Anti-C5 antibodies with improved pharmacokinetics

Abstract

This disclosure provides antibodies useful for inhibiting terminal complement (e.g., the assembly and / or activity of C5b-9 TCCs) and C5a anaphylatoxin-mediated inflammation, and thus for treating complement-related disorders. [Solution] The antibody has several improved properties compared to eculizumab, including, for example, an increased serum half-life in humans. This disclosure also shows that the novel antibody described herein has reduced sensitivity to target-mediated clearance and thus has a longer serum efflux half-life (half-life) in the blood compared to known anti-C5 antibodies.

Description

[Technical Field]

[0001] Related applications This application claims priority and interest to U.S. Provisional Patent Application No. 61 / 949,932 (filed March 7, 2014), the disclosure of which is incorporated herein by reference in its entirety.

[0002] The fields of this invention are medicine, immunology, molecular biology, and protein chemistry. [Background technology]

[0003] The complement system works in conjunction with the body's other immune systems to defend against the invasion of cells and viral pathogens. There are at least 25 complement proteins, found as a collection of complexes of plasma proteins and membrane cofactors. Plasma proteins constitute approximately 10% of the globulins in vertebrate serum. Complement components achieve their immunodefense functions by interacting in a series of complex but precise enzymatic cleavage and membrane-binding events. The resulting complement cascade leads to the production of products with opsonin, immunomodulatory, and lytic functions. A brief overview of the biological activities associated with complement activity can be found, for example, in The Merck Manual, 16. th This is provided in the Edition.

[0004] The complement cascade can proceed via the classical pathway (CP), the lectin pathway, or the alternative pathway (AP). The lectin pathway is typically initiated by the binding of mannose-binding lectins (MBLs) to high-mannose substrates. The AP can be antibody-independent and can be initiated by specific molecules on the pathogen surface. The CP is typically initiated by antibody recognition of an antigen site on a target cell and binding to that site. These pathways converge at the C3 convertase, the point where complement component C3 is cleaved by an active protease to produce C3a and C3b.

[0005] AP C3 convertase is initiated by the spontaneous hydrolysis of complement component C3, which is abundant in the plasma fraction of blood. This process, also known as "tick-over," occurs through the spontaneous cleavage of thioester bonds in C3, forming C3i or C3(H2O). Tick-over is facilitated by the presence of a surface (e.g., bacterial cell surface) that supports and / or has neutral or positively charged properties for the binding of activated C3. The formation of this C3(H2O) allows for the binding of the plasma protein factor B, which in turn allows factor D to cleave factor B into Ba and Bb. The Bb fragment remains bound to C3, forming a complex containing C3(H2O)Bb, i.e., the "fluid phase" or "initiating" C3 convertase. Although produced in small amounts, the fluid phase C3 convertase can cleave multiple C3 proteins into C3a and C3b, resulting in the formation of C3b and subsequent covalent bonding of C3b to a surface (e.g., bacterial surface). Factor B bound to surface-bound C3b is cleaved by factor D, thereby forming a surface-bound AP C3 convertase complex containing C3b and Bb. (See, for example, Muller-Eberhard (1988) Ann Rev Biochem 57:321-347.)

[0006] AP C5 convertase, i.e., (C3b)2,Bb, is the second C3b monomer, AP It is formed upon addition to the C3 convertase. (See, for example, Medicus et al. (1976) J Exp Med 144:1076-1093 and Fearon et al. (1975) J Exp Med 142:856-863.) The role of the second C3b molecule is to bind to C5 and present it for cleavage by Bb. (See, for example, Isenman et al. (1980) J Immunol 124:326-331.) AP C3 and C5 convertases are stabilized by the addition of the trimer protein propagin, as described, for example, in Medicus et al. (1976) above. However, propagination is not required to form a functional alternative pathway C3 or C5 convertase. (See, for example, Schreiber et al. (1978) Proc Natl Acad Sci USA 75:3948-3952 and Sissons et al. (1980) Proc Natl Acad Sci USA 77:559-562).

[0007] CP C3 convertase is formed when complement component C1, a complex of C1q, C1r, and C1s, interacts with an antibody bound to a target antigen (e.g., a microbial antigen). Binding of the C1q portion of C1 to the antibody-antigen complex triggers a conformational change in C1 that activates Clr. The activated C1r then cleaves the C1-associated C1s, thereby producing an activated serine protease. The activated C1s cleaves complement component C4 into C4b and C4a. Similar to C3b, the newly generated C4b fragment contains a highly reactive thiol that readily forms amide or ester bonds with suitable molecules on the target surface (e.g., a microbial cell surface). C1s also cleaves complement component C2 into C2b and C2a. The complex formed by C4b and C2a is the CP C3 convertase, which can process C3 into C3a and C3b. CP C5 convertases, namely C4b, C2a, and C3b, are formed when the C3b monomer is added to CP C3 convertase. (e.g., Muller-Eb) See Erhard (1988), the above, and Cooper et al. (1970) J Exp Med 132:775-793.

[0008] In addition to its role in C3 and C5 convertases, C3b also functions as an opsonin through its interaction with complement receptors present on the surface of antigen-presenting cells such as macrophages and dendritic cells. The opsonin function of C3b is generally considered one of the most important anti-infective functions of the complement system. Patients with genetic lesions that block C3b function are susceptible to a wide range of pathogens, while patients with lesions later in the complement cascade sequence, i.e., lesions that block C5 function, are susceptible only to Neisseria infections and are only somewhat more susceptible.

[0009] AP and CP C5 convertases cleave C5 into C5a and C5b. Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotactic, and C5b, which enables the formation of the lysation terminal complement complex C5b-9. C5b combines with C6, C7, and C8 to form the C5b-8 complex on the surface of the target cell. Upon binding of several C9 molecules, a membrane attack complex (MAC, C5b-9, terminal complement complex, i.e., TCC) is formed. When a sufficient number of MACs are inserted into the target cell membrane, the openings they create (MAC pores) mediate the rapid osmotic lysis of the target cell.

[0010] A properly functioning complement system provides robust defense against infectious microorganisms, but improper modulation or activation of the complement pathway has been linked to the development of various disorders, including, for example, rheumatoid arthritis (RA), lupus nephritis, asthma, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD), paroxysmal nocturnal hemoglobinuria (PNH), macular degeneration (e.g., age-related macular degeneration (AMD)), hemolysis, hyperhepatic enzyme and hypothrombocytopenic (HELLP) syndrome, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, minor immune vasculitis, epidermolysis bullosa, recurrent miscarriage, multiple sclerosis (MS), traumatic brain injury, and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis. (e.g., Holers et al.) See al. (2008) Immunological Reviews 223:300-316. Downregulation of complement activation has been demonstrated to be effective in treating several disease indications in various animal models. For example, Rother et al. (2007) Nature Biotechnology 25(11):1256-1264, Wang et al. (1996) Proc Natl Acad Sci USA 93:8563-8568, Wang et al. (1995) Proc Natl Acad Sci USA 92:8955-8959, Rinder et al. (1995) J Clin Invest 96:1564-1572, Kroshus et al. (1995) Transplantation 60:1194-1202, Homeister et al. (1993) J Immunol 150:1055-1064, Weisman et al. (1990) Science 249:146-151, Amsterdam et al. (1995) Am J See Physiol 268:H448-H457 and Rabinovici et al. (1992) J Immunol 149:1744-1750. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Muller-Eberhard (1988) Ann Rev Biochem 57:321-347 [Overview of the project] [Means for solving the problem]

[0012] This disclosure relates to an anti-C5 antibody having one of the improved properties compared to, for example, known anti-C5 antibodies used for therapeutic purposes. For example, the anti-C5 antibodies described herein exhibit an increased serum lifespan compared to the serum efflux half-life of eculizumab. Due to their improved pharmacokinetic properties, the antibodies described herein feature many advantages, such as advantages over other anti-C5 antibodies that bind to full-length or mature C5 and inhibit its cleavage. Similar to such anti-C5 antibodies, the antibodies described herein can inhibit C5a-mediated inflammatory responses and C5b (MAC)-dependent cytolysis resulting from C5 cleavage. However, since the concentration of C5 in human plasma is approximately 0.37 μM (Rawal and Pangburn (2001) J Immunol 166(4):2635-2642), high concentrations and / or frequent administration of anti-C5 antibodies such as eculizumab are often necessary to effectively inhibit C5 in humans. This disclosure describes experimental data demonstrating, in examples, that anti-C5 antibodies are highly effective in inhibiting complement in vitro and in vivo (see, for example, Hillmen et al. (2004) N Engl J Med 350(6):552), but that the antibodies are particularly sensitive to target-mediated clearance due to high concentrations of C5 in the blood. This disclosure also shows that the novel antibodies described herein have reduced sensitivity to target-mediated clearance and, consequently, have a longer serum efflux half-life (half-life) in the blood compared to known anti-C5 antibodies.

[0013] Considering their long half-lives, the antibodies described herein can be administered to humans at much lower doses and / or less frequently than known anti-C5 antibodies (e.g., eculizumab), while still effectively achieving the same or higher C5 inhibition in humans. For example, the ability to administer lower doses of antibodies compared to eculizumab also allows for additional delivery routes, such as subcutaneous, intramuscular, intrapulmonary delivery, and administration via the use of biodegradable microbodies.

[0014] Accordingly, in one embodiment, the present disclosure features an anti-C5 antibody having one or more improved properties (e.g., improved pharmacokinetic properties) compared to eculizumab. The antibody or its C5 binding fragment (a) binds to complement component C5, (b) inhibits the cleavage of C5 into fragments C5a and C5b, and (c) comprises a heavy chain CDR1 containing the amino acid sequence shown in (i) SEQ ID NO: 1, (ii) a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, (iii) a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, (iv) a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, (v) a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (vi) a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight) amino acids (plural) of (i) to (vi) are substituted with different amino acids. In some embodiments, C5 is human C5.

[0015] In some embodiments of the antibodies or fragments described herein, at least one amino acid of the heavy chain CDR1 is substituted with a different amino acid. In some embodiments of the antibodies or fragments described herein, at least one amino acid of the heavy chain CDR2 is substituted with a different amino acid. In some embodiments of the antibodies or fragments described herein, at least one amino acid of the heavy chain CDR3 is substituted with a different amino acid.

[0016] In some embodiments of any of the antibodies or fragments described herein, at least one amino acid of the light chain CDR1 is substituted with a different amino acid. In some embodiments of any of the antibodies or fragments described herein, the glycine at position 8 of SEQ ID NO: 4 is substituted with a different amino acid (e.g., histidine).

[0017] In some embodiments of the antibodies or fragments described herein, at least one amino acid of the light chain CDR2 is substituted with a different amino acid. In some embodiments of the antibodies or fragments described herein, at least one amino acid of the light chain CDR3 is substituted with a different amino acid.

[0018] In some embodiments of any antibody or fragment described herein, the substitution is glycine at position 1 for SEQ ID NO: 1, tyrosine at position 2 for SEQ ID NO: 1, isoleucine at position 3 for SEQ ID NO: 1, phenylalanine at position 4 for SEQ ID NO: 1, serine at position 5 for SEQ ID NO: 1, asparagine at position 6 for SEQ ID NO: 1, tyrosine at position 7 for SEQ ID NO: 1, tryptophan at position 8 for SEQ ID NO: 1, isoleucine at position 9 for SEQ ID NO: 1, glutamine at position 10 for SEQ ID NO: 1, glutamic acid at position 1 for SEQ ID NO: 2, isoleucine at position 2 for SEQ ID NO: 2, leucine at position 3 for SEQ ID NO: 2, proline at position 4 for SEQ ID NO: 2, glycine at position 5 for SEQ ID NO: 2, serine at position 6 for SEQ ID NO: 2, glycine at position 7 for SEQ ID NO: 2, serine at position 8 for SEQ ID NO: 2, threonine at position 9 for SEQ ID NO: 2, glutamic acid at position 10 for SEQ ID NO: 2, The process is performed at an amino acid position selected from the group consisting of tyrosine at position 11 for sequence number 2, threonine at position 12 for sequence number 2, glutamic acid at position 13 for sequence number 2, asparagine at position 14 for sequence number 2, phenylalanine at position 15 for sequence number 2, lysine at position 16 for sequence number 2, aspartic acid at position 17 for sequence number 2, tyrosine at position 1 for sequence number 3, phenylalanine at position 2 for sequence number 3, phenylalanine at position 3 for sequence number 3, glycine at position 4 for sequence number 3, serine at position 5 for sequence number 3, serine at position 6 for sequence number 3, proline at position 7 for sequence number 3, asparagine at position 8 for sequence number 3, tryptophan at position 9 for sequence number 3, tyrosine at position 10 for sequence number 3, phenylalanine at position 11 for sequence number 3, aspartic acid at position 12 for sequence number 3, and valine at position 13 for sequence number 3.

[0019] In some embodiments of any of the antibodies or fragments described herein, the substitution is made at an amino acid position selected from the group consisting of glycine at position 8 for SEQ ID NO: 4, leucine at position 10 for SEQ ID NO: 4, valine at position 3 for SEQ ID NO: 6, and threonine at position 6 for SEQ ID NO: 6.

[0020] In some embodiments of any of the antibodies or fragments described herein, the substitution is made at an amino acid position selected from the group consisting of tyrosine at position 2 for SEQ ID NO: 1, isoleucine at position 9 for SEQ ID NO: 1, leucine at position 3 for SEQ ID NO: 2, and serine at position 8 for SEQ ID NO: 2.

[0021] In some embodiments of any of the antibodies or fragments described herein, both the tyrosine at position 2 in SEQ ID NO: 1 and the leucine at position 3 in SEQ ID NO: 2 are substituted with a different amino acid. In some embodiments of any of the antibodies or fragments described herein, the different amino acid is histidine.

[0022] In some embodiments of the antibodies or fragments described herein, both the isoleucine at position 9 in SEQ ID NO: 1 and the serine at position 8 in SEQ ID NO: 2 are substituted with a different amino acid. In some embodiments of the antibodies or fragments described herein, both the isoleucine at position 9 in SEQ ID NO: 1 and the leucine at position 3 in SEQ ID NO: 2 are substituted with a different amino acid. In some embodiments of the antibodies or fragments described herein, the different amino acid is histidine.

[0023] In some embodiments of the antibodies or fragments described herein, both the tyrosine at position 2 in SEQ ID NO: 1 and the serine at position 8 in SEQ ID NO: 2 are substituted with different amino acids. In some embodiments of the antibodies or fragments described herein, the antibody or antigen-binding fragment comprises a combination of amino acid substitutions selected from Table 1. In some embodiments of the antibodies or fragments described herein, the different amino acid is histidine.

[0024] In some embodiments of any of the antibodies or fragments described herein, the amino acid substitution combinations include (ii) substitution of a different amino acid for glycine at position 8 in SEQ ID NO: 4 in the light chain polypeptide of the antibody or its antigen-binding fragment, (ii) substitution of a different amino acid for glycine at position 2 in SEQ ID NO: 1 in the heavy chain polypeptide of the antibody or its antigen-binding fragment, and (iii) substitution of a different amino acid for serine at position 8 in SEQ ID NO: 2 in the heavy chain polypeptide of the antibody or its antigen-binding fragment. In some embodiments of any of the antibodies or fragments described herein, the different amino acid is histidine.

[0025] In some embodiments of any of the antibodies or fragments described herein, the tyrosine at position 2 in SEQ ID NO: 1 and the serine at position 8 in SEQ ID NO: 2 are substituted with histidine. In some embodiments of any of the antibodies or fragments described herein, the different amino acid is histidine.

[0026] In some embodiments, any of the antibodies or fragments described herein have a concentration of 0.1 nM ≤ K at pH 7.4 and 25°C. D Affinity dissociation constant (K) is within the range of ≤1nM. D ) binds to C5. In some embodiments, any of the antibodies or fragments described herein have a concentration of 0.2 nM ≤ K at pH 7.4 and 25°C. D K is within the range of ≤1nM D It binds to C5. In some embodiments, any of the antibodies or fragments described herein have a concentration of 0.5 nM ≤ K at pH 7.4 and 25°C. D K is within the range of ≤1nM D This is used to join C5.

[0027] In some embodiments, any of the antibodies or fragments described herein is K at pH 6.0 and 25°C with a K content of ≥1 nM (e.g., ≥50 nM, ≥100 nM, or ≥1 μM). D This is used to join C5.

[0028] In some embodiments of any of the antibodies or fragments described herein, [(K of the antibody or its antigen-binding fragment to C5 at pH 6.0 and 25°C D ) / (K of the antibody or its antigen-binding fragment to C5 at pH 7.4 and 25°C D )] exceeds 25. In some embodiments of any of the antibodies or fragments described herein, [(K of the antibody or its antigen-binding fragment to C5 at pH 6.0 and 25°C D ) / (K of the antibody or its antigen-binding fragment to C5 at pH 7.4 and 25°C D )] exceeds 100 (e.g., exceeds 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, or 8500).

[0029] In some embodiments of any of the antibodies or fragments described herein, K of the antibody or its antigen-binding fragment to C5 at pH 7.4 and 25°C D is less than 1 nM (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM).

[0030] Based on the characteristics of some mutant eculizumab molecules described in the examples, the inventors discovered a new genus of antibodies with improved pharmacokinetic properties compared to eculizumab. Antibodies within this genus have an affinity for C5 that is weaker than the affinity of eculizumab for C5 at pH 7.4. Nevertheless, these antibodies have an affinity dissociation constant (K for C5 at pH 7.4 that is 1 nM or less D) has. This disclosure is not bound by any particular theory or mechanism of action, but the inventors believe that the slight reduction in the affinity of eculizumab to C5 at pH 7.4 and the subsequent effect on the affinity of the antibody to C5 at pH 6.0 substantially reduces the C5-mediated clearance of the antibody without substantially affecting the resulting complement inhibitory function of the antibody in the patient, while maintaining the same / similar affinity ratios at pH 7.4 and pH 6.0. Therefore, the inventors have defined an optimal affinity range for an anti-C5 antibody that yields improved pharmacokinetic properties while retaining the desired pharmacokinetic properties compared to eculizumab, respectively. Thus, in another embodiment, this disclosure provides (a) an affinity dissociation constant (K) ≤ 1 nM at pH 7.4 and 25°C. D (b) binds to complement component C5 with (b) a K of 10 nM or higher at pH 6.0 and 25°C. D The isolated antibody or its antigen-binding fragment is characterized by binding to C5 with (c)C5 and inhibiting the cleavage of C5 into fragments C5a and C5b, [(K of the antibody or its antigen-binding fragment against C5 at pH 6.0 and 25°C] D ) / (K of antibody or antigen-binding fragment against C5 at pH 7.4 and 25°C) D )] is 25 or more.

[0031] In some embodiments, the antibody or its antigen-binding fragment has a concentration of 0.1 nM ≤ K at pH 7.4 and 25°C. D Affinity dissociation constant (K) within the range of ≤1nM D ) binds to C5. In some embodiments, the antibody or its antigen-binding fragment is 0.2 nM ≤ K at pH 7.4 and 25°C. D K within the range of ≤1nM D It binds to C5. In some embodiments, the antibody or its antigen-binding fragment has a concentration of 0.5 nM ≤ K at pH 7.4 and 25°C. D K within the range of ≤1nM D It binds to C5. In some embodiments, the antibody or its antigen-binding fragment is ≥1 nM K at pH 6.0 and 25°C. DIt binds to C5. In some embodiments, the antibody or its antigen-binding fragment is ≥50 nM K at pH 6.0 and 25°C. D It binds to C5. In some embodiments, the antibody or its antigen-binding fragment is ≥100 nM K at pH 6.0 and 25°C. D It binds to C5. In some embodiments, the antibody or its antigen-binding fragment is subjected to ≥1 μM K at pH 6.0 and 25°C. D This is used to join C5.

[0032] In some embodiments, [(K of an antibody or antigen-binding fragment against C5 at pH 6.0 and 25°C] D ) / (K of antibody or antigen-binding fragment against C5 at pH 7.4 and 25°C) D )] refers to numbers greater than 50 (for example, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, or greater than 8500).

[0033] In some embodiments, the antibody or its antigen-binding fragment is prepared at pH 7.4 and 25°C, K D It binds to C5 with a <1nM concentration. In some embodiments, the antibody or its antigen-binding fragment is prepared at pH 7.4 and 25°C, K D It binds to C5 with a concentration of <0.8 nM. In some embodiments, the antibody or its antigen-binding fragment is prepared at pH 7.4 and 25°C, K D It binds to C5 with a concentration of <0.5 nM. In some embodiments, the antibody or its antigen-binding fragment is prepared at pH 7.4 and 25°C, K D It binds to C5 with a molecular weight of <0.2nM.

[0034] In some embodiments, the antibody or its antigen-binding fragment comprises (ii) a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, (iii) a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, (iv) a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, (v) a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (vi) a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, wherein at least one amino acid from (i) to (vi) is substituted with a different amino acid. The different amino acid may be any amino acid (e.g., histidine). In some embodiments, at least one amino acid in the heavy chain CDR1 is substituted with a different amino acid. In some embodiments, at least one amino acid in the heavy chain CDR2 is substituted with a different amino acid. In some embodiments, at least one amino acid in the heavy chain CDR3 is substituted with a different amino acid. In some embodiments, at least one amino acid in the light chain CDR1 is substituted with a different amino acid. In some embodiments, at least one amino acid in the light chain CDR2 is substituted with a different amino acid. In some embodiments, at least one amino acid in the light chain CDR3 is substituted with a different amino acid.

[0035] In some embodiments, the substitution is made at an amino acid position selected from the group consisting of glycine at position 8 for SEQ ID NO: 4, leucine at position 10 for SEQ ID NO: 4, valine at position 3 for SEQ ID NO: 6, and threonine at position 6 for SEQ ID NO: 6. In some embodiments, the substitution is made at an amino acid position selected from the group consisting of tyrosine at position 2 for SEQ ID NO: 1, isoleucine at position 9 for SEQ ID NO: 1, leucine at position 3 for SEQ ID NO: 2, and serine at position 8 for SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment contains a combination of amino acid substitutions selected from Table 1.

[0036] In some embodiments, the combination of amino acid substitutions introduced into the CDR includes (i) substitution of a different amino acid for glycine at position 8 in SEQ ID NO: 4 in the light chain polypeptide of the antibody or its antigen-binding fragment, (ii) substitution of a different amino acid for glycine at position 2 in SEQ ID NO: 1 in the heavy chain polypeptide of the antibody or its antigen-binding fragment, and (iii) substitution of a different amino acid for serine at position 8 in SEQ ID NO: 2 in the heavy chain polypeptide of the antibody or its antigen-binding fragment.

[0037] In some embodiments, the amino acid substitution combinations include (i) substitution of a different amino acid for glycine at position 2 in SEQ ID NO: 1 of the heavy chain polypeptide of the antibody or its antigen-binding fragment, and (ii) substitution of a different amino acid for serine at position 8 in SEQ ID NO: 2 of the heavy chain polypeptide of the antibody or its antigen-binding fragment.

[0038] In some embodiments, the tyrosine at position 2 of SEQ ID NO: 1 and the serine at position 8 of SEQ ID NO: 2 are substituted (for example, with histidine).

[0039] In some embodiments, one of the antibodies or fragments thereof binds to the mutant human Fc constant region (e.g., mutant human IgG) with higher affinity than the affinity of the native human Fc constant region in which the mutant human Fc constant region was induced. The mutant Fc constant region includes the Fc constant region. The mutant Fc constant region may contain one or more (e.g., two, three, four, or five or more) amino acid substitutions compared to the native human Fc constant region from which the mutant human Fc constant region was induced. This substitution may occur, for example, at amino acid positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) relative to the natural human Fc constant region. These substitutions are all EU numbered as follows: methionine for glycine at position 237, alanine for proline at position 238, lysine for serine at position 239, isoleucine for lysine at position 248, alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250, phenylalanine, tryptophan, or tyrosine for methionine at position 252, threonine for serine at position 254, glutamic acid for arginine at position 255, aspartic acid, glutamic acid, or glutamine for threonine at position 256, alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine for proline at position 257, 25 Histidine for glutamic acid at position 8, alanine for aspartic acid at position 265, phenylalanine for aspartic acid at position 270, alanine or glutamic acid for asparagine at position 286, histidine for threonine at position 289, alanine for asparagine at position 297, glycine for serine at position 298, alanine for valine at position 303, alanine for valine at position 305, alanine for threonine at position 307, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine, alanine for valine at position 308, phenylalanine, isoleucine, leucine,Methionine, proline, glutamine, or threonine; alanine for leucine or valine at position 309; aspartic acid, glutamic acid, proline, or arginine; alanine for glutamine at position 311; histidine, or isoleucine; alanine or histidine for aspartic acid at position 312; lysine or arginine for leucine at position 314; alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine for asparagine at position 325; valine for isoleucine at position 332; leucine for lysine at position 334; histidine for lysine at position 360; alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; asparagine at position 384 Alternatively, it may be selected from the group consisting of alanine relative to serine, aspartic acid or histidine relative to glycine at position 385, proline relative to glutamine at position 386, glutamic acid relative to proline at position 387, alanine or serine relative to asparagine at position 389, alanine relative to serine at position 424, alanine relative to methionine at position 428, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine, lysine relative to histidine at position 433, alanine relative to asparagine at position 434, phenylalanine, histidine, serine, tryptophan, or tyrosine, and histidine relative to tyrosine or phenylalanine at position 436.

[0040] In some embodiments of any of the antibody or antigen-binding fragments described herein, the mutant human Fc constant region comprises, respectively, methionine at position 428 and asparagine at position 434, with EU numbering.

[0041] In some embodiments, the antibody or any of its antigen-binding fragments may include, or consist of, a heavy chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12 or 14, and a light chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 or 11.

[0042] This disclosure also features antibodies containing the heavy chain variable region of eculizumab (SEQ ID NO: 7) or the CDR of the heavy chain region of eculizumab (SEQ ID NOs: 1-3), which include any of the mutant human Fc constant regions described herein, for example, each mutant human Fc constant region, with EU numbering, containing methionine at position 428 and asparagine at position 434.

[0043] In one embodiment, an antibody or antigen-binding fragment has an increased half-life in humans compared to the serum half-life of eculizumab. As used herein, half-life is defined as the time it takes for the plasma concentration of an antibody drug in the body to be reduced by half or 50%. This 50% reduction in serum concentration reflects the amount of drug circulating that is not removed by natural antibody clearance. The half-life of eculizumab has been determined to be 272+82 hours or 11.3 days in PNH patients and 12.1 days in aHUS patients (see Soliris prescribing information). The human half-lives of the antibodies or fragments described herein may be increased compared to the human half-life of eculizumab. The increase in half-life compared to eculizumab may be at least 1.5 times, at least 2 times, at least 2.5 times, or at least 3 times.

