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 present invention also provides pharmaceutical preparations comprising the antibodies, and methods of treating an individual suffering from a complement-mediated disease or condition, comprising administering the antibodies to the individual. The binding specificity and C1q substitution function of the anti-C1s and anti-C1r antibodies were evaluated. The time-dependent complement neutralization function of the antibodies and binding to native and truncated C1s or C1r proteins were also shown.

Description

Technical Field

[0001] The present invention relates to anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods of using the same. Background Art

[0002] The C1 complex is a large protein complex that functions as a key initiator of the classical pathway cascade reaction. The C1 complex consists of three components, C1q, C1r, and C1s, in a molar ratio of 1:2:2, respectively (NPL 1). The classical pathway is initiated when the C1 complex binds to an antibody-bound target. C1q, which has six globular heads, mediates the binding of the C1 complex to the antibody through avidity interactions with the Fc region. Once tightly bound to the target, C1r in the C1 complex automatically activates and becomes enzymatically active. The activated C1r then cleaves and activates the zymogen C1s in the C1 complex (NPL 2). Subsequently, the active C1s cleaves its substrate complement components C2 and C4 into C2a / C2b, and C4a / C4b fragments, respectively. This leads to the assembly of the C3 convertase C4b2a on the target surface, which cleaves C3 to form C3b. C3b in turn cleaves C5 to initiate the formation of the terminal membrane attack complex, C5b, C6, C7, C8, and C9, which cleaves the target through the formation of a pore.

[0003] 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 similarly, the interaction between C1r2s2 and C1q (NPL 5). In contrast, the CCP1-CCP2-serine protease domains of C1r and C1s are responsible for the proteolytic cleavage of their respective substrates (NPL 6, NPL 7). The C1r2s2 tetramer interacts with the six stems in C1q through the six binding sites in the CUB1-EGF-CUB2 domain of the tetramer (NPL 5).

[0004] Although a normally functioning complement system can defend the host against pathogen invasion, dysregulation or inappropriate activation of the classical pathway can lead to a variety of complement-mediated disorders, such as, but not limited to, autoimmune hemolytic anemia (AIHA), Behcet's disease, bullous pemphigus (BP), immune thrombocytopenic purpura (ITP), etc. Therefore, inhibition of excessive or uncontrolled activation of the classical pathway may provide clinical benefits for patients suffering from such disorders.

[0005] An antibody, HI532, that binds to the beta domain of C1s was reported to inhibit the interaction of C1r2s2 with C1q (NPL 8). However, this antibody was unable to completely neutralize the hemolytic activity of human serum and retained 30% of the activity even after incubation of serum with the antibody for 24 hours.

[0006] Antibodies are highly attractive drugs because they are stable in plasma, highly specific for their targets, and generally exhibit good pharmacokinetic profiles. However, due to their large molecular size, the dose of therapeutic antibodies is generally high. In cases where the target is present in high abundance, the required therapeutic dose of the antibody is even higher. As a result, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive approaches to reduce the dose and high production costs associated with therapeutic antibodies.

[0007] It is reported that antibodies that bind to antigens in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies") can neutralize multiple antigen molecules with a single antibody molecule (NPL 9, PTL 1). pH-dependent antibodies firmly bind to their antigens under neutral pH conditions in plasma, but dissociate from the antigen in endosomes under acidic pH conditions. Once dissociated from the antigen, the antibody circulates back to the plasma through the FcRn receptor, and the dissociated antibody is degraded in the lysosomes of the cell. Then, the circulating antibody is free to bind and neutralize the antigen molecule again, and the process continues to repeat as long as the antibody keeps circulating.

[0008] Reference List

[0009] Patent Literature

[0010] [PTL 1] WO2009 / 125825

[0011] Non-patent literature

[0012] [NPL 1]Wang et.al.Mol Cell.2016Jul 7;63(1):135-45

[0013] [NPL 2]Mortensen et.al.Proc Natl Acad Sci US A.2017Jan 31;114(5):986-991

[0014] [NPL 3]Gal et.al.Mol Immunol.2009Sep;46(14):2745-52

[0015] [NPL 4]Almitairi et.al.Proc Natl Acad Sci US A.2018Jan 23;115(4):768-773

[0016] [NPL 5]Bally et.al.J Biol Chem.2009Jul 17;284(29):19340-8

[0017] [NPL 6]Rossi et.al.1998J Biol Chem.1998Jan 9;273(2):1232-9

[0018] [NPL 7]Lacroix et.al.J Biol Chem.2001Sep 28;276(39):36233-40

[0019] [NPL 8]Tseng et.al.Mol Immunol.1997Jun;34(8-9):671-9

[0020] [NPL 9]Igawa et.al.Nat Biotechnol.2010Nov;28(11):1203-7 SUMMARY OF THE INVENTION

[0022] Technical issues

[0023] The present invention provides anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods of using the same.

[0024] Solution to the problem

[0025] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody having a substitution function that allows the antibody to bind to the C1qrs complex and promote the dissociation of C1q from the C1qrs complex.

[0026] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is an antibody that binds to the C1qrs complex on the BIACORE (registered trademark) chip and promotes the dissociation of C1q from the C1qrs complex. In a further embodiment, when 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 after a sufficient period of time, as determined by the BIACORE (registered trademark) assay, the antibody of the present invention can be determined as an antibody with a substitution function.

[0027] In some embodiments, when the time point of crossing is within 60s, 100s, 150s, 200s, 500s, 700s, 1000s, 1500s or 2000s after the time point of the start of antibody injection, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex can be determined as an antibody with replacement function, as determined by 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 (RU), respectively, and the antibody as the analyte is injected at 500nM, 10 microliters (μL) / min.

[0028] In some embodiments, when substantially all of the C1q is separated from the C1qrs complex within 100s, 300s, 500s, 700s, 1000s, 1500s, 2000s, 3000s, 5000s, 7000s, or 10000s after the time point at which the antibody injection is started, the isolated antibody of the present invention that inhibits the interaction between the C1q and the C1r2s2 complex can be determined as an antibody having a substitution function, as determined by BIACORE (registered trademark) 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.

[0029] In some embodiments, an isolated antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that has at least 70% neutralizing activity against human serum complement in an RBC assay.

[0030] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that specifically binds C1s or an antibody that specifically binds C1r.

[0031] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is an antibody that specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s. In further embodiments, the antibody of the present invention competes with an antibody selected from the group consisting of 1)-5) below for binding to the epitope:

[0032] 1) an antibody comprising an HVR-H1 sequence of SEQ ID NO:32, an HVR-H2 sequence of SEQ ID NO:33, an HVR-H3 sequence of SEQ ID NO:34, an HVR-L1 sequence of SEQ ID NO:35, an HVR-L2 sequence of SEQ ID NO:36, and an HVR-L3 sequence of SEQ ID NO:37,

[0033] 2) an antibody comprising an HVR-H1 sequence of SEQ ID NO:38, an HVR-H2 sequence of SEQ ID NO:39, an HVR-H3 sequence of SEQ ID NO:40, an HVR-L1 sequence of SEQ ID NO:41, an HVR-L2 sequence of SEQ ID NO:42, and an HVR-L3 sequence of SEQ ID NO:43,

[0034] 3) an antibody comprising an HVR-H1 sequence of SEQ ID NO:44, an HVR-H2 sequence of SEQ ID NO:45, an HVR-H3 sequence of SEQ ID NO:46, an HVR-L1 sequence of SEQ ID NO:47, an HVR-L2 sequence of SEQ ID NO:48, and an HVR-L3 sequence of SEQ ID NO:49,

[0035] 4) an antibody comprising an HVR-H1 sequence of SEQ ID NO:50, an HVR-H2 sequence of SEQ ID NO:51, an HVR-H3 sequence of SEQ ID NO:52, an HVR-L1 sequence of SEQ ID NO:53, an HVR-L2 sequence of SEQ ID NO:54, and an HVR-L3 sequence of SEQ ID NO:55, and

[0036] 5) An antibody comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:59, an HVR-L2 sequence of SEQ ID NO:60 and an HVR-L3 sequence of SEQ ID NO:61.

[0037] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is an antibody that specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r. In further embodiments, the antibody of the present invention competes with an antibody selected from the group consisting of 6)-13) below for binding to the epitope:

[0038] 6) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 119, an HVR-H2 sequence of SEQ ID NO: 127, an HVR-H3 sequence of SEQ ID NO: 135, an HVR-L1 sequence of SEQ ID NO: 143, an HVR-L2 sequence of SEQ ID NO: 151, and an HVR-L3 sequence of SEQ ID NO: 159,

[0039] 7) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 120, an HVR-H2 sequence of SEQ ID NO: 128, an HVR-H3 sequence of SEQ ID NO: 136, an HVR-L1 sequence of SEQ ID NO: 144, an HVR-L2 sequence of SEQ ID NO: 152, and an HVR-L3 sequence of SEQ ID NO: 160,

[0040] 8) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 121, an HVR-H2 sequence of SEQ ID NO: 129, an HVR-H3 sequence of SEQ ID NO: 137, an HVR-L1 sequence of SEQ ID NO: 145, an HVR-L2 sequence of SEQ ID NO: 153, and an HVR-L3 sequence of SEQ ID NO: 161,

[0041] 9) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 122, an HVR-H2 sequence of SEQ ID NO: 130, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 146, an HVR-L2 sequence of SEQ ID NO: 154, and an HVR-L3 sequence of SEQ ID NO: 162,

[0042] 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,

[0043] 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,

[0044] 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

[0045] 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.

[0046] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody having an antigen binding activity that varies depending on ion concentration. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody having a C1s binding activity that varies depending on ion concentration. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody having a C1r binding activity that varies depending on ion concentration.

[0047] In some embodiments, the isolated antibody of the present 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 present 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 present 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.

[0048] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to the antigen with a higher affinity under high calcium concentration conditions than under low calcium concentration conditions. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1s with a higher affinity under high calcium concentration conditions than under low calcium concentration conditions. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is an antibody that binds to C1r with a higher affinity under high calcium concentration conditions than under low calcium concentration conditions.

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

[0050] In certain embodiments, in the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, 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 certain embodiments, in the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, when measured at high calcium concentrations at both neutral and acidic pH, the ratio of its 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.

[0051] In some embodiments, in the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, when measured at low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, wherein the anti-C1s antibody binds to the dimeric state of C1s. In some embodiments, in the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, when measured at low calcium concentrations 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.

[0052] In certain embodiments, in the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, when measured at a neutral pH at a high calcium concentration and at an acidic pH at a low calcium concentration, 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 antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex, when measured at a neutral pH at a high calcium concentration and at an acidic pH at a low calcium concentration, the ratio of its 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.

[0053] In some embodiments, an anti-C1s antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex comprises a histidine residue at one or more of the following Kabat numbering system positions:

[0054] 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

[0055] 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.

[0056] In some embodiments, an anti-C1r antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex comprises a histidine residue at one or more of the following Kabat numbering system positions:

[0057] 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

[0058] 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.

[0059] In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex comprises at least one histidine substituted at one or more of the following Kabat numbering system positions:

[0060] 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

[0061] 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.

[0062] In some embodiments, the anti-C1r antibody of the invention that inhibits the interaction between C1q and C1r2s2 complex comprises at least one histidine substituted at one or more of the following Kabat numbering system positions:

[0063] 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

[0064] 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.

[0065] In a further embodiment, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex and has pH dependence competes for binding to C1s with an antibody selected from the following 1)-5) under neutral pH conditions:

[0066] 1) an antibody comprising an HVR-H1 sequence of SEQ ID NO:32, an HVR-H2 sequence of SEQ ID NO:33, an HVR-H3 sequence of SEQ ID NO:34, an HVR-L1 sequence of SEQ ID NO:35, an HVR-L2 sequence of SEQ ID NO:36, and an HVR-L3 sequence of SEQ ID NO:37,

[0067] 2) an antibody comprising an HVR-H1 sequence of SEQ ID NO:38, an HVR-H2 sequence of SEQ ID NO:39, an HVR-H3 sequence of SEQ ID NO:40, an HVR-L1 sequence of SEQ ID NO:41, an HVR-L2 sequence of SEQ ID NO:42, and an HVR-L3 sequence of SEQ ID NO:43,

[0068] 3) an antibody comprising an HVR-H1 sequence of SEQ ID NO:44, an HVR-H2 sequence of SEQ ID NO:45, an HVR-H3 sequence of SEQ ID NO:46, an HVR-L1 sequence of SEQ ID NO:47, an HVR-L2 sequence of SEQ ID NO:48, and an HVR-L3 sequence of SEQ ID NO:49,

[0069] 4) an antibody comprising an HVR-H1 sequence of SEQ ID NO:50, an HVR-H2 sequence of SEQ ID NO:51, an HVR-H3 sequence of SEQ ID NO:52, an HVR-L1 sequence of SEQ ID NO:53, an HVR-L2 sequence of SEQ ID NO:54, and an HVR-L3 sequence of SEQ ID NO:55,

[0070] 5) an antibody comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:59, an HVR-L2 sequence of SEQ ID NO:60, and an HVR-L3 sequence of SEQ ID NO:61,

[0071] In a further embodiment, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex and has pH dependence competes for binding to C1r with an antibody selected from the following 6)-13) under neutral pH conditions:

[0072] 6) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 119, an HVR-H2 sequence of SEQ ID NO: 127, an HVR-H3 sequence of SEQ ID NO: 135, an HVR-L1 sequence of SEQ ID NO: 143, an HVR-L2 sequence of SEQ ID NO: 151, and an HVR-L3 sequence of SEQ ID NO: 159,

[0073] 7) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 120, an HVR-H2 sequence of SEQ ID NO: 128, an HVR-H3 sequence of SEQ ID NO: 136, an HVR-L1 sequence of SEQ ID NO: 144, an HVR-L2 sequence of SEQ ID NO: 152, and an HVR-L3 sequence of SEQ ID NO: 160,

[0074] 8) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 121, an HVR-H2 sequence of SEQ ID NO: 129, an HVR-H3 sequence of SEQ ID NO: 137, an HVR-L1 sequence of SEQ ID NO: 145, an HVR-L2 sequence of SEQ ID NO: 153, and an HVR-L3 sequence of SEQ ID NO: 161,

[0075] 9) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 122, an HVR-H2 sequence of SEQ ID NO: 130, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 146, an HVR-L2 sequence of SEQ ID NO: 154, and an HVR-L3 sequence of SEQ ID NO: 162,

[0076] 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,

[0077] 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,

[0078] 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

[0079] 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.

