Anti-C1s Antibodies and Methods of Use

Abstract

The present invention provides an anti-C1s antibody and methods for using the antibody. The affinity of the antibody for C1s depends on pH and other conditions. The present invention also provides pharmaceutical formulations containing the antibody and methods for treating an individual with a complement-mediated disease or disorder, comprising administering the antibody to the individual. In addition, the binding affinity of the antibody at high and low pH was measured, and mouse PK studies were performed with the antibody to evaluate the pharmacokinetic parameters of the antibody.

Description

[Technical Field]

[0001] The present invention relates to anti-C1s antibodies and methods of using them. [Background technology]

[0002] background The complement system contains approximately 25–30 complement proteins that play a key role in host defense against pathogens, foreign antigens, and tumor cells. The complement system also contributes to maintaining homeostasis by removing immune complexes and apoptotic cells from the body. Complement components accomplish their functions by interacting in a cascade of enzymatic processes and membrane-binding events. The end result of these processes is the generation of products with lytic, immunoregulatory, and opsonic functions.

[0003] It is widely known that the complement system can be divided into three distinct pathways: the classical pathway, the leucine pathway, and the alternative pathway. Although the initiation of each pathway is different, all three pathways share the same terminal complement components (C5-C9) that converge and are ultimately responsible for the destruction of target cells.

[0004] The C1 complex is a large protein complex that serves as the primary initiator of the classical pathway cascade. It consists of three components: C1q, C1r, and C1s, in a molar ratio of 1:2:2 (Non-Patent Document 1). The classical pathway is initiated when the C1 complex binds to an antibody-bound target. C1q, which has six globular heads, mediates the binding of the C1 complex to the antibody through an avidity interaction with the Fc region. Upon tight binding to the target, C1r within the C1 complex autoactivates and becomes enzymatically active. Activated C1r then cleaves and activates the proenzyme C1s within the C1 complex (Non-Patent Document 2). Activated C1s then cleaves its substrates, complement components C2 and C4, into C2a / C2b and C4a / C4b fragments, respectively. This allows the assembly of the C3 convertase C4b2a on the target surface, which cleaves C3 to form C3b. C3b then cleaves C5, initiating the formation of the terminal membrane attack complex of C5b, C6, C7, C8, and C9, which lyses the target through pore formation.

[0005] C1s forms a calcium-dependent homodimer (Non-Patent Document 3). It has been reported that at a calcium ion concentration of 1 mM, C1s is mostly in a dimeric state, whereas at a calcium concentration of 1 nM, it is primarily in a monomeric state (Non-Patent Document 4). In blood, C1s and C1r are predominantly associated as calcium-dependent C1r2s2 heterotetramers, which reversibly bind to C1q in a 1:1 ratio to form the C1 complex. In the absence or low concentration of calcium, the C1r2s2 tetramer dissociates into one C1r dimer and two C1s monomers (Non-Patent Document 5). C1s is a 79 kDa glycoprotein, and 5–6% of its mass is due to glycosylation (Non-Patent Document 6). The serum C1s concentration has been reported to be approximately 55 μg / mL (0.7 μM) (Non-Patent Document 7).

[0006] Although a properly functioning complement system protects the host from pathogens, dysregulation or inappropriate activation of the classical pathway leads to various complement-mediated disorders, including, but not limited to, autoimmune hemolytic anemia (AIHA), Behçet's disease, bullous pemphigoid (BP), immune thrombocytopenic purpura (ITP), etc. Therefore, inhibition of excessive or uncontrolled activation of the classical pathway may provide clinical benefit to patients with such disorders.

[0007] Antibodies are very attractive pharmaceuticals because they are stable in plasma, highly specific for their targets, and generally exhibit favorable pharmacokinetic profiles. However, due to their large molecular size, the doses of therapeutic antibodies are typically high. If the target is present in high abundance, even higher therapeutic doses of antibodies are required. Consequently, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive ways to reduce the doses and high manufacturing costs associated with therapeutic antibodies.

[0008] Several previous reports have described anti-C1s antibodies. For example, Matsumoto et al. (1986) (Non-Patent Document 8) described three antibodies that bind to different epitopes on C1s. One clone preferentially bound to the active form of C1s, while the other two bound to both the proenzyme and the active form of C1s. Only one of these two clones was able to inhibit the cleavage of C2 and C4 by C1s. Patent Document 1 describes an antibody that inhibits the cleavage of C4 but not C2 through C1s. In addition, Patent Document 2 describes several antibodies that bind to conformational epitopes on C1s that are selective for the active form of C1s compared to the proenzyme form. Patent Document 3 describes two clones of anti-C1s antibodies that can block the cleavage of C4.

[0009] The affinity of an antibody for its antigen determines how effectively the antibody can neutralize its target. Various affinity maturation methods (Non-Patent Document 9) have been used to increase the affinity of antibodies so as to reduce the dose required for therapeutic effect. However, one antibody molecule typically has two binding sites and is therefore limited in that it can only neutralize two targets (one antigen per binding site) after administration. Even if an antibody can bind to a target with infinite affinity through covalent interactions, the maximum number of targets neutralized by the antibody is still limited to two.

[0010] Antibodies that bind to antigens in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies") have been reported to enable the neutralization of multiple antigen molecules by a single antibody molecule (Non-Patent Document 10, Patent Document 4). pH-dependent antibodies bind strongly to their antigens under neutral pH conditions in plasma but dissociate from the antigen under acidic pH conditions in cellular endosomes. Upon dissociation from the antigen, the antibody is recycled into plasma by the FcRn receptor, and the dissociated antigen is degraded in cellular lysosomes. The recycled antibody is then free to bind to and neutralize antigen molecules again, and this process is repeated as long as the antibody remains in the blood. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2014 / 071206 [Patent Document 2] WO2014 / 066744 [Patent Document 3] WO2014 / 186599 [Patent Document 4] WO2009 / 125825 [Non-patent literature]

[0012] [Non-Patent Document 1] Wang et. al. Mol Cell. 2016 Jul 7;63(1):135-45 [Non-patent document 2] Mortensen et. al. Proc Natl Acad Sci US A. 2017 Jan 31;114(5):986-991 [Non-patent document 3] Arlaud et. al. Biochim Biophys Acta. 1980 Nov 6;616(1):105-15 [Non-patent document 4] Rivas et. al. Biochemistry. 1992 Dec 1;31(47):11707-12 [Non-Patent Document 5] Rossi et.al. Methods Mol Biol. 2014;1100:43-60 [Non-patent document 6] Petillot et. al. FEBS Lett. 1995 Jan 30;358(3):323-8 [Non-Patent Document 7] Shi et. al. Blood. 2014 Jun 26;123(26):4015-22 [Non-patent document 8] Matsumoto et. al. J Immunol. 1986 Nov 1;137(9):2907-12 [Non-Patent Document 9] Kim et. al. Methods Mol Biol. 2014;1131:407-20 [Non-Patent Document 10] Igawa et. al. Nat Biotechnol. 2010 Nov;28(11):1203-7 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides anti-C1s antibodies and methods of using same. [Means for solving the problem]

[0014] In addition to binding to C1s in a pH-dependent manner, the effect of calcium on the affinity of pH-dependent antibodies for C1s may be another important characteristic. C1s forms dimers at high calcium concentrations but dissociates into monomers at low calcium concentrations. When C1s is in a dimeric state, bivalent antibodies can form immune complexes by cross-linking multiple C1s molecules. This allows antibodies to bind to C1s molecules in the complex through both affinity and avidity interactions, thereby increasing the apparent affinity of the antibody. In contrast, when C1s is in a monomeric state, antibodies bind to C1s only through affinity interactions. This means that pH-dependent C1s antibodies can form immune complexes with dimeric C1s in plasma, but C1s dissociates into monomers upon entry into acidic endosomes. This disassembles the immune complex, which enhances the pH-dependent dissociation of the antibody from the antigen.

[0015] In some embodiments, the isolated anti-C1s antibody of the present invention has a C1s-binding activity that varies depending on ion concentration. In some embodiments, the isolated anti-C1s antibody binds to C1s with higher affinity at neutral pH than at acidic pH. In some embodiments, the anti-C1s antibody binds to C1s with higher affinity under high calcium concentration conditions than under low calcium concentration conditions. In some embodiments, the isolated anti-C1s antibody binds to C1s with higher affinity under neutral pH and high calcium concentration conditions than under acidic pH and low calcium concentration conditions.

[0016] In some embodiments, when an isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In some embodiments, when an isolated anti-C1s antibody of the present invention is measured under low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to C1s in a dimeric state. In some embodiments, for an isolated anti-C1s antibody of the present invention, when measured under high calcium concentrations at both neutral and acidic pH, the ratio of the koff value of its C1s binding activity at acidic pH to the koff value of its C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater. In some embodiments, for an isolated anti-C1s antibody of the present invention, when measured under low calcium concentrations at both neutral and acidic pH, the ratio of the koff value of its C1s binding activity at acidic pH to the koff value of its C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to C1s in a dimeric state. In some embodiments, for an isolated anti-C1s antibody of the present invention, when measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater.

[0017] In some embodiments, the isolated anti-C1s antibody of the present invention has the following position according to the Kabat numbering system: Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97 One or more of the amino acids contain a histidine residue. In some embodiments, the isolated anti-C1s antibody of the present invention has the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 One or more of the amino acids contain a histidine residue.

[0018] In some embodiments, the isolated anti-C1s antibody of the present invention has the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 It contains one, two, three, four or five histidines.

[0019] In some embodiments, the isolated anti-C1s antibody of the present invention has a C1s residue at the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 and a histidine residue at one or more amino acid positions of one or more CDRs or one or more amino acid positions of one or more FRs.

[0020] In some embodiments, the isolated anti-C1s antibody of the present invention has the following positions according to the Kabat numbering system: 1) L92 and L94 2) L92 and L95 3) L94 and L95 4) L92, L94, and L95 5) H65 and L92 6) H65 and L94 7) H65 and L95 8) H65, L92, and L94 9) H65, L92, and L95 10) H65, L94, and L95 11) H65, L92, L94, and L95 12) H99 and L92 13) H99 and L94 14) H99 and L95 15) H99, L92, and L94 16) H99, L92, and L95 17) H99, L94, and L95 18) H99, L92, L94, and L95 19) H65 and H99 20) H65, H99, and L92 21) H65, H99, and L94 22) H65, H99, and L95 23) H65, H99, L92, and L94 24) H65, H99, L92, and L95 25) H65, H99, L94, and L95 26) H65, H99, L92, L94, and L95, or 27) H27, H99, and L95 contains a histidine residue.

[0021] In some embodiments, the anti-C1s antibodies of the invention have the following positions according to the Kabat numbering system: Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97 and at least one histidine substituted in one or more of:

[0022] In some embodiments, the anti-C1s antibodies of the invention have the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 and at least one histidine substituted in one or more of:

[0023] In some embodiments, the isolated anti-C1s antibody of the present invention has the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 and containing one, two, three, four, or five substituted histidines.

[0024] In some embodiments, the isolated anti-C1s antibody of the present invention has a C1s residue at the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 and at least one histidine residue as a substituted residue at one or more amino acid positions of one or more CDRs or at one or more amino acid positions of one or more FRs.

[0025] In some embodiments, the isolated anti-C1s antibody of the present invention has the following positions according to the Kabat numbering system: 1) L92 and L94 2) L92 and L95 3) L94 and L95 4) L92, L94, and L95 5) H65 and L92 6) H65 and L94 7) H65 and L95 8) H65, L92, and L94 9) H65, L92, and L95 10) H65, L94, and L95 11) H65, L92, L94, and L95 12) H99 and L92 13) H99 and L94 14) H99 and L95 15) H99, L92, and L94 16) H99, L92, and L95 17) H99, L94, and L95 18) H99, L92, L94, and L95 19) H65 and H99 20) H65, H99, and L92 21) H65, H99, and L94 22) H65, H99, and L95 23) H65, H99, L92, and L94 24) H65, H99, L92, and L95 25) H65, H99, L94, and L95 26) H65, H99, L92, L94, and L95, or 27) H27, H99, and L95 contains at least one histidine as a substituted residue.

[0026] In some embodiments, the isolated anti-C1s antibody of the present invention exhibits, with respect to binding to C1s under neutral pH conditions, (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:23, the HVR-H2 sequence of SEQ ID NO:24, the HVR-H3 sequence of SEQ ID NO:25, the HVR-L1 sequence of SEQ ID NO:26, the HVR-L2 sequence of SEQ ID NO:27, and the HVR-L3 sequence of SEQ ID NO:28; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:29, the HVR-H2 sequence of SEQ ID NO:30, the HVR-H3 sequence of SEQ ID NO:31, the HVR-L1 sequence of SEQ ID NO:32, the HVR-L2 sequence of SEQ ID NO:33, and the HVR-L3 sequence of SEQ ID NO:34; (c) human monoclonal anti-C1s antibody M241 or human monoclonal anti-C1s antibody M81; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (f) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (g) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (h) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:85. wherein the antibody binds to C1s with higher affinity at neutral pH than at acidic pH, as described in (i) or (ii) below: (i) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater; (ii) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the koff value of C1s binding activity at acidic pH to the koff value of C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater. In some embodiments, the isolated anti-C1s antibody of the present invention has, with respect to binding to C1s, (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (c) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:85; The antibody competes with an antibody selected from the group consisting of:

[0027] In some embodiments, the present disclosure provides an isolated anti-C1s antibody that specifically binds to an epitope within a region comprising domains IV and V of complement component I (C1s). In some instances, the antibody inhibits C1s from binding to complement component 4 (C4). In some instances, the epitope bound by an isolated anti-C1s antibody of the present disclosure is a conformational epitope. In some embodiments, the epitope of C1s is an epitope of human C1s.

[0028] In some embodiments, the anti-C1s antibodies of the invention have the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 wherein at least one amino acid in one or more of SEQ ID NO:35 or 36 is substituted with histidine.

[0029] In some embodiments, the anti-C1s antibody of the present invention has at least one amino acid in the variable region substituted with an amino acid selected from the group consisting of D, E, K, R and Q to reduce the non-specific binding activity of the antibody at acidic pH.

[0030] In some embodiments, the anti-C1s antibodies of the present invention have at least one amino acid in the variable region substituted with an amino acid selected from the group consisting of D, E, K, R, and Q so as to increase the ratio of the KD values ​​of C1s binding activity at acidic pH to C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)).

[0031] In some embodiments, an isolated anti-C1s antibody of the invention comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:39, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:40, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:41, and comprises framework regions of human or primate origin. In some embodiments, an isolated anti-C1s antibody of the invention comprises (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:43, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:44, and comprises framework regions of human or primate origin.

[0032] In some embodiments, anti-C1s antibodies of the invention comprise (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:19, 17, or 22; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20, 18, or 21; or (c) the VH sequence of (a) and the VL sequence of (b). In some embodiments, anti-C1s antibodies of the invention comprise the VH sequence of SEQ ID NO:19, 17, or 22. In some embodiments, anti-C1s antibodies of the invention comprise the VL sequence of SEQ ID NO:20, 18, or 21. In further embodiments, anti-C1s antibodies of the invention comprise the VH sequence of SEQ ID NO:19, 17, or 22 and the VL sequence of SEQ ID NO:20, 18, or 21. In further embodiments, anti-C1s antibodies of the invention comprise the VH sequence of SEQ ID NO:19 and the VL sequence of SEQ ID NO:20. In a further embodiment, an anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 19 and the VL sequence of SEQ ID NO: 21. In a further embodiment, an anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO: 21.

[0033] In some embodiments, the isolated anti-C1s antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-C1s antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the isolated anti-C1s antibody of the present invention is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In further embodiments, the isolated anti-C1s antibody of the present invention is an antibody fragment that binds to C1s. In some specific embodiments, the isolated anti-C1s antibody of the present invention is a human IgG1 or humanized IgG1.

[0034] The present invention also provides isolated nucleic acids encoding anti-C1s antibodies of the present invention. The present invention also provides host cells comprising nucleic acids of the present invention. The present invention also provides methods for producing antibodies, comprising culturing host cells of the present invention so that the antibodies are produced.

[0035] The present invention also provides a pharmaceutical formulation comprising an antibody of the present invention and a pharmaceutically acceptable carrier.

