid/100:0.1 [v/v] and mobile phase B: acetonitrile:formic acid/100:0.1 [v/v]). about.]). Retention time and peak area were determined using Masslynx Analyst Data (Waters) software. C5 analyte concentrations were calculated from a calibration curve that was generated by plotting the peak area ratio of the C5 reference standard (LQGTLPVEAR- ¹² C6 ¹⁴ N4 unlabeled C5 peptide generated by tryptic digestion of hC5) and the internal standard (C5 peptide labeled with a stable isotope) in depending on the nominal concentration of the C5 reference standard. Concentrations were calculated using linear regression. The lowest concentration of the C5 reference standard (3.9 μg/mL) was within the dynamic range of the assay and was determined as an LLOQ assay.

Results.

Human total C5 serum concentrations were assessed for samples taken from both pre-dose tail blood draws (pre-dose) and terminal blood draws on days 10, 30 and 35 in the respective animals. Total hC5 concentrations post-dosing with H4H12166P were similar (ranging ~1 to 0.9-fold) to pre-dose levels on days 10, 30, and 35 post-dosing. The small differences observed were not statistically significant as assessed by the Mann-Whitney test using GraphPad Prism software. Molar ratio analysis of C5/H4H12166P showed that H4H12166P remained in molar excess relative to C5 until day 35 post dosing (Table 31).

Table 31

Summary of PD characteristics for H4H12166R

days after dose % CP haemolys for mean e ± SD % inhibitors ania C5 (µg/ml) H4H1216 6R (µg/ml ) the average ± SD Molar ratio of terminal C5: H4N12166R Pere D dose terminal Multiples changed AND I 0 77.2 0 n/d n/a n/a ten 5.2 93 34.2 30.0 1, oh 78.8 0.3 thirty 7.4 90 31.9 28.4 0.9 30.4 0.8 35 6.0 92 36, 1 32.7 1.0 29.6 0.9 40 37.1 52 n/d 20.9 n/d fifty All animals in the group are excluded due to MANA credits > 1000 60 73.57 ⁵ n/d G' | n/d

- 68 039858 ^a The percentage of CP inhibition of hemolytic activity was calculated from the average % CP hemolysis on the indicated day after dosing relative to the average % CP hemolysis on day 0.

^b Frequency rate of change - terminal (indicated days after dosing) C5:C5 before the dose.

SD - standard deviation.

MANA is a mouse anti-human antibody. n/d - not determined.

Animals with data affected by MANA were completely excluded from the calculations (2x day 30, 1x day 35, 2x day 40 and all 4 mice on day 50).

Example 13 Mapping of epitopes for binding H4H12166P to C5 by hydrogen-deuterium exchange.

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

HDX-MS experiments were performed on an integrated Waters HDX/MS platform, consisting of a Leaptec HDX PAL system for deuterium labeling, Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, Waters Acquity M-Class (p,Binary solvent manager ) for the analytical column gradient and a Synapt G2-Si mass spectrometer for measuring the masses of peptic peptides.

The labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 µl of C5 (6 pmol/µl) or C5 premixed with antibody at a 1:1 molar ratio was incubated with 56.2 µl of D2O labeling solution for various time points (e.g., non-deuterated control = 0 s, labeled for 1 and 20 min). Deuteration was quenched by transferring 50 µl of the sample to 50 µl of pre-chilled quenching buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0°C for 2 min . The quenched sample was then injected into the Waters HDX Manager for inline pepsin/protease XIII digestion. Cleaved peptides were captured on an ACQUITY UPLC VEN C18 VanGuard 1.7-µm, 2.1 x5 mm guard column at 0°C and eluted onto an analytical column.

ACQUITY UPLC VEN C18 1.7-µm, 1.0x50 mm for 9-minute gradient separation 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 37 V cone voltage, a scan time of 0.5 s, and a mass/charge range of 50-1700 Thomson units (Th).