[0080] In some embodiments, the present disclosure provides an isolated anti-C1s antibody that specifically binds to an epitope within a region encompassing a CUB1-EGF-CUB2 domain (also referred to as an interaction domain or CUB domain), which is composed of CUB1, EGF, and CUB2 of complement component 1s (Cls), which 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 a 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 of C1s 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 disclosure 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 epitope of C1s is an epitope of human C1s, or preferably an epitope of human C1s and an epitope of cynomolgus monkey C1s.

[0081] In some embodiments, the present disclosure provides an isolated anti-C1r antibody that specifically binds to an epitope within a region encompassing a CUB1-EGF-CUB2 domain, which is composed of CUB1, EGF, and CUB2 of complement component 1r (Clr), which 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-mentioned epitope of C1r is an epitope of human C1r, or preferably an epitope of human C1r and an epitope of cynomolgus monkey C1r.

[0082] In some embodiments, the isolated anti-C1s antibody of the present invention 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 a framework region of human or primate origin. In some embodiments, the isolated anti-C1s antibody of the present invention 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 a framework region of human or primate origin.

[0083] In some embodiments, an isolated anti-C1r antibody of the invention comprises (a) an HVR-H1 comprising an amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) an HVR-H2 comprising an amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) an HVR-H3 comprising an amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142, wherein the antibody comprises a framework region of human or primate origin. In some embodiments, an isolated anti-C1r antibody of the invention comprises (a) an HVR-L1 comprising an amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) an HVR-L2 comprising an amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) an HVR-L3 comprising an amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166, wherein the antibody comprises a framework region of human or primate origin.

[0084] In some embodiments, the anti-C1s antibody of the present invention comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, 20, 21, 23, or 24; (b) a VL sequence having at least 95% sequence identity to 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 present invention comprises the VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24. In some embodiments, the anti-C1s antibody of the present invention comprises the VL sequence of SEQ ID NO: 26, 27, 28, 30, or 31. In further embodiments, the anti-C1s antibody of the present 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 a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence of SEQ ID NO: 19 and a VL sequence of SEQ ID NO: 26. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 27. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence of SEQ ID NO: 21 and a VL sequence of SEQ ID NO: 28. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence of SEQ ID NO: 23 and a VL sequence of SEQ ID NO: 30. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence of SEQ ID NO: 24 and a VL sequence of SEQ ID NO: 31.

[0085] In some embodiments, the anti-C1r antibody of the invention comprises (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 antibody of the invention comprises the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109 or 110. In some embodiments, the anti-C1r antibody of the invention comprises the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117 or 118. In a further embodiment, the anti-C1r antibody of the present invention comprises 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. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 103 and a VL sequence of SEQ ID NO: 111. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 104 and a VL sequence of SEQ ID NO: 112. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 105 and a VL sequence of SEQ ID NO: 113. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 106 and a VL sequence of SEQ ID NO: 114. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 107 and a VL sequence of SEQ ID NO: 115. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 108 and a VL sequence of SEQ ID NO: 116. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 109 and a VL sequence of SEQ ID NO: 117. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence of SEQ ID NO: 110 and a VL sequence of SEQ ID NO: 118.

[0086] In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is a monoclonal antibody. In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is a human antibody, a humanized antibody, or a chimeric antibody. In a further embodiment, the anti-C1s antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In a further embodiment, the anti-C1s antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes 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 the C1q and C1r2s2 complexes is a human IgG1 or a humanized IgG1.

[0087] In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is a monoclonal antibody. In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is a human antibody, a humanized antibody or a chimeric antibody. In a further embodiment, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In a further embodiment, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and 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 C1r2s2 complex is a human IgG1 or a humanized IgG1.

[0088] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complexes 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.

[0089] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between the C1q and C1r2s2 complexes has a human Fc region having a binding activity selected from the group consisting of:

[0090] a) The binding activity to activated Fc gamma receptors is stronger than that of the Fc region of native human IgG1,

[0091] b) has a stronger binding activity to inhibitory Fc gamma receptors than to activating Fc gamma receptors, and

[0092] c) The binding activity to FcRn at neutral pH is stronger than that of the Fc region of native human IgG1.

[0093] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and 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 of the present invention that inhibits the interaction between C1q and C1r2s2 complex binds to at least human C1r or preferably binds to both cynomolgus monkey C1r and human C1r.

[0094] The present invention also provides an isolated nucleic acid encoding an anti-C1s antibody of the present invention. The present invention also provides an isolated nucleic acid encoding an 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 of producing an antibody, comprising culturing the host cell of the present invention to produce the antibody.

[0095] The present invention also provides a pharmaceutical preparation comprising the antibody of the present invention and a pharmaceutically acceptable carrier thereof.

[0096] The anti-C1s antibodies of the present invention can be used as a medicament. The anti-C1s antibodies of the present invention can be used to treat or prevent complement-mediated diseases or conditions. The anti-C1s antibodies of the present invention can be used to enhance the clearance (or removal) of C1s from plasma. The anti-C1s antibodies of the present invention can be used to enhance the clearance (or removal) of C1r2s2 from plasma. The anti-C1s antibodies of the present invention can be used to enhance the clearance (or removal) of C1r2s2 from plasma instead of Clq from plasma. In some cases, the antibody inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls.

[0097] The anti-C1r antibodies of the present invention can be used as a medicament. The anti-C1r antibodies of the present invention can be used to treat or prevent complement-mediated diseases or conditions. The anti-C1r antibodies of the present invention can be used to enhance the clearance (or removal) of C1r from plasma. The anti-C1r antibodies of the present invention can be used to enhance the clearance (or removal) of C1r2s2 from plasma. The anti-C1r antibodies of the present invention can be used to enhance the clearance (or removal) of C1r2s2 from plasma instead of Clq from plasma. In some cases, the antibody inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Clr.

[0098] The anti-Cls antibodies of the present invention can be used to prepare drugs. In some embodiments, the drug is used to treat or prevent complement-mediated diseases or disorders. 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 instead of Clq from plasma. In this case, the enhanced level of clearance of C1q from plasma is not necessarily empty (zero). That is, the enhanced level of clearance of C1q from plasma may be zero, or may not be zero but close to zero, or may be insignificant or low enough to be technically ignored by those skilled in the art. In some cases, the drug inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls.

[0099] For example, enhancement of C1s / C1q clearance (CL) can be measured as follows.

[0100] The total concentration of human C1s and C1q in mouse plasma was measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting human C1s and C1q in defined amounts in mouse plasma, obtaining 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 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 (hen egg white) in 50 mmol / L ammonium bicarbonate and incubated at 56°C for 45 minutes. Then, 2 μL of 500 mmol / L iodoacetamide was added and incubated at 37 ° C for 30 minutes in the dark. Next, 160 μL of 0.5 μg / mL sequencing-grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37 ° C. Finally, 5 μL of 10% trifluoroacetic acid was added to inactivate any residual trypsin. 40 μL of digested samples were analyzed by LC / ESI-MS / MS. LC / ESI-MS / MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D I-level UPLC (Waters). Human C1s-specific peptide LLEVPEGR and human C1q-specific peptide IAFSATR were monitored by selected reaction monitoring (SRM). For human C1s, the SRM transition is [M+2H]2+ (m / z 456.8) to y6 ion (m / z 686.3), and for human C1q, the SRM transition is [M+2H]2+ (m / z 383.2) to y5 ion (m / z 581.3). Calibration curves were constructed by weighted (1 / x2) linear regression using peak areas plotted against concentration. Concentrations in mouse plasma were calculated from the calibration curves using analysis software Masslynx Ver.4.1 (Waters).

[0101] The pharmacokinetics of total hC1s and hC1q following administration of anti-C1s antibodies in mice were evaluated as follows.

[0102] The in vivo pharmacokinetics of hC1s, hC1q, and anti-C1s antibodies were evaluated after administration of antigens (hC1q, recombinant C1r2s2, and a mixture of hC1q and rC1r2s2) alone or together with anti-C1s antibodies to mice (CB17 / Icr-Prkdcscid / CrlCrl: Charles River Japan). Three mice were assigned to each dosing group.

[0103] First, mice were intravenously injected with hC1q solution (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) at a dose of 10 mL / kg. Immediately after the antigen solution was administered, an anti-C1s antibody solution (2.5 mg / mL) was administered to the same individual in the same manner.

[0104] The dose settings for C1q and rC1r2s2 were designed to provide physiological concentrations in human plasma immediately after administration. During the study, the dose of anti-C1s antibody was adjusted to exceed the concentration of both antigens, thus assuming that nearly all hC1s was in the bound form in the circulation.

[0105] Blood was collected at 5, 30 minutes, 2, 7 hours, 3, 7, 14, 21 and 28 days after injection. The blood was immediately centrifuged to separate the plasma samples. The plasma concentrations of hC1s and hC1q were measured by LC / ESI-MS / MS at each sampling point. The PK parameters of hC1s and hC1q were estimated by non-compartmental analysis (Phoenix WinNonlin version 8.0, Certara).

[0106] An antibody having (i) an Fc comprising a mutation to reduce C1q and Fc gamma receptor binding, or (ii) an Fc comprising a mutation to reduce C1q binding while maintaining Fc gamma receptor binding is administered to a mouse. For example, in the present invention, the Fc of "SG136" comprises a mutation that reduces C1q and Fc gamma receptor binding, while the Fc of "SG1148" comprises a mutation that reduces C1q binding while maintaining Fc gamma receptor binding.

[0107] The above mouse PK studies are performed on the test antibodies (e.g., CCP1-CCP2-SP or CUB1-EGF-CUB2 binders), 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 antibodies of the 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.

[0108] The anti-Clr antibodies of the present invention can be used to prepare a medicament. In some embodiments, the medicament is used to treat or prevent a complement-mediated disease or condition. In some embodiments, the medicament is used to enhance the clearance (or removal) of C1r from plasma. In some embodiments, the medicament is used to enhance the clearance (or removal) of C1r2s2 from plasma. In some embodiments, the medicament is used to enhance the clearance (or removal) of C1r2s2 from plasma instead of Clq from plasma. In some cases, the medicament inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Clr.

[0109] The present invention also provides methods for treating or preventing an individual suffering from a complement-mediated disease or condition. In some embodiments, the method comprises administering to an individual an effective amount of an 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 an individual an effective amount of an 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 but not Clq from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma but not Clq from plasma. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma but not Clq from plasma. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma but not Clq from plasma. In some cases, the antibody inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls.

[0110] The present invention also provides methods for treating or preventing an individual suffering from a complement-mediated disease or condition. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1r antibody of the present invention. The present invention also provides a method for enhancing the clearance (or removal) of C1r from plasma in an individual. In some embodiments, the method comprises administering to an 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 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 but not C1q from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1r antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma but not C1q from plasma. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1r antibody of the present invention to enhance the clearance (or removal) of C1r2s2 from plasma but not C1q from plasma. In some cases, the antibody inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Clr.

[0111] More specifically, the present invention provides the following:

[0112] [1] An isolated antibody that inhibits the interaction between C1q and C1r2s2 complex, wherein the antibody has a substitution function such that the antibody binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex.

[0113] [2] The antibody of [1], wherein the antibody binds to the C1qrs complex on the Biacore chip and promotes the dissociation of C1q from the C1qrs complex, wherein, after a sufficient period of 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.

[0114] [3] The antibody described in [2], wherein the time point of crossing over in the Biacore assay is within 60s, 100s, 150s, 200s, 500s, 700s, or 1000s after the time point of the start of antibody injection, as determined by Biacore assay using the following conditions: the capture levels of C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the antibody as the analyte is injected at 500nM, 10μL / min.

[0115] [4] The antibody of [2], wherein substantially all of the C1q is dissociated from the C1qrs complex within 100 s, 300 s, 500 s, 700 s, 1000 s, 1500 s, or 2000 s after 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] [5] An isolated antibody that inhibits the interaction between C1q and C1r2s2 complexes, wherein the antibody has at least 70% neutralizing activity against human serum complement in an RBC assay.

[0117] [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.

[0118] [7] An isolated antibody that inhibits the interaction between the C1q and C1r2s2 complexes,

[0119] The antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s and competes with an antibody selected from the group consisting of 1) to 5) below for binding to the epitope:

[0120] 1) an antibody comprising an HVR-H1 sequence of SEQ ID NO:32, an HVR-H2 sequence of SEQ ID NO:33, an HVR-H3 sequence of SEQ ID NO:34, an HVR-L1 sequence of SEQ ID NO:35, an HVR-L2 sequence of SEQ ID NO:36, and an HVR-L3 sequence of SEQ ID NO:37,

[0121] 2) an antibody comprising an HVR-H1 sequence of SEQ ID NO:38, an HVR-H2 sequence of SEQ ID NO:39, an HVR-H3 sequence of SEQ ID NO:40, an HVR-L1 sequence of SEQ ID NO:41, an HVR-L2 sequence of SEQ ID NO:42, and an HVR-L3 sequence of SEQ ID NO:43,

[0122] 3) an antibody comprising an HVR-H1 sequence of SEQ ID NO:44, an HVR-H2 sequence of SEQ ID NO:45, an HVR-H3 sequence of SEQ ID NO:46, an HVR-L1 sequence of SEQ ID NO:47, an HVR-L2 sequence of SEQ ID NO:48, and an HVR-L3 sequence of SEQ ID NO:49,

[0123] 4) an antibody comprising an HVR-H1 sequence of SEQ ID NO:50, an HVR-H2 sequence of SEQ ID NO:51, an HVR-H3 sequence of SEQ ID NO:52, an HVR-L1 sequence of SEQ ID NO:53, an HVR-L2 sequence of SEQ ID NO:54, and an HVR-L3 sequence of SEQ ID NO:55, and

[0124] 5) an antibody comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:59, an HVR-L2 sequence of SEQ ID NO:60, and an HVR-L3 sequence of SEQ ID NO:61, or

[0125] The antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r and competes with an antibody selected from the group consisting of the following 6)-13) for binding to the epitope:

[0126] 6) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 119, an HVR-H2 sequence of SEQ ID NO: 127, an HVR-H3 sequence of SEQ ID NO: 135, an HVR-L1 sequence of SEQ ID NO: 143, an HVR-L2 sequence of SEQ ID NO: 151, and an HVR-L3 sequence of SEQ ID NO: 159,

[0127] 7) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 120, an HVR-H2 sequence of SEQ ID NO: 128, an HVR-H3 sequence of SEQ ID NO: 136, an HVR-L1 sequence of SEQ ID NO: 144, an HVR-L2 sequence of SEQ ID NO: 152, and an HVR-L3 sequence of SEQ ID NO: 160,

[0128] 8) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 121, an HVR-H2 sequence of SEQ ID NO: 129, an HVR-H3 sequence of SEQ ID NO: 137, an HVR-L1 sequence of SEQ ID NO: 145, an HVR-L2 sequence of SEQ ID NO: 153, and an HVR-L3 sequence of SEQ ID NO: 161,

[0129] 9) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 122, an HVR-H2 sequence of SEQ ID NO: 130, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 146, an HVR-L2 sequence of SEQ ID NO: 154, and an HVR-L3 sequence of SEQ ID NO: 162,

[0130] 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,

[0131] 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,

[0132] 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

[0133] 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.