[0036] The anti-C1s antibodies of the present invention may be for use as pharmaceuticals. The anti-C1s antibodies of the present invention may be for use in treating complement-mediated diseases or disorders. The anti-C1s antibodies of the present invention may be for use in enhancing the clearance (or removal) of C1s from plasma. The anti-C1s antibodies of the present invention may be for use in enhancing the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma. In some embodiments, the anti-C1s antibodies of the present invention may be for use in inhibiting the cleavage of complement component C4, where the antibody does not inhibit the cleavage of complement component C2. In some examples, the antibody inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s.

[0037] The anti-C1s antibodies of the present invention can be used in the manufacture of a pharmaceutical. In some embodiments, the pharmaceutical is for treating a complement-mediated disease or disorder. In some embodiments, the pharmaceutical is for enhancing the clearance (or removal) of C1s from plasma. In some embodiments, the pharmaceutical is for enhancing the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma. The pharmaceutical is for inhibiting cleavage of complement component C4, where the antibody does not inhibit cleavage of complement component C2. In some examples, the pharmaceutical inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s.

[0038] The present invention also provides methods for treating an individual having a complement-mediated disease or disorder. In some embodiments, the methods comprise administering to the individual an effective amount of an anti-C1s antibody of the present invention. The present invention also provides methods for enhancing the clearance (or removal) of C1s from plasma in an individual. In some embodiments, the methods comprise administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance (or removal) of C1s from plasma. The present invention also provides methods for enhancing the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma in an individual. In some embodiments, the methods comprise administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma. The present invention also provides methods for inhibiting the cleavage of complement component C4, wherein the antibody does not inhibit the cleavage of complement component C2. In some instances, the antibody inhibits a component of the classical complement pathway, and in some instances, the component of the classical complement pathway is C1s.

[0039] More specifically, the present invention provides: [1] An isolated antibody that binds to C1s, wherein the antibody binds to C1s with higher affinity at neutral pH than at acidic pH, as described in (i) or (ii) below: (i) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater; (ii) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the koff value of C1s binding activity at acidic pH to the koff value of C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater. [2] Position in the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 The antibody of [1], wherein one or more of the following residues contain histidine residues: [3] Position in the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 The antibody of [1] or [2], wherein at least one amino acid in one or more of: [4] Under neutral pH conditions, binding to C1s: (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:23, the HVR-H2 sequence of SEQ ID NO:24, the HVR-H3 sequence of SEQ ID NO:25, the HVR-L1 sequence of SEQ ID NO:26, the HVR-L2 sequence of SEQ ID NO:27, and the HVR-L3 sequence of SEQ ID NO:28; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:29, the HVR-H2 sequence of SEQ ID NO:30, the HVR-H3 sequence of SEQ ID NO:31, the HVR-L1 sequence of SEQ ID NO:32, the HVR-L2 sequence of SEQ ID NO:33, and the HVR-L3 sequence of SEQ ID NO:34; (c) human monoclonal anti-C1s antibody M241; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (f) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (g) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (h) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:85. An antibody according to any one of [1] to [3], which competes with an antibody selected from the group consisting of: [5] At least one amino acid in the variable region is located at the following position according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96 The antibody of any of [1] to [4], comprising a VH sequence of SEQ ID NO: 35 or 36 and / or a VL sequence of SEQ ID NO: 37 or 38, wherein one or more of the following are substituted with histidine: [6] Furthermore, at least one amino acid in the variable region is 1) to reduce the nonspecific binding activity of the antibody at acidic pH, or 2) The ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is increased. The antibody of any one of [1] to [5], wherein the antibody is substituted with an amino acid selected from the group consisting of D, E, K, R, and Q. [7] An isolated antibody that binds to C1s, comprising (a) an HVR-H1 having the amino acid sequence of SEQ ID NO:39, (b) an HVR-H2 having the amino acid sequence of SEQ ID NO:40, and (c) an HVR-H3 having the amino acid sequence of SEQ ID NO:41, and comprising framework regions of human or primate origin. [8] An isolated antibody that binds to C1s, comprising (a) HVR-L1 having the amino acid sequence of SEQ ID NO:42, (b) HVR-L2 having the amino acid sequence of SEQ ID NO:43, and (c) HVR-L3 having the amino acid sequence of SEQ ID NO:44, and comprising framework regions of human or primate origin. [9] An antibody according to any of [1] to [8], comprising: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, 17 or 22; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20, 18 or 21; or (c) the VH sequence of (a) and the VL sequence of (b).

[10] The antibody of [9], comprising a VH sequence of SEQ ID NO: 19, 17 or 22.

[11] The antibody of [9], comprising a VL sequence of SEQ ID NO: 20, 18 or 21.

[12] An antibody comprising the VH sequence of SEQ ID NO: 19 and the VL sequence of SEQ ID NO: 20.

[13] A pharmaceutical formulation comprising any one of the antibodies according to [1] to

[12] and a pharmaceutically acceptable carrier.

[14] A method for treating an individual with a complement-mediated disease or disorder, comprising the step of administering to the individual an effective amount of any of the antibodies [1] to

[12] . Additionally, the present invention provides:

[15] A method for removing C1s from plasma, comprising: (a) identifying an individual in need of removal of C1s from their plasma; (b) providing an antibody, wherein the antibody binds to C1s through its C1s-binding domain and has a KD(pH 5.8) / KD(pH 7.4) value of 2 to 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of KD for C1s at pH 5.8 to KD for C1s at pH 7.4, as determined using surface plasmon resonance technology, and the antibody binds to C1s in plasma in vivo and dissociates from the bound C1s under conditions present in endosomes in vivo, and the antibody is a human IgG or a humanized IgG; and (c) administering the antibody to the individual. A method comprising:

[16] A method for removing C1s from plasma in a subject, comprising: (a) identifying a first antibody, wherein the first antibody binds to C1s through the C1s binding domain of the first antibody; (b) identifying a second antibody, wherein the second antibody is: (1) binding to C1s through the C1s-binding domain of the second antibody; (2) has the same amino acid sequence as the first antibody, except that at least one amino acid in the variable region of the first antibody has been substituted with histidine and / or at least one histidine has been inserted into the variable region of the first antibody; (3) having a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the first antibody and is between 2 and 10,000, wherein KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4, as determined using surface plasmon resonance technology; (4) Binds to C1s in plasma in vivo (5) Under conditions present in endosomes in vivo, it dissociates from bound C1s, and (6) Human IgG or humanized IgG. process; (c) identifying a subject in need of reducing plasma C1s levels; and (d) administering a second antibody to the subject such that the subject's plasma C1s level is reduced. A method comprising:

[17] A method for removing C1s from plasma in a subject, comprising: (a) identifying a first antibody, wherein the first antibody is: (1) binding to C1s through the C1s-binding domain of the first antibody; (2) at least one variable region of the first antibody has the same amino acid sequence as a second antibody that binds to C1s through the antigen-binding domain of the second antibody, except that the variable region of the first antibody has at least one more histidine residue than the corresponding variable region of the second antibody; (3) having a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the second antibody and is between 2 and 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4, as determined using surface plasmon resonance technology; (4) Binds to C1s in plasma in vivo (5) Under conditions present in endosomes in vivo, it dissociates from bound C1s, and (6) Human IgG or humanized IgG. process; (b) identifying a subject in need of reducing plasma C1s levels; and (c) administering the first antibody to the subject at least once so as to reduce the subject's plasma C1s level. A method comprising:

[18] The antibody was immobilized, the antigen was used as the analyte, and the following conditions were used: 10 mM MES buffer, 0.05% polyoxyethylenesorbitan monolaurate, and 150 mM NaCl at 37 degrees Celsius (°C). The KD is determined using surface plasmon resonance technology using any one of the methods

[15] -

[17] .

[19] An isolated antibody that binds to the C1 complex consisting of C1q, C1r, and C1s, wherein the antibody binds to the C1 complex with higher affinity at neutral pH than at acidic pH, as described in (i) or (ii) below: (i) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the KD value of C1 complex binding activity at acidic pH to the KD value of C1 complex binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more; (ii) When measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of the KD value of C1 complex binding activity at acidic pH to the KD value of C1 complex binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more.

[20] An isolated antibody that binds to C1s, wherein the antibody binds to C1s with higher affinity at neutral pH than at acidic pH, and when measured under low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of the C1s binding activity at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more, and the anti-C1s antibody binds to C1s in a dimeric state. [Brief explanation of the drawings]

[0040] [Figure 1]FIG. 1 shows the correlation between the improvement in sweeping index and the KD(5.8+) / KD(7.4+) ratio for all antibodies listed in Table 10 except for antibody IPN92H0286 / IPN93L0205-SG136. [Figure 2] Figure 2 shows the correlation between the improvement in sweeping index and the koff (5.8+ of 775+) / koff (7.4+) ratio for all antibodies listed in Table 10 except for antibody IPN92H0286 / IPN93L0205-SG136. DETAILED DESCRIPTION OF THE INVENTION

[0041] The techniques and procedures described or cited herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993), using conventional techniques commonly used by those skilled in the art.

[0042] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0043] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0044] An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0045] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd or KD). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. The term "avidity" refers to the strength of the total non-covalent interactions between a single or multiple binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, avidity is not strictly limited to activity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). When members of a binding pair can bind to each other in both monovalent and multivalent binding modes, avidity is the strength of the sum of these bonds. The avidity of molecule X to its partner Y can usually be expressed by dissociation constant (KD). Alternatively, the on-rate and off-rate (Kon and Koff) can be used to evaluate binding. Avidity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0046] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0047] The term "anti-C1S antibody" or "antibody that binds to C1S" refers to an antibody that can bind to C1S with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to C1S. In one embodiment, the extent of binding of the anti-C1S antibody to an unrelated, non-C1S protein is less than about 10% of the antibody's binding to C1S, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C1S has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-C1S antibody binds to an epitope of C1S that is conserved among C1S from different species.

[0048] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0049] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0050] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0051] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0052] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0053] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212Pb, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor or anticancer agents, as disclosed below.

[0054] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0055] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0056] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen and includes specific amino acids that directly contact the antibody. Epitopic determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0057] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0058] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0059] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0060] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0061] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0062] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0063] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0064] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0065] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0066] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0067] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0068] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0069] "Isolated nucleic acid encoding an anti-C1S antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell.

[0070] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0071] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0072] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0073] The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0074] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0075] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be said that a given amino acid sequence A has or contains a certain percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0076] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0077] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0078] As used herein, the term "C1S," unless otherwise indicated, refers to any native C1S from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed C1S, as well as any form of C1S resulting from processing in cells. The term also encompasses naturally occurring variants of C1S, such as splice variants and allelic variants. An exemplary amino acid sequence of human C1S is set forth in SEQ ID NO:1. Exemplary amino acid sequences of cynomolgus monkey and rat C1S are set forth in SEQ ID NOs:3 and 2, respectively.

[0079] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0080] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0081] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0082] II. Compositions and Methods In one aspect, the present invention is based in part on anti-C1S antibodies and uses thereof. In certain embodiments, antibodies that bind to C1S are provided. The antibodies of the present invention are useful, for example, for the diagnosis or treatment of complement-mediated diseases or disorders.

[0083] A. Exemplary Anti-C1s Antibodies In one aspect, the present invention provides an isolated antibody that binds to C1s. In one aspect, the present invention provides an isolated antibody that binds to C1s, wherein the binding activity varies depending on ion concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on pH, i.e., hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on calcium concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on both pH and calcium concentration. Such antibodies are expected to be particularly advantageous as pharmaceuticals, since they allow for reduced dosage and administration frequency in patients, thereby reducing the total dose.

[0084] In one aspect, when an isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In one aspect, when an isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, the ratio of the kD value of its C1s binding activity at acidic pH to the kD value of its C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater. In one aspect, when an isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater. In some embodiments, when an isolated anti-C1s antibody of the present invention is measured under low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to dimeric C1s. In some embodiments, when an isolated anti-C1s antibody of the present invention is measured under low calcium concentrations at both neutral and acidic pH, the ratio of the koff value of its C1s binding activity at acidic pH to the koff value of its C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to dimeric C1s.

[0085] Without being bound by any particular theory, if 1) the epitope structure of C1s bound by the antibody of the present invention may undergo conformational changes in the absence of calcium, thereby changing the affinity of the antibody, or 2) the interaction (affinity type or avidity type) of the antibody of the present invention may change depending on the state of C1s (monomer state or dimer state), measurements using specific conditions (under high calcium concentration at neutral pH and under low calcium concentration at acidic pH) can be used to evaluate the ratio of KD values ​​(KD (acidic pH) / KD (neutral pH)).

[0086] In other words, the antibodies of the present invention bind to C1s with higher affinity at neutral pH than at acidic pH as described in (i) or (iii) below: (i) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater; (ii) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the koff value of C1s binding activity at acidic pH to the koff value of C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater; (iii) When measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater.

[0087] More generally, without being bound by any particular theory, if 1) the epitope structure of a particular antigen bound by an antibody of the present invention may conformationally change in the absence of calcium, thereby altering the affinity of the antibody, or 2) the interaction (affinity type or avidity type) of an antibody of the present invention may change depending on the state of the antigen (monomer state or dimer state), measurements using specific conditions (high calcium concentration at neutral pH and low calcium concentration at acidic pH) can be used to evaluate the ratio of KD values ​​(KD(acidic pH) / KD(neutral pH)). If this ratio is high, the affinity at acidic pH is lower than the affinity at neutral pH. Alternatively, as mentioned below, KD can be calculated by the following equation: off / k on It is defined as the ratio of k between acidic and neutral conditions. off The ratio of values, ie, (koff(acidic pH) / koff(neutral pH)), can also be used for comparison between affinity at acidic pH and affinity at neutral pH.

[0088] Thus, the antibodies of the present invention bind to antigens with higher affinity at neutral pH than at acidic pH as follows: when measured at high calcium concentrations at neutral pH and at low calcium concentrations at acidic pH, the ratio of the KD value of antigen-binding activity at acidic pH to the KD value of antigen-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater.

[0089] In one aspect, for an isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured under high calcium concentrations at both neutral and acidic pH, and the antibody does not contain CDR-H1 (SEQ ID NO:23), CDR-H2 (SEQ ID NO:24), CDR-H3 (SEQ ID NO:25), CDR-L1 (SEQ ID NO:26), CDR-L2 (SEQ ID NO:27) and CDR-L3 (SEQ ID NO:28).

[0090] In one aspect, for an isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater when measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH, and the antibody does not contain CDR-H1 (SEQ ID NO:23), CDR-H2 (SEQ ID NO:24), CDR-H3 (SEQ ID NO:25), CDR-L1 (SEQ ID NO:26), CDR-L2 (SEQ ID NO:27) and CDR-L3 (SEQ ID NO:28). In one aspect, for an isolated anti-C1s antibody of the present invention, when measured under low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to C1s in a dimeric state, and wherein the antibody does not comprise CDR-H1 (SEQ ID NO:23), CDR-H2 (SEQ ID NO:24), CDR-H3 (SEQ ID NO:25), CDR-L1 (SEQ ID NO:26), CDR-L2 (SEQ ID NO:27) and CDR-L3 (SEQ ID NO:28).

[0091] The above KD ratio, i.e., KD(acidic pH) / KD(neutral pH), can be compared between a parent antibody (i.e., the original antibody before the modification of the present invention) and an antibody into which one or more amino acid mutations (e.g., addition, insertion, deletion, or substitution) have been introduced into the original (parent) antibody. The original (parent) antibody can be any known antibody or a newly isolated antibody, as long as it specifically binds to C1s. Thus, in one aspect, for an isolated anti-C1s antibody of the present invention, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, or 10-fold higher than the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) of its original (parent) antibody. In other words, the present invention provides isolated anti-C1s antibodies in which one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) have been introduced into a parent (original) antibody, and the ratio of (i) to (ii) below is at least 1.2, 1.4, 1.6, 1.8, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.5, 2.7, 2.8, 2.9, 3, 3.5, 4, 5, 8 or 10: (i) the ratio of the KD value of the C1s binding activity of the isolated anti-C1s antibody at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)); (ii) the ratio of the KD value of the C1s binding activity of the parent (original) antibody at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)). These KD ratios can be measured under any (high or low) calcium concentration, for example, under high calcium concentrations at both neutral and acidic pH, or under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD to assess pH and / or Ca dependence.