To identify peptides from human C5, LC-MSE data from a non-deuterated sample were processed and searched in a database containing human C5, pepsin and their randomized sequences via Waters ProteinLynx Global Server (PLGS) software.

The identified peptides were imported into the DynamX software and filtered by two criteria: (1) minimum products per amino acid = 0.3 and (2) replication files threshold = 3. The DynamX software then automatically determined the deuterium uptake for each peptide based on the retention time and high mass accuracy (<10 ppm) for multiple time points with three repetitions for each time.

Using an inline pepsin/protease XIII column coupled to MSE data recording, a total of 189 peptides from human C5 were identified in the absence or presence of antibody, representing a sequence coverage of 62%. In table. 32 illustrates five peptides that had significantly reduced deuteration uptake (centroid delta values >0.9 Da with p values <0.05) when bound to H4H12166P.

Table 32

Deuteration of human C5 peptides upon binding to H4H12166P

Remainder Deuteration 1 min Deuteration 20 min C5 centroid H+ С5+Н4Н12166Р centroid МН+ Δ C5 centroid MN+ С5+Н4Н12166Р centroid МН+ Δ 591- 599 1015.38± 0.09 1014.44± 0.16 -0.93 1015.64± 0.04 1014.60 ± 0.08 -1.04 593- 599 769.41± 0.11 768.33± 0.05 -1.08 769.65± 0.01 768.30± 0.004 -1.35

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775- 787 1693.81 0.11 ± 1692.85 0.07 ± -0.96 1694.06 0.04 ± 1692.96 0.02 ± -1.10 775- 794 2439.62 0.29 ± 2438.42 0.20 ± -1.20 2440.16 0.06 ± 2439.17 0.21 ± -0.99 779- 787 1141.14 0.04 ± 1140.21 0.05 ± -0.93 1141.23 0.03 ± 1140.21 0.02 ± -1.02

The registered mass of the peptide corresponds to the average value of the centroid MH+ mass from three repetitions. These peptides, corresponding to amino acids 591-599 and 775-794, had a lower rate of deuteration when bound to H4H12166P. These identified residues also correspond to residues 591-599 and 775-794 in human C5 as determined by Uniprot entry P01031 (CO5_HUMAN; SEQ ID NO: 359).

Example 14 Effect of anti-C5 antibodies on ocular inflammation in experimental autoimmune uveitis in mice.

This study was conducted to evaluate the role of C5 in experimental autoimmune uveitis (EAU). Both genetic [C5 knockout (KO), dual C3/C5 KO mice] and pharmacological (anti-C5 antibody) experimental approaches were used.

Ways.

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

Results.

Compared to wild-type mice, the occurrence of inflammation (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, dual C3/C5 KO mice have significantly more vitreous cell clusters and higher disease scores at day 28 compared to wild-type mice (p<0.05). In animals that received anti-mC5 Ab (50 mg/kg), the incidence of inflammation and vitreous cell clusters were significantly reduced compared to no treatment or isotype control group by day 21 (p<0.01). At weeks 3 and 4, the OCT scores in the anti-C5 antibody treatment group were significantly lower compared to no treatment or isotype control (p<0.0001), (Fig. 7). week 4 (Fig. 8).

Conclusion.

Eye inflammation due to EAU was alleviated by inhibition of C5 activity, through genetic deletion, or pharmacological inhibition using a specific anti-C5 antibody. Depletion of C5 delayed the onset of EAU and reduced the OST score of the disease. These results indicate that C5 is a potential therapeutic target in autoimmune uveitis. The anti-C5 antibody has a protective effect on EAU disease in wild-type mice. The inventors' findings also suggest that C3 may be beneficial for EAU disease in mice.

Example 15 Effect of anti-human C5 antibodies on experimental autoimmune uveitis.

