Anti-complement component antibodies and methods of use

Abstract

The present invention provides anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods of using the same. The invention also provides pharmaceutical formulations comprising the antibodies, and methods of treating a subject having a complement-mediated disease or disorder comprising administering the antibodies to the subject. And evaluating the binding specificity and C1q substitution function of the anti-C1s antibody and the anti-C1r antibody. The time dependent complement neutralizing function of the antibodies as well as binding to native and truncated C1s or C1r proteins are also shown.

Description

[0001] This application is a divisional application of a patent application with an application date of April 12, 2019, a Chinese patent application number of 201980038684.5, and an invention title of "Anti-complement component antibodies and methods of use". Technical Field

[0002] The present invention relates to anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods of use thereof. Background Art

[0003] The C1 complex is a large protein complex that serves as a key initiator of the classical pathway cascade. The C1 complex is composed of three components, C1q, C1r, and C1s, in a molar ratio of 1:2:2, respectively (NPL 1). When the C1 complex binds to an antibody-bound target, the classical pathway is initiated. C1q, which has six globular heads, mediates the binding of the C1 complex to the antibody through affinity interactions with the Fc region. Once tightly bound to the target, C1r in the C1 complex autoactivates and becomes enzymatically active. Then, the activated C1r cleaves and activates the zymogen C1s in the C1 complex (NPL 2). Subsequently, active C1s cleaves its substrates, complement components C2 and C4, into C2a / C2b and C4a / C4b fragments, respectively. This results in the assembly of the C3 convertase C4b2a on the target surface, which cleaves C3 to form C3b. C3b then cleaves C5 to initiate the formation of the terminal membrane attack complex, C5b, C6, C7, C8, and C9, which lyses the target by pore formation.

[0004] Both C1s and C1r proteins have the same domain architecture, namely CUB1-EGF-CUB2-CCP1-CCP2-serine protease (NPL 3). The CUB1-EGF-CUB2 domain mediates the interaction between C1r and C1s to form the C1r2s2 tetramer (NPL 4), and likewise, mediates the interaction between C1r2s2 and C1q (NPL 5). In contrast, the CCP1-CCP2-serine protease domain of C1r and C1s is responsible for the proteolytic cleavage of their respective substrates (NPL 6, NPL 7). The C1r2s2 tetramer interacts with the six stalks in C1q through six binding sites in the CUB1-EGF-CUB2 domain of the tetramer (NPL 5).

[0005] Although the complement system with normal function can defend the host against pathogen invasion, the dysregulation or inappropriate activation of the classical pathway can lead to various complement-mediated disorders, such as but not limited to, autoimmune hemolytic anemia (AIHA), Behcet's disease, bullous pemphigoid (BP), immune thrombocytopenic purpura (ITP), etc. Therefore, the inhibition of the excessive or uncontrolled activation of the classical pathway can provide clinical benefits to patients suffering from such disorders.

[0006] An antibody HI532 that binds to the β-domain of C1s has been reported to be able to inhibit the interaction of C1r2s2 with C1q (NPL 8). However, this antibody cannot completely neutralize the hemolytic activity of human serum, and even after incubating the serum with this antibody for 24 hours, 30% of the activity remains.

[0007] Antibodies are highly attractive drugs because they are stable in plasma, highly specific for their targets, and generally exhibit good pharmacokinetic characteristics. However, due to their large molecular size, the doses of therapeutic antibodies are usually very high. In the case of the presence of targets in high abundance, the required therapeutic doses of antibodies are even higher. As a result, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive ways to reduce the doses associated with therapeutic antibodies and the high production costs.

[0008] 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 be able to neutralize multiple antigen molecules with a single antibody molecule (NPL 9, PTL 1). pH-dependent antibodies bind tightly to their antigens under neutral pH conditions in plasma, but dissociate from the antigens in endosomes under acidic pH conditions. Once dissociated from the antigens, the antibodies recycle back to the plasma through the FcRn receptor, while the dissociated antigens are degraded in the lysosomes of the cells. Then, the recycled antibodies freely bind and neutralize antigen molecules again, and this process continues to repeat as long as the antibodies remain in circulation.

[0009] Citation List

[0010] Patent Literature

[0011] [PTL 1] WO2009 / 125825

[0012] Non-Patent Literature

[0013] [NPL 1] Wang et. al. Mol Cell. 2016 Jul 7;63(1):135-45

[0014] [NPL 2] Mortensen et. al. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):986-991

[0015] [NPL 3] Gal et. al. Mol Immunol. 2009 Sep;46(14):2745-52

[0016] [NPL 4] Almitairi et. al. Proc Natl Acad Sci U S A. 2018 Jan 23;115(4):768-773

[0017] [NPL 5] Bally et. al. J Biol Chem. 2009 Jul 17;284(29):19340-8

[0018] [NPL 6] Rossi et. al. 1998 J Biol Chem. 1998 Jan 9;273(2):1232-9

[0019] [NPL 7] Lacroix et. al. J Biol Chem. 2001 Sep 28;276(39):36233-40

[0020] [NPL 8] Tseng et. al. Mol Immunol. 1997 Jun;34(8-9):671-9

[0021] [NPL 9] Igawa et. al. Nat Biotechnol. 2010 Nov;28(11):1203-7 Summary of the Invention Overview of the Invention

[0023] Technical Problem

[0024] The present invention provides anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods of using the same.

[0025] Solution to the Problem

[0026] In some embodiments, the isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having a replacement function, the replacement function enabling the antibody to bind to the C1qrs complex and promoting the dissociation of C1q from the C1qrs complex.

[0027] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to the C1qrs complex on a BIACORE® chip and promotes the dissociation of C1q from the C1qrs complex. In further embodiments, when after a sufficient time, as determined by BIACORE® assay, the value of the response unit (Ru) is lower in the presence of the antibody than in the absence of the antibody, the antibody of the present invention can be determined to be an antibody with a displacing function.

[0028] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex can be determined to be an antibody with a displacing function when the cross time point is within 60 s, 100 s, 150 s, 200 s, 500 s, 700 s, 1000 s, 1500 s, or 2000 s after the start time point of antibody injection, as determined by BIACORE® assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500 nM, 10 microliters (μL) / min.

[0029] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex can be determined to be an antibody with a displacing function when almost all of the C1q dissociates from the C1qrs complex within 100 s, 300 s, 500 s, 700 s, 1000 s, 1500 s, 2000 s, 3000 s, 5000 s, 7000 s, or 10000 s after the start time point of antibody injection, as determined by BIACORE® assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units, respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.

[0030] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that has at least 70% neutralizing activity against human serum complement in an RBC assay.

[0031] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that specifically binds to C1s or an antibody that specifically binds to C1r.

[0032] In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s. In further embodiments, the antibody of the invention competes for binding to the epitope with an antibody selected from the group consisting of the following 1)-5):

[0033] 1) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37,

[0034] 2) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43,

[0035] 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49,

[0036] 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55, and

[0037] 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61.

[0038] In some embodiments, the isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r. In further embodiments, the antibody of the invention competes for binding to the epitope with an antibody selected from the group consisting of 6)-13) below:

[0039] 6) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159,

[0040] 7) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160,

[0041] 8) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161,

[0042] 9) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162,

[0043] 10) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 123, an HVR-H2 sequence of SEQ ID NO: 131, an HVR-H3 sequence of SEQ ID NO: 139, an HVR-L1 sequence of SEQ ID NO: 147, an HVR-L2 sequence of SEQ ID NO: 155, and an HVR-L3 sequence of SEQ ID NO: 163,

[0044] 11) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 124, an HVR-H2 sequence of SEQ ID NO: 132, an HVR-H3 sequence of SEQ ID NO: 140, an HVR-L1 sequence of SEQ ID NO: 148, an HVR-L2 sequence of SEQ ID NO: 156, and an HVR-L3 sequence of SEQ ID NO: 164,

[0045] 12) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 125, an HVR-H2 sequence of SEQ ID NO: 133, an HVR-H3 sequence of SEQ ID NO: 141, an HVR-L1 sequence of SEQ ID NO: 149, an HVR-L2 sequence of SEQ ID NO: 157, and an HVR-L3 sequence of SEQ ID NO: 165, and

[0046] 13) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 126, an HVR-H2 sequence of SEQ ID NO: 134, an HVR-H3 sequence of SEQ ID NO: 142, an HVR-L1 sequence of SEQ ID NO: 150, an HVR-L2 sequence of SEQ ID NO: 158, and an HVR-L3 sequence of SEQ ID NO: 166.

[0047] In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having antigen-binding activity that varies depending on the ionic concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having C1s-binding activity that varies depending on the ionic concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having C1r-binding activity that varies depending on the ionic concentration.

[0048] In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to the antigen with higher affinity at neutral pH than at acidic pH. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1s with higher affinity at neutral pH than at acidic pH. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1r with higher affinity at neutral pH than at acidic pH.

[0049] In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to the antigen with higher affinity under conditions of high calcium concentration than under conditions of low calcium concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1s with higher affinity under conditions of high calcium concentration than under conditions of low calcium concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1r with higher affinity under conditions of high calcium concentration than under conditions of low calcium concentration.

[0050] In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to the antigen with higher affinity under the conditions of both neutral pH and high calcium concentration than under the conditions of both acidic pH and low calcium concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1s with higher affinity under the conditions of both neutral pH and high calcium concentration than under the conditions of both acidic pH and low calcium concentration. In some embodiments, the isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1r with higher affinity under the conditions of both neutral pH and high calcium concentration than under the conditions of both acidic pH and low calcium concentration.

[0051] In certain embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at high calcium concentration 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 certain embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at high calcium concentration at both neutral and acidic pH, the ratio of the KD value of its C1r binding activity at acidic pH to the KD value of its C1r binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater.

[0052] In some embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at low calcium concentration 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 the dimeric state of C1s. In some embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at low calcium concentration at both neutral and acidic pH, the ratio of the KD value of its C1r binding activity at acidic pH to the KD value of its C1r binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to the dimeric state of C1r.

[0053] In certain embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at high calcium concentration at neutral pH and at low calcium concentration 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 2 or greater. In certain embodiments, in the isolated antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex, when measured at high calcium concentration at neutral pH and at low calcium concentration at acidic pH, the ratio of the KD value of its C1r binding activity at acidic pH to the KD value of its C1r binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater.

[0054] In some embodiments, the anti-C1s antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complex comprise histidine residues at one or more of the following Kabat numbering system positions:

[0055] Heavy chains: 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

[0056] Light chains: 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.

[0057] In some embodiments, the anti-C1r antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complexes comprise histidine residues at one or more of the following Kabat numbering system positions:

[0058] Heavy chains: 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

[0059] Light chains: 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.

[0060] In some embodiments, the anti-C1s antibodies of the invention that inhibit the interaction between C1q and C1r2s2 complexes comprise at least one histidine substituted at one or more of the following Kabat numbering system positions:

[0061] Heavy chains: 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

[0062] Light chains: 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.

[0063] In some embodiments, the anti-C1r antibody that inhibits the interaction between C1q and the C1r2s2 complex of the present invention comprises at least one histidine substituted at one or more of the following Kabat numbering system positions:

[0064] Heavy chains: 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

[0065] Light chains: 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.

[0066] In a further embodiment, the pH-dependent anti-C1s antibody that inhibits the interaction between C1q and the C1r2s2 complex of the present invention competes for binding to C1s at neutral pH with an antibody selected from the following 1)-5):

[0067] 1) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37,

[0068] 2) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43,

[0069] 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49,

[0070] 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55,

[0071] 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61,

[0072] In a further embodiment, the pH-dependent anti-C1r antibody of the invention that inhibits the interaction between C1q and the C1r2s2 complex competes with the antibodies selected from 6)-13) below for binding to C1r under neutral pH conditions:

[0073] 6) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159,

[0074] 7) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160,

[0075] 8) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161,

[0076] 9) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162,

[0077] 10) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 123, the HVR-H2 sequence of SEQ ID NO: 131, the HVR-H3 sequence of SEQ ID NO: 139, the HVR-L1 sequence of SEQ ID NO: 147, the HVR-L2 sequence of SEQ ID NO: 155, and the HVR-L3 sequence of SEQ ID NO: 163,

[0078] 11) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 124, the HVR-H2 sequence of SEQ ID NO: 132, the HVR-H3 sequence of SEQ ID NO: 140, the HVR-L1 sequence of SEQ ID NO: 148, the HVR-L2 sequence of SEQ ID NO: 156, and the HVR-L3 sequence of SEQ ID NO: 164,

[0079] 12) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 125, the HVR-H2 sequence of SEQ ID NO: 133, the HVR-H3 sequence of SEQ ID NO: 141, the HVR-L1 sequence of SEQ ID NO: 149, the HVR-L2 sequence of SEQ ID NO: 157, and the HVR-L3 sequence of SEQ ID NO: 165, and

[0080] 13) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 126, the HVR-H2 sequence of SEQ ID NO: 134, the HVR-H3 sequence of SEQ ID NO: 142, the HVR-L1 sequence of SEQ ID NO: 150, the HVR-L2 sequence of SEQ ID NO: 158, and the HVR-L3 sequence of SEQ ID NO: 166.

[0081] In some embodiments, the present disclosure provides isolated anti-C1s antibodies that specifically bind to an epitope within a region encompassing the CUB1-EGF-CUB2 domain (also referred to as the interaction domain or CUB domain), which is composed of CUB1, EGF, and CUB2 of complement component 1s (C1s), and is also referred to herein as the "CUB1-EGF-CUB2 domain of C1s". In some embodiments, the antibody does not bind to the CCP1-CCP2-SP domain (also referred to as the catalytic domain, or CCP-SP domain) of C1s. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is an epitope that is not located in the beta domain of C1s. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is an epitope located in the alpha domain or the gamma domain of C1s. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is a linear epitope. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present invention is an epitope within the following amino acids: amino acids 16-291 of complement C1s protein, amino acids 16-172 of complement C1s protein, amino acids 16-210 of complement C1s protein, amino acids 16-111 of complement C1s protein, amino acids 112-210 of complement C1s protein, amino acids 131-172 of complement C1s protein, or amino acids 16-130 of complement C1s protein. In some embodiments, the above epitopes of C1s are epitopes of human C1s, or preferably epitopes of human C1s and cynomolgus monkey C1s.

[0082] In some embodiments, the present disclosure provides isolated anti-C1r antibodies that specifically bind to an epitope within a region encompassing the CUB1-EGF-CUB2 domain, which is composed of CUB1, EGF, and CUB2 of complement component 1r (C1r), and is also referred to herein as the "CUB1-EGF-CUB2 domain of C1r". In some embodiments, the antibody does not bind to the CCP1-CCP2-SP domain (also referred to as the catalytic domain) of C1r. In some cases, the epitope bound by the isolated anti-C1r antibody of the present disclosure is a linear epitope or a conformational epitope. In some embodiments, the above epitopes of C1r are epitopes of human C1r, or preferably epitopes of human C1r and cynomolgus monkey C1r.

[0083] In some embodiments, the isolated anti-C1s antibody of the invention comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58, wherein the antibody comprises framework regions of human or primate origin. In some embodiments, the isolated anti-C1s antibody of the invention comprises (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61, wherein the antibody comprises framework regions of human or primate origin.

[0084] In some embodiments, the isolated anti-C1r antibody of the invention comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142, wherein the antibody comprises framework regions of human or primate origin. In some embodiments, the isolated anti-C1r antibody of the invention comprises (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166, wherein the antibody comprises framework regions of human or primate origin.

[0085] In some embodiments, the anti-C1s antibody of the invention comprises (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 19, 20, 21, 23, or 24; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 26, 27, 28, 30, or 31; or (c) the VH sequence of (a) and the VL sequence of (b). In some embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24. In some embodiments, the anti-C1s antibody of the invention comprises the VL sequence of SEQ ID NO: 26, 27, 28, 30, or 31. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24 and the VL sequence of SEQ ID NO: 26, 27, 28, 30, or 31. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 19 and the VL sequence of SEQ ID NO: 26. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 27. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 21 and the VL sequence of SEQ ID NO: 28. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 30. In further embodiments, the anti-C1s antibody of the invention comprises the VH sequence of SEQ ID NO: 24 and the VL sequence of SEQ ID NO: 31.

[0086] In some embodiments, the anti-C1r antibodies of the invention comprise (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118; or (c) the VH sequence of (a) and the VL sequence of (b). In some embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110. In some embodiments, the anti-C1r antibodies of the invention comprise the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110 and the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 103 and the VL sequence of SEQ ID NO: 111. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 104 and the VL sequence of SEQ ID NO: 112. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 105 and the VL sequence of SEQ ID NO: 113. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 106 and the VL sequence of SEQ ID NO: 114. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 107 and the VL sequence of SEQ ID NO: 115. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 108 and the VL sequence of SEQ ID NO: 116. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 109 and the VL sequence of SEQ ID NO: 117. In further embodiments, the anti-C1r antibodies of the invention comprise the VH sequence of SEQ ID NO: 110 and the VL sequence of SEQ ID NO: 118.

[0087] In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a monoclonal antibody. In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In further embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody fragment that binds to C1s. In some specific embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is human IgG1 or humanized IgG1.

[0088] In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a monoclonal antibody. In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In further embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody fragment that binds to C1r. In some specific embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is human IgG1 or humanized IgG1.

[0089] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody comprising an Fc region having at least one amino acid modification in this region, thereby enhancing the reduction of plasma antigen concentration and / or improving the pharmacokinetics of the antibody.

[0090] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex has a human Fc region having binding activity selected from the group consisting of:

[0091] a) Binding activity to activated Fc gamma receptors that is stronger than the binding activity of the Fc region of natural human IgG1,

[0092] b) Binding activity to inhibitory Fc gamma receptors that is stronger than the binding activity to activated Fc gamma receptors, and

[0093] c) has a stronger binding activity to FcRn at neutral pH than the Fc region of natural human IgG1.

[0094] In some embodiments, the isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex binds to at least human C1s or preferably binds to both cynomolgus monkey C1s and human C1s. In some embodiments, the isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex binds to at least human C1r or preferably binds to both cynomolgus monkey C1r and human C1r.

