Anti-complement antibody and method of use

Abstract

This invention provides anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, as well as methods for using them. [Solution] The present invention provides anti-C1s antibodies and anti-C1r antibodies, a pharmaceutical formulation containing the antibodies, and a method for treating an individual with a complement-mediated disease or disorder, comprising the step of administering the antibodies to the individual. The binding specificity and C1q dissociation-promoting function of the anti-C1s antibodies and anti-C1r antibodies have been evaluated. With respect to the antibodies, time-dependent complement neutralization function and binding to native and cleaved C1s or C1r proteins have also been demonstrated.

Description

[Technical Field]

[0001] The present invention relates to anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, and methods for using them. [Background technology]

[0002] background The C1 complex is a large protein complex that functions as a major initiator in the classical pathway cascade. The C1 complex consists of three components: C1q, C1r, and C1s, in a molar ratio of 1:2:2 (Non-Patent Literature 1). The classical pathway is initiated when the C1 complex binds to an antibody-bound target. C1q, which has six spherical heads, mediates the binding of the C1 complex to the antibody through avidity-type interactions with the Fc region. Upon tight binding to the target, C1r within the C1 complex self-activates and becomes enzymatically active. The activated C1r then cleaves and activates the proenzyme C1s within the C1 complex (Non-Patent Literature 2). Subsequently, the activated C1s cleaves its substrates, complement components C2 and C4, into C2a / C2b and C4a / C4b fragments, respectively. This causes an aggregate of the C3 convertase C4b2a to appear on the target surface, which cleaves C3 to form C3b. C3b then cleaves C5, initiating the formation of terminal membrane invasion complexes of C5b, C6, C7, C8, and C9, which dissolve the target through pore formation.

[0003] Both C1s and C1r proteins have the same domain structure: CUB1-EGF-CUB2-CCP1-CCP2-serine protease (Non-Patent Literature 3). The CUB1-EGF-CUB2 domain mediates the interaction between C1r and C1s to form the C1r2s2 tetramer (Non-Patent Literature 4), and also mediates the interaction between C1r2s2 and C1q (Non-Patent Literature 5). In contrast, the CCP1-CCP2-serine protease domains of C1r and C1s are responsible for the proteolytic cleavage of their respective substrates (Non-Patent Literature 6, Non-Patent Literature 7). The C1r2s2 tetramer interacts with the six stem portions of C1q through six binding sites within the CUB1-EGF-CUB2 domain of this tetramer (Non-Patent Literature 5).

[0004] A properly functioning complement system protects the host from pathogens, but abnormal regulation or inappropriate activation of classical pathways can lead to various complement-mediated disorders, such as, non-limited, autoimmune hemolytic anemia (AIHA), Behçet's disease, bullous pemphigoid (BP), and immune thrombocytopenic purpura (ITP). Therefore, inhibiting excessive or uncontrolled activation of classical pathways may provide clinical benefit to patients with such disorders.

[0005] It has been reported that HI532, an antibody that binds to the beta domain of C1s, can inhibit the interaction between C1r2s2 and C1q (Non-Patent Literature 8). However, this antibody could not completely neutralize the hemolytic activity of human serum, and 30% of the activity remained even after incubating the serum with this antibody for 24 hours.

[0006] Antibodies are highly attractive pharmaceuticals because they are stable in plasma, highly specific to their targets, and generally exhibit good pharmacokinetic profiles. However, due to their large molecular size, therapeutic antibody doses are typically high. If the target is abundant, the required therapeutic dose of antibody becomes even higher. Therefore, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive ways to reduce the doses and high manufacturing costs associated with therapeutic antibodies.

[0007] Antibodies that bind to antigens in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies" in this specification) have been reported to enable the neutralization of multiple antigen molecules by a single antibody molecule (Non-Patent Document 9, Patent Document 1). pH-dependent antibodies strongly bind to their antigens under neutral pH conditions in plasma, but dissociate from the antigen under acidic pH conditions in the endosomes of cells. After dissociation from the antigen, the antibody is recycled into the plasma by the FcRn receptor, and the dissociated antigen is degraded in the lysosomes of cells. The recycled antibody then freely binds to the antigen molecule again and neutralizes it, and this process is repeated as long as the antibody remains in the bloodstream. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO2009 / 125825 [Non-patent literature]