[0134] [8] An isolated antibody that inhibits the interaction between C1q and C1r2s2 complex, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than that at pH 7.4.

[0135] [9] The antibody of any one of [1] to [8], wherein the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s or C1r, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than its antigen-binding activity at pH 7.4.

[0136]

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

[0137] (i) when measured at high calcium concentrations at both neutral and acidic pH, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more,

[0138] (ii) When measured at a neutral pH at a high calcium concentration and at an acidic pH at a low calcium concentration, the ratio of the KD value of the C1s binding activity at an acidic pH to the KD value of the C1s binding activity at a neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more.

[0139]

[11] The antibody of 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.

[0140]

[12] The antibody of

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

[0141] a) The binding activity to activated Fc gamma receptors is stronger than that of the Fc region of native human IgG1,

[0142] b) has a stronger binding activity to inhibitory Fc gamma receptors than to activating Fc gamma receptors, and

[0143] c) The binding activity to FcRn at neutral pH is stronger than that of the Fc region of native human IgG1.

[0144]

[13] The antibody of 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.

[0145]

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

[13] and a pharmaceutically acceptable carrier.

[0146]

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

[13] . BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0150] [ Figure 2A ] Figure 2A Shown is the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on the BIACORE (registered trademark) sensor surface. The antibody displacement of native human C1q is depicted by overwriting 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. Sensorgram Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. Figure 2A The RU at time 0 is normalized (ie, set to be the same) in .

[0151] [ Figure 2B ] Figure 2B Shown is the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on the BIACORE (registered trademark) sensor surface. The antibody displacement of native human C1q is depicted by overwriting 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. Sensorgram Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. Figure 2B The RU at time 0 is normalized (ie, set to be the same) in .

[0152] [ Figure 2C ] Figure 2C Figure 2 shows antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on a BIACORE (registered trademark) sensor surface. The displacement of native human C1q by the antibody is depicted by overwriting 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. To compare these sensorgrams, Figure 2C The RU at the Ab injection sites were normalized (ie, set to be the same) in .

[0153] [ Figure 2D ] Figure 2D Figure 2 shows antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer immobilized on a BIACORE (registered trademark) sensor surface. The displacement of native human C1q by the antibody is depicted by overwriting 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. To compare these sensorgrams, Figure 2D The RU at the Ab injection sites were normalized (ie, set to be the same) in .

[0154] [ Figure 3 ] Figure 3 Shown is the antibody-mediated displacement of recombinant human C1r2s2 Flag / His tetramer from biotinylated native human C1q that has been immobilized on the BIACORE (registered trademark) sensor surface. Recombinant human C1r2s2 Flag / His tetramer was flowed over to bind to immobilized native human C1q, and then the dissociation rate of C1r2s2 was monitored by flowing over a buffer alone (solid line), or by flowing over an antibody to dissociate C1r2s2 (dashed line).

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

[0156] [ Figure 5 ] Figure 5 Neutralization of human serum complement activity is shown.

[0157] [ Figure 6 ] Figure 6Shown are competitive epitope binning results for antibodies binding to the CUB1-EGF-CUB2 domain of C1s.

[0158] [ Figure 7 ] Figure 7 Shown are the pharmacokinetics of human C1s and human C1q following administration of anti-C1s antibodies in mice.

[0159] [ Figure 8 ] Figure 8 Time-dependent neutralization of human serum complement activity by anti-C1s antibodies is shown.

[0160] [ Fig. 9 ] Fig. 9 Antibody binding to native human zymogen C1s in reducing and non-reducing Western blot analysis is shown.

[0161] [ Fig.10 ] Fig.10 The antibody is shown to bind to the truncated C1s protein in a reducing Western blot.

[0162] [ Fig.11A ] Fig.11A The binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1r protein is shown. BIACORE (registered trademark) sensorgram of the anti-C1r antibody against the recombinant human C1r CCP1-CCP2-SP-FLAG protein.

[0163] [ Fig. 11B ] Fig. 11B The binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1r protein is shown. BIACORE (registered trademark) sensorgram of anti-C1r antibody against native human C1r enzyme.

[0164] [ Fig. 12A ] Fig. 12A Shown is the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer captured on a BIACORE (registered trademark) sensor surface. The 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. Sensorgram Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. Fig. 12A The RU at time 0 is normalized (ie, set to be the same) in .

[0165] [ Fig. 12B ] Fig. 12B Shown is the antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramer captured on a BIACORE (registered trademark) sensor surface. The 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. Sensorgram Figure 3 (solid line) depicts the baseline when only the antibody binds to C1r2s2 in the absence of any C1q. Fig. 12B The RU at time 0 is normalized (ie, set to be the same) in .

[0166] [ Fig. 12C ] Fig. 12C Figure 2 shows antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on a BIACORE (registered trademark) sensor surface. Displacement of native human C1q by antibody is depicted by overlaying two sensorgrams. Sensorgram 1 (solid line) depicts stable capture of C1qrs on the sensor surface. Sensorgram 2 (dashed line) depicts binding of the antibody to C1qrs and displacement of C1q from C1r2s2. To compare these sensorgrams, Fig. 12C The RU at the Ab injection sites were normalized (ie, set to be the same) in .

[0167] [ Fig.12D ] Fig.12D Figure 2 shows antibody-mediated displacement of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on a BIACORE (registered trademark) sensor surface. Displacement of native human C1q by antibody is depicted by overlaying two sensorgrams. Sensorgram 1 (solid line) depicts stable capture of C1qrs on the sensor surface. Sensorgram 2 (dashed line) depicts binding of the antibody to C1qrs and displacement of C1q from C1r2s2. To compare these sensorgrams, Fig.12D The RU at the Ab injection sites were normalized (ie, set to be the same) in .

[0168] [ Fig.13 ] Fig.13 Neutralization of human serum complement activity is shown.

[0169] Description of Implementation Methods

[0170] The techniques and methods described or referenced herein are generally well understood and routinely used by those skilled in the art using conventional methodology, such as, for example, the widely used methods described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, Humana Press; Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RIFreshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (eds. DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (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 JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0171] I. Definitions

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

[0173] For the purpose of interpreting this application, the following definitions will apply and when 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 of the definitions given below conflict with any document incorporated herein by reference, the definitions given below shall prevail.

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

[0175] "Affinity" refers to the sum of the strength 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 noted, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a 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 as follows. "Affinity", "binding affinity", "binding capacity" and "binding activity" can be used interchangeably. The term "binding activity" refers to the sum of the strength 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 this article, binding activity is not strictly limited to reflecting the activity of 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). When the members of the 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 to its partner Y can generally be represented by a dissociation constant (KD). Alternatively, the association and dissociation rates (Kon and Koff) can be used for the assessment of binding. Binding activity 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.

[0176] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess such alterations which result in an improvement in the affinity of the antibody for antigen.

[0177] The terms "anti-C1s antibody" and "antibody that binds to C1s" refer to an antibody that is capable of binding to C1s with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent for targeting C1s. In one embodiment, the extent of binding of the anti-C1s antibody to unrelated, non-C1s proteins is 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 antibody that binds to C1s 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, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13M). In certain embodiments, an anti-C1s antibody binds to an epitope of C1s that is conserved between C1s derived 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 is useful as a diagnostic and / or therapeutic agent for targeting C1r. In one embodiment, the extent of binding of the anti-C1r antibody to unrelated, non-C1r proteins is 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 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). In certain embodiments, an anti-C1r antibody binds to an epitope of C1r that is conserved between C1r derived 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 (eg, 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, which comprises a portion of an intact antibody that binds to an antigen that the intact antibody binds to. 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 major 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 classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0184] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular 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 radioisotopes 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 nucleolytic enzymes; 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 below.

[0185] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0186] An "effective amount" of an agent (eg, a pharmaceutical agent) refers to an amount effective at dosages and times necessary to achieve the desired therapeutic or prophylactic result.

[0187] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, and includes specific amino acids that are in direct contact with the antibody. An epitope determinant can include chemically active surface clusters of molecules (such as amino acids, sugar side chains, phosphoryl or sulfonyl groups), and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Typically, an antibody that is specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0188] In this article, the term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain containing 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 end of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) in the Fc region may or may not exist. Unless otherwise noted herein, the amino acid residue numbering in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0189] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, in VH (or VL), HVR and FR sequences typically appear 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 to refer to antibodies having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0191] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as 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 human cell, or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0193] "People's consensus framework" is such a framework, which 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 as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is as in subgroup κI in Kabat et al. above. In one embodiment, for VH, the subgroup is as in subgroup III in Kabat et al. above.

[0194] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise 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 non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

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

[0196] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0197] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0198] (c) antigenic contact points occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0199] (d) a combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

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

[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] An "isolated" antibody is one 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 reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0204] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

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

[0206] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, that is, the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants are usually present in small amounts. In contrast to polyclonal antibody preparations (usually including different antibodies for different determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation is for a single determinant on an antigen. Therefore, the attributive "monoclonal" indicates that the nature of the antibody is obtained from a substantially homologous antibody population, and is not considered to require the antibody to be prepared by any specific method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, 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 (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0208] "Natural antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, referred to as κ and λ.

[0209] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0210] " Percentage (%) amino acid sequence identity " with respect to reference polypeptide sequence is defined as the percentage of the amino acid residues in the candidate sequence identical with the amino acid residues in the reference polypeptide sequence after comparing sequences and introducing gaps (gap) when necessary to achieve maximum percentage sequence identity, without any conservative substitutions being considered as a part of sequence identity. Comparisons for determining percentage amino acid sequence identity can be achieved in a variety of ways within the art, for example, 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 comparing sequences, including any algorithm required for achieving maximum comparison over the full length of the sequence being compared.

[0211] The author of the ALIGN-2 sequence comparison computer program is Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington DC, 20559, together with user files, and it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program can be compiled from the source code. The ALIGN-2 program should be compiled for UNIX operating systems, 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 amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a particular % amino acid sequence identity to or relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by a 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's 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 expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraphs.

[0212] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation would be administered.

[0213] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical preparation other than the active ingredient, 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 "specific binding" refers to the activity or feature of an antibody that binds to 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, "specific binding" refers to the activity or feature of an antibody that binds to a target antigen at a binding level that includes or replaces significant (i.e., specific) binding in addition to background (i.e., non-specific) binding. Specificity can be measured by any method mentioned in this specification or known in the art. The above-mentioned level of non-specific or background binding can be zero, or can be close to zero instead of zero, or can be low enough to be technically ignored by those skilled in the art. For example, when a technician cannot detect or observe any significant (or relatively strong) antibody and non-target antigen in a suitable binding assay, it can be said that the antibody "does not specifically bind" the non-target antigen. On the contrary, when a technician can detect or observe any significant (or relatively strong) antibody and target antigen in a suitable binding assay, it can be said that the antibody "specifically binds" the target antigen.

[0215] Unless otherwise indicated, 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 as well as 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 an 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 Nos: 3 and 2, respectively.

[0216] Unless otherwise indicated, 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 as well as any form of C1r resulting from processing in a cell. The term also encompasses naturally occurring variants of C1r, such as splice variants or allelic variants. The amino acid sequence of an 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 Nos: 5 and 6, respectively.

[0217] As used herein, "treatment" (and grammatical variants such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving 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 chain or light chain that is involved in antibody antigen binding. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6 th ed., WH Freeman & Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to 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 that is capable of propagating another nucleic acid to which it is attached. 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 to which they are operably attached. 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 the C1q and C1r2s2 complexes and their uses. In certain embodiments, antibodies that bind to C1s are provided. In certain embodiments, antibodies that bind to C1r are provided. The antibodies of the invention can be used, for example, to diagnose or treat complement-mediated diseases or conditions.

[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 C1r2s2 complexes. In one aspect, the present invention provides an isolated antibody having a substitution function, which allows 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 its binding activity varies depending on ion concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on pH, that is, hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on calcium concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies depending on both pH and calcium concentration. 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 its binding activity varies depending on ion concentration. In certain embodiments, the binding activity of the anti-C1r antibody varies depending on pH, that is, hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1r antibody varies depending on 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 the Embodiments" section, the term "C1s" may be replaced by "C1r", except for descriptions related to sequences specific to anti-C1s antibodies and sequences and domains specific to C1s protein.

[0225] Such antibodies are expected to be particularly advantageous as drugs because the dosage and frequency of administration to patients can be reduced, resulting in a lower total dose. Anti-C1s antibodies are expected to have a higher safety profile than antibodies that bind to and remove C1qrs complexes from plasma because they only remove C1r2s2 from plasma (by binding to C1s) and not C1q from plasma. As a result, side effects associated with C1q depletion can be avoided. In addition, antibodies with rapid substitution of C1q are expected to have faster complement neutralization activity, which can translate into faster therapeutic efficacy.