[0092] In one aspect, the antibodies of the present invention have antigen-binding activity that differs between intracellular and extracellular conditions. Intracellular and extracellular conditions refer to the different conditions inside and outside a cell. Categories of conditions include, for example, ion concentration, more specifically, metal ion concentration, hydrogen ion concentration (pH), and calcium ion concentration. "Intracellular conditions" preferably refer to an environment characteristic of the endosomal environment, and "extracellular conditions" preferably refer to an environment characteristic of the plasma environment. Antibodies with the property of having antigen-binding activity that varies depending on ion concentration can be obtained by screening a large number of antibodies for domains that have such properties. For example, antibodies with the above properties can be obtained by producing a large number of antibodies whose sequences differ from each other using the hybridoma method or antibody library method and measuring their antigen-binding activity under different ion concentrations. B cell cloning is an example of a method for screening such antibodies. Furthermore, as described below, at least one characteristic amino acid residue that can confer the property of having an antigen-binding activity that varies depending on ion concentration to an antibody is identified, and a library of multiple antibodies having different sequences but sharing the characteristic amino acid residue as a common structure is prepared. Such a library can be screened to effectively isolate antibodies with the above-mentioned properties.

[0093] In one aspect, the present invention provides antibodies that bind to C1s with higher affinity at neutral pH than at acidic pH. In another aspect, the present invention provides anti-C1s antibodies that exhibit pH-dependent binding to C1s. As used herein, the phrase "pH-dependent binding" means "reduced binding at acidic pH compared to binding at neutral pH," and the two phrases can be interchangeable. For example, anti-C1s antibodies "having pH-dependent binding properties" include antibodies that bind to C1s with higher affinity at neutral pH than at acidic pH.

[0094] In certain embodiments, when measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is greater than or equal to 2. In certain embodiments, antibodies of the invention bind to C1s with at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more greater affinity at neutral pH than at acidic pH.

[0095] In certain embodiments, when measured under high calcium concentrations at both neutral and acidic pH, the ratio of the Koff value of C1s binding activity at acidic pH to the Koff value of C1s binding activity at neutral pH (Koff(acidic pH) / Koff(neutral pH)) is greater than or equal to 2. In certain embodiments, antibodies of the present invention bind to C1s with at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more greater affinity at neutral pH than at acidic pH.

[0096] In certain aspects, when measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In certain embodiments, antibodies of the invention bind to C1s with at least 2, 3, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more greater affinity at neutral pH than at acidic pH.

[0097] In the above example, for example, the acidic pH is 5.8 and the neutral pH is 7.4, and therefore, KD(acidic pH) / KD(neutral pH) is KD(pH 5.8) / KD(pH 7.4). In this regard, examples of acidic pH and neutral pH are described in detail later in this specification. In some embodiments, KD(acidic pH) / KD(neutral pH), for example, KD(pH 5.8) / KD(pH 7.4), can be 2 to 10,000. In the above example, for example, the acidic pH is 5.8 and the neutral pH is 7.4, and therefore, koff(acidic pH) / koff(neutral pH) is koff(pH 5.8) / koff(pH 7.4). In this regard, examples of acidic pH and neutral pH are described in detail later in this specification. In some embodiments, koff(acidic pH) / koff(neutral pH), for example koff(pH 5.8) / koff(pH 7.4), can be between 2 and 10,000.

[0098] When an antigen is a soluble protein, the binding of an antibody to the antigen can result in an extension of the antigen's plasma half-life (i.e., a reduction in the clearance of the antigen from plasma) because the antibody may have a longer half-life in plasma than the antigen itself and may function as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex by FcRn via the intracellular endosomal pathway (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties, which bind to antigens in the neutral extracellular environment but release the antigen into acidic endosomal compartments after intracellular entry, are expected to have superior antigen neutralization and clearance properties compared to counterparts that bind in a pH-independent manner (Igawa et al (2010) Nature Biotechnol 28(11); 1203-1207; Devanaboyina et al (2013) mAbs 5(6): 851-859; International Patent Application Publication No.: WO 2009 / 125825).

[0099] In one aspect, the present invention provides antibodies that bind to C1s with higher affinity under high calcium concentration conditions than under low calcium concentration conditions.

[0100] In the present invention, preferred metal ions include, for example, calcium ions. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction (e.g., skeletal, smooth, and cardiac muscle contraction); activation of leukocytes (e.g., motility, phagocytosis, etc.); activation of platelets (e.g., platelet shape change, secretion, etc.); activation of lymphocytes; activation of mast cells (e.g., histamine secretion); cellular responses mediated by catecholamine alpha receptors or acetylcholine receptors; exocytosis; release of transmitters from neuronal terminals; and axonal flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have several calcium ion-binding sites and are thought to have derived from a common origin in molecular evolution. Furthermore, many calcium-binding motifs have been described. Such well-known motifs include, for example, cadherin domains, the EF hands of calmodulin, the C2 domain of protein kinase C, the Gla domain of the blood clotting protein factor IX, the C-type lectins of the asialoglycoprotein receptor and the mannose-binding receptor, the A domain of the LDL receptor, annexins, the thrombospondin type 3 domain, and the EGF-like domain.

[0101] In the present invention, when the metal ion is a calcium ion, it is desirable that the antigen-binding activity under a low calcium ion concentration condition is lower than that under a high calcium ion concentration condition. Meanwhile, the intracellular calcium ion concentration is lower than the extracellular calcium ion concentration. Conversely, the extracellular calcium ion concentration is higher than the intracellular calcium ion concentration. In the present invention, the low calcium ion concentration is preferably 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. In the present invention, the high calcium ion concentration is preferably 100 μM to 10 μM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). In the present invention, it is preferable that the low calcium ion concentration is the calcium ion concentration in endosomes, and the high calcium ion concentration is the calcium ion concentration in plasma. When comparing the antigen-binding activity levels at low and high calcium ion concentrations, the binding of the antibodies of the present invention is preferably stronger at high calcium ion concentrations than at low calcium ion concentrations. In other words, the antigen-binding activity of the antibodies of the present invention is preferably lower at low calcium ion concentrations than at high calcium ion concentrations. When the level of binding activity is expressed as a dissociation constant (KD), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or greater, even more preferably 10 or greater, and even more preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or greater. There is no particular upper limit to the value of KD (low calcium ion concentration) / KD (high calcium ion concentration), and any value, such as 100, 400, 1000, or 10,000, may be used as long as it is within the skill of those skilled in the art. The dissociation rate constant (kd) can be used instead of KD. If it is difficult to calculate the KD value, the activity can be evaluated based on the level of binding reactivity when the analyte is run at the same concentration on a Biacore.When an antigen is applied to a chip on which an antigen-binding molecule of the present invention is immobilized, the binding reactivity at a low calcium concentration is preferably 1 / 2 or less, more preferably 1 / 3 or less, even more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less of the binding reactivity at a high calcium concentration. It is generally known that the extracellular (e.g., in plasma) calcium ion concentration in a living body is high, while the intracellular (e.g., in endosome) calcium ion concentration is low. Therefore, in the present invention, a high calcium ion concentration is preferred as the extracellular condition, and a low calcium ion concentration is preferred as the intracellular condition. When an antigen-binding molecule (e.g., an antibody) of the present invention is conferred with the property of lower antigen-binding activity under intracellular calcium ion concentration conditions compared to extracellular calcium ion concentration conditions, the antigen bound to the antigen-binding molecule of the present invention extracellularly dissociates from the antigen-binding molecule of the present invention intracellularly, thereby enhancing antigen uptake from extracellular to intracellular. When such an antibody is administered to a living body, it is possible to reduce the antigen concentration in plasma and thereby reduce the physiological activity of the antigen in the living body, making the antibody of the present invention useful. Methods for screening for antigen-binding domains or antibodies with lower antigen-binding activity under low calcium ion concentration conditions than under high calcium ion concentration conditions include, for example, the methods described in WO2012 / 073992 (e.g., paragraphs 0200 to 0213). The method for conferring weaker antigen-binding activity under low calcium ion concentration conditions than under high calcium ion concentration conditions to the antigen-binding domain of the present invention is not particularly limited and may be performed by any method. Specifically, such methods are described in Japanese Patent Application No. 2011-218006 and include, for example, a method of substituting at least one amino acid residue in the antigen-binding domain with an amino acid residue having metal chelating activity and / or a method of inserting at least one amino acid residue having metal chelating activity into the antigen-binding domain. A preferred embodiment of the antigen-binding molecule of the present invention is an antigen-binding molecule in which at least one amino acid residue in the antigen-binding domain has been substituted with an amino acid residue having metal chelating activity and / or in which at least one amino acid residue having metal chelating activity has been inserted into the antigen-binding domain.

[0102] Preferred examples of amino acid residues having metal chelating activity include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. Furthermore, preferred examples of amino acid residues that change the antigen-binding activity of the antigen-binding domain depending on calcium ion concentration include amino acid residues that form a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been reported in detail (e.g., Springer et al., (Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490); Moncrief et al., (J. Mol. Evol. (1990) 30, 522-562); Chauvaux et al., (Biochem. J. (1990) 265, 261-265); Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol. (1990) 2, 410-419); Schaefer et al., (Genomics (1995) 25, 638 to 643); Economou et al., (EMBO J. (1990) 9, 349-354); Wurzburg et al., (Structure. (2006) 14, 6, 1049-1058)). The EF hand of troponin C, calmodulin, parvalbumin, and myosin light chain; the C2 domain of protein kinase C; the Gla domain of blood coagulation protein factor IX; the C-type lectins of asialoglycoprotein receptor and mannose-binding receptor, ASGPR, CD23, and DC-SIGN; the A domain of LDL receptor; annexin domain; cadherin domain; thrombospondin type 3 domain; and EGF-like domain are preferably used as calcium-binding motifs.

[0103] The antigen-binding domain of the present invention may contain amino acid residues whose antigen-binding activity changes depending on calcium ion concentration, such as the aforementioned amino acid residues with metal chelating activity and amino acid residues forming a calcium-binding motif. The position of such amino acid residues within the antigen-binding domain is not particularly limited, and may be any position as long as the antigen-binding activity changes depending on calcium ion concentration. Furthermore, as long as the antigen-binding activity changes depending on calcium ion concentration, such amino acid residues may be contained alone or in combination of two or more. Suitable examples of such amino acid residues include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. When the antigen-binding domain is an antibody variable region, these amino acid residues may be contained in the heavy chain variable region and / or light chain variable region. In a preferred embodiment, these amino acid residues may be contained in the CDR3 of the heavy chain variable region, more preferably at positions 95, 96, 100a, and / or 101 according to the Kabat numbering system in the CDR3 of the heavy chain variable region.

[0104] In another preferred embodiment, these amino acid residues may be contained in CDR1 of the light chain variable region, more preferably at positions 30, 31, and / or 32 according to the Kabat numbering of CDR1 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR2 of the light chain variable region, more preferably at position 50 according to the Kabat numbering of CDR2 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR3 of the light chain variable region, more preferably at position 92 according to the Kabat numbering of CDR3 of the light chain variable region.

[0105] Furthermore, the above embodiments may be combined. For example, the amino acid residue may be contained in two or three CDRs selected from CDR1, CDR2, and CDR3 of the light chain variable region, and more preferably, the amino acid residue may be contained in one or more of positions 30, 31, 32, 50, and / or 92 according to the Kabat numbering system in the light chain variable region.

[0106] A library of many antigen-binding domains with different sequences that share the same amino acid residue that changes its antigen-binding activity in response to calcium ion concentration as described above is prepared, and screening from this library can be performed to efficiently obtain antigen-binding domains that have binding activity to a desired antigen and whose antigen-binding activity changes in response to calcium ion concentration.

[0107] For purposes of this disclosure, the "affinity" of an antibody for C1s is expressed in terms of the antibody's KD. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody's binding to that antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, the phrase "higher affinity at neutral pH than at acidic pH" (or the equivalent phrase "pH-dependent binding") means that the antibody's KD at acidic pH is higher than the antibody's KD at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to C1s with higher affinity at neutral pH than at acidic pH if the antibody's KD for binding to C1s at acidic pH is at least two-fold higher than the antibody's KD for binding to C1s at neutral pH. Thus, the invention includes antibodies that bind to C1s at acidic pH with a KD that is at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more times greater than the KD for binding of the antibody to C1s at neutral pH. -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the KD value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even larger.

[0108] The binding properties for a particular antigen can also be expressed in terms of the antibody's kd, which refers to the dissociation rate constant of an antibody for a particular antigen and is expressed in reciprocal seconds (i.e., sec -1 The kd is expressed in units of 100 kcal / 1000 kcal. An increased kd value indicates weaker binding of the antibody to its antigen. Thus, the present invention includes antibodies that bind to C1s with a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies that bind to C1s at acidic pH with a kd that is at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000, or more times greater than the kd of the antibody's binding to C1s at neutral pH. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0109] In certain instances, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's KD value at acidic pH to the antibody's KD value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral KD ratio of 2 or greater, then for purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH." In certain exemplary embodiments, the acidic / neutral KD ratio for antibodies of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000-fold or more. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the KD value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even larger.

[0110] Alternatively, "reduced binding at acidic pH compared to binding at neutral pH" can be expressed as the ratio of the antibody's koff value at acidic pH to the antibody's koff value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral koff ratio of 2 or greater, then for purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH." In certain exemplary embodiments, the acidic / neutral koff ratio for antibodies of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000-fold or more.

[0111] In certain instances, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's kd value at acidic pH to the antibody's kd value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral kd ratio of 2 or greater, then for purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH." In certain exemplary embodiments, the acidic / neutral kd ratio for antibodies of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000-fold or more. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0112] In a specific embodiment, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) of an anti-C1s antibody of the present invention having pH and / or Ca dependence is the same as or greater than that of a reference antibody selected from the group consisting of 1) to 5) below. 1) an antibody comprising the VH and VL sequences in SEQ ID NO:19 and SEQ ID NO:20, respectively; 2) an antibody comprising the VH and VL sequences in SEQ ID NO:17 and SEQ ID NO:18, respectively; 3) an antibody comprising the VH and VL sequences in SEQ ID NO:22 and SEQ ID NO:20, respectively; 4) an antibody comprising the VH and VL sequences in SEQ ID NO: 19 and SEQ ID NO: 21, respectively; and 5) An antibody comprising the VH and VL sequences in SEQ ID NO:22 and SEQ ID NO:21, respectively. In a further embodiment, anti-C1s antibodies such as IPN92H0288-SG4GK / IPN93L0211-SK1, IPN92H0288-SG4GK / IPN93L0058-SK1, and IPN92H0307-SG4GK / IPN93L0058-SK1 disclosed in Example 4 can be used as the reference antibody.

[0113] As used herein, the term "acidic pH" refers to a pH between 4.0 and 6.5. The term "acidic pH" includes pH values ​​of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain aspects, the "acidic pH" is 5.8 or 6.0.

[0114] As used herein, the phrase "neutral pH" refers to a pH of 6.7 to about 10.0. The phrase "neutral pH" includes pH values ​​of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In certain aspects, a "neutral pH" is 7.0 or 7.4.

[0115] As used herein, the expression "under high calcium concentration conditions" or "under high calcium concentrations" refers to 100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). The expression "under high calcium concentration conditions" or "at high calcium concentrations" refers to 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 0.5 mM, 0.7 mM, 0.9 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2.0 mM, 2.2 mM, 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM Ca. 2+ In certain aspects, "high calcium concentration conditions" or "high calcium concentration" refers to a calcium concentration of 1.2 mM Ca. 2+ Represents.

[0116] As used herein, the expression "under low calcium concentration conditions" or "under low calcium concentrations" refers to 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. The expression "under low calcium concentration conditions" or "at low calcium concentrations" refers to 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2.0 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM Ca 2+ In certain aspects, "under low calcium concentration conditions" or "under low calcium concentration" includes calcium concentration values ​​of 3.0 μM Ca 2+ Represents.