This example describes the effects of anti-human C5 antibodies in an experimental autoimmune uveitis (EAU) mouse model. The mice used in this study were humanized to express human C5 protein using Velocigene® technology (Valenzuela et al. 2003, Nat. Biotechnol. 21:652-659). Humanized mice were created with the replacement of exons 2-41 of the mouse C5 gene with exons 2-42 of the human C5 gene (disclosed in US Patent Application Publication 2015/0313194, incorporated herein in its entirety).

Ways.

Adult male mice were immunized subcutaneously in each thigh with 150 μg of peptide 1-20 (GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO: 364) human interphotoreceptor retinoid-binding protein (IRBP) (Avichezer et al. 2000, Invest. Ophthalmol. Vis. Sci. 41: 127-131) in 0.2 ml CFA emulsion, supplemented with Mycobacterium tuberculosis H37RA strain up to 2.5 mg/ml. Mice were then intraperitoneally inoculated with 1.0 μg of pertussis toxin (PTX) to help induce cell-mediated autoimmunity by promoting Thi polarization of the immune response (Thurau et al. 1997, Clin. Exp. Immunol. 109:370 -376; Silver et al. 1999, Invest Ophthalmol Vis Sci 40:28982905). The body weight of the animals was monitored twice a week.

Ophthalmic 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 with 0.5% tropicamide ophthalmic solution and the fundus of the eye was examined using a fundus camera contact lens in the Spectralis Heidelberg+OCT retinal angiography (HRA) platform system (Heidelberg Engineering, Carlsbad, CA, USA).

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

Also on OCT images, the severity of vitreous infiltration with inflammatory cells was ranked by assessing the average number of inflammatory cell clusters in the vitreous, in four lateral OCT scans that crossed the optic nerve, for each eye.

A 4-degree scale was developed to assess disease severity on OCT images (OST scores) (Table 33).

Table 33

EAU assessment in vivo using optical coherence tomography (OCT)

Degree Criteria 0 Without changes 0.5 (trace) Small infiltration of inflammatory cells into the vitreous, primarily near the optic nerve head (<15 clusters) one Small infiltration of inflammatory cells in the vitreous, primarily near the optic nerve head (<25 clusters); small focal subretinal lesions (gray spots) on the periphery; small folds of the retina on the periphery; expansion of retinal vessels; perivasculitis and vasculitis 2 Moderate inflammatory cell infiltration in the vitreous is more diffuse, but not in the far periphery (<50 clusters); destruction of the retinal layer; the formation of small or medium-sized granulomas with retinal folds, primarily on the periphery; vasodilation; small focal choroidal neovascularization; perivasculitis and vasculitis; slight retinal edema (<10 µm) 3 Moderate or severe diffuse infiltration

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inflammatory cells in the vitreous body (>50 clusters); diffuse destruction of the retinal layer and dilated vessels in the inner nuclear layer; the formation of medium or large granulomas with retinal folds throughout the retina; severe vasodilation of the retina; perivasculitis and vasculitis; moderate diffuse choroidal neovascularization; moderate or severe retinal edema (10-40 microns); small retinal detachments 4 Severe diffuse infiltration of inflammatory cells into the vitreous (>70 clusters); diffuse destruction of layers and dilated vessels in the inner nuclear layer; diffuse formation of severe granulomas with retinal folds; severe diffuse choroidal neovascularization; perivasculitis and vasculitis; retinal degeneration or severe retinal edema (decrease >20 µm or increase >40 µm, respectively); extensive retinal detachment

Statistical analysis.

Statistical analysis of parametric data (body weight, inflammatory cell clusters in the vitreous body and retinal thickness) was performed by one-way analysis of variance and Tukey's multiple comparison test. For non-parametric data (OCT scores and histological scores), analysis was performed by Kruskal-Wallis test and Dunn test using GraphPad Prism version 5.0d software against isotype controls or no treatment groups. Data represent means ± SEM. p-values less than 0.05 were considered statistically significant.

Results.