[0044] In some embodiments of any of the antibodies or fragments described herein, the antibody has a serum half-life of at least 10 days in humans (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days or more). This half-life (or extension of half-life compared to eculizumab) can be achieved in some embodiments by antibodies described herein containing a naturally occurring human Fc constant region. In some embodiments, this half-life is measured compared to antibodies containing a mutant human Fc constant region described herein. The human half-life of the antibodies or fragments described herein may be increased compared to the human half-life of eculizumab. The half-lives in humans of the antibodies described herein are at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, or at least 35 days.

[0045] In some embodiments, any of the antibodies or fragments described herein are humanized, fully human, deimmunized, or chimeric. In some embodiments, the antibodies or fragments described herein may be, for example, recombinant antibodies, single-chain antibodies, diabodies, intracellular antibodies, Fv fragments, Fd fragments, Fab fragments, Fab' fragments, and F(ab')2 fragments.

[0046] In some embodiments, any of the antibodies or fragments thereof described herein may contain heterologous portions, such as sugars. For example, the antibody or fragment may be glycosylated. This heterologous portion may also be a detectable label, such as a fluorescent label, a luminescent label, a heavy metal label, a radioactive label, or an enzymatic label.

[0047] In some embodiments, any of the antibodies or antigen-binding fragments described herein may be produced in CHO cells. In some embodiments, the antibodies or antigen-binding fragments do not contain detectable sialic acid residues.

[0048] In some embodiments, any of the antibodies or antigen-binding fragments described herein may be modified with a moiety that enhances either or both of (a) stabilization of the antibody or antigen-binding fragment in circulation, and (b) retention of the antibody or antigen-binding fragment in circulation. Such moiety may be PEG (PEGylated).

[0049] In another embodiment, the Disclosure features nucleic acids encoding one or both of the heavy and light chain polypeptides of any of the antibodies or antigen-binding fragments described herein. It also features vectors (e.g., cloning or expression vectors) containing nucleic acids and cells (e.g., insect cells, bacterial cells, fungal cells, or mammalian cells). The Disclosure further provides methods for generating any of the antibodies or antigen-binding fragments described herein. These methods optionally include providing the cells (or cell cultures) containing an expression vector (embedded or extrachromosomal), the vector containing nucleic acids encoding one or both of the heavy and light chain polypeptides of any of the antibodies or antigen-binding fragments described herein. The cells or cell cultures are cultured under conditions and for a sufficient amount of time to enable the expression of the nucleic acid-encoded antibody or its antigen-binding fragment by the cells (or cell cultures). The methods may also include isolating the antibody or its antigen-binding fragment from the cells (or cells of the culture) or from the medium in which the cells(s) were cultured.

[0050] In another embodiment, the Disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more antibodies or antigen-binding fragments described herein.

[0051] In another embodiment, the present disclosure features a therapeutic kit comprising (i) one or more antibodies or antigen-binding fragments described herein, and (ii) means for delivering the antibodies or antigen-binding fragments to a human. These means may be, for example, a syringe or a pump.

[0052] In another embodiment, the Disclosure features a product comprising a container including a label and one or more antibodies or antigen-binding fragments described herein, the label indicating that the composition should be administered to a person having, suspected of having, or at risk of developing a complement-related condition. The product may further comprise one or more additional active therapeutic agents for use in treating a person having, suspected of having, or at risk of developing a complement-related condition.

[0053] In another embodiment, the present disclosure features a method for treating a patient suffering from a complement-related condition, the method comprising administering to one or more of the antibodies or antigen-binding fragments described herein in an amount effective for treating the complement-related condition. Complement-related conditions include, for example, rheumatoid arthritis, antiphospholipid syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, minor immune vasculitis, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, and immune complex hematoma. This may be selected from the group consisting of angitis, Takayasu's arteriovenous artery, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous gas embolism, restenosis following stent placement, rotational atherosclerosis, percutaneous coronary intervention, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, transplant rejection (e.g., kidney transplant), Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, Degos disease, and fulminant antiphospholipid syndrome.

[0054] As used herein, the term “antibody” refers to a whole antibody comprising two light-chain polypeptides and two heavy-chain polypeptides. A whole antibody comprises different antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” also includes polyclonal antibodies, monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, primate-derived antibodies, deimmunized antibodies, and fully human antibodies. Antibodies may be produced in or derived from any of the following mammals of various species, e.g., humans, non-human primates (e.g., orangutans, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies may be purified antibodies or recombinant antibodies.

[0055] As used herein, the terms “antibody fragment,” “antigen-binding fragment,” or similar terms refer to a fragment of an antibody that has the ability to bind to a target antigen (e.g., human C5) and inhibit the activity of that target antigen. Examples of such fragments include single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. An scFv fragment is a single polypeptide chain containing both the heavy and light chain variable regions of the antibody from which the scFv is induced. In addition, intracellular antibodies, minibodies, triabodies, and diabodies are also included in the definition of antibodies and are suitable for use in the methods described herein. For example, see Todorovska et al. (2001) J Immunol Methods 248(1):47-66, Hudson and Kortt (1999) J Immunol Methods 231(1):177-189, Poljak (1994) Structure 2(12):1121-1123, and Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283 (each disclosure is incorporated herein by reference in its entirety).

[0056] As used herein, the term “antibody fragment” also includes single-domain antibodies, such as camelized single-domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem Sci 26:230-235, Nuttall et al. (2000) Curr Pharm Biotech 1:253-263, Reichmann et al. (1999) J Immunol Meth 231:25-38, PCT applications published 94 / 04678 and 94 / 25591, and U.S. Patent No. 6,005,079 (all of which are incorporated herein by reference in their entirety). In some embodiments, this disclosure provides single-domain antibodies comprising two VH domains having modifications such that a single-domain antibody is formed.

[0057] In some embodiments, the antigen-binding fragment comprises a variable region of a heavy chain polypeptide and a variable region of a light chain polypeptide. In some embodiments, the antigen-binding fragment described herein comprises the light chain and the CDR of the heavy chain polypeptide of the antibody. The present invention also provides, for example, the following items: (Item 1) An isolated antibody or its antigen-binding fragment, (a) Binds to human C5 complement component, (b) Inhibit the cleavage of C5 into fragments C5a and C5b, (c) The isolated antibody or its antigen-binding fragment, comprising (i) a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 23, (ii) a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 19, (iii) a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, (iv) a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, (v) a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (vi) a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. (Item 2) The isolated antibody or its antigen-binding fragment according to item 1, comprising the heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 8. (Item 3) The isolated antibody or antigen-binding fragment according to item 1 or 2, further comprising a mutant human Fc constant region, wherein the mutant human Fc constant region binds to the human neonatal Fc receptor (FcRn) with a higher affinity than that of the native human Fc constant region from which the mutant human Fc constant region was induced. (Item 4) The isolated antibody or antigen-binding fragment described in any one of items 1 to 3, wherein the mutated human Fc constant region contains methionine at position 428 and asparagine at position 434, respectively, according to EU numbering. (Item 5) The isolated antibody or its antigen-binding fragment according to any one of items 1 to 4, further comprising the heavy chain constant region shown in Sequence ID No. 13. (Item 6) The isolated antibody or antigen-binding fragment according to any one of items 1 to 5, comprising a heavy chain polypeptide containing the amino acid sequence shown in SEQ ID NO: 14 and a light chain polypeptide containing the amino acid sequence shown in SEQ ID NO: 11. (Item 7) The isolated antibody or its antigen-binding fragment according to any one of items 1 to 6, wherein the antibody has a serum half-life of at least 25 days in humans. (Item 8) The antibody or its antigen-binding fragment has a concentration of 0.1 nM ≤ K at pH 7.4 and 25°C. D Affinity dissociation constant (K) is within the range of ≤1nM. D The isolated antibody or antigen-binding fragment described in any one of items 1 to 7, which binds to human C5 via ) (Item 9) The antibody or its antigen-binding fragment, at pH 6.0 and 25°C, K D The isolated antibody or its antigen-binding fragment according to any one of items 1 to 8, which binds to human C5 with a strength of ≥10 nM. (Item 10) [(K of the antibody or antigen-binding fragment against human C5 at pH 6.0 and 25°C D ) / (K of the antibody or antigen-binding fragment against human C5 at pH 7.4 and 25°C) D)] The isolated antibody or antigen-binding fragment described in any one of items 1 to 9, which is greater than 25. (Item 11) An isolated antibody or its antigen-binding fragment, (a) Binds to human C5 complement component, (b) Inhibit the cleavage of human C5 into fragments C5a and C5b, (c) comprising (i) a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 23, (ii) a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 19, (iii) a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, (iv) a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, (v) a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (vi) a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. [(K of the antibody or antigen-binding fragment against human C5 at pH 6.0 and 25°C D ) / (K of the antibody or antigen-binding fragment against human C5 at pH 7.4 and 25°C) D )] exceeds 25, The aforementioned mutant human Fc constant region contains methionine at position 428 and asparagine at position 434, respectively, according to EU numbering. The aforementioned antibody is an isolated antibody or its antigen-binding fragment, wherein the antibody has a serum half-life of at least 25 days in humans. (Item 12) The isolated antibody or antigen-binding fragment according to item 11, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region polypeptide having the amino acid sequence shown in SEQ ID NO: 12 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. (Item 13) The isolated antibody or antigen-binding fragment according to item 11, further comprising the heavy chain constant region shown in Sequence ID No. 13. (Item 14) An isolated antibody wherein the antibody or its antigen-binding fragment comprises a heavy chain polypeptide containing the amino acid sequence shown in SEQ ID NO: 14 and a light chain polypeptide containing the amino acid sequence shown in SEQ ID NO: 11. (Item 15) The aforementioned antibody is the isolated antibody described in item 14, which is produced in CHO cells. (Item 16) The aforementioned isolated antibody as described in item 15, wherein the antibody does not contain any detectable sialic acid residues. (Item 17) A nucleic acid encoding the heavy chain polypeptide of the antibody or its antigen-binding fragment as described in item 14. (Item 18) A nucleic acid encoding both the heavy chain polypeptide and the light chain polypeptide of the antibody or antigen-binding fragment described in item 14. (Item 19) A vector comprising the nucleic acid described in item 17 or 18. (Item 20) An expression vector comprising the nucleic acid described in item 17 or 18. (Item 21) A cell containing the expression vector described in item 20. (Item 22) A method for generating an antibody or an antigen-binding fragment thereof, comprising culturing the cells under conditions and for a period of time sufficient to enable the expression of the antibody or antigen-binding fragment encoded by the nucleic acid by the cells described in item 21. (Item 23) The method according to item 22, further comprising isolating the antibody or an antigen-binding fragment thereof. (Item 24) A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the antibody or antigen-binding fragment described in any one of items 1 to 16. (Item 25) (i) the isolated antibody or antigen-binding fragment described in any one of items 1 to 16, and (ii) means for delivering the antibody or antigen-binding fragment to a human. (Item 26) The treatment kit according to item 19, wherein the means is a syringe. (Item 27) It is a product, A container including a label, (i) A product comprising a composition comprising the isolated antibody or an antigen-binding fragment described in any one of items 1 to 16, wherein the label indicates that the composition should be administered to a person who has, is suspected of having, or is at risk of developing a complement-related condition. (Item 28) A method for treating a patient suffering from a complement-related condition, comprising administering to the subject an amount of the isolated antibody or antigen-binding fragment described in any one of items 1 to 16 that is effective in treating the complement-related condition. (Item 29) A method for treating a patient suffering from a complement-related condition, comprising administering to the subject an amount of the pharmaceutical composition described in item 24 that is effective for treating the complement-related condition. (Item 30) The aforementioned complement-related conditions include rheumatoid arthritis, antiphospholipid syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, minor immune vasculitis, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, and immune complex The method described in item 28, selected from the group consisting of vasculitis, Takayasu's arteritis, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous gas embolism, restenosis following stent placement, rotational atherosclerosis, percutaneous coronary intervention, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, Degos disease, and fulminant antiphospholipid syndrome.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. Preferred methods and materials are described below, but similar or equivalent methods and materials may also be used in the implementation or testing of the methods and compositions of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference.

[0059] Other features and advantages of this disclosure, such as methods for treating or preventing complement-related conditions, will become apparent from the following description, examples, and claims. A brief explanation of arrays

[0060] Sequence ID 1 shows the amino acid sequence of the heavy chain CDR1 of eculizumab (as defined under the mixed Kabat-Chothia definition). Sequence ID 2 shows the amino acid sequence of the heavy chain CDR2 of eculizumab (as defined under the Kabat definition). Sequence ID 3 shows the amino acid sequence of the heavy chain CDR3 of eculizumab (as defined under the mixed Kabat definition). Sequence ID 4 shows the amino acid sequence of the eculizumab light chain CDR1 (as defined under the Kabat definition). Sequence ID 5 shows the amino acid sequence of the eculizumab light chain CDR2 (as defined under the Kabat definition). Sequence ID 6 shows the amino acid sequence of the eculizumab light chain CDR3 (as defined under the Kabat definition). Sequence ID 7 shows the amino acid sequence of the heavy chain variable region of eculizumab. Sequence ID 8 shows the amino acid sequences of the light chain variable regions of eculizumab and the BNJ441 antibody. Sequence ID 9 shows the amino acid sequence of the heavy chain constant region of eculizumab. Sequence ID 10 shows the amino acid sequence of the entire heavy chain of eculizumab. Sequence ID 11 shows the amino acid sequences of the entire light chains of eculizumab and the BNJ441 antibody. Sequence ID 12 shows the amino acid sequence of the heavy chain variable region of the BNJ441 antibody. Sequence ID 13 shows the amino acid sequence of the heavy chain constant region of the BNJ441 antibody. Sequence ID 14 shows the amino acid sequence of the entire heavy chain of the BNJ441 antibody. Sequence ID 15 shows the amino acid sequence of an IgG double-chain constant region mutation containing a YTE substitution. Sequence ID 16 shows the entire amino acid sequence of the eculizumab mutant heavy chain, including the heavy chain constant region shown in Sequence ID 15 (above). Sequence ID 17 shows the amino acid sequence of the eculizumab light chain CDR1, which has a glycine-to-histidine substitution at position 8 compared to Sequence ID 4 (as defined under the Kabat definition). Sequence ID 18 shows the amino acid sequence of the light chain variable region of the EHG303 antibody. Sequence ID 19 shows the amino acid sequence of the heavy chain CDR2 of eculizumab, in which the serine at position 8 is replaced with histidine compared to Sequence ID 2. Sequence ID 20 shows the amino acid sequence of the so-called "FLAG" tag. Sequence ID 21 shows a polyhistidine sequence commonly used as an antigen tag. Sequence ID 22 shows the amino acid sequence of the so-called hemagglutinin tag. Sequence ID 23 shows the amino acid sequence of the eculizumab heavy chain CDR1, in which the tyrosine at position 2 is replaced with histidine (compared to Sequence ID 1). Sequence ID 24 shows the amino acid sequence of the heavy chain polypeptide of the EHG303 antibody. Sequence ID 25 shows the light chain polypeptide amino acid sequence of the EHG303 antibody. Sequence ID 26 shows the amino acid sequence of the heavy chain polypeptide of the EHL049 antibody. Sequence ID 27 shows the amino acid sequence of the light chain polypeptide of the EHL049 antibody. Sequence ID 28 shows the amino acid sequence of the EHL000 heavy chain polypeptide. Sequence ID 29 shows the amino acid sequence of the light chain polypeptide of the EHL000 antibody. Sequence ID 30 shows the amino acid sequence of the light chain polypeptide BHL006. Sequence ID 31 shows the amino acid sequence of the heavy chain polypeptide of the BHL006 antibody. Sequence ID 32 shows the amino acid sequence of the light chain polypeptide of the BHL009 antibody. Sequence ID 33 shows the amino acid sequence of the heavy chain of the BHL009 antibody. Sequence ID 34 shows the amino acid sequence of the light chain of the BHL0011 antibody. Sequence ID 35 shows the amino acid sequence of the heavy chain of the BHL011 antibody. [Brief explanation of the drawing]

[0061] [Figure 1] This line graph shows the clearance of eculizumab from the serum of human FcRn transgenic mice, with or without exogenous human C5. The Y-axis represents the percentage of antibody remaining in the serum, and the X-axis represents time in days. [Figure 2] This line graph shows the clearance of eculizumab variants containing the constant region of IgG2 (Ecu-IgG2) and the Ecu-IgG2 antibody (Ecu-IgG2(YTE)) with YTE substitution from mouse serum. The Y-axis represents the percentage of antibody remaining in the serum, and the X-axis represents time in days. [Figure 3] This line graph shows the clearance of the eculizumab variant of the IgG2 constant region (Ecu-IgG2) and the Ecu-IgG2 antibody containing the YTE substitution (Ecu-IgG2(YTE)) from mouse serum. Experiments were performed in or without the presence of exogenous human C5. The Y-axis represents the percentage of antibody remaining in the serum, and the X-axis represents time in days. [Figure 4] This is a sensorogram plot showing the dynamics of association (at pH 7.4) and dissociation (at pH 7.4 and pH 6.0) of three anti-C5 antibodies, namely EHL000, EHG303, and EHL049. The Y-axis represents arbitrary units, while the X-axis represents time (in seconds). [Figure 5A]This is a sensorogram plot showing the dissociation dynamics at pH 7.4 and pH 6.0 for EHG303 (Y27H-S57H double substitution) antibody, the Y27H single substitution mutant of eculizumab, and eculizumab (ecu, Ec293F). The Y-axis represents nanometers (nm), while the X-axis represents time (seconds). [Figure 5B] This is a sensorogram plot showing the dynamics of dissociation at pH 7.4 and pH 6.0 for EHG304 (I34H-L52H double substitution) antibody, the I34H single substitution mutant of eculizumab, and eculizumab (ecu, Ec293F). The Y-axis represents nanometers (nm), while the X-axis represents time (seconds). The EHG304 antibody did not meet the second threshold for selection, meaning it exceeded the maximum acceptable variation (from eculizumab) for dissociation at pH 7.4. [Figure 5C] This is a sensorogram plot showing the dissociation dynamics of EHG303 (Y27H-S57H double substitution) antibody and eculizumab (ecu, Ec293F) at pH 7.4 and pH 6.0. The Y-axis represents nanometers (nm), while the X-axis represents time (seconds). [Figure 5D] This is a sensorogram plot showing the dissociation dynamics of the EHL049 [G31H (light chain) / Y27H-S57H double substitution (heavy chain)] antibody, the Y27H-S57H (EHG303) double substitution mutant of eculizumab, and eculizumab (ecu) at pH 7.4 and pH 6.0. The Y-axis represents nanometers (nm), while the X-axis represents time (seconds). [Figure 5E] This is a sensorogram plot showing the dynamics of dissociation at pH 7.4 and pH 6.0 for the EHL058 [G31H (light chain) / L52H-S57H double substitution (heavy chain)] antibody, the L52H-S57H double substitution (heavy chain) mutant of eculizumab, and eculizumab (ecu). The Y-axis is in nanometers (nm), while the X-axis represents time (seconds). The EHL058 antibody did not meet the second threshold for selection, meaning it exceeded the maximum acceptable variation (from eculizumab) for dissociation at pH 7.4. [Figure 6]This line graph shows the clearance of EHL000, BNJ421, and BNJ423 from the serum of NOD / scid / C5-deficient mice. The Y-axis represents the percentage of antibodies remaining in the serum, and the X-axis represents time in days. [Figure 7] This line graph shows the clearance of EHL000, BNJ421, and BNJ423 from the serum of NOD / scid / C5-deficient mice, in the presence or absence of human C5. The Y-axis represents the percentage of antibodies remaining in the serum, and the X-axis represents time in days. [Figure 8] This line graph shows the activity of EHL000, BNJ423, and BNJ421 antibodies in an in vitro hemolysis assay. The Y-axis represents the percentage of hemolysis, and the X-axis represents time in days. [Figure 9A] This line graph shows the pharmacokinetics of the BHL011 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents antibody concentration in μg / mL. The X-axis represents time in days. [Figure 9B] This line graph shows the pharmacokinetics of the BHL011 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents the percentage of antibody concentration remaining in the serum on day 1 at each time point. The X-axis represents time in days. [Figure 10A] This line graph shows the pharmacokinetics of the BHL006 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents antibody concentration in μg / mL. The X-axis represents time in days. [Figure 10B] This line graph shows the pharmacokinetics of the BHL006 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents the percentage of antibody concentration remaining in the serum on day 1 at each time point. The X-axis represents time in days. [Figure 11A] This line graph shows the pharmacokinetics of the BHL009 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents antibody concentration in μg / mL. The X-axis represents time in days. [Figure 11B]This line graph shows the pharmacokinetics of the BHL009 antibody in hFcRn transgenic mice. Each line represents a different animal. The Y-axis represents the percentage of antibody concentration remaining in the serum on day 1 at each time point. The X-axis represents time in days. [Figure 12] This line graph shows logarithmic plots of the mean pharmacokinetics of BHL011, BHL006, and BHL009 antibodies in hFcRn transgenic mice. Each line represents a different antibody as indicated. The Y-axis represents the percentage of antibody concentration remaining in the serum on day 1 at each time point. The X-axis represents time in days. [Figure 13] This line graph shows linear plots of the mean pharmacokinetics of BHL011, BHL006, and BHL009 antibodies in hFcRn transgenic mice. Each line represents a different antibody as indicated. The Y-axis represents the percentage of antibody concentration remaining in the serum on day 1 at each time point. The X-axis represents time in days. [Figure 14] This line graph shows the blocking ability of BHL011 antibody in an in vitro serum hemolysis assay after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents time in days. [Figure 15] This line graph shows the blocking ability of BHL006 antibody in an in vitro serum hemolysis assay after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents time in days. [Figure 16] This line graph shows the blocking ability of BHL009 antibody in an in vitro serum hemolysis assay after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents time in days. [Figure 17] This graph shows the correlation between BHL011 serum concentration and in vitro serum hemolysis assay results after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents the antibody concentration in μg / mL. [Figure 18] This graph shows the correlation between BHL006 serum concentration and in vitro serum hemolytic activity after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents the antibody concentration in μg / mL. [Figure 19] This graph shows the correlation between BHL009 serum concentration and in vitro serum hemolytic activity after a single dose. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents the antibody concentration in μg / mL. [Figure 20] This line graph shows the mean in vitro hemolytic activity after a single dose of BHL011, BHL009, or BHL006 in hFcRn transgenic mice. Each line represents a different antibody, as indicated. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents time in days. [Figure 21A] Figure 21B is a pair of line graphs showing semi-logarithmic (Figure 21A) and linear (Figure 21B) plots of affinity for BNJ441 and eculizumab as a function of pH. The Y-axis represents dissociation % and the X-axis represents pH. [Figure 21B] Figure 21B is a pair of line graphs showing semi-logarithmic (Figure 21A) and linear (Figure 21B) plots of affinity for BNJ441 and eculizumab as a function of pH. The Y-axis represents dissociation % and the X-axis represents pH. [Figure 22] This line graph shows the pharmacokinetics of BNJ441 and eculizumab in NOD / scid mice in the absence of human C5. The Y-axis represents antibody concentration in μg / mL, and the X-axis represents time in days. [Figure 23] This line graph shows the pharmacokinetics of BNJ441 and eculizumab in NOD / scid mice in the presence of human C5. The Y-axis represents antibody concentration in μg / mL, and the X-axis represents time in days. [Figure 24] This line graph shows the percentage of BNJ441 and eculizumab remaining in the serum of NOD / scid mice in the presence of human C5 as a function of time. The Y-axis represents antibody concentration in μg / mL, and the X-axis represents time in days. [Figure 25] This line graph shows the extracorporeal serum hemolysis-blocking activity of BNJ441 antibody and eculizumab after a single dose, as a function of time. The Y-axis represents the percentage of hemolysis (compared to pre-administration levels), and the X-axis represents time in days. [Figure 26]The mean serum BNJ441 concentration-time profiles following intravenous administration of 200 mg or 400 mg doses to healthy volunteers are shown (upper panel - linear scale, lower panel - log-linear scale). [Figure 27] The mean chicken erythrocyte hemolysis-time profiles following intravenous administration of placebo, 200 mg of BNJ441, or 400 mg of BNJ441 to healthy volunteers are shown. [Figure 28] This shows the relationship between BNJ441 concentration and chicken erythrocyte hemolysis rate following intravenous administration of BNJ441 to healthy human volunteers. [Figure 29] This shows the performance of BNJ441 compared to eculizumab in terminal complement activity assays. [Figure 30] The structure of BNJ441 is shown. [Figure 31] This shows the interchain disulfide bonds in BNJ441. [Modes for carrying out the invention]

[0062] This disclosure provides, in particular, antibodies useful for inhibiting terminal complement (e.g., the assembly and / or activity of C5b-9 TCCs) and C5a anaphylatoxin-mediated inflammation, and thus for treating complement-related disorders. The antibodies have several improved properties compared to eculizumab, including, for example, an increased serum half-life in humans. Exemplary antibodies, conjugates, pharmaceutical compositions, and formulations, as well as methods for using any of the above, are described in detail below and illustrated in the examples.

[0063] antibody The anti-C5 antibodies described herein bind to complement component C5 (e.g., human C5) and inhibit the cleavage of C5 into fragments C5a and C5b. As described above, such antibodies also have improved pharmacokinetic properties compared to other anti-C5 antibodies used for therapeutic purposes (e.g., eculizumab).

[0064] In some embodiments, the anti-C5 antibody described herein comprises (i) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (ii) a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, (iii) a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (iv) a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (v) a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and (vi) a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6, wherein at least one (for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids of (i) to (vi) is substituted with a different amino acid.

[0065] The precise boundaries of the CDR were defined differently according to different methods. In some embodiments, the location of the CDR or framework region within the light chain or heavy chain variable domain may be as defined by Kabat et al. [(1991) "Sequences of Proteins of Immunological Interest," NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, MD]. In such cases, the CDR is the "Kabat CDR" (e.g., "Kabat CDR"). These regions may be referred to as "LCDR2" or "Kabat HCDR1". In some embodiments, the location of the CDR in the light-chain or heavy-chain variable region may be as defined by Chothia et al. (1989) Nature 342:877-883. Thus, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR3"). In some embodiments, the location of the CDRs in the light-chain and heavy-chain variable regions may be as defined by the Kabat-Chothia mixed definition. In such embodiments, these regions may be referred to as "mixed Kabat-Chothia CDRs". Thomas et al. [(1996) Mol Immunol 33(17 / 18):1389-1401] illustrate the identification of CDR boundaries by Kabat and Chothia definitions.