[0095] The present invention also provides an isolated nucleic acid encoding the anti-C1s antibody of the present invention. The present invention also provides an isolated nucleic acid encoding the anti-C1r antibody of the present invention. The present invention also provides a host cell comprising the nucleic acid of the present invention. The present invention also provides a method for producing an antibody, comprising culturing the host cell of the present invention to produce the antibody.

[0096] The present invention also provides a pharmaceutical preparation comprising the antibody of the present invention and a pharmaceutically acceptable carrier thereof.

[0097] The anti-C1s antibody of the present invention can be used as a drug. The anti-C1s antibody of the present invention can be used for treating or preventing complement-mediated diseases or disorders. The anti-C1s antibody of the present invention can be used for enhancing the clearance (or removal) of C1s from plasma. The anti-C1s antibody of the present invention can be used for enhancing the clearance (or removal) of C1r2s2 from plasma. The anti-C1s antibody of the present invention can be used for enhancing the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In some cases, the antibody inhibits components of the classical complement pathway; in some cases, the classical complement pathway component is C1s.

[0098] The anti-C1r antibody of the present invention can be used as a drug. The anti-C1r antibody of the present invention can be used for treating or preventing complement-mediated diseases or disorders. The anti-C1r antibody of the present invention can be used for enhancing the clearance (or removal) of C1r from plasma. The anti-C1r antibody of the present invention can be used for enhancing the clearance (or removal) of C1r2s2 from plasma. The anti-C1r antibody of the present invention can be used for enhancing the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In some cases, the antibody inhibits components of the classical complement pathway; in some cases, the classical complement pathway component is C1r.

[0099] The anti-Cls antibody of the present invention can be used to prepare a drug. In some embodiments, the drug is used for treating or preventing a complement-mediated disease or disorder. In some embodiments, the drug is used to enhance the clearance (or removal) of Cls from plasma. In some embodiments, the drug is used to enhance the clearance (or removal) of C1r2s2 from plasma. In some embodiments, the drug is used to enhance the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In this case, the enhanced level of C1q clearance from plasma is not necessarily nil (zero). That is to say, the enhanced level of C1q clearance from plasma can be zero, or can be non-zero but close to zero, or can be insignificant or low enough to be technically ignored by those skilled in the art. In certain cases, the drug inhibits components of the classical complement pathway; in certain cases, the component of the classical complement pathway is Cls.

[0100] For example, the enhancement of C1s / C1q clearance rate (CL) can be measured as follows.

[0101] The total concentrations of human C1s and C1q in mouse plasma were measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting human C1s and C1q in defined amounts in mouse plasma to obtain human C1s concentrations of 0.477, 0.954, 1.91, 3.82, 7.64, 15.3, 30.5 micrograms (μg) / mL and human C1q concentrations of 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, and 62.5 μg / mL, respectively. 2 μL of the calibration standards and plasma samples were mixed with 25 μL of 6.8 mol / L urea, 9.1 mmol / L dithiothreitol, and 0.4 μg / mL lysozyme (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 any remaining trypsin. 40 μL of the digested samples were analyzed by LC / ESI-MS / MS. LC / ESI-MS / MS was performed using an Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D class I UPLC (Waters). The human C1s-specific peptide LLEVPEGR and the human C1q-specific peptide IAFSATR were monitored by selected reaction monitoring (SRM). For human C1s, the SRM transition was [M+2H]2+ (m / z 456.8) to the y6 ion (m / z 686.3), and for human C1q, the SRM transition was [M+2H]2+ (m / z 383.2) to the y5 ion (m / z 581.3). The calibration curve was constructed by weighted (1 / x2) linear regression using the peak areas plotted against the concentrations. The concentrations in mouse plasma were calculated according to the calibration curve using the analytical software Masslynx Ver. 4.1 (Waters).

[0102] The pharmacokinetics of total hC1s and hC1q were evaluated in mice after administration of the anti-C1s antibody as follows.

[0103] The in vivo pharmacokinetics of hC1s, hC1q, and anti-C1s antibody were evaluated after administration of antigen (hC1q, recombinant C1r2s2, and a mixture of hC1q and rC1r2s2) alone or in combination with anti-C1s antibody to mice (CB17 / Icr-Prkdcscid / CrlCrl: Charles River Japan). Three mice were assigned to each treatment group.

[0104] First, a solution of hC1q (0.84 mg / mL), rC1r2s2 (0.47 mg / mL), or a mixture solution containing 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 individual in the same manner.

[0105] The dose settings of C1q and rC1r2s2 were designed to be the physiological concentrations in human plasma immediately after administration. During the study, the dose of anti-C1s antibody was adjusted to exceed the concentrations of the two antigens, and thus, it was assumed that almost all hC1s in the circulation were in the bound form.

[0106] Blood was collected at 5 and 30 minutes, 2 and 7 hours, 3, 7, 14, 21, and 28 days after injection. The blood was immediately centrifuged to separate plasma samples. The plasma concentrations of hC1s and hC1q were measured at each sampling point by LC / ESI-MS / MS. The PK parameters of hC1s and hC1q were estimated by non-compartmental analysis (Phoenix WinNonlin version 8.0, Certara).

[0107] Mice were administered antibodies having: (i) an Fc containing mutations to reduce C1q and Fc gamma receptor binding, or (ii) an Fc containing mutations to reduce C1q binding while maintaining Fc gamma receptor binding. For example, in the present invention, the Fc of "SG136" contains mutations that reduce C1q and Fc gamma receptor binding, while the Fc of "SG1148" contains mutations that reduce C1q binding while maintaining Fc gamma receptor binding.

[0108] The above-described murine PK study is conducted on a test antibody (such as a CCP1-CCP2-SP or CUB1-EGF-CUB2 binder), and the PK parameters of hC1q and hC1s are calculated. Then, the C1s CL ratio (SG1148 / SG136) of the binder or the C1q CL ratio (SG1148 / SG136) of the binder can be evaluated. In some embodiments, the C1q CL ratio of the antibody of the present invention is 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, or 1.0 or less.

[0109] The anti-Clr antibody of the present invention can be used to prepare a medicament. In some embodiments, the medicament is used for treating or preventing a complement-mediated disease or disorder. In some embodiments, the medicament is used for enhancing the clearance (or removal) of C1r from plasma. In some embodiments, the medicament is used for enhancing the clearance (or removal) of C1r2s2 from plasma. In some embodiments, the medicament is used for enhancing the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In certain cases, the medicament inhibits a component of the classical complement pathway; in certain cases, the classical complement pathway component is Clr.

[0110] The present invention also provides a method for treating or preventing an individual suffering from a complement-mediated disease or disorder. In some embodiments, the method comprises administering to the individual an effective amount of the anti-C1s antibody of the present invention. The present invention also provides a method for enhancing the clearance (or removal) of C1s from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of the anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1s from plasma. The present invention also provides a method for enhancing the clearance (or removal) of C1r2s2 from plasma in an individual. The present invention also provides a method for enhancing the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of the anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma. In some embodiments, the method comprises administering to the individual an effective amount of the anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In certain cases, the antibody inhibits a component of the classical complement pathway; in certain cases, the classical complement pathway component is Cls.

[0111] The present invention also provides methods of treating or preventing an individual having a complement-mediated disease or disorder. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1r antibody of the present invention. The present invention also provides methods of enhancing the clearance (or removal) of C1r from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1r antibody of the present invention to enhance the clearance (or removal) of C1r from plasma. The present invention also provides methods of enhancing the clearance (or removal) of C1r2s2 from plasma in an individual. The present invention also provides methods of enhancing the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1r antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1r antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma rather than C1q from plasma. In certain cases, the antibody inhibits components of the classical complement pathway; in certain cases, the classical complement pathway component is C1r.

[0112] More specifically, the present invention provides the following:

[0113] [1] An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has a displacing function such that the antibody binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex.

[0114] [2] The antibody of [1], wherein the antibody binds to the C1qrs complex on a Biacore chip and promotes the dissociation of C1q from the C1qrs complex, wherein, after sufficient time, as determined by Biacore assay, the value of the response unit (RU) in the presence of the antibody is lower than the value of the response unit (RU) in the absence of the antibody.

[0115] [3] The antibody of [2], wherein the cross time point in the Biacore assay is within 60 s, 100 s, 150 s, 200 s, 500 s, 700 s, or 1000 s after the time point of the start of antibody injection, as determined by Biacore assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.

[0116] [4] The antibody according to [2], wherein almost all of the C1q dissociates from the C1qrs complex within 100 s, 300 s, 500 s, 700 s, 1000 s, 1500 s, or 2000 s after the time point of the start of antibody injection, as determined by Biacore assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.

[0117] [5] An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has at least 70% neutralizing activity against human serum complement in the RBC assay.

[0118] [6] The antibody according to any one of [1] to [5], wherein the antibody is an antibody that specifically binds to C1s or an antibody that specifically binds to C1r.

[0119] [7] An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex,

[0120] wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s and competes for binding to the epitope with an antibody selected from the group consisting of the following 1)-5):

[0121] 1) An antibody that comprises the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37,

[0122] 2) An antibody that comprises the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43,

[0123] 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49,

[0124] 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55, and

[0125] 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61, or

[0126] wherein said antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r and competes for binding to said epitope with an antibody selected from the group consisting of 6)-13):

[0127] 6) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159,

[0128] 7) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160,

[0129] 8) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161,

[0130] 9) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162,

[0131] 10) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 123, the HVR-H2 sequence of SEQ ID NO: 131, the HVR-H3 sequence of SEQ ID NO: 139, the HVR-L1 sequence of SEQ ID NO: 147, the HVR-L2 sequence of SEQ ID NO: 155, and the HVR-L3 sequence of SEQ ID NO: 163,

[0132] 11) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 124, the HVR-H2 sequence of SEQ ID NO: 132, the HVR-H3 sequence of SEQ ID NO: 140, the HVR-L1 sequence of SEQ ID NO: 148, the HVR-L2 sequence of SEQ ID NO: 156, and the HVR-L3 sequence of SEQ ID NO: 164,

[0133] 12) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 125, the HVR-H2 sequence of SEQ ID NO: 133, the HVR-H3 sequence of SEQ ID NO: 141, the HVR-L1 sequence of SEQ ID NO: 149, the HVR-L2 sequence of SEQ ID NO: 157, and the HVR-L3 sequence of SEQ ID NO: 165, and

[0134] 13) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 126, an HVR-H2 sequence of SEQ ID NO: 134, an HVR-H3 sequence of SEQ ID NO: 142, an HVR-L1 sequence of SEQ ID NO: 150, an HVR-L2 sequence of SEQ ID NO: 158, and an HVR-L3 sequence of SEQ ID NO: 166.

[0135] [8] An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.4.

[0136] [9] The antibody according to any one of [1] to [8], wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s or C1r, and wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.4.

[0137]

[10] The antibody according to [9], wherein the affinity of the antibody for binding to C1s or C1r at acidic pH is lower than its affinity for binding to C1s or C1r at neutral pH, as described in (i) or (ii) below:

[0138] (i) When measured at high calcium concentration 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 2 or greater.

[0139] (ii) When measured at high calcium concentration at neutral pH and at low calcium concentration 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.

[0140]

[11] The antibody according to any one of [8] to

[10] , wherein the antibody comprises an Fc region having at least one amino acid modification within the region, thereby enhancing the reduction of plasma antigen concentration and / or improving the pharmacokinetics of the antibody.

[0141]

[12] The antibody according to

[11] , wherein the Fc region is a human Fc region having binding activity selected from the group consisting of:

[0142] a) Binding activity to activated Fc gamma receptors that is stronger than the binding activity of the Fc region of natural human IgG1.

[0143] b) It has a stronger binding activity to inhibitory Fc gamma receptors than to activating Fc gamma receptors, and

[0144] c) It has a stronger binding activity to FcRn at neutral pH than the Fc region of natural human IgG1.

[0145]

[13] The antibody according to any one of [1] to

[12] , wherein the antibody binds to both cynomolgus monkey C1s and human C1s, or to both cynomolgus monkey C1r and human C1r.

[0146]

[14] A pharmaceutical composition comprising the antibody according to any one of [1] to

[13] and a pharmaceutically acceptable carrier.

[0147]

[15] A method of treating an individual suffering from a complement-mediated disease or disorder, comprising administering to the individual a therapeutically effective amount of the antibody according to any one of [1] to

[13] . Description of the Drawings

[0148] Figure 1A Figure 1A Shows the binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1s protein. BIACORE (registered trademark) sensorgram of the anti-C1s antibody against the recombinant human C1s CCP1-CCP2-SP-His protein.

[0149] Figure 1B Figure 1B Shows the binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1s protein. BIACORE (registered trademark) sensorgram of the anti-C1s antibody against the native zymogen human C1s protein.

[0150] Figure 2A Figure 2A Shows antibody-mediated displacement of native human C1q from the recombinant human C1r2s2 Flag / His tetramer immobilized on the surface of a BIACORE (registered trademark) sensor. Antibody displacement of native human C1q is described by overwriting 3 sensorgrams. Sensorgram 1 (small dashed line) describes the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) describes the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. The sensor Figure 3 (solid line) describes the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. To compare these sensorgrams, at Figure 2A time 0 the RU is normalized (i.e., set to the same).

[0151] Figure 2B ​​​​​Figure 2B Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on the surface of a BIACORE (registered trademark) sensor. The antibody displacement of native human C1q is described by overwriting three sensorgrams. Sensorgram 1 (small dashed line) describes the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) describes the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. The sensor Figure 3 (solid line) describes the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. To compare these sensorgrams, at Figure 2B the RU at time 0 is normalized (i.e., set to the same).

[0152] Figure 2C Figure 2C Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on the surface of a BIACORE (registered trademark) sensor. The antibody displacement of native human C1q is described by overwriting three sensorgrams. Sensorgram 1 (small dashed line) describes the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) describes the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. To compare these sensorgrams, at Figure 2C the RU at the Ab injection is normalized (i.e., set to the same).

[0153] Figure 2D Figure 2D Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on the surface of a BIACORE (registered trademark) sensor. The antibody displacement of native human C1q is described by overwriting three sensorgrams. Sensorgram 1 (small dashed line) describes the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) describes the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. To compare these sensorgrams, at Figure 2D the RU at the Ab injection is normalized (i.e., set to the same).

[0154] Figure 3 Figure 3 ​​​Shows the replacement of the antibody-mediated recombinant human C1r2s2 Flag / His tetramer from biotinylated native human C1q, which has been immobilized on the BIACORE® sensor surface. The recombinant human C1r2s2 Flag / His tetramer was flowed through to bind to the immobilized native human C1q, and then the dissociation rate of C1r2s2 was monitored by flowing through a separate buffer (solid line), or C1r2s2 was dissociated by flowing through an antibody (dashed line).

[0155] Figure 4 Figure 4 Shows antibody-mediated blockade of the binding of native human C1q to the recombinant human C1r2s2 Flag / His tetramer. Antibodies with C1q-blocking function compete with C1q for binding to C1r2s2.

[0156] Figure 5 Figure 5 Shows the neutralization of human serum complement activity.

[0157] Figure 6 Figure 6 Shows the competitive epitope binning results of antibodies that bind to the CUB1-EGF-CUB2 domain of C1s.

[0158] Figure 7 Figure 7 Shows the pharmacokinetics of human C1s and human C1q after administration of anti-C1s antibody in mice.

[0159] Figure 8 Figure 8 Shows the time-dependent neutralization of human serum complement activity by anti-C1s antibody.

[0160] Figure 9 Figure 9 Shows antibodies that bind to native human zymogen C1s in reducing and non-reducing Western blot analyses.

[0161] Figure 10 Figure 10 Shows antibody binding to truncated C1s protein in a reducing Western blot.

[0162] Figure 11A Figure 11A Shows the binding specificity of an antibody to the CUB1-EGF-CUB2 domain of the C1r protein. BIACORE® sensorgram of an anti-C1r antibody against recombinant human C1r CCP1-CCP2-SP-FLAG protein.

[0163] ​​​​​​​​​Figure 11B Figure 11B Shows the binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1r protein. BIACORE® sensorgram of an anti-C1r antibody against native human C1r enzyme.

[0164] Figure 12A Figure 12A Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer captured on the surface of a BIACORE® sensor. Antibody displacement of native human C1q is depicted by overlaying three sensorgrams. Sensorgram 1 (small dashed line) depicts the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) depicts the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. The Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. To compare these sensorgrams, at Figure 12A time 0 the RUs are normalized (i.e., set to be the same).

[0165] Figure 12B Figure 12B Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer captured on the surface of a BIACORE® sensor. Antibody displacement of native human C1q is depicted by overlaying three sensorgrams. Sensorgram 1 (small dashed line) depicts the stable capture of C1qrs on the sensor surface. Sensorgram 2 (large dashed line) depicts the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. The Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. To compare these sensorgrams, at Figure 12B time 0 the RUs are normalized (i.e., set to be the same).

[0166] Figure 12C Figure 12C Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer captured on the surface of a BIACORE® sensor. Antibody displacement of native human C1q is depicted by overlaying two sensorgrams. Sensorgram 1 (solid line) depicts the stable capture of C1qrs on the sensor surface. Sensorgram 2 (dashed line) depicts the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. To compare these sensorgrams, at Figure 12C the RU at the point of Ab injection is normalized (i.e., set to be the same).

[0167] ​​​​Figure 12D Figure 12D Shows the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on the surface of a BIACORE® sensor. Antibody displacement of native human C1q is depicted by overlaying two sensorgrams. Sensorgram 1 (solid line) describes the stable capture of C1qrs on the sensor surface. Sensorgram 2 (dashed line) describes the binding of the antibody to C1qrs and the displacement of C1q from C1r2s2. To compare these sensorgrams, the RU at the point of Ab injection was normalized (i.e., set to be the same) in Figure 12D .