[0009] [Non-Patent Document 1] Wang et. al. Mol Cell. 2016 Jul 7;63(1):135-45 [Non-Patent Document 2] Mortensen et. al. Proc Natl Acad Sci US A. 2017 Jan 31;114(5):986-991 [Non-Patent Document 3] Gal et. al. Mol Immunol. 2009 Sep;46(14):2745-52 [Non-Patent Document 4] Almitairi et. al. Proc Natl Acad Sci US A. 2018 Jan 23;115(4):768-773 [Non-Patent Document 5] Bally et. al. J Biol Chem. 2009 Jul 17;284(29):19340-8 [Non-Patent Document 6] Rossi et. al. 1998 J Biol Chem. 1998 Jan 9;273(2):1232-9 [Non-Patent Document 7] Lacroix et. al. J Biol Chem. 2001 Sep 28;276(39):36233-40 [Non-Patent Document 8] Tseng et. al. Mol Immunol. 1997 Jun;34(8-9):671-9 [Non-Patent Document 9] Igawa et. al. Nat Biotechnol. 2010 Nov;28(11):1203-7 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention provides anti-complement component antibodies, such as anti-C1s antibodies and anti-C1r antibodies, as well as methods for using them. [Means for solving the problem]

[0011] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex are antibodies that have a displacement function, which involves binding to the C1qrs complex and promoting the dissociation of C1q from the C1qrs complex.

[0012] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to the C1qrs complex on a BIACORE® chip and promotes the dissociation of C1q from the C1qrs complex. In further embodiments, the antibody of the present invention can be determined to have a dissociation-promoting function if, in a BIACORE® assay, the value of the resonance units (RU) in the presence of the antibody is lower than the value of the resonance units (RU) in the absence of the antibody after a sufficient amount of time has elapsed.

[0013] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex can be determined to have a dissociation-promoting function if, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RUs) and 200 resonance units (RUs), respectively, and a 500 nM antibody is injected as an analyte at a rate of 10 microliters (μL) / min," the crossover time is within 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection.

[0014] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex can be determined to have a dissociation-promoting function if, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RUs) and 200 resonance units (RUs), respectively, and a 500 nM antibody as analyte is injected at 10 μL / min," almost all of the C1q dissociates from the C1qrs complex within 100 seconds, 300 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, 2000 seconds, 3000 seconds, 5000 seconds, 7000 seconds, or 10000 seconds after the start of antibody injection.

[0015] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and C1r2s2 complexes are antibodies that have neutralizing activity against at least 70% of human serum complement in an RBC assay.

[0016] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex are antibodies that specifically bind to C1s or antibodies that specifically bind to C1r.

[0017] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex are antibodies that specifically bind to an epitope within the CUB1-EGF-CUB2 domain of C1s. In further embodiments, the antibodies of the present invention compete with antibodies selected from the group consisting of 1) to 5) below for binding to the epitope: 1) An antibody containing the HVR-H1 sequence of SEQ ID NO:32, the HVR-H2 sequence of SEQ ID NO:33, the HVR-H3 sequence of SEQ ID NO:34, the HVR-L1 sequence of SEQ ID NO:35, the HVR-L2 sequence of SEQ ID NO:36, and the HVR-L3 sequence of SEQ ID NO:37. 2) An antibody containing the HVR-H1 sequence with SEQ ID NO:38, the HVR-H2 sequence with SEQ ID NO:39, the HVR-H3 sequence with SEQ ID NO:40, the HVR-L1 sequence with SEQ ID NO:41, the HVR-L2 sequence with SEQ ID NO:42, and the HVR-L3 sequence with SEQ ID NO:43. 3) An antibody containing the HVR-H1 sequence with SEQ ID NO:44, the HVR-H2 sequence with SEQ ID NO:45, the HVR-H3 sequence with SEQ ID NO:46, the HVR-L1 sequence with SEQ ID NO:47, the HVR-L2 sequence with SEQ ID NO:48, and the HVR-L3 sequence with SEQ ID NO:49. 4) Antibodies containing the HVR-H1 sequence of SEQ ID NO:50, the HVR-H2 sequence of SEQ ID NO:51, the HVR-H3 sequence of SEQ ID NO:52, the HVR-L1 sequence of SEQ ID NO:53, the HVR-L2 sequence of SEQ ID NO:54, and the HVR-L3 sequence of SEQ ID NO:55, as well as 5) An antibody containing the HVR-H1 sequence with SEQ ID NO: 56, the HVR-H2 sequence with SEQ ID NO: 57, the HVR-H3 sequence with SEQ ID NO: 58, the HVR-L1 sequence with SEQ ID NO: 59, the HVR-L2 sequence with SEQ ID NO: 60, and the HVR-L3 sequence with SEQ ID NO: 61.