[0066] Any amino acid can be substituted with any other amino acid. In some embodiments, this substitution is a conservative substitution. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine. In some embodiments, one or more amino acids are substituted with histidine.

[0067] In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of heavy chain CDR1 are substituted with different amino acids. In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of heavy chain CDR2 are substituted with different amino acids. In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of heavy chain CDR3 are substituted with different amino acids.

[0068] In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of light chain CDR1 are substituted with different amino acids. In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of light chain CDR2 are substituted with different amino acids. In some embodiments, at least one (e.g., at least two, three, four, or five) amino acids of light chain CDR3 are substituted with different amino acids.

[0069] In some embodiments, the substitution is glycine at position 1 relative to SEQ ID NO: 1, tyrosine at position 2 relative to SEQ ID NO: 1, isoleucine at position 3 relative to SEQ ID NO: 1, phenylalanine at position 4 relative to SEQ ID NO: 1, serine at position 5 relative to SEQ ID NO: 1, asparagine at position 6 relative to SEQ ID NO: 1, tyrosine at position 7 relative to SEQ ID NO: 1, tryptophan at position 8 relative to SEQ ID NO: 1, isoleucine at position 9 relative to SEQ ID NO: 1, glutamine at position 10 relative to SEQ ID NO: 1, glutamic acid at position 1 relative to SEQ ID NO: 2, isoleucine at position 2 relative to SEQ ID NO: 2, leucine at position 3 relative to SEQ ID NO: 2, proline at position 4 relative to SEQ ID NO: 2, glycine at position 5 relative to SEQ ID NO: 2, serine at position 6 relative to SEQ ID NO: 2, glycine at position 7 relative to SEQ ID NO: 2, serine at position 8 relative to SEQ ID NO: 2, threonine at position 9 relative to SEQ ID NO: 2, glutamic acid at position 10 relative to SEQ ID NO: 2, and 11 relative to SEQ ID NO: 2 The process is performed at an amino acid position selected from the group consisting of tyrosine, threonine at position 12 relative to SEQ ID NO: 2, glutamic acid at position 13 relative to SEQ ID NO: 2, asparagine at position 14 relative to SEQ ID NO: 2, phenylalanine at position 15 relative to SEQ ID NO: 2, lysine at position 16 relative to SEQ ID NO: 2, aspartic acid at position 17 relative to SEQ ID NO: 2, tyrosine at position 1 relative to SEQ ID NO: 3, phenylalanine at position 3 relative to SEQ ID NO: 3, glycine at position 4 relative to SEQ ID NO: 3, serine at position 5 relative to SEQ ID NO: 3, serine at position 6 relative to SEQ ID NO: 3, proline at position 7 relative to SEQ ID NO: 3, asparagine at position 8 relative to SEQ ID NO: 3, tryptophan at position 9 relative to SEQ ID NO: 3, tyrosine at position 10 relative to SEQ ID NO: 3, phenylalanine at position 11 relative to SEQ ID NO: 3, aspartic acid at position 12 relative to SEQ ID NO: 3, and valine at position 13 relative to SEQ ID NO: 3.

[0070] In some embodiments, the glycine at position 31 in SEQ ID NO: 8 is substituted with a different amino acid. For example, the underlined glycine in CDR1 of the eculizumab light chain, i.e., [ka] This can be substituted with a different amino acid. This substitution is histidine instead of glycine, i.e., [ka] It is possible.

[0071] In some embodiments, the anti-C5 antibody described herein is glycine at position 26 relative to SEQ ID NO: 7, tyrosine at position 27 relative to SEQ ID NO: 7, isoleucine at position 28 relative to SEQ ID NO: 7, phenylalanine at position 29 relative to SEQ ID NO: 7, serine at position 30 relative to SEQ ID NO: 7, asparagine at position 31 relative to SEQ ID NO: 7, tyrosine at position 32 relative to SEQ ID NO: 7, tryptophan at position 33 relative to SEQ ID NO: 7, isoleucine at position 34 relative to SEQ ID NO: 7, glutamine at position 35 relative to SEQ ID NO: 7, glutamic acid at position 50 relative to SEQ ID NO: 7, isoleucine at position 51 relative to SEQ ID NO: 7, leucine at position 52 relative to SEQ ID NO: 7, proline at position 53 relative to SEQ ID NO: 7, glycine at position 54 relative to SEQ ID NO: 7, serine at position 55 relative to SEQ ID NO: 7, glycine at position 56 relative to SEQ ID NO: 7, serine at position 57 relative to SEQ ID NO: 7, threonine at position 58 relative to SEQ ID NO: 7, glutamic acid at position 59 relative to SEQ ID NO: 7, and 60 relative to SEQ ID NO: 7. The amino acid substitution is made at an amino acid position selected from the group consisting of tyrosine at position 1, threonine at position 61 relative to SEQ ID NO: 7, glutamic acid at position 62 relative to SEQ ID NO: 7, asparagine at position 63 relative to SEQ ID NO: 7, phenylalanine at position 64 relative to SEQ ID NO: 7, lysine at position 65 relative to SEQ ID NO: 7, aspartic acid at position 66 relative to SEQ ID NO: 7, tyrosine at position 99 relative to SEQ ID NO: 7, phenylalanine at position 100 relative to SEQ ID NO: 7, phenylalanine at position 101 relative to SEQ ID NO: 7, glycine at position 102 relative to SEQ ID NO: 7, serine at position 103 relative to SEQ ID NO: 7, serine at position 104 relative to SEQ ID NO: 7, proline at position 105 relative to SEQ ID NO: 7, asparagine at position 106 relative to SEQ ID NO: 7, tryptophan at position 107 relative to SEQ ID NO: 7, tyrosine at position 108 relative to SEQ ID NO: 7, phenylalanine at position 109 relative to SEQ ID NO: 7, aspartic acid at position 110 relative to SEQ ID NO: 7, and valine at position 111 relative to SEQ ID NO: 7. In some embodiments, the anti-C5 antibody comprises two or more of the above substitutions (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) and any combination thereof.

[0072] In some embodiments, the anti-C5 antibody comprises at least one substitution that satisfies the following criteria with respect to eculizumab. (1) The maximum variation in association kinetics at pH 7.4 was 33% smaller in peak phase shift over 800 seconds compared to the averaged peak phase shift over 800 seconds observed for eculizumab. (2) The maximum variation in dissociation dynamics at pH 7.4 was less than three times the peak phase shift observed for eculizumab at 800 seconds, and (3) The minimum change in dissociation dynamics at pH 6.0 is at least three times lower in peak phase shift beyond 800 seconds compared to the averaged peak phase shift observed for eculizumab at 800 seconds. For example, with respect to criterion (1) above, if the averaged peak phase shift 800 seconds after association with eculizumab is approximately 0.75 nM, then a test antibody with a phase shift of less than 0.5 nM (e.g., reproduced two or more times) does not meet the above criterion. In contrast, an anti-C5 antibody with a peak phase shift greater than 0.5 nM at 800 seconds meets the first criterion. Such substitution is equivalent to the k of eculizumab at pH 7.4. a and k d It produces anti-C5 antibodies that deviate slightly from the pH, but eculizumab's k at pH 6.0 d It deviates significantly from the norm.

[0073] In some embodiments, the anti-C5 antibodies described herein include at least one (e.g., at least two, three, or four) amino acid substitutions at an amino acid position selected from the group consisting of glycine at position 31, leucine at position 33, valine at position 91, and threonine at position 94 of SEQ ID NO: 8. In some embodiments, the anti-C5 antibodies described herein include at least one (e.g., at least two, three, four, or five) amino acid substitutions at an amino acid position selected from the group consisting of tyrosine at position 27, isoleucine at position 34, leucine at position 52, and serine at position 57 of SEQ ID NO: 7.

[0074] In some embodiments, the anti-C5 antibodies described herein contain in their light chain variable region at least one substitution selected from the following: glycine at position 31 relative to SEQ ID NO: 8, leucine at position 33 relative to SEQ ID NO: 8, valine at position 91 relative to SEQ ID NO: 8, and threonine at position 94 relative to SEQ ID NO: 8. See Table 1 below. In some embodiments, the anti-C5 antibodies described herein contain in their heavy chain variable region at least one substitution selected from the following: tyrosine at position 27 relative to SEQ ID NO: 7, isoleucine at position 34 relative to SEQ ID NO: 7, leucine at position 52 relative to SEQ ID NO: 7, and serine at position 57 relative to SEQ ID NO: 7. See Table 1 below.

[0075] In some embodiments, the antibody contains at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions relative to the CDR set defined by SEQ ID NOs: 1-6. Thus, in some embodiments, the anti-C5 antibodies described herein contain the combinations listed in Table 1 and two or more substitutions at the amino acid positions. [Table 1-1] [Table 1-2] [Table 1-3]

[0076] The substitutions listed in Table 1 may be for any amino acid other than the indicated amino acid residue. In some embodiments, the different amino acid is histidine.

[0077] In some embodiments, the anti-C5 antibodies described herein include substitutions made at amino acid positions selected from the group consisting of tyrosine at position 27 of SEQ ID NO: 7, isoleucine at position 34 of SEQ ID NO: 7, leucine at position 52 of SEQ ID NO: 7, and serine at position 57 of SEQ ID NO: 7. In some embodiments, both tyrosine at position 27 of SEQ ID NO: 7 and leucine at position 52 of SEQ ID NO: 7 are each substituted with different amino acids. In some embodiments, both isoleucine at position 34 of SEQ ID NO: 7 and serine at position 57 of SEQ ID NO: 7 are each substituted with different amino acids. In some embodiments, isoleucine at position 34 of SEQ ID NO: 7 and leucine at position 52 of SEQ ID NO: 7 are each substituted with different amino acids. In some embodiments, both tyrosine at position 27 of SEQ ID NO: 7 and serine at position 57 of SEQ ID NO: 7 are each substituted with different amino acids. In some embodiments of any of the anti-C5 antibodies described herein, the different amino acid is histidine. For example, tyrosine at position 27 and serine at position 57 may each be substituted with histidine.

[0078] In some embodiments, the anti-C5 antibody described herein has the following amino acid sequence, i.e., [ka] It contains or comprises a heavy chain CDR1 comprising the following amino acid sequence: [ka] It contains or comprises a heavy chain CDR2 comprising the following amino acid sequence: [ka] It includes a heavy chain variable region.

[0079] In some embodiments, the anti-C5 antibody described herein has the following amino acid sequence, i.e., [ka] Includes a light chain variable region.

[0080] The anti-C5 antibodies described herein have an affinity dissociation constant (K) of at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM at pH 7.4 and 25°C (and otherwise under physiological conditions). D ) can bind to C5. In some embodiments, the K of the anti-C5 antibody D The saturation is 1 nM or less (for example, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM or less).

[0081] In some embodiments of any anti-C5 antibody described herein, the K of the antibody against C5 at pH 6.0 C D / (K of antibody against C5 at pH 7.4, 25°C) D ) is 21 (for example, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 2 (Exceeding 50, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, or 8000).

[0082] Methods for determining whether an antibody binds to a protein antigen and / or the affinity of an antibody to a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blotting, dot blotting, surface plasmon resonance (SPR) methods (e.g., BIAcore system, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). For example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Benny KCLo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN:1588290921), Borrebaek (1992) "Antibody Engineering, A Practical Guide," WHFreeman and Co., NY, Borrebaek(1995)″Antibody Engineering,″2 nd Edition, Oxford University Press, NY, Oxford, Johne et al. (1993) J Immunol Meth 160:191-198, Jonsson et al. (1993) Ann Biol Clin 51:19-26, and Jonsson et al. (1991) Biotechniques. See 11:620-627. In addition, methods for measuring affinity (e.g., dissociation constant and association constant) are described in the examples.

[0083] When used herein, "k a The term "k" refers to the rate constant of antibody association with an antigen. dThe term "K" refers to the rate constant of antibody dissociation from antigen / antigen complex. D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of antibody-antigen interactions. The equilibrium dissociation constant is estimated from the ratio of the kinetic rate constants. D =k a / k d Such determinations are preferably measured at 25°C or 37°C (see Examples). For example, the kinetics of an antibody binding to human C5 can be determined via surface plasmon resonance (SPR) on a BIAcore 3000 instrument using an anti-Fc capture method for immobilizing the antibody at pH 8.0, 7.4, 7.0, 6.5, and 6.0.

[0084] The anti-C5 antibodies described herein may be active in blocking the generation or activity of C5a and / or C5b active fragments of the C5 protein (e.g., human C5 protein). Through this blocking effect, the antibodies inhibit the pro-inflammatory effects of C5a and the generation of the C5b-9 membrane invasion complex (MAC) on the cell surface.

[0085] Methods for determining whether the specific antibodies described herein inhibit C5 cleavage are known in the art. Inhibition of human complement component C5 can reduce the cytolytic capacity of complement in the body fluid of interest. Such reduction of the cytolytic capacity of complement present in body fluid(s) can be achieved by methods well known in the art, for example, Kabat and Mayer (eds.), "Experimental Immunochemistry, 2 nd Conventional hemolysis assays, such as the hemolysis assay described by Thomas (1961), pages 135-139, Edition, 135-240, Springfield, IL, CC, or, for example, Hillmen et al. (2004)N This can be measured by variations of conventional assays, such as the chicken erythrocyte hemolysis method described in Engl J Med 350(6):552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 to C5a and C5b forms are known in the art, for example, Moongkarndi et al. (1982) Immunobiol 162:397, Moongkarndi et al. (1983) Immunobiol 165:323, and Isenman et al. (1980) J Immunol 124(1):326-31, Thomas et al. (1996) Mol Immunol 33(17-18):1389-401, and Evans This is described in et al. (1995) Mol Immunol 32(16):1183-95. For example, the concentrations and / or physiological activities of C5a and C5b in body fluids can be measured by methods well known in the art. Methods for measuring C5a concentration or activity include, for example, chemotactic assays, RIA, or ELISA (see, for example, Ward and Zvaifler (1971) J Clin Invest 50(3):606-16 and Wurzner et al. (1991) Complement Inflamm 8:328-340). For C5b, the hemolysis assays or assays for soluble C5b-9 discussed herein can be used. Other assays known in the art can also be used. These or other suitable types of assays can be used to screen candidate drugs that can inhibit human complement component C5.

[0086] Immunological techniques such as ELISA can be used to measure the protein concentrations of C5 and / or its reconstitution products and to determine the ability of anti-C5 antibodies to inhibit the conversion of C5 to biologically active products. In some embodiments, the production of C5a is measured. In some embodiments, terminal complement formation is detected using C5b-9 novel epitope-specific antibodies.

[0087] A hemolysis assay can be used to determine the inhibitory activity of an anti-C5 antibody against complement activation. To determine the effect of the anti-C5 antibody against classical complement pathway-mediated hemolysis in a serum test solution in vitro, target cells are used, for example, hemolysin-coated sheep erythrocytes or chicken erythrocytes sensitized with an anti-chicken erythrocyte antibody. The percentage of hemolysis is normalized by considering 100% hemolysis, which is equivalent to the hemolysis that occurs in the absence of the inhibitor. In some embodiments, the classical complement pathway is activated by a human IgM antibody, for example, as used in the Wieslab® Classical Complement Pathway Kit (Wieslab® COMPL CP310, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with an anti-C5 antibody in the presence of a human IgM antibody. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-complex anti-C5b-9 antibody and a fluorescent substrate, as well as by measuring the absorbance at an appropriate wavelength. As a control, the test serum is incubated in the absence of anti-C5 antibody. In some embodiments, the test serum is C5-deficient serum reconstituted with C5 polypeptide.

[0088] To determine the effect of anti-C5 antibodies on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig erythrocytes are used as target cells. In some embodiments, the serum test solution is C5-deficient serum reconstituted with C5 polypeptide. The percentage of hemolysis is normalized by considering 100% hemolysis, which is equivalent to hemolysis occurring in the absence of the inhibitor. In some embodiments, the alternative complement pathway is activated by a lipopolysaccharide molecule, for example, as used in the Wieslab® Alternative Complement Pathway Kit (Wieslab® COMPL AP330, Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with anti-C5 antibody in the presence of lipopolysaccharide. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-complex anti-C5b-9 antibody and a fluorescent substrate, and by measuring the fluorescence at an appropriate wavelength. As a control, the test serum is incubated in the absence of anti-C5 antibody.

[0089] In some embodiments, C5 activity, or its inhibition, is quantified using the CH50eq assay. The CH50eq assay is a method for measuring total classical complement activity in serum. This assay is a lysis assay that determines the amount required to produce 50% lysis (CH50) using antibody-sensitized erythrocytes as activators of the classical complement pathway and various dilutions of the test serum. The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides an indirect measure of TCC formation because terminal complement complexes (TCCs) themselves are directly involved in the hemolysis being measured.

[0090] This assay is well known and commonly performed by those skilled in the art. Briefly, to activate the classical complement pathway, an undiluted serum sample (e.g., a reconstituted human serum sample) is added to microassay wells containing antibody-sensitized erythrocytes, thereby generating TCCs. The activated serum is then diluted in microassay wells coated with a capture reagent (e.g., an antibody that binds to one or more components of TCCs). TCCs present in the activated sample bind to monoclonal antibodies coating the surface of the microassay wells. The wells are washed, and a detection reagent, which is detectably labeled and recognizes the bound TCCs, is added to each well. The detectable label may be, for example, fluorescent or enzymatic. The assay results are expressed in units of CH50 equivalents per milliliter (CH50U Eq / mL).

[0091] Inhibition, for example, when relating to terminal complement activity, includes a reduction of at least 5% (e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%) of terminal complement activity in a hemolysis assay or CH50eq assay, compared to the effect of a control antibody (or its antigen-binding fragment) under similar conditions and at equimolar concentrations. As used herein, substantial inhibition refers to inhibition of a given activity (e.g., terminal complement activity) by at least 40% (e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%). In some embodiments, the anti-C5 antibodies described herein contain one or more amino acid substitutions to the CDR of eculizumab (i.e., SEQ ID NOs: 1-6) but retain at least 30% of the complement inhibitory activity of eculizumab in a hemolysis assay or CH50eq assay (e.g., at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%).

[0092] The anti-C5 antibodies described herein have a serum half-life in humans of at least 20 days (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days). Methods for measuring the serum half-life of antibodies are known in the art and are illustrated in the examples. See, for example, Dall'Acqua et al. (2006) J Biol Chem 281:23514-23524, Hinton et al. (2004) J Biol Chem 279:6213-6216, Hinton et al. (2006) J Immunol 176:346-356, and Petkova et al. (2006) Int Immunol 18(12):1759-69 (each disclosure is incorporated herein by reference in its entirety). In some embodiments, the anti-C5 antibodies described herein have a serum half-life at least 20% longer than the serum half-life of eculizumab (e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500%), as measured in one of the mouse model systems described in the Examples (e.g., C5-deficient / NOD / scid mouse or hFcRn transgenic mouse model system).

[0093] Modification of the Fc region The anti-C5 antibodies described herein may, in some embodiments, include a mutant human Fc constant region that binds to the human neonatal Fc receptor (FcRn) with superior affinity to the naturally occurring human Fc constant region than the naturally occurring human Fc constant region in which the mutant human Fc constant region was induced. For example, the Fc constant region may contain one or more (e.g., 2, 3, 4, 5, 6, 7, or 8 or more) amino acid substitutions compared to the naturally occurring human Fc constant region in which the mutant human Fc constant region was induced. These substitutions can increase the binding affinity of the IgG antibody containing the mutant Fc constant region to FcRn at pH 6.0 while maintaining the pH-dependent interaction. See, for example, Hinton et al. (2004) J Biol Chem 279:6213-6216 and Datta-Mannan et al. (2007) Drug Metab Dispos 35:1-9. Methods for testing whether one or more substitutions within the Fc constant region of an antibody increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and are illustrated in the examples. See, for example, Datta-Mannan et al. (2007) J Biol Chem 282(3):1709-1717, International Publication No. 98 / 23289, International Publication No. 97 / 34631, and U.S. Patent No. 6,277,375 (each disclosure is incorporated herein by reference in its entirety).

[0094] Substitutions that enhance the binding affinity of the antibody Fc constant region to FcRn are known in the art, for example, (1) Dall'Acqua et al. (2006) J M252Y / S254T / T256E triple substitution as described in Biol Chem 281:23514-23524, (2) Hinton et al. (2004) J Biol Examples include the M428L or T250Q / M428L substitutions described in Chem 279:6213-6216 and Hinton et al. (2006) J Immunol 176:346-356, and the N434A or T307 / E380A / N434A substitutions described in Petkova et al. (2006) Int Immunol 18(12):1759-69. Additional substitution pairs: P257I / Q311I, P257I / N434H, and D376V / N434H are, for example, Datta-Mannan This is described in et al. (2007) J Biol Chem 282(3):1709-1717 (the disclosure is incorporated herein by reference in its entirety).

[0095] In some embodiments, the mutant constant region has a substitution at EU amino acid residue 255 for valine. In some embodiments, the mutant constant region has a substitution at EU amino acid residue 309 for asparagine. In some embodiments, the mutant constant region has a substitution at EU amino acid residue 312 for isoleucine. In some embodiments, the mutant constant region has a substitution at EU amino acid residue 386.

[0096] In some embodiments, the mutant Fc constant region contains 30 or fewer amino acid substitutions, insertions, or deletions (e.g., 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer) compared to the induced native constant region. In some embodiments, the mutant Fc constant region contains one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the mutant human Fc constant region contains methionine at position 428 and asparagine at position 434, respectively, with EU numbering. In some embodiments, the mutant Fc constant region includes a 428L / 434S double substitution, as described, for example, in U.S. Patent No. 8,088,376.

[0097] In some embodiments, the mutant constant region includes substitutions at amino acid positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) compared to the natural human Fc constant region. In some embodiments, this substitution is all EU numbered as follows: methionine for glycine at position 237, alanine for proline at position 238, lysine for serine at position 239, isoleucine for lysine at position 248, alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250, phenylalanine, tryptophan, or tyrosine for methionine at position 252, threonine for serine at position 254, glutamic acid for arginine at position 255, aspartic acid, glutamic acid, or glutamine for threonine at position 256, alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine for proline at position 257, histidine for glutamic acid at position 258, and asparagine at position 265. Alanine for aspartic acid, phenylalanine for aspartic acid at position 270, alanine or glutamic acid for asparagine at position 286, histidine for threonine at position 289, alanine for asparagine at position 297, glycine for serine at position 298, alanine for valine at position 303, alanine for valine at position 305, alanine for threonine at position 307, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine, alanine for valine at position 308, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine, alanine for leucine or valine at position 309, aspartic acid, glutamic acid, proline,or arginine, alanine, histidine, or isoleucine for glutamine at position 311, alanine or histidine for aspartic acid at position 312, lysine or arginine for leucine at position 314, alanine or histidine for asparagine at position 315, alanine for lysine at position 317, glycine for asparagine at position 325, valine for isoleucine at position 332, leucine for lysine at position 334, histidine for lysine at position 360, alanine for aspartic acid at position 376, alanine for glutamic acid at position 380, alanine for glutamic acid at position 382, ​​alanine for asparagine or serine at position 384, The group is selected from the following: aspartic acid or histidine for glycine at position 385, proline for glutamine at position 386, glutamic acid for proline at position 387, alanine or serine for asparagine at position 389, alanine for serine at position 424, alanine for methionine at position 428, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine, lysine for histidine at position 433, alanine for asparagine at position 434, phenylalanine, histidine, serine, tryptophan, or tyrosine, and histidine for tyrosine or phenylalanine at position 436.

[0098] In some embodiments, the anti-C5 antibodies described herein may include a heavy chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12 or 14, and / or a light chain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 or 11.

[0099] Method for producing anti-C5 antibody and its antigen-binding fragment This disclosure also features methods for producing any of the anti-C5 antibodies and their antigen-binding fragments described herein. In some embodiments, the methods for preparing the antibodies described herein may include immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for producing any of the antibodies described herein are described herein. For example, to produce an antibody that binds to C5, an expert may immunize a suitable subject (e.g., a non-human mammal such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate) with a polypeptide to full-length C5, such as a full-length human C5 polypeptide. In some embodiments, non-human mammals are C5 deficient, such as the C5-deficient mice described in Levy and Ladda (1971) Nat New Biol 229(2):51-52, Crocker et al. (1974) J Clin Pathol 27(2):122-124, Wetsel et al. (1990) J Biol Chem 265:2435-2440, and Jungi and Pepys (1981) Immunology 43(2):271-279.

[0100] Suitable subjects (e.g., non-human mammals) can be immunized with appropriate antigens, along with a sufficient number of subsequent booster immunizations to induce antibody production in the mammal. Immunogens can be administered to subjects (e.g., non-human mammals) together with adjuvants. Adjuvants useful in generating antibodies in subjects include, but are not limited to, protein adjuvants, bacterial adjuvants such as whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota), as well as bacterial components including the cell wall skeleton of Mycobacterium tuberculosis, trehalose dimycolate, monophosphoryl lipid A, methanol-extractable residues (MERs), complete or incomplete Freund's adjuvants, viral adjuvants, and chemical adjuvants such as aluminum hydroxide, iodoacetate, and cholesteryl hemisuccinate. Other adjuvants that may be used in methods to induce an immune response include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. For example, see Lodmell et al. (2000) Vaccine 18:1059-1066, Johnson et al. (1999) J Med Chem 42:4640-4649, Baldridge et al. (1999) Methods 19:103-107, and Gupta See also et al. (1995) Vaccine 13(14):1263-1276.

[0101] In some embodiments, these methods involve preparing hybridoma cell lines that secrete monoclonal antibodies bound to an immunogen. For example, a suitable mammal, such as a laboratory mouse, is immunized with a C5 polypeptide as described above. Antibody-producing cells from the immunized mammal (e.g., spleen B cells) can be isolated 2-4 days after at least one additional immunization with the immunogen and then briefly grown in culture medium before fusion with cells of a suitable myeloma cell line. These cells can be fused in the presence of a fusion promoter, such as vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones that secrete the desired antibody are selected. For example, spleen cells from a Balb / c mouse immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag14. After fusion, the cells are grown in a suitable culture medium supplemented at regular intervals with a selective medium, such as HAT medium, to prevent normal myeloma cells from over-proliferating the desired hybridoma cells. Next, the resulting hybrid cells are screened for the secretion of a desired antibody, for example, an antibody that binds to C5 and inhibits its cleavage into C5a and C5b fragments.