In the first study (study A), mice were treated subcutaneously every 3 days from day 5 with isotype control antibody (50 mg/kg) or 10 mg/kg or 50 mg/kg H4H12170P. Treatment with 10 mg/kg H4H12170P led to a reduction in inflammation and retinal damage (FIG. 9). Mice treated with 10 mg/kg H4H12170P also showed a statistically significant decrease in OCT scores by day 21 and day 28 (FIG. 10).

In the second study (study B), mice were treated subcutaneously every 3 days from day 6 using an isotype control (10 mg/kg), 3 mg/kg or 10 mg/kg H4H12166P or using comparator 2 (see Example 2 herein). description; control constructs used in the following examples). Treatment with either H4H12166P at either 3 mg/kg or 10 mg/kg caused a dose-dependent decrease in OCT scores that was statistically significant on days 14-28 (FIG. 11). Treatment with 10 mg/kg H4H12166P in C5 humanized mice from day 6 post-EAU induction resulted in a dose-dependent reduction in inflammation and retinal damage as measured by OCT performed on days 14-28 (FIG. 12).

For both studies, non-invasive ante-mortem evaluation with OCT demonstrated progressive inflammation, increased retinal thickness, and morphological abnormalities in control animals after immunization with IRBP.

Conclusion.

These experiments provide additional pharmacological evidence that C5 plays a role in the pathogenesis of autoimmune uveitis. Pharmacological depletion of human C5 with a fully human anti-human C5 antibody delayed the incidence of EAU and reduced disease severity, confirming the effect of these antibodies in autoimmune uveitis.

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

This study was carried out in order to evaluate the role of C5 in reperfusion injury in renal ischemia. Both genetic (using C3 knockout and C5 knockout mice) and pharmacological approaches (using anti-C5 antibodies) were used. The model of reperfusion during ischemia was created by bilateral clamping of the legs of the renal vessels on

- 72 039858 min, followed by 48 hours of reperfusion. A simulated laparotomy served as a control. Anti-C5 antibody was administered at 50 mg/kg intravenously in a single dose immediately after ischemia (therapeutic) or subcutaneously in two doses, on day -1 and day 1 of surgery (prophylactically). In this study, the anti-C5 antibody M1M17628N containing HCVR/LCVR of SEQ ID NO: 362/363 was used. Markers blood urea nitrogen (BUN) and serum creatinine were used to assess disease and protection levels in mice.

Table 34

Percent change in blood urea nitrogen levels in mice treated with anti-C5 antibody (M1M17628N) in prophylactic and therapeutic regimens

BUN, % change avg. RIRI+carrier RIRI + isotype control Day 2 Day 2 RIRI+M1M17628N (prof.) -37.19 -34.68 RIRI+M1M17628N (medical) - 53.70 -51.85

Table 35

Percent change in serum creatinine levels in mice treated with anti-C5 antibody (M1M17628N) in prophylactic and therapeutic regimens

SCr,% change avg. RIRI+carrier RIRI + isotype control Day 2 Day 2 RIRI+M1M17628N (prof.) -53.09 -49.34 RIRI+M1M17628N (medical) -59.40 -56.16

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 levels of blood urea nitrogen and serum creatinine. The anti-C5 antibody showed functional protection in the RIRI model in both prophylactic and therapeutic regimens (Tables 34, 35).

Example 17 Effect of anti-C5 antibodies on lupus nephritis.

This example describes the effect of anti-C5 antibodies in the treatment of lupus nephritis in a mouse model.

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

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

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

Example 18 Effect of Anti-C5 Antibodies on Astrocyte Cell Death.

Optocomyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS) that predominantly affects the optic nerve and spinal cord. In NMO, anti-aquaporin-4 autoantibodies (AQP4-Ab) cause damage to astrocytes through activation of complement-dependent cytotoxicity (CDC). The objectives of this study were to evaluate the role of the complement system in the progression of NMO and the use of anti-complement antibody as a potential therapeutic treatment for NMO.