[0117] The KD and kd values ​​presented herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, for example, Example 2 herein.) The KD and kd values ​​can be determined at 25 degrees Celsius (°C) or 37°C. This determination can be carried out in the presence of 150 mM NaCl. In some embodiments, this determination can be carried out using surface plasmon resonance technology, immobilizing the antibody and using the antigen as an analyte, using the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37 degrees Celsius (°C).

[0118] In one aspect, the present invention provides an anti-C1s antibody having pH dependence, which comprises at least one histidine in the variable region, and wherein at least one amino acid in the variable region is 1) to reduce the nonspecific binding activity of the antibody at acidic and / or neutral pH, or 2) The ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is increased. Antibodies are provided in which the amino acids have been substituted with other amino acids. In one aspect, the present invention provides an anti-C1s antibody having pH dependence, which comprises at least one histidine in the variable region, and wherein at least one amino acid in the variable region is 1) to reduce the nonspecific binding activity of the antibody at acidic and / or neutral pH, or 2) The ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is increased. Antibodies are provided in which the amino acid residues are substituted with amino acids selected from the group consisting of D, E, K, R and Q. In certain embodiments, the phrase "non-specific binding activity" refers to the extracellular matrix (ECM) binding activity of an antibody. In certain aspects, the phrase "non-specific binding activity" refers to the ECM binding activity of an antibody at acidic pH. In certain embodiments, at least one amino acid in the anti-C1s antibody of the present invention may be substituted with one or more amino acids to reduce the ECM binding activity at acidic pH. In certain embodiments, at least one amino acid in the anti-C1s antibody of the present invention may be substituted with one or more amino acids to reduce the ECM binding activity at neutral pH.

[0119] In one aspect, the present invention provides an anti-C1s antibody having pH dependence, wherein at least one amino acid is substituted in the variable region such that the antibody's ECM-binding activity is reduced. In certain embodiments, the ECM-binding activity at acidic pH is reduced. In certain embodiments, the ECM-binding activity at neutral pH is reduced. In certain embodiments, such an antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations, where such alterations improve the ECM-binding activity of the antibody to an antigen, i.e., reduce the ECM-binding activity.

[0120] The method for measuring "binding to the extracellular matrix" is not particularly limited. Measurements can be performed using an ELISA system, in which a polypeptide is added to a plate on which the extracellular matrix is ​​immobilized, followed by the addition of a labeled antibody against the polypeptide, thereby detecting binding between the polypeptide and the extracellular matrix. Measurements using electrochemiluminescence (ECL) are particularly preferred because they enable more sensitive detection of extracellular matrix binding ability. Specifically, binding between the polypeptide and the extracellular matrix can be measured by adding a mixture of the polypeptide and a ruthenium antibody to a plate on which the extracellular matrix is ​​immobilized, followed by an ECL system that measures the electrochemiluminescence of ruthenium. The concentration of the polypeptide to be added can be set at any desired level, but adding a high concentration is preferred to increase the detection sensitivity of extracellular matrix binding. The extracellular matrix used in the present invention may be derived from either plants or animals, as long as it contains glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin, and perlecan. However, animal-derived extracellular matrices are preferred in the present invention. For example, extracellular matrices derived from animals such as humans, mice, rats, monkeys, rabbits, and dogs can be used. In particular, natural human extracellular matrix derived from humans is preferred for monitoring improved pharmacokinetics in humans. Furthermore, conditions for evaluating the binding of a polypeptide to an extracellular matrix are preferably in the neutral range (physiological conditions) of around pH 7.4, but do not necessarily need to be within the neutral range; evaluation can also be performed in the acidic range (around pH 6.0). Furthermore, when evaluating the binding of a polypeptide to an extracellular matrix, an antigen molecule to which the polypeptide binds can be coexisted in order to evaluate the binding of the polypeptide / antigen molecule complex to the extracellular matrix.

[0121] In some embodiments, whether the nonspecific binding activity of an antibody at an acidic pH is reduced can be measured, for example, by using the ELISA or ECL mentioned in other paragraphs (see, for example, Example 4). In a further embodiment, for an isolated anti-C1s antibody of the present invention, the ECM binding value can be compared between the parent antibody (i.e., the original antibody before the D, E, K, R, and / or Q substitutions) and an antibody into which one or more amino acid substitutions (D, E, K, R, and / or Q) have been introduced into the original (parent) antibody (provided that the antibody contains at least one histidine in the variable region). The original (parent) antibody can be any known antibody or a newly isolated antibody, as long as it specifically binds to C1s. Thus, in one aspect, for an isolated anti-C1s antibody of the present invention, the ECM binding value of the displaced antibody is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, or 10-fold lower than the ECM binding value of the original (parent) antibody.

[0122] Without being bound by any particular theory, histidine residues of an antibody may interact with various residues surrounding the histidine residue of the antibody. Such interactions may affect the structure of the antibody or the conformation of the CDR. Histidine is protonated and positively charged at acidic pH. Introducing a positively charged residue (e.g., arginine or lysine) into a position surrounding the histidine causes repulsion between the positively charged residue and the protonated histidine at acidic pH, thereby inducing a structural or conformational change in the antibody or CDR. Similarly, introducing a negatively charged residue (e.g., aspartic acid or glutamic acid) into a position surrounding the histidine causes an interaction between the negatively charged residue and the protonated histidine at acidic pH, thereby inducing a structural or conformational change in the antibody or CDR. These structural or conformational changes in the antibody or CDR that occur at acidic pH may affect the antigen binding of the antibody and reduce the binding affinity of the antibody to the antigen at acidic pH. In summary, the introduction of charged residues (e.g., arginine, lysine, aspartic acid, or glutamic acid) into positions surrounding histidine residues of an antibody can reduce the binding affinity of the antibody to the antigen at acidic pH, thereby improving the pH dependence of the antibody-antigen interaction through a unique mechanism.

[0123] In one aspect, the present invention provides a method for increasing the ratio of the KD value of an antibody's antigen-binding activity at acidic pH to the KD value of the antigen-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)), comprising the steps of: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) substituting at least one amino acid in the variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q, and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for enhancing the clearance (removal) of an antigen from plasma, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) substituting at least one amino acid in the variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q, and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for promoting antigen uptake into cells via an antigen-binding molecule, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) substituting at least one amino acid in the variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q, and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for increasing the number of antigens that can be bound by a single antigen-binding molecule, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) substituting at least one amino acid in the variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q, and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for enhancing the ability of an antigen-binding molecule to remove an antigen from plasma, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) substituting at least one amino acid in the variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q, and H; The present invention provides a method comprising:

[0124] In certain embodiments, in the above-described methods of the present invention, the distance between the histidine residue contained in the variable region and the substituted amino acid (i.e., D, E, K, R, Q, or H) is less than 20 angstroms, less than 18 angstroms, less than 16 angstroms, less than 14 angstroms, less than 12 angstroms, less than 10 angstroms, less than 8 angstroms, less than 6 angstroms, less than 4 angstroms, or less than 2 angstroms.

[0125] In one aspect, the present invention provides methods for enhancing the clearance of C1s from plasma in an individual. In some embodiments, the methods comprise administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of C1s from plasma. The present invention also provides methods for enhancing the clearance of a complex of C1r and C1s from plasma in an individual. In some embodiments, the methods comprise administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of a complex of C1r and C1s from plasma. The present invention also provides methods for enhancing the clearance of a complex of C1q, C1r, and C1s from plasma in an individual. In some embodiments, the methods comprise administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of a complex of C1q, C1r, and C1s from plasma.

[0126] In another aspect, the present invention provides a method for removing C1s from plasma, the method comprising the steps of: (a) identifying an individual in need of C1s removal from their plasma; (b) providing an antibody that binds to C1s, wherein the antibody binds to C1s through its antigen-binding (C1s-binding) domain and has a KD(pH 5.8) / KD(pH 7.4) value of 2 to 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4, as determined using surface plasmon resonance technology; the antibody binds to C1s in plasma in vivo and dissociates from the bound C1s under conditions present in endosomes in vivo; and the antibody is a human IgG or humanized IgG; and (c) administering the antibody to the individual. In a further aspect, such surface plasmon resonance techniques can be used at 37° C. and 150 mM NaCl. In a further aspect, such surface plasmon resonance techniques can be used with immobilized antibodies and antigens as analytes, and under the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37° C. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD.

[0127] In another aspect, the present invention provides a method for removing C1s from plasma in a subject, the method comprising: (a) identifying a first antibody, wherein the first antibody binds to C1s through the antigen-binding domain of the first antibody; and (b) identifying a second antibody, wherein the second antibody (1) binds to C1s through the antigen-binding (C1s-binding) domain of the second antibody, (2) has the same amino acid sequence as the first antibody except for substitution of at least one amino acid in the variable region of the first antibody with histidine and / or insertion of at least one histidine into the variable region of the first antibody, and (3) has a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the first antibody and is between 2 and 10,000, wherein (7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4, when the KD is determined using surface plasmon resonance technology; (4) binds to C1s in plasma in vivo, (5) dissociates from the bound C1s under conditions present in endosomes in vivo, and (6) is a human IgG or a humanized IgG; (c) identifying a subject in need of reduced plasma C1s levels; and (d) administering a second antibody to the subject such that the subject's plasma C1s level is reduced. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance techniques can be used with immobilized antibodies, antigen as the analyte, and the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37° C. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD.

[0128] In another aspect, the present invention provides a method for removing C1s from plasma in a subject, comprising the steps of: (a) identifying a first antibody, the first antibody (1) binding to C1s through an antigen-binding domain of the first antibody; (2) having an amino acid sequence identical to that of a second antibody that binds to C1s through an antigen-binding (C1s-binding) domain of the second antibody, except that at least one variable region of the first antibody has at least one more histidine residue than the corresponding variable region of the second antibody; and (3) having a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the second antibody and is between 2 and 10,000, wherein KD(pH 5.8) / KD(pH 7.4) is the ratio of the KD for C1s at pH 5.8 and pH 7.4 when the KD is determined using surface plasmon resonance technology. (b) identifying a subject in need of a reduction in plasma C1s level; and (c) administering the first antibody at least once to the subject so that the subject's plasma C1s level is reduced. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance technology can be used with an immobilized antibody, an antigen as an analyte, and the following conditions: 37°C, 10 mM MES buffer, 0.05% polyoxyethylenesorbitan monolaurate, and 150 mM NaCl. The present invention also provides a method for inhibiting cleavage of complement component C4, wherein the antibody does not inhibit cleavage of complement component C2. In some instances, the antibody inhibits a component of the classical complement pathway, and in some instances, the classical complement pathway component is C1s. In a further aspect, the dissociation rate constant (kd) can be used in place of the KD.

[0129] In one aspect, the present disclosure provides a method for modulating complement activation. In some embodiments, the method inhibits complement activation, for example, to reduce the production of C4b2a. In some embodiments, the present disclosure provides a method for modulating complement activation in an individual having a complement-mediated disease or disorder, comprising administering to the individual an anti-C1s antibody of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of the present disclosure. In some embodiments, such a method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is human. Administration can be by any route known to those of skill in the art, including those disclosed herein. In some embodiments, administration is intravenous administration. In some embodiments, administration is intrathecal administration.

[0130] In certain embodiments, the anti-C1s antibody of the present invention binds to C1s from two or more species. In certain embodiments, the anti-C1s antibody binds to C1s from humans and non-human animals. In certain embodiments, the anti-C1s antibody binds to C1s from humans, rats, and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, and baboons).

[0131] In one aspect, the present invention provides anti-C1s antibodies comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:44.

[0132] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:41. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:41 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:44. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:41, HVR-L3 comprising the amino acid sequence of SEQ ID NO:44, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:40. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:41.

[0133] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:43; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:44. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:43; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:44.

[0134] In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:43; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:44.

[0135] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:44.

[0136] In some embodiments, anti-C1s antibody variants are provided that are prepared by introducing amino acid modifications into an antibody comprising the VH sequence of SEQ ID No. 35 or 36 and the VL sequence of SEQ ID No. 37 or 38. In some embodiments, anti-C1s antibody variants are provided that are prepared by introducing amino acid modifications into antibodies VH1 / Vk1, VH1 / Vk2, VH1 / Vk3, VH2 / Vk1, VH2 / Vk2, VH2 / Vk3, VH3 / Vk1, VH3 / Vk2, VH3 / Vk3, VH4 / Vk1, VH4 / Vk2, or VH4 / Vk3 disclosed in WO2014 / 071206.

[0137] In some embodiments, the anti-C1s antibodies of the invention comprise a histidine at one or more of the following positions according to the Kabat numbering system: Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97.

[0138] In some embodiments, the anti-C1s antibodies of the invention comprise a histidine at one or more of the following positions according to the Kabat numbering system: Heavy chain: H26, H27, H28, H29, H30, H32, H33, H34, H50, H51, H52a, H54, H57, H58, H59, H60, H61, H65, H93, H95, H99, H100 and H100a; and Light chain: L25, L28, L91, L92, L94, L95, L96 and L97.

[0139] In some embodiments, the anti-C1s antibodies of the invention comprise at least one histidine substituted for one or more amino acid residues at positions selected from the following positions according to the Kabat numbering system: Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97.

[0140] In some embodiments, any one or more amino acids of the aforementioned anti-C1s antibodies are substituted with a histidine at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0141] In some embodiments, the isolated anti-C1s antibody of the invention comprises one, two, three, four, or five histidines substituted for amino acid residues at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0142] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine substituted residue at one or more of the following positions and CDR or FR amino acid positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0143] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine substituted residue at the following position according to the Kabat numbering system: 1) L92 and L94 2) L92 and L95 3) L94 and L95 4) L92, L94, and L95 5) H65 and L92 6) H65 and L94 7) H65 and L95 8) H65, L92, and L94 9) H65, L92, and L95 10) H65, L94, and L95 11) H65, L92, L94, and L95 12) H99 and L92 13) H99 and L94 14) H99 and L95 15) H99, L92, and L94 16) H99, L92, and L95 17) H99, L94, and L95 18) H99, L92, L94, and L95 19) H65 and H99 20) H65, H99, and L92 21) H65, H99, and L94 22) H65, H99, and L95 23) H65, H99, L92, and L94 24) H65, H99, L92, and L95 25) H65, H99, L94, and L95 26) H65, H99, L92, L94, and L95, or 27)H27, H99, and L95.

[0144] In any of the above-described embodiments, the anti-C1s antibody is a humanized antibody. In one embodiment, the anti-C1s antibody comprises the HVR of any of the above-described embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1s antibody comprises the HVR of any of the above-described embodiments and further comprises a VH or VL comprising FR sequences. In a further embodiment, the anti-C1s antibody of the invention comprises the following heavy or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO:4 or 12, FR2 comprises the amino acid sequence of SEQ ID NO:5, FR3 comprises the amino acid sequence of SEQ ID NO:6, and FR4 comprises the amino acid sequence of SEQ ID NO:7. For the light chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO:8, FR2 comprises the amino acid sequence of SEQ ID NO:9, FR3 comprises the amino acid sequence of SEQ ID NO:10, and FR4 comprises the amino acid sequence of SEQ ID NO:11.