[0102] In some embodiments, any of the antibodies or antigen-binding fragments described herein may be produced in CHO cells. In some embodiments, the antibodies or antigen-binding fragments do not contain detectable sialic acid residues.

[0103] In some embodiments, skilled individuals can identify anti-C5 antibodies from non-immune-dominant libraries, for example, as described in U.S. Patent No. 6,300,064 (Knappik et al., Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0104] Subpopulations of antibodies screened using the above method may be characterized for their specificity and binding affinity to a particular immunogen (e.g., C5) using any immunological or biochemical method known in the art. For example, the specific binding of an antibody to natural full-length C5 may be determined, compared to C5a, using immunological or biochemical methods such as, but not limited to, ELISA assays, SPR assays, immunoprecipitation assays, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that may be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradioquantification assays, fluorescence immunoassays, and protein A immunoassays. Such assays are commonplace and well known in the art.

[0105] Antibodies can also be assayed using any SPR-based assay known in the art to characterize the dynamic parameters of the antibody's interaction with C5. Examples include BIAcore instruments (Biacore AB, Uppsala, Sweden), lAsys instruments (Affinity Sensors, Franklin, Massachusetts), IBIS systems (Windsor Scientific Limited, Berks, UK), SPR-CELLIA systems (Nippon Laser and Electronics Lab, Hokkaido, Japan), and SPR Any commercially available SPR instrument, including but not limited to the Detector Spreeta (Texas Instruments, Dallas, Texas), may be used in the methods described herein. See, for example, Mullett et al. (2000) Methods 22:77-91, Dong et al. (2002) Reviews in Mol Biotech 82:303-323, Fivash et al. (1998) Curr Opin Biotechnol 9:97-101, and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0106] It is understood that the above method can also be used to determine, for example, whether an anti-C5 antibody does not bind to full-length native C3 and / or C4 proteins.

[0107] As described in the above references, after phage selection, the antibody-coding region from the phage is isolated and used to generate a whole antibody containing human antibodies, or any desired fragment, which can then be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as detailed below. For example, techniques for generating Fab, Fab', and F(ab')2 fragments by recombination are known methods in the art, e.g., PCT Publication 92 / 22324, Mullinax et al. (1992) BioTechniques 12(6):864-869, and Sawai et al. Methods disclosed in al. (1995) Am J Repr Immunol 34:26-34 and Better et al. (1988) Science 240:1041-1043 may also be used. Examples of techniques that can be used to generate single-chain Fv and antibodies include U.S. Patent Nos. 4,946,778 and 5,258,498 and Huston et al. (1991) Methods in Examples include those described in Enzymology 203:46-88, Shu et al. (1993) Proc Nat Acad Sci USA 90:7995-7999, and Skerra et al. (1988) Science 240:1038-1040.

[0108] In some embodiments, epitope mapping can be used to identify, for example, C5 regions that interact with antibodies. Methods for identifying epitopes to which specific antibodies bind are also known in the art and are described above.

[0109] The antibodies and fragments thereof identified herein may or may be made into “chimeric.” Chimeric antibodies and their antigen-binding fragments comprise portions from two or more different species (e.g., mouse and human). Chimeric antibodies may be generated using a mouse variable region of desired specificity fused to a human constant domain (e.g., U.S. Patent No. 4,816,567). This method can be used to modify non-human antibodies to make them more suitable for human clinical application (e.g., methods for treating or preventing complement-mediated disorders in a subject).

[0110] The monoclonal antibodies of this disclosure include “humanized” forms of non-human (e.g., mouse) antibodies. Humanized or CDR-implanted mAbs are particularly useful as therapeutic agents for humans because they are not rapidly cleared from circulation like mouse antibodies and do not typically cause reverse immune responses. Generally, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as “implant” residues, and they are typically taken from “implant” variable domains. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be carried out essentially by substituting a rodent framework or CDR sequence with the corresponding sequence of a human antibody, following the methods of Winter and colleagues (see, e.g., Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-327, and Verhoeyen et al. (1988) Science 239:1534-1536). See, for example, Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, the humanized form of a non-human (e.g., mouse) antibody is a human antibody (recipient antibody), and amino acid residues from the CDR region of a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate antibody) having the desired specificity, affinity, and binding ability are transplanted onto a framework scaffold of the human antibody.

[0111] In some cases, one or more amino acid residues in the framework region of human immunoglobulins are also substituted by the corresponding amino acid residues of non-human antibodies (so-called "reverse mutations"). In addition, phage presentation libraries can be used to alter amino acids at selected positions within the antibody sequence. The properties of humanized antibodies are also influenced by the selection of the human framework. Furthermore, humanized and chimeric antibodies can be modified to include residues not found in the recipient or donor antibody in order to further improve antibody properties, such as affinity or effector function.

[0112] Fully human antibodies are also provided in this disclosure. The term “human antibody” includes antibodies having variable and constant regions (if any) derived from human immunoglobulin sequences, preferably human germline sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo). However, the term “human antibody” does not include antibodies (i.e., humanized antibodies) in which CDR sequences derived from another mammalian species, such as mouse, are transplanted onto human framework sequences. Fully human or human antibodies may be derived from transgenic mice carrying human antibody genes (carrying variable (V), diverse (D), linked (J), and constant (C) exons) or from human cells.

[0113] Human sequences can encode both the heavy and light chains of human antibodies and are rearranged in mice to function correctly and provide a broad antibody repertoire similar to that in humans. Transgenic mice can be immunized with target protein immunogens to produce diverse arrays of specific antibodies and their encoding RNAs. The nucleic acids encoding the antibody chain components of such antibodies may then be cloned from the animals into a presentation vector. Typically, separate populations of nucleic acids encoding the heavy and light chain sequences are cloned, and these separate populations are then recombined upon insertion into the vector so that any given replication of the vector receives a random combination of heavy and light chains. The vector is designed to express antibody chains so that the antibody chains can be assembled and presented on the outer surface of a presentation package containing the vector. For example, the antibody chains may be expressed as a fusion protein with a phage coat protein from the outer surface of a phage. The presentation packages can then be selected and screened for presentation of antibodies that bind to a target.

[0114] In some embodiments, the anti-C5 antibodies described herein include a modified heavy chain constant region having (or not having) reduced effector function compared to its corresponding unmodified constant region. Effector function requiring the constant region of the anti-C5 antibody can be modulated by modifying the properties of the constant region or the Fc region. Modified effector function includes, for example, the modulation of one or more of the following activities: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and pro-inflammatory responses. Modification refers to an increase, decrease, or elimination of effector function activity exhibited by the target antibody containing the modified constant region compared to the activity of the constant region in its unmodified form. In certain embodiments, modification includes situations in which activity is abolished or completely absent.

[0115] A modified constant region having modified FcR binding affinity and / or ADCC activity and / or modified CDC activity is a polypeptide having enhanced or reduced FcR binding activity and / or ADCC activity and / or CDC activity compared to the unmodified constant region. A modified constant region exhibiting increased binding to FcR binds to at least one FcR with better affinity than the unmodified polypeptide. A modified constant region exhibiting decreased binding to FcR binds to at least one FcR with lower affinity than the unmodified constant region. Such variants exhibiting reduced binding to FcR may have little to no preferred binding to FcR, for example, 0-50% of the binding to FcR (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1%) compared to the binding level to the native sequence immunoglobulin constant region or Fc region. Similarly, a modified constant region exhibiting regulated ADCC and / or CDC activity may show either increased or decreased ADCC and / or CDC activity compared to the unmodified constant region. For example, in some embodiments, an anti-C5 antibody containing a modified constant region may exhibit approximately 0–50% of the ADCC and / or CDC activity of the unmodified constant region (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1%). Anti-C5 antibodies described herein, which include a modified constant region that displays reduced ADCC and / or CDC activity, may exhibit reduced ADCC and / or CDC activity or no activity at all.

[0116] In certain embodiments, the modified constant region has at least one amino acid substitution, insertion, and / or deletion compared to the natural sequence constant region or the unmodified constant region, for example, about 1 to about 100 amino acid substitutions, insertions, and / or deletions within the natural sequence constant region or the constant region of the parent polypeptide. In some embodiments, the modified constant region as herein has at least about 70% homology (similarity) or identity with the unmodified constant region, and in some examples at least about 75%, in other examples at least about 80%, and in other embodiments at least about 85%, 90%, or 95% homology or identity with it. The modified constant region may include one or more amino acid deletions or insertions. Additionally, the modified constant region may include one or more amino acid substitutions, deletions, or insertions (e.g., addition of one or more sugar components, loss of one or more sugar components, or a change in the composition of one or more sugar components compared to the unmodified constant region) resulting in a modified post-translational modification, for example, including a modified glycosylation pattern.

[0117] Antibodies with or without modified effector function can be produced by manipulating or generating antibodies having heterologous constant, Fc, or heavy chain regions, and antibodies with modified function and / or activity can be produced using recombinant DNA technology and / or cell culture and expression conditions. For example, recombinant DNA technology can be used to manipulate one or more amino acid substitutions, deletions, or insertions in regions affecting antibody function, including effector function (e.g., Fc or constant region). Alternatively, post-translational modifications, such as changes in glycosylation patterns, can be achieved by manipulating cell cultures and expression conditions under which antibodies are produced. Preferred methods for introducing one or more substitutions, additions, or deletions into the Fc region of an antibody are well known in the art, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2 ndEdition, "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), Borrebaek (1992), John et al. (1993), PCT Publication No. 06 / 53301, and U.S. Patent No. 7,704,497, including, for example, standard DNA mutagenesis techniques.

[0118] In some embodiments, the anti-C5 antibodies described herein exhibit reduced or no effector function. In some embodiments, the anti-C5 antibody includes a hybrid constant region or a portion thereof such as a G2 / G4 hybrid constant region (see, for example, Burton et al. (1992) Adv Immun 51:1-18, Canfield et al. (1991) J Exp Med 173:1483-1491, and Mueller et al. (1997) Mol Immunol 34(6):441-452). See above.

[0119] In addition to using the G2 / G4 constructs described above, anti-C5 antibodies described herein having reduced effector function can be generated by introducing other types of changes into the amino acid sequence of a specific region of the antibody. Such amino acid sequence changes include, but are not limited to, the Ala-Ala mutation described, for example, in PCT Publications 94 / 28027 and 98 / 47531, and in Xu et al. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, anti-C5 antibodies having one or more mutations in a constant region including an Ala-Ala mutation have reduced effector function or none at all. According to these embodiments, the constant region of the antibody may include a substitution for alanine at position 234 or a mutation for alanine at position 235. Additionally, the modified constant region may include a double mutation, i.e., a mutation for alanine at position 234 and a second mutation for alanine at position 235. In one embodiment, the anti-C5 antibody comprises an IgG4 framework, and the Ala-Ala mutation describes a mutation(s) from phenylalanine to alanine at position 234 and / or a mutation from leucine to alanine at position 235. In another embodiment, the anti-C5 antibody comprises an IgG1 framework, and the Ala-Ala mutation describes a mutation(s) from leucine to alanine at position 234 and / or a mutation from leucine to alanine at position 235. The anti-C5 antibody may, as an alternative or additional measure, carry other mutations, including a point mutation K322A within the CH2 domain (Hezareh et al. (2001) J Virol 75:12161-12168). Antibodies having the aforementioned mutation(s) within the constant region may further be blocking or non-blocking antibodies.

[0120] When introduced into the heavy chain constant region, the additional substitution results in a reduction of effector function, as described, for example, Shields et al. (2001) J Biol Chem 276(9):6591-6604. See Table 1, in particular, of Shields et al. (binding of human IgG1 variants to human FcRn and FcγR (the disclosure is incorporated herein in its entirety by reference)). By screening a library of anti-IgE antibodies (each antibody in the library differing in one or more substitutions within the heavy chain constant region) for binding to a population of Fc receptors (including FcRn, FcγRI, FcγRIIA, and FcγRIIIA), the authors identified several substitutions that modulate specific Fc-Fc receptor interactions. For example, a mutant IgG2a heavy chain constant region in which the CH2 domain contains a D265A substitution (heavy chain amino acid numbering by Kabat et al. (above)) results in a complete loss of interaction between the mutant constant region and the IgG Fc receptors FcγRIIB, FcγRIII, FcγRI, and FcγRIV. Table 1, Shields et al. (2001), pp. 6595. Baudino et al. (2008), J Immunol See also 181:6664-6669 (above).

[0121] Changes within the hinge region also affect effector function. For example, deletion of the hinge region may reduce affinity for Fc receptors and thus reduce complement activation (Klein et al. (1981) Proc Natl Acad Sci). (USA 78:524-528). Therefore, this disclosure also relates to antibodies having modifications within the hinge region.

[0122] In some embodiments, anti-C5 antibodies may contain a modified constant region exhibiting enhanced or reduced complement-dependent cytotoxicity (CDC). Modified CDC activity can be achieved by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region of the antibody. See, for example, U.S. Patent No. 6,194,551. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds within this region. Thus, the resulting homodimeric antibodies may have improved or reduced internalization ability and / or increased or decreased complement-mediated cytotoxicity. For example, see Caron et al. (1992) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922, PCT Publication Nos. 99 / 51642 and 94 / 29351, Duncan and Winter (1988) Nature 322:738-40, and U.S. Patents Nos. 5,648,260 and 5,624,821.

[0123] Another potential means of modulating antibody effector function is alteration of glycosylation, summarized, for example, in Raju (2003) BioProcess International 1(4):44-53. Wright and Morrison showed that minute heterogeneity of human IgG oligosaccharides can affect biological functions such as CDC and ADCC, binding to various Fc receptors, and binding to Clq proteins (1997) TIBTECH 15:26-32. Antibody glycosylation patterns can vary depending on productive cells and cell culture conditions (Raju, above). Such differences can lead to changes in both effector function and pharmacokinetics. For example, Israel et al. (1996) Immunology 89(4):573-578, and Newkirk et al. See al. (1996) Clin Exp Immunol 106(2):259-264. Differences in effector function may be related to the ability of IgG to bind to the Fcγ receptor (FcγR) on effector cells. Shields et al. showed that IgG with amino acid sequence modifications that improve binding to FcγR can exhibit up to 100% enhanced ADCC using human effector cells. (2001) J Biol Chem 276(9):6591-6604. These modifications include amino acid changes not found at the binding interface, but both the properties of the sugar components and their structural patterns may also contribute to the observed differences. In addition, the presence or absence of fucose in the oligosaccharide component of IgG can improve binding and ADCC. See, for example, Shields et al. (2002) J Biol Chem 277(30):26733-26740. Asn 297 IgG lacking the fucosylated carbohydrate bound to the receptor exhibited normal receptor binding to the FcγRI receptor. In contrast, binding to the FcγRIIIIA receptor was 50-fold improved, especially at lower antibody concentrations, and was accompanied by enhanced ADCC.

[0124] Further methods exist for modifying the effector function of antibodies. For example, antibody-producing cells may be highly mutagenic, thereby producing antibodies with randomly modified polypeptide residues throughout the antibody molecule. See, for example, PCT Publication 05 / 011735. Examples of highly mutagenic host cells include those deficient in DNA mismatch repair. Antibodies produced by this method may have low antigenicity and / or beneficial pharmacokinetic properties. Additionally, such antibodies may be selected for properties such as enhanced or reduced effector function. Further details of molecular biological techniques useful for preparing the antibodies or antigen-binding fragments described herein are provided below.

[0125] Recombinant antibody expression and purification The antibodies or antigen-binding fragments described herein can be produced using a variety of techniques known in the arts of molecular biology and protein chemistry. For example, nucleic acids encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector containing transcription and translation control sequences, including, for example, a promoter sequence, a ribosome binding site, transcription start and stop sequences, translation start and stop sequences, a transcription terminator signal, a polyadenylation signal, and an enhancer or activator sequence. The control sequences include the promoter, as well as the transcription start and stop sequences. In addition, the expression vector may include multiple replication systems so that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression, and in a prokaryotic host for cloning and amplification.

[0126] Various modifications, such as substitutions, can be introduced into the DNA sequences encoding the heavy and / or light chain polypeptides described herein using standard methods known to those skilled in the art. For example, the introduction of histidine substitutions at one or more CDR sites of an antibody can be carried out using standard methods such as PCR-mediated mutagenesis, where the mutant nucleotide is incorporated into a PCR primer so that the PCR product contains the desired mutation or site-directed mutagenesis. The substitution is introduced into one or more of the CDR regions, for example, the K of the antibody against the antigen at pH 7.4 or pH 6.0. D This can be increased or decreased. Techniques for site-directed mutagenesis are well known in the art. See, for example, Sambrook et al. (above).

[0127] Several possible vector systems can be used for the expression of cloned heavy and light chain polypeptides from nucleic acids in mammalian cells. One group of vectors relies on the integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). Selectable marker genes can either be bound to the DNA gene sequence to be expressed or introduced into the same cell by simultaneous translocation (Wigler et al. (1979) Cell 16:77). A second group of vectors utilizes DNA elements that confer self-replication capability to extrachromosomal plasmids. These vectors include bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, and polyomavirus (Deans et al. (1984) Proc It can be derived from animal viruses such as Natl Acad Sci USA 81:1292, or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0128] Expression vectors can be introduced into cells in a manner suitable for subsequent nucleic acid expression. The method of introduction is primarily determined by the target cell type, which is discussed below. Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfusion, polybren-mediated transfusion, protoplast fusion, and direct microinjection.

[0129] Suitable host cells for the expression of antibodies or their antigen-binding fragments include yeast, bacteria, insects, plants, and mammalian cells. Of particular interest are bacteria such as Escherichia coli, fungi such as Saccharomyces cerevisiae and Pichia pastris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primate cell lines.

[0130] In some embodiments, antibodies or fragments thereof may be expressed in transgenic animals (e.g., transgenic mammals) and purified therefrom. For example, antibodies may be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk, as described, for example, in Houdebine (2002) Curr Opin Biotechnol 13(6):625-629, van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159, and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157.

[0131] Antibodies and their fragments can be generated from cells by culturing host cells converted with an expression vector containing the nucleic acid encoding the antibody or fragment, under conditions and for a sufficient period of time to enable protein expression. Such conditions for protein expression vary depending on the choice of expression vector and host cell and are readily verifiable by those skilled in the art through routine experiments. For example, antibodies expressed in *E. coli* can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors, and suitable host cells varies depending on several factors and can be readily optimized as needed. The antibodies (or fragments thereof) described herein may be expressed in mammalian cells or in other expression systems, including but not limited to yeast, baculovirus, and in vitro expression systems (e.g., Kaszubska et al. (2000) Protein Expression). See also Purification 18:213-220).

[0132] Following expression, antibodies and their fragments can be isolated. The terms “purified” or “isolated” as applied to any of the proteins (antibodies or fragments) described herein refer to polypeptides that have been separated or purified from components (e.g., proteins or other naturally occurring biomolecules or organic molecules) that naturally accompany, for example, other proteins, lipids, and nucleic acids in prokaryotes that express proteins. Typically, a polypeptide is purified when it constitutes at least 60% by weight of the total protein in the sample (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99% by weight).

[0133] Antibodies or their fragments can be isolated or purified by various methods known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoresis, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reversed-phase HPLC chromatography. For example, antibodies can be purified using standard anti-antibody columns (e.g., protein-A or protein-G columns). Ultrafiltration and dialysis are also useful in conjunction with protein concentration. For example, Scopes (1994) "Protein Purification, 3 rd See edition, "Springer-Verlag, New York City, New York." The required degree of purification varies depending on the desired application. In some cases, purification of the expressed antibody or its fragments is not essential.

[0134] Methods for determining the yield or purity of purified antibodies or fragments thereof are known in the art and include, for example, the Bradford assay, ultraviolet spectroscopy, Biuret protein assay, Lowry protein assay, Amido Black protein assay, high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (using protein stains such as Coomassie blue or colloidal silver staining).

[0135] In some embodiments, endotoxins can be removed from antibodies or fragments. Methods for removing endotoxins from protein samples are known in the art. For example, endotoxins can be removed from protein samples using a variety of commercially available reagents, including, but not limited to, the ProteoSpin® Endotoxin Removal Kit (Norgen Biotek Corporation), Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific, Pierce Protein Research Products), MiraCLEAN® Endotoxin Removal Kit (Mirus), or Acrodisc®-Mustang® E-Membrane (Pall Corporation).

[0136] Methods for detecting and / or measuring the amount of endotoxins present in a sample (both before and after purification) are known in the art, and commercially available kits are available. For example, the concentration of endotoxins in a protein sample can be determined using the QCL-1000 chromogenic kit (BioWhittaker) or Limulus morphological cell lysate (LAL) kits, such as the Pyrotell®, Pyrotell®-T, Pyrochrome®, Chromo-LAL, and CSE kits available from Associates of Cape Cod Incorporated.

[0137] Modification of antibodies or their antigen-binding fragments Antibodies or their antigen-binding fragments may be modified after their expression and purification. The modifications may be covalent or non-covalent. Such modifications may be introduced into the antibody or fragment, for example, by reacting a target amino acid residue of a polypeptide with an organic inducer that can react with selected side-chain or terminal residues. Suitable modification sites may be selected using any of a variety of criteria, including, for example, structural analysis or amino acid sequence analysis of the antibody or fragment.

[0138] In some embodiments, an antibody or its antigen-binding fragment may be compounded with a heterologous moiety. This heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or, but not limited to, a detectable label such as a radiolabel, enzyme label, fluorescent label, heavy metal label, luminescence label, or affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, for example, antigen tags for use in purifying antibodies or fragments. [ka] Polyhistidine [ka] hemagglutinin [ka] Examples include glutathione-S-transferase (GST) or maltose-binding protein (MBP). Heterogeneous polypeptides also include polypeptides useful as diagnostic or detectable markers (e.g., enzymes), such as luciferase, fluorescent proteins (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyltransferase (CAT). Suitable radiolabels include, for example, 32 P, 33 P, 14 C, 125 I, 131 I, 35 S, and 3H is one example. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight® 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Fluorescent labels include, for example, any of various luminescent lanthanides (e.g., europium or terbium) chelates. For example, suitable europium chelates include diethylenetriamipentaacetic acid (DTPA) or europium ichtherate of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0139] Two proteins (e.g., an antibody and a heterologous portion) can be crosslinked using one of several known chemical crosslinking agents. An example of such a crosslinking agent is one that links two amino acid residues via a bond containing a "hindered" disulfide bond. In these bonds, the disulfide bond within the crosslinking unit is protected from reduction (by hindering the group on either side of the disulfide bond) by, for example, reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes the lysine at one terminal of the protein and the cysteine ​​at the other to form such a bond between the two proteins. Heterobifunctional reagents that crosslink by different coupling moieties on each protein can also be used. Other useful crosslinking agents include reagents that bind two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamide]butylamine), and a guanidinium group present in the side chain of an amino group and arginine (e.g., p-azidophenylglyoxal monohydrate).

[0140] In some embodiments, the radiolabel can be directly compounded into the amino acid backbone of the antibody. Alternatively, the radiolabel can be compounded into a larger molecule (e.g., meta-iodophenyl (mIP) derivatives of the associated protein, which bind to free amino groups). 125 I) In iodophenyl-N-hydroxysuccinimide 125 I([ 125[I]mIPNHS) (see, for example, Rogers et al. (1997) J Nucl Med 38:1221-1229) or may be included as part of a chelate (e.g., DOTA or DTPA) that is sequentially bound to the protein backbone. Methods for conjugating radiolabels, or larger molecules / chelates containing them, to the antibody or antigen-binding fragments described herein are known in the art. Such methods require incubating the radiolabeled protein under conditions that promote the binding of the radiolabel or chelate to the protein (e.g., pH, salt concentration, and / or temperature) (see, for example, U.S. Patent No. 6,001,329).

[0141] Methods for conjugating fluorescent labels (sometimes referred to as "fluorophores") to proteins (e.g., antibodies) are known in the field of protein chemistry. For example, a fluorophore can be conjugated to a free amino group (e.g., of lysine) or sulfhydryl group (e.g., of cysteine) of a protein using a succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moiety attached to the fluorophore. In some embodiments, the fluorophore can be conjugated to a heterobifunctional crosslinking agent moiety such as sulfo-SMCC. A preferred conjugation method requires incubation of the antibody protein or a fragment with the fluorophore under conditions that promote the binding of the fluorophore to the protein. For example, Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications," John Wiley and Please refer to Sons (ISBN 0471495603).

[0142] In some embodiments, the antibody or fragment may be modified with a moiety that improves the stabilization and / or retention of the antibody in circulation, for example, in blood, serum, or other tissues. For example, the antibody or fragment may be PEGylated or HES-modified as described, for example, Lee et al. (1999) Bioconjug Chem 10(6):973-8, Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485, and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 (see Fresenius Kabi, Germany, e.g., Pavisic et al. (2010) Int J Pharm 387(1-2):110-119). The stabilizing portion can improve the stability or retention of the antibody (or fragment) by at least 1.5 times (for example, at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, or 50 times or more).

[0143] In some embodiments, the antibodies or antigen-binding fragments described herein can be glycosylated. In some embodiments, the antibodies or antigen-binding fragments described herein can be subjected to enzyme therapy or chemotherapy, or can be generated from cells, so that the antibody or fragment has reduced or absent glycosylation. Methods for generating antibodies having reduced glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368, Wright et al. (1991) EMBO J 10(10):2717-2723, and Co et al. (1993) Mol Immunol 30:1361.

[0144] Pharmaceutical compositions and preparations The compositions described herein may be formulated as pharmaceutical solutions for administration to a subject, for example, for the treatment or prevention of complement-related disorders. Pharmaceutical compositions generally include pharmaceutically acceptable carriers. As used herein, “pharmaceutically acceptable carriers” means, and includes, any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and physiologically compatible equivalents. Compositions may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, for example, Berge et al. (1977) J Pharm Sci 66:1-19).

[0145] The composition may be formulated according to standard methods. Pharmaceutical formulation is a well-established field, for example, Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20 th Edition, Lippincott, Williams & Wilkins (ISBN:0683306472), Ansel et al. (1999)″Pharmaceutical Dosage Forms and Drug Delivery Systems,″7 th Edition, Lippincott Williams & Wilkins Publishers (ISBN:0683305727), and Kibbe (2000)″Handbook of Pharmaceutical Excipients American Pharmaceutical Association,″3 rdFurther details are provided in Edition (ISBN: 091733096X). In some embodiments, the composition may be formulated, for example, as a buffer solution suitable for storage at 2–8°C (e.g., 4°C) at a preferred concentration. In some embodiments, the composition may be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition may be formulated for storage at 2–8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, in some embodiments, the compositions described herein are stable for storage at 2–8°C (e.g., 4°C) for at least 1 year.