[0018] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex are antibodies that specifically bind to an epitope within the CUB1-EGF-CUB2 domain of C1r. In further embodiments, the antibodies of the present invention compete with antibodies selected from the group consisting of 6) to 13) below for binding to the epitope: 6) An antibody containing the HVR-H1 sequence with SEQ ID NO:119, the HVR-H2 sequence with SEQ ID NO:127, the HVR-H3 sequence with SEQ ID NO:135, the HVR-L1 sequence with SEQ ID NO:143, the HVR-L2 sequence with SEQ ID NO:151, and the HVR-L3 sequence with SEQ ID NO:159. 7) An antibody containing the HVR-H1 sequence with SEQ ID NO:120, the HVR-H2 sequence with SEQ ID NO:128, the HVR-H3 sequence with SEQ ID NO:136, the HVR-L1 sequence with SEQ ID NO:144, the HVR-L2 sequence with SEQ ID NO:152, and the HVR-L3 sequence with SEQ ID NO:160. 8) An antibody containing the HVR-H1 sequence with SEQ ID NO:121, the HVR-H2 sequence with SEQ ID NO:129, the HVR-H3 sequence with SEQ ID NO:137, the HVR-L1 sequence with SEQ ID NO:145, the HVR-L2 sequence with SEQ ID NO:153, and the HVR-L3 sequence with SEQ ID NO:161. 9) An antibody containing the HVR-H1 sequence with SEQ ID NO:122, the HVR-H2 sequence with SEQ ID NO:130, the HVR-H3 sequence with SEQ ID NO:138, the HVR-L1 sequence with SEQ ID NO:146, the HVR-L2 sequence with SEQ ID NO:154, and the HVR-L3 sequence with SEQ ID NO:162. 10) An antibody containing the HVR-H1 sequence with SEQ ID NO:123, the HVR-H2 sequence with SEQ ID NO:131, the HVR-H3 sequence with SEQ ID NO:139, the HVR-L1 sequence with SEQ ID NO:147, the HVR-L2 sequence with SEQ ID NO:155, and the HVR-L3 sequence with SEQ ID NO:163. 11) An antibody containing the HVR-H1 sequence with SEQ ID NO:124, the HVR-H2 sequence with SEQ ID NO:132, the HVR-H3 sequence with SEQ ID NO:140, the HVR-L1 sequence with SEQ ID NO:148, the HVR-L2 sequence with SEQ ID NO:156, and the HVR-L3 sequence with SEQ ID NO:164. 12) Antibodies containing the HVR-H1 sequence with SEQ ID NO:125, the HVR-H2 sequence with SEQ ID NO:133, the HVR-H3 sequence with SEQ ID NO:141, the HVR-L1 sequence with SEQ ID NO:149, the HVR-L2 sequence with SEQ ID NO:157, and the HVR-L3 sequence with SEQ ID NO:165, as well as 13) An antibody containing the HVR-H1 sequence with SEQ ID NO:126, the HVR-H2 sequence with SEQ ID NO:134, the HVR-H3 sequence with SEQ ID NO:142, the HVR-L1 sequence with SEQ ID NO:150, the HVR-L2 sequence with SEQ ID NO:158, and the HVR-L3 sequence with SEQ ID NO:166.

[0019] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having antigen-binding activity that changes in an ion concentration-dependent manner. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having C1s-binding activity that changes in an ion concentration-dependent manner. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody having C1r-binding activity that changes in an ion concentration-dependent manner.

[0020] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to the 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 the 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 the C1r2s2 complex is an antibody that binds to C1r with higher affinity at neutral pH than at acidic pH.

[0021] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to the antigen with 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 the C1r2s2 complex is an antibody that binds to C1s with 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 the C1r2s2 complex is an antibody that binds to C1r with higher affinity under high calcium concentration conditions than under low calcium concentration conditions.