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

[0227] In one aspect, the isolated antibody that inhibits the interaction between the C1q and C1r2s2 complexes of the present invention is an antibody that binds to the C1qrs complex on a chip for surface plasmon resonance assay (e.g., a BIACORE (registered trademark) chip) and promotes the dissociation of C1q from the C1qrs complex. In some aspects, the above-mentioned function of binding to the C1qrs complex and promoting the dissociation of C1q from the C1qrs complex is referred to herein as "substitution function / activity" or "C1q substitution function / activity". The function / activity can be suitably assessed qualitatively or quantitatively using a surface plasmon resonance assay, such as the BIACORE (registered trademark) assay described herein. In other aspects, when a sufficient period of time has passed, the value of the response unit (RU) when the antibody is present is lower than the value of the response unit (RU) when the antibody is not present, as determined by a surface plasmon resonance assay (e.g., a BIACORE (registered trademark) assay), the antibody of the present invention can be determined as an antibody with a substitution function. In the sensorgram obtained by such an assay, a "time point of intersection" can be identified, where the curve in the presence of C1q but not the antibody intersects the curve in the presence of C1q and the antibody at this "time point of intersection" (see the Examples for details). Strictly speaking, even in a single sensorgram, multiple time points of intersection may be observed due to noise or oscillation of the latter curve when crossing the previous curve. In this case, any one of the multiple time points of intersection can be selected as the "time point of intersection". "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 "time point of intersection". 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 time point when the antibody injection starts. Alternatively, the time point of measurement may be at least 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 3000s, 5000s, 7000s, or 10000s after the time point of intersection.

[0228] In one aspect, an isolated antibody of the present invention that inhibits the interaction between C1q and C1r2s2 complex can be identified as an antibody having a replacement function when the time point of crossover (e.g., in a BIACORE (registered trademark) assay) is within 60s, 100s, 150s, 200s, 500s, 700s, 1000s, 1500s or 2000s after the time point of start of antibody injection, as determined by, for example, a 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 analyte is injected at 500nM, 10 microliters (μL) / min.

[0229] In one aspect, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex can be determined as an antibody with substitution function when substantially all (or all) of the C1q is separated from the C1qrs complex within 100s, 300s, 500s, 700s, 1000s, 1500s, 2000s, 3000s, 5000s, 7000s, or 10000s after the start of antibody injection, as determined by, for example, BIACORE (registered trademark) 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. For example, in the sensorgram obtained from such an assay, when C1q and antibody are present, the value (RU) approaches or reaches the value (RU) when the antibody is present but C1q is not present, and it can be determined that "almost all (or all) C1q is dissociated from the C1qrs complex". In this article, "almost all (C1q)" refers to 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 percentages; "(all C1q)" refers to a percentage of 100%. The percentage of dissociated C1q can be quantitatively determined by any assay described herein. In some aspects, the present invention provides a method for screening an antibody that replaces C1q from a C1r2s2 complex using the above-mentioned method for measuring the "replacement function / activity" of such an antibody. In one embodiment, the screening method includes selecting an antibody that inhibits the interaction between the C1q and C1r2s2 complexes; that is, selecting an antibody that binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex. Surface plasmon resonance assays, such as the BIACORE (registered trademark) assay described herein, can be used to appropriately select antibodies with replacement functions / activities. In some embodiments, the screening method includes, after a sufficient period of time, determining by surface plasmon resonance assays (e.g., BIACORE (registered trademark) assays) the value of the response unit (RU) when (i) the antibody is present and the value of the response unit (RU) when (ii) the antibody is not present. The screening method may include comparing the value of (i) above and the value of (ii) above. The screening method may include selecting an antibody when the value of (i) above is lower than the value of (ii) above. The screening method may involve identifying a "crossover time point" where the curve for the presence of CIq in the absence of antibody intersects the curve for the presence of CIq and antibody.As described above, multiple intersecting time points can be observed even in a single sensorgram, and any one of the multiple intersecting time points can be selected as the "intersecting time point". In some embodiments, the screening method may include measuring the value of the response unit (RU) at least 60s, 100s, 150s, 200s, 500s, 700s, 1000s, 1500s or 2000s after the time point at which the antibody injection begins. Alternatively, the screening method may include measuring the value of the response unit (RU) at least 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 3000s, 5000s, 7000s or 10000s after the time point of intersection. In some embodiments, the screening method may include: selecting an antibody that inhibits the interaction between the C1q and C1r2s2 complex or an antibody with a replacement function when the time point of antibody crossing is within 60s, 100s, 150s, 200s, 500s, 700s, 1000s, 1500s or 2000s after the time point of start of antibody injection, as determined by, for example, BIACORE (registered trademark) 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 500nM, 10 microliters (μL) / min. In some embodiments, the screening method may include: selecting an antibody that inhibits the interaction between C1q and the C1r2s2 complex or an antibody having a replacement function when substantially all (or all) of the C1q dissociates from the C1qrs complex within 100s, 300s, 500s, 700s, 1000s, 1500s, 2000s, 3000s, 5000s, 7000s, or 10000s after the start of antibody injection, as determined by, for example, BIACORE (registered trademark) 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 500nM, 10μL / min. As described above, "substantially all (CIq)" refers to 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 more, and "all (CIq)" refers to 100%, and the percentage of dissociated CIq can be quantitatively determined by any assay described herein, including the BIACORE (registered trademark) assay.

[0230] (BIACORE (registered trademark) / blocking concept)

[0231] In one aspect, the present invention provides an isolated antibody that inhibits the interaction between the C1q and C1r2s2 complexes, wherein the antibody has a blocking function that allows 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 more. The blocking function / activity or blocking rate can be determined by using BIACORE (registered trademark) assay. The following conditions can be used to evaluate the level of C1q blocking: 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 variants) is injected at 50, 100, 200 nM. The blocking rate was calculated by the following formula: [1-(human C1q binding reaction in the presence of the antibody variant / human C1q binding reaction in the absence of the antibody variant)]×100%.

[0232] (pH dependence)

[0233] In one aspect, the antibodies of the invention bind to an antigen (e.g., C1s) or to a 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., antigen binding activity to C1s) is lower than the antigen binding activity at pH 7.4. In a preferred embodiment, wherein 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 pH-dependent antibodies 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 a dimeric state, the bivalent antibody is able to form an immune complex by cross-linking multiple C1s molecules. This allows the antibody to bind to the C1s molecules within the complex through both affinity and avidity interactions, thereby increasing the apparent affinity of the antibody. In contrast, when C1s is in a monomeric state, the antibody binds C1s only through avidity interactions. This means that in plasma, pH-dependent C1s antibodies can form immune complexes with dimeric C1s, but once entering acidic endosomes, C1s will dissociate into monomers. This leads to the disassembly 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 antibody of the present invention, 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 more. In one aspect, in the isolated anti-C1s antibody of the present invention, when measured at high calcium concentration at neutral pH and at low calcium concentration at acidic pH, the ratio of its 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 more. In some embodiments, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at high calcium concentration at both neutral and acidic pH, 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 change the epitope structure of C1s to which the antibody of the present invention binds, thereby changing the affinity of the antibody, or 2) the interaction (affinity or avidity) of the antibody of the present invention can vary depending on the state of C1s (monomer state or dimer state), measurements using specific conditions (at neutral pH at high calcium concentration and at acidic pH at low calcium concentration) can be used to assess the ratio of KD values ​​(KD (acidic pH) / KD (neutral pH)).

[0237] In other words, the antibodies of the 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 concentrations at both neutral and acidic pH, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more,

[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 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 more.

[0240] More generally, without being bound by a particular theory, in the following situations: 1) the absence of calcium can change the epitope structure of certain antigens bound by the antibodies of the present invention through conformational changes, thereby changing the affinity of the antibody, or 2) the interaction (affinity or avidity) of the antibodies of the present invention can vary depending on the state of the antigen (monomer 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 assess the ratio of KD values ​​(KD (acidic pH) / KD (neutral pH)).

[0241] Therefore, the antibodies of the present invention bind to 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 for antigen-binding activity at acidic pH to the KD value for antigen-binding activity at neutral pH (KD (acidic pH) / KD (neutral pH)) is 2 or more.

[0242] The above KD ratio, i.e. (KD (acidic pH) / KD (neutral pH)), can be compared between a parent antibody (i.e., the original antibody before modification of the present invention) and an antibody into which one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) have been introduced relative to the original (parent) antibody. The original (parent) antibody may 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 for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, 8 times, 10 times higher than the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) of the original (parent) 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 a parent (original) antibody, and the ratio of the following (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 the C1s binding activity at acidic pH of the isolated anti-C1s antibody to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)); (ii) the ratio of the KD value of the C1s binding activity at acidic pH of the parent (original) antibody to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)). These KD ratios can be measured at any (high or low) calcium concentration, for example, at neutral and acidic pH at high calcium concentration, or at neutral pH at high calcium concentration and at acidic pH at low calcium concentration.

[0243] In one aspect, the antibody of the present invention has different antigen binding activity between intracellular conditions and extracellular conditions. Intracellular and extracellular conditions refer to different conditions between the inside and outside of the cell. The categories of conditions include, for example, ion concentration, more specifically, metal ion concentration, hydrogen ion concentration (pH) and calcium ion concentration. "Intracellular conditions" preferably refer to the characteristic environment of the internal environment of the endosome, while "extracellular conditions" preferably refer to the characteristic environment of the environment in the plasma. Antibodies with the property that the antigen binding activity changes according to the ion concentration can be obtained by screening a large number of antibodies with domains having such properties. For example, antibodies with the above properties can be obtained by using hybridoma methods or antibody library methods to produce a large number of antibodies with different sequences from each other, and measuring their antigen binding activity under different ion concentrations. The B cell cloning method is one of the examples of the method for screening such antibodies. In addition, as described below, at least one unique amino acid residue that can give antibodies the characteristic of antigen binding activity that changes according to ion concentration is specified to prepare a large number of antibody libraries, wherein the antibodies have different sequences and have unique amino acid residues as common structures. Such libraries can be screened to effectively separate antibodies with the above properties.

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

[0245] In certain embodiments, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at high calcium concentrations at both neutral and acidic pH. In specific embodiments, the antibodies of the invention bind to 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 for C1s binding activity at acidic pH to the KD value for 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, an antibody of the invention binds 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 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., a reduced clearance rate of the antigen from the plasma) because the antibody can have a longer half-life in plasma than the antigen itself and can act as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in the cell (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties (binding to antigen in the neutral extracellular environment while releasing the antigen into the acidic endosomal compartment after entering the cell) are expected to have superior 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 invention provides antibodies that bind 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 contraction of muscles such as skeletal muscle, smooth muscle and cardiac muscle; activation of leukocyte movement, phagocytosis, etc.; activation of platelet shape change, secretion, etc.; lymphocyte activation; mast cell activation, including histamine secretion; cell response mediated by catecholamine α receptor or acetylcholine receptor; exocytosis; release of transmitters from neuronal terminals; and axoplasmic flow of neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin and myosin light chain, which have several calcium ion binding sites, which are believed to be derived from the 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 the coagulation protein Factor IX, the C-type lectins of the acyaroglycoprotein receptor and the mannose binding receptor, the A domain of the LDL receptor, annexins, thrombospondin type 3 domains and EGF-like domains.

[0251] In the present invention, when the metal ion is a calcium ion, it is expected that the antigen binding activity under low calcium ion concentration conditions is lower than the antigen binding activity under high calcium ion concentration conditions. At the same time, the intracellular calcium ion concentration is lower than the extracellular calcium ion concentration. On the contrary, the extracellular calcium ion concentration is higher than the intracellular calcium ion concentration. In the present invention, the low calcium ion concentration is preferably 0.1 μM (micro M) to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in the early endosome in vivo. At the same time, in the present invention, the high calcium ion concentration is preferably 100 μM to 10 μM, more preferably 200 μM to 5mM, and particularly preferably 0.5mM to 2.5mM, which is close to the calcium ion concentration in plasma (in blood). In the present invention, it is preferred that the low calcium ion concentration is the calcium ion concentration in the endosome, and the high calcium ion concentration is the calcium ion concentration in the plasma. When the level of antigen binding activity is compared 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 represented by a dissociation constant (KD), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or greater, and 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. The upper limit of the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is not particularly limited, and can be any value such as 100, 400, 1000 or 10000, as long as those skilled in the art can prepare. The dissociation rate constant (kd) can be used instead of KD. When it is difficult to calculate KD value, the level of binding reaction in Biacore can be evaluated based on the same concentrations by analyte.When antigen is by being fixed on the chip of antigen binding molecules of the present invention, the binding reaction under low calcium concentration is preferably 1 / 2 of the binding reaction under high calcium concentration or less, more preferably 1 / 3 or less, more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less.As is well known, usually, extracellular calcium ion concentration (for example, in blood plasma) is high in vivo, while intracellular calcium ion concentration (for example, in endosome) is low.Therefore, in the present invention, preferably extracellular condition is high calcium ion concentration, and intracellular condition is low calcium ion concentration.When the characteristic of antigen binding activity lower than under extracellular calcium ion concentration condition is given to antigen binding molecules of the present invention (for example, antibody), the antigen combined with antigen binding molecules of the present invention in extracellular is dissociated with antigen binding molecules of the present invention in cell, thereby enhancing antigen from extracellular mixing into cell.When this antibody is applied to a living body, the antigen concentration in the plasma can be reduced and the physiological activity of the antigen in the body can be reduced. Therefore, the antibody of the present invention is useful. The method of screening the antigen-binding domain or antibody having 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 giving the antigen-binding domain of the present invention the characteristic of being more weakly bound to the antigen under low calcium ion concentration conditions than under high calcium ion concentration conditions, and any method can be implemented. Specifically, the method is described in Japanese Patent Application No. 2011-218006 and includes, for example, replacing 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 in the antigen-binding domain. The antigen-binding molecule of the present invention, wherein at least one amino acid residue of the antigen-binding domain has been replaced by 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, is a preferred embodiment of the antigen-binding molecule of the present invention.

[0252] The amino acid residue with metal chelating activity preferably includes, for example, serine, threonine, asparagine, glutamine, aspartic acid and glutamic acid. In addition, the amino acid residue that changes the antigen-binding activity of the antigen-binding domain according to the calcium ion concentration preferably includes, for example, the amino acid residue that forms the 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 the coagulation protein factor IX; the C-type lectins of the acylglycoprotein 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 the calcium binding motif.