[0146] Pharmaceutical compositions can take various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends, in part, on the intended mode of administration and therapeutic application. For example, compositions containing a composition intended for systemic or topical delivery may take the form of an injection solution or infusion solution. Thus, compositions may be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). When used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent expressions refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions (see below).

[0147] The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions may be prepared by incorporating the required amount of the compositions described herein, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the compositions described herein into a sterile medium containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the method for preparation includes vacuum drying and lyophilization, in addition to the powder of the compositions described herein, to produce any additional desired components (see below) from its previously sterile filtered solution. Appropriate fluidity of the solutions may be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Long-term absorption of injectable compositions may be achieved by including absorption-delaying reagents in the composition, such as monostearate and gelatin.

[0148] The compositions described herein may also be incorporated into immunoliposome compositions. Such formulations may be prepared by methods known in the art, such as those described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688, Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030, and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes having increased circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0149] In certain embodiments, the composition may be formulated with a controlled-release formulation comprising a carrier that protects the compound from immediate release, such as an embedding piece and a microencapsulation delivery system. Biodegradable, biocompatible polymers such as vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0150] In some embodiments, compositions may be incorporated into compositions suitable for intrapulmonary administration (e.g., administration via inhaler or nebulizer) to mammals such as humans. Methods for formulating such compositions are well known in the art and are described, for example, in U.S. Patent Application Publication No. 20080202513, U.S. Patents No. 7,112,341 and No. 6,019,968, and PCT Publications No. 00 / 061178 and No. 06 / 122257 (each disclosure is incorporated herein by reference in its entirety). Dry powder inhaler formulations and systems suitable for administration of the formulations are described, for example, in U.S. Patent Application Publication No. 20070235029, PCT Publication No. 00 / 69887, and U.S. Patent No. 5,997,848. Additional formulations suitable for intrapulmonary administration (and methods for formulating polypeptides) are described, for example, in U.S. Patent Application Publications 20050271660 and 20090110679.

[0151] In some embodiments, compositions may be formulated for delivery to the eye. As used herein, the term “eye” refers to any and all anatomical tissues and structures associated with the eye. The eye has a wall consisting of three distinct layers: the outer sclera, the middle choroidal layer, and the inner retina. Behind the lens is a chamber filled with a gel-like fluid called the vitreous fluid. At the back of the eye is the retina, which detects light. The cornea is an optically transparent tissue that transmits images to the back of the eye. The cornea contains one pathway for the penetration of drugs into the eye. Other anatomical tissue structures associated with the eye include the lacrimal system, which includes the secretory, distributing, and excretory systems. The secretory system includes lacrimal secretions stimulated by blinking and temperature changes, resulting from reflex secretions that have a tear evaporation and efferent parasympathetic supply and secrete tears in response to physical or emotional stimuli. The distributing system includes the lacrimal meniscus around the eyelids and the margins of the open eyelid, which spreads the tears across the entire ocular surface by blinking, thus reducing the expansion of the dry area.

[0152] In some embodiments, the composition may be administered topically, for example, by topical application or intravitreal injection. For example, in some embodiments, the composition may be formulated for administration by eye drops.

[0153] Therapeutic preparations for treating the eyes may contain one or more active agents in a pharmaceutically acceptable solution, suspension, or ointment at a concentration of about 0.01% to about 1% by weight, preferably about 0.05% to about 0.5%. The preparations preferably take the form of a sterile aqueous solution containing additional components such as, for example, but not limited to, preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, and thickeners.

[0154] Suitable preservatives for use in such solutions include benzalkonium chloride, benzethonium chloride, chlorobutanol, and thimerosal. Suitable buffers include, for example, boric acid, sodium bicarbonate and potassium, sodium borate and potassium, sodium carbonate and potassium, sodium acetate, and sodium diphosphate in amounts sufficient to maintain the pH between approximately pH 6 and pH 8, preferably between approximately pH 7 and pH 7.5. Suitable isotonic agents include dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, and sodium chloride.

[0155] Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, and thiourea. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282, and tyroxapol. Suitable thickeners include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethylcellulose. The preparations can be administered topically to subjects requiring conventional treatment (e.g., subjects suffering from AMD), for example, in the form of droplets or by immersing the eyes in a therapeutic solution containing one or more of the compositions.

[0156] In addition, various devices have been developed to deliver drugs into the vitreous cavity of the eye. For example, U.S. Patent Application Publication No. 20020026176 describes a drug-containing plug that can be inserted through the pleura to protrude into the vitreous cavity and deliver the drug to the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for delivery into the suprachoroidal space or avascular area for sustained release of a drug into the inside of the eye. U.S. Patents No. 5,773,019 and No. 6,001,386 disclose implantable drug delivery devices that can be attached to the pleural surface of the eye, respectively. The device comprises an internal core containing an effective amount of a poorly soluble drug coated with a non-biodegradable polymer that is permeable to the poorly soluble drug. During operation, the poorly soluble drug permeates the biodegradable polymer coating for sustained release from the device. Additional methods and devices for the delivery of therapeutic drugs to the eye (e.g., delivery via transpleural patches and contact lenses) are described, for example, Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151, Ranta and Urtti (2006) Adv This information is found in Drug Delivery Rev 58(11):1164-1181, Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10), Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347, Kim et al. (2007) Ophthalmic Res 39:244-254, and PCT Publication No. 04 / 073551, the entirety of which is incorporated herein by reference.

[0157] As described above, compositions with relatively high concentrations can be prepared. For example, these compositions may be formulated at concentrations of approximately 10 mg / mL to 100 mg / mL (e.g., approximately 9 mg / mL to 90 mg / mL, approximately 9 mg / mL to 50 mg / mL, approximately 10 mg / mL to 50 mg / mL, approximately 15 mg / mL to 50 mg / mL, approximately 15 mg / mL to 110 mg / mL, approximately 15 mg / mL to 100 mg / mL, approximately 20 mg / mL to 100 mg / mL, approximately 20 mg / mL to 80 mg / mL, approximately 25 mg / mL to 100 mg / mL, approximately 25 mg / mL to 85 mg / mL, approximately 20 mg / mL to 50 mg / mL, approximately 25 mg / mL to 50 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 30 mg / mL to 50 mg / mL, approximately 40 mg / mL to 100 mg / mL, or approximately 50 mg / mL to 100 mg / mL). In some embodiments, the composition may be formulated at a concentration greater than 5 mg / mL but less than 50 mg / mL. Methods for formulating proteins in aqueous solutions are known in the art and are described, for example, in U.S. Patent No. 7,390,786, McNally and Hastedt (2007), "Protein Formulation and Delivery," Second Edition, Drugs and the Pharmaceutical Sciences, Volume 175, CRC Press, and Banga (2005), "Therapeutic peptides and proteins: formulation, processing, and delivery systems, Second Edition," CRC Press. In some embodiments, the aqueous solution has a neutral pH, for example, 6.5 to 8 (e.g., 7 to 8, including both ends). In some embodiments, the aqueous solution has a pH of about 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the aqueous solution has a pH greater than (or equal to) 6 (e.g., 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9 or greater) but a pH less than 8.

[0158] Nucleic acids encoding therapeutic polypeptides can be incorporated into gene constructs used as part of gene therapy protocols such that they are expressed intracellularly and deliver nucleic acids that can be used to generate agents. Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Techniques include the insertion of the target gene in viral vectors including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1 (HSV-1), or recombinant bacteria or eukaryotic plasmids. Viral vectors can directly transform cells, and plasmid DNA can be delivered, for example, with the aid of cationic liposomes (lipofectins) or induced polylysine complexes, gramicidin S, artificial viral envelopes or other such intracellular carriers, and by direct injection of gene constructs or CaPO4 precipitation (see, e.g., WO 04 / 060407) carried out in vivo. (See also the "ex vivo techniques" below.) Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM known to those of skill in the art (e.g., Eglitis et al. (1985) Science 230:1395-1398, Danos and Mulligan (1988) Proc Natl Acad Sci USA 85:6460-6464, Wilson et al. (1988) Proc Natl Acad Sci USA 85:3014-3018, Armentano et al. (1990) Proc Natl Acad Sci USA 87:6141-6145, Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043, Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644, Kay et al. (1992) Human Gene See Therapy 3:641-647, Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895, Hwu et al. (1993) J Immunol 150:4104-4115, U.S. Patents Nos. 4,868,116 and 4,980,286, and PCT Publications Nos. 89 / 07136, 89 / 02468, 89 / 05345 and 92 / 07573. Another viral gene delivery system utilizes adenovirus-derived vectors (see, for example, Berkner et al. (1988) BioTechniques 6:616, Rosenfeld et al. (1991) Science 252:431-434, and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenovirus vectors derived from adenovirus strain Ad type 5 dl324 or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Yet another viral vector system useful for delivering target genes is adeno-associated virus (AAV). For example, see Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356, Samulski et al. (1989) J Virol 63:3822-3828, and McLaughlin et al. (1989) J Virol 62:1963-1973.

[0159] In some embodiments, the composition may be combined with one or more additional therapeutic agents, for example, additional therapies for treating or preventing complement-related disorders (e.g., AP-related disorders or CP-related disorders) in the subject. Additional agents for treating complement-related disorders in the subject may vary depending on the specific disorder being treated, but may include, without limitation, antihypertensive agents (e.g., angiotensin-converting enzyme inhibitors) [e.g., for use in treating HELLP syndrome], anticoagulants, corticosteroids (e.g., prednisone), or immunosuppressants (e.g., vincristine or cyclosporine A). Examples of anticoagulants include, for example, warfarin (coumadin), aspirin, heparin, phenindione, fondaparinux, hydraparinux, and thrombin inhibitors (e.g., argatroban, repiridine, bivalirudine, or dabigatran). The compositions described herein may also be combined with fibrinolytic agents for the treatment of complement-related disorders (e.g., Anklod, ε-aminocaproic acid, anti-plasmin-α1, prostacyclin, and defibrotide). In some embodiments, the compositions may be combined with lipid-lowering agents such as inhibitors of hydroxymethylglutaryl-CoA reductase. In some embodiments, the compositions may be combined with or used in conjunction with anti-CD20 agents such as rituximab (Rituxan®, Biogen Idec, Cambridge, MA). In some embodiments, for example, in the treatment of rheumatoid arthritis (RA), the compositions may be combined with one or both of infliximab (Remicade®, Centocor, Inc.) and methotrexate (Rheumatrex®, Trexall®). In some embodiments, the compositions described herein may be combined with nonsteroidal anti-inflammatory drugs (NSAIDs).Many different NSAIDs are available, and some over-the-counter medications include ibuprofen (Advil®, Motrin®, Nuprin®) and naproxen (Alleve®). Many others are available by prescription, including meloxicam (Mobic®), etodolac (Lodine®), nabumetone (Relafen®), sulindac (Clinoril®), tolmetin (Tolectin®), choline magnesium trisalicylate (Trilasate®), diclofenac (Cataflam®, Voltaren®, Arthrotec®), diflunisal (Dolobid®), indomethacin (Indocin®), ketoprofen (Orudis®, Oruvail®), oxaprozin (Daypro®), and piroxicam (Feldene®). In some embodiments, the composition may be formulated for use with an antihypertensive agent, an antiepileptic agent (e.g., magnesium sulfate), or an antithrombotic agent. Examples of antihypertensive agents include, for example, labetalol, hydralazine, nifedipine, calcium channel blockers, nitroglycerin, or sodium nitroprusside. (See, e.g., Mihu et al. (2007) J Gastrointestin Liver Dis 16(4):419-424.) Examples of antithrombotic agents include, for example, heparin, antithrombin, prostacyclin, or low-dose aspirin.

[0160] In some embodiments, a composition formulated for intrapulmonary administration may include at least one additional activator for treating lung injury. This at least one activator may be, for example, an anti-IgE antibody (e.g., omalizumab), an anti-IL-4 antibody or anti-IL-5 antibody, an anti-IgE inhibitor (e.g., montelukast sodium), a sympathetic neuron mimetic (e.g., albuterol), an antibiotic (e.g., tobramycin), a deoxyribonuclease (e.g., Pulmozyme®), an anticholinergic (e.g., ipratropium bromide), a corticosteroid (e.g., dexamethasone), a β-adrenergic receptor agonist, a leukotriene inhibitor (e.g., dileuton), a 5-lipoxygenase inhibitor, a PDE inhibitor, a CD23 antagonist, an IL-13 antagonist, a cytokine release inhibitor, a histamine H1 receptor antagonist, an antihistamine, an anti-inflammatory agent (e.g., cromolyn sodium), or a histamine release inhibitor.

[0161] In some embodiments, the composition may be formulated for administration with one or more additional therapeutic agents for use in treating complement-related disorders of the eye. Such additional therapeutic agents may be, for example, bevacizumab or a Fab fragment of bevacizumab, or ranibizumab (all marketed by Roche Pharmaceuticals, Inc.), and pegaptanib sodium (Mucogen®, Pfizer, Inc.). Such kits may optionally include instructions for administering the composition.

[0162] In some embodiments, the composition may be formulated for administration to a subject in conjunction with intravenous gamma globulin therapy (IVIG), plasma exchange therapy, plasma replacement, or plasma exchange. In some embodiments, the composition may be formulated before, during, or after kidney transplantation.

[0163] When a composition is to be used in combination with a second activator, the composition may be co-formulated with the second agent, or it may be formulated separately from the second agent. For example, each pharmaceutical composition may be mixed and administered together, for example, immediately before administration, or administered separately, for example, at the same time or at different times (see below).

[0164] Applicable The compositions described herein may be used in several diagnostic and therapeutic applications. For example, detectably labeled antigen-binding molecules can be used in assays to detect the presence or amount of a target antigen in a sample (e.g., a biological sample). The compositions may be used in in vitro assays to study the inhibition of target antigen function. For example, in some embodiments in which the compositions bind to and inhibit complement proteins, the compositions may be used as a positive control in assays designed to identify additional novel compounds that inhibit complement activity or otherwise are useful for treating complement-related disorders. For example, a C5 inhibitory composition may be used as a positive control in assays to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that reduce or suppress C5 production or MAC formation. The compositions may also be used in therapeutic methods detailed below.

[0165] Treatment method The compositions described herein may be administered to subjects, such as human subjects, by various methods that are partially dependent on the route of administration. The route may be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection (IM).

[0166] Subcutaneous administration can be achieved by a device. This device means may be a syringe, a pre-filled syringe, a single-use or reusable auto-injector, a pen syringe, a patch syringe, a attachable syringe, a morphological syringe injection pump with a subcutaneous injection set, or other devices for compounding antibody drugs for subcutaneous injection.

[0167] Administration may be achieved, for example, by local injection, injection, or graft. The graft may be a membrane such as a silastic membrane, or a porous, non-porous, or gel-like material containing fibers. The graft may be configured for sustained or periodic release of the composition to the target. See, for example, U.S. Patent Application Publication No. 20080241223, U.S. Patents No. 5,501,856, 4,863,457, and 3,710,795, European Patent No. 488401, and 430539 (each disclosure is incorporated herein by reference in its entirety). The compositions described herein may be delivered to the target by implantable devices based on diffusion, erosion, or convection systems, such as osmotic pumps, biodegradable grafts, electron diffusion systems, electron osmotic pumps, vapor pressure pumps, electrolytic pumps, foaming pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

[0168] In some embodiments, the compositions described herein are therapeutically delivered to a subject by topical administration. As used herein, “topical administration” or “topical delivery” refers to delivery that does not rely on the transport of the composition or drug to its intended target tissue or site via the vascular system. For example, a composition may be delivered by injection or implantation of the composition or drug, or by injection or implantation of a device containing the composition or drug. Following topical administration near the target tissue or site, the composition or drug, or one or more of its components, may diffuse into the intended target tissue or site.

[0169] In some embodiments, the compositions described herein may be administered topically to a joint (e.g., a junction). For example, in embodiments where the disorder is arthritis, a therapeutically appropriate composition may be administered directly to or near the joint (e.g., intra-articular). Examples of intra-articular joints to which the compositions described herein may be administered topically include, for example, the buttocks, knee, elbow, wrist, sternoclavicular, temporomandibular, carpal bones, tarsal bones, ankle, and any other joints that are subject to arthritis. The compositions described herein may also be administered to sacs such as, for example, the acromion, biceps radius, cubital radius, deltoid muscle, infrapatella, ischium, and any other sacs known in the medical field.

[0170] In some embodiments, the compositions described herein may be administered topically to the eye. As used herein, the term “eye” refers to any and all anatomical tissues and structures associated with the eye. The eye has a wall consisting of three distinct layers: the outer sclera, the middle choroidal layer, and the medial retina. Behind the lens is a chamber filled with a gel-like fluid called the vitreous humor. At the back of the eye is the retina, which detects light. The cornea is an optically transparent tissue that transmits images to the back of the eye. The cornea contains one pathway for the penetration of drugs into the eye. Other anatomical tissue structures associated with the eye include the lacrimal system, which includes the secretory, distributing, and excretory systems. The secretory system includes secretions stimulated by blinking and temperature changes, resulting from reflex secretions that have a tear evaporation and efferent parasympathetic supply and secrete tears in response to physical or emotional stimuli. The distributing system includes the lacrimal meniscus around the eyelids and the margins of the open eyelid, which spreads the tears across the entire ocular surface by blinking, thus reducing the expansion of the dry area.

[0171] In some embodiments, the compositions described herein are administered into the posterior chamber of the eye. In some embodiments, the compositions described herein are administered intravitreously. In some embodiments, the compositions described herein are administered transsclerally.

[0172] In some embodiments, for example, embodiments for the treatment or prevention of disorders such as COPD or asthma, the compositions described herein may be administered to a target by the lungs. Pulmonary drug delivery may be achieved by inhalation, and administration by inhalation as described herein may be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include constant-dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, the compositions described herein may be administered to the lungs of a target by a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the medical field and include, but are not limited to, TurboHaler® (AstraZeneca, London, England), AIR® inhaler (Alkermes®, Cambridge, Massachusetts), Rotahaler® (GlaxoSmithKline, London, England), and Eclipse® (Sanofi-Aventis, Paris, France). See also, for example, PCT Publications 04 / 026380, 04 / 024156, and 01 / 78693. DPI devices are used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, the compositions described herein may be administered intrapulmonaryly by a constant-dose inhaler. These inhalers rely on a propellant to deliver distinct doses of the compound to the lungs. Examples of compounds administered by a constant-dose inhaler include, for example, Astovent® (Boehringer-Ingelheim, Ridgefield, Connecticut) and Flovent® (GlaxoSmithKline). See also, for example, U.S. Patents 6,170,717, 5,447,150, and 6,095,141.

[0173] In some embodiments, the compositions described herein may be administered to the target lungs by a sprayer. The sprayer uses compressed air to deliver the compound as a liquefied aerosol or mist. The sprayer may be, for example, a jet sprayer (e.g., an air or liquid jet sprayer) or an ultrasonic sprayer. Additional devices and methods of intrapulmonary administration are described, for example, in U.S. Patent Application Publications 20050271660 and 20090110679 (each disclosure is incorporated herein by reference in its entirety).

[0174] In some embodiments, the compositions provided herein exist in unit dosage forms and may be particularly suitable for self-administration. The formulations of the Disclosure may typically be contained in containers such as vial cartridges, filled syringes, or disposable pens. Dispensing devices, such as the dispensing device described in U.S. Patent No. 6,302,855, may be used, for example, in conjunction with the injection systems of the Disclosure.

[0175] The injection system of this disclosure may utilize a delivery pen such as that described in U.S. Patent No. 5,308,341. Pen devices most commonly used for self-delivery of insulin to diabetic patients are well known in the art. Such a device may comprise at least one injection needle (e.g., a 31-gauge needle with a length of approximately 5–8 mM) and is typically pre-filled with one or more therapeutic unit doses of therapeutic solution, and is useful for rapid delivery of the solution to the target with as little pain as possible.

[0176] A drug delivery pen includes a vial holder from which a vial of insulin or other drug can be received. The vial holder is an elongated, substantially tubular structure having a proximal end and a distal end. The distal end of the vial holder includes mounting means for engaging with a double-ended needle cannula. The proximal end also includes mounting means for engaging with a pen body, which includes a drive and a dose setting device. A single-use drug (e.g., a high-concentration solution of the composition described herein) containing a vial for use with a prior art vial holder includes a distal end having a puncturable elastic septum that can be punctured by one end of a double-ended needle cannula. The proximal end of the vial includes a stopper slidably disposed in liquid-tight engagement with the cylindrical wall of the vial. The drug delivery pen is used by inserting a vial of drug into the vial holder. The pen body is then connected to the proximal end of the vial holder. The pen body includes a dose setting device for specifying the dose of drug to be delivered by the pen, and a drive for advancing the vial stopper distally by a distance corresponding to the selected dose. The pen user places the bilateral needle cannulas on both ends of the vial holder, ensuring the proximal tips of the needle cannulas puncture the septum on the vial. The patient then selects a dose and operates the pen to advance the stopper distally, delivering the selected dose. The dose selection device returns to zero after the selected dose has been injected. The patient then removes and discards the needle cannulas and holds the drug delivery pen in a convenient location for the next required drug administration. The drug in the vial is used up after several doses of such drug. The patient then separates the vial holder from the pen body. The empty vial can then be removed and discarded. A new vial can be inserted into the vial holder, and the vial holder and pen body can be reassembled and used as described above. Thus, drug delivery pens generally have a driving mechanism for accurate drug delivery and ease of use.

[0177] Dosage mechanisms such as a rotating knob enable the user to accurately adjust the amount of drug to be injected by a pen from a pre-packaged vial of drug. To inject the dosage of the drug, the user inserts the needle subcutaneously and presses the knob once as far as it can be pushed. The pen may be an overall mechanical device or may be combined with an electronic circuit to accurately set and / or indicate the dosage of the drug to be injected into the user. See, for example, U.S. Patent No. 6,192,891.

[0178] In some embodiments, the needle of the pen device is disposable, and the kit includes one or more disposable replacement needles. Devices suitable for the delivery of any one of the compositions discussed herein are also described in, for example, U.S. Patent Nos. 6,277,099, 6,200,296, and 6,146,361 (the disclosures of each are incorporated herein by reference in their entirety). Microneedle-based pen devices are described in, for example, U.S. Patent No. 7,556,615 (the disclosure of which is incorporated herein by reference in its entirety). See also the Precision Pen Injector (PPI) device, Molly (trademark), manufactured by Scandinavian Health Ltd.

[0179] The present disclosure also presents controlled release or long-term release formulations suitable for the chronic and / or self-administration of drugs such as the compositions described herein. The various formulations can be administered to patients who use the drug as a bolus or who require treatment by continuous infusion over a long period of time.

[0180] In some embodiments, the high-concentration compositions described herein are formulated for sustained release, long-term release, timed release, controlled release, or continuous release administration. In some embodiments, depot formulations are used to administer the composition to a subject that requires it. In this method, the composition is formulated with one or more carriers that provide a stepwise release of the active agent over a period of hours or days. Such formulations often are based on a degradable matrix that disperses in the body in a stepwise manner to release the active agent.

[0181] In some embodiments, the compositions described herein are administered to subjects requiring them by a sprayer or inhaler. For example, the compositions described herein may be delivered by a sprayer or inhaler to subjects (e.g., humans) suffering from conditions such as asthma or COPD.

[0182] The preferred dose of the composition described herein that can treat or prevent a disorder in a subject may depend on various factors, including, for example, the age, sex, and weight of the subject being treated, and the specific inhibitory compound used. For example, a different dose of one composition (e.g., an anti-C5 composition) may be required to treat a subject with RA compared to the doses of different compositions (e.g., anti-TNFα compositions) required to treat that subject. Other factors that may influence the dose administered to a subject include, for example, the type or severity of the disorder. For example, a subject with RA may require a different dose of the anti-C5 composition described herein than a subject with PNH. Other factors include, for example, other medical disorders the subject currently has or has had in the past, the subject's general health, the subject's genetic predisposition, diet, administration time, excretion rate, drug combinations, and any other additional therapeutic agents administered to the subject. It should also be understood that a specific medication and treatment plan for any particular subject may also depend on the judgment of the treating healthcare practitioner (e.g., a physician or nurse).

[0183] The compositions described herein may be administered as a fixed dose or in milligrams per kilogram (mg / kg) doses. In some embodiments, the dose may also be selected to reduce or avoid antibody production or other host immune responses to one or more antigen-binding molecules in the composition. Exemplary doses of antibodies, such as those of the compositions described herein, are, for example, 1 to 1000 mg / kg, 1 to 100 mg / kg, 0.5 to 50 mg / kg, 0.1 to 100 mg / kg, 0.5 to 25 mg / kg, 1 to 20 mg / kg, and 1 to 10 mg / kg. Exemplary doses of the compositions described herein are, without limitation, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg.

[0184] A pharmaceutical solution may contain a therapeutically effective amount of the composition described herein. Such an effective amount can be readily determined by those skilled in the art, based in part on the effect of the composition administered, or, if multiple agents are used, the combined effect of the composition and one or more additional activators. A therapeutically effective amount of the composition described herein may also vary depending on the individual's disease state, age, sex, and weight, as well as factors such as the ability of the composition (and one or more additional activators) to elicit a desired response in that individual, e.g., improvement of at least one state parameter, e.g., improvement of at least one symptom of complement-mediated disorder. For example, a therapeutically effective amount of the composition described herein can inhibit (reduce the severity of or eliminate the occurrence of) and / or prevent any one of the symptoms of a particular disorder and / or a particular disorder known in the art or described herein. A therapeutically effective amount is also such that the therapeutically beneficial effects outweigh any harmful or adverse effects of the composition.

[0185] A preferred human dose of any of the compositions described herein may be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718, Hanouska et al. (2007) Clin Cancer Res 13(2,part 1):523-531, and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0186] The terms “therapeutic amount” or “therapeutic dose,” or similar terms as used herein, are intended to mean an amount of an agent (e.g., a composition described herein) that elicits a desired biological or medical response (e.g., improvement of one or more symptoms of a complement-related disorder). In some embodiments, the pharmaceutical solution described herein contains at least one of the compositions in a therapeutic amount. In some embodiments, the solution contains one or more compositions and one or more additional therapeutic agents (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) so that the composition as a whole is therapeutically effective. For example, the solution may contain an anti-C5 composition and an immunosuppressant described herein, each of which, when combined, is at a therapeutically effective concentration for treating or preventing a complement-related disorder (e.g., COPD, asthma, sepsis, or complement-related inflammatory disorders such as RA) in a subject.