[0145] In another aspect, an anti-C1s antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, 17, or 22. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C1s antibody comprising that sequence retains the ability to bind to C1s. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 19, 17, or 22. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C1s antibody comprises a VH sequence of SEQ ID NO:19, 17, or 22, including post-translational modifications of said sequences. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0146] In another aspect, an anti-C1s antibody is provided that comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20, 18, or 21. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C1s antibody comprising that sequence retains the ability to bind to C1s. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 20, 18, or 21. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C1s antibody comprises a VL sequence of SEQ ID NO:20, 18, or 21, including post-translational modifications of said sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:42 or 45, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:43, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:44. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0147] In another aspect, a C1s antibody is provided, comprising the VH of any of the above embodiments and the VL of any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:19 and SEQ ID NO:20, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:17 and SEQ ID NO:18, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:22 and SEQ ID NO:20, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:19 and SEQ ID NO:21, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:22 and SEQ ID NO:21, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0148] In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-C1s antibodies provided herein. For example, in certain embodiments, (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:23, the HVR-H2 sequence of SEQ ID NO:24, the HVR-H3 sequence of SEQ ID NO:25, the HVR-L1 sequence of SEQ ID NO:26, the HVR-L2 sequence of SEQ ID NO:27, and the HVR-L3 sequence of SEQ ID NO:28; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:29, the HVR-H2 sequence of SEQ ID NO:30, the HVR-H3 sequence of SEQ ID NO:31, the HVR-L1 sequence of SEQ ID NO:32, the HVR-L2 sequence of SEQ ID NO:33, and the HVR-L3 sequence of SEQ ID NO:34; (c) human monoclonal anti-C1s antibody M241 (HycultBiotech, catalog number HM2109) or human monoclonal anti-C1s antibody M81 (HycultBiotech, catalog number HM2108); (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (f) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (g) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (h) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:85; and binds to C1s with higher affinity at neutral pH than at acidic pH, as described in (i) or (ii) below: (i) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater; (ii) When measured at both neutral and acidic pH under high calcium concentrations, the ratio of the koff value of C1s binding activity at acidic pH to the koff value of C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater. In some embodiments, the isolated anti-C1s antibody of the present invention has, with respect to binding to C1s, (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (c) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:85. The antibody competes with an antibody selected from the group consisting of:

[0149] In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-C1s antibodies provided herein. For example, in certain embodiments, the present invention provides antibodies that bind to the same epitope as an antibody selected from the group consisting of IPN-M1, IPN-M2, IPN-M3, IPN-M8, IPN-M9, IPN-M10, IPN-M11, IPN-M13, IPN-M14, IPN-M15, IPN-M18, IPN-M23, IPN-M24, IPN-M27, IPN-M28, IPN-M29, and IPN-M33, which are disclosed in WO2014 / 066744.

[0150] In some embodiments, the isolated anti-C1s antibody of the present invention exhibits, with respect to binding to C1s at neutral pH: (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:23, the HVR-H2 sequence of SEQ ID NO:24, the HVR-H3 sequence of SEQ ID NO:25, the HVR-L1 sequence of SEQ ID NO:26, the HVR-L2 sequence of SEQ ID NO:27, and the HVR-L3 sequence of SEQ ID NO:28; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:29, the HVR-H2 sequence of SEQ ID NO:30, the HVR-H3 sequence of SEQ ID NO:31, the HVR-L1 sequence of SEQ ID NO:32, the HVR-L2 sequence of SEQ ID NO:33, and the HVR-L3 sequence of SEQ ID NO:34; and (c) human monoclonal anti-C1s antibody M241 (HycultBiotech, catalog number HM2109) or human monoclonal anti-C1s antibody M81 (HycultBiotech, catalog number HM2108); The antibody competes with an antibody selected from the group consisting of:

[0151] In some embodiments, an isolated anti-C1s antibody of the invention competes for binding to C1s at neutral pH with an antibody selected from the group consisting of IPN-M1, IPN-M2, IPN-M3, IPN-M8, IPN-M9, IPN-M10, IPN-M11, IPN-M13, IPN-M14, IPN-M15, IPN-M18, IPN-M23, IPN-M24, IPN-M27, IPN-M28, IPN-M29, and IPN-M33 disclosed in WO2014 / 066744.

[0152] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to an epitope within a region comprising domains IV and V of complement component Is (C1s) and has pH-dependent binding properties. In some examples, the antibody inhibits binding of C1s to complement component 4 (C4). In some examples, the antibody does not inhibit the protease activity of C1s. In some examples, the epitope bound by the isolated humanized monoclonal antibody of the present disclosure is a conformational epitope.

[0153] In one aspect, the present disclosure provides an isolated antibody having pH-dependent binding that specifically binds to an epitope within the complement C1s protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to activated C1s protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to the inactive form of C1s. In other examples, the isolated anti-C1s antibody of the present disclosure binds to both activated C1s protein and the inactive form of C1s.

[0154] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding that specifically binds to an epitope within a region comprising domains IV and V of C1s. For example, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 272 to 422 of the amino acid sequence set forth in SEQ ID NO:1. In some examples, the isolated humanized monoclonal antibody specifically binds to an epitope within amino acids 272 to 422 of the amino acid sequence set forth in SEQ ID NO:1 and inhibits C4 binding to C1s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, comprising administering to an individual in need thereof an effective amount of an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 272 to 422 of the amino acid sequence set forth in SEQ ID NO:1 and inhibits C4 binding to C1s.

[0155] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to an epitope comprising aspartic acid at position 357 of human C1s antigen and has pH-dependent binding properties.

[0156] In a further aspect of the invention, the anti-C1S antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-C1S antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, a complete IgG1, IgG2, IgG3, or IgG4 antibody, or other antibody class or isotype as defined herein.

[0157] In further aspects, anti-C1S antibodies according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1-7 below.

[0158] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd or KD) of M.

[0159] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured using the lowest concentration ( 125I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0160] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0161] In some embodiments, the binding affinity of each histidine substitution mutant of the present invention at pH 7.4 and pH 5.8 is determined using a Biacore T200 instrument (GE Healthcare) at 37° C. Recombinant Protein A / G (Pierce) can be immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). The antibodies and analytes can be prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl, 0.05% Tween 20, 0.005% NaN, pH 7.4), 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl, 0.05% Tween 20, 0.005% NaN, pH 5.8), or 5(-) buffer (20 mM ACES, 150 mM NaCl, 3 μM CaCl, 0.05% Tween 20, 0.005% NaN, pH 5.8). Each antibody can be captured on the sensor surface using Protein A / G. The antibody capture amount is targeted at 200 resonance units (RU). Serum-derived human C1s (CompTech) or prepared recombinant C1s can be injected at, for example, 50 or 200 nM, followed by dissociation. The sensor surface is regenerated every cycle, for example, with 10 mM glycine-HCl, pH 1.5. Binding affinity can be determined, for example, by processing the data and fitting it to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0162] A specific example of the steps of the Biacore assay of the present invention is as follows. The binding affinities of histidine-substituted mutants at pH 7.4 and pH 5.8 were determined using a Biacore T200 instrument (GE Healthcare) at 37°C. Recombinant Protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured onto the sensor surface by Protein A / G. The antibody capture amount was targeted at 200 resonance units (RU). Serum-derived human C1s is injected at 12.5 and 50 nM at pH 7.4, or at 50 and 200 nM at pH 5.8, or at 200 and 800 nM at pH 5.8, followed by dissociation. After each cycle, the sensor surface is regenerated with 10 mM glycine-HCl, pH 1.5. The binding affinity is determined by processing the data and fitting it to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). An additional dissociation region at pH 5.8 is incorporated immediately after the dissociation region at pH 7.4. The dissociation rate in 5(+) buffer is determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software. Alternatively, the binding affinity of histidine-substituted mutants at pH 7.4 and pH 5.8 was determined using a Biacore T200 instrument (GE Healthcare) at 37°C. Recombinant Protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured onto the sensor surface by Protein A / G. The antibody capture amount was targeted at 200 resonance units (RU). Serum-derived human C1s is injected at 50 nM and then dissociated. After each cycle, the sensor surface is regenerated with 10 mM glycine-HCl, pH 1.5. The binding affinity is determined by processing the data and fitting it to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). The dissociation region at pH 7.4 is immediately followed by an additional dissociation region at pH 5.8. The dissociation rate in 5(+) buffer is determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0163] In some embodiments, an additional dissociation region at pH 5.8 is optionally incorporated immediately after the dissociation region at pH 7.4. This dissociation rate in 5(+) buffer can be determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0164] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-lives in vivo.

[0165] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0166] Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0167] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0168] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0169] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0170] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Further described in Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0171] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0172] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0173] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0174] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005).

[0175] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0176] 5. Library-derived antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0177] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0178] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0179] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for C1S and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of C1S. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing C1S. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0180] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the "knob-in-hole" technique (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1994)). see, for example, Tutt et al., J. Immunol. 147: 60 (1991).

[0181] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0182] As used herein, the antibody or fragment also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to C1S and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0183] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0184] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0185] [Table 1]

[0186] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0187] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further testing will have modified (e.g., improved) specific biological properties compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0188] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0189] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0190] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989), Science 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or excluded from the list. Mutants can be screened to determine whether they contain desired properties.

[0191] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing to the N- or C-terminus of the antibody an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0192] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0193] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0194] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0195] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0196] c) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0197] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg MS et al., Blood 101:1045-1052 (2003); and Cragg MS and MJ Glennie et al., Blood 103:2738-2743 (2004)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0198] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0199] Certain antibody variants with improved or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0200] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0201] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0202] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0203] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0204] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0205] e) antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3 dioxolane, poly-1,3,6 trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if two or more polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0206] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0207] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-C1S antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or SP2 / 0 cell)). In one aspect, a method for producing an anti-C1S antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-C1S antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0208] For recombinant production of an anti-C1S antibody, nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0209] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0210] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0211] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0212] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0213] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells; MRC5 cells; and FS4 cells. -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0214] Antibodies with pH dependence can be obtained by using screening and / or mutagenesis methods, for example, as described in WO 2009 / 125825. The screening method can include any process for identifying antibodies with pH-dependent binding properties from a population of antibodies specific to a particular antigen. In certain embodiments, the screening method can include measuring one or more binding parameters (e.g., KD or kd) of individual antibodies in the initial antibody population at both acidic and neutral pH. Antibody binding parameters can be measured using, for example, surface plasmon resonance or any other analytical method that allows for quantitative or qualitative assessment of the binding properties of an antibody for a particular antigen. In certain embodiments, the screening method can include identifying antibodies that bind to the antigen with an acidic KD / neutral KD ratio of 2 or greater. Alternatively, the screening method can include identifying antibodies that bind to the antigen with an acidic kD / neutral kD ratio of 2 or greater.

[0215] In another embodiment, the mutagenesis method may involve incorporating amino acid deletions, substitutions, or additions within the heavy and / or light chains of an antibody to enhance the pH-dependent binding of the antibody to an antigen. In certain embodiments, mutagenesis may be performed within one or more variable domains of an antibody, such as one or more HVRs (e.g., CDRs). For example, mutagenesis may involve substituting an amino acid within one or more HVRs (e.g., CDRs) of an antibody with another amino acid. In certain embodiments, mutagenesis may involve substituting one or more amino acids within at least one HVR (e.g., CDR) of an antibody with histidine. In certain embodiments, "enhanced pH-dependent binding" means that a mutant antibody exhibits a greater acidic KD / neutral KD ratio or a greater acidic KD / neutral KD ratio than the original "parent" antibody (i.e., an antibody with reduced pH dependence) prior to mutagenesis. In certain embodiments, a mutant antibody has an acidic KD / neutral KD ratio of 2 or greater. Alternatively, a mutant antibody has an acidic KD / neutral KD ratio of 2 or greater.

[0216] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R. 1 N=C=NR (where R and R 1 are different alkyl groups).

[0217] Animals (usually non-human mammals) are immunized against an antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animal is bled and the serum is assayed for antibody titer. The animal is boosted until the titer plateaus. Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Aggregating agents such as alum are also suitably used to enhance the immune response.

[0218] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0219] For example, monoclonal antibodies can be produced using the hybridoma method first described in Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster, is immunized as described hereinabove to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0220] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0221] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus generated are seeded and grown in an appropriate culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0222] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and SP-2 cells (and their derivatives, such as X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0223] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson, Anal Biochem. 107(1):220-239 (1980).

[0224] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0225] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0226] C. Assay The anti-C1S antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0227] 1. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, and the like.

[0228] In another aspect, a competitive assay can be used to identify antibodies that compete with any of the anti-C1S antibodies described herein for binding to C1S. In certain embodiments, when such a competing antibody is present in excess, it inhibits (e.g., reduces) the binding of a reference antibody to C1S by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as that bound by any of the anti-C1S antibodies described herein. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). In certain embodiments, such competitive assays can be performed under neutral pH conditions.

[0229] In an exemplary competitive assay, immobilized C1S is incubated in a solution containing a first labeled antibody (e.g., one of those described herein) that binds to C1S and a second, unlabeled antibody to be tested for its ability to compete with the first antibody for binding to C1S. The second antibody may be present in hybridoma supernatant. As a control, immobilized C1S is incubated in a solution containing the first labeled antibody but not the second, unlabeled antibody. After incubation under conditions that allow binding of the first antibody to C1S, excess unbound antibody is removed and the amount of label bound to immobilized C1S is measured. If the amount of label bound to immobilized C1S is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to C1S. See Harlow and Lane (1988), Antibodies: A Laboratory Manual, ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0230] In another aspect, antibodies that bind to the same epitope as the anti-C1s antibodies provided herein or that compete with the anti-C1s antibodies provided herein for binding to C1s can be identified using a sandwich assay. A sandwich assay involves using two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein to be detected. In a sandwich assay, a test sample analyte binds to a first antibody immobilized on a solid support, followed by binding of a second antibody to the analyte, thereby forming an insoluble ternary complex. See David & Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. An antibody that binds to C1s simultaneously with the anti-C1s antibodies provided herein may be determined to be an antibody that binds to a different epitope than the anti-C1s antibody. Therefore, an antibody that does not bind to C1s at the same time as the anti-C1s antibody provided herein can be determined to be an antibody that binds to the same epitope as the anti-C1s antibody or competes with the anti-C1s antibody for binding to C1s.

[0231] 2. Activity Assay In one aspect, an assay is provided for identifying anti-C1s antibodies with biological activity. Biological activity can include blocking classical pathway activation and blocking the generation of the cleavage products C2a, C2b, C3a, C3b, C4a, C4b, C5a, and C5b that result from classical pathway activation. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0232] In certain embodiments, antibodies of the present invention are tested for such biological activity. In some embodiments, antibodies of the present invention can be evaluated for their ability to inhibit complement-mediated hemolysis of chicken red blood cells (cRBCs) sensitized with antibodies against cRBC antigens. Using human serum as a source of complement proteins, the activity of antibodies of the present invention can be determined by measuring the amount of released hemoglobin spectrophotometrically. In some embodiments, antibodies of the present invention can be evaluated for their ability to inhibit cleavage of purified C4 (but not C2) via activated C1s. This activity of the antibody is determined by measuring the amount of cleaved C4 or C2 by gel electrophoresis or Western blotting. Cleaved C4 or C2 can be detected by its smaller molecular weight compared to its native, uncleaved form.

[0233] 3. Mouse PK study to evaluate antigen (C1s) removal In a particular embodiment, the promotion of antigen (eg, human C1s (also referred to as hC1s)) clearance by antibodies of the invention can be assessed in vivo (eg, in mice) as follows. Measurement of C1s concentrations in mouse plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS) The concentration of hC1s (or anti-C1s antibody) in mouse plasma can be measured by LC / ESI-MS / MS. Calibration standards are prepared by mixing and diluting a defined amount of hC1s (or anti-C1s antibody) in mouse plasma. The calibration standards and plasma samples are mixed with urea, dithiothreitol, and lysozyme (chicken egg white), for example, in ammonium bicarbonate, and incubated. Iodoacetamide is then added and incubated in the dark. Trypsin in ammonium bicarbonate is then added and incubated. Finally, trifluoroacetic acid is added to inactivate any remaining trypsin. The sample is then analyzed by LC / ESI-MS / MS. Human C1s-specific peptides (e.g., LLEVPEGR) are monitored by selected reaction monitoring (SRM). The SRM transition for human C1s can be [M+2H]2+ (m / z 456.8) to the y6 ion (m / z 686.4). A calibration curve can be generated by weighted (1 / x2) linear regression using peak areas plotted against concentration, from which concentrations in mouse plasma are calculated. Evaluation of the pharmacokinetics of total hC1s after administration of anti-C1s antibody in mice The in vivo pharmacokinetics of hC1s, hC1q, or anti-C1s antibody can be evaluated after administering antigen alone or together with anti-C1s antibody to mice. A solution / mixture containing hC1s (or the like) is intravenously injected into mice. Following administration of the antigen solution, the anti-C1s antibody solution is immediately administered to the same individual in the same manner. The dose setting can be appropriately designed so that almost all of the hC1s is in a bound form in the blood. Blood is collected over time, for example, at 5, 30 minutes, 2, 7 hours, 3, 7, 14, 21, and 28 days after injection. The blood is immediately centrifuged to separate plasma samples. The plasma concentration of hC1s (or the like) is measured by LC / ESI-MS / MS at each sample collection time point. The PK parameters of hC1s (or the like) are estimated by non-compartmental analysis. For example, the hC1s CL (clearance) ratio is calculated for anti-C1s antibodies. If this ratio is high, this means that hC1s removal can be more promoted.