[0253] The antigen-binding domains of the present invention may contain amino acid residues that change antigen-binding activity according to calcium ion concentration, such as the above-mentioned amino acid residues with metal chelating activity and amino acid residues that form calcium binding motifs. The position of such amino acid residues in the antigen-binding domain is not particularly limited, as long as the antigen-binding activity changes according to the calcium ion concentration, they can be located at any position. At the same time, as long as the antigen-binding activity changes according to the calcium ion concentration, such amino acid residues can be included alone or in a 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 the CDR3 of the heavy chain variable region, the amino acid residues may be included in the CDR3 of the heavy chain variable region, more preferably at position 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 residue may be comprised in CDR1 of the light chain variable region, more preferably at position 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 residue may be comprised 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 residue may be comprised in CDR3 of the light chain variable region, more preferably at position 92.

[0255] In addition, the above embodiments can be combined. For example, according to the Kabat numbering in the light chain variable region, the amino acid residue can be contained 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 large number of antigen-binding domains having different sequences while sharing the above-mentioned amino acid residues that change antigen-binding activity according to calcium ion concentration as a common structure are prepared as a library. The library can be screened to efficiently obtain antigen-binding domains having binding activity to a desired antigen, wherein their antigen-binding activity changes according to calcium ion concentration.

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

[0258] The binding properties of an antibody to a specific antigen can also be expressed as the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody with respect to the specific antigen and is expressed in reciprocal seconds (i.e., sec -1 ). An increase in the kd value indicates that the antibody binds to its antigen less strongly. The invention thus includes antibodies that bind to C1s at an acidic pH with a higher kd value than at a neutral pH. The invention includes antibodies that bind to C1s at an acidic pH with a kd that 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 times greater than the kd of the antibody binding to C1s at a neutral pH. In another embodiment, the antibody may have a kd value of 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 antibody may have a kd value of 10 at acidic pH. -3 1 / s,10 -2 1 / s,10 -1 1 / s, or larger.

[0259] In some cases, "reduced 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, an antibody may be considered to exhibit "reduced binding to C1s at acidic pH compared to its binding at neutral pH" if it exhibits an acidic / neutral KD ratio of 2 or greater. In certain exemplary embodiments, the acidic / neutral KD ratio of the antibodies of the invention may 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 more. In another embodiment, the KD value of the antibody at neutral pH may be 10 -7 M,10 -8 M,10 -9 M,10 -10 M,10 -11 M,10 -12 In another embodiment, the KD value of the antibody at acidic pH can be 10 -9 M,10 -8 M,10 -7 M,10 -6 M, or larger.

[0260] In some cases, "reduced binding at acidic pH compared to neutral pH" is expressed as the ratio of the antibody's kd value at acidic pH to the antibody's kd value at neutral pH (or vice versa). For example, for the purposes of the present invention, an antibody may be considered to exhibit "reduced binding to C1s at acidic pH compared to its binding at neutral pH" if it exhibits an acidic / neutral kd ratio of 2 or more. In certain exemplary embodiments, the acidic / neutral kd ratio of an antibody of the invention may 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 more. In another embodiment, the antibody may have a kd value of 10 at neutral pH. -2 1 / s, 10 -3 1 / s, 10 -41 / s, 10 -5 1 / s, 10 -6 1 / s, or less. In another embodiment, the antibody may have a kd value of 10 at acidic pH. -3 1 / s,10 -2 1 / s,10 -1 1 / s, or larger.

[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 a specific aspect, "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 a specific aspect, "neutral pH" is 7.0 or 7.4.

[0263] As used herein, the expression "under high calcium concentration conditions" or "at a high calcium concentration" means 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 expression "under high calcium concentration conditions" or "at high calcium concentration" includes 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 0.5 mM, 0.7 mM, 0.9 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2.0 mM, 2.2 mM, 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM Ca. 2+ In certain aspects, "under high calcium concentration conditions" or "at high calcium concentration" refers to 1.2 mM Ca 2+ .

[0264] As used herein, the expression "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 the early endosome in vivo. The expression "under low calcium concentration conditions" or "at low calcium concentration" includes 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+ calcium concentration values. In certain aspects, "under low calcium concentration conditions" or "at low calcium concentration" refers to 3.0 μM Ca 2+ .

[0265] As expressed herein, KD values ​​and kd values ​​can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, e.g., Example 2 herein). KD values ​​and kd values ​​can be determined at 25 degrees Celsius (°C) or 37°C. This assay can be performed in the presence of 150 mM NaCl. In some embodiments, the assay can be performed using surface plasmon resonance technology, wherein the antibody is immobilized, the antigen is used as an 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 a method for enhancing the clearance of Cls from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of Cls from plasma. The present invention also provides a method for enhancing the clearance of a complex of Clr and Cls from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of a complex of Clr and Cls from plasma. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of Clr2s2 from plasma. In some embodiments, the method comprises administering to an individual an effective amount of an anti-C1s antibody of the present invention to enhance the clearance of Clr2s2 from plasma rather than Clq 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 Cls from the individual's plasma; (b) providing an antibody that binds to Cls via the antigen binding (Cls binding) domain of the antibody and has a KD (pH 5.8) / KD (pH 7.4) value, which is defined as a ratio of the KD for Cls at pH 5.8 to the KD for Cls at pH 7.4 of 2 to 10,000 when the KD is determined using surface plasmon resonance technology, wherein the antibody binds to Cls in plasma in vivo and dissociates from the bound Cls under conditions present in endosomes in vivo, and wherein the antibody is human IgG or 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 150mM NaCl. In another aspect, the surface plasmon resonance technique 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 the antigen binding domain of the first antibody; (b) identifying a second antibody that: (1) binds to C1s through the 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 in the variable region of the first antibody is replaced by histidine and / or at least one histidine is inserted into the variable region of the first antibody, and (3) has a KD(pH5.8) / KD(pH7.4) value that is higher than the KD(pH5.8) / KD(pH7.4) value of the first antibody and is between 2 and 10,000, wherein 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 the KD is determined using surface plasmon resonance technology. 7.4 for C1s, (4) binds to C1s in plasma in vivo, (5) dissociates from bound C1s under conditions present in endosomes in vivo, and (6) is human IgG or humanized IgG; (c) identifying a subject in need of lowering his or her plasma levels of C1s; and (d) administering the second antibody to the subject to lower the plasma levels of C1s in the subject. In another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technique can be used wherein 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 via an antigen binding domain of the first antibody, (2) has an amino acid sequence identical to a second antibody that binds to C1s via an antigen binding (C1s binding) domain of a second antibody, except that at least one variable region of the first antibody has at least one more histidine residue than the corresponding variable region of the second antibody, and (3) has a KD(pH5.8) / KD(pH7.4) value that is higher than the KD(pH5.8) / KD(pH7.4) value of the second antibody and is between 2 and 10,000, wherein 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 the KD is determined using surface plasmon resonance technology. 7.4 for C1s, (4) binds to C1s in plasma in vivo, (5) dissociates from bound C1s under conditions present in endosomes in vivo, and (6) is human IgG or humanized IgG; (b) identifying a subject in need of lowering his or her plasma levels of C1s; and (c) administering at least one of the first antibodies to the subject to lower the plasma levels of C1s in the subject. In another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In another aspect, this surface plasmon resonance technique can be used wherein 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 a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls.

[0270] In one aspect, the present disclosure provides a method for regulating 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 regulating complement activation in an individual suffering from a complement-mediated disease or condition, 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 may be performed by any route known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, 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 humans and non-human animals. In specific embodiments, the anti-C1s antibodies bind to C1s from humans, rats, and monkeys (e.g., cynomolgus monkeys, macaques, monkeys, chimpanzees, and baboons).

[0272] In one aspect, the present invention provides an anti-C1s antibody comprising at least one, two, three, four, five or six HVRs selected from the following: (a) an HVR-H1 comprising an amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (b) an HVR-H2 comprising an amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; (c) an HVR-H3 comprising an amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58; (d) an HVR-L1 comprising an amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (e) an HVR-L2 comprising an amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (f) an HVR-L3 comprising an amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61.

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

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

[0275] In another aspect, an anti-C1s 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 group consisting of: (i) an HVR-H1 comprising an amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56, (ii) an HVR-H2 comprising an amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57, and (iii) an HVR-H3 comprising an 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 group consisting of: (i) an HVR-L1 comprising an amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59, (ii) an HVR-L2 comprising an amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60, and (c) an HVR-L3 comprising an amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. HVR-L3 having the amino acid sequence of 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 an antibody comprising a VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24 and a VL sequence of SEQ ID NO: 26, 27, 28, 30, or 31.

[0277] In some embodiments, an anti-C1s antibody of the invention comprises a 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, an anti-C1s antibody of the invention comprises at least one histidine substituted with one or more amino acid residues at a position 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 in any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1s antibody comprises the HVRs in any of the above embodiments and further comprises a VH or VL comprising a FR sequence. In another embodiment, the anti-C1s antibody of the invention comprises the following heavy chain or light chain variable domain FR sequence

[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 comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to the reference sequence, but the anti-C1s antibody comprising the sequence retains the ability to bind to 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 substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-C1s antibody comprises a VH sequence of SEQ ID NO: 19, 20, 21, 23, or 24, including post-translational modifications of said sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from the following: (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56, (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57, and (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. Post-translational modifications include, but are not limited to, modifying the N-terminal glutamine or glutamic acid of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0285] In another aspect, an anti-C1s antibody is 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 comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to the reference sequence, but the anti-C1s antibody comprising the sequence retains the ability to bind to 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 substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-C1s antibody comprises a VH sequence of SEQ ID NO: 26, 27, 28, 30, or 31, including post-translational modifications of said sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from the following: (a) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2 comprising an amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. Post-translational modifications include, but are not limited to, modifying the N-terminal glutamine or glutamic acid of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0286] In another aspect, an anti-C1s antibody is provided, wherein the antibody comprises a VH as in any embodiment provided above, and a VL as in any embodiment provided above. In one embodiment, the antibody comprises the VH and VL sequences in 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

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

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

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

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

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

[0292] 5) An antibody comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:59, an HVR-L2 sequence of SEQ ID NO:60 and an HVR-L3 sequence of SEQ ID NO:61.

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

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

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

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

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

[0298] 5) An antibody comprising an HVR-H1 sequence of SEQ ID NO:56, an HVR-H2 sequence of SEQ ID NO:57, an HVR-H3 sequence of SEQ ID NO:58, an HVR-L1 sequence of SEQ ID NO:59, an HVR-L2 sequence of SEQ ID NO:60 and an HVR-L3 sequence of SEQ ID NO:61.

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

[0302] In another aspect, an 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 group consisting of: (i) an HVR-H1 comprising an amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126, (ii) an HVR-H2 comprising an amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134, and (iii) an HVR-H3 comprising an 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 group consisting of: (i) an HVR-H1 comprising an amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126, (ii) an HVR-H2 comprising an amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134 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, an anti-C1r antibody of the invention comprises a 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 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.

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

[0308] 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

[0309] 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.

[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 an acceptor 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 comprising a FR sequence. In another embodiment, the anti-C1r antibody of the invention comprises the following heavy chain or light chain variable domain FR sequence.

[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 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 to 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, substitutions, insertions, or deletions occur in regions outside of the HVRs (ie, in the FRs). Optionally, the anti-C1s antibody comprises the VH sequence in SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110, including post-translational modifications of said sequence. In a specific embodiment, the VH comprises one, two or three 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, 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, modifying the N-terminal glutamine or glutamic acid of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0312] In another aspect, an anti-C1r antibody is 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 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 to 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, substitutions, insertions, or deletions occur in regions outside of the HVRs (ie, in the FRs). Optionally, the anti-C1r antibody comprises the VL sequence in SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (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, modifying the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0313] In another aspect, an anti-C1r antibody is provided, wherein the antibody comprises a VH as in any embodiment provided above, and a VL as in any embodiment provided above. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences in 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, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 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 the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

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

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

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

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

[0318] 9) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 122, an HVR-H2 sequence of SEQ ID NO: 130, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 146, an HVR-L2 sequence of SEQ ID NO: 154, and an 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 present invention competes for binding to C1r with an antibody selected from the group consisting of 6) to 13) below. In some embodiments, the isolated anti-C1r antibody of the present invention competes for binding to C1r with an antibody selected from the group consisting of 6) to 13) below at neutral pH.

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

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

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

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

[0328] 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,

[0329] 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,

[0330] 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

[0331] 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,

[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 (Cls). 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 of C1s 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 amino acids 16-291 of the complement C1s protein, amino acids 16-172 of the complement C1s protein as set forth in SEQ ID NO: 1, amino acids 16-210 of the complement C1s protein as set forth in SEQ ID NO: 1, amino acids 16-111 of the complement C1s protein as set forth in SEQ ID NO: 1, amino acids 112-210 of the complement C1s protein as set forth in SEQ ID NO: 1, amino acids 131-172 of the complement C1s protein as set forth in SEQ ID NO: 1, or amino acids 16-130 of the complement C1s protein as set forth 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 both 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 (Clr), the CUB1-EGF-CUB2 domain consisting 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-mentioned epitope of C1r is an epitope of human C1r.

[0334] In another aspect of the 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 may comprise any of the features described in Sections 1-7 below, either alone 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, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0338] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab form of the antibody of interest and its antigen. For example, solution binding affinity of the Fab for the antigen is measured by titrating the antibody in the presence of unlabeled antigen with a minimum concentration of ( 125Fab was equilibrated with I) labeled antigen and then bound antigen was captured with an anti-Fab antibody coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were 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 at room temperature (about 23° C.) for two to five hours. In a non-adsorbent plate (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 the anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, the incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is achieved. Thereafter, the mixture is transferred to the capture plate for room temperature incubation (e.g., for one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate has dried, 150 μl / well of scintillant (MICROSCINT-20TM; Packard) is added and the plate is placed on the TOPCOUNT TM The counts were performed on a gamma counter (Packard) for tens of minutes. The concentration of each Fab that resulted in less than or equal to 20% of maximal binding was selected for competitive binding assays.