[0187] The toxic and therapeutic effects of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., any animal model of complement-mediated disorders described herein). These procedures may include, for example, LD 50 (A lethal dose for 50% of the population) and ED 50 It can be used to determine the dose that is therapeutically effective in 50% of the population. The dose-to-injury ratio is the therapeutic index, and the ratio LD 50 / ED50 It can be expressed as follows. Compositions described herein that exhibit a high therapeutic index are preferred. Compositions that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue and to minimize potential damage to character cells, thereby reducing side effects.

[0188] Data obtained from cell culture assays and animal studies can be used when formulating dosage ranges for use in humans. The dosages of the compositions described herein are generally minimally or completely non-toxic, and ED-free. 50 The circulating concentration of the composition containing [the substance] is within this range. The dosage may vary within this range depending on the form of administration and route of administration used. In the case of the compositions described herein, the therapeutically effective dose can first be estimated from a cell culture assay. The dose is determined in the cell culture in an animal model. 50 The dosage can be formulated to achieve a circulating plasma concentration range that includes (i.e., the antibody concentration that achieves half of the maximum inhibition of symptoms). Using such information, a useful dose in humans can be determined more accurately. The levels in plasma may be measured, for example, by high-performance liquid chromatography. For example, in some embodiments where local administration (e.g., to the eye or junction) is desired, cell cultures or animal modeling can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

[0189] In some embodiments, these methods can be administered in conjunction with other therapies for complement-related disorders. For example, the composition may be administered to the subject concurrently with, before, or after plasmapheresis, IVIG therapy, or plasmapheresis. See, for example, Appel et al. (2005) J Am Soc Nephrol 16:1392-1404. In some embodiments, the composition may be administered to the subject concurrently with, before, or after kidney transplantation.

[0190] "Subject" as used herein may be any mammal. For example, the subject may be a human, a non-human primate (e.g., an orangutan, gorilla, macaque, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In some embodiments, the subject may be an infant (e.g., a human infant).

[0191] As used herein, the subjects “requiring prevention,” “requiring treatment,” or “needing it” refer to those who, in the judgment of an appropriate medical practitioner (e.g., a physician, nurse, or nursing practitioner in the case of humans, or a veterinarian in the case of non-human mammals), would benefit to some extent from a given treatment.

[0192] The term “prevent” is recognized in the art and, when used in relation to a pathological condition, is well understood in the art and includes the administration of a composition that reduces or delays the onset of symptoms of a medical condition in a subject compared to a subject that does not receive the composition described herein. Therefore, prevention of complement-related disorders such as asthma includes, for example, reducing the degree or frequency of cough, wheezing, or chest pain in a group of patients receiving prophylactic treatment compared to an untreated control group, and / or delaying the onset of cough or wheezing by, for example, a statistically and / or clinically significant amount in the treated group versus the unresponsive control group.

[0193] As described above, the compositions described herein (e.g., anti-C5 compositions) can be used to treat a variety of complement-related conditions, including but not limited to rheumatoid arthritis (RA), lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis (MS), macular degeneration (e.g., age-related macular degeneration (AMD)), hemolysis, hyperhepatic enzyme and hypothrombocytopenic (HELLP) syndrome, sepsis, dermatomyositis, diabetic retinopathy, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, minor immune vasculitis, epidermolysis bullosa, recurrent miscarriage, multiple sclerosis (MS), and traumatic brain injury. For example, see Holers (2008) Immunological Reviews 223:300-316 and Holers and Thurman (2004) Molecular Immunology 41:147-152. In some embodiments, complement-mediated disorders include, but are not limited to, cardiovascular disorders, myocarditis, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renal vascular disorders, mesenteric / intestinal vascular disorders, vascular regeneration to grafts and / or regrafts, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, organ or tissue transplantation, Takayasu's arteriovenous arthritis, capillary leak syndrome, dilated cardiomyopathy, diabetic vascular disorders, thoracoabdominal aortic aneurysms, Kawasaki disease (arteritis), congestive basal embolism (VGE), and restenosis following stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA). (See, for example, U.S. Patent Application Publication No. 20070172483.) In some embodiments, complement-related disorders include myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome, antiphospholipid syndrome (APS), Degos disease, and catastrophe APS (CAPS).

[0194] In some embodiments, the compositions described herein can be used alone or in combination with a second anti-inflammatory agent to treat inflammatory disorders such as, but not limited to, rheumatoid arthritis (RA) (above), inflammatory bowel disease, sepsis (above), septic shock, acute lung injury, disseminated intravascular coagulation (DIC), or Crohn's disease. In some embodiments, the second anti-inflammatory agent may be selected from the group consisting of NSAIDs, corticosteroids, anti-TNF agents such as methotrexate, hydroxychloroquine, etanercept, and infliximab, B-cell depressants such as rituximab, interleukin-1 antagonists, or T-cell costimulators such as abatacept.

[0195] In some embodiments, complement-related disorders include, but are not limited to, amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease, and chronic inflammatory demyelinating neuropathy.

[0196] Complement-related disorders include, but are not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, alpha-1 antitrypsin deficiency, emphysema, bronchiectasis, bronchiolitis obstructive, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular disorders, as well as other complement-related lung disorders.

[0197] In some embodiments, the compositions described herein are administered to a subject to treat, prevent, or improve at least one symptom of a complement-related inflammatory response (e.g., a complement-related inflammatory response of complement-related disorder). For example, the compositions can be used to treat, prevent, and / or improve one or more symptoms associated with complement-related inflammatory responses, such as graft rejection / graft-versus-host disease (GVHD), reperfusion injury (e.g., following cardiopulmonary bypass or tissue transplantation), and tissue injury following other forms of trauma such as burns (e.g., severe burns), blunt trauma, spinal cord injury, or frostbite. For example, see Park et al. (1999) Anesth Analg 99(1):42-48, Tofukuji et al. (1998) J Thorac Cardiovasc Surg 116(6):1060-1068, Schmid et al. (1997) Shock 8(2):119-124, and Bless et al. (1999) Am J Physiol 276(1):L57-L63.

[0198] In some embodiments, the compositions described herein may be administered to a subject as monotherapy. Alternatively, as described above, the compositions may be administered to a subject as combination therapy with another treatment, e.g., another treatment for complement-related disorders or complement-related inflammatory responses. For example, this combination therapy may include administering one or more additional agents (e.g., anticoagulants, antihypertensives, or anti-inflammatory drugs (e.g., steroids)) to a subject (e.g., a human patient) that provide a therapeutic benefit to a subject having or being at risk of developing sepsis. In another example, the combination therapy may include administering one or more additional agents (e.g., anti-IgE antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, or antihistamines) to a subject that provide a therapeutic benefit to a subject having, being at risk of developing, or suspected of having, a complement-related lung disorder such as COPD or asthma. In some embodiments, the compositions and one or more additional activators are administered simultaneously. In other embodiments, the compositions are administered first, and one or more additional activators are administered second. In some embodiments, one or more additional activators are administered first, and the composition is administered second.

[0199] The compositions described herein may replace or enhance previously administered or currently administered therapies. For example, when treating with the compositions described herein, the administration of one or more additional activators may be discontinued or reduced, for example, at lower levels, such as a low level of eculizumab following the administration of the anti-C5 composition described herein. In some embodiments, the administration of the previous therapy may be maintained. In some embodiments, the previous therapy is maintained until the level of the composition reaches a level sufficient to produce a therapeutic effect. The two therapies may be administered in combination.

[0200] Monitoring a subject (e.g., a human patient) for improvement of a disorder (e.g., sepsis, severe burns, rheumatoid arthritis, lupus nephritis, Goodpasture syndrome, or asthma) as defined herein means evaluating the subject for changes in disease parameters, e.g., improvement of one or more symptoms of a given disorder. Many of the symptoms of the above disorders (e.g., complement-related disorders) are well known in the field of medicine. In some embodiments, the evaluation is performed for at least one hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours after administration of the composition described herein, or for at least 1, 2, 4, 10, 13, or 20 days or more, or for at least 1, 2, 4, 10, 13, or 20 weeks or more. The subject may be evaluated during one or more of the following periods: before the start of treatment, during treatment, or after the administration of one or more elements of the treatment. The evaluation may include evaluating the need for further treatment, e.g., whether the dosage, frequency of administration, or duration of treatment should be changed. This may include assessing the need to add or remove selected treatments, for example, any of the treatments for complement-related disorders described herein.

[0201] The following examples are illustrative only, and many variations and equivalents will become apparent to those skilled in the art by reading this disclosure; therefore, they should not be considered to limit the scope of this disclosure in any way. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. [Examples]

[0202] Example 1. The half-life of eculizumab is a combination of several clearance rates. The mean half-life of eculizumab in PNH and aHUS patients receiving a prescribed dosing regimen is approximately 11–12 days, while the expected half-life for humanized monoclonal antibodies with IgG2 / 4 Fc is predicted to be similar to that of antibodies containing IgG2 or IgG4 Fc, approximately 21–28 days. Morell et al. (1970) J Clin Invest 49(4):673-680. To understand the potential impact of antigen-mediated clearance on the overall clearance rate of eculizumab, a human neonatal Fc receptor (hFcRn) mouse model was used (the mice lack endogenous FcRn but are transgenic to hFcRn (B6.Cg-Fcgrt)). tm1Dcr The following experiments were performed using Tg(FCGRT)32Dcr / DcrJ, storage number 014565, Jackson Laboratories, Bar Harbor, Maine. Transgenic FcRn models were used, for example, Petkova et al. (2006) Int Immunology 18(12):1759-1769, Oiao This is described in et al. (2008) Proc Natl Acad Sci USA 105(27):9337-9342 and Roopenian et al. (2010) Methods Mol Biol 602:93-104.

[0203] A single dose of 100 μg of eculizumab in 200 μL of phosphate-buffered saline (PBS) was administered intravenously (iv) to each of five hFcRn transgenic mice. Approximately 100 μL of blood samples were collected from each mouse on days 1, 3, 7, 14, 21, 28, and 35 post-administration. The concentration of eculizumab in serum was measured by ELISA. Briefly, assay plates were coated and blocked with sheep anti-human Igκ light chain capture antibody. The wells of the plates were then brought into contact with serum samples under conditions that would allow eculizumab, if present in the serum, to bind to the capture antibody. The relative amount of eculizumab bound to each well was detected using detectably labeled anti-human IgG4 antibody and quantified compared to a standard curve generated from naive mouse serum containing known amounts of eculizumab.

[0204] The half-life of the antibody serum was calculated using the following formula.

number

[0205] The experimental results are shown in Figure 1. The half-life of eculizumab in the hFcRn mouse model was 13.49 ± 0.93 days.

[0206] To determine the effect of human C5 on the half-life of eculizumab using the hFcRn model, the antibody was pre-mixed with human C5 (Complement Technology Inc., catalog number: A120) in a 4:1 molar ratio before administration, and a dose of 100 μg of eculizumab was administered intravenously (iv) on day 0. Approximately 100 μL of blood was collected in 1.5 mL Eppendorf tubes for serum due to posterior orbital hemorrhage on days 1, 3, 7, 14, 21, 28, and 35.

[0207] As shown in Figure 1, the half-life of eculizumab in the hFcRn mouse model in the presence of C5 was 4.55 ± 1.02 days. These results suggest that, in addition to the endocytosis-mediated antibody clearance mechanism, which is primarily controlled by FcRn-mediated regeneration, the half-life of eculizumab can be significantly influenced by antigen-mediated clearance via human C5.

[0208] Example 2. Amino acid substitutions within the Fc domain of eculizumab increase the half-life of eculizumab, but are not sufficient to outweigh the effect of C5 on eculizumab clearance. Certain amino acid substitutions within the Fc region of IgG antibodies have been shown to reduce the rate of antibody elimination from circulation. Substitutions that increase the binding affinity of IgG antibodies to FcRn at pH 6.0 are examples of such biological effects. See, for example, Dall'Acqua et al. (2006) J Immunol 117:1129-1138 and Ghetie et al. (1997) Nat Biotech 15:637-640. Zalevsky et al. [(2010) Nat Biotech 28:157-159] describe several amino acid substitutions that can increase the half-life of IgG antibodies in serum, e.g., M428L / N434S. Other amino acid substitutions that extend the half-life include, for example, T250Q / M428L and M252Y / S254T / T256E. For example, see International Patent Application Publication No. 2008 / 048545 and Dall'Acqua et al. (2006) J Biol Chem 281:23514-23524. To determine whether one or more amino acid substitutions in the Fc constant region of eculizumab can extend the half-life of eculizumab in serum, the following substitution, namely M252Y / S254T / T256E, was introduced into eculizumab based on the EU numbering index (hereinafter, this variant of eculizumab will be referred to as the YTE variant). The heavy chain constant region consists of the following amino acid sequence. [ka] The amino acid sequence for the full-length heavy-chain polypeptide of the YTE variant of eculizumab is shown in SEQ ID NO: 16.

[0209] The YTE variant was evaluated in conjunction with eculizumab in the hFcRn mouse model described in Example 1. Specifically, 100 μg of eculizumab (IgG2 / 4 Fc region) and eculizumab variants containing the Fc or YTE variant in 200 μL of phosphate-buffered saline (PBS) were administered intravenously (iv) to each of eight hFcRn transgenic mice. Serum was collected from each mouse on days 1, 3, 7, 14, 21, 28, and 35 post-administration. The concentration of each antibody in the serum was measured by ELISA, and the half-life was calculated as described in Example 1. The results are shown in Figure 2 and Table 2. [Table 2]

[0210] As shown in Figure 2 and Table 2, YTE substitution more than doubled the mean half-life of eculizumab from 14.28 ± 1 day to 29.07 ± 4.7 days.

[0211] To determine the effect of human C5 on the half-life of eculizumab YTE variants, mice were administered human C5 as described above in Example 1. A dose of 100 μg of eculizumab, eculizumab-IgG2 variant, or eculizumab-IgG2 YTE variant was administered intravenously on day 0. As shown in Figure 3 and Table 3, the half-lives of eculizumab, eculizumab-IgG2 variant, and eculizumab-IgG2 YTE variant were significantly reduced in the presence of molar excess human C5. Therefore, amino acid substitutions within the FcRn-binding domain of eculizumab were insufficient to outweigh the contribution of C5-mediated clearance to the half-life of eculizumab. [Table 3]

[0212] Example 3. Effect of amino acid substitutions in the CDR of eculizumab on half-life As described above, the half-life of eculizumab in mice is significantly shortened in the presence of its antigen, human C5 (hC5). While not bound by any specific theory or mechanism of action, the accelerated clearance in the presence of the antigen may be partly due to eculizumab's very high affinity (K) for C5. D It is assumed that the results (approximately 30 pM at pH 7.4 and approximately 600 pM at pH 6.0) do not allow for the efficient dissociation of antibody:C5 complexes in the early endosomal compartment after action. Without dissociation, the antibody:antigen complex is either reclaimed in the extracellular compartment via the neonatal Fc receptor (FcRn) or targeted for lysosomal degradation. In either case, the antibody cannot bind more than two C5 molecules during its lifetime.

[0213] Strong affinity of eculizumab for C5 (K DA C5 concentration of approximately 30 pM allows for nearly complete binding of all C5 in the blood, ensuring that even a small amount of C5 is activated to form C5a and TCCs. Therefore, the affinity of eculizumab for C5 is related to the in vivo efficacy of that antibody in patients treated with the antibody. The inventors present a method to weaken the affinity of eculizumab for C5 without impairing the in vivo efficacy of eculizumab. This disclosure is not limited to such methods, but this was achieved by introducing histidine at one or more positions within the CDR of eculizumab. Histidine has a pKa of 6.04. This means that histidine gains a proton when the pH value decreases from 7.4 (blood) to less than 6.0 (early endosomes). Thus, within endosomes, histidine becomes more positively charged. The inventors hypothesized that introducing histidine to or near the C5 binding site within eculizumab could disrupt endosomal binding by shifting the charge within the endosome while maintaining high affinity for C5 in the blood at neutral pH. Such substitution is hypothesized to increase the half-life by promoting the dissociation of the antibody from the antibody:C5 complex in the acidic environment of the endosome, thereby allowing C5 to be degraded in the lysosome while the free antibody is regenerated.

[0214] Using eculizumab as the parent antibody, a series of mutant antibodies were generated in which all CDR positions were replaced with histidine. The heavy chain variable region of eculizumab has the following amino acid sequence. [ka] (The CDR region of the heavy chain variable region is underlined.) The light chain variable region of eculizumab has the following amino acid sequence. [ka]

[0215] Attempts to scan for histidine yielded 66 single histidine substitution variants of eculizumab. The light and heavy chain coding sequences for these antibody variants were cloned into separate "single gene construct" plasmids suitable for expression in identified mammalian cells and sequences. Antibodies containing single amino acid substitutions were transiently expressed in HEK293F cells by simultaneous translocation of single gene constructs encoding a single light or heavy chain. Simultaneous translocation of "wild-type" heavy and light chains representing the unmodified eculizumab CDR sequence was also performed (EHL000). Tissue culture supernatants were normalized for antibody expression levels and used to evaluate antibody binding to human C5 compared to EHL000 using biolayer interferometry on an Octet Red instrument (ForteBio Inc.). In short, antibodies were captured on an anti-human IgG Fc biosensor (ForteBio, catalog number 18-5001). Next, the loaded chips were exposed to a pH 7.4 buffer solution containing 12.5 nM of naturally purified human C5 for 800 seconds to evaluate the association kinetics compared to the parent antibody. Dissociation kinetics were evaluated by transferring the chips to either a pH 7.4 buffer solution or a pH 6.0 buffer solution for 800 seconds. All measurements were repeated to ensure consistency of readings.

[0216] Single histidine substitution mutants of eculizumab were selected based on a set of three characteristics compared to eculizumab. The preferred histidine mutant was the k of eculizumab at pH 7.4. a and k d Although it deviates only slightly, the k of eculizumab at pH 6.0 d It deviated significantly from the standard. The relative threshold selection criteria were as follows: (1) The maximum variation in association kinetics at pH 7.4 was 33% smaller in peak phase shift over 800 seconds compared to the averaged peak phase shift over 800 seconds observed for eculizumab. (2) The maximum variation in dissociation dynamics at pH 7.4 was less than three times the peak phase shift observed for eculizumab at 800 seconds, and (3) The minimum change in dissociation dynamics at pH 6.0 is at least three times lower in peak phase shift beyond 800 seconds compared to the averaged peak phase shift observed for eculizumab at 800 seconds. For example, with respect to item (1) above, if the mean peak phase shift after association with eculizumab at 800 seconds is approximately 0.75 nM, then a test antibody with a phase shift of less than 0.5 nM (e.g., replicated two or more times) does not meet the above criterion. In contrast, a test antibody with a peak phase shift greater than 0.5 nM at 800 seconds meets the first criterion.

[0217] The single substitutions within the light chain variable region that satisfy these thresholds were G31H, L33H, V91H, and T94H for SEQ ID NO: 8. The single substitutions within the heavy chain variable region that satisfy these thresholds were Y27H, I34H, L52H, and S57H for SEQ ID NO: 7. See Figures 5A, 5B, 5C, and 5D.

[0218] A second set of antibodies was generated containing all possible combinations of two histidine substitutions at positions where a single substitution met the threshold criteria. See Table 1. The association and dissociation kinetics of these antibodies were analyzed by the same method and compared with both the original parent antibody and the single histidine substitution. Similarly, third and fourth sets of antibodies containing three or four histidine substitutions were generated, and their association and dissociation kinetics were analyzed against the associated two or three histidine substitution precedents. See Table 1. At each stage, the same criteria were used for the minimum threshold of association kinetics at pH 7.4, the maximum threshold of dissociation kinetics at pH 7.4, and the minimum threshold of dissociation kinetics at pH 6. Eight substitution combinations met the above criteria and were selected for affinity determination at pH 7.4 and pH 6.0 by SPR. Affinity is listed in Table 4. [Table 4]

[0219] In these substitution combinations, the affinity of eculizumab for C5 was reduced by more than 1000-fold at pH 6.0, but at pH 7.0, the affinity was reduced by less than 20-fold. From these results, EHG303 (Table 4) exhibits high affinity at pH 7.4 (0.146 nM) and a K2-8,000-plus affinity at pH 6.0. D ) / (K at pH 7.4 D It was selected for further analysis due to the ratio of ). The heavy chain polypeptide of the EHG303 antibody contains the following amino acid sequence. [ka] The light chain polypeptide of the EHG303 antibody contains the following amino acid sequence. [ka] In the above sequence, the underlined portion corresponds to the leader sequence of each polypeptide, and the italicized portion is a heterologous amino acid introduced by cloning.

[0220] The EHL049 antibody was also selected. Its heavy chain polypeptide contains the following amino acid sequence. [ka] The light chain polypeptide of the EHL049 antibody contains the following amino acid sequence. [ka] In the above sequence, the underlined portion corresponds to the leader sequence of each polypeptide, and the italicized portion is a heterologous amino acid introduced by cloning.

[0221] Finally, the EHL000 heavy chain polypeptide contains the following amino acid sequence. [ka] The light chain polypeptide of the EHL000 antibody contains the following amino acid sequence. [ka] In the above sequence, the underlined portion corresponds to the leader sequence of each polypeptide, and the italicized portion is a heterologous amino acid introduced by cloning.

[0222] Example 4. Histidine substitution extends the half-life of eculizumab in serum. The light and heavy chain polypeptides of the EHL and EHG antibodies described above were expressed from a single gene construct. The heavy and light chain coding sequences from EHG303 were incorporated into a dual gene expression vector, similar to the light and heavy chain sequences for the EHL049 antibody. The resulting EHG303 clone was designated BNJ421, and the resulting EHL049 clone was designated BNJ423. The amino acid sequence of the heavy chain variable region of BNJ421 is as follows: [ka] The amino acid sequence of the light chain variable region of BNJ421 is as follows: [ka] The heavy chain variable region of the BNJ423 antibody contains the following amino acid sequence. [ka] The light chain amino acid sequence of BNJ423 is as follows: [ka]

[0223] These two molecules were evaluated together with EHL000 in immunodeficient (NOD / scid) and C5-deficient mice. A single dose of 100 μg of EHL000, BNJ421, or BNJ423 in 200 μL of phosphate-buffered saline (PBS) was administered to each of 8 mice by intravenous (i.v.) injection. Serum was collected from each mouse on days 1, 3, 7, 14, 21, 28, and 35 after administration. The concentration of each antibody in the serum was measured by ELISA. The antibody serum half-life was calculated using Pharsight Phoenix® WinNonlin® version 6.3 software, by using non-compartmental analysis (NCA) and direct response Emax. The percentage of antibody remaining in the serum was calculated as follows. [Number] where C t= is the antibody concentration on a given day, and C1 = the antibody concentration on day 1. The results are shown in Figure 6 and Table 5. [Table 5]

[0224] To determine the effect of human C5 on the half-life of these antibodies using the same mouse model, human C5 was administered subcutaneously to the mice at a loading dose of 250 μg on day -1 (the day before the antibody was administered to the mice), followed by twice-daily doses of 50 μg of C5 to maintain the serum C5 concentration at approximately 20 μg / mL (as described in Example 1).

[0225] As shown in Figure 7 (and Table 6 below), the half-life of EHL000 (eculizumab-IgG1) in a mouse model in the presence of human (hC5) (at concentrations where the molar ratio of C5 to eculizumab was greater than 1:1) was 2.49 ± 0.34 days, while the half-lives of BNJ421 and BNJ423 antibodies (including histidine substitution) were substantially better at 15.25 ± 0.90 days and 22.71 ± 0.71 days, respectively. These results indicate that histidine substitution within the CDR of eculizumab, and the resulting pH-dependent affinity for C5, significantly reduces the rate of clearance of eculizumab variants from serum compared to eculizumab. [Table 6]

[0226] Example 5. Histidine-substituted eculizumab mutants do not lose complement inhibitory activity. Furthermore, the serum hemolytic activity of each human C5-containing sample from the experiment described in Example 4 was also evaluated. Terminal complement activity in mouse serum was determined by evaluating its ability to lyse chicken erythrocytes. Since the mice used were C5 deficient, the hemolytic activity directly reflects the activity of human C5 in the sample. Briefly, antibodies at concentrations of 50, 3, and 0 μg / mL in gelatin-veronal buffered saline (GVBS) (Comptech catalog number B100) containing 0.1% gelatin, 141 mM NaCl, 0.5 mM MgCl2, 0.15 mM CaCl2, and 1.8 mM sodium barbital were used as low, moderate, and 100% lysis controls, respectively. Experimental samples were prepared by diluting mouse test serum 1:10 in GVBS. Equal volumes of 20% mouse C5-deficient serum and 20% human serum in GVBS (Bioreclamation, catalog number HMSRM-COMP+) were placed in the control wells, and equal volumes of 20% mouse C5-deficient serum and 20% human C5-deficient serum in GVBS (Complement Technologies, catalog number A320) were placed in the test sample wells. Sample volume (50 μL) was dispensed into the corresponding triple wells of a 96-well plate (Corning, Tewksbury, MA catalog number 3799). EDTA (500 mM, Sigma, catalog number E-9884) was added to the third well of both the control and sample triples to create a "no hemolysis" serum color control. Chicken red blood cells were washed in GVBS, sensitized, and the classical complement pathway was activated by incubation with anti-chicken RBC polyclonal antibody (Intercell Technologies, 0.1% v / v) at 4°C for 15 minutes. After washing again, approximately 7.5 × 10⁻⁶ cells were obtained. 7 The sensitized chicken erythrocytes (approximately 2.5 × 10⁶) were resuspended in GVBS at the final cell / mL concentration. 6Cells were added to plates containing the control and sample, briefly mixed on a plate agitator, and incubated at 37°C for 30 minutes. The reagents were mixed again, centrifuged at 845 × g for 3 minutes, and 85 μL of supernatant was transferred to the wells of a 96-well flat-bottom microtiter plate (Nunc, Penfield, NY, catalog no. 439454). Absorbance at 415 nM was measured using a microplate reader, and the percentage of hemolysis was determined using the following formula.

number

[0227] As shown in Figure 8, despite a slight decrease in affinity at pH 7.4 compared to eculizumab, both BNJ421 and BNJ423 were still able to bind to almost all human C5 present in circulation and inhibit hemolysis. These results indicate that the affinity of eculizumab for C5 can be weakened without compromising the efficacy of the antibody in vivo, conferring an increased serum half-life to the antibody.