[0234] D. Immunoconjugates The present invention also provides immunoconjugates comprising an anti-C1S antibody herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0235] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342). (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0236] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0237] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 When a radioconjugate is used for detection, the radioconjugate is a radioactive atom (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0238] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0239] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0240] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-C1S antibodies provided herein are useful for detecting the presence of C1S in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ophthalmic lens fluid, or mucus.

[0241] In one embodiment, an anti-C1S antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of C1S in a biological sample is provided. In a specific embodiment, the method comprises contacting a biological sample with an anti-C1S antibody described herein under conditions that allow binding of the anti-C1S antibody to C1S, and detecting whether a complex is formed between the anti-C1S antibody and C1S. Such a method can be an in vitro method or an in vivo method. In one embodiment, the anti-C1S antibody is used to select subjects suitable for treatment with an anti-C1S antibody, for example, when C1S is a biomarker for patient selection.

[0242] Examples of disorders that can be diagnosed using the antibodies of the invention include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangle disease with calcifications, discoid lupus erythematosus, Down's syndrome, focal segmental glomerular vasculopathy, and others. Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker syndrome, Guillain-Barré syndrome, Hallervorden-Spatz syndrome, hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g., bacterial (e.g., meningococcal or streptococcal), viral (e.g., Diseases caused by human immunodeficiency virus (HIV) or other infectious agents), inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy -, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic Parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (E.These include, but are not limited to, STEC-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementemic urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractory state.

[0243] In certain embodiments, labeled anti-C1S antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands), for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H and 131Fluorophores such as I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0244] F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-C1S antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of use thereof (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0245] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0246] The formulations herein may contain more than one active ingredient as needed for the particular indication being treated. Preferably, they have complementary activities that do not adversely affect each other. For example, it may be desirable to provide a formulation for use in combination therapy. Such active ingredients are present in a suitable combination in amounts that are effective for the intended purpose.

[0247] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0248] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0249] Preparations to be used for in vivo administration are generally sterile, and sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0250] G. Therapeutic Methods and Compositions Any of the anti-C1S antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-C1S antibody is provided for use as a pharmaceutical. In a further aspect, an anti-C1S antibody is provided for use in treating a complement-mediated disease or disorder. In a particular embodiment, an anti-C1S antibody is provided for use in a method of treatment. In a particular embodiment, the invention provides an anti-C1S antibody for use in a method of treating an individual with a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of an anti-C1S antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the above embodiments is preferably a human.

[0251] In a further embodiment, the present invention provides an anti-C1s antibody for use in treating a complement-mediated disease or disorder. In a further embodiment, the anti-C1s antibody of the present invention can be used to enhance the clearance of C1s from plasma. In a further embodiment, the anti-C1s antibody of the present invention can be used to enhance the clearance of a complex of C1q, C1r, and C1s from plasma. In a further embodiment, the anti-C1s antibody of the present invention can be used to inhibit the cleavage of complement component C4, where the antibody does not inhibit the cleavage of complement component C2. In some examples, the antibody inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for treating a complement-mediated disease or disorder. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for enhancing the clearance of C1s from plasma. In certain embodiments, the present invention provides anti-C1s antibodies for use in methods of enhancing clearance of C1q, C1r, and C1s complexes from plasma. In certain embodiments, the present invention provides anti-C1s antibodies for use in methods of inhibiting cleavage of complement component C4, where the antibody does not inhibit cleavage of complement component C2. In certain embodiments, the present invention provides anti-C1s antibodies for use in methods of inhibiting a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s. In any of the above embodiments, the "individual" is preferably a human.

[0252] In one aspect, the present disclosure provides a method for modulating complement activation. In some embodiments, the method inhibits complement activation, for example, to reduce the production of C4b2a. In some embodiments, the present disclosure provides a method for modulating complement activation in an individual having a complement-mediated disease or disorder, comprising administering to the individual an anti-C1s antibody of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of the present disclosure. In some embodiments, such a method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is human. Administration can be by any route known to those of skill in the art, including those disclosed herein. In some embodiments, administration is intravenous or subcutaneous. In some embodiments, administration is intrathecal.

[0253] A complement-mediated disease or disorder is a disorder characterized by abnormal amounts of complement C1s or abnormal levels of complement C1s proteolytic activity in the cells, tissues, or body fluids of an individual.

[0254] In some instances, a complement-mediated disease or disorder is characterized by the presence of an increased (higher than normal) amount of C1s or elevated levels of complement C1s activity in cells, tissues, or body fluids. For example, in some instances, a complement-mediated disease or disorder is characterized by the presence of an increased amount of C1s and / or elevated activity of C1s in brain tissue and / or cerebrospinal fluid. A "higher than normal" amount of C1s in a cell, tissue, or body fluid indicates that the amount of C1s in that cell, tissue, or body fluid is higher than a normal control level, e.g., higher than a normal control level for an individual or population of individuals of the same age group. A "higher than normal" level of C1s activity in a cell, tissue, or body fluid indicates that the proteolytic cleavage caused by C1s in that cell, tissue, or body fluid is higher than a normal control level, e.g., higher than a normal control level for an individual or population of individuals of the same age group. In some instances, an individual with a complement-mediated disease or disorder exhibits one or more additional symptoms of such a disease or disorder.

[0255] In other examples, complement-mediated diseases or disorders are characterized by the presence of lower-than-normal amounts of C1s or low levels of complement C1s activity in cells, tissues, or body fluids. For example, in some examples, complement-mediated diseases or disorders are characterized by the presence of lower amounts of C1s and / or low activity of C1s in brain tissue and / or cerebrospinal fluid. A "lower-than-normal" amount of C1s in a cell, tissue, or body fluid indicates that the amount of C1s in that cell, tissue, or body fluid is lower than a normal control level, e.g., lower than a normal control level for an individual or population of individuals of the same age group. A "lower-than-normal" level of C1s activity in a cell, tissue, or body fluid indicates that the proteolytic cleavage caused by C1s in that cell, tissue, or body fluid is lower than a normal control level, e.g., lower than a normal control level for an individual or population of individuals of the same age group. In some examples, an individual with a complement-mediated disease or disorder exhibits one or more additional symptoms of such a disease or disorder.

[0256] A complement-mediated disease or disorder is a disease or disorder in which the amount or activity of complement C1s is sufficient to cause the disease or disorder in an individual. In some embodiments, the complement-mediated disease or disorder is selected from the group consisting of an autoimmune disease, cancer, a blood disorder, an infectious disease, an inflammatory disease, an ischemia-reperfusion injury, a neurodegenerative disease, a neurodegenerative disorder, an eye disease, a kidney disease, a transplant rejection, a vascular disease, and a vasculitic disease. In some embodiments, the complement-mediated disease or disorder is an autoimmune disease. In some embodiments, the complement-mediated disease or disorder is cancer. In some embodiments, the complement-mediated disease or disorder is an infectious disease. In some embodiments, the complement-mediated disease or disorder is an inflammatory disease. In some embodiments, the complement-mediated disease or disorder is a blood disorder. In some embodiments, the complement-mediated disease or disorder is an ischemia-reperfusion disease. In some embodiments, the complement-mediated disease or disorder is an eye disease. In some embodiments, the complement-mediated disease or disorder is a kidney disease. In some embodiments, the complement-mediated disease or disorder is transplant rejection. In some embodiments, the complement-mediated disease or disorder is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease or disorder is a vascular disease. In some embodiments, the complement-mediated disease or disorder is a vasculitic disease. In some embodiments, the complement-mediated disease or disorder is a neurodegenerative disease or disorder. In some embodiments, the complement-mediated disease is a neurodegenerative disease. In some embodiments, the complement-mediated disorder is a neurodegenerative disorder. In some embodiments, the complement-mediated disease or disorder is a tauopathy.

[0257] Examples of complement-mediated diseases or disorders include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, and Barraquer-Simons syndrome. syndrome), Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangle disease with calcifications, discoid lupus erythematosus, Down's syndrome, focal segmental glomerular Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker syndrome, Guillain-Barré syndrome, Hallervorden-Spatz syndrome, hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g., bacterial (e.g., meningococcal or streptococcal), viral (e.g., Diseases caused by human immunodeficiency virus (HIV) or other infectious agents), inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy -, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic Parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (E.These include, but are not limited to, STEC-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementemic urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractory state.

[0258] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with parkinsonism linked to chromosome 17) are the most common forms of tauropathy. Thus, the present invention relates to any of the above methods in which the tauropathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia, or frontotemporal dementia with parkinsonism linked to chromosome 17. Other tauropathies include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

[0259] Neurodegenerative tauropathies include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinsonism complex, argyrophilic grain dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle disease with calcifications, Down's syndrome, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, and Gerstmann-Straussler-Scheinker syndrome. These include: Hallervorden-Spatz syndrome, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, neurofibrillary dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0260] The present disclosure also provides methods of treating synucleinopathies, such as Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiple system atrophy (MSA), etc. For example, PD with dementia (PDD) can be treated using the methods of the present disclosure.

[0261] In some embodiments, the complement-mediated disease or disorder comprises Alzheimer's disease. In some embodiments, the complement-mediated disease or disorder comprises Parkinson's disease. In some embodiments, the complement-mediated disease or disorder comprises transplant rejection. In some embodiments, the complement-mediated disease or disorder is antibody-mediated transplant rejection.

[0262] In some embodiments, the anti-C1s antibodies of the present disclosure prevent or delay the onset of at least one symptom of a complement-mediated disease or disorder in an individual. In some embodiments, the anti-C1s antibodies of the present disclosure reduce or eliminate at least one symptom of a complement-mediated disease or disorder in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, hematological diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion diseases, neurodegenerative diseases, neurodegenerative disorders, kidney diseases, transplant rejection, eye diseases, vascular diseases, or vasculitic diseases. The symptom can be a neurological symptom, such as cognitive impairment, memory impairment, loss of motor function, etc. The symptom can also be the activity of C1s protein in the cells, tissues, or bodily fluids of an individual. The symptom can also be the degree of complement activation in the cells, tissues, or bodily fluids of an individual.

[0263] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual modulates complement activation in the individual's cells, tissues, or bodily fluids. In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual inhibits complement activation in the individual's cells, tissues, or bodily fluids. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, inhibits complement activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to complement activation in the individual before treatment with the anti-C1s antibody.

[0264] In some embodiments, the anti-C1s antibodies of the present disclosure reduce C3 deposition on red blood cells, e.g., in some embodiments, the anti-C1s antibodies of the present disclosure reduce deposition of C3b, iC3b, etc. on RBCs. In some embodiments, the anti-C1s antibodies of the present disclosure inhibit complement-mediated red blood cell lysis.

[0265] In some embodiments, the anti-C1s antibodies of the present disclosure reduce the deposition of C3 on platelets. For example, in some embodiments, the anti-C1s antibodies of the present disclosure reduce the deposition of C3b, iC3b, etc. on platelets.

[0266] In some embodiments, administration of an anti-C1s antibody of the present disclosure results in an outcome selected from the group consisting of: (a) reduced complement activation, (b) improved cognitive function, (c) reduced neuronal loss, (d) reduced phosphorylated Tau levels in neurons, (e) reduced glial cell activation, (f) reduced lymphocyte infiltration, (g) reduced macrophage infiltration, (h) reduced antibody deposition, (i) reduced glial cell loss, (j) reduced oligodendrocyte loss, (k) reduced dendritic cell infiltration, (l) reduced neutrophil infiltration, (m) reduced red blood cell lysis, (n) reduced red blood cell lysis. (o) reduced leukocyte phagocytosis, (o) reduced platelet phagocytosis, (p) reduced platelet lysis, (q) improved graft survival, (r) reduced macrophage-mediated phagocytosis, (s) improved vision, (t) improved motor control, (u) improved thrombus formation, (v) improved coagulation, (w) improved kidney function, (x) reduced antibody-mediated complement activation, (y) reduced autoantibody-mediated complement activation, (z) improved anemia, (aa) reduced demyelination, (ab) reduced eosinophilia, (ac) reduced C3 deposition on red blood cells (e.g., reduced deposition of C3b, iC3b, etc. on RBCs), and and (ad) reduced C3 deposition on platelets (e.g., reduced deposition of C3b, iC3b, etc. on platelets), and (ae) reduced anaphylatoxin production, (af) reduced autoantibody-mediated blister formation, (ag) reduced autoantibody-induced pruritus, (ah) reduced autoantibody-induced lupus erythematosus, (ai) reduced autoantibody-mediated skin erosion, (aj) reduced red blood cell destruction due to transfusion reactions, (ak) reduced red blood cell lysis due to alloantibodies, (al) reduced hemolysis due to transfusion reactions, (am) reduced alloantibody-mediated reduced platelet lysis, (an) reduced platelet lysis due to transfusion reactions, (ao) reduced mast cell activation, (ap) reduced mast cell histamine release, (aq) reduced vascular permeability, (ar) reduced edema, (as) reduced complement deposition on graft endothelium, (at) reduced anaphylatoxin production in graft endothelium, (au) reduced dermal-epidermal junction separation, (av) reduced anaphylatoxin production at the dermal-epidermal junction, (aw) reduced alloantibody-mediated complement activation in graft endothelium, (ax) reduced antibody-mediated neuromuscular junction loss,(ay) reduced complement activation at the neuromuscular junction, (az) reduced anaphylatoxin production at the neuromuscular junction, (ba) reduced complement deposition at the neuromuscular junction, (bb) reduced paralysis, (be) reduced numbness, (bd) improved bladder control, (be) improved bowel management, (bf) reduced autoantibody-associated mortality, and (bg) reduced autoantibody-associated morbidity.

[0267] In some embodiments, the anti-C1s antibodies of the disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, can promote the following outcomes: (a) complement activation; (b) cognitive decline; (c) neuronal loss; (d) phosphorylated Tau levels in neurons; (e) glial activation; (f) lymphocyte infiltration; (g) macrophage infiltration; (h) antibody deposition; (i) glial cell loss; (j) oligodendrocyte loss; (k) dendritic cell infiltration; (l) neutrophil infiltration; (m) erythrocyte lysis; (n) erythrocyte phagocytosis; (o) platelet phagocytosis; (p) platelet lysis; ( (q) graft rejection; (r) macrophage-mediated phagocytosis; (s) vision loss; (t) antibody-mediated complement activation; (u) autoantibody-mediated complement activation; (v) demyelination; (w) eosinophilia by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the level or degree of the outcome in the individual before treatment with the anti-C1s antibody.

[0268] In some embodiments, the anti-C1s antibodies of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, are effective in improving one or more of the following outcomes: a) cognitive function; b) graft survival; c) vision; d) motor control; e) clot formation; f) coagulation; g) renal function, and h) hematocrit (red blood cell count) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level or degree of the outcome in the individual before treatment with the anti-C1s antibody.

[0269] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces complement activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to complement activation in the individual before treatment with the anti-C1s antibody.

[0270] In some embodiments, administration of an anti-C1s antibody of the present disclosure improves cognitive function in an individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, improves cognitive function in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the cognitive function in the individual before treatment with the anti-C1s antibody.

[0271] In some embodiments, administration of an anti-C1s antibody of the present disclosure reduces the rate of cognitive decline in an individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces the rate of cognitive decline in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the rate of cognitive decline in the individual before treatment with the anti-C1s antibody.

[0272] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces neuronal loss in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces neuronal loss in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to neuronal loss in the individual before treatment with the anti-C1s antibody.

[0273] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces phosphorylated Tau levels in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces phosphorylated Tau in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the phosphorylated Tau levels in the individual before treatment with the anti-C1s antibody.

[0274] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces glial cell activation in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces glial activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to glial cell activation in the individual before treatment with the anti-C1s antibody. In some embodiments, the glial cells are astrocytes or microglia.

[0275] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces lymphocyte infiltration in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces lymphocyte infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the lymphocyte infiltration in the individual before treatment with the anti-C1s antibody.