[0339] According to another embodiment, Kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, at 25 ° C, the determination using BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (GE Healthcare) is performed using immobilized antigen CM5 chips with ~10 response units (RU). In one embodiment, according to the instructions for use of the provider, the carboxymethylated dextran biosensor chip (CM5, GE Healthcare) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Antigen is diluted to 5 micrograms (μg) / ml (~0.2μM) with 10mM sodium acetate pH 4.8, and then injected with a flow rate of 5 microliters (μl) / minute to achieve about 10 response units (RU) of coupled protein. After the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into a 4% paraformaldehyde-containing solution with 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant-free PBS (PBST). The association rate (k) was calculated using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) was calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate measured by the above surface plasmon resonance assay exceeds 10 6 M -1 s -1 , the binding rate can be determined by measuring the binding rate in the presence of increasing concentrations of the antigen (e.g., in a spectrometer such as a spectrophotometer equipped with a stop-flow (Aviv Instruments) or a 8000-series SLM-AMINCO with a stirred chamber). TM When measured in a spectrophotometer (ThermoSpectronic), a fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) was used that measured the increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C.

[0340] In some embodiments, the binding affinity of each histidine-substituted variant of the present invention at pH 7.4 and pH 5.8 is determined using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37° C. Recombinant protein A / G (Pierce) can be immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes can be prepared in 7(+) buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05% Tween20, 0.005% NaN3, pH 7.4), 5(+) buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05% Tween20, 0.005% NaN3, pH 5.8), or 5(-) buffer (20mM ACES, 150mM NaCl, 3μM CaCl2, 0.05% Tween20, 0.005% NaN3, pH 5.8). Each antibody can be captured to the sensor surface by protein A / G. The target for antibody capture level is 200 resonance units (RU). Prepared native zymogen human C1s (CompTech) or recombinant human C1s can be injected at, for example, 50nM and then dissociated.

[0341] Specific examples of the steps of the Biacore assay of the present invention are as follows.

[0342] The binding specificity of C1s CUB1-EGF-CUB2 binders was determined using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37°C. Recombinant protein A / G (Pierce) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7(+) buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4). Each antibody was captured on the sensor surface by protein A / G. The target for antibody capture level was 100 resonance units (RU). Native zymogen human C1s (Comptech A103) (as a monomer at 50nM) or recombinant human C1s CCP1-CCP2-SP-His (as a monomer at 100nM) was injected and then dissociated. The sensor surface was regenerated after each cycle with 10 mM glycine-HCl pH 1.5. It could be determined that the C1sCUB1-EGF-CUB2 binder bound to the native zymogen human C1s but not to the recombinant human C1s CCP1-CCP2-SP-His, a truncated protein lacking the CUB1-EGF-CUB2 domains.

[0343] The C1q replacement function of the antibody was confirmed at 37 degrees Celsius by using the C1r2s2 capture method of a BIACORE (registered trademark) T200 instrument (GE Healthcare). Anti-His antibodies (GE-Healthcare) were immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies, recombinant human C1r2s2 Flag / His tetramers and native human C1q (Comptech A099) were prepared in pH 7.4 buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 1mg / mL BSA (IgG-free), 1mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH7.4). Recombinant human C1r2s2 Flag / His tetramers were first captured on the sensor surface by anti-His antibodies ("hc1r2s2"). The capture level was targeted to 200 resonance units (RU). Native human C1q was injected at 100 nM to capture 200 RU ("hc1q"), followed immediately by an injection of 500 nM antibody at 10 micro L / min for 1200 sec. The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 for each cycle. For antibodies with C1q substituted functionality, the response units of sensorgram 2 (in the presence of C1r2s2, C1q, and antibody) were lower than the response units in sensorgram 1 (in the presence of C1r2s2, C1q, and buffer but no antibody) after the time point at which sensorgrams 1 and 2 intersected ("time point of intersection"). The time point of intersection was determined by subtracting the buffer response (sensogram 1) from the antibody (Ab) response (sensogram 2) and referencing the time point at which the difference changed from positive to negative.

[0344] The C1q replacement function of the antibody was confirmed by the C1q capture method using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37 degrees Celsius. Antibodies, recombinant human C1r2s2Flag / His tetramers and biotinylated native human C1q (Comptech A099) were prepared in pH 7.4 buffer (20mM ACES, 150mM NaCl, 1.2mM CaCl2, 1mg / mL BSA (IgG-free), 1mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). The biotinylated native human C1q was first captured into a flow cell of a CAP sensor chip (GE-Healthcare). The capture level was targeted to be in the range of 800 to 1000 resonance units (RU). Recombinant human C1r2s2 Flag / His tetramer was injected at 300 nM, followed by injection of the antibody at 500 nM for 180 s at 10 micro L / min. In each cycle, the sensor surface was regenerated with 8 M guanidine hydrochloride and 1 M NaOH in a ratio of 8:1. Antibodies with C1q substitution function enhanced the dissociation rate of C1r2s2, i.e., the curve in the presence of the antibody was lower than the curve in the absence of the antibody.

[0345] To evaluate the blocking effect of antibodies on the binding of C1q to C1r2s2, blocking experiments were performed using a BIACORE (registered trademark) T200 instrument (GE Healthcare) at 37 degrees Celsius. Anti-his antibodies (GE Healthcare) were immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies, recombinant human C1r2s2 Flag / His tetramers, and native human C1q were prepared in pH 7.4 buffer (20mMACES, 150mM NaCl, 1.2mM CaCl2, 1mg / mL BSA (IgG-free), 1mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). Recombinant human C1r2s2 Flag / His tetramers were first captured onto the sensor surface by anti-His antibodies ("hc1r2s2"). The capture level was targeted to 200 resonance units (RU). The antibody variants were injected at 500 nM, followed by native human CIq at 100 nM ("hclq"). The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 for each cycle. Antibodies with CIq blocking function are those that compete with CIq for binding to C1r2s2.

[0346] In some embodiments, an additional dissociation phase at pH 5.8 is integrated immediately after the dissociation phase at pH 7.4, if necessary. The dissociation rate in 5(+) buffer can be determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[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, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, ed., (Springer-Verlag, New York), pp. 269-315 (1994); see also, WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0349] Diabodies are antibody fragments with two antigen binding sites that 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] A single domain antibody is an antibody fragment that contains 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, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1.

[0351] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, 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 another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from the class or subclass of the parent 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 immunogenicity to people while retaining the specificity and affinity of the parent non-human antibodies. Usually, humanized antibodies include one or more variable domains, wherein HVR, for example, CDR (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (for example, antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0355] Humanized antibodies and methods for their preparation are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); 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 the consensus sequence of human antibodies having a particular subgroup of light chain 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. 151:262). 3 (1993)); human mature (somatic) 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 immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or they are present outside the chromosomes or are randomly integrated into the chromosomes of the animals. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, which describes HUMAB (registered trademark) technology; U.S. Pat. No. 7,041,870, which describes KM MOUSE (registered trademark) technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes VELOCIMOUSE (registered trademark) technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0360] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for preparing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86 (1991). Human antibodies prepared via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 (2006). Additional methods include, e.g., U.S. Pat. No. 7,189,826 (describing the preparation of monoclonal human IgM antibodies by hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005).

[0361] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The following describes the technology for selecting human antibodies from antibody libraries.

[0362] 5. Antibodies from the library

[0363] The antibodies of the present invention can be isolated by screening combinatorial libraries of antibodies having one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies having desired binding properties. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, for example, 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, the VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for phage binding to the 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, a naive library (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self antigens as well as self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. 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] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0366] 6. Multispecific Antibodies

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

[0368] Techniques for making 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. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic guidance for preparing antibody Fc-heterodimers (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. 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 "diabody" technology 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, e.g., 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] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which comprise an antigen binding site that binds to C1s as well as another, different antigen (see, eg, US 2008 / 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. The amino acid sequence variants of the antibody can be prepared by introducing suitable 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 the antibody amino acid sequence and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has desired characteristics, for example, antigen-binding.

[0373] a) Substitution, insertion and deletion variants

[0374] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. More changes are provided under the heading of "exemplary substitutions" in Table 1 and are further described below regarding the classification of amino acid side chains. Amino acid substitutions can be introduced into the target antibody and the product screened for desired activity (e.g., maintained / improved antigen binding, reduced immunogenicity or improved ADCC or CDC).

[0375] [Table 1]

[0376] Original residue Exemplary substitutions Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

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

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

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

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

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

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

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

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

[0385] A substitution variant includes replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, the resulting variant selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will substantially maintain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can, for example, be routinely prepared using affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0386] Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigens, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and reselecting therefrom has been described, for example, 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 with the desired affinity. Another method for introducing diversity includes an HVR directed approach, in which several HVR residues (e.g., 4-6 residues at the same time) are randomized. HVR residues that are involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0387] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not significantly reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions described herein) that do not significantly reduce binding affinity may be made in HVRs. Such changes may, for example, be outside of the residues in the HVR that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, 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 in Cunningham and Wells (1989) Science 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitutions. Alternatively, or additionally, 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 amino-terminal and / or carboxyl-terminal fusions of polypeptides ranging from one residue to more than one hundred residues in length, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertion variants of antibody molecules include fusions of enzymes (e.g., for ADEPT) or polypeptides that increase the plasma half-life of antibodies to the N- or C-termini of antibodies.

[0390] b) Glycosylation variants

[0391] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Adding glycosylation sites to antibodies or deleting glycosylation sites can be readily achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0392] When antibody comprises Fc district, it is possible to change the carbohydrate connected thereto.Natural antibodies produced by mammalian cells typically comprise branched, biparting (biantennary) oligosaccharides, which are usually attached to the Asn297 of the CH2 domain of the Fc district by N-connection.See, e.g., Wright et al., TIBTECH 15:26-32 (1997).Oligosaccharides can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose connected to the GlcNAc in the "stem" of the biparting oligosaccharide structure.In some embodiments, the modification of the oligosaccharides in the antibody of the present invention can be carried out to produce antibody variants with specific improved properties.

[0393] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose that is attached to the Fc region (directly or indirectly). For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546, for example. Asn297 refers to an aspartic acid residue located near position 297 (EU numbering of Fc region residues) in the Fc region; however, due to small sequence variations in antibodies, Asn297 may also be located about + / -3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such a fucosylated variant may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Disclosed example variants of "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; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 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 defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003 / 085107).

[0394] Also provided are antibody variants having oligosaccharides bisected, for example, wherein the two-branched oligosaccharides connected to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharides connected to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0395] c. Fc region variants

[0396] (Cleaning 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. The Fc region variants can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. In some embodiments, the Fc region is the Fc region of human IgG1.

[0398] In order to enhance the reduction of plasma antigen concentration and / or improve the pharmacokinetics of antibodies, amino acid residues at the site of binding to FcRn in the Fc region of IgG can be modified to enhance its uptake by cells. When an antibody with pH dependence is modified in this way, the mutant will be a "sweeping" antibody that can bind FcRn more firmly and effectively transfer the antigen to the endosome (where the pH is acidic) and then degrade, but can itself be more efficiently recovered to the cell surface. Compared with the unmodified original (parent) antibody, this modified "sweeping" antibody can bind to FcRn firmly at neutral pH and on the cell surface, and enhance the uptake and degradation of antigen. (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 said region that enhances reduction of plasma antigen concentration and / or improves the pharmacokinetics of the antibody.

[0400] In some embodiments, the Fc region is a human Fc region having a stronger binding activity to an activated Fc gamma receptor than the binding activity of the Fc region of native human IgG1. As mentioned in, for example, WO 2013 / 047752, in order to enhance the binding activity to an activated Fc gamma receptor, one or more amino acids selected from the group consisting of amino acids at the following positions in the Fc region may be modified: 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245 45,246,247,249,250,251,254,255,256,258,260,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,278,279,280,281,282,283,284,285,2 86,288,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,311,313,315,317,318,320,322,323,324,325,326,327,328,329,330,331, 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) which differ from the amino acids at the corresponding positions in the Fc region of native human IgG1, which is the parent (original) antibody.

[0401] In some embodiments, the Fc region is a human Fc region having a stronger binding activity to an inhibitory Fc gamma receptor than to an activated Fc gamma receptor. As mentioned in, for example, WO 2013 / 125667, in order to enhance the binding activity to an inhibitory Fc gamma receptor, one or more amino acids selected from the group consisting of amino acids at the following positions in the Fc region may be modified: 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, 315 435, 436, 438, 439, 440, 442, and 447 (EU numbering) that differ from the corresponding positions in the native human IgG1 Fc region by an amino acid.

[0402] In some embodiments, the Fc region is a human Fc region having a stronger binding activity to FcRn at neutral pH than that of the Fc region of native human IgG1. As mentioned in, for example, WO 2011 / 122011, in order to enhance the binding activity to FcRn at neutral pH, one or more amino acids selected from the group consisting of amino acids at the following positions in the Fc region may be modified to differ from native human IgG1. The amino acids at the corresponding positions in the Fc region.

[0403] In certain embodiments, the present invention contemplates antibody variants that have some but not all effector functions, which makes them ideal candidates for the in vivo half-life of antibodies important and certain effector functions (such as complement and ADCC) that are not necessary or harmful. 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 therefore may lack ADCC activity), but maintains FcRn binding ability. The main cells that mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) Table 3 on page 464. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 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 assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo, for example in an animal model as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, 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, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0404] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with 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. Pat. No. 7,332,581).

[0405] Certain antibody variants with increased or decreased binding to FcRs are described. (See, e.g., U.S. Pat. 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 with one or more amino acid substitutions that improve 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 (ie, increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0408] US2005 / 0014934A1 (Hinton et al.) describes antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus (J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The antibody comprises 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., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also, Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0409] d. Cysteine ​​engineered antibody variants

[0410] In certain embodiments, it may be desirable to prepare a cysteine ​​engineered antibody, e.g., a "thioMAb," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In a particular embodiment, the replaced residue appears at the access site of the antibody. By replacing the residue with cysteine, a reactive thiol group is thereby placed at the access site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, thereby producing an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described in, e.g., 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 other non-protein moieties known in the art and readily available. Suitable parts 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 / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in preparation due to its stability in water. Polymers can have any molecular weight and can be branched or non-branched. The number of polymers connected to an antibody can vary, and if more than one polymer is connected, it can be the same or different molecules. Generally, the amount and / or type of polymer to be derivatized can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative is useful for treatment of a defined condition, and the like.