[0022] In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to the antigen with higher affinity under neutral pH and high calcium concentration conditions than under acidic pH and low calcium concentration conditions. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to C1s with higher affinity under neutral pH and high calcium concentration conditions than under acidic pH and low calcium concentration conditions. In some embodiments, the isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody that binds to C1r with higher affinity under neutral pH and high calcium concentration conditions than under acidic pH and low calcium concentration conditions.

[0023] In some embodiments, for isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex, when measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In some embodiments, for isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex, when measured under high 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.

[0024] In some embodiments, for isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex, when measured under low calcium concentrations at both neutral and acidic pH, the ratio of the KD value of its C1s-binding activity at acidic pH to the KD value of its C1s-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, where the anti-C1s antibody binds to dimeric C1s. In some embodiments, for isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex, when measured under 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, where the anti-C1s antibody binds to dimeric C1r.

[0025] In some embodiments, when an isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of its C1s binding activity KD value at acidic pH to its C1s binding activity KD value at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In some embodiments, when an isolated antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of its C1r binding activity KD value at acidic pH to its C1r binding activity KD value at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater.

[0026] In some embodiments, the anti-C1s antibody of the present invention, which inhibits the interaction between the C1q and C1r2s2 complex, is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 One or more of these contain a histidine residue.

[0027] In some embodiments, the anti-C1r antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 One or more of these contain a histidine residue.

[0028] In some embodiments, the anti-C1s antibody of the present invention, which inhibits the interaction between the C1q and C1r2s2 complex, is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 It contains at least one histidine substituted in one or more of the following.

[0029] In some embodiments, the anti-C1r antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 It contains at least one histidine substituted in one or more of the following.

[0030] In a further embodiment, the pH-dependent anti-C1s antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, competes with antibodies selected from the group consisting of 1) to 5) below for binding to C1s under neutral pH conditions: 1) An antibody containing the HVR-H1 sequence of SEQ ID NO:32, the HVR-H2 sequence of SEQ ID NO:33, the HVR-H3 sequence of SEQ ID NO:34, the HVR-L1 sequence of SEQ ID NO:35, the HVR-L2 sequence of SEQ ID NO:36, and the HVR-L3 sequence of SEQ ID NO:37. 2) An antibody containing the HVR-H1 sequence with SEQ ID NO:38, the HVR-H2 sequence with SEQ ID NO:39, the HVR-H3 sequence with SEQ ID NO:40, the HVR-L1 sequence with SEQ ID NO:41, the HVR-L2 sequence with SEQ ID NO:42, and the HVR-L3 sequence with SEQ ID NO:43. 3) An antibody containing the HVR-H1 sequence with SEQ ID NO:44, the HVR-H2 sequence with SEQ ID NO:45, the HVR-H3 sequence with SEQ ID NO:46, the HVR-L1 sequence with SEQ ID NO:47, the HVR-L2 sequence with SEQ ID NO:48, and the HVR-L3 sequence with SEQ ID NO:49. 4) Antibodies containing the HVR-H1 sequence of SEQ ID NO:50, the HVR-H2 sequence of SEQ ID NO:51, the HVR-H3 sequence of SEQ ID NO:52, the HVR-L1 sequence of SEQ ID NO:53, the HVR-L2 sequence of SEQ ID NO:54, and the HVR-L3 sequence of SEQ ID NO:55, as well as 5) An antibody containing the HVR-H1 sequence with SEQ ID NO: 56, the HVR-H2 sequence with SEQ ID NO: 57, the HVR-H3 sequence with SEQ ID NO: 58, the HVR-L1 sequence with SEQ ID NO: 59, the HVR-L2 sequence with SEQ ID NO: 60, and the HVR-L3 sequence with SEQ ID NO: 61.

[0031] In a further embodiment, the pH-dependent anti-C1r antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, competes with antibodies selected from the group consisting of 6) to 13) below for binding to C1r under neutral pH conditions: 6) An antibody containing the HVR-H1 sequence with SEQ ID NO:119, the HVR-H2 sequence with SEQ ID NO:127, the HVR-H3 sequence with SEQ ID NO:135, the HVR-L1 sequence with SEQ ID NO:143