[0228] Example 6. pH-dependent binding to C5 and enhanced FcRn-mediated regeneration are additional factors for the serum half-life of eculizumab variants. As shown above, in the presence of human C5, the half-life of histidine-substituted eculizumab variants is significantly prolonged in transgenic mice. To evaluate the potential additional effects of pH-dependent binding to C5 and FcRn on constitutive C5 synthesis and the pharmacokinetics (PK) and pharmacodynamics (PD) of anti-C5 antibodies in the presence of human FcRn, a series of PK / PD experiments were performed using anti-mouse C5 antibodies with a human constant region within human FcRn expressed in transgenic mice. These mouse anti-C5 antibodies were engineered from the variable region of BB5.1, and the mouse antibodies act as a pharmacological surrogate of eculizumab by binding to mouse C5 and preventing its cleavage into active metabolic fragments C5a and C5b [De Vries et al. (2003) Transplantation 3:375-382]. A high-affinity anti-mouse C5 antibody (designated BHL011) was engineered using affinity-optimized mutants of the BB5.1 mouse variable region, as well as the human Igκ and human IgG2 / G4 constant regions. A pH-dependent mutant of BHL011 was engineered by incorporating three histidine substitutions into the mouse variable region (this mutant was designated BHL006). A third antibody was engineered by incorporating two amino acid substitutions into the human constant region heavy chain (M428L, N434S) to increase affinity for hFcRn (this mutant was designated BHL009).

[0229] The amino acid sequence of the light chain polypeptide of BHL006 is as follows: [ka] The amino acid sequence of the heavy chain polypeptide of the BHL006 antibody is as follows: [ka]

[0230] The amino acid sequence of the light chain polypeptide of BHL009 is as follows: [ka] The amino acid sequence of the heavy chain polypeptide BHL009 is as follows: [ka]

[0231] The amino acid sequence of the BHL011 light chain polypeptide is as follows: [ka] The amino acid sequence of the heavy chain polypeptide BHL011 is as follows: [ka]

[0232] The kinetics of BHL011, BHL006, and BHL009 bound to purified mice were determined via SPR on a BIACore 3000 apparatus using an anti-Fc human capture method. Briefly, anti-human Fc (KPL, catalog number: 01-10-20), diluted to 0.1 mg / mL in 10 mM sodium acetate at pH 5.0, was immobilized on two flow cells of a CM5 tip by amine coupling for 8 minutes. The antibody was then diluted to 0.25 μg / mL in electrophoresis buffer (HBS-EP, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, GE Life Sciences, catalog number: BR1001-88). The diluted antibody was then injected into one flow cell, followed by injection of 6 nM mouse C5 into both cells. The second flow cell was used as a reference surface. Binding was evaluated at pH 7.4 and pH 6.0. The surface was regenerated each time with 20 mM HCl and 0.01% P20. A 1:1 Langmuir model was used, and data were processed using BIA evaluation 4.1 software with "dual reference". Dissociation of BHL011, BHL006, and BHL009 compounded into mouse C5 at pH 6.0 was similarly evaluated using injection of 6 nM mouse C5 (pH 7.4) followed by injection of HBS-EP buffer (pH 6.0). The results of these experiments are shown in Table 7. [Table 7]

[0233] To determine the effect of pH-dependent binding to C5 on the pharmacokinetics (PK) of anti-C5 antibodies in the presence of constitutive C5 synthesis, and the possibility of enhanced FcRn reuse to confer an additional increase in half-life, total serum concentrations of BHL011, BHL006, and BHL009 were analyzed using the transgenic FcRn mouse model described in Example 1. Total antibody serum concentrations and serum concentrations as a percentage of day 1 concentrations are shown in Figures 9–11. Male mice are represented by solid lines, and females by dotted lines. Total antibody serum concentrations on day 1 were higher in females than in males, proportional to the difference in body weight and distribution. This sex difference likely contributed to the inter-animal variability of BHL011 pharmacokinetics, possibly due to dose-dependent nonlinearity arising from C5-mediated clearance (Figures 9A and 9B). In general, inter-animal variability was lower for BHL006 (Figures 10A and 10B) and BHL009 (Figures 11A and 11B), with the exception of one female (2939) in the BHL006 dose group that showed accelerated clearance. The reason for the accelerated clearance in animal 2939 is unknown.

[0234] In the presence of constitutive synthesis of C5 and hFcRn, the high-affinity IgG2 / 4 anti-C5 antibody (BHL011) had an average terminal half-life of 6 days, with approximately 98% being cleared from circulation by day 21 (Figures 12 and 13, Table 8). The average clearance rate for the pH-dependent anti-C5 antibody with an IgG2 / 4 Fc region (BHL006) was attenuated, with an average β-phase half-life of 16–19 days. For the pH-dependent anti-C5 antibody with an IgG2 / 4 Fc region, an additional approximately twofold increase in half-life was observed, along with improved affinity for hFcRn (BHL009 half-life of approximately 36 days). These parameters are consistent with those observed for IgG2 / 4 antibodies in hFcRn mice in the absence of antigen, with or without M428L and N434S substitutions. These results demonstrate that the increased affinity for pH-dependent C5 binding and FcRn confers an additional effect in extending PK exposure with anti-C5 antibodies. [Table 8]

[0235] Pharmacodynamics of anti-mouse C5 antibodies in human FcRn transgenic mice The pharmacological activity of anti-mouse C5 antibodies in serum samples was evaluated in vitro using a complement-classical pathway-mediated chicken erythrocyte (cRBC) hemolysis assay. Hemolytic activity was calculated as a percentage of the activity in the pre-administration sample and is shown in Figures 14-16. Males are represented by solid lines, and females by dotted lines. The antagonism of in vitro hemolytic activity is proportional to the total antibody concentration in the sample. Sex differences in the duration of hemolytic activity antagonism were significant for BHL011 (Figure 14) and correspond to the weight-dependent inter-animal variability of BHL011 PK (Figure 9). In general, inter-animal variability was lower for BHL006 (Figure 15) and BHL009 (Figure 16), with the exception of females (2939) in the BHL006 dose group that showed accelerated antibody clearance (Figure 10).

[0236] The difference in the correlation between total serum antibody concentration and the antagonism of in vitro hemolytic activity is proportional to the affinity of the antibody for C5. High-affinity antibody (BHL011) almost completely suppressed hemolytic activity at approximately 200 μg / mL (Figure 17), while weak-affinity, pH-dependent anti-C5 antibodies required concentrations 2 to 3 times higher to achieve complete antagonism in vitro (Figures 18 and 19).

[0237] Despite this loss of ability in pH-dependent anti-C5 antibodies, the average activity levels against cRBC hemolysis across animals from each group suggest that they may support extended dosing intervals. On day 14, animals treated with high-affinity anti-C5 (BHL011) had an average hemolytic activity level of over 40%, while animals treated with pH-dependent anti-C5 (BHL006 and BHL009) maintained an average hemolytic activity level of less than 40% until days 21 and 28, respectively (Figure 20).

[0238] Compared to high-affinity anti-mouse C5 antibodies (BHL011), the significant extension of half-life and corresponding duration of antagonism of antibodies with pH-dependent binding to mouse C5 (BHL006 and BHL009) was consistent with the studies described in Examples 4 and 7, in which pH-dependent anti-human C5 antibodies (BNJ421, BNJ423, or BNJ441) exhibited a similar increase in half-life compared to their high-affinity counterparts (EHL000 or eculizumab) in mice co-administered with human C5. These findings further validate the concept that manipulating pH-dependent antigen binding through selective histidine substitution within the CDR can significantly reduce antigen-mediated clearance through C5, allowing free antibodies to return to circulation and be recycled. Furthermore, the combination of pH-dependent antigen binding in BHL009 and enhanced affinity for FcRn is an additional factor influencing the PK properties, doubling the half-life compared to pH-dependent binding alone (BHL006). These observations are consistent with the hypothesis that pH-dependent binding to C5, combined with improved affinity for FcRn, can lead to a significant extension of the PK parameters and therapeutic PD duration observed with eculizumab, enabling administration more than once a month.

[0239] Example 7. Generation of mutant eculizumab with pH-dependent binding to C5 and enhancement of FcRn-mediated regeneration. Antibodies were generated using eculizumab as the parent molecule. Compared to eculizumab, the mutant antibody (designated BNJ441) contained four amino acid substitutions in its heavy chain: Tyr-27-His, Ser-57-His, Met-429-Leu, and Asn-435-Ser (note that positions 429 and 435 of BNJ441 correspond to positions 428 and 434 under the EU numbering system). The amino acid sequence of the heavy chain polypeptide is shown in SEQ ID NO: 14. The amino acid sequence of the light chain polypeptide is shown in SEQ ID NO: 11. Manipulating these mutations allows for an extension of the dosing interval of BNJ441 (compared to eculizumab) by two distinct mechanisms: (1) reducing antibody clearance through target-mediated antibody clearance, and (2) increasing the circulating half-life by increasing the efficiency of FcRn-mediated antibody regeneration.

[0240] The two amino acid substitutions, Tyr-27-His and Ser-57-His, within the first and second complementarity-determining regions (CDRs) of the heavy chain variable region, affect the affinity dissociation constant (K) of BNJ441 for C5. D Compared to eculizumab, it weakens the affinity of BNJ441 by approximately 17 times at pH 7.4 and by approximately 36 times at pH 6.0. Two mutations in the third heavy chain constant domain (CH3), Met-429-Leu and Asn-435-Ser, increase the affinity of BNJ441 for FcRn by approximately 10 times at pH 6.0 compared to eculizumab.

[0241] Binding kinetics (antibodies against C5) The kinetics of BNJ441 or eculizumab bound to C5 were determined via surface plasmon resonance (SPR) on a BIAcore 3000 instrument using an anti-Fc capture method at pH 8.0, 7.4, 7.0, 6.5, and 6.0. Goat anti-human IgG(Fc) polyclonal antibody (KPL number 01-10-20) was diluted to 0.1 mg / mL in 10 mM sodium acetate at pH 5.0 and immobilized on two flow cells of a CM5 tip by amine coupling for 8 minutes. Test antibodies (BNJ441 or eculizumab) were diluted to 0.20 μg / mL in electrophoresis buffer (HBS-EP, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, GE Life Sciences, catalog number: BR1001-88). Next, the diluted antibody was injected into one flow cell (20 μL for the pH 7.4 experiment and 40 μL for the pH 6.0 experiment), followed by injection of C5 at different concentrations into both cells. The electrophoresis buffer was titrated with 3 M HCl for pH 7.0, 6.5, and 6.0 dynamics, and with 0.5 M NaOH for pH 8.0 dynamics. The surface was regenerated with 20 mM HCl and 0.01% P2O after each cycle. Data were processed using a 1:1 Langmuir model with BIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden) in "dual reference" mode.

[0242] The C5 dissociation rates from BNJ441 or eculizumab at pH 8.0, 7.4, 7.0, 6.5, and 6.0 were determined via SPR on a BIAcore 3000 instrument using the anti-Fc capture method described above with the following modifications. Diluted test antibody was injected into one flow cell, followed by injection of 6 nM C5 into both cells. Immediately after C5 injection, 250 μL of electrophoresis buffer at various pH levels was injected. The electrophoresis buffer was prepared as described above. Data were processed using BIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden) with "dual reference". The C5 dissociation % of BNJ441 and eculizumab was calculated by taking the difference of dissociation at t=0 and t=300 seconds.

[0243] Binding kinetics (antibodies against FcRn) The kinetics of BNJ441 or eculizumab bound to human FcRn were determined via SPR on a BIAcore 3000 instrument using F(ab')2 capture methods at pH 7.4 and 6.0. Goat F(ab')2 anti-human IgG F(ab')2 (Rockland Immunochemicals, catalog number: 709-1118), diluted to 0.04 mg / mL in 10 mM sodium acetate at pH 5.0, was immobilized on two flow cells with CM5 tips by amine coupling for 7 minutes. The test antibody (BNJ441 or eculizumab) was diluted to 2 μg / mL in electrophoresis buffer (HBS-EP, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, GE Life Sciences, catalog no. BR1001-88). The diluted antibody was then injected into one flow cell, followed by injection of FcRn into both cells. The electrophoresis buffer was titrated for pH 6.0 kinetics using 3 M HCl. Surface regeneration was performed with 10 mM glycine HCl, pH 1.5. Data were processed using a 1:1 Langmuir model with BIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden) in "dual reference" mode.

[0244] Results of combined research The dynamics of antibody C5 binding were found to be pH-dependent, and the effects on both association and dissociation rates are shown in Table 9. [Table 9]

[0245] In an attempt to model the relative rates of early endosome suppositories and antibody:C5 complex dissociation after acidification, antibody:C5 complexes were allowed to form in pH 7.4 buffer, and the pH state of the buffer was then switched during dissociation. The percentage of antibody complex dissociation after 300 seconds (estimated by the decrease in resonance units [RU]) was calculated for each pH condition (Table 10). Only BNJ441 at pH 6.0 resulted in more than 50% antibody:C5 complex dissociation after 5 minutes. [Table 10]

[0246] Figures 21A and 21B show semi-logarithmic and linear plots of the percentage of dissociation of the BNJ441:C5 complex or eculizumab:C5 complex as a function of pH.

[0247] The two amino acid substitutions, Tyr-27-His and Ser-57-His, within the first and second complementarity-determining regions (CDRs) of the heavy chain variable region, affect the affinity dissociation constant (K) of BNJ441 for C5. DThis weakens BNJ441 affinity for C5 by approximately 17-fold at pH 7.4 and approximately 36-fold at pH 6.0 compared to eculizumab. It is unclear whether the pH dependence of BNJ441 affinity for C5 is a result of altered protonation status of histidine introduced at positions 27 and / or 57, or simply an overall weakening of affinity for C5. However, in other anti-C5 antibodies, these mutations, in combination with additional histidine substitutions, were observed to result in a much more pronounced loss of affinity at pH levels below 6.5. Two mutations in the third heavy chain constant domain (CH3), Met-429-Leu and Asn-435-Ser, enhance BNJ441 affinity for FcRn by approximately 10-fold at pH 6.0 compared to eculizumab.

[0248] PK properties of the BNJ441 antibody BNJ441 antibody and eculizumab were evaluated in immunodeficient (NOD / scid) and C5-deficient mice. A single dose of 100 μg of BNJ441 or eculizumab in 200 μL of phosphate-buffered saline (PBS) was administered intravenously (iv) to each of eight mice. Serum was collected from each mouse on days 1, 3, 7, 14, 21, 28, and 35 post-administration. The concentrations of each antibody in the serum were measured by ELISA.

[0249] As shown in Figure 22, in the absence of human C5, serum antibody concentrations decreased similarly in mice administered with BNJ441 and eculizumab over a 35-day period. However, in the presence of human C5, eculizumab serum concentrations decreased rapidly to undetectable levels after 14 days, while BNJ441 serum concentrations decreased more slowly and at a constant rate over the study period (Figure 23).

[0250] When comparing the PK profiles of the two antibodies in and out of human C5, eculizumab clearance was accelerated in the presence of human C5 compared to that in the absence of human C5, but the PK profile of BNJ441 in the presence of human C5 was similar to that of BNJ441 in the absence of human C5 at day 28, with clearance only being accelerated from day 28 to day 35 (Figure 24). The half-lives of BNJ441 and eculizumab were equivalent in the absence of human C5 (25.37 ± 1.02 days for BNJ441 and 27.65 ± 2.28 days for eculizumab). However, in the presence of human C5, BNJ441 demonstrated a more than threefold increase in half-life compared to eculizumab (13.40 ± 2.18 days for BNJ441 vs. 3.93 ± 0.54 days for eculizumab). It should be noted that the clearance rate of BNJ441 did not differ significantly up to day 28, with or without human C5. See Table 11. [Table 11-1] [Table 11-2]

[0251] Serum hemolytic activity To determine the effect of histidine substitution on antibody hemolytic activity, an in vitro hemolysis assay was performed as described in Example 6. In the presence of BNJ441 or eculizumab, terminal complement activity was consistent with the respective PK profiles of each antibody (Figure 25), meaning that the level of inhibition of serum hemolytic activity was proportional to the concentration of each antibody remaining in the serum. Both antibodies conferred almost complete inhibition of hemolysis up to day 3. However, eculizumab did not show antagonistic activity up to day 14, while BNJ441 maintained approximately 83% inhibition up to day 14 and partial complement inhibition up to day 28.

[0252] conclusion Findings from this study suggest that BNJ441 exhibited a more than threefold extension of half-life compared to eculizumab in the presence of human C5. Furthermore, the serum half-life of BNJ441 compared to eculizumab translated into an extended pharmacodynamic profile, as evidenced by the longer hemolytic inhibition. Example 8. Safety, tolerance, PK, and PD of BNJ441 in healthy human subjects.

[0253] The safety, tolerance, pharmacokinetics (PK), and disease progression (PD) of BNJ441 were evaluated in a Phase 1 randomized, blinded, placebo-controlled single-elevation dose (SAD) human clinical study in which BNJ441 was administered intravenously to healthy subjects.

[0254] BNJ441 was compounded with excipients in an aqueous solution without sterile storage volume. The BNJ441 formulation contained no unusual excipients or excipients of animal or human origin. The formulation was phosphate-buffered to pH 7.0. The components consisted of 10 mg / mL of BNJ441, 3.34 mM of mononucleotide sodium phosphate, 6.63 mM of dinucleotide sodium phosphate, 150 mM of sodium chloride, 0.02% of polysorbate 80, and QS water.

[0255] The BNJ441 formulation is supplied as a 10 mg / mL antibody solution in a 20 mL single-use vial and is designed for injection by diluting it with commercially available saline solution (0.9% sodium chloride injection, Ph Eur) for IV administration. [Table 12-1]

[0256] Ten healthy subjects received a single dose of BNJ441. Four subjects received a 200 mg dose, and six subjects received a 400 mg dose. The pharmacokinetic and safety data for this study were determined and discussed below.

[0257] Pharmacokinetics The serum BNJ441 concentration-time profiles after IV administration at doses of 200 mg and 400 mg are shown in Figure 26. Concentration-time data were available up to 90 days (2136 hours) and 57 days (1344 hours) after the 200 mg and 400 mg doses, respectively. The mean serum concentration was maintained above 50 μg / mL for 2 to 4 days (48 to 96 hours) after the 200 mg dose and for 14 to 21 days (336 to 504 hours) after the 400 mg dose.

[0258] A summary of the BNJ441 PK parameters is reported in Table 12 below. The geometric mean (CV) C 最大 of BNJ441 was 78.5 (10.2%) μg / mL after the 200 mg dose and 139 (16.2%) μg / mL after the 400 mg dose. The observed median (range) t 最大 was 2.4 (0.79 to 8.0) hours after the start of infusion for the 200 mg dose and 0.58 (0.58 to 1.1) hours for the 400 mg dose. The geometric mean (CV) AUC (0~56日) was 32,800 (8.6%) μ-hr / mL for the 200 mg dose and 58,100 (18.9%) μg-hr / mL for the 400 mg dose. The geometric mean C 最大 and AUC (0~56日) showed that exposure increased in an apparent dose-proportional manner. The geometric mean t 1 / 2 (CV) was 38.5 (18.4%) days and 32.9 (13.3%) days for the 200 mg and 400 mg doses, respectively.

[0259] In summary, the PK data suggest that the mean BNJ441 C 最大 and AUC (0~56日) increased in a dose-proportional manner and support a mean (standard deviation [SD]) t of 35.5 ± 6.1 days after IV administration. Analysis of chicken red blood cell (cRBC) hemolysis data showed that terminal complement was completely inhibited for up to 2 days after a single 400 mg IV dose when the BNJ441 concentration exceeded 100 μg / mL. 1 / 2

Table 12-2

[0260] Pharmacodynamics As shown in Figure 27, the ability of BNJ441 to inhibit cRBC hemolysis over a long period was also evaluated. Mean cRBC hemolytic activity was relatively stable in subjects receiving placebo. The onset of cRBC hemolysis inhibition was rapid, with complete terminal complement inhibition observed at the end of the infusion (0.29 hours for the 200 mg dose and 0.58 hours for the 400 mg dose). BNJ441 had a dose-dependent duration of action, lasting 4 to 14 days.

[0261] The relationship between BNJ441 concentration and cRBC hemolysis was plotted and is shown in Figure 28. As shown in Figure 28, complete terminal complement inhibition occurred at BNJ441 concentrations above 50 μg / mL, and no inhibition was observed at BNJ441 concentrations below 25 μg / mL.

[0262] Example 9. Single-dose study in cynomolgus monkeys A single IV dose of BNJ441 was administered to cynomolgus monkeys at a dose of 60 or 150 mg / kg (n=4 for each dose group, 2 males and 2 females per dose group) as a 2-hour infusion. Blood samples for BNJ441 analysis were collected from day 1 to day 112.

[0263] All monkeys treated with BNJ441 were screened for the presence of cynomolgus monkey anti-human antibodies (CAHA) before administration (0 hours) and on days 8, 14, 28, 56, 84, and 112.

[0264] All monkeys in the 60 and 150 mg / kg dose groups were confirmed to be positive at least once, with the exception of animal 2002 in the 150 mg / kg dose group. The presence of CAHA in animal 2002, or the non-positive time point in other animals, cannot be ruled out due to the possibility of buffering of administered BNJ441 by biotinylated BNJ441 and ruthenylated BNJ441 crosslinking assays. Positive CAHA results were observed from day 56 to 112 post-administration in the 60 mg / kg dose group and from day 28 to 112 post-administration in the 150 mg / kg dose group. The first confirmed CAHA-positive samples in the 60 mg / kg group were at day 56 (animals 1002 and 1503), two at day 84 (animals 1002 and 1503), and three at day 112 (animals 1001, 1002, and 1502). Animal 1503, which was CAHA-positive on days 56 and 84, was no longer CAHA-positive on day 112. The first CAHA-positive sample confirmed in the 150 mg / kg dose group was animal 2502 on day 28, followed by two monkeys (animals 2001 and 2502) on day 56, three monkeys (animals 2001, 2501, and 2502) on day 84, and three monkeys (animals 2001, 2501, and 2502) on day 112.

[0265] Individual BNJ441 concentration-time profiles were calculated. In the 60 mg / kg dose group, all monkeys had quantifiable plasma BNJ441 concentrations up to day 112 of the PK sample. However, in the 150 mg / kg dose group, only one monkey (animal 2002) had quantifiable plasma BNJ441 concentrations up to day 112. The concentration-time data indicated a long retention of BNJ441 in the systemic circulation of the monkeys.

[0266] Non-compartmental PK parameters and summary statistics for BNJ441 were calculated for each dose level for all monkeys and are shown in Tables 13 and 14 for the 60 mg / kg and 150 mg / kg dose levels, respectively. The median t is consistent with the infusion period. 最大 The response time was 2 hours for the 60 mg / kg and 150 mg / kg dose levels. In one monkey (animal 2501) in the 150 mg / kg dose group, the response time 12 hours after administration was 2 hours.最大 The sample concentration at 12 hours post-administration was approximately 5% higher than that observed at 2 hours post-administration. Geometric mean C 最大 AUC ∞ , and AUC 最後 All values ​​increased with gradual dose increases. Geometric mean dose-normalized C 最大 The values ​​are similar across the two doses, showing a dose-proportional increase in peak BNJ441 concentration with increasing dose, but the geometric mean dose-normalized AUC ∞ The values ​​differed between dose groups. This difference may be due to the CAHA-mediated increase in BNJ441CL in the 150 mg / kg dose group, where BNJ441 clearance was approximately 37% higher in monkeys administered 150 mg / kg compared to monkeys administered 60 mg / kg. Geometric mean V ss The results were similar between the two dose groups (within 12%). [Table 13] [Table 14]

[0267] Example 10. Comparative evaluation of BNJ441, eculizumab, and h5G1.1 binding to the Fc-γ receptor C1q in vitro. The binding of three humanized antibodies, BNJ441, eculizumab, and h5G1.1-IgG1, to molecules known to be mediators of antibody effector function was investigated. BNJ441, eculizumab, and h5G1.1-IgG1 each have unique functions and therapeutic profiles. However, all three are humanized antibody antagonists of terminal complement, binding to very similar epitopes on human complement component C5 and preventing cleavage to its active metabolites C5a and C5b during complement activity.

[0268] BNJ441, eculizumab, and h5G1.1-IgG1 are identical in their light chain sequences, each possessing a humanized variable region and a human Igκ constant region. Both BNJ441 and eculizumab contain a human hybrid IgG2-G4 Fc, which includes the CH1 region, hinge, and the first 29 amino acids of the CH2 region from human IgG2 fused to the remainder of the CH2 and CH3 of human IgG4. This chimeric Fc combines the stable disulfide bond pairing of IgG2 with the effector-free properties of IgG4. Because BNJ441 and eculizumab are directed towards soluble antigens, it was not possible to directly evaluate their ability to initiate antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Instead, direct measurements of BNJ441 or eculizumab binding to the Fcγ receptor (FcγR) and complement component C1q were performed, and it was inferred that in the absence of binding, they could not mediate ADCC or CDC, respectively. h5G1.1-IgG1 (an IgG1 isotype antibody with the same humanized variable region as eculizumab) was included as a control. While the IgG1 isotype Fc region is expected to fully bind effector functional molecules, h5G1.1-IgG1 itself would not induce ADCC or CDC in the absence of cell-associated antigens.

[0269] As described above in Example 7, BNJ441 was modified from eculizumab to increase its in vivo half-life by introducing four amino acid substitutions into the heavy chain. Two amino acid changes in the humanized heavy chain variable region, Tyr-27-His and Ser-57-His (heavy chain amino acid numbering by Kabat et al.), were introduced to destabilize binding to C5 at pH 6.0, but had minimal effect on binding to C5 at pH 7.4. Mutations in the third heavy chain constant region domain (CH3), Met-428-Leu and Asn-434-Ser, were introduced to enhance binding to the human neonatal Fc receptor (FcRn). Together, these mutations were designed to significantly attenuate antigen-mediated drug clearance by increasing the dissociation of the antibody:C5 complex into free antibody in the acidic environment of early endosomes after ingestion, and to increase the amount of antibody fragments recycled back into the vascular compartment by FcRn from early endosomes.

[0270] In these studies, multimer interactions with all three antibodies of the FcγR subclass (FcγR1, FcγRIIa, FcγRIIb, FcγRIIb / c, FcγRIIIa, and FcγRIIIb) were evaluated using enzyme-linked immunosorbent assay (ELISA), while monomer interactions with FcγR were evaluated using surface plasmon resonance (SPR). Binding of C1q to the three antibodies was investigated using biolayer interferometry and SPR. The reagents used for these analyses are shown in Table 15. [Table 15]

[0271] Binding of multivalent antibody complexes to FcγR Antibody conjugates were prepared by incubating BNJ441, eculizumab, or h5G1.1-hG1 overnight in phosphate-buffered saline (PBS) in a 1.5 mL microcentrifuge tube with goat anti-human F(ab')2-biotin (Jackson Immunolabs) in a 2:1 antibody:F(ab')2 molar ratio.