[0413] In another embodiment, a conjugate of an antibody with a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength, and includes but is not limited to such a wavelength, which does not damage normal cells, but heats the non-protein moiety to a temperature at which cells adjacent to the antibody-non-protein moiety 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. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-C1s antibody described herein. Such nucleic acid may 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 chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp2 / 0 cell). 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 as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0416] For recombinant production of anti-C1s antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0417] Host cells suitable for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be prepared in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also, Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in Escherichia coli). After expression, antibodies can be separated from bacterial cell paste into soluble fractions and can be further purified.

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

[0419] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0420] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[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 monkey kidney CV1 cell lines transformed by SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); as described, for example, in Mather et al., Annals TRI cells described in NY Acad. Sci. 383: 44-68 (1982); MRC5 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, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, 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 antibody populations that are specific to a particular antigen. In certain embodiments, screening methods can include measuring one or more binding parameters (e.g., KD or kd) of a single antibody in an initial antibody population at acidic pH and neutral pH. For example, surface plasmon resonance can be used, or any other analytical method that allows quantitative or qualitative assessment of the binding properties of an antibody to a particular antigen can be used to measure the binding parameters of an antibody. In certain embodiments, screening methods can include identifying antibodies that bind to an antigen at an acidic KD / neutral KD ratio of 2 or greater. Alternatively, screening methods can include identifying antibodies that bind to an antigen at an acidic kd / neutral kd ratio of 2 or greater.

[0423] In another embodiment, the mutagenesis method may include the deletion, replacement or addition of amino acids incorporated into the heavy chain and / or light chain of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In certain embodiments, mutagenesis may be carried out in one or more variable domains of the antibody, such as in one or more HVRs (e.g., CDRs). For example, mutagenesis may include replacing an amino acid in one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In certain embodiments, mutagenesis may include replacing 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" refers to a mutant form of an antibody that exhibits a greater acidic KD / neutral KD ratio than the original "parent" (i.e., pH-dependent) form of the antibody before mutagenesis, or a greater acidic kd / neutral kd ratio. 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 an 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 bifunctional or derivatizing agents, e.g., maleimidobenzoic acid succinimidyl ester (conjugated through cysteine ​​residues), N-hydroxysuccinimidyl (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, wherein R and R1 are different alkyl groups.

[0425] Animals (usually non-human mammals) are immunized against antigens, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the antibody titer of the serum is determined. The animals are boosted until the titer plateaus. Preferably, the animals are boosted with conjugates of the same antigen conjugated to different proteins and / or conjugated by different cross-linking agents. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum are suitable for enhancing immune responses.

[0426] Monoclonal antibodies are obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Thus, the attributive term "monoclonal" indicates the characteristic of an antibody 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, mice or other suitable host animals (such as hamsters) are immunized as described above to cause lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0428] The immune reagent will typically include an antigen protein or a fusion variant thereof. Typically, if cells of human origin are required, peripheral blood lymphocytes (PBL) are used, or if non-human mammalian sources are required, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortal cell lines using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp.59-103).

[0429] Immortal cell lines are usually transformed mammalian cells, particularly rodents, myeloma cells of cattle and human origin. Usually, rat or mouse myeloma cell lines are adopted. The hybridoma cells thus prepared are inoculated and cultivated in a suitable culture medium, and the culture medium preferably contains one or more substances that suppress the growth or survival of non-fused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically comprise hypoxanthine, aminopterin and thymidine (HAT culture medium), which is a substance that prevents the growth of HGPRT defective cells.

[0430] Preferred immortalized myeloma cells are those cells, which effectively fuse, support the selected antibody production cells to stabilize high levels of production antibodies, and are sensitive to culture medium such as HAT culture medium. Among them, preferred are mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from American Type Culture Collection (American Type Culture Collection, Manassas, Virginia USA). Human myeloma and mouse-human hybrid myeloma cell lines are also described as being used to produce 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 antigens, the culture medium of the hybridoma cells is determined. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107 (1): 220-239 (1980).

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

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

[0434] C. Assay

[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, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, and the like.

[0438] In another aspect, competition 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) binding of a reference antibody to C1s by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as 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 competition assay can be conducted under neutral pH conditions. In some embodiments, competition assays are performed using, for example, Octet TM Systematic tandem competition assay.

[0439] In an exemplary competition assay, immobilized C1s is incubated in a solution comprising a first labeled antibody and a second unlabeled antibody, the first labeled antibody binding to C1s (e.g., one of those described herein), 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 the 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 allow the first antibody to bind to C1s, excess unbound antibody is removed and the amount of label bound to immobilized C1s is measured. If the amount of label bound to immobilized C1s is 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 to the same epitope as the anti-C1s antibodies provided herein or that compete with the anti-C1s antibodies provided herein for binding to C1s. A sandwich assay involves the use of two antibodies, each of which is capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, a 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. Pat. No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay), or an anti-immunoglobulin antibody labeled with a detectable moiety can be used for measurement (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. It can be determined that an antibody that binds to C1s simultaneously with an anti-C1s antibody provided herein is an antibody that binds to a different epitope than the anti-C1s antibody. Therefore, an antibody that is not able to bind to C1s simultaneously with an anti-C1s antibody provided herein can be determined to be an antibody that binds to the same epitope as the anti-C1s antibody or competes with the anti-C1s antibody for binding to C1s.

[0441] 2. Activity Assay

[0442] In one aspect, an assay for identifying an anti-C1s antibody having biological activity is provided. The biological activity may include blocking activation of the classical pathway and the generation of cleavage products C2a, C2b, C3a, C3b, C4a, C4b, C5a and C5b resulting from activation of the 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 present invention is tested. In some embodiments, the ability of the antibodies of the present invention to inhibit complement-mediated hemolysis of sheep red blood cells (RBC) sensitized by antibodies to sheep RBC antigens can be evaluated, i.e., using the RBC assay. In some embodiments, the ability of the antibodies of the present invention to inhibit complement-mediated hemolysis of chicken red blood cells (cRBC) sensitized by antibodies to cRBC antigens can be evaluated. Using human serum as a source of complement proteins, the activity of the antibodies of the present invention can be determined by measuring the amount of released hemoglobin by spectrophotometry.

[0444] The RBC assay can be suitably performed using known methods (e.g., methods disclosed in J. Vis. Exp. 2010; (37): 1923). This article describes how to perform a 50% hemolytic complement (CH50) assay as an RBC lysis assay. In short, the assay measures the activation of the classical complement pathway and detects the reduction, loss, or inactivation of any component in the pathway. It assesses the activity of complement components in serum to lyse red blood cells. When antibodies are incubated with test serum, the 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 of inhibition of cell lysis by complement components; however, the concept and basic setup are substantially the same as 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). The serum is then added to an equal volume of sensitized sheep erythrocytes and incubated (e.g., at 37°C for 1 hour) to allow erythrocyte lysis. The reaction is then stopped. The mixture is centrifuged to precipitate undissolved cells, the supernatant is removed, and the release of hemoglobin is analyzed using the absorbance (OD) at 415nm minus the OD at 630nm. In order to calculate the percentage inhibition of erythrocyte lysis, 0% inhibition is set to the condition of not adding antibody (buffer only), and 100% inhibition is set to the condition of adding EDTA at a final concentration of 5mM (see, e.g., Example 7). When an antibody shows a percentage of inhibition of erythrocyte lysis, this means that the antibody has neutralizing activity on human serum complement, e.g., the activity of inhibiting the interaction between C1q and C1r2s2 complexes.

[0445] Therefore, to assess the activity of inhibiting the interaction between C1q and C1r2s2 complexes, the RBC assay can be used to evaluate the neutralizing activity of the antibodies of the invention against human serum complement. In one embodiment, the invention provides an isolated antibody that inhibits the interaction between C1q and C1r2s2 complexes, 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 conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0448] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or more 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. Pat. 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 described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca Americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

[0450] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to prepare radioconjugates. Examples include 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. When the radioconjugate is used for detection, it may contain a radioactive atom for scintillation 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 the antibody and cytotoxic agent 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 imido esters (such as dimethyladipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and di-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0452] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared using cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[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 "detection" includes quantitative detection or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph fluid, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, lens fluid or mucus.

[0455] In one embodiment, an anti-C1s antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of C1s in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-C1s antibody as described herein under conditions that allow 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 may be an in vitro or in vivo method. In one embodiment, the anti-C1s antibody is used to select subjects suitable for treatment with the anti-C1s antibody, for example, where C1s is a biomarker for selecting patients.

[0456] Exemplary disorders that can be diagnosed using the antibodies of the invention include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, allergic reactions, argyrophilic dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, English amyloid angiopathy, bullous pemphigoid, Buerger's disease, Clq nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, t-Jakob disease, Crohn's disease, cryoglobulin vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangles with calcifications, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism associated with chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome, Hallervorden-Spa Tz disease, hemolytic-uremic syndrome, hereditary angioedema, hypophosphastasis, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infectious diseases (e.g., those 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 sediment disease), membranous nephritis, 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 parkinsonism, polymyositis, prion 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, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementary urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion failure.

[0457] In certain embodiments, labeled anti-C1s antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescent, chromophore, electron dense, chemiluminescent, and radioactive labels), as well as indirectly detectable moieties such as enzymes or ligands, for example, via an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactose peroxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0458] F. Pharmaceutical preparations

[0459] Pharmaceutical formulations of anti-C1s antibodies as described herein are prepared in the form of lyophilized formulations or aqueous solutions 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)). Pharmaceutical carriers are generally nontoxic to recipients at the dosages and concentrations used, 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 octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 The invention also includes a polypeptide (e.g., a polypeptide having a residue of 100,000 amino acids); a protein such as serum albumin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine; a monosaccharide, a disaccharide, and other carbohydrates, including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a nonionic surfactant such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs 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 chondroitinases.

[0460] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0461] When necessary, the preparations herein may also contain more than one active ingredient for a specific indication for treatment, preferably those with complementary activities that do not adversely affect each other. For example, it may be necessary to further provide a preparation for combined 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 microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0463] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0464] Preparations useful 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 of the anti-C1s antibodies provided herein can be used in therapeutic methods.

[0467] In one aspect, an anti-C1s antibody for use as a medicament is provided. In another aspect, an anti-C1s antibody for use in treating a complement-mediated disease or condition is provided. In certain embodiments, an anti-C1s antibody for use in a method of treatment is provided. 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 condition, 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 a further embodiment, the present invention provides an anti-C1s antibody for use in treating a complement-mediated disease or condition. In a further embodiment, the anti-C1s antibody of the present invention can be used to enhance the clearance of C1s from plasma. In a further embodiment, the anti-C1s antibody of the present invention can be used to enhance the clearance of C1r2s2 from plasma. In a further embodiment, the anti-C1s antibody of the present invention can be used to enhance the clearance of C1r2s2 from plasma instead of C1q from plasma. In some cases, the antibody inhibits a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for treating a complement-mediated disease or condition. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for enhancing the clearance of C1s from plasma. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for enhancing the clearance of C1r2s2 from plasma. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method for enhancing the clearance of C1r2s2 from plasma instead of C1q from plasma. In certain embodiments, the invention provides an anti-C1s antibody for use in a method of inhibiting a component of the classical complement pathway; in certain cases, the classical complement pathway component is Cls. An "individual" according to any of the above embodiments is preferably a human.

[0469] In one aspect, the present disclosure provides a method for regulating 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 regulating complement activation in an individual suffering from a complement-mediated disease or condition, 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 performed by any route known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous or subcutaneous. In some embodiments, administration is intrathecal.

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

[0471] In some cases, a complement-mediated disease or condition is characterized by the presence of elevated (higher than normal) amounts of Cls or elevated levels of complement Cls activity in a cell, tissue, or fluid. For example, in some cases, a complement-mediated disease or condition is characterized by elevated amounts of Cls and / or elevated activity 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 a normal control level, such as higher than a normal control level for an individual or population of the same age group. An "above normal level" of Cls activity in a cell, tissue, or fluid means that proteolytic cleavage affected by Cls in the cell, tissue, or fluid is higher than a normal control level, such as higher than a normal control level for an individual or population of the same age group. In some cases, an individual suffering from a complement-mediated disease or condition exhibits one or more other symptoms of the disease or condition.

[0472] In other cases, the complement-mediated disease or condition is characterized by the presence of a lower than normal amount of Cls or a lower level of complement Cls activity in a cell, tissue or fluid. For example, in some cases, the complement-mediated disease or condition is characterized by the presence of a lower amount and / or lower activity of Cls in brain tissue and / or cerebrospinal fluid. A "lower 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 a normal control level, such as a normal control level for an individual or population of the same age group. A "lower 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 a normal control level, such as a normal control level for an individual or population of the same age group. In some cases, an individual suffering from a complement-mediated disease or condition exhibits one or more other symptoms of the disease or condition.

[0473] A complement-mediated disease or condition is a disease or condition in which the amount or activity of complement C1s causes a disease or condition in an individual. In some embodiments, a complement-mediated disease or condition is 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, a complement-mediated disease or condition is an autoimmune disease. In some embodiments, a complement-mediated disease or condition is cancer. In some embodiments, a complement-mediated disease or condition is an infectious disease. In some embodiments, a complement-mediated disease or condition is an inflammatory disease. In some embodiments, a complement-mediated disease or condition is a blood disease. In some embodiments, a complement-mediated disease or condition is ischemia-reperfusion injury. In some embodiments, a complement-mediated disease or condition is an eye disease. In some embodiments, a complement-mediated disease or condition is kidney disease. In some embodiments, the complement-mediated disease or condition is transplant rejection. In some embodiments, the complement-mediated disease or condition is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease or condition is a vascular disease. In some embodiments, the complement-mediated disease or condition is a vasculitis condition. In some embodiments, the complement-mediated disease or condition is a neurodegenerative disease or condition. 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 condition 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, allergic reactions, argyrophilic dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, English 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, cryoglobulin vasculitis, Pugh's dementia, and leukemia. pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangles with calcifications, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism associated with 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 disease, inclusion body myositis, infectious diseases (e.g., those caused by bacteria (e.g., Neisseria meningitidis or Streptococcus), viruses (e.g., human immunodeficiency virus (HIV)), or other infectious agents) , Inflammatory disorders, Ischemia / reperfusion injury, Mild cognitive impairment, Immune thrombocytopenic purpura (ITP), Molybdenum cofactor deficiency (MoCD) type A, Membranoproliferative glomerulonephritis (MPGN) I, Membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), Membranous nephritis, Multi-infarct dementia, Lupus (eg, 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 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, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementary urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion failure.