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Rat primary cortical astrocytes were obtained from the cerebral cortex of postnatal rat pups and cultured with AQP4-Ab (antibody rAb-53 from US Patent Application Publication 2014/0170140; Bennett et al 2009, Ann. Neurol. 66: 617-629) and complement proteins in order to demonstrate cell-mediated cytotoxicity. Then the experiments were repeated with the addition of antibodies against C5 in order to demonstrate the blocking of the destruction of astrocyte cells.

In order to quantify cell death, a CytoTox-Glo™ fluorescent cytotoxic assay was performed. Various concentrations of anti-C5 antibody (0.001, 0.01, 0.1, 1, 10, 100 or 1000 μg/ml) or an isotype control antibody were used in the assay.

In order to determine if anti-C5 antibody could block CDC induced by AQP4-Ab, astrocytes were seeded and the CytoTox-Glo™ cytotoxic assay was repeated to find the optimal dose of AQP4-Ab for this seeding. The optimal concentration of AQP4-Ab was found to be 50 μg/ml, and in the following experiment, a constant dose of AQP4-Ab (50 μg/ml) was used, while the dose of anti-C5 antibody was varied. As shown in FIG. 16, a decrease in RLU was observed (average 300k to average 100k) with increasing anti-C5 antibody, demonstrating that anti-C5 blocked astrocyte cell death. For both experiments, RLU did not change depending on the isotype control antibody. As shown in FIG. 16, anti-C5 antibodies inhibited AQP4-Ab induced cytotoxicity on primary cortical astrocytes with an IC _{50 of} 15-17 nM.

In a future study, anti-AQP4 antibody and anti-C5 antibody should be injected into the rat brain in order to assess the therapeutic effect on complement-mediated cytotoxicity against astrocytes in the CNS.

Example 19 Endothelial Analysis.

This example describes an in vitro glomerular endothelial assay to investigate blocking of C5b-9 and C3 deposition by an anti-C5 antibody.

Reproducible methods for evaluating the inhibitory effects of drug candidates on complement activation are important for preclinical testing. Due to the complexity of complement activation pathways, the assay should use relevant cells and endpoints for a given therapeutic indication. Here, using an immortal human glomerular endothelial cell line (HGEC), a C3 and C5 complement deposition model was validated for use in assessing the blocking activity of an anti-C3 or C5 mAb.

Ways.

Primary human renal glomerular endothelial cells (HGEC; Cell Biologies) were seeded overnight in complete medium in collagen I-coated black 96-well clean-bottomed plates. Cells were treated with either PBS (control) or activated for 10 min with 10 μM ADP. After washing in PBS, 50% human serum (complement conserved, C3-depleted, or C5-depleted) was added for 4 hours. Anti-C5 antibodies were added at 1 mg/ml to the serum before treatment. Cells were washed, fixed and examined with anti-C3b antibodies (Thermofisher) and/or anti-C5b-9 antibodies (Abcam), secondary antibodies and counterstained with DAPI. Images were captured on ImagExpress and multiparametric image analysis was used to quantify fluorescent staining for each image and averaged for each condition.

Results.

Deposition of C3 and C5b-9 was observed in ADP-activated HGEC exposed to normal human serum, but not in non-activated HGEC (C3: 1.5χ10 ⁷ ±1.0χ10 ⁷ ; C5: 7.9χ10 ⁶ ±6.6χ10 ⁶ , P<0.05 vs. non-ADP-activated HGECs). C3 and C5b-9 deposits were significantly reduced in ADP-activated HGEC exposed to C3 or C5 depleted serum (C3: 3.3χ105±4.8χ10 ⁴ ; C5: 1.5χ10 ⁶ ±6.0χ10 ⁵ , P<0 .05). The addition of a blocking anti-C5 mAb significantly reduced deposition of C5b-9 from normal human serum on ADP-activated HGECs, deposition comparable to C5-depleted sera (C5 mAb: 1.02χ10 ⁶ ±6.0χ10 ⁵ , control mAb 3.7χ10 ⁶ ±1 .6χ10 ⁶ , P<0.05 vs control mAb).