[0168] Figure 13 Figure 13 Shows the neutralization of human serum complement activity. Detailed Description

[0169] Description of Embodiments

[0170] ​The technologies and methods described or cited herein are generally well understood and routinely used by those skilled in the art using conventional methodologies, such as, for example, the widely used methods described below: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (ed. by F.M. Ausubel et al., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (ed. by M.J. MacPherson, B.D. Hames and G.R. Taylor. (1995)), Antibodies, A Laboratory Manual, ed. by Harlow and Lane (1988), and Animal Cell Culture (ed. by R.I. Freshney (1987)); Oligonucleotide Synthesis (ed. by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (ed. by R.I. Freshney) 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (ed. by A. Doyle, J.B. Griffiths and D.G. Newell, 1993 - 8) J. Wiley and Sons; Handbook of Experimental Immunology (ed. by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M.Edited by Miller and M.P. Calos, 1987); PCR: The Polymerase Chain Reaction, (Edited by Mullis et al., 1994); Current Protocols in Immunology (Edited by J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (Edited by D. Catty., IRL Press, 1988 - 1989); Monoclonal Antibodies: A Practical Approach (Edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (Edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (Edited by V.T. DeVita et al., J.B. Lippincott Company, 1993).

[0171] I. Definitions

[0172] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992) provide one of ordinary skill in the art with a general guide to many of the terms used in this application. All documents cited herein (including patent applications and publications) are incorporated by reference in their entirety.

[0173] For the purposes of interpreting this application, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the techniques used herein are only for the purpose of describing particular embodiments and are not intended to be limiting. If any definition given below conflicts with any document incorporated by reference herein, the definition given below shall control.

[0174] "Acceptor human framework" for purposes herein is a framework that comprises an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that is derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework that is "derived from" a human immunoglobulin framework or a human consensus framework may comprise the identical amino acid sequence thereof, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0175] "Affinity" refers to the sum of the strengths of 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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the 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. "Affinity", "binding affinity", "binding capacity", and "binding activity" can be used interchangeably. The term "binding activity" refers to the sum of the strengths of non-covalent interactions between a single or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In the present context, binding activity is not strictly limited to the activity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). When the members of a binding pair can bind to each other in a monovalent and multivalent binding manner, the binding activity is the sum of the strengths of these bindings. The binding activity of molecule X for its partner Y can generally be expressed by the dissociation constant (KD). Alternatively, the association and dissociation rates (Kon and Koff) can be used for the evaluation of binding. Binding activity can be measured by conventional methods known in the art, including those described herein. The following describes specific illustrative and exemplary embodiments for measuring binding affinity.

[0176] An "affinity matured" antibody is an antibody that has one or more alterations in one or more hypervariable regions (HVRs) (compared to a parental antibody that does not have such alterations), which result in an increase in the affinity of the antibody for an antigen.

[0177] The terms "anti-C1s antibody" and "C1s-binding antibody" refer to an antibody that is capable of binding C1s with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent for targeting C1s. In one embodiment, the anti-C1s antibody binds to an unrelated, non-C1s protein to an extent less than about 10% of the binding of the antibody to C1s, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the C1s-binding antibody is 1 (micromolar) 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 to 10 -13 M, e.g., 10 -9 M to 10 -13(M). In certain embodiments, the anti-C1s antibody binds to an epitope of C1s that is conserved among C1s from different species.

[0178] The terms “anti-C1r antibody” and “antibody that binds to C1r” refer to an antibody that is capable of binding to C1r with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent for targeting C1r. In one embodiment, the anti-C1r antibody binds to an unrelated, non-C1r protein to an extent less than about 10% of the binding of the antibody to C1r, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C1r has a dissociation constant (Kd) of: 1 (micromolar) 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 μM or less, e.g., 10 -8 μM to 10 -13 μM, e.g., 10 -9 μM to 10 -13 μM). In certain embodiments, the anti-C1r antibody binds to an epitope of C1r that is conserved among C1r from different species.

[0179] The term “antibody” is used herein in the broadest sense and encompasses various 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.

[0180] “Antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds antigen. 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.

[0181] 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 antigen by 50% or more in a competition assay, whereas conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0182] 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.

[0183] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different types of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0184] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as ribonucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed hereinafter.

[0185] "Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with 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; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0186] An "effective amount" of a reagent (e.g., a pharmaceutical formulation) is an amount effective for achieving the desired therapeutic or prophylactic result for a given dose and time.

[0187] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is the region of an antigen that is bound by an antibody that targets the antigen and includes specific amino acids that directly contact the antibody. Epitope determinants can include clusters of chemically reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features, and / or specific charge features. Generally, an antibody specific for a particular target antigen will preferentially recognize the epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0188] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446 - 447) of the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residue numbering in the Fc region or constant region is according to the EU numbering system, which is also known 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.

[0189] "Framework" or "FR" refers to variable domain residues that are different from the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences in VH (or VL) generally occur in the following order: FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4.

[0190] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody that has a structure substantially similar to a native antibody structure or has a heavy chain that contains an Fc region as defined herein.

[0191] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to that of the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

[0192] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or an amino acid sequence of an antibody of non-human origin derived from a human antibody library or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0193] A "human consensus framework" is a framework that represents the most common amino acid residues in the 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 such as those in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κ I as in Kabat et al. above. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. above.

[0194] A "humanized" antibody is an antibody that refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will contain substantially all of at least one (and typically, two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may contain at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0195] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain in which the sequence is hypervariable ("complementary determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0196] (a) Hypervariable loops that occur 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));

[0197] (b) CDRs that occur 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));

[0198] (c) Antigen - contact sites that occur 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

[0199] (d) Combinations of (a), (b) and / or (c), including 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).

[0200] Unless otherwise indicated, herein, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al. above.

[0201] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecules including but not limited to a cytotoxic agent.

[0202] An “individual” or “subject” is a mammal. Mammals include but are not limited to domestic animals (e.g., cows, sheep, cats, dogs, and 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 a human.

[0203] A "separated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, 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, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0204] A "separated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Separated nucleic acids include nucleic acid molecules that are contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0205] A "separated nucleic acid encoding an anti-C1s antibody" or "a separated nucleic acid encoding an anti-C1r antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules at one or more locations in a host cell.

[0206] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homologous, i.e., the individual antibodies that make up the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the preparation of the monoclonal antibody preparation, such variants being present usually in small amounts. In contrast to polyclonal antibody preparations (which typically include different antibodies directed against different determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the adjective "monoclonal" indicates the nature of the antibody as being obtained from a substantially homologous population of antibodies and is not to be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used according to the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0207] 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 can be present in a pharmaceutical formulation.

[0208] "Natural antibody" refers to immunoglobulin molecules that naturally occur and have multiple structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), which is also referred to as the variable heavy chain 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), which is also referred to as the variable light chain domain or light chain variable domain, followed by the light chain constant (CL) domain. The light chains of antibodies can be assigned to one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0209] The term "package insert" is used to refer to the instructions for use that are typically included in the commercial packaging of a therapeutic product and that contain information about the indications, uses, dosage, administration, combination therapies, contraindications, and / or warnings for the use of such therapeutic product.

[0210] "Percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after the sequences are aligned and gaps are introduced, if necessary, to achieve maximum percentage sequence identity, with no conservative substitutions being considered part of the sequence identity. The alignment for determining percentage amino acid sequence identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0211] The author of the ALIGN-2 sequence comparison computer program is Genentech, Inc., and the source code has been deposited with the United States Copyright Office, Washington D.C., 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program may be compiled from the source code. The ALIGN-2 program should be compiled for use with the UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not need to be changed. In the case of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to or vice versa a given amino acid sequence B (which may alternatively be stated as a given amino acid sequence A has or contains a particular % amino acid sequence identity relative to or vice versa 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 the program alignment of A and B, and Y is the total number of amino acid residues in B. It is to be understood that when 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 be equal to the % amino acid sequence identity of B to A. Unless otherwise expressly indicated, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0212] The term "pharmaceutical composition" refers to a composition which has a form that permits the biological activity of the active ingredient contained therein to be effective and which contains no other components that are unacceptably toxic to the subject to which the composition is to be administered.

[0213] "Pharmaceutically acceptable carrier" refers to components other than the active ingredient in a pharmaceutical composition which are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0214] As used herein, the phrase "specifically binds" refers to the activity or property of an antibody to bind a non-target antigen at a binding level that includes background (i.e., non-specific) binding but does not include significant (i.e., specific) binding. In other words, "specifically binds" refers to the activity or property of an antibody to bind a target antigen at a binding level that includes significant (i.e., specific) binding in addition to or in place of background (i.e., non-specific) binding. Specificity can be measured by any method mentioned in this specification or known in the art. The above level of non-specific or background binding can be zero, or it can be non-zero but close to zero, or it can be low enough to be technically ignored by those skilled in the art. For example, when a skilled person cannot detect or observe any significant (or relatively strong) signal of binding between an antibody and a non-target antigen in a suitable binding assay, it can be said that the antibody "does not specifically bind" the non-target antigen. In contrast, when a skilled person can detect or observe any significant (or relatively strong) signal of binding between an antibody and a target antigen in a suitable binding assay, it can be said that the antibody "specifically binds" the target antigen.

[0215] Unless otherwise specified, the term "C1s" as used herein refers to any native C1s from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (such as mice and rats). The term encompasses "full-length" unprocessed C1s and any form of C1s derived from processing in cells. The term also encompasses naturally occurring variants of C1s, such as splice variants or allelic variants. The amino acid sequence of exemplary human C1s is shown in SEQ ID NO: 1. The amino acid sequences of exemplary cynomolgus monkey and rat C1s are shown in SEQ ID No: 3 and 2, respectively.

[0216] Unless otherwise specified, the term "C1r" as used herein refers to any native C1r from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (such as mice and rats). The term encompasses "full-length" unprocessed C1r and any form of C1r derived from processing in cells. The term also encompasses naturally occurring variants of C1r, such as splice variants or allelic variants. The amino acid sequence of exemplary human C1r is shown in SEQ ID NO: 4. The amino acid sequences of exemplary cynomolgus monkey and rat C1r are shown in SEQ ID No: 5 and 6, respectively.

[0217] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of an individual being treated and can be performed for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving the prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

[0218] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antigen binding by the antibody. The heavy and light chain variable domains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6 th th ed., W.H. Freeman & Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using, respectively, the VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0219] As used herein, the term "vector" refers to a nucleic acid molecule capable of causing the proliferation of another nucleic acid molecule to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0220] II. Compositions and Methods

[0221] In one aspect, the invention is based in part on antibodies that inhibit the interaction between C1q and the C1r2s2 complex and their use. In certain embodiments, antibodies that bind C1s are provided. In certain embodiments, antibodies that bind C1r are provided. The antibodies of the invention can be used, for example, to diagnose or treat complement-mediated diseases or disorders.

[0222] A. Exemplary Anti-Complement Component Antibodies

[0223] In one aspect, the present invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex. In one aspect, the present invention provides an isolated antibody having a replacement function that enables the antibody to bind to the C1qrs complex and promote the dissociation of C1q from the C1qrs complex. 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, and whose binding activity varies depending on the ion concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on pH, i.e., the hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on the calcium concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on both pH and calcium concentration. In another aspect, the present invention provides an isolated antibody that binds to C1r. In one aspect, the present invention provides an isolated antibody that binds to C1r, and whose binding activity varies depending on the ion concentration. In certain embodiments, the binding activity of the anti-C1r antibody varies depending on pH, i.e., the hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1r antibody varies depending on the calcium concentration. In certain embodiments, the binding activity of the anti-C1r antibody varies depending on both pH and calcium concentration.

[0224] Throughout the "Description of Embodiments" section, the term "C1s" may be replaced with "C1r", except for descriptions related to sequences specific to anti-C1s antibodies and sequences and domains specific to the C1s protein.

[0225] Such antibodies are expected to be particularly superior as drugs because the dosage and frequency of administration to patients can be reduced, and as a result, the total dose can be decreased. Compared with antibodies that bind to the C1qrs complex from plasma and remove the C1qrs complex from plasma, anti-C1s antibodies are expected to have higher safety because they only remove C1r2s2 (by binding to C1s) from plasma and do not remove C1q from plasma. As a result, side effects associated with Clq depletion can be avoided. Additionally, antibodies with rapid replacement of C1q are expected to have faster neutralizing complement activity, which can translate into faster therapeutic efficacy.

[0226] (BIACORE (registered trademark) / Replacement concept)

[0227] In one aspect, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to the C1qrs complex on a chip for surface plasmon resonance assay (e.g., BIACORE (registered trademark) chip) and promotes the dissociation of C1q from the C1qrs complex. In some aspects, the functions of binding to the C1qrs complex and promoting the dissociation of C1q from the C1qrs complex mentioned above are referred to herein as "displacement function / activity" or "C1q displacement function / activity". This function / activity can be suitably evaluated qualitatively or quantitatively using surface plasmon resonance assay, such as the BIACORE (registered trademark) assay described herein. In other aspects, when after sufficient time, the value of the response unit (RU) in the presence of the antibody is lower than the value of the response unit (RU) in the absence of the antibody, as determined by surface plasmon resonance assay (e.g., BIACORE (registered trademark) assay), the antibody of the present invention can be determined to be an antibody with displacement function. In the sensorgram obtained by such an assay, a "crossing time point" can be identified, where the curve in the presence of C1q without the antibody intersects with the curve in the presence of C1q and the antibody at this "crossing time point" (see Examples for details). Strictly speaking, even in a single sensorgram, multiple crossing time points may be observed due to the noise or oscillation of the latter curve when crossing the former curve. In this case, any one of the multiple crossing time points can be selected as the "crossing time point". "After sufficient time" means that the measurement time point of the value of the response unit (RU) is sufficient for the purpose of measurement after the "crossing time point". In some embodiments, the measurement time point of the value of the response unit (RU) is at least 60s, 100s, 150s, 200s, 500s, 700s, 1000s, 1500s or 2000s after the start time point of antibody injection. Alternatively, the measurement time point can be at least 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 3000s, 5000s, 7000s, or 10000s after the crossing time point.

[0228] In one aspect, when the crossing time point (e.g., in a BIACORE® assay) is within 60 s, 100 s, 150 s, 200 s, 500 s, 700 s, 1000 s, 1500 s or 2000 s after the start time point of antibody injection, the isolated antibody that inhibits the interaction between C1q and C1r2s2 complex of the present invention can be determined as an antibody with substitution function, as determined by, for example, a BIACORE® assay using the following conditions: the capture levels of C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units respectively, and the antibody as an analyte is injected at 500 nM, 10 microliters (μL) / min.

[0229] In one aspect, when almost all (or all) of the C1q dissociates from the C1qrs complex within 100 s, 300 s, 500 s, 700 s, 1000 s, 1500 s, 2000 s, 3000 s, 5000 s, 7000 s, or 10000 s after the time point when antibody injection begins, the isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex can be determined to be an antibody with replacement function, as determined, for example, using the following conditions in a BIACORE (registered trademark) assay: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units, respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min. For example, in the sensorgram obtained from such an assay, when C1q and the antibody are present, the value (RU) approaches or reaches the value (RU) when the antibody is present and C1q is absent, and "almost all (or all) of the C1q dissociates from the C1qrs complex" can be determined. As used herein, "almost all (of the C1q)" refers to a percentage of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher; "(all of the C1q)" refers to a percentage of 100%. The percentage of dissociated C1q can be quantitatively determined by any assay method described herein. In some aspects, the present invention provides a method for screening antibodies that replace C1q from the C1r2s2 complex using the above method for measuring the "replacement function / activity" of such antibodies. In one embodiment, the screening method includes selecting an antibody that inhibits the interaction between C1q and the C1r2s2 complex; that is, selecting an antibody that binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex. A surface plasmon resonance assay, such as the BIACORE (registered trademark) assay described herein, can be used to appropriately select an antibody with replacement function / activity. In some embodiments, the screening method includes, after a sufficient time, determining (i) the value of the response unit (RU) in the presence of the antibody and (ii) the value of the response unit (RU) in the absence of the antibody by a surface plasmon resonance assay (such as the BIACORE (registered trademark) assay). The screening method can include comparing the value of (i) above with the value of (ii) above. The screening method can include selecting the antibody when the value of (i) above is lower than the value of (ii) above.The screening method can include identifying a "crossing time point" at which the curve in the absence of antibody but presence of C1q intersects with the curve in the presence of both C1q and antibody. As described above, multiple crossing time points can be observed even in a single sensorgram, and any one of the multiple crossing time points can be selected as the "crossing time point". In some embodiments, the screening method can include measuring the value of the response unit (RU) at least 60 s, 100 s, 150 s, 200 s, 500 s, 700 s, 1000 s, 1500 s, or 2000 s after the time point of the start of antibody injection. Alternatively, the screening method can include measuring the value of the response unit (RU) at least 100 s, 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, 1000 s, 3000 s, 5000 s, 7000 s, or 10000 s after the crossing time point. In some embodiments, the screening method can include: when the crossing time point of the antibody is within 60 s, 100 s, 150 s, 200 s, 500 s, 700 s, 1000 s, 1500 s, or 2000 s after the time point of the start of antibody injection, selecting an antibody that inhibits the interaction between C1q and C1r2s2 complex or an antibody with a substitution function, such as determined by, for example, BIACORE (registered trademark) assay using the following conditions: the capture levels of C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units, respectively, and the antibody as the analyte is injected at 500 nM, 10 microliters (μL) / min. In some embodiments, the screening method can include: when almost all (or all) of C1q dissociates from the C1qrs complex within 100 s, 300 s, 500 s, 700 s, 1000 s, 1500 s, 2000 s, 3000 s, 5000 s, 7000 s, or 10000 s after the time point of the start of antibody injection, selecting an antibody that inhibits the interaction between C1q and C1r2s2 complex or an antibody with a substitution function, such as determined by, for example, BIACORE (registered trademark) assay using the following conditions: the capture levels of C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units, respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.As described above, "substantially all (C1q)" refers to a percentage of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, and "all (C1q)" refers to 100%, and the percentage of dissociated C1q can be quantitatively determined by any assay described herein (including the BIACORE® assay).