[0272] Microtiter plates pre-coated with Ni-NTA (Qiagen) were incubated overnight at 4°C with 50 μL / well of 6X histidine-tagged human FcγR (FcγRI, FcγRIIa, FcγRIIb / c, FcγRIIIa, or FcγRIIIb) at a receptor concentration of 5 μg / mL in PBS. The plates were then washed three times with PBS / 0.05% Tween-20. After washing, 50 μL of antibody conjugates in PBS / 0.05% Tween-20 were incubated in the plates at room temperature (RT) for 60 minutes. After washing the plates with PBS / 0.05% Tween-20, 50 μL of streptavidin-HRP (Invitrogen) in PBS / 0.05% Tween-20 was added to the plates and incubated at room temperature for 60 minutes. Following incubation and washing, 75 μL of TMB-ELISA substrate (3,3',5,5'-tetramethylbenzidine, Thermo Scientific) was added. The reaction was stopped with 75 μL of 2 M H2SO4, and the absorbance was read at 450 nM.

[0273] The samples were experimented with twice, and the data were presented as averages. The results were entered into a spreadsheet program. The absorbance of antibody-immune complexes at 450 nM for each concentration, or the absorbance in the absence of antibody-immune complexes, was plotted graphically. The major dissociation constants were calculated and summarized in Table 16, and discussed below.

[0274] Binding of monovalent antibody to FcγR The kinetics of BNJ441, eculizumab, and h5G1.1-IgG binding to FcγR were determined via SPR on a BIAcore 3000 instrument using direct fixation. BNJ441, eculizumab, and h5G1.1 were diluted in 10 mM sodium acetate at pH 5.0 and fixed on one flow cell of a CM5 tip by amine coupling. A second flow cell was used as a reference surface. A concentrated FcγR solution diluted in electrophoresis buffer (HBS-EP, pH 7.4) was injected into both cells. The surface was regenerated with 20 mM HCl and 0.01% P20 after each cycle. Using a stable-state affinity model, data were analyzed with "dual reference" in BIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden).

[0275] The kinetics of h5G1.1-IgG1 binding to FcγRI were evaluated via single-cycle kinetics due to its potent affinity. The antibody was diluted in 10 mM sodium acetate at pH 5.0 and immobilized directly on one flow cell of a CM5 tip by amine coupling. A second flow cell was used as a reference surface. A concentrate of FcγR1 diluted in electrophoresis buffer (HBS-EP, pH 7.4) was injected into both cells. This assay did not require regeneration. Data were analyzed using a 1:1 titration kinetics model in BIAevaluation4.1 (Biacore AB, Uppsala, Sweden) software with "dual reference". [Table 16]

[0276] An ELISA assay was performed to detect multimeric interactions driven by the binding activity of antibody-immune complexes and FcγR. The results are summarized in Table 16. BNJ441 and eculizumab did not show detectable binding to FcγRI, FcγRIIb / c, FcγRIIIa, or FcγRIIIb, and each showed 4- to 8-fold weak association with FcγRIIa. The dissociation constant (K) for monomeric FcγR binding to BNJ441 and eculizumab induced by SPR was calculated.D The FcγR interaction was very weak, and the two antibodies were almost indistinguishable. FcγRI (approx. 4 μM), FcγRIIa (approx. 2 μM), FcγRIIb (approx. 9 μM), FcγRIIIa (approx. 7 μM), and FcγRIIIb (approx. 3 μM). The dissociation constants relative to the IgG1 isotype control (h5G1.1-IgG1) were consistent with high affinity interaction with FcγR1 (123 pM) and a moderate increase in binding to low affinity FcγR compared to the IgG2-G4 isotype antibodies, namely FcγRIIa (approx. 1 μM), FcγRIIb (approx. 3 μM), FcγRIIIa (approx. 1 μM), and FcγRIIIb (approx. 2 μM). See Table 16. Interactions between C1q and BNJ441 or eculizumab could not be detected by biolayer interferometry. These results are consistent with the idea that the chimeric human IgG2-G4 Fc of eculizumab has little to no ability to evoke effector function through FcγR or C1q, and mediate ADCC or CDC, respectively. Furthermore, these results indicate that the heavy-chain amino acid substitutions incorporated into BNJ441 do not significantly alter their binding compared to eculizumab.

[0277] Example 11. Tissue cross-reactivity study 1. GLP Human Cross-Reactivity Study Potential cross-reactivity with human tissues was determined using fluorescently activated BNJ441 (designated as BNJ441-FITC) and a control antibody with different antigen specificity (OX-90G2G4-FITC).

[0278] BNJ441-FITC resulted in moderate to strong staining of the positive control material (purified human complement protein C5 ultraviolet [UV]-resin spot slide, designated hC5) but did not specifically react with the negative control material (human hypercalcemia of malignant peptide, amino acid residues 1-34, UV-resin spot slide, designated PTHrP 1-34). The control article, OX-90G2G4-FITC, did not specifically react with either the positive or negative control material. The excellent specific reaction of BNJ441-FITC with the positive control material, the lack of specific reactivity with the negative control material, and the lack of reactivity with the control article demonstrated that the assay was sensitive, specific, and replicable.

[0279] Staining with BNJ441-FITC was observed in the human tissue panel, as summarized below. • Protein material in most human tissues • Cytoplasm and / or cytoplasmic granules in the following tissue elements - Mononuclear cells in the colon, esophagus, lymph nodes, parathyroid gland, spleen, and tonsils - Platelets in blood smear and bone marrow - Megakaryocytes in bone marrow - Epithelium of the fallopian tubes, liver (hepatocytes), pancreatic duct, and cervix - Mesothelium in the lungs

[0280] Since C5 is a circulating serum protein, staining of proteinaceous materials was expected. Since mononuclear cells such as monocytes, macrophages, and dendritic cells, as well as platelets, have been reported to secrete C5, staining of these cell types with BNJ441-FITC was also expected. Additionally, mesothelial cell lines have been shown to produce C5. However, no literature describing C5 expression by epithelial cell types stained with BNJ441-FITC or megakaryocytes was available in this study, although platelets shown to produce C5 are derived from megalococytes. Therefore, staining of epithelial cell types may represent either previously unrecognized sites of C5 expression or tissue cross-reactivity with protein sequences or structures from similar but unrelated proteins or other constituents of a tissue segment. However, with the exception of staining of proteinaceous materials, all staining observed in this study is cytoplasmic by nature, and it is unlikely that the cytoplasm and cytoplasmic structures are accessible in vivo to the test material. In summary, no specific cross-reactivity with BNJ441-FITC staining, which would be relevant to predicting treatment-related toxicity, was observed.

[0281] 2. Cross-reactivity study of GLP cynomolgus monkey tissues Cross-reactivity studies of standard GLP tissues were also conducted using a population of cynomolgus monkey tissues, and both off-target and on-target binding were examined using the same reagents as those used in human tissue binding studies.

[0282] As summarized below, some staining with BNJ441-FITC was observed in cynomolgus monkey tissue populations. • Protein material in most cynomolgus monkey tissues • Cytoplasm and / or cytoplasmic granules in the following tissue elements - Mononuclear cells in lymph nodes, spleen, and tonsils - Epithelium of the fallopian tubes

[0283] BNJ441-FITC staining observed in the cynomolgus monkey tissue population was generally lower in intensity and frequency than that observed in the human tissue population in the companion human tissue cross-reactivity study. Furthermore, staining was observed in platelets, megakaryocytes, pancreatic duct epithelium, cervical epithelium, hepatocytes, and mesothelium in the human tissue aggregate, but these tissue elements were not stained in the cynomolgus monkey tissue aggregate. In addition, with the exception of the staining of proteinaceous materials, the staining observed in this study was cytoplasmic by nature, and it is unlikely that the cytoplasm and cytoplasmic structures were accessible in vivo to the test material. Since BNJ441 has been shown to be highly specific to human C5 (and not cross-reactive to C5 from non-human primates), the limited binding observed in this study was likely due to nonspecific binding with unspecified cross-reactive materials.

[0284] Example 12: Capabilities of BNJ441 compared to eculizumab in terminal complement activity assays. Mutations engineered within BNJ441 to result in pH-dependent binding to C5 can be expected to reduce its affinity at pH 7.4 (approximately 491 pM) by about 17 times compared to eculizumab (approximately 29.3 pM), thus reducing its ability to inhibit C5-mediated terminal complement activity compared to eculizumab. To estimate the capabilities of BNJ441 and eculizumab under physiologically relevant conditions, the antagonistic effects of complement-mediated hemolysis in erythrocytes (RBCs) from three commonly used animal models (chickens, sheep, and rabbits) were evaluated in 90% normal human serum.

[0285] RBCs and sheep erythrocytes (sRBCs) were pre-sensitized with antibodies to initiate activation of the classical complement pathway (CCP). Rabbit erythrocytes (rRBCs) were not pre-sensitized and used as a model for activation of the alternative complement pathway (CAP). Antibodies were pre-incubated in 100, 200, and 400 nM serum, resulting in antigen-binding site to C5 molar ratios of approximately 0.5:1, 1:1, and 2:1, respectively. Antibody BNJ430, which contains the same Fc region as BNJ441 but does not bind to human C5, was included as a negative control. Hemolysis percentages were measured at 0, 1, 2, 3, 4, 5, 6, and 8 minutes to ensure that the reaction was observed under initial rate conditions.

[0286] As shown in Figure 29, neither BNJ441 nor eculizumab showed antagonistic activity in cRBC hemolysis at 100 nM. Both antibodies showed partial antagonistic activity at 200 nM, with BNJ441 exhibiting reduced efficacy compared to eculizumab (antigen-binding site vs. C5 in approximately 1:1 molar ratio). Inhibition of hemolysis was nearly complete for either antibody when incubated with antigen-binding site vs. C5 in a 2:1 molar ratio (400 nM). The results of the sRBC hemolysis assay were similar, showing less than 20% hemolysis at 200 nM in the presence of BNJ441 and nearly complete inhibition by each antibody at 400 nM (data not shown). The CAP-mediated rRBC hemolysis assay showed higher levels of hemolysis in the presence of the anti-C5 antibody, with no detectable inhibition at 200 nM and only partial inhibition at 400 nM (data not shown).

[0287] In conclusion, the moderate loss of ability of BNJ441 compared to eculizumab in these in vitro complement activity assays is consistent with its weak affinity for C5. The affinity of BNJ441 for C5 is approximately 1000 times lower in vivo than the concentration of C5 and the targeted therapeutic level of BNJ441, and therefore it is unlikely to impair its therapeutic effect.

[0288] Example 13: Selectivity of BNJ441 compared to eculizumab in terminal complement activity assays To evaluate the pharmacological activity of BNJ441 in non-human animal models, we measured BNJ441's ability to antagonize complement-mediated hemolysis of antibody-sensitized cRBCs in serum from chimpanzees, baboons, rhesus monkeys, cynomolgus monkeys, beagles, rabbits, guinea pigs, rats, and mice. Eculizumab and anti-mouse-C5 antibodies containing human IgG2 / G4 Fc (BNJ430) were used as isotype controls.

[0289] For each assay, sensitized cRBCs were prepared from 400 μL of chicken whole blood in Alsever (Lampire Biologicals), washed four times at 4°C with 1 mL of GVBS, and 5 × 10⁶ cRBCs were added to the GVBS. 7 The cells were resuspended in a volume of cells / mL. To sensitize chicken erythrocytes, polyclonal anti-chicken RBC antibody (Rockland) was added to the cells at a concentration of 150 μg / mL and incubated on ice for 15 minutes. After one wash with GVBS, the cells were resuspended in GVBS to a final volume of 3.6 mL.

[0290] Complement-preserved serum was obtained from bioreclamation containing serum from the following mammals: humans, chimpanzees, baboons, rhesus monkeys, cynomolgus monkeys, beagles, rabbits, guinea pigs, and rats. Antibodies BNJ441 (10 mg / mL), eculizumab (10 mg / mL), and BNJ430 (0.873 mg / mL) were diluted to final concentrations of 0, 60, 300, and 600 nM in 30% serum in GVBS and incubated at room temperature for 30 minutes. Sensitized cRBCs were incubated in 30 μL / wells (2.5 × 10⁶). 6 The antibody / serum mixture was added to the cells and incubated at 37°C for 30 minutes. The reaction was stopped by adding 30 μL of 0.5 M EDTA to each well. The plate was centrifuged at 1800 Xg for 3 minutes, and 80 μL of the supernatant was transferred to a new flat-bottom 96-well plate. Absorbance was measured at 415 nM.

[0291] Because mouse serum is a poor source of classical pathway complement activity, mouse serum was mixed 1:1 with C5-deficient human serum to evaluate the potential pharmacological activity of BNJ441 in mice. Antibodies were diluted to final concentrations of 0, 60, 300, and 600 nM in 50% total serum (25% mouse serum, 25% C5-deficient human serum) in GVBS and incubated at room temperature for 30 minutes. Sensitized cRBCs were then placed in 30 μL / well (2.5 × 10⁶) of the antibody / serum mixture. 6 The cells were added and incubated at 37°C for 30 minutes. The reaction was stopped by adding 30 μL of 0.5 M EDTA to each well. The plate was centrifuged at 1800 Xg for 3 minutes, and 80 μL of the supernatant was transferred to a new flat-bottom 96-well plate. Absorbance was measured at 415 nM.

[0292] Samples containing serum without anti-C5 antibody were used with or without 10 mM EDTA, either undissolved or completely dissolved. Sample conditions were used for three or two experiments.

[0293] The results are entered into a spreadsheet, enabling background removal from the undissolved control, normalization of the hemolysis rate compared to the complete hemolysis control, calculation of the mean (±sd), and graphical display of the data. The mean background absorbance from the undissolved control was subtracted from each replica, and the absorbance of the sample was expressed as a percentage of dissolution in the complete hemolysis control according to the following equation: % of cRBC hemolysis = (A415 value in each sample replica - mean A415 value in the undissolved control) / (mean A415 value in the complete hemolysis control - mean A415 value in the undissolved control) × 100.

[0294] The mean and standard deviation of the cRBC hemolysis percentage in the sample replication were plotted as a graphic (data is not shown).

[0295] BNJ441 was shown to exhibit no detectable binding to native C5 from cynomolgus monkeys, nor any in vitro pharmacological activity, in any non-human serum tested with an 8-fold molar excess of the antigen-binding site for C5. In summary, these data are consistent with the conclusion that BNJ441 has no relevant pharmacological activity in any readily accessible non-human species suitable for modeling pharmacokinetics or pharmacodynamics in humans.

[0296] Example 14: Physicochemical characterization of BNJ441 The BNJ441 antibody is a recombinant, humanized antibody composed of two identical 448-amino acid heavy chains and two identical 214-amino acid light chains. See Figure 30. The constant region of BNJ441 includes a human κ-light chain constant region and a hybrid human IgG2-IgG4 heavy chain constant region (also referred to as "G2 / G4"). The IgG2 / G4 constant region is appropriately designed to reduce the antibody's effector activity, complement activity, and immunogenicity. The first five amino acids of the heavy chain CH1 domain, hinge region, and CH2 domain correspond to the human IgG2 amino acid sequence, residues 6-36 within the CH2 domain, which are common to both human IgG2 and IgG4 amino acid sequences, while the remainder of the CH2 and CH3 domains correspond to the human IgG4 amino acid sequence. The heavy chain and light chain variable regions, which form the human C5 binding site consisting of a human framework region, were transplanted into the mouse complementarity-determining region. Interchain disulfide bonds within the BNJ441 antibody are shown in Figure 31. Figure 31 shows the residue numbers for all disulfide bond pairings and N-linked glycan moieties.

[0297] Table 17 lists the general properties of the BNJ441 antibody. The theoretical chemical formulas and theoretical average molecular weights of the main components presented below assume that the antibody contains 18 disulfide bonds, two heavy chain N-terminal pyroglutamine oxidations, two heavy chain C-terminal lysine clippings, and the addition of two G0F glycan residues. The number of amino acid residues in BNJ441 was predicted by amino acid analysis. [Table 17]

[0298] A stable Chinese hamster ovary (CHO) cell line expressing BNJ441 was developed for the production of BNJ441. The CHOK1SV cell source used to generate this cell line was obtained from the Lonza Biologics CHOK1SV master cell bank 269-M. This cell source was verified to be free of bacterial and fungal contaminants, as well as all detectable viruses except for non-infectious intracellular retroviral particles. Host CHOK1SV cells were transduced with plasmid pBNJ441.1, and stable clones were selected by MSX. Primary clone 3A5 was selected as the generating cell line for the production of BNJ441.

[0299] The BNJ441 bulk drug batch was prepared in both manipulated and GMP forms and physicochemically characterized by the tests listed in Table 18. The manipulated batch was produced in a pilot plant using CHO cells grown in a 200L bioreactor, and the purified material was used in the PK study. The GMP batch was produced using CHO cells grown in the pilot plant, with a 200L bioreactor. The BNJ441 manipulated and GMP bulk drug batches were formulated and tested at approximately 10 mg / mL. The physicochemical properties of these batches are summarized in Table 19. [Table 18] [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6]

[0300] Table 19 shows that the intact molecular weight determined for the operational batch was 147830.80 Da, and for the GMP batch it was 147830.72 Da. These values ​​are consistent with the principal component molecular weight of 147,827.62 Da calculated for BNJ441 in Table 17 and were within the 100 ppm mass precision of an externally calibrated ESI-ToF-MS. No major peaks were observed beyond the 147,000–149,500 Da range. This method identified molecules based on intact molecular weight. The test sample was injected onto a C4 RP-HPLC column and eluted with an aqueous:organic solvent gradient. The eluate was then electrosprayed onto a ToF mass spectrometer, and the spectra from the upper half of the chromatographic peaks were inversely superimposed to provide the intact molecular weight.

[0301] Table 19 shows the N-terminal sequences determined for the BNJ441 batch. The determined N-terminal sequences of the heavy and light chains were consistent with the amino acid sequences for the BNJ441 batch. The heavy chain was found to be blocked by PyroQ, as expected, and unblocked by pyrograltamate aminopeptidase (PGAP). The primary protein sequences at the N-terminus of the polypeptide chains were determined by serial Edman degradation and HPLC analysis.

[0302] Table 19 shows the amino acid analysis residues per molecule determined for the BNJ441 batch. These values ​​all matched the number of residues per mole calculated for the BNJ441 batch based on the primary sequence shown in the first column of Table 19. Amino acid analysis data was obtained three times. This method evaluates the primary structure of a molecule by acidic hydrolysis of the protein into its individual amino acid constituents. This method does not detect cysteine ​​or tryptophan. Asparagine and aspartate were detected in a single peak and labeled as Asx. Glutamine and glutamate were also detected in a single peak and labeled as Glx. Of the 20 standard amino acids, 14 were uniquely detected by this method, totaling 16 amino acids with the addition of Asx and Glx groups. Of those shown, BNJ441 has a total of 1262 residues that can be detected by these methods.

[0303] Table 19 shows the circular dichroism (CD) near-UV and far-UV local features and inverse superposition results for batches of BNJ441. The inverse superposition explains the α-helical, 3 / 10-helical, β-sheet, rotation, poly(Pro)II content, and disordered structure determined by CDPro software for a given set of references. Near-UV (tertiary structure) and far-UV (secondary structure) CD spectra were determined for each batch. This method uses the differential absorption of left and right circularly polarized light exhibited within the absorption band of optically active (chiral) molecules such as proteins to reveal higher-order molecular structures within the molecule (2 o and 3 o The following was evaluated: the CD spectrum was superimposed in reverse, and the results are shown in Table 19.

[0304] Table 19 shows the average molecular weight of each glycan determined. The N-linked oligosaccharide or glycan molecular weights observed for the BNJ441 batch were consistent with the theoretical glycan molecular weights shown in Table 20. Free glycan molecular weight spectra were determined by MALDI-TOF mass spectrometry. This method identified glycans associated with drug molecules by molecular weight. The glycans were previously enzymatically cleaved from the antibody by PNGase F. The glycans were then solid-phase extracted and mixed with a 3,4-dihydroxybenzoic acid matrix solution for co-precipitation on a MALDI target. This dried sample was ionized in a TOF mass spectrometer using a nitrogen laser. m / z(M+Na) + The spectrum was collected. [Table 20]

[0305] Table 19 shows the oligosaccharide percentages determined for batch BNJ441. The total for various types of N-linked oligosaccharides, i.e., (total of G0F and G1F), acidic, high-mannose, neutral, monosialylated, and diciallylated, was calculated. Only N-linked oligosaccharides contained neutral oligosaccharides. The levels of neutral oligosaccharides were 99.99% and 100.0% for the operation and GMP batches, respectively. Oligosaccharides were detected using HPLC, and chromatograms were quantitatively evaluated. This method evaluates the glycosylation pattern by identifying N-linked oligosaccharides associated with drug molecules based on the retention time of enzymatically released and fluorescently tagged oligosaccharides. This method provided a relatively abundant sample of each oligosaccharide species. Briefly, oligosaccharides were enzymatically cleaved from the antibody using PNGase F and tagged with anthranilic acid. Excess anthranilic acid was removed using a HILIC filtration step. Samples were injected onto a WAX-HPLC system equipped with a Showa Denko Asahipak amino column, and tagged oligosaccharides were detected using a fluorescence detector (360 nM excitation and 420 nM emission).

[0306] The monosaccharide percentages for BNJ441 were determined and are shown in Table 19. Monosaccharide percentages were determined for five monosaccharides (GlcNAc, GalNAc, galactose, mannose, and fucose) using reverse-phase high-pressure chromatography (RP-HPLC) following fluorescent labeling. This assay characterizes the glycosylation pattern by determining the monosaccharide associated with the drug molecule based on the retention time of the fluorescently labeled monosaccharide. Briefly, acid hydrolysis removes oligosaccharides from the protein, leaving its constituent monosaccharides. The free monosaccharides were then labeled with anthranilic acid (AA) by reductive amination. The sample was then injected onto an RP-HPLC system with a Waters Symmetry® C-18 column, and the AA-tagged monosaccharides were detected by a fluorescence detector (360 nM excitation, 420 nM emission). The sample was tested twice, and the reported value was the average of the two results.

[0307] Next, N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) sialic acids were determined. In each case, the determined NANA and NGNA sialic acid content of the BNJ441 batch was below the limit of quantification shown in Table 19 (less than 6 mMol / mol). NGNA was not observed in any batch. Sialic acid was measured and separated on RP-HPLC using multi-point calibration according to fluorescent labeling. This method evaluates the glycosylation pattern by determining the type and relative amount of sialic acid associated with the drug molecule. Sialic acid was chemically cleaved from the antibody by incubation with sodium bisulfate and then tagged with O-phenylenediamine. The sample was injected onto an RP-HPLC system with a Beckman C18 Ultrasphere column, and the tagged sialic acid was detected by a fluorescence detector (230 nM excitation, 425 nM emission). The sample was tested twice, and the average of the two results was reported.

[0308] As shown in Table 19, the determined T for each BNJ441 batch mThe value was 67.0°C. Differential scanning calorimetry (DSC) scanning was performed, and calorimetry data was acquired using a Micro-Cal VP-DSC by scanning upward at a rate of 75°C / hour from 20°C to 95°C. Y-axis and temperature calibration was performed before testing the sample. The Y-axis deflection % error was less than 1%, and the transition midpoint was within the acceptable range of ±0.2°C at both 28.2°C and 75.9°C. The sample was scanned against a blank of the same buffer composition and volume. DSC measures the enthalpy (ΔH) of expansion due to thermal denaturation. Biomolecules in solution are in equilibrium between their native (folded) structure and their denatured (unfolded) state. Transition midpoint (T m ) is the temperature at which 50% of a protein retains its native structure and 50% denatures. T for each sample m This is determined by measuring ΔH over a temperature gradient in the sample cells, compared to that of the tribe cells.

[0309] The affinity (KD) values ​​for BNJ441 operation and GMP batch material were 461 pM and 421 pM, respectively, indicating good compatibility. The binding kinetics of each BNJ441 batch are shown in Table 19. The binding kinetics of the anti-C5 antibody (BNJ441) to human C5 were evaluated using surface plasmon resonance (Biacore 3000). Sensorgrams are not shown. The kinetics of BNJ441 to C5 were determined using an anti-Fc human capture method. Anti-Fc human (KPL#01-10-20), diluted to 0.1 mg / mL in 10 mM sodium acetate at pH 5.0, was immobilized on two flow cells of a CM5 tip for 8 minutes by amine coupling. Anti-C5 antibody (BNJ441) was diluted to 0.35 μg / mL in electrophoresis buffer (HBS-EP, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). The diluted antibody was then injected into another flow cell, followed by injection of C5 (0.19–6 nM) into both flow cells. A secondary flow cell was used as a reference. The surface was regenerated with 20 mM HCl and 0.01% P20 each time (100 μL / min, 200 μL injection). A 1:1 Langmuir model was used, and data were processed using BIAevaluation 4.1 software with "dual reference".

[0310] The affinity (KD) for self-assembly of BNJ441 for the BNJ441 operation and GMP batch material was 7.1 mM and 0.27 mM, respectively. See Table 19. Poor compatibility was attributed to the low levels of binding observed for both the BNJ441 operation and GMP batch material, and the self-assembly and measured affinity were below the detection limit of the instrument. Low levels of self-assembly are beneficial for manufacturability and ultimately for administration to patients. Sensorgrams are not shown. The self-assembly kinetics of the anti-C5 antibody (BNJ441) were evaluated using surface plasmon resonance (Biacore 3000). The self-assembly kinetics of BNJ441 were determined by direct fixation of the antibody (BNJ441). BNJ441 diluted to approximately 31 μg / mL in 10 mM sodium acetate at pH 5.0 was fixed on one flow cell of a CM5 tip, and 2000 RU was obtained by amine coupling. A secondary flow cell was used as a reference. Next, diluted anti-C5 antibody BNJ441 (1.6–50 μM in electrophoresis buffer, HBS-EP, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) was injected into both flow cells. Regeneration was not necessary due to poor binding. Using a stable-state affinity model, the data was processed using BIAevaluation 4.1 software with "dual reference".

[0311] Physicochemical characterization of BNJ441 was performed using manipulation and GMP batches, and it was shown to be consistent with the amino acid sequence of the antibody. The physicochemical data summarized in this example encompass a range of properties including purity, molecular size, identity, structure, glycosylation, thermal stability, kinetics, and self-assembly, and is expected to serve as a basis for characterizing the BNJ441 bulk drug.

[0312] While this disclosure has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of this disclosure. In addition, many modifications can be made to adapt specific circumstances, materials, substance compositions, processes, or one or more process steps to the purposes, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of this disclosure.

Claims

[Claim 1] The kit or method described in the specification.

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