Conclusion.

These data demonstrate the utility of in vitro human glomerular endothelial analysis for modeling complement C3 and C5 deposition. In addition to in vitro screening, this assay has the potential as a translational model for evaluating anti-complement strategies in kidney disease using patient-derived sera.

The scope of the present invention is not to be limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the above description and accompanying figures. Such modifications are intended to be within the scope of the appended claims.

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CLAIM

Claims (22)

CLAIM 1. An antibody or antigen-binding fragment thereof that specifically binds to complement factor 5 (C5) protein, wherein the antibody or antigen-binding fragment thereof contains: HCDR1, which is SEQ ID NO: 100; contains amino acid subsequence, submitted in HCDR2, which is SEQ ID NO: 102; contains amino acid subsequence, submitted in HCDR3, which is SEQ ID NO: 104; contains amino acid subsequence, submitted in LCDR1, which is SEQ ID NO: 108; contains amino acid subsequence, submitted in LCDR2, which is SEQ ID NO: 110; and contains amino acid subsequence, submitted in LCDR3, which is SEQ ID NO: 112. contains amino acid subsequence, submitted in 2. An antibody or antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 98 and/or a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 98. SEQ ID NO: 106. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NO: 98/106. 4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 353. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, containing a heavy chain and a light chain, where the light chain contains the amino acid sequence of SEQ ID NO: 354. 6. An antibody or antigen-binding fragment according to any one of claims 1 to 5, comprising the heavy chain/light chain amino acid sequence pair of SEQ ID NO: 353/354. 7. An antibody or antigen-binding fragment according to any one of claims 1 to 6, which is a complete antibody molecule. 8. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to C5 according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or diluent. 9. A pen-type delivery device containing the pharmaceutical composition of claim 8. 10. Autoinjection device containing the pharmaceutical composition according to claim 8. 11. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes the heavy chain variable region (HCVR) of an antibody or antigen-binding fragment thereof according to any one of claims 1-7. 12. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof according to any one of claims 1-7. 13. An expression vector that contains the polynucleotide molecule according to paragraphs. 11 and 12. 14. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, expressing the vector according to claim 13. 15. A method of preventing or treating at least one symptom or indication of a disease or disorder associated with C5, wherein the method includes administering a pharmaceutical composition containing a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-7, the subject in need. 16. The method of claim 15, wherein the disease or disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, geographic atrophy, uveitis, optomyelitis, multiple sclerosis, stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, inappropriate or unwanted complement activation disorders, complications of hemodialysis, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammatory disorders, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal injury, including burns or frostbite, postischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, you culitis, bullous pemphigoid, C3 glomerulopathy, membranoproliferative glomerulonephritis, diabetic nephropathy, Alport syndrome, progressive renal failure, proteinuric kidney disease, - 75 039858 reperfusion injury in renal ischemia, lupus nephritis, balloon angioplasty, post-hemodialysis syndrome in cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease, sepsis, immune complex disorders, autoimmune disease, renal impairment, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism, pulmonary infarction, pneumonia, and myasthenia gravis. 17. The method of claim 15 wherein the disease or disorder is an atypical hemolytic uremic syndrome. 18. The method of claim 15 wherein the disease or disorder is paroxysmal nocturnal hemoglobinuria. 19. The method according to any one of claims 15-18, wherein the pharmaceutical composition is administered prophylactically to a subject in need thereof. 20. The method according to any one of claims 15 to 19, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent. 21. The method of claim 20 wherein the second therapeutic agent is selected from the group consisting of an anticoagulant, an anti-inflammatory drug, an antihypertensive agent, an immunosuppressive agent, a lipid lowering agent, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab , an anticonvulsant, a C3 inhibitor, a second anti-C5 antibody, and an antithrombotic agent. 22. The method according to any one of claims 15-21, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or inside the skull.