[0230] (BIACORE® / blocking concept)

[0231] In one aspect, the present invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has a blocking function that enables the antibody to bind to C1r2s2 and inhibit the binding of C1q to C1r2s2. In another aspect, the antibody of the present invention has a blocking rate of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher. The blocking function / activity or blocking rate can be determined by using the BIACORE® assay. The level of C1q blocking can be evaluated using the following conditions: the capture level of C1r2s2 is targeted at 50, 100, 200, 400 resonance units (RU). Antibody variants are injected at 250, 500, 1000, 2000 nM to saturate antibody binding, and then human C1q (with or without 250, 500, 1000, 2000 nM antibody variant) is injected at 50, 100, 200 nM. The blocking rate is calculated by the formula: [1 - (human C1q binding reaction in the presence of antibody variant / human C1q binding reaction in the absence of antibody variant)] × 100%.

[0232] (pH-dependent)

[0233] In one aspect, the antibodies of the invention bind to an antigen (such as C1s) or to the C1r2s2 complex in a pH-dependent manner. In a preferred embodiment, the invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex (by binding to C1s), wherein the antigen-binding activity at pH 5.8 (i.e., the antigen-binding activity against C1s) is lower than the antigen-binding activity at pH 7.4. In a preferred embodiment, the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.4.

[0234] In addition to binding C1s in a pH-dependent manner, the effect of calcium on the affinity of the pH-dependent antibody for C1s may be another important property. C1s forms dimers at high calcium concentrations but dissociates into monomers at low calcium concentrations. When C1s is in the dimeric state, bivalent antibodies can form immune complexes by crosslinking multiple C1s molecules. This allows the antibody to bind to the C1s molecules within the complex through avidity and affinity interactions, thereby increasing the apparent affinity of the antibody. In contrast, when C1s is in the monomeric state, the antibody binds to C1s only through affinity interactions. This means that pH-dependent C1s antibodies in plasma can form immune complexes with dimeric C1s, but once they enter acidic endosomes, C1s will dissociate into monomers. This leads to the breakdown of the immune complex, which then enhances the pH-dependent dissociation of the antibody from the antigen.

[0235] In one aspect, in the isolated anti-C1s antibodies of the invention, when measured at 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, in the isolated anti-C1s antibodies of the invention, when measured at high calcium concentrations at neutral pH and at 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 2 or greater. In some embodiments, in the isolated anti-C1s antibodies of the invention, when measured at 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, wherein the anti-C1s antibody binds to the dimeric state of C1s.

[0236] Without being bound by a particular theory, in the following cases: 1) the absence of calcium can conformationally alter the epitope structure of C1s bound by the antibodies of the present invention, thereby altering the antibody affinity, or 2) the interaction (affinity or avidity) of the antibodies of the present invention can vary depending on the state of C1s (monomeric state or dimeric state), measurements using specific conditions (at neutral pH at high calcium concentration and at acidic pH at low calcium concentration) can be used to evaluate the ratio of KD values (KD(acidic pH) / KD(neutral pH)).

[0237] 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 (ii) below:

[0238] (i) When measured at high calcium concentration 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 2 or greater.

[0239] (ii) When measured at high calcium concentration at neutral pH and at low calcium concentration 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.

[0240] More generally, without being bound by a particular theory, in the following cases: 1) the absence of calcium can conformationally alter the epitope structure of certain antigens bound by the antibodies of the present invention, thereby altering the antibody affinity, or 2) the interaction (affinity or avidity) of the antibodies of the present invention can vary depending on the state of the antigen (monomeric state or dimeric state), measurements using specific conditions (at neutral pH at high calcium concentration and at acidic pH at low calcium concentration) can be used to evaluate the ratio of KD values (KD(acidic pH) / KD(neutral pH)).

[0241] Accordingly, the antibodies of the present invention bind to the antigen with higher affinity at neutral pH than at acidic pH, as follows: when measured at high calcium concentration at neutral pH and at low calcium concentration 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 2 or greater.

[0242] The above KD ratio, i.e., (KD(acidic pH) / KD(neutral pH)), can be compared between the parental antibody (i.e., the original antibody before modification of the present invention) and an antibody in which one or more amino acid mutations (e.g., addition, insertion, deletion, or substitution) have been introduced relative to the original (parental) antibody. The original (parental) antibody can be any known or newly isolated antibody as long as it specifically binds to C1s. Thus, in one aspect, in the 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, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, 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 the original (parental) antibody. In other words, the present invention provides an isolated anti-C1s antibody, wherein one or more amino acid mutations (e.g., addition, insertion, deletion, or substitution) have been introduced into the isolated anti-C1s antibody from the parental (original) antibody, and the ratio of (i) to (ii) is at least 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 3.5, 4, 5, 8, or 10: (i) 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 the isolated anti-C1s antibody; (ii) 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 the parental (original) antibody. These KD ratios can be measured at any (high or low) calcium concentration, e.g., at high calcium concentration at neutral and acidic pH, or at high calcium concentration at neutral pH and at low calcium concentration at acidic pH.

[0243] In one aspect, the antibodies of the present invention have different antigen-binding activities between intracellular and extracellular conditions. Intracellular and extracellular conditions refer to different conditions between the inside and outside of a cell. Categories of conditions include, for example, ionic concentrations, and more specifically, metal ion concentrations, hydrogen ion concentration (pH), and calcium ion concentration. "Intracellular conditions" preferably refer to the characteristic environment of the internal environment of endosomes, while "extracellular conditions" preferably refer to the characteristic environment of the environment in plasma. Antibodies having the property that antigen-binding activity varies according to ionic concentration can be obtained by screening a large number of antibodies of domains having such a property. For example, antibodies having the above property can be obtained by generating a large number of antibodies with different sequences from each other using the hybridoma method or antibody library method, and measuring their antigen-binding activities at different ionic concentrations. The B cell cloning method is one example of a method for screening such antibodies. In addition, as described below, at least one unique amino acid residue that can confer on an antibody the property of having antigen-binding activity that varies according to ionic concentration is specified to prepare a library of a large number of antibodies having different sequences while sharing the unique amino acid residue as a common structure. Such libraries can be screened to efficiently isolate antibodies having the above property.

[0244] In one aspect, the present invention provides antibodies that bind 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 expression "pH-dependent binding" means "decreased binding at acidic pH compared to at neutral pH", and the two expressions can be used interchangeably. For example, an anti-C1s antibody "having a pH-dependent binding characteristic" includes an antibody that binds C1s with higher affinity at neutral pH than at acidic pH.

[0245] In certain embodiments, when measured at high calcium concentration 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 or greater. In a specific embodiment, the antibodies of the present invention bind C1s with at least 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, 10000, or more times higher affinity at neutral pH than at acidic pH.

[0246] In certain embodiments, 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 or greater when measured at high calcium concentration at neutral pH and at low calcium concentration at acidic pH. In specific embodiments, the 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 or more times higher affinity at neutral pH than at acidic pH.

[0247] In the above case, for example, the acidic pH is 5.8 and the neutral pH is 7.4, so 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 below. In some embodiments, KD(acidic pH) / KD(neutral pH) such as KD(pH 5.8) / KD(pH 7.4) can be from 2 to 10,000.

[0248] When the antigen is a soluble protein, the binding of the antibody to the antigen can result in an extended half-life of the antigen in plasma (i.e., reduced clearance rate of the antigen from plasma), because the antibody can have a longer half-life in plasma than the antigen itself and can act as a carrier for the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in cells (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties (binding to the antigen in the neutral extracellular environment while releasing the antigen into the acidic endosomal compartment after entering the cell) are expected to have excellent properties in antigen neutralization and clearance relative to their counterparts that bind in a pH-dependent 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).

[0249] In one aspect, the present invention provides an antibody that binds to C1s with higher affinity under conditions of high calcium concentration than under conditions of low calcium concentration.

[0250] In the present invention, preferred metal ions include, for example, calcium ions. Calcium ions are involved in the regulation of many biological phenomena, including the contraction of muscles such as skeletal muscle, smooth muscle and cardiac muscle; the activation of leukocyte movement, phagocytosis, etc.; the activation of platelet shape change, secretion, etc.; lymphocyte activation; mast cell activation including histamine secretion; cell responses mediated by catecholamine α receptors or acetylcholine receptors; exocytosis; the release of neurotransmitters from neuron terminals; and axoplasmic flow in neurons. Known intracellular calcium receptors include troponin C, calmodulin, parvalbumin and myosin light chain, which have several calcium binding sites and are believed to have originated from a common origin of molecular evolution. There are also many known calcium binding motifs. Such well-known motifs include, for example, the cadherin domain, the EF-hand of calmodulin, the C2 domain of protein kinase C, the Gla domain of blood coagulation factor IX, the C-type lectins of the acylaroglycoprotein receptor and the mannose binding receptor, the A domain of the LDL receptor, annexin, the thrombospondin type 3 domain and the EGF-like domain.

[0251] In the present invention, when the metal ion is calcium ion, it is desired that the antigen-binding activity under low calcium ion concentration conditions is lower than that under high calcium ion concentration conditions. At the same time, 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 from 0.1 μM (micro M) to 30 μM, more preferably from 0.5 μM to 10 μM, and particularly preferably from 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. At the same time, in the present invention, the high calcium ion concentration is preferably from 100 μM to 10 mM, more preferably from 200 μM to 5 mM, and particularly preferably from 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (in blood). In the present invention, preferably, 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 between low and high calcium ion concentrations, it is preferred that the antibody of the present invention has a stronger binding strength at high calcium ion concentration than at low calcium ion concentration. In other words, preferably, the antigen-binding activity of the antibody of the present invention is lower at low calcium ion concentration than at high calcium ion concentration. When the level of binding activity is expressed by the dissociation constant (KD), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or greater, still more preferably 10 or greater, and more preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. There is no particular limitation on the upper limit of the value of KD (low calcium ion concentration) / KD (high calcium ion concentration), and it can be any value such as 100, 400, 1000 or 10000 as long as those skilled in the art can prepare it. The dissociation rate constant (kd) can be used instead of KD. When it is difficult to calculate the KD value, the activity can be evaluated based on the level of the binding reaction in Biacore when the analyte passes through at the same concentration. When the antigen passes through the chip immobilized with the antigen-binding molecule of the present invention, the binding reaction at low calcium concentration is preferably 1 / 2 or less, more preferably 1 / 3 or less, more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less of the binding reaction at high calcium concentration. As is well known, generally, the extracellular calcium ion concentration in vivo (e.g., in plasma) is high, while the intracellular calcium ion concentration (e.g., in endosomes) is low. Therefore, in the present invention, it is preferred that the extracellular condition is a high calcium ion concentration and the intracellular condition is a low calcium ion concentration. When the property that the antigen-binding activity under intracellular calcium ion concentration conditions is lower than that under extracellular calcium ion concentration conditions is imparted to the antigen-binding molecule (e.g., antibody) of the present invention, 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 the incorporation of the antigen from extracellular to intracellular.When such an antibody is administered to a living body, it can reduce the antigen concentration in plasma and reduce the physiological activity of the antigen in the body. Therefore, the antibody of the present invention is useful. A method for screening an antigen-binding domain or an antibody that has a lower antigen-binding activity under low calcium ion concentration conditions than under high calcium ion concentration conditions includes, for example, the method described in WO2012 / 073992 (for example, paragraphs 0200-0213). There is no particular limitation on the method for imparting the antigen-binding domain of the present invention with the property of binding less strongly to an antigen under low calcium ion concentration conditions than under high calcium ion concentration conditions, and it can be implemented by any method. Specifically, the method is described in Japanese Patent Application No. 2011-218006 and includes, 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 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 one in which at least one amino acid residue of the antigen-binding domain has been substituted with an amino acid residue having metal chelating activity and / or at least one amino acid residue having metal chelating activity has been inserted into the antigen-binding domain.

[0252] Amino acid residues having metal chelating activity preferably include, for example, serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. In addition, amino acid residues that change the antigen-binding activity of the antigen-binding domain according to the calcium ion concentration preferably include, for example, amino acid residues that form a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described 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 in troponin C, calmodulin, parvalbumin, and myosin light chain; the C2 domain in protein kinase C; the Gla domain in blood coagulation factor IX; the C-type lectins of the asialoglycoprotein receptor and the mannose-binding receptor, ASGPR, CD23, and DC-SIGN; the A domain in the LDL receptor; the annexin domain; the cadherin domain; the thrombospondin type 3 domain; and the EGF-like domain are preferably used as calcium-binding motifs.

[0253] The antigen-binding domain of the present invention may contain amino acid residues that alter antigen-binding activity according to the calcium ion concentration, such as the amino acid residues having metal chelating activity and the amino acid residues forming a calcium-binding motif described above. The position of such amino acid residues in the antigen-binding domain is not particularly limited, and they may be located at any position as long as the antigen-binding activity changes according to the calcium ion concentration. At the same time, as long as the antigen-binding activity changes according to the calcium ion concentration, such amino acid residues may be included alone or in combination of two or more. The amino acid residues preferably include, for example, serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. When the antigen-binding domain is an antibody variable region, the amino acid residues may be included in the heavy chain variable region and / or the light chain variable region. In a preferred embodiment, according to the Kabat numbering in CDR3 of the heavy chain variable region, the amino acid residues may be included in CDR3 of the heavy chain variable region, more preferably at positions 95, 96, 100a, and / or 101.

[0254] In another preferred embodiment, according to the Kabat numbering in CDR1 of the light chain variable region, the amino acid residues may be included in CDR1 of the light chain variable region, more preferably at positions 30, 31, and / or 32. In yet another preferred embodiment, according to the Kabat numbering in CDR2 of the light chain variable region, the amino acid residues may be included in CDR2 of the light chain variable region, more preferably at position 50. In yet another preferred embodiment, according to the Kabat numbering in CDR3 of the light chain variable region, the amino acid residues may be included in CDR3 of the light chain variable region, more preferably at position 92.

[0255] In addition, the above embodiments may be combined. For example, according to the Kabat numbering in the light chain variable region, the amino acid residues may be included in two or three CDRs selected from CDR1, CDR2, and CDR3 of the light chain variable region, more preferably at any one or more of positions 30, 31, 32, 50, and / or 92.

[0256] A library of a large number of antigen-binding domains having different sequences while sharing the above amino acid residues that alter antigen-binding activity according to the calcium ion concentration as a common structure is prepared. The library can be screened to effectively obtain antigen-binding domains having binding activity to a desired antigen, wherein their antigen-binding activity changes according to the calcium ion concentration.

[0257] For the purposes of the present disclosure, the "affinity" of an antibody for C1s is expressed as the KD of the antibody. The KD of an antibody is the equilibrium dissociation constant of the antibody-antigen interaction. The larger the KD value of an antibody for binding its antigen, the weaker its binding affinity for the specific antigen. Thus, as used herein, the statement "higher affinity at neutral pH than at acidic pH" (or the equivalent statement "pH-dependent binding") means that the KD of the antibody is greater at acidic pH than at neutral pH. For example, in the context of the present invention, if the KD of an antibody for binding C1s at acidic pH is at least 2-fold greater than the KD of the antibody for binding C1s at neutral pH, the antibody is considered to bind C1s with higher affinity at neutral pH than at acidic pH. Accordingly, the present invention encompasses antibodies wherein the KD of the antibody for binding C1s at acidic pH is at least 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, 10000 or more fold greater than the KD of the antibody for binding C1s at neutral pH. In another embodiment, the KD value of the antibody at neutral pH can be 10 -7 M, 10 -8 M, 10 -9 M,10 -10 M, 10 -11 M, 10 -12 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or greater.

[0258] The binding characteristics of an antibody for a specific antigen can also be expressed as the kd of the antibody. The kd of an antibody is the dissociation rate constant of the antibody with respect to a specific antigen and is expressed in reciprocal seconds (i.e., sec -1 ). An increase in the kd value indicates weaker binding of the antibody to its antigen. The present invention thus encompasses antibodies wherein the antibody binds C1s with a higher kd value at acidic pH than at neutral pH. The present invention encompasses antibodies wherein the kd of the antibody for binding C1s at acidic pH is at least 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, 10000, or more fold greater than the kd of the antibody for binding C1s at neutral pH. In another embodiment, the kd value of the antibody at neutral pH can be 10-2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or greater.

[0259] In some cases, "decreased binding at acidic pH compared to neutral pH" is expressed as the ratio of the KD value of the antibody at acidic pH to the KD value of the antibody at neutral pH (or vice versa). For example, for the purposes of the present invention, if an antibody exhibits an acidic / neutral KD ratio of 2 or greater, the antibody can be considered to exhibit "decreased binding to C1s at acidic pH compared to its binding at neutral pH". In some exemplary embodiments, the acidic / neutral KD ratio of the antibodies of the present invention can be 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, 10000, or greater. In another embodiment, the KD value of the antibody at neutral pH can be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or less. In another embodiment, the KD value of the antibody at acidic pH can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or greater.

[0260] In some cases, "decreased binding at acidic pH compared to neutral pH" is expressed as the ratio of the kd value of the antibody at acidic pH to the kd value of the antibody at neutral pH (or vice versa). For example, for the purposes of the present invention, if an antibody shows an acidic / neutral kd ratio of 2 or greater, the antibody can be considered to show "decreased binding to C1s at acidic pH compared to its binding at neutral pH". In certain exemplary embodiments, the acidic / neutral kd ratio of the antibodies of the present invention can be 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, 10000, or greater. In another embodiment, the kd value of the antibody at neutral pH can be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s, or less. In another embodiment, the kd value of the antibody at acidic pH can be 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s, or greater.

[0261] As used herein, the expression "acidic pH" refers to a pH of 4.0 to 6.5. The expression "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, "acidic pH" is 5.8 or 6.0.

[0262] As used herein, the expression "neutral pH" refers to a pH of 6.7 to about 10.0. The expression "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, "neutral pH" is 7.0 or 7.4.

[0263] As used herein, the phrase "under high calcium concentration conditions" or "at high calcium concentration" refers to 100 μM to 10 mM, more preferably 200 μM to 5 mM, particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (in blood). The phrase "under high calcium concentration conditions" or "at high calcium concentration" includes the calcium concentration values of 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+ The calcium concentration values of. In certain aspects, "under high calcium concentration conditions" or "at high calcium concentration" refers to 1.2 mM Ca 2 + .

[0264] As used herein, the phrase "under low calcium concentration conditions" or "at low calcium concentration" refers to 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. The phrase "under low calcium concentration conditions" or "at low calcium concentration" includes the calcium concentration values of 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 Ca2+. In certain aspects, "under low calcium concentration conditions" or "at low calcium concentration" refers to 3.0 μM Ca 2+ .