[0475] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia and frontotemporal dementia associated with chromosome 17 with Parkinson's disease) are the most common forms of tauopathy. Accordingly, 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 associated with chromosome 17 with Parkinson's disease. Other tauopathy 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, argyrophilic grain dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, frontotemporal dementia, frontotemporal dementia with Parkinsonism associated with chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinke r disease, Hallevorden-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, dementia with tangles only, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0477] The present disclosure also provides methods of treating synucleinopathies such as Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiple system atrophy (MSA), etc. For example, PD with dementia (PDD) can be treated using the methods of the present disclosure.

[0478] In some embodiments, the complement-mediated disease or condition comprises Alzheimer's disease. In some embodiments, the complement-mediated disease or condition comprises Parkinson's disease. In some embodiments, the complement-mediated disease or condition comprises transplant rejection. In some embodiments, the complement-mediated disease or condition 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 condition 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 condition in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, hematological diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative conditions, kidney diseases, transplant rejection, eye diseases, vascular diseases, or vasculitic conditions. Symptoms can be neurological symptoms, such as impaired cognitive function, memory impairment, loss of motor function, etc. Symptoms can also be C1s protein activity in cells, tissues, or fluids of an individual. Symptoms can also be the degree of complement activation in cells, tissues, or fluids of an individual.

[0480] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual modulates complement activation in cells, tissues, or fluids of the individual. In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual can inhibit complement activation in cells, tissues, or fluids of the individual. For example, in some embodiments, when an anti-C1s antibody of the present disclosure is administered to an individual suffering from a complement-mediated disease or condition at one or more doses as a monotherapy or in combination therapy, 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 complement activity in the individual prior to 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 lysis of red blood cells.

[0482] In some embodiments, an anti-C1s antibody of the present disclosure reduces deposition of C3 on platelets; for example, in some embodiments, an anti-C1s antibody of the present disclosure reduces 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) a reduction in complement activation; (b) an improvement in cognitive function; (c) a reduction in neuronal loss; (d) a reduction in the level of phosphorylated Tau protein in neurons; (e) a reduction in glial cell activation; (f) a reduction in lymphocyte infiltration; (g) a reduction in macrophage infiltration; (h) a reduction in antibody deposition; (i) a reduction in glial cell loss; (j) a reduction in oligodendrocyte loss; (k) a reduction in dendritic cell infiltration; (l) a reduction in neutrophil infiltration; (m) a reduction in red blood cell lysis; (n) a reduction in red blood cell phagocytosis; (o) a reduction in platelet phagocytosis; ( p) reduction in platelet lysis; (q) improved graft survival; (r) reduction in macrophage-mediated phagocytosis; (s) improved vision; (t) improved motor control; (u) improved thrombosis; (v) improved coagulation; (w) improved renal function; (x) reduction in antibody-mediated complement activation; (y) reduction in autoantibody-mediated complement activation; (z) improvement in anemia; (aa) reduction in demyelination; (ab) reduction in eosinophilia; (ac) reduction in C3 deposition on erythrocytes (e.g., reduced deposition of C3b, iC3b, etc. on erythrocytes); (ad) reduction in C3 deposition on platelets (e.g., reduced deposition of C3b, iC3b, etc. on platelets); (ae ) decreased production of anaphylatoxins; (af) decreased autoantibody-mediated blister formation; (ag) decreased autoantibody-induced pruritus; (ah) decreased autoantibody-induced lupus erythematosus; (ai) decreased autoantibody-mediated skin erosions; (aj) decreased red blood cell destruction due to infusion reactions; (ak) decreased red blood cell lysis due to alloantibodies; (al) decreased hemolysis due to infusion reactions; (am) decreased alloantibody-mediated platelet lysis; (an) decreased platelet lysis due to infusion reactions; (ao) decreased mast cell activation; (ap) decreased mast cell histamine release; (aq) decreased vascular permeability; (ar) decreased edema; (as ) reduction in complement deposition on the graft endothelium; (at) reduction in the production of anaphylatoxins in the graft vascular endothelium; (au) reduction in separation of the dermal-epidermal junction; (av) reduction in the production of anaphylatoxins at the dermal-epidermal junction; (aw) reduction in alloantibody-mediated complement activation in the graft vascular endothelium; (ax) reduction in antibody-mediated loss of the neuromuscular junction; (ay) reduction in complement activation at the neuromuscular junction; (az) reduction in the production of anaphylatoxins at the neuromuscular junction; (ba) reduction in complement deposition at the neuromuscular junction; (bb) reduction in paralysis; (be) reduction in numbness; (bd) enhanced bladder control; (be) enhanced bowel control; (bf) reduction in mortality associated with autoantibodies;(bg) Decreased morbidity associated with autoantibodies. ;

[0484] In some embodiments, when an anti-C1s antibody of the present disclosure is administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, a reduction of 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 of one or more of the following outcomes can be achieved as compared to the level or extent of the outcome in the individual prior to treatment with the anti-C1s antibody: (a) complement activation; (b) decreased cognitive function; (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) red blood cell lysis; (n) red blood cell phagocytosis; (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 an anti-C1s antibody of the disclosure is administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, 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 improvement in one or more of the following outcomes can be achieved as compared to the level or extent of the outcome in the individual prior to treatment with the anti-C1s antibody: a) cognitive function; b) graft survival; c) vision; d) motor control; e) thrombosis; f) coagulation; g) renal function; and h) hematocrit (red blood cell 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 an anti-C1s antibody of the present disclosure is administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or in combination therapy, 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 complement activation in the individual prior to treatment with the anti-C1s antibody.

[0487] In some embodiments, administration of an anti-C1s antibody of the present disclosure improves cognitive function in the individual. For example, in some embodiments, when an anti-C1s antibody of the present disclosure is administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or in combination therapy, 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 cognitive function in the individual before treatment with the anti-C1s antibody.

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

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

[0493] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces macrophage infiltration in the individual. For example, in some embodiments, when an anti-C1s antibody of the present disclosure is administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or in combination therapy, 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 macrophage infiltration in the individual before treatment with the anti-C1s antibody.

[0494] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, when an anti-C1s antibody of the present disclosure is administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or in combination therapy, 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 antibody deposition in the individual prior to treatment with the anti-C1s antibody.

[0495] In some embodiments, administering an anti-C1s antibody of the present disclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a) production in the individual. For example, in some embodiments, when an anti-C1s antibody of the present disclosure is administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, anaphylatoxin production 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 anaphylatoxin production in the individual prior to 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 an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient in treating an individual with a complement-mediated disease or condition. In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure in treating an individual with a complement-mediated disease or condition. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient in treating an individual with a complement-mediated disease or condition.

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

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

[0499] In some embodiments, the disclosure provides a use of an anti-C1s antibody of the disclosure in the manufacture of a medicament for modulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in an individual with a complement-mediated disease or condition.

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

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

[0502] In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure for regulating complement activation or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides an anti-C1s antibody of the present disclosure for regulating complement activation. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for regulating complement activation. In some embodiments, the anti-C1s antibody inhibits complement activation.

[0503] In another aspect, the present invention provides the use of anti-C1s antibodies in the production or preparation of drugs. In one embodiment, the drug is used to treat a complement-mediated disease or condition. In another embodiment, the drug is used in a method for treating a complement-mediated disease or condition, the method comprising administering an effective amount of the drug to an individual suffering from a complement-mediated disease or condition. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, such as a therapeutic agent as described below. In another embodiment, the drug is used to enhance the clearance (or removal) of Cls from plasma. In another embodiment, the drug is used to enhance the clearance (or removal) of the C1r2s2 complex from plasma. In another embodiment, the drug is used to enhance the clearance (or removal) of C1r2s2 from plasma rather than Clq from plasma. In another embodiment, the drug is used to inhibit a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls.

[0504] In another embodiment, the medicament is 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 medicament.The "individual" according to any of the above embodiments may be a human.

[0505] In another aspect, the present invention provides a method for treating a complement-mediated disease or condition. In one embodiment, the method comprises administering an effective amount of an anti-C1s antibody to an individual suffering from the complement-mediated disease or condition. 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 may 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 in an individual but not C1q from the plasma. In some cases, the present invention provides a method for inhibiting a component of the classical complement pathway in an individual; in some 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, e.g., for use in any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-C1s antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-C1s antibodies provided herein and at least one additional therapeutic agent, e.g., a therapeutic agent as described below.

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

[0509] Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-C1s antibody and 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 invention may also be used in combination with radiation therapy.

[0510] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. A variety of dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse injections.

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

[0512] For the prevention or treatment of disease, the appropriate dose of the antibody of the present 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 administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g...

Claims

1. An isolated antibody that inhibits the interaction between the C1q and C1r2s2 complexes, The antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s and is selected from the group consisting of the following 1)-5): 1) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 32, an HVR-H2 sequence of SEQ ID NO: 33, an HVR-H3 sequence of SEQ ID NO: 34, an HVR-L1 sequence of SEQ ID NO: 35, an HVR-L2 sequence of SEQ ID NO: 36, and an HVR-L3 sequence of SEQ ID NO: 37, 2) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 38, an HVR-H2 sequence of SEQ ID NO: 39, an HVR-H3 sequence of SEQ ID NO: 40, an HVR-L1 sequence of SEQ ID NO: 41, an HVR-L2 sequence of SEQ ID NO: 42, and an HVR-L3 sequence of SEQ ID NO: 43, 3) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 44, an HVR-H2 sequence of SEQ ID NO: 45, an HVR-H3 sequence of SEQ ID NO: 46, an HVR-L1 sequence of SEQ ID NO: 47, an HVR-L2 sequence of SEQ ID NO: 48, and an HVR-L3 sequence of SEQ ID NO: 49, 4) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 50, an HVR-H2 sequence of SEQ ID NO: 51, an HVR-H3 sequence of SEQ ID NO: 52, an HVR-L1 sequence of SEQ ID NO: 53, an HVR-L2 sequence of SEQ ID NO: 54, and an HVR-L3 sequence of SEQ ID NO: 55, and 5) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 56, an HVR-H2 sequence of SEQ ID NO: 57, an HVR-H3 sequence of SEQ ID NO: 58, an HVR-L1 sequence of SEQ ID NO: 59, an HVR-L2 sequence of SEQ ID NO: 60, and an HVR-L3 sequence of SEQ ID NO: 61, or The antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1r and is selected from the group consisting of the following 6)-13): 6) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 119, an HVR-H2 sequence of SEQ ID NO: 127, an HVR-H3 sequence of SEQ ID NO: 135, an HVR-L1 sequence of SEQ ID NO: 143, an HVR-L2 sequence of SEQ ID NO: 151, and an HVR-L3 sequence of SEQ ID NO: 159, 7) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 120, an HVR-H2 sequence of SEQ ID NO: 128, an HVR-H3 sequence of SEQ ID NO: 136, an HVR-L1 sequence of SEQ ID NO: 144, an HVR-L2 sequence of SEQ ID NO: 152, and an HVR-L3 sequence of SEQ ID NO: 160, 8) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 121, an HVR-H2 sequence of SEQ ID NO: 129, an HVR-H3 sequence of SEQ ID NO: 137, an HVR-L1 sequence of SEQ ID NO: 145, an HVR-L2 sequence of SEQ ID NO: 153, and an HVR-L3 sequence of SEQ ID NO: 161, 9) an antibody comprising an HVR-H1 sequence of SEQ ID NO: 122, an HVR-H2 sequence of SEQ ID NO: 130, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 146, an HVR-L2 sequence of SEQ ID NO: 154, and an HVR-L3 sequence of SEQ ID NO: 162, 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, 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, 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 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.

2. The antibody of claim 1, wherein the antibody has a substitution function such that the antibody binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex.

3. The antibody of claim 2, wherein the antibody binds to the C1qrs complex on the BIACORE (registered trademark) chip and promotes the dissociation of C1q from the C1qrs complex, wherein: When a sufficient period of time has passed, 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 BIACORE (registered trademark) assay.

4. The antibody of claim 3, wherein the time point of crossing in the BIACORE (registered trademark) assay is within 1000 s after the time point of the start of antibody injection, determined by the BIACORE (registered trademark) 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. The antibody of claim 3, wherein substantially all of the C1q dissociates from the C1qrs complex within 2000 s after the time point at which the antibody injection is initiated, as determined by BIACORE (registered trademark) 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.

6. The antibody of claim 1, wherein the antigen-binding activity of the antibody at pH 5.8 is lower than that at pH 7.

4.

7. The antibody of claim 1, wherein the antibody binds to C1s or C1r with lower affinity at acidic pH than at neutral pH, as described in (i) or (ii) below: (i) when measured at high calcium concentrations at both neutral and acidic pH, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more, (ii) When measured at a neutral pH at a high calcium concentration and at an acidic pH at a low calcium concentration, the ratio of the KD value of the C1s binding activity at an acidic pH to the KD value of the C1s binding activity at a neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more.

8. The antibody of claim 6, 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.

9. The antibody of claim 8, wherein the antibody enhances reduction in plasma antigen concentration, wherein the Fc region is a human Fc region having a binding activity selected from the group consisting of: a) The binding activity to activated Fc gamma receptors is stronger than that of the Fc region of native human IgG1, b) has stronger binding activity to inhibitory Fc gamma receptors than to activating Fc gamma receptors, and c) The binding activity to FcRn at neutral pH is stronger than that of the Fc region of native human IgG1.

10. The antibody of claim 1, wherein the antibody binds to both cynomolgus monkey C1s and human C1s, or to both cynomolgus monkey C1r and human C1r.

11. A pharmaceutical formulation comprising the antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

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