[0276] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces macrophage infiltration in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces macrophage infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the macrophage infiltration in the individual before treatment with the anti-C1s antibody.

[0277] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces antibody deposition in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to antibody deposition in the individual before treatment with the anti-C1s antibody.

[0278] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a) production in the individual. For example, in some embodiments, an anti-C1s antibody of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces anaphylatoxin production in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the anaphylatoxin production level in the individual before treatment with the anti-C1s antibody.

[0279] In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient to treat an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure to treat an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient to treat an individual having a complement-mediated disease or disorder.

[0280] In some embodiments, the present disclosure provides use of an anti-C1s antibody of the present disclosure in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

[0281] In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient to inhibit complement activation. In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient to inhibit complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure to inhibit complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient to inhibit complement activation in an individual having a complement-mediated disease or disorder.

[0282] In some embodiments, the present disclosure provides use of an anti-C1s antibody of the present disclosure in the manufacture of a medicament for modulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in an individual with a complement-mediated disease or disorder.

[0283] In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in medical therapy. In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure for use in medical therapy. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in medical therapy.

[0284] In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure for treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder.

[0285] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure or an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure for modulating complement activation. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the anti-C1s antibody inhibits complement activation.

[0286] In a further aspect, the present invention provides use of an anti-C1s antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a complement-mediated disease or disorder. In a further embodiment, the medicament is for use in a method of treating a complement-mediated disease or disorder, comprising administering an effective amount of the medicament to an individual having a complement-mediated disease or disorder. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). In a further embodiment, the medicament is for use in enhancing the clearance (or removal) of C1s from plasma. In a further embodiment, the medicament is for use in enhancing the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma. In a further embodiment, the medicament is for use in inhibiting the cleavage of complement component C4, wherein the antibody does not inhibit the cleavage of complement component C2. In a further embodiment, the medicament is for use in inhibiting a component of the classical complement pathway, and in some instances, the component of the classical complement pathway is C1s.

[0287] In a further aspect, the medicament is for use in a method of treating an individual having a complement-mediated disease or disorder, comprising administering to the individual an effective amount of the medicament. In any of the above aspects, the "individual" may be a human.

[0288] In a further aspect, the present invention provides a method of treating a complement-mediated disease or disorder. In one aspect, the method comprises administering an effective amount of an anti-C1s antibody to an individual having such a complement-mediated disease or disorder. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (described below). In any of the above aspects, the "individual" may be a human.

[0289] In a further aspect, the present invention provides a method for enhancing the clearance (or removal) of C1s from plasma in an individual. In a further aspect, the present invention provides a method for enhancing the clearance (or removal) of the complex of C1q, C1r, and C1s from plasma in an individual. In some embodiments, the anti-C1s antibodies of the present invention provide a method in an individual, wherein the antibody inhibits the cleavage of complement component C4 without inhibiting the cleavage of complement component C2. In some examples, the present invention provides a method for inhibiting a component of the classical complement pathway in an individual, and in some examples, the component of the classical complement pathway is C1s. In one embodiment, the "individual" is a human.

[0290] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the anti-C1s antibodies provided herein, e.g., for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-C1s antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-C1s antibodies provided herein and at least one additional therapeutic agent (e.g., as described below).

[0291] The antibodies of the invention can be used in therapy either alone or in combination with other agents, for example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent.

[0292] The combination therapy described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, in which administration of the antibody of the present invention can precede, be simultaneous with, and / or follow administration of the additional therapeutic agent. In one embodiment, administration of the anti-C1S antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibody of the present invention can be used in combination with radiation therapy.

[0293] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, single administration or repeated administration over various time periods, bolus administration, and pulse infusion.

[0294] The antibodies of the present invention are formulated, administered, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of agent delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will typically be used in the same dosages and by any route of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0295] The appropriate dose of an antibody of the invention (whether used alone or in combination with one or more other additional therapeutic agents) for disease prevention or treatment will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous medical history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors discussed above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs. One exemplary dose of antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about 20, or, for example, about six doses of antibody). A high initial loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and measurements.

[0296] It will be appreciated that any of the above-described formulations or therapeutic methods may be practiced using an immunoconjugate of the invention instead of, or in addition to, an anti-C1S antibody.

[0297] H.Product In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label on or package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. The article of manufacture may further include (a) a first container with a composition comprising the antibody of the present invention contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture of this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0298] It will be appreciated that any of the above-described products may contain an immunoconjugate of the present invention instead of or in addition to an anti-C1S antibody. [Example]

[0299] III. Working Examples The following are examples of the methods and compositions of the present invention. In light of the above general description, it will be understood that various other embodiments may be practiced.

[0300] While the foregoing invention has been described in detail by way of illustration and illustration for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0301] Example 1: Expression and purification of human C1s Recombinant human C1s (SEQ ID NO:1) with a C-terminal Flag tag (hC1s-Flag) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Culture supernatant expressing human recombinant hC1s-Flag was applied to a column packed with anti-Flag M2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). Fractions containing hC1s-Flag were collected and subsequently applied to a Superdex 200 gel filtration column (GE healthcare Uppsala, Sweden). Fractions containing hC1s-Flag were then collected, concentrated, and stored at -80°C.

[0302] Recombinant C1s (SEQ ID NO:1) with an 8x histidine tag at the carboxyl terminus (hC1s-His) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). The culture supernatant containing hC1s-His was applied to a HisTrap Excel column (GE Healthcare, Uppsala, Sweden) and eluted with imidazole. Fractions containing hC1s-His were collected and applied to a Superdex 200 gel filtration column (GE Healthcare, Uppsala, Sweden). Fractions containing hC1s-His were collected, concentrated, and stored at -80°C.

[0303] Example 2: Preparation of anti-C1s antibody Polynucleotides encoding the heavy and light chain variable regions of the anti-C1s antibody, IPN009VH2 (SEQ ID NO: 13) and IPN009VK3 (SEQ ID NO: 14), (described in WO2016164358) were synthesized by GenScript Inc. The heavy and light chain variable regions were cloned into expression vectors containing the heavy chain constant region SG4GK (SEQ ID NO: 15) and the light chain constant region SK1 (SEQ ID NO: 16), respectively. The anti-C1s antibody C1_IPN009VH2-SG4GK / IPN009VK3-SK1 (IPN009VH2 / IPN009VK3) was transiently expressed using FreeStyle FS293-F cells and 293fectin (Life Technologies) according to the manufacturer's instructions. Recombinant antibodies were purified using Protein A (GE Healthcare) and eluted in D-PBS, Tris-buffered saline (TBS), or His buffer (20 mM histidine, 150 mM NaCl, pH 6.0). If necessary, size exclusion chromatography was performed to remove high- and / or low-molecular-weight components.

[0304] Histidine scans were performed at specific positions within the CDRs and FRs of IPN009VH2 / IPN009VK3. The positions of mutations in the mutants are shown in Table 2 (upper part, columns 4 and 6). The same applies to Tables 3-1 to 3-6. Each amino acid was individually mutated to histidine using the In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio Company) according to the manufacturer's instructions. All mutants were transiently expressed and purified as described above. The binding affinity of each histidine-substituted mutant at pH 7.4 and pH 5.8 was determined at 37°C using a Biacore T200 instrument (GE Healthcare). Recombinant Protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4), 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8), or 5(-) buffer (20 mM ACES, 150 mM NaCl, 3 μM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured onto the sensor surface using Protein A / G. The antibody capture amount was targeted at 200 resonance units (RU). Serum-derived human C1s (CompTech) or recombinant C1s prepared as described in Example 1 was injected at 50 nM followed by dissociation. The sensor surface was regenerated after each cycle with 10 mM glycine-HCl, pH 1.5. Binding affinities were determined by processing the data and fitting them to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).Some of the single histidine substitutions had a higher KD in 5(+) buffer ("KD 5.8+") and / or a higher KD in 5(-) buffer ("KD 5.8-") compared to the KD in 7(+) buffer ("KD 7.4+"). The ratio of KD 5.8+ to KD 7.4+ ("KD 5+ / 7+") and the ratio of KD 5.8- to KD 7.4+ ("KD 5- / 7+") were calculated. If the KD 5+ / 7+ or KD 5- / 7+ of a variant is higher than that of the parent antibody IPN009VH2-SG4GK / IPN009VK3-SK1, the variant is considered to be "pH"-dependent or "pH and Ca"-dependent, respectively. If the binding reactivity of a mutant in 5(+) buffer and / or 5(-) buffer is much lower than that of IPN009VH2 / IPN009VK3, the mutant is also considered to be pH-dependent and / or pH and Ca-dependent. Therefore, such mutations are effective in generating pH-dependent and / or pH and Ca-dependent antibodies. The results of the Biacore assay are shown in Table 2. If the binding reactivity is much lower than that of IPN009VH2 / IPN009VK3, it is indicated as "low" in the table. To further enhance pH-dependent and / or pH and Ca-dependent reactivity, combinations of histidine mutations were performed. Antibodies with combinations of histidine mutations were evaluated using Biacore using the method described above. The results of the Biacore assay are shown in Tables 3-1 and 3-2. Many mutants showed much better pH-dependent and pH and Ca-dependent reactivity than IPN009VH2 / IPN009VK3.

[0305] [Table 2]

[0306] [Table 3-1]

[0307] [Table 3-2]

[0308] Example 3: Further optimization of the pH and / or pH and Ca dependence of anti-C1s antibodies C1_IPN92H0033 (SEQ ID NO: 17)-SG4GK / IPN93L0024 (SEQ ID NO: 18)-SK1 (IPN92H0033 / IPN93L0024) was selected for further optimization. Histidine, lysine, arginine, aspartic acid, glutamic acid, and glutamine scans were performed at specific positions in the CDRs and FRs of IPN92H0033 / IPN93L0024. Mutants with mutations were generated and purified using the method described above and evaluated using Biacore. All mutants were evaluated using Biacore as described above. Mutants with improved pH and / or pH and Ca dependence were selected for combination. Mutants with improved KD at 7(+) were also selected for combination. After several combinations, mutants with multiple mutations acquired significant pH and / or pH and Ca dependence. The Biacore results are shown in Tables 4-1 and 4-2.

[0309] As shown in Tables 4-1 and 4-2, surprisingly, the introduction of a charged residue (arginine, lysine, aspartic acid, or glutamic acid) into an antibody that already had a histidine residue significantly improved the pH dependence of the antibody-antigen interaction.

[0310] [Table 4-1]

[0311] [Table 4-2]

[0312] Without being bound by any particular theory, histidine residues in an antibody may interact with various residues surrounding the histidine residue within the antibody. Such interactions may affect the structure of the antibody or the conformation of the CDR. Histidine is protonated and positively charged at acidic pH. Introduction of a positively charged residue (e.g., arginine or lysine) at a position surrounding the histidine may cause repulsion between the positively charged residue and the protonated histidine at acidic pH, thereby inducing a change in the structure or conformation of the antibody or CDR. Similarly, introduction of a negatively charged residue (e.g., aspartic acid or glutamic acid) at a position surrounding the histidine may cause an interaction between the negatively charged residue and the protonated histidine at acidic pH, thereby inducing a change in the structure or conformation of the antibody or CDR. These structural or conformational changes in the antibody or CDR that occur at acidic pH may affect the antigen binding of the antibody and reduce the binding affinity of the antibody to the antigen at acidic pH. In summary, the introduction of charged residues (e.g., arginine, lysine, aspartic acid, or glutamic acid) at positions surrounding histidine residues in an antibody can reduce the binding affinity of the antibody to its antigen at acidic pH, thereby improving the pH dependence of the antibody-antigen interaction through a unique mechanism.

[0313] Example 4: Reduction of non-specific binding Nonspecific binding is an important factor in predicting the pharmacokinetics of antibodies (MABS 2017, VOL. 9, NO. 5, 756-766). High nonspecific binding leads to rapid clearance of the antibody and poor pharmacokinetics, making antibodies with high nonspecific binding undesirable for therapeutic antibody development. The extracellular matrix binding assay (ECM binding assay) is one assay method for predicting nonspecific binding (WO 2012 / 093704).

[0314] pH-dependent and pH- and Ca-dependent antibodies were selected for the ECM binding assay. The ECM binding assay incorporates Meso Scale Discovery (MSD) technology and electrochemiluminescence (ECL). First, the surface of a Multi-Array High Bind 96-well plate (MSD) was coated with ECM (BD Matrigel) overnight at 4°C. The ECM-coated plate was then blocked with ECL blocking buffer at either pH 7.4 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, containing 0.05% Tween 20 and 0.5% BSA, pH 7.4) or pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, containing 0.05% Tween 20 and 0.5% BSA, pH 5.8) for 2 hours at 30°C. Selected variants and assay controls were then diluted in dilution buffer at either pH 7.4 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, pH 7.4, containing 0.01% Tween 20 and 0.1% BSA) or pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, pH 5.8, containing 0.01% Tween 20 and 0.1% BSA). The diluted samples and assay controls were incubated on the plate at 600 rpm and 30°C for 1 hour, followed by incubation with 0.25% glutaraldehyde (Sigma) for 10 minutes at room temperature. The plate was then washed with 1x PBST (Sigma) and incubated with sulfo-tagged goat anti-human IgG (Invitrogen) at 600 rpm and 30°C for 1 hour. The plate was washed again with 1x PBST, and Read Buffer T (2x) containing detergent (MSD) was added to each well. The plate was read using a MESO SECTOR S 600. The results of the ECM binding assay are shown in Table 5. The binding of selected variants to ECM at pH 5.8 was higher than that of IPN009VH2 / IPN009VK3. To improve the pharmacokinetics of the antibody, further optimization was performed to reduce binding to ECM.Negatively charged residues were introduced into selected mutants. Binding affinity and ECM binding were assessed using Biacore and ECM binding assays, respectively, as described above. Several residues were identified that reduced ECM binding while maintaining or enhancing pH and / or pH and Ca dependence. By combining these identified mutations, three antibodies were successfully constructed: C1_IPN92H0288(SEQ ID NO: 19)-SG4GK / IPN93L0211(SEQ ID NO: 20)-SK1, C1_IPN92H0288-SG4GK / IPN93L0058(SEQ ID NO: 21)-SK1, and C1_IPN92H0307(SEQ ID NO: 22)-SG4GK / IPN93L0058-SK1. These three mutants showed superior pH and pH and Ca dependence, with lower ECM binding at both pH 7.4 and pH 5.8. The results of the Biacore and ECM binding assays for these three mutants are shown in Table 6.

[0315] [Table 5]

[0316] [Table 6]

[0317] Example 5: Affinity Measurement The binding affinities of all histidine-substituted mutants at pH 7.4 and pH 5.8 were determined using a Biacore T200 instrument (GE Healthcare) at 37°C. Recombinant Protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured onto the sensor by Protein A / G. The antibody capture amount was targeted at 200 resonance units (RU). Serum-derived human C1s was injected at 12.5 and 50 nM at pH 7.4, and at 50 and 200 nM for all samples except IPN92H0281 / IPN93L0024-SG136 and IPN92H0286 / IPN93L0205-SG136 at pH 5.8, and at 200 and 800 nM for IPN92H0281 / IPN93L0024-SG136 and IPN92H0286 / IPN93L0205-SG136, followed by dissociation. After each cycle, the sensor surface was regenerated with 10 mM glycine-HCl, pH 1.5. Binding affinities were determined by processing the data and fitting them to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare) (Table 7). Immediately after the dissociation region at pH 7.4, an additional dissociation region at pH 5.8 was incorporated. This dissociation rate in 5(+) buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software. The asterisk in the affinity value at pH 5.8 indicates that the affinity measurement value could not be accurately determined due to low binding reactivity.