[0265] As expressed herein, the KD and kd values can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, e.g., 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 performed by using surface plasmon resonance technology, where the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl, 37 degrees Celsius (°C).

[0266] In one aspect, the present invention provides methods for enhancing the clearance of Cls from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of C1s from plasma. The present invention also provides methods for enhancing the clearance of the complex of C1r and C1s from plasma in an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of the complex of C1r and C1s from plasma. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of C1r2s2 from plasma. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of C1r2s2 from plasma rather than C1q from plasma.

[0267] In another aspect, the present invention provides a method for removing Cls from plasma, the method comprising: (a) identifying an individual in need of removing C1s from the individual's plasma; (b) providing an antibody that binds to C1s through the antigen-binding (C1s-binding) domain of the antibody and has a KD(pH5.8) / KD(pH7.4) value, which 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, wherein the ratio is from 2 to 10,000, and wherein the antibody binds to C1s in vivo plasma and dissociates from the bound C1s under conditions present in in vivo endosomes, and wherein the antibody is a human IgG or a humanized IgG; and (c) administering the antibody to the individual. In another aspect, this surface plasmon resonance technology can be used at 37 °C and 150 mM NaCl. In another aspect, this surface plasmon resonance technology can be used, where the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl, 37 °C.

[0268] In another aspect, the present invention provides a method for removing C1s from the plasma of a subject, the method comprising: (a) identifying a first antibody that binds to C1s through an antigen-binding domain of the first antibody; (b) identifying a second antibody that: (1) binds to C1s through an antigen-binding (C1s-binding) domain of the second antibody, (2) has an amino acid sequence identical to that of the first antibody except that at least one amino acid of the variable region of the first antibody is replaced with histidine and / or at least one histidine is inserted into the variable region of the first antibody, (3) has a KD(pH5.8) / KD(pH7.4) value higher than that of the first antibody and between 2 and 10,000, where KD(pH5.8) / KD(pH7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4 when determining KD using surface plasmon resonance technology, (4) binds to C1s in the plasma in vivo, (5) dissociates from the bound C1s under conditions present in endosomes in vivo, and (6) is human IgG or humanized IgG; (c) identifying a subject in need of reducing his or her plasma level of C1s; and (d) administering the second antibody to the subject to reduce the plasma level of C1s in the subject. In another aspect, this surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technology can be used, where the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl, 37°C.

[0269] In another aspect, the present invention provides a method for removing C1s from the plasma of a subject, the method comprising: (a) identifying a first antibody that: (1) binds to C1s through the antigen-binding domain of the first antibody, (2) has the same amino acid sequence as a second antibody that binds to C1s through the 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, (3) has a KD(pH5.8) / KD(pH7.4) value higher than that of the second antibody and between 2 and 10,000, where KD(pH5.8) / KD(pH7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4 when determining KD 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; (b) identifying a subject in need of reducing his or her plasma level of C1s; and (c) administering the first antibody to the subject at least once to reduce the plasma level of C1s in the subject. In another aspect, this surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technology can be used, where the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl, 37°C. In some cases, the antibody inhibits components of the classical complement pathway; in some cases, the component of the classical complement pathway is Cls.

[0270] In one aspect, the present disclosure provides a method for modulating complement activation. In some embodiments, the method inhibits complement activation, such as reducing 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, the method 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 the 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 a human. Administration can be by any route known to those of skill in the art, including those disclosed herein. In some embodiments, the administration is intravenous. In some embodiments, the administration is intrathecal injection.

[0271] In certain embodiments, the anti-C1s antibodies of the invention bind to C1s from more than one species. In particular embodiments, the anti-C1s antibodies bind to C1s from human and non-human animals. In specific embodiments, the anti-C1s antibodies bind to C1s from human, rat, and monkey (e.g., cynomolgus monkey, rhesus monkey, gibbon, chimpanzee, and baboon).

[0272] In one aspect, the 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: 32, 38, 44, 50, or 56; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61.

[0273] In one aspect, the present invention provides an anti-C1s antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58.

[0274] In another aspect, the present invention provides anti-C1s antibodies comprising at least one, at least two, or all three VL HVR sequences selected from the following: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61.

[0275] In another aspect, the anti-C1s antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the following: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50 or 56, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the following: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61.

[0276] In some embodiments, anti-C1s antibody variants are provided, which are prepared by introducing amino acid modifications into antibodies comprising the VH sequences of SEQ ID No: 19, 20, 21, 23, or 24 and the VL sequences of SEQ ID NO: 26, 27, 28, 30, or 31.

[0277] In some embodiments, the anti-C1s antibodies of the invention comprise histidine at one or more of the following Kabat numbering system positions:

[0278] 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

[0279] 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.

[0280] In some embodiments, the anti-C1s antibodies of the invention comprise at least one histidine substituting for one or more amino acid residues at positions selected from the following Kabat numbering system positions:

[0281] 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

[0282] 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.

[0283] In any of the above embodiments, the anti-C1s antibody is humanized. In one embodiment, the anti-C1s antibody comprises the HVRs of any of the above embodiments and further comprises a recipient human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1s antibody comprises the HVRs of any of the above embodiments and further comprises a VH or VL containing an FR sequence. In another embodiment, the anti-C1s antibody of the invention comprises the following heavy or light chain variable domain FR sequences

[0284] In another aspect, the 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, 20, 21, 23, or 24. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, provided that the anti-C1s antibody comprising the sequence retains the ability to bind C1s. In certain embodiments, in SEQ ID NO: 19, 20, 21, 23, or 24, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. 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 the VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from the following: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56, (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57, and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.

[0285] In another aspect, anti-C1s antibodies are provided, wherein the antibody 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: 26, 27, 28, 30, or 31. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C1s antibody comprising the sequence retains the ability to bind C1s. In certain embodiments, in SEQ ID NO: 26, 27, 28, 30 or 31, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-C1s antibody comprises a VH sequence of SEQ ID NO: 26, 27, 28, 30, or 31, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.

[0286] In another aspect, anti-C1s antibodies are provided, wherein the antibodies comprise a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 19 and SEQ ID NO: 26, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 20 and SEQ ID NO: 27, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 21 and SEQ ID NO: 28, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 23 and SEQ ID NO: 30, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 24 and SEQ ID NO: 31, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.

[0287] In another aspect, the present invention provides antibodies that bind the same epitope as the anti-C1s antibodies provided herein. In a preferred aspect, the antibodies specifically bind the same epitope as the anti-C1s antibodies provided herein. For example, in certain embodiments, antibodies are provided that (specifically) bind the same epitope as an antibody selected from the group consisting of:

[0288] 1) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37,

[0289] 2) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43,

[0290] 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49,

[0291] 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55, and

[0292] 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61.

[0293] In some embodiments, the isolated anti-C1s antibody of the invention competes with the antibodies selected from the group consisting of the following 1) to 5) for binding to C1s. In some embodiments, the isolated anti-C1s antibody of the invention competes with the antibodies selected from the group consisting of the following 1) to 5) for binding to C1s at neutral pH.

[0294] 1) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37,

[0295] 2) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43,

[0296] 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49,

[0297] 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55, and

[0298] 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61.

[0299] In one aspect, the present invention provides anti-C1r antibodies that comprise at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166.

[0300] In one aspect, the present invention provides anti-C1r antibodies that comprise at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142. In one embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142.

[0301] In another aspect, the present invention provides an anti-C1r antibody comprising at least one, at least two, or all three of the VL HVR sequences selected from the following: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166.

[0302] In another aspect, the anti-C1r antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the following: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the following: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166.

[0303] In some embodiments, anti-C1s antibody variants are provided, which are prepared by introducing amino acid modifications into an antibody comprising a VH sequence of SEQ ID No: 103, 104, 105, 106, 107, 108, 109, or 110 and a VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118.

[0304] In some embodiments, the anti-C1r antibody of the invention comprises histidine at one or more of the following Kabat numbering system positions:

[0305] 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

[0306] Light chains: 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.

[0307] In some embodiments, the anti-C1r antibody of the invention comprises at least one histidine substituting one or more amino acid residues at positions selected from the following Kabat numbering system positions:

[0308] Heavy chains: 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

[0309] Light chains: 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.

[0310] In any of the above embodiments, the anti-C1r antibody is humanized. In one embodiment, the anti-C1r antibody comprises the HVRs in any of the above embodiments and further comprises a recipient human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1r antibody comprises the HVRs in any of the above embodiments and further comprises a VH or VL containing the FR sequence. In another embodiment, the anti-C1r antibody of the invention comprises the following heavy or light chain variable domain FR sequences.

[0311] In another aspect, the anti-C1r 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: 103, 104, 105, 106, 107, 108, 109, or 110. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C1r antibody comprising the sequence retains the ability to bind C1r. In certain embodiments, in SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-C1s antibody comprises a VH sequence in SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.

[0312] In another aspect, anti-C1r antibodies are provided, wherein the antibody 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: 111, 112, 113, 114, 115, 116, 117, or 118. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-C1r antibody comprising the sequence retains the ability to bind C1r. In certain embodiments, in SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-C1r antibody comprises the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from the following: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.

[0313] In another aspect, anti-C1r antibodies are provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 103 and SEQ ID NO: 111, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 104 and SEQ ID NO: 112, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 105 and SEQ ID NO: 113, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 106 and SEQ ID NO: 114, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 107 and SEQ ID NO: 115, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 108 and SEQ ID NO: 116, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 109 and SEQ ID NO: 117, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, the modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamate by pyroglutamylation.In one embodiment, the antibodies comprise the VH and VL sequences of SEQ ID NO: 110 and SEQ ID NO: 118, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0314] In another aspect, the present invention provides antibodies that bind the same epitope as the anti-C1r antibodies provided herein. In a preferred aspect, the antibodies specifically bind the same epitope as the anti-C1r antibodies provided herein. For example, in certain embodiments, antibodies are provided that specifically bind the same epitope as an antibody selected from the group consisting of:

[0315] 6) an antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159,

[0316] 7) an antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160,

[0317] 8) an antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161,

[0318] 9) an antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162,

[0319] 10) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 123, an HVR-H2 sequence of SEQ ID NO: 131, an HVR-H3 sequence of SEQ ID NO: 139, an HVR-L1 sequence of SEQ ID NO: 147, an HVR-L2 sequence of SEQ ID NO: 155, and an HVR-L3 sequence of SEQ ID NO: 163,

[0320] 11) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 124, an HVR-H2 sequence of SEQ ID NO: 132, an HVR-H3 sequence of SEQ ID NO: 140, an HVR-L1 sequence of SEQ ID NO: 148, an HVR-L2 sequence of SEQ ID NO: 156, and an HVR-L3 sequence of SEQ ID NO: 164,

[0321] 12) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 125, an HVR-H2 sequence of SEQ ID NO: 133, an HVR-H3 sequence of SEQ ID NO: 141, an HVR-L1 sequence of SEQ ID NO: 149, an HVR-L2 sequence of SEQ ID NO: 157, and an HVR-L3 sequence of SEQ ID NO: 165, and

[0322] 13) An antibody comprising an HVR-H1 sequence of SEQ ID NO: 126, an HVR-H2 sequence of SEQ ID NO: 134, an HVR-H3 sequence of SEQ ID NO: 142, an HVR-L1 sequence of SEQ ID NO: 150, an HVR-L2 sequence of SEQ ID NO: 158, and an HVR-L3 sequence of SEQ ID NO: 166.

[0323] In some embodiments, the isolated anti-C1r antibody of the invention competes with antibodies selected from the group consisting of 6) to 13) below for binding to C1r. In some embodiments, the isolated anti-C1r antibody of the invention competes with antibodies selected from the group consisting of 6) to 13) below for binding to C1r at neutral pH.

[0324] 6) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159,

[0325] 7) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160,

[0326] 8) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161,

[0327] 9) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162,

[0328] 10) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 123, the HVR-H2 sequence of SEQ ID NO: 131, the HVR-H3 sequence of SEQ ID NO: 139, the HVR-L1 sequence of SEQ ID NO: 147, the HVR-L2 sequence of SEQ ID NO: 155, and the HVR-L3 sequence of SEQ ID NO: 163,

[0329] 11) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 124, the HVR-H2 sequence of SEQ ID NO: 132, the HVR-H3 sequence of SEQ ID NO: 140, the HVR-L1 sequence of SEQ ID NO: 148, the HVR-L2 sequence of SEQ ID NO: 156, and the HVR-L3 sequence of SEQ ID NO: 164,

[0330] 12) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 125, the HVR-H2 sequence of SEQ ID NO: 133, the HVR-H3 sequence of SEQ ID NO: 141, the HVR-L1 sequence of SEQ ID NO: 149, the HVR-L2 sequence of SEQ ID NO: 157, and the HVR-L3 sequence of SEQ ID NO: 165, and

[0331] 13) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 126, the HVR-H2 sequence of SEQ ID NO: 134, the HVR-H3 sequence of SEQ ID NO: 142, the HVR-L1 sequence of SEQ ID NO: 150, the HVR-L2 sequence of SEQ ID NO: 158, and the HVR-L3 sequence of SEQ ID NO: 166,

[0332] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody with pH-dependent binding that specifically binds to an epitope within a region encompassing the CUB1-EGF-CUB2 domain, which is composed of CUB1, EGF, and CUB2 of complement component 1s (C1s). In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is an epitope that is not located in the beta domain of C1s. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is an epitope located in the alpha domain or the gamma domain of C1s. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present disclosure is a linear epitope. In some embodiments, the epitope bound by the isolated anti-C1s antibody of the present invention is amino acids 16-291 of the complement C1s protein, amino acids 16-172 of the complement C1s protein as shown in SEQ ID NO: 1, amino acids 16-210 of the complement C1s protein as shown in SEQ ID NO: 1, amino acids 16-111 of the complement C1s protein as shown in SEQ ID NO: 1, amino acids 112-210 of the complement C1s protein as shown in SEQ ID NO: 1, amino acids 131-172 of the complement C1s protein as shown in SEQ ID NO: 1, or an epitope within amino acids 16-130 of the complement C1s protein as shown in SEQ ID NO: 1. In some embodiments, the above epitope of C1s is an epitope of human C1s. In some embodiments, the isolated anti-C1s antibody of the present invention can bind to the activated C1s protein and the inactivated C1s form.

[0333] In some embodiments, the present disclosure provides an isolated anti-C1r antibody that specifically binds to an epitope within a region encompassing the CUB1-EGF-CUB2 domain of complement component 1r (C1r), which is composed of CUB1, EGF, and CUB2. In some cases, the epitope bound by the isolated anti-C1r antibody of the present disclosure is a linear epitope or a conformational epitope. In some embodiments, the above epitope of C1r is an epitope of human C1r.

[0334] In another aspect of the present invention, the anti-C1s antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-C1s antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., a complete IgG1, IgG2, IgG3, or IgG4 antibody or other antibody classes or isotypes defined herein.

[0335] In another aspect, an anti-C1s antibody according to any of the above embodiments can comprise any of the features described in Sections 1-7 below (individually or in combination).

[0336] 1. Antibody affinity

[0337] In certain embodiments, the antibodies provided herein have a dissociation constant (Kd or KD) 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 to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0338] In one embodiment, the Kd is measured by radio-labeled antigen binding assay (RIA). In one embodiment, the Fab form of the antibody of interest and its antigen are used for RIA. For example, the solution binding affinity of the Fab for the antigen is measured by: in the presence of a titration series of unlabeled antigen with the minimum concentration of ( 125I) Label the antigen-equilibrated Fab, and then capture the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) was coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) fetal bovine serum albumin in PBS for two to five hours at room temperature (about 23°C). In non-adsorptive plates (Nunc #269620), 100 pM or 26 pM [125I]-antigen was mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, the mixture was transferred to the capture plate for incubation at room temperature (e.g., for one hour). Then the solution was removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate was dry, 150 μl / well of scintillant (MICROSCINT-20TM; Packard) was added, and the plate was counted for ten minutes on a TOPCOUNT TM γ counter (Packard). The concentration of each Fab that resulted in less than or equal to 20% of the maximum binding was selected for the competitive binding assay.

[0339] According to another embodiment, Kd is measured using BIACORE® surface plasmon resonance assay. For example, at 25 °C, the assay using BIACORE®-2000 or BIACORE®-3000 (GE Healthcare) is performed with immobilized antigen CM5 chip at ~10 response units (RU). In one embodiment, according to the provider's instructions, the carboxymethylated dextran biosensor chip (CM5, GE Healthcare) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 micrograms (μg) / ml (~0.2 μM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 microliters (μl) / minute to achieve ~10 response units (RU) of the coupled protein. After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serial two-fold dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / minute at 25 °C into PBS (PBST) with 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant. The association rate (k on ) and dissociation rate (k off ) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio k off / k on . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate measured by the above surface plasmon resonance assay exceeds 10 6 M -1 s -1 , then the association rate can be determined by the following method: in the presence of elevated concentrations of antigen (as measured in a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) equipped with a mixing chamber), using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in 25 °C PBS pH 7.2 (excitation = 295 nm; emission = 340 nm, 16 nm bandpass).

[0340] In some embodiments, the binding affinity of each histidine-substituted variant of the invention 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) can be immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes can be 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 can be captured onto the sensor surface by Protein A / G. The target antibody capture level is 200 resonance units (RU). Native zymogen human C1s (CompTech) or recombinant human C1s prepared, for example, at 50 nM can then be injected and dissociated.

[0341] A specific example of the steps of the Biacore assay of the invention is as follows.

[0342] The binding specificity of the C1s CUB1-EGF-CUB2 binder 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). Each antibody was captured onto the sensor surface by Protein A / G. The target antibody capture level was 100 resonance units (RU). Native zymogen human C1s (Comptech A103) (as a monomer, at 50 nM) or recombinant human C1s CCP1-CCP2-SP-His (as a monomer, at 100 nM) was injected and then dissociated. The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 for each cycle. It was determined that the C1s CUB1-EGF-CUB2 binder binds to native zymogen human C1s but not to recombinant human C1s CCP1-CCP2-SP-His, which is a truncated protein lacking the CUB1-EGF-CUB2 domain.

[0343] The C1q replacement function of the antibody was confirmed at 37 °C by the C1r2s2 capture method using a BIACORE® T200 instrument (GE Healthcare). Anti-His antibody (GE-Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies, recombinant human C1r2s2 Flag / His tetramer, and native human C1q (Comptech A099) were prepared in a pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). The recombinant human C1r2s2 Flag / His tetramer was first captured onto the sensor surface via the anti-His antibody (“hc1r2s2”). The target capture level was 200 resonance units (RU). Native human C1q was injected at 100 nM to capture 200 RU (“hc1q”), and then immediately, an antibody at 500 nM was injected at 10 μL / min for 1200 sec. The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 for each cycle. For antibodies with C1q replacement function, after the time point where sensorgrams 1 and 2 cross (“the time point of crossing”), the response units of sensorgram 2 (in the presence of C1r2s2, C1q, and the antibody) were lower than those of sensorgram 1 (in the presence of C1r2s2, C1q, and buffer but without the antibody). The time point of crossing was determined by subtracting the buffer response (sensorgram 1) from the antibody (Ab) response (sensorgram 2) and referring to the time point when the difference changed from positive to negative.

[0344] The C1q replacement function of the antibody was confirmed at 37 °C by the C1q capture method using a BIACORE® T200 instrument (GE Healthcare). Antibodies, recombinant human C1r2s2 Flag / His tetramer, and biotinylated native human C1q (Comptech A099) were prepared in a pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). Biotinylated native human C1q was first captured into one flow cell of a CAP sensor chip (GE-Healthcare). The target capture level was in the range of 800 to 1000 resonance units (RU). Recombinant human C1r2s2 Flag / His tetramer was injected at 300 nM, and then the antibody was injected at 500 nM for 180 s at 10 μL / min. In each cycle, the sensor surface was regenerated with 8 M guanidine hydrochloride and 1 M NaOH in an 8:1 ratio. Antibodies with C1q replacement function enhanced the dissociation rate of C1r2s2, i.e., the curve in the presence of the antibody was lower than that in the absence of the antibody.

[0345] To evaluate the blocking effect of the antibody on the binding of C1q to C1r2s2, a blocking assay was performed at 37 °C using a BIACORE® T200 instrument (GE Healthcare). Anti-His antibody (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies, recombinant human C1r2s2 Flag / His tetramer, and native human C1q were prepared in a pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). Recombinant human C1r2s2 Flag / His tetramer was first captured onto the sensor surface via the anti-His antibody (“hc1r2s2”). The target capture level was 200 resonance units (RU). Antibody variants were injected at 500 nM, and then native human C1q was injected at 100 nM (“hc1q”). The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 in each cycle. Antibodies with C1q blocking function are those that compete with C1q for binding to C1r2s2.

[0346] In some embodiments, if necessary, after the dissociation step at pH 7.4, an additional dissociation step at pH 5.8 was immediately incorporated. By using Scrubber 2.0 (BioLogic software) curve fitting software to process and fit the data, the dissociation rate in 5(+) buffer could be determined.

[0347] 2. Antibody fragments

[0348] 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, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenberg and Moore, (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. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0349] Diabodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and 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).

[0350] Single-domain antibodies are antibody fragments that contain all or part of the heavy-chain variable domain or all or part 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.

[0351] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and preparation by recombinant host cells (e.g., E. coli or phage), as described herein.

[0352] 3. Chimeric and Humanized Antibodies

[0353] In certain embodiments, the antibodies provided herein are chimeric antibodies. 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 mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In additional examples, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0354] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody 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 further comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or enhance antibody specificity or affinity.

[0355] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in 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 specific determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); 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 the "directed selection" method for FR shuffling).

[0356] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies having a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al. J. Immunol. 151:2623 (1993)); human mature (somatic 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 FR library screening (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0357] 4. Human Antibodies

[0358] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0359] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present extrachromosomally or is randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSETM technology; U.S. Patent No. 5,770,429, which describes the HUMAB® technology; U.S. Patent No. 7,041,870, which describes the K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes the VELOCIMOUSE® technology). The human variable regions from the complete antibodies produced by such animals can be further modified, e.g., by combining them with different human constant regions.

[0360] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (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 prepared via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include those described in, e.g., U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) has also been 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).

[0361] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0362] 5. Antibodies from Libraries

[0363] The antibodies of the invention can be isolated by screening combinatorial libraries of antibodies for one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and are 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, in 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).

[0364] In certain phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and the library can then be screened for phage that bind antigen, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive libraries (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthetically prepared by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and effect rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373 and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0365] As used herein, an antibody or antibody fragment isolated from a human antibody library is considered a human antibody or human antibody fragment.

[0366] 6. Multispecific Antibodies

[0367] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different epitopes. In certain embodiments, one binding specificity is for C1s and the other is for another antigen. In certain embodiments, a bispecific antibody can bind two different epitopes of C1s. Bispecific antibodies can also be used to localize a cytotoxic agent to cells expressing C1s. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0368] Techniques for preparing 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)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering the electrostatic steering effect for the preparation of antibody Fc-heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to prepare bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553(1992)); using the "diabody" technique for preparing bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0369] Also included herein are engineered antibodies having more than three functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576A1).

[0370] Antibodies or fragments herein also include "dual action Fab" or "DAF" which contain antigen-binding sites that bind C1s as well as another different antigen (see, e.g., US2008 / 0069820).

[0371] 7. Antibody Variants

[0372] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from the antibody amino acid sequence, and / or insertions into and / or substitutions of residues within the antibody amino acid sequence. 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.

[0373] a) Substitution, Insertion, and Deletion Variants

[0374] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include the HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More extensive changes are provided under the heading "Exemplary Substitutions" in Table 1 and are further described below in terms of amino acid side-chain classifications. Amino acid substitutions can be introduced into the target antibody and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0375] [Table 1]

[0376]

[0377] Amino acids can be grouped according to common side-chain properties:

[0378] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0379] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0380] (3) Acidic: Asp, Glu;

[0381] (4) Basic: His, Lys, Arg;

[0382] (5) Residues that affect chain orientation: Gly, Pro;

[0383] (6) Aromatic: Trp, Tyr, Phe.

[0384] Non-conservative substitutions entail the exchange of a member of one of these classes for a member of another class.

[0385] A substitution variant comprises substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have an alteration (e.g., an improvement) of certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be routinely prepared, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0386] Alterations (e.g., substitutions) can be made in the HVRs, e.g., to improve antibody affinity. Such alterations can be made in HVR "hotspots", i.e., residues encoded by codons that mutate at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by constructing a secondary library and rescreening therefrom has been described, e.g., in Hoogenboom et al., 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. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4-6 residues simultaneously) are randomized. HVR residues involved in antigen binding can be specifically identified, e.g., using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0387] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, so long as such alterations do not significantly decrease the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as described herein) that do not significantly decrease binding affinity can be made in the HVRs. Such alterations can be, e.g., outside of residues in the HVRs that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0388] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as 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 Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether it affects the interaction of the antibody with the antigen. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex can be analyzed to determine the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they have the desired properties.

[0389] Amino acid sequence insertions include N-terminal and / or C-terminal fusions of polypeptides ranging in length from one residue to polypeptides of more than one hundred residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody to the N- or C-terminus of the antibody.

[0390] b) Glycosylation Variants

[0391] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Adding glycosylation sites to or removing glycosylation sites from an antibody can be readily accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0392] When an antibody contains an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include a variety of carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibodies of the invention can be carried out to produce antibody variants having certain improved properties.

[0393] In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose that is (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) linked to Asn 297, as measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546, for example. Asn297 refers to an asparagine residue located near position 297 (EU numbering of Fc region residues); however, due to small sequence variations in the antibody, Asn297 can also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have enhanced ADCC function. See, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Exemplary variants that are disclosed and relate to "defucosylated" or "fucose-deficient" antibodies include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cell line (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as α-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).

[0394] Antibody variants having bisected oligosaccharides are also provided, e.g., wherein the branched oligosaccharide attached to the antibody Fc region is bisected with GlcNAc. Such antibody variants can have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 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 can have enhanced CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0395] c. Fc Region Variants

[0396] (Sweeping technology)

[0397] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc region is the Fc region of human IgG1.

[0398] To enhance the reduction of plasma antigen concentration and / or improve the pharmacokinetics of an antibody, amino acid residues at the site of binding to FcRn in the Fc region of IgG can be modified to enhance its cellular uptake. When an antibody with pH-dependence is modified in this way, the mutant will be a "sweeping" antibody that can bind more firmly to FcRn and enable efficient transfer of the antigen into endosomes (where the pH is acidic), followed by degradation, but can itself be recycled more efficiently to the cell surface. Compared with the unmodified original (parental) antibody, this modified "sweeping" antibody can bind firmly to FcRn at neutral pH and on the cell surface and enhance antigen uptake and degradation. (Semin Immunopathol. 2018; 40(1): 125-140).

[0399] In some aspects, the antibody comprises an Fc region having at least one amino acid modification within the region to enhance the reduction of plasma antigen concentration and / or improve the pharmacokinetics of the antibody.

[0400] In some embodiments, the Fc region is a human Fc region that has a stronger binding activity to activated Fc gamma receptors than the Fc region of native human IgG1. As mentioned, for example, in WO 2013 / 047752, to enhance the binding activity to activated Fc gamma receptors, one or more amino acids selected from the group consisting of the amino acids at the following positions in the Fc region can be modified: 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 (EU numbering) to amino acids different from the corresponding positions in the Fc region of native human IgG1, which is the parental (original) antibody.

[0401] In some embodiments, the Fc region is a human Fc region that has a stronger binding activity to inhibitory Fc gamma receptors than to activating Fc gamma receptors. As mentioned, for example, in WO 2013 / 125667, to enhance the binding activity to inhibitory Fc gamma receptors, one or more amino acids selected from the group consisting of the amino acids at the following positions can be modified in the Fc region: 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) with amino acids different from the corresponding positions in the native human IgG1 Fc region.

[0402] In some embodiments, the Fc region is a human Fc region that has a stronger binding activity to FcRn at neutral pH than the Fc region of native human IgG1. As mentioned, for example, in WO 2011 / 122011, to enhance the binding activity to FcRn at neutral pH, one or more amino acids selected from the group consisting of the amino acids at the following positions can be modified in the Fc region: 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) with amino acids different from the corresponding positions in the native human IgG1 Fc region.

[0403] In certain embodiments, the present invention contemplates antibody variants that have some but not all effector functions, making them ideal candidates for applications where the antibody in vivo half-life is important while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / elimination of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind FcRn. The major cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is outlined in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the ADCC activity of a test molecule are described in U.S. Patent 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACT1 for flow cytometry TMNon-radioactive cytotoxicity assays (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model as described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0404] Antibodies with reduced effector function include antibodies having substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted to alanine (U.S. Patent No. 7,332,581).

[0405] Certain antibody variants with increased or decreased binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).)

[0406] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that enhance ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0407] In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0408] Antibodies having an increased half-life and increased binding to the neonatal Fc receptor (FcRn) (J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) responsible for transporting maternal IgG to the fetus are described in US2005 / 0014934A1 (Hinton et al.). The antibodies comprise an Fc region having one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also, Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relates to other examples of Fc region variants.

[0409] d. Cysteine Engineered Antibody Variants

[0410] In certain embodiments, it may be desirable to prepare cysteine-engineered antibodies, e.g., "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the replaced residues occur at accessible sites of the antibody. By replacing the residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, thereby generating immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced 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 can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0411] e) Antibody Derivatives

[0412] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-protein moieties known in the art and readily available. Moieties suitable for antibody derivatization 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 / propanediol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in preparation due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of derivatized polymers can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used for therapy under defined conditions, and the like.

[0413] In another embodiment, conjugates of antibodies with non-protein moieties that can be selectively heated by exposure to radiation are 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 have any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein moiety to a temperature at which adjacent antibody-non-protein moiety-containing cells are killed.

[0414] B. Recombinant Methods and Compositions

[0415] Antibodies can be prepared using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-C1s antibody as described herein. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In another embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with): (1) a vector that comprises a nucleic acid that encodes 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 that comprises a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector that comprises a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method for preparing an anti-C1s antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell medium).

[0416] For recombinant preparation of an anti-C1s antibody, a nucleic acid encoding the antibody, such as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such a nucleic acid can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to the genes encoding the antibody heavy and light chains).

[0417] Host cells suitable for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be prepared in bacteria, particularly when glycosylation and Fc effector functions are not required. For the 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 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste into the soluble fraction and further purified.

[0418] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains in which the glycosylation pathways have been "humanized" such that antibodies with partial or complete human glycosylation patterns are produced. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0419] Host cells suitable for the expression of glycosylated antibodies also are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains have been identified which can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0420] Plant cell cultures also can 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 (which describe the PLANTIBODIES TM technology for the production of antibodies in transgenic plants).

[0421] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension culture can be useful. Other examples of useful mammalian host cell lines are the SV40 (COS-7) transformed monkey kidney CV1 cell line; human embryonic kidney cell lines (293 or 293 cells as described, for example, by Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, for example, by Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, for example, by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-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 certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0422] Antibodies with pH-dependent characteristics can be obtained by using screening methods and / or mutagenesis methods, for example, as described in WO2009 / 125825. Screening methods can include any method for identifying antibodies with pH-dependent binding characteristics in a population of antibodies specific for 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 acidic pH and neutral pH. The binding parameters of the antibodies can be measured using, for example, surface plasmon resonance, or any other analytical method that allows for quantitative or qualitative assessment of the binding characteristics of the antibody to the 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.

[0423] In another embodiment, the mutagenesis method may include incorporating deletions, substitutions, or additions of amino acids within the heavy and / or light chains of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In certain embodiments, the mutagenesis can be performed within one or more variable domains of the antibody, such as within one or more HVRs (e.g., CDRs). For example, the mutagenesis may include substituting an amino acid within one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In certain embodiments, the mutagenesis may include substituting one or more amino acids in at least one HVR (e.g., CDR) of the antibody with histidine. In certain embodiments, "enhanced pH-dependent binding" means that the mutant form of the antibody exhibits a greater acidic KD / neutral KD ratio, or a greater acidic kd / neutral kd ratio, than the original "parental" (i.e., less pH-dependent) form of the antibody prior to mutagenesis. In certain embodiments, the mutant form of the antibody has an acidic KD / neutral KD ratio of 2 or greater. Alternatively, the mutant form of the antibody has an acidic kd / neutral kd ratio of 2 or greater.

[0424] Polyclonal antibodies are preferably prepared in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing reagent such as maleimidobenzoyl succinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups.

[0425] The animal (usually a non-human mammal) is immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 µg or 5 µg of the 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 the original amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animal is bled and the antibody titer of the serum is determined. The animal is boosted until the titer plateaus. Preferably, the animal is boosted with conjugates of the same antigen conjugated to different proteins and / or conjugated through different cross-linking agents. The conjugate can also be prepared as a protein fusion in recombinant cell culture. In addition, aggregating agents such as alum are suitable for enhancing the immune response.

[0426] A monoclonal antibody is obtained from a substantially homogeneous population of 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 minor amounts. Thus, the adjective "monoclonal" indicates the antibody characteristic of not being a mixture of discrete antibodies.

[0427] For example, monoclonal antibodies can be prepared using the hybridoma method, which was first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal (such as a hamster) is immunized as described above to cause the production of lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro.

[0428] The immunizing agent will typically include the antigen protein or a fusion variant thereof. Generally, if human-derived cells are desired, peripheral blood lymphocytes (PBL) are used, or if non-human mammalian-derived cells are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortal cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0429] The immortal cell line is typically a transformed mammalian cell, particularly myeloma cells of rodent, bovine, and human origin. Generally, a rat or mouse myeloma cell line is employed. The hybridoma cells thus prepared are inoculated and cultured in a suitable medium, which preferably contains 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 medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which is a substance that blocks the growth of HGPRT-deficient cells.

[0430] Preferred immortalized myeloma cells are those cells that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, murine myeloma cell lines are preferred, such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and the SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human hybrid myeloma 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)).

[0431] For the production of monoclonal antibodies against an antigen, the medium in which the hybridoma cells are cultured is assayed. 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, the binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107(1):220-239 (1980).

[0432] After identifying the hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution and cultured by standard methods (Goding, supra). Media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo as tumors in mammals.

[0433] The monoclonal antibodies secreted by the subclones are suitably separated from the medium, ascites, or serum by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0434] C. Assays

[0435] The anti-C1s antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0436] 1. Binding Assays and Other Assays

[0437] In one aspect, for example, the antigen-binding activity of the antibodies of the invention can be tested by known methods such as ELISA, Western blotting, etc.

[0438] In another aspect, competitive assays can be used to identify antibodies that compete with any of the anti-C1s antibodies described herein for binding to C1s, or to identify antibodies that bind to the same epitope as any of the anti-C1s antibodies described herein. In certain embodiments, when such a competing antibody is present in excess, it blocks (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., linear or conformational epitope) bound by any of the anti-C1s antibodies described herein. Detailed exemplary methods for epitope mapping of antibody binding are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In certain embodiments, such a competitive assay can be performed under neutral pH conditions. In some embodiments, the competitive assay is a tandem competitive assay using, for example, the Octet TM system.

[0439] In an exemplary competition assay, immobilized C1s is incubated in a solution comprising a first labeled antibody that binds C1s (e.g., one of those described herein) and a second unlabeled antibody, and the ability of the second unlabeled antibody to compete with the first antibody for binding to C1s is tested. The second antibody can be present in a hybridoma supernatant. As a control, immobilized C1s is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that permit the first antibody to bind to C1s, excess unbound antibody is removed and the amount of label bound to the immobilized C1s is measured. If the amount of label bound to the immobilized C1s is significantly reduced in the test sample relative 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).

[0440] In another aspect, a sandwich assay can be used to identify antibodies that bind the same epitope as the anti-C1s antibodies provided herein or that compete with the anti-C1s antibodies provided herein for binding to C1s. A sandwich assay involves the use of two antibodies, each of which binds a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thereby forming an insoluble three-part complex. See David & Greene, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay), or it can be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. Antibodies that bind C1s simultaneously with the anti-C1s antibodies provided herein can be identified as antibodies that bind a different epitope from the anti-C1s antibodies. Thus, antibodies that do not bind C1s simultaneously with the anti-C1s antibodies provided herein can be identified as antibodies that bind the same epitope as the anti-C1s antibodies or that compete with the anti-C1s antibodies for binding to C1s.