[0318] [Table 7]

[0319] Example 6: Mouse PK study using pH- and / or Ca-dependent anti-C1s antibodies Measurement of anti-C1s antibody and total hC1s concentrations in plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS) The concentrations of anti-C1s antibody and human C1s in mouse plasma were measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting a specified amount of anti-C1s antibody and human C1s in mouse plasma to achieve anti-C1s concentrations of 12.5, 25, 50, 100, 200, 400, and 800 micrograms (μg) / mL and human C1s concentrations of 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, and 62.5 μg / mL. Two μL of calibration standard and plasma sample were mixed with 25 μL of 6.8 mol / L urea, 9.1 mmol / L dithiothreitol, and 0.45 μg / mL lysozyme (hen egg white) in 50 mmol / L ammonium bicarbonate and incubated at 56°C for 45 minutes. Then, 2 μL of 500 mmol / L iodoacetamide was added and incubated at 37°C for 30 minutes in the dark. Next, 160 μL of 0.5 μg / mL sequencing-grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37°C. Finally, 5 μL of 10% trifluoroacetic acid was added to inactivate residual trypsin. 50 μL of the digested sample was subjected to LC / ESI-MS / MS analysis. LC / ESI-MS / MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D I-class UPLC (Waters). The anti-C1s antibody-specific peptide GLPSSIEK and the human C1s-specific peptide LLEVPEGR were monitored by selected reaction monitoring (SRM). The SRM transitions were [M+2H]2+ (m / z 415.7) to the y6 ion (m / z 660.3) for the anti-C1s antibody and [M+2H]2+ (m / z 456.8) to the y6 ion (m / z 686.3) for human C1s. Calibration curves were generated by weighted (1 / x2) linear regression using peak areas plotted against concentration. Mouse plasma concentrations were calculated from the calibration curves using the analytical software Masslynx version 4.1 (Waters).

[0320] Evaluation of the pharmacokinetics of total hC1s and pH- and / or Ca-dependent anti-C1s antibodies in mice The in vivo pharmacokinetics of hC1s (human complement component 1s prepared as described in Example 1) and the pH- and / or Ca-dependent antibodies prepared in Example 2 were investigated in mice (CB17 / Icr-Prkdc) with hC1s alone or in combination with anti-C1s antibodies. scid / CrlCrlj: Charles River Japan). Three mice were assigned to each treatment group. Mice were intravenously injected once at a dose of 10 mL / kg with either hC1s solution (0.23 mg / mL) or a mixture containing hC1s and anti-C1s antibody (0.23 and 2.5 mg / mL, respectively). In this study, the dose was set so that the concentration of anti-C1s antibodies was in excess of C1s, and it was therefore assumed that almost all C1s in the blood was in a bound form. Blood was collected 5 minutes, 30 minutes, 2 hours, 7 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after injection. The blood was immediately centrifuged to separate plasma samples. At each sample collection time, the plasma concentrations of anti-C1s antibody, hC1s, were measured by LC / ESI-MS / MS. PK parameters of anti-C1s antibody and hC1s were estimated by non-compartmental analysis (Phoenix WinNonlin version 8.0, Certara). The following antibodies were administered to mice as anti-C1s antibodies: 1. IPN009VH2 / IPN009VK3-SG136, 2. IPN92H0033 / IPN009VK3-SG136, 3. IPN009VH2 / IPN93L0021-SG136, 4. IPN009VH2 / IPN93L0023-SG136, 5. IPN009VH2 / IPN93L0024-SG136, 6. IPN92H0038 / IPN93L0024-SG136, 7. IPN92H0033 / IPN93L0024-SG136, 8. IPN92H0281 / IPN93L0024-SG136, 9. IPN92H0286 / IPN93L0205-SG136, 10. IPN92H0288 / IPN93L0211-SG136, 11. IPN92H0288 / IPN93L0058-SG136, 12. IPN92H0307 / IPN93L0058-SG136. The plasma antibody concentration profiles of IPN92H0286 / IPN93L0205-SG136 were rapidly eliminated from the blood. This rapid elimination was likely due to its superior ECM binding. Other pH- and / or Ca-dependent anti-C1s antibodies also showed similar plasma concentration profiles. The CL of IPN92H0286 / IPN93L0205-SG136 was 56.5 mL / day / kg, while the CL of other anti-C1s antibodies was within a two-fold range (4.8–8.1 mL / day / kg). These results indicate that pH and / or Ca-dependence do not affect the pharmacokinetics of antibodies in plasma. The time course of plasma hC1s concentrations in the presence of pH- and / or Ca-dependent anti-C1s antibodies showed a more rapid disappearance than that of the non-pH / Ca-dependent anti-C1s antibodies IPN009VH2 / IPN009VK3-SG136. The CL of hC1s in the presence of pH / Ca-dependent anti-C1s antibodies tended to be greater than that in the presence of either Ca- or pH-dependent anti-C1s antibodies. These phenomena suggest that pH- and Ca-dependent binding properties, or a combination of these, are useful for promoting C1s clearance in vivo.

[0321] Pharmacokinetic parameters in mice co-injected with a mixture of human C1S and antibodies and affinity values ​​of antibody mutants The sweeping index of each antibody represents the ability of the antibody variant to remove antigen from the blood. It was calculated by dividing the antigen clearance rate by the antibody clearance rate (Table 8). The improvement in sweeping index represents the relative ability of the antibody variant to remove antigen from the blood compared to the parent antibody. It was calculated by dividing the sweeping index of each antibody variant by the sweeping index of the parent antibody, which therefore has a sweeping index of 1. koff (5.8+ of 775+) represents the dissociation rate of C1s in the additional dissociation region at pH 5.8 immediately following the dissociation region at pH 7.4. The "+" indicates the presence of 1.2 mM CaCl2 in both the pH 7.4 and pH 5.8 regions. The koff (5.8+ of 775+) / koff (7.4) column indicates the ratio of the dissociation rate at pH 5.8 to the dissociation rate at pH 7.4 in the 775+ assay. The KD(5.8+) / KD(7.4+) column indicates the ratio of the antibody affinity at pH 5.8 to the affinity at pH 7.4. The "+" indicates the presence of 1.2 mM CaCl2 during affinity measurements at pH 5.8 and pH 7.4. The asterisk symbol for the affinity value at pH 5.8 indicates that the affinity measurement could not be accurately determined due to low binding reactivity.

[0322] [Table 8]

[0323] The correlation between the improvement in sweeping index and the KD(5.8+) / KD(7.4+) ratio was plotted for all antibodies (Figure 1). However, antibody IPN92H0286 / IPN93L0205-SG136 was excluded from the plot because it exhibited rapid clearance, likely due to its high ECM binding (Table 5). The dotted line indicates the best fit using linear regression, and the R-squared value indicates the goodness of fit. The KD(5.8+) / KD(7.4+) value indicates the ratio of antibody affinity at pH 5.8 to affinity at pH 7.4. The "+" indicates the presence of 1.2 mM CaCl2 in affinity measurements at pH 5.8 and pH 7.4. The correlation between the improvement in sweeping index and the koff (5.8+ of 775+) / koff (7.4+) ratio was plotted for all antibodies (Figure 2). However, antibody IPN92H0286 / IPN93L0205-SG136 was excluded from the plot because it exhibited rapid clearance, likely due to high ECM binding (Table 5). The dotted line indicates the line of best fit using linear regression, and the R-squared value indicates the goodness of fit. The koff (5.8+ of 775+) / koff (7.4+) value indicates the ratio of the dissociation rate at pH 5.8 to the dissociation rate at pH 7.4 in the 775+ assay. The "+" indicates the presence of 1.2 mM CaCl2 in both the pH 5.8 and pH 7.4 regions.

[0324] Example 7 Histidine-substituted variants IPN93L0026 and IPN92H0012 (Table 9) contain variable regions with one histidine substitution (e.g., I96H, a substitution at position 96 of the light chain, and I51H, a substitution at position 51 of the heavy chain, according to the Kabat numbering system, respectively). The binding affinities of all histidine-substituted variants at pH 7.4 and pH 5.8 were determined at 37°C using a Biacore T200 instrument (GE Healthcare). Recombinant Protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured onto the sensor surface using Protein A / G. The antibody capture amount was targeted at 200 resonance units (RU). Serum-derived human C1s was injected at 50 nM and subsequently dissociated. The sensor surface was regenerated after each cycle with 10 mM glycine-HCl, pH 1.5. Binding affinities were determined by processing the data and fitting them to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). The dissociation region at pH 7.4 was immediately followed by an additional dissociation region at pH 5.8. The dissociation rate in 5(+) buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software (Table 9). As shown in Table 9, the single histidine substitution mutants IPN93L0026 and IPN92H0012 showed improved ratios of koff (5.8+ of 775+) / koff (7.4+). Referring to the correlation between the improvement in sweeping index and the ratio of koff (5.8+ of 775+) / koff (7.4+) (Table 8), these substitutions, either alone or in combination with other substitutions, have the ability to enhance the improvement in sweeping index.

[0325] [Table 9]

[0326] Example 8: Mouse PK study using CCP1-CCP2-SP conjugate Measurement of total human C1s concentrations in mouse plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS) Total concentrations of human C1s in mouse plasma were measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting a defined amount of human C1s in mouse plasma to yield concentrations of 0.477, 0.954, 1.91, 3.82, 7.64, 15.3, and 30.5 μg / mL. Two μL of the calibration standard and plasma sample were mixed with 25 μL of 7.5 mol / L urea, 8 mmol / L dithiothreitol, and 1 μg / mL lysozyme (hen egg white) in 50 mmol / L ammonium bicarbonate and incubated at 56°C for 45 minutes. Then, 2 μL of 500 mmol / L iodoacetamide was added, and the mixture was incubated at 37°C for 30 minutes in the dark. Next, 160 μL of 0.5 μg / mL sequencing-grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37°C. Finally, 5 μL of 10% trifluoroacetic acid was added to inactivate residual trypsin. 40 μL of the digested sample was subjected to LC / ESI-MS / MS analysis. LC / ESI-MS / MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D I-class UPLC (Waters). The human C1s-specific peptide LLEVPEGR was monitored by selected reaction monitoring (SRM). The SRM transition for human C1s was [M+2H]2+ (m / z 456.8) to the y6 ion (m / z 686.4). A calibration curve was generated by weighted (1 / x2) linear regression using peak areas plotted against concentration. The concentrations in mouse plasma were calculated from this calibration curve using analytical software Masslynx version 4.1 (Waters).

[0327] Evaluation of the pharmacokinetics of total hC1s after anti-C1s antibody administration in mice The antigen was injected either alone (a mixture of hC1q and rC1r2s2) or together with anti-C1s antibody (CB17 / Icr-Prkdc scid The in vivo pharmacokinetics of hC1s and anti-C1s antibodies was evaluated after administration of 1000 mg / kg of IgG1 antibody to 1000 mg / kg of mice (CrlCrlj: Charles River Japan). Three mice were assigned to each treatment group. First, a mixture of hC1q and rC1r2s2 (0.84 and 0.47 mg / mL, respectively) was intravenously injected into mice at a dose of 10 mL / kg. Immediately after administration of the antigen solution, an anti-C1s antibody solution (2.5 mg / mL) was administered to the same mice in the same manner. The doses of C1q and rC1r2s2 were set to achieve physiological concentrations in human plasma immediately after administration. During this study, the anti-C1s antibody dose was adjusted to ensure excess concentrations of anti-C1s antibody over both antigens, so that it was estimated that almost all hC1s in the blood would be in bound form. Blood was collected 5 minutes, 30 minutes, 2 hours, 7 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after injection. The blood was immediately centrifuged to separate plasma samples. At each sample collection time, the plasma concentration of hC1s was measured by LC / ESI-MS / MS. The PK parameters of hC1s were estimated by non-compartmental analysis (Phoenix WinNonlin version 8.0, Certara). The following antibodies were administered to mice as anti-C1s antibodies (Table 10): 1. COS0098bb-SG1148 / SG136 2. COS0112gg-SG1148 / SG136 3. COS0127bb-SG1148 / SG136 4. COS0158ee-SG1148 / SG136 5. COS0182hh-SG1148 / SG136. SG136 Fc contains mutations that reduce both C1q and Fc gamma receptor binding. SG1148 Fc contains mutations that reduce C1q binding while maintaining Fc gamma receptor binding. PK parameters of hC1s are shown in Table 11. The hC1s CL ratios (SG1148 / SG136) of five CCP1-CCP2-SP conjugates (COS0098bb, COS0112gg, COS0127bb, COS0158ee, and COS0182hh) were 9.2, 6.9, 5.6, 3.8, and 6.6, respectively. This value indicates the ability to promote hC1s clearance.

[0328] [Table 10] Constant region names: SG1148 (CH: SEQ ID NO: 86 and CL: SEQ ID NO: 88), SG136 (CH: SEQ ID NO: 87 and CL: SEQ ID NO: 88)

[0329] [Table 11]

Claims

1. An isolated bivalent antibody that binds to C1s, wherein when measured at a calcium concentration of 100 μM to 10 mM at pH 7.4 and at a calcium concentration of 0.1 μM to 30 μM at pH 5.8, the ratio of the KD value of its C1s binding activity at pH 5.8 to the KD value of its C1s binding activity at pH 7.4 (KD(pH5.8) / KD(pH7.4)) is 5 or greater, and the antibody forms an immune complex with dimeric C1s in plasma and dissociates into monomers of C1s in endosomes, thereby disassembling the immune complex, thereby enhancing the clearance of C1s from plasma. Under pH 7.4 conditions, the antibody: (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (c) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:

85. An antibody that competes with an antibody selected from the group consisting of:

2. The antibody described in claim 1, which is an antibody selected from the group consisting of the following (a) to (e): (a) an antibody comprising the HVR-H1 sequence of SEQ ID NO:56, the HVR-H2 sequence of SEQ ID NO:57, the HVR-H3 sequence of SEQ ID NO:58, the HVR-L1 sequence of SEQ ID NO:71, the HVR-L2 sequence of SEQ ID NO:72, and the HVR-L3 sequence of SEQ ID NO:73; (b) an antibody comprising the HVR-H1 sequence of SEQ ID NO:59, the HVR-H2 sequence of SEQ ID NO:60, the HVR-H3 sequence of SEQ ID NO:61, the HVR-L1 sequence of SEQ ID NO:74, the HVR-L2 sequence of SEQ ID NO:75, and the HVR-L3 sequence of SEQ ID NO:76; (c) an antibody comprising the HVR-H1 sequence of SEQ ID NO:62, the HVR-H2 sequence of SEQ ID NO:63, the HVR-H3 sequence of SEQ ID NO:64, the HVR-L1 sequence of SEQ ID NO:77, the HVR-L2 sequence of SEQ ID NO:78, and the HVR-L3 sequence of SEQ ID NO:79; (d) an antibody comprising the HVR-H1 sequence of SEQ ID NO:65, the HVR-H2 sequence of SEQ ID NO:66, the HVR-H3 sequence of SEQ ID NO:67, the HVR-L1 sequence of SEQ ID NO:80, the HVR-L2 sequence of SEQ ID NO:81, and the HVR-L3 sequence of SEQ ID NO:82; and (e) an antibody comprising the HVR-H1 sequence of SEQ ID NO:68, the HVR-H2 sequence of SEQ ID NO:69, the HVR-H3 sequence of SEQ ID NO:70, the HVR-L1 sequence of SEQ ID NO:83, the HVR-L2 sequence of SEQ ID NO:84, and the HVR-L3 sequence of SEQ ID NO:

85.

3. The antibody described in claim 1, which is an antibody selected from the group consisting of the following (a) to (e): (a) an antibody comprising the VH sequence of SEQ ID NO:46 and the VL sequence of SEQ ID NO:51; (b) an antibody comprising the VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:52; (c) an antibody comprising the VH sequence of SEQ ID NO:48 and the VL sequence of SEQ ID NO:53; (d) an antibody comprising the VH sequence of SEQ ID NO: 49 and the VL sequence of SEQ ID NO: 54; and (e) an antibody comprising the VH sequence of SEQ ID NO:50 and the VL sequence of SEQ ID NO:55

4. An antibody described in claim 1, comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:71, an HVR-L2 sequence of SEQ ID NO:72, and an HVR-L3 sequence of SEQ ID NO:

73.

5. An antibody described in claim 1, comprising a VH sequence of SEQ ID NO:46 and a VL sequence of SEQ ID NO:

51.

6. A pharmaceutical formulation for enhancing the clearance of C1s from plasma, comprising an antibody described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier, based on the characteristics of the antibody's KD value ratio and its characteristics of forming an immune complex with dimeric C1s in plasma and dissociating C1s into monomers in endosomes, thereby disassembling the immune complex.

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