[0441] 2. Activity assays

[0442] In one aspect, assays are provided for identifying anti-C1s antibodies that have biological activity. Biological activity can include blocking activation of the classical pathway and generation of the cleavage products C2a, C2b, C3a, C3b, C4a, C4b, C5a, and C5b resulting from activation of that pathway. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0443] In certain embodiments, such biological activity of the antibodies of the invention is tested. In some embodiments, the ability of the antibodies of the invention to inhibit complement-mediated hemolysis of sheep red blood cells (RBCs) that have been sensitized with antibodies against sheep RBC antigens, i.e., using an RBC assay, can be evaluated. In some embodiments, the ability of the antibodies of the invention to inhibit complement-mediated hemolysis of chicken red blood cells (cRBCs) that have been sensitized with antibodies against cRBC antigens can be evaluated. Using human serum as a source of complement proteins, the activity of the antibodies of the invention can be determined by measuring the amount of hemoglobin released spectrophotometrically.

[0444] The RBC assay can be appropriately performed using known methods (e.g., the method disclosed in J. Vis. Exp. 2010; (37): 1923). How to perform a 50% hemolytic complement (CH50) assay as an RBC lysis assay is described herein. Briefly, this assay measures the activation of the classical complement pathway and detects a reduction, absence, or inactivation of any component in this pathway. It assesses the activity of complement components in serum to lyse red blood cells. When an antibody is incubated with the test serum, this pathway is activated and causes hemolysis. If one or more components of the classical pathway are reduced, the CH50 value is reduced. The CH50 assay is not exactly the same as the assay used in the examples herein, but rather measures the percentage inhibition of complement components on cell lysis; however, the concept and basic setup are essentially the same as those of the present invention. In the present invention, in one embodiment, the RBC assay is performed as follows. Human serum is pre-incubated with the antibody of interest (e.g., incubated at 37 degrees Celsius (°C) for 3 hours). Then the serum is added to an equal volume of sensitized sheep red blood cells and incubated (e.g., for 1 hour at 37°C) to allow lysis of the red blood cells. Then the reaction is stopped. The mixture is centrifuged to precipitate the undissolved cells, the supernatant is taken out, and the release of hemoglobin is analyzed by subtracting the OD at 630 nm from the OD at 415 nm. To calculate the percentage inhibition of red blood cell lysis, 0% inhibition is set as the condition without adding an antibody (only buffer), and 100% inhibition is set as the condition with the addition of EDTA at a final concentration of 5 mM (see, e.g., Example 7). When the antibody shows a percentage of inhibition of red blood cell lysis, this means that the antibody has neutralizing activity against human serum complement, e.g., the activity of inhibiting the interaction between C1q and C1r2s2 complexes.

[0445] Thus, to evaluate the activity of inhibiting the interaction between C1q and C1r2s2 complex, the RBC assay can be used to assess the neutralizing activity of the antibodies of the present invention against human serum complement. In one embodiment, the present invention provides an isolated antibody that inhibits the interaction between C1q and C1r2s2 complex, wherein the antibody has at least 70% neutralizing activity against human serum complement in the RBC assay.

[0446] D. Immunoconjugates

[0447] The present invention also provides immunoconjugates comprising an anti-C1s antibody herein, which is conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes.

[0448] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Patent 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 Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see 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; trichothecene; and CC1065.

[0449] In another embodiment, the immunoconjugate comprises an antibody as described herein, the antibody conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments 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, enomycin, phenomycin, and tricothecene.

[0450] In another embodiment, the immunoconjugate comprises an antibody as described herein, the antibody conjugated to a radioactive atom to form a radio conjugate. A variety of radioisotopes can be used to prepare radio conjugates. Examples include 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioisotopes of Pb and Lu. When the radio conjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, such as Tc-99m or 123I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0451] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane hydrochloride (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazo compounds (such as bis(p-azidobenzoyl)hexanediamine), di-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and di-active fluorine compounds (such as 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-methyl-diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" which facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0452] Immunoconjugates or ADCs herein are expressly contemplated, but not limited to, conjugates prepared using cross-linking agent reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC and thio-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0453] E. Methods and Compositions for Diagnosis and Detection

[0454] In certain embodiments, any of the anti-C1s antibodies provided herein can be used to detect the presence of C1s in a biological sample. As used herein, the term "detect" includes quantitative detection or qualitative detection. In certain embodiments, the biological sample includes cells or tissues such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, breast secretion, lymph fluid, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, aqueous humor of the eye or mucus.

[0455] In one embodiment, an anti-C1s antibody is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of C1s in a biological sample is provided. In certain embodiments, the method includes contacting the biological sample with an anti-C1s antibody as described herein under conditions that permit the anti-C1s antibody to bind to C1s, and detecting whether a complex is formed between the anti-C1s antibody and C1s. Such a method can be an in vitro or in vivo method. In one embodiment, the anti-C1s antibody is used to select a subject suitable for treatment with the anti-C1s antibody, for example, where C1s is a biomarker for selecting a patient.

[0456] Exemplary disorders that can be diagnosed using the antibodies of the present invention include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argentophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, Clq nephropathy, cancer, catastrophic 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 tangles with calcification, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, hemolytic-uremic syndrome, hereditary angioedema, hypophosphastasis, idiopathic pneumonia syndrome, immune complex diseases, inclusion body myositis, infectious diseases (e.g., diseases caused by bacteria (e.g., Neisseria meningitidis or Streptococcus), viruses (e.g., 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 nephropathy, multi-infarct dementia, lupus (e.g., 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-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic parkinsonism, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis,Tangle only dementia, transplant rejection, vasculitis (e.g., ANCA - associated vasculitis), Wegner'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, acquired epidermolysis bullosa, delayed hemolytic transfusion reaction, low - complement urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractoriness.

[0457] In certain embodiments, a labeled anti - C1s antibody is provided. Labels include, but are not limited to, labels or moieties for direct detection (such as fluorescence, chromophore, electron density, chemiluminescence, and radiolabels), as well as moieties for indirect detection such as enzymes or ligands, e.g., by enzyme reaction or molecular interaction. Exemplary labels include, but are not limited to, radioactive isotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3 - dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β - galactosidase, glucoamylase, lysozyme, glucose oxidases such as glucose oxidase, galactose oxidase, and glucose - 6 - phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those enzymes that are coupled to utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0458] F. Pharmaceutical Formulations

[0459] The pharmaceutical formulation of the anti-C1s antibody as described herein is prepared in the form of a lyophilized formulation or an aqueous solution by mixing the antibody having the desired degree of purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)). The pharmaceutical carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as cetrimonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (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, aspartic acid, 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 or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0460] 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 WO 2006 / 044908, the latter formulation containing a histidine-acetate buffer.

[0461] When needed, the formulations herein may also contain more than one active ingredient for a particular indication for treatment, preferably those having complementary activities that do not have an adverse effect on each other. For example, it may be desirable to further provide a formulation for combination therapy. Such active ingredients are suitably present in the combination in an amount effective for the intended use.

[0462] The active ingredient can be encapsulated in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization respectively, such as hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in a coarse emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980).

[0463] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices being in the form of a physical article, e.g., a film or a microcapsule.

[0464] Formulations suitable for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0465] G. Methods of Treatment and Compositions

[0466] Any anti-C1s antibody provided herein can be used in a method of treatment.

[0467] In one aspect, there is provided an anti-C1s antibody for use as a medicament. In a further aspect, there is provided an anti-C1s antibody for the treatment of a complement-mediated disease or disorder. In certain embodiments, there is provided an anti-C1s antibody for use in a method of treatment. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method of treating an individual suffering from a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of the 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.

[0468] In further embodiments, the invention provides anti-C1s antibodies for treating complement-mediated diseases or disorders. In further embodiments, the anti-C1s antibodies of the invention can be used to enhance the clearance of C1s from plasma. In further embodiments, the anti-C1s antibodies of the invention can be used to enhance the clearance of C1r2s2 from plasma. In further embodiments, the anti-C1s antibodies of the invention can be used to enhance the clearance of C1r2s2 from plasma rather than C1q from plasma. In some cases, the antibody inhibits components of the classical complement pathway; in some cases, the classical complement pathway component is Cls. In certain embodiments, the invention provides anti-C1s antibodies in methods for treating complement-mediated diseases or disorders. In certain embodiments, the invention provides anti-C1s antibodies in methods for enhancing the clearance of C1s from plasma. In certain embodiments, the invention provides anti-C1s antibodies in methods for enhancing the clearance of C1r2s2 from plasma. In certain embodiments, the invention provides anti-C1s antibodies in methods for enhancing the clearance of C1r2s2 from plasma rather than the clearance of C1q from plasma. In certain embodiments, the invention provides anti-C1s antibodies in methods for inhibiting components of the classical complement pathway; in certain cases, the classical complement pathway component is Cls. The "individual" according to any of the above embodiments is preferably a human.

[0469] In one aspect, the present disclosure provides a method of modulating complement activation. In some embodiments, the method inhibits complement activation, such as reducing the production of C4b2a. In some embodiments, the present disclosure provides a method of modulating complement activation in an individual having a complement-mediated disease or disorder, the method 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 a human. Administration can be by any route known to those of skill in the art, including those disclosed herein. In some embodiments, the administration is intravenous or subcutaneous. In some embodiments, the administration is intrathecal.

[0470] A complement-mediated disease or disorder is a disorder characterized by an abnormal amount of complement C1s or an abnormal level of complement C1s proteolytic activity in the cells, tissues or fluids of an individual.

[0471] In some cases, complement-mediated diseases or disorders are characterized by the presence of elevated (above normal levels) Cls or elevated complement Cls activity in cells, tissues or fluids. For example, in certain cases, complement-mediated diseases or disorders are characterized by the presence of elevated levels and / or activity of Cls in brain tissue and / or cerebrospinal fluid. An "above normal" amount of Cls in a cell, tissue or fluid means that the amount of Cls in the cell, tissue or fluid is higher than the normal control level, e.g., higher than the normal control level of an individual or population of the same age group. An "above normal level" of Cls activity in a cell, tissue or fluid means that the proteolytic cleavage affected by Cls in the cell, tissue or fluid is higher than the normal control level, e.g., higher than the normal control level of an individual or population of the same age group. In certain cases, an individual suffering from a complement-mediated disease or disorder exhibits one or more other symptoms of such disease or disorder.

[0472] In other cases, complement-mediated diseases or disorders are characterized by the presence of less than normal amounts of Cls or the presence of lower levels of complement Cls activity in cells, tissues or fluids. For example, in some cases, complement-mediated diseases or disorders are characterized by the presence of lower amounts and / or lower activity of Cls in brain tissue and / or cerebrospinal fluid. A "less than normal" amount of Cls in a cell, tissue or fluid means that the amount of Cls in the cell, tissue or fluid is lower than the normal control level, e.g., lower than the normal control level of an individual or population of the same age group. A "less than normal" level of Cls activity in a cell, tissue or fluid means that the proteolytic cleavage affected by Cls in the cell, tissue or fluid is lower than the normal control level, e.g., lower than the normal control level of an individual or population of the same age group. In certain cases, an individual suffering from a complement-mediated disease or disorder exhibits one or more other symptoms of such disease or disorder.

[0473] Complement-mediated diseases or disorders are diseases or disorders in which the amount or activity of complement C1s causes it to cause a disease or disorder in an individual. In some embodiments, complement-mediated diseases or disorders are selected from the group consisting of: autoimmune diseases, cancers, blood diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, eye diseases, kidney diseases, transplant rejection, vascular diseases, and vasculitis diseases. In some embodiments, the complement-mediated disease or disorder is an autoimmune disease. In some embodiments, the complement-mediated disease or disorder is a 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 disease. In some embodiments, the complement-mediated disease or disorder is ischemia-reperfusion injury. 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 vasculitis disorder. 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 disease is a neurodegenerative disorder. In some embodiments, the complement-mediated disease or disorder is a tauopathy.

[0474] Examples of complement-mediated diseases or disorders include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argentophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, Clq nephropathy, cancer, catastrophic 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 tangles with calcification, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, hemolytic-uremic syndrome, hereditary angioedema, hypophosphastasis, idiopathic pneumonia syndrome, immune complex diseases, inclusion body myositis, infectious diseases (e.g., diseases caused by bacteria (e.g., Neisseria meningitidis or Streptococcus), viruses (e.g., 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 nephropathy, multi-infarct dementia, lupus (e.g., 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-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic parkinsonism, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis,Tangle only dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner'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, acquired epidermolysis bullosa, delayed hemolytic transfusion reaction, low complement urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractoriness.

[0475] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with parkinsonism associated with chromosome 17) are the most common forms of tauopathies. Correspondingly, the present invention relates to any method as described above, wherein the tauopathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with parkinsonism associated with chromosome 17. Other tauopathies include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

[0476] Neurodegenerative tauopathies include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinsonism-dementia complex, argentophilic grain dementia, British-type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, pugilistic dementia, diffuse neurofibrillary tangles with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism associated with chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, 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 parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0477] The present disclosure also provides methods for 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 by the methods of the present disclosure.

[0478] In some embodiments, complement-mediated diseases or disorders include Alzheimer's disease. In some embodiments, complement-mediated diseases or disorders include Parkinson's disease. In some embodiments, complement-mediated diseases or disorders include transplant rejection. In some embodiments, the complement-mediated disease or disorder is antibody-mediated transplant rejection.

[0479] 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, cancers, hematological diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, kidney diseases, transplant rejection, eye diseases, vascular diseases, or vasculitis disorders. Symptoms can be neurological symptoms, e.g., impaired cognitive function, memory impairment, loss of motor function, etc. Symptoms can also be the C1s protein activity in the cells, tissues, or fluids of an individual. Symptoms can also be the degree of complement activation in the cells, tissues, or fluids of an individual.

[0480] In some embodiments, administering the anti-C1s antibodies of the present disclosure to an individual modulates complement activation in the cells, tissues, or fluids of the individual. In some embodiments, administering the anti-C1s antibodies of the present disclosure to an individual inhibits complement activation in the cells, tissues, or fluids of the individual. For example, in some embodiments, when the anti-C1s antibodies of the present disclosure are administered to an individual suffering from a complement-mediated disease or disorder in one or more doses in the form of monotherapy or combination therapy, the complement activation in the individual is inhibited 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 complement activity in the individual before treatment with the anti-C1s antibody.

[0481] In some embodiments, the anti-C1s antibodies of the present disclosure reduce the deposition of C3 on red blood cells; for example, in some embodiments, the anti-C1s antibodies of the present disclosure reduce the 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.

[0482] 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.

[0483] In some embodiments, administration of an anti-C1s antibody of the present disclosure results in a result selected from the group consisting of: (a) reduced complement activation; (b) improved cognitive function; (c) reduced neuronal loss; (d) reduced levels of phosphorylated Tau protein 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 erythrocyte lysis; (n) reduced erythrophagocytosis; (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 thrombosis; (v) improved coagulation; (w) improved renal function; (x) reduced antibody-mediated complement activation; (y) reduced autoantibody-mediated complement activation; (z) improved anemia; (aa) reduced demyelination; (ab) reduced eosinophilia; (ac) reduced deposition of C3 on erythrocytes (e.g., reduced deposition of C3b, iC3b, etc. on erythrocytes); (ad) reduced deposition of C3 on platelets (e.g., reduced deposition of C3b, iC3b, etc. on platelets); (ae) reduced generation of anaphylatoxins; (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 erythrocyte destruction due to infusion reactions; (ak) reduced erythrocyte lysis due to alloantibodies; (al) reduced hemolysis due to infusion reactions; (am) reduced alloantibody-mediated platelet lysis; (an) reduced platelet lysis due to infusion reactions; (ao) reduced mast cell activation; (ap) reduced mast cell histamine release; (aq) reduced vascular permeability; (ar) reduced edema; (as) reduced deposition of complement on graft endothelium; (at) reduced generation of anaphylatoxins in graft vascular endothelium; (au) reduced separation of the dermal-epidermal junction; (av) reduced generation of anaphylatoxins at the dermal-epidermal junction; (aw) reduced alloantibody-mediated complement activation in transplanted vascular endothelium; (ax) reduced antibody-mediated loss of neuromuscular junctions; (ay) reduced complement activation at the neuromuscular junction; (az) reduced generation of anaphylatoxins at the neuromuscular junction; (ba) reduced complement deposition at the neuromuscular junction; (bb) reduced paralysis; (be) reduced numbness; (bd) enhanced bladder control; (be) enhanced bowel control; (bf) reduced mortality associated with autoantibodies;(bg)Reduced incidence associated with autoantibodies.;

[0484] In some embodiments, when the anti-C1s antibodies of the present disclosure are administered to an individual having a complement-mediated disease or disorder in one or more doses, either as monotherapy or in combination therapy, 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% reduction in one or more of the following outcomes can be achieved compared to the level or degree of the outcome in the individual prior to treatment with the anti-C1s antibody: (a) Complement activation; (b) Cognitive decline; (c) Neuronal loss; (d) Phosphorylated Tau levels in neurons; (e) Glial cell 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) Erythrophagocytosis; (o) Platelet phagocytosis; (p) Platelet lysis; (q) Graft rejection; (r) Macrophage-mediated phagocytosis; (s) Visual loss; (t) Antibody-mediated complement activation; (u) Autoantibody-mediated complement activation; (v) Demyelination; (w) Eosinophilia.

[0485] In some embodiments, when the anti-C1s antibodies of the present disclosure are administered to an individual having a complement-mediated disease or disorder in one or more doses, either as monotherapy or in combination therapy, 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% improvement in one or more of the following outcomes can be achieved compared to the level or degree of the outcome in the individual prior to treatment with the anti-C1s antibody: a) Cognitive function; b) Graft survival rate; c) Vision; d) Motor control; e) Thrombosis; f) Coagulation; g) Renal function; and h) Hematocrit (erythrocyte count).

[0486] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual having a complement-mediated disease or disorder in one or more doses in a monotherapy or combination therapy, the complement activation in the individual is reduced 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 complement activation in the individual before treatment with the anti-C1s antibody.

[0487] In some embodiments, administering an anti-C1s antibody of the present disclosure improves cognitive function in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual having a complement-mediated disease or disorder in one or more doses in a monotherapy or combination therapy, the cognitive function in the individual is improved 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.

[0488] In some embodiments, administering an anti-C1s antibody of the present disclosure reduces the rate of decline in cognitive function in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual having a complement-mediated disease or disorder in one or more doses in a monotherapy or combination therapy, the rate of decline in cognitive function in the individual is reduced 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 decline in cognitive function in the individual before treatment with the anti-C1s antibody.

[0489] In some embodiments, administering an anti-C1s antibody of the present disclosure reduces neuronal loss in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual having a complement-mediated disease or disorder in one or more doses in a monotherapy or combination therapy, the neuronal loss in the individual is reduced 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 neuronal loss in the individual before treatment with the anti-C1s antibody.

[0490] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces the level of phosphorylated Tau in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in a single therapy or combination therapy in one or more doses, the phosphorylated Tau in the individual is reduced 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 of phosphorylated Tau in the individual before treatment with the anti-C1s antibody.

[0491] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces glial cell activation in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in a single therapy or combination therapy in one or more doses, the glial cell activation in the individual is reduced 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 glial cell activation in the individual before treatment with the anti-C1s antibody. In some embodiments, the glial cells are astrocytes or microglia.

[0492] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces lymphocyte infiltration in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in a single therapy or combination therapy in one or more doses, the lymphocyte infiltration in the individual is reduced 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.

[0493] 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, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in one or more doses in the form of monotherapy or combination therapy, the macrophage infiltration in the individual is reduced 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.

[0494] 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, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in one or more doses in the form of monotherapy or combination therapy, the antibody deposition in the individual is reduced 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 antibody deposition in the individual before treatment with the anti-C1s antibody.

[0495] In some embodiments, administration of an anti-C1s antibody of the present disclosure to an individual reduces the production of anaphylatoxins (e.g., C3a, C4a, C5a) in the individual. For example, in some embodiments, when the anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or disorder in one or more doses as monotherapy or combination therapy, the production of anaphylatoxins in the individual is reduced 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 production of anaphylatoxins in the individual before treatment with the anti-C1s antibody.

[0496] In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising the anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient in the treatment of 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 in the treatment of an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising the anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient in the treatment of an individual having a complement-mediated disease or disorder.

[0497] In some embodiments, the present disclosure provides the use of the anti-C1s antibodies of the present disclosure in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

[0498] In some embodiments, the present disclosure provides the use of the anti-C1s antibodies of the present disclosure or a pharmaceutical composition comprising the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient in inhibiting complement activation. In some embodiments, the present disclosure provides the use of the anti-C1s antibodies of the present disclosure or a pharmaceutical composition comprising the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient in inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of the anti-C1s antibodies of the present disclosure in inhibiting 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 the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient in inhibiting complement activation in an individual having a complement-mediated disease or disorder.

[0499] In some embodiments, the present disclosure provides the use of the anti-C1s antibodies of the present disclosure in the manufacture of a medicament for regulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in an individual having a complement-mediated disease or disorder.

[0500] In some embodiments, the present disclosure provides the anti-C1s antibodies of the present disclosure or a pharmaceutical composition comprising the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient for medical treatment. In some embodiments, the present disclosure provides the anti-C1s antibodies of the present disclosure for medical treatment. In some embodiments, the present disclosure provides a pharmaceutical composition comprising the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient for medical treatment.

[0501] In some embodiments, the present disclosure provides the anti-C1s antibodies of the present disclosure or a pharmaceutical composition comprising the anti-C1s antibodies 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 the anti-C1s antibodies 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 the anti-C1s antibodies of the present disclosure and a pharmaceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder.

[0502] In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure or a pharmaceutical composition comprising the 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 the 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.

[0503] In another aspect, the present invention provides the 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 another embodiment, the medicament is for a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual suffering from a complement-mediated disease or disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as a therapeutic agent as described below. In another embodiment, the medicament is for enhancing the clearance (or removal) of Cls from plasma. In another embodiment, the medicament is for enhancing the clearance (or removal) of the C1r2s2 complex from plasma. In another embodiment, the medicament is for enhancing the clearance (or removal) of C1r2s2 from plasma rather than Clq from plasma. In another embodiment, the medicament is for inhibiting a component of the classical complement pathway; in certain cases, the classical complement pathway component is Cls.

[0504] In another embodiment, the medicament is for a method of treating an individual suffering from a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of the medicament. The "individual" according to any of the above embodiments can be a human.

[0505] In another aspect, the present invention provides a method for treating a complement-mediated disease or disorder. In one embodiment, the method comprises administering to an individual suffering from the complement-mediated disease or disorder 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, as described below. The "individual" according to any of the above embodiments can be a human.

[0506] In another aspect, the present invention provides a method for enhancing the clearance (or removal) of Cls from the plasma of an individual. In another aspect, the present invention provides a method for enhancing the clearance (or removal) of C1r2s2 from the plasma in an individual. In another aspect, the present invention provides a method for enhancing the clearance (or removal) of C1r2s2 from the plasma rather than Clq from the plasma in an individual. In certain cases, the present invention provides a method for inhibiting components of the classical complement pathway in an individual; in certain cases, the component of the classical complement pathway is Cls. In one embodiment, the "individual" is a human.

[0507] In another aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-C1s antibodies provided herein for use in any of the above treatment methods, for example. 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, such as the therapeutic agents described below.

[0508] The antibodies of the present invention can be used alone for treatment or in combination with other reagents for treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent.

[0509] Such combination treatments as described above include co-administration (wherein more than two therapeutic agents are included in the same or separate formulations) and separate administration, in which case the administration of the antibody of the present invention can occur before, simultaneously with, and / or after the administration of the additional therapeutic agent or reagent. In one embodiment, the administration of the anti-C1s antibody and the administration of the additional therapeutic agent occur within about one month of each other, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days. The antibodies of the present invention can also be used in combination with radiotherapy.

[0510] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral administration, intratracheal administration, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Medication can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of medication regimens are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse injection.

[0511] The antibodies of the invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered herein include the particular disease to be treated, the particular mammal being treated, the clinical condition of the individual patient, the cause, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the medical practitioner. The antibodies need not, but optionally, be formulated with one or more additional agents currently used to prevent or treat the targeted disease. The effective amount of such other agent depends on the amount of antibody present in the formulation, the type of disease or treatment, and the other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and any route empirically / clinically determined to be appropriate.

[0512] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibodies are suitably administered to the patient either as a single dose or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage of antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) can be used for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage can be about 1 μg / kg to over 100 mg / kg, depending on the factors mentioned above. For repeated administration over several days or longer, the treatment is generally continued until a desired suppression of disease symptoms occurs, depending on the circumsta...

Claims

1. An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has a displacing function such that the antibody binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex.

2. The antibody according to claim 1, wherein the antibody binds to the C1qrs complex on a BIACORE (registered trademark) chip and promotes the dissociation of C1q from the C1qrs complex, wherein, When sufficient time has elapsed, as determined by BIACORE® assay, the value of the response unit (RU) in the presence of the antibody is lower than the value of the response unit (RU) in the absence of the antibody.

3. The antibody of claim 2, wherein the cross time point in the BIACORE® assay is within 1000 s after the start time point of antibody injection, as determined by BIACORE® assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.

4. The antibody of claim 2, wherein almost all of the C1q dissociates from the C1qrs complex within 2000 s after the start time point of antibody injection, as determined by BIACORE® assay using the following conditions: the capture levels of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500 nM, 10 μL / min.

5. An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has at least 70% neutralizing activity against human serum complement in an RBC assay.

6. The antibody of any one of claims 1 to 5, wherein the antibody is an antibody that specifically binds to C1s or an antibody that specifically binds to C1r.

7. An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s and competes for binding to the epitope with an antibody selected from the group consisting of the following 1)-5): 1) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 32, the HVR-H2 sequence of SEQ ID NO: 33, the HVR-H3 sequence of SEQ ID NO: 34, the HVR-L1 sequence of SEQ ID NO: 35, the HVR-L2 sequence of SEQ ID NO: 36, and the HVR-L3 sequence of SEQ ID NO: 37, 2) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 38, the HVR-H2 sequence of SEQ ID NO: 39, the HVR-H3 sequence of SEQ ID NO: 40, the HVR-L1 sequence of SEQ ID NO: 41, the HVR-L2 sequence of SEQ ID NO: 42, and the HVR-L3 sequence of SEQ ID NO: 43, 3) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 44, the HVR-H2 sequence of SEQ ID NO: 45, the HVR-H3 sequence of SEQ ID NO: 46, the HVR-L1 sequence of SEQ ID NO: 47, the HVR-L2 sequence of SEQ ID NO: 48, and the HVR-L3 sequence of SEQ ID NO: 49, 4) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 50, the HVR-H2 sequence of SEQ ID NO: 51, the HVR-H3 sequence of SEQ ID NO: 52, the HVR-L1 sequence of SEQ ID NO: 53, the HVR-L2 sequence of SEQ ID NO: 54, and the HVR-L3 sequence of SEQ ID NO: 55, and 5) 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: 59, the HVR-L2 sequence of SEQ ID NO: 60, and the HVR-L3 sequence of SEQ ID NO: 61, or wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r and competes for binding to the epitope with an antibody selected from the group consisting of 6)-13): 6) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 119, the HVR-H2 sequence of SEQ ID NO: 127, the HVR-H3 sequence of SEQ ID NO: 135, the HVR-L1 sequence of SEQ ID NO: 143, the HVR-L2 sequence of SEQ ID NO: 151, and the HVR-L3 sequence of SEQ ID NO: 159, 7) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 120, the HVR-H2 sequence of SEQ ID NO: 128, the HVR-H3 sequence of SEQ ID NO: 136, the HVR-L1 sequence of SEQ ID NO: 144, the HVR-L2 sequence of SEQ ID NO: 152, and the HVR-L3 sequence of SEQ ID NO: 160, 8) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 121, the HVR-H2 sequence of SEQ ID NO: 129, the HVR-H3 sequence of SEQ ID NO: 137, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 153, and the HVR-L3 sequence of SEQ ID NO: 161, 9) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 122, the HVR-H2 sequence of SEQ ID NO: 130, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 146, the HVR-L2 sequence of SEQ ID NO: 154, and the HVR-L3 sequence of SEQ ID NO: 162, 10) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 123, the HVR-H2 sequence of SEQ ID NO: 131, the HVR-H3 sequence of SEQ ID NO: 139, the HVR-L1 sequence of SEQ ID NO: 147, the HVR-L2 sequence of SEQ ID NO: 155, and the HVR-L3 sequence of SEQ ID NO: 163, 11) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 124, the HVR-H2 sequence of SEQ ID NO: 132, the HVR-H3 sequence of SEQ ID NO: 140, the HVR-L1 sequence of SEQ ID NO: 148, the HVR-L2 sequence of SEQ ID NO: 156, and the HVR-L3 sequence of SEQ ID NO: 164, 12) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 125, the HVR-H2 sequence of SEQ ID NO: 133, the HVR-H3 sequence of SEQ ID NO: 141, the HVR-L1 sequence of SEQ ID NO: 149, the HVR-L2 sequence of SEQ ID NO: 157, and the HVR-L3 sequence of SEQ ID NO: 165, and 13) An antibody comprising the HVR-H1 sequence of SEQ ID NO: 126, the HVR-H2 sequence of SEQ ID NO: 134, the HVR-H3 sequence of SEQ ID NO: 142, the HVR-L1 sequence of SEQ ID NO: 150, the HVR-L2 sequence of SEQ ID NO: 158, and the HVR-L3 sequence of SEQ ID NO:

166.

8. An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.

4.

9. The antibody according to any one of claims 1 to 8, wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s or C1r, and wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.

4.

10. The antibody according to claim 9, wherein the antibody has a lower affinity for C1s or C1r at acidic pH than at neutral pH, as described in (i) or (ii) below: (i) When measured at high calcium concentration 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 2 or greater, (ii) When measured at high calcium concentration at neutral pH and at low calcium concentration 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.

11. The antibody according to any one of claims 8 to 10, wherein the antibody comprises an Fc region having at least one amino acid modification within the region, thereby enhancing the reduction of plasma antigen concentration and / or improving the pharmacokinetics of the antibody.

12. The antibody according to claim 11, wherein the antibody enhances the reduction of plasma antigen concentration, and wherein the Fc region is a human Fc region having binding activity selected from the group consisting of: a) Binding activity to activated Fc gamma receptor stronger than that of the Fc region of natural human IgG1, b) Binding activity to inhibitory Fc gamma receptor stronger than binding activity to activated Fc gamma receptor, and c) Binding activity to FcRn at neutral pH stronger than that of the Fc region of natural human IgG1.

13. The antibody according to any one of claims 1 to 12, wherein the antibody binds to both cynomolgus monkey C1s and human C1s, or to both cynomolgus monkey C1r and human C1r.

14. A pharmaceutical formulation comprising the antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A method of treating an individual suffering from a complement-mediated disease or disorder, comprising administering to the individual an effective amount of the antibody according to any one of claims 1 to 13.

16. An isolated anti-complement component 1s (Cls) antibody that specifically binds to an epitope within a region encompassing the CUB1-EGF-CUB2 domain, which CUB1-EGF-CUB2 domain is composed of CUB1, EGF, and CUB2 of Cls.

17. The antibody according to claim 16, which does not bind to the CCP1-CCP2-SP domain of C1s.

18. The antibody according to claim 16, wherein the epitope bound by the antibody is not an epitope located in the beta domain of C1s.

19. The antibody according to claim 16, wherein the epitope bound by the antibody is an epitope located in the alpha domain or the gamma domain of C1s.

20. The antibody according to claim 16, wherein the epitope bound by the antibody is a linear epitope.

21. The antibody according to claim 16, wherein the epitope to which the antibody binds is an epitope within any of the following: amino acids 16 - 291 of C1s, amino acids 16 - 172 of C1s, amino acids 16 - 210 of C1s, amino acids 16 - 111 of C1s, amino acids 112 - 210 of C1s, amino acids 131 - 172 of C1s, or amino acids 16 - 130 of C1s.

22. The antibody according to claim 16, wherein the epitope to which the antibody binds is an epitope of human C1s, or an epitope of human C1s and an epitope of cynomolgus monkey C1s.

23. The antibody according to claim 16, which comprises (a) HVR - H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50 or 56; (b) HVR - H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51 or 57; and (c) HVR - H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52 or 58, wherein the antibody comprises framework regions of human or primate origin.

24. The antibody according to claim 16, which comprises (a) HVR - L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53 or 59; (b) HVR - L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54 or 60; and (c) HVR - L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55 or 61, wherein the antibody comprises framework regions of human or primate origin.

25. The antibody according to claim 16, which comprises (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 19, 20, 21, 23 or 24; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 26, 27, 28, 30 or 31; or (c) the VH sequence of (a) and the VL sequence of (b).

26. The antibody according to claim 16, which comprises the VH sequence of SEQ ID NO: 19, 20, 21, 23 or 24.

27. The antibody according to claim 16, which comprises the VL sequence of SEQ ID NO: 26, 27, 28, 30 or 31.

28. The antibody according to claim 25, which comprises the VH sequence of SEQ ID NO: 19, 20, 21, 23 or 24 and the VL sequence of SEQ ID NO: 26, 27, 28, 30 or 31.

29. The antibody according to claim 28, which comprises any one of the following combinations of VH and VL sequences: (a) the VH sequence of SEQ ID NO:19 and the VL sequence of SEQ ID NO:26; (b) The VH sequence of SEQ ID NO:20 and the VL sequence of SEQ ID NO:27; (c) The VH sequence of SEQ ID NO:21 and the VL sequence of SEQ ID NO:28; (d) The VH sequence of SEQ ID NO:23 and the VL sequence of SEQ ID NO:30; and (e) The VH sequence of SEQ ID NO:24 and the VL sequence of SEQ ID NO:

31.

30. An isolated anti-complement component 1r (Clr) antibody that specifically binds to an epitope within a region encompassing the CUB1-EGF-CUB2 domain, wherein the CUB1-EGF-CUB2 domain is composed of CUB1, EGF, and CUB2 of Clr.

31. The antibody according to claim 30, which does not bind to the CCP1-CCP2-SP domain of C1r.

32. The antibody according to claim 30, wherein the epitope to which the antibody binds is a linear epitope or a conformational epitope.

33. The antibody according to claim 30, wherein the epitope to which the antibody binds is an epitope of human C1r, or an epitope of human C1r and an epitope of cynomolgus monkey C1r.

34. The antibody according to claim 30, which comprises (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142, wherein the antibody comprises framework regions of human or primate origin.

35. The antibody according to claim 30, which comprises (a) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 comprising an amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166, wherein the antibody comprises framework regions of human or primate origin.

36. The antibody according to claim 30, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118; or (c) the VH sequence of (a) and the VL sequence of (b).

37. The antibody according to claim 30, comprising the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110.

38. The antibody according to claim 30, comprising the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118.

39. The antibody according to claim 36, comprising the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110 and the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118.

40. The antibody according to claim 39, comprising any one of the following combinations of VH and VL sequences: (a) the VH sequence of SEQ ID NO: 103 and the VL sequence of SEQ ID NO: 111; (b) the VH sequence of SEQ ID NO: 104 and the VL sequence of SEQ ID NO: 112; (c) the VH sequence of SEQ ID NO: 105 and the VL sequence of SEQ ID NO: 113; (d) the VH sequence of SEQ ID NO: 106 and the VL sequence of SEQ ID NO: 114; (e) the VH sequence of SEQ ID NO: 107 and the VL sequence of SEQ ID NO: 115; (f) the VH sequence of SEQ ID NO: 108 and the VL sequence of SEQ ID NO: 116; (g) the VH sequence of SEQ ID NO: 109 and the VL sequence of SEQ ID NO: 117; and (h) the VH sequence of SEQ ID NO: 110 and the VL sequence of SEQ ID NO: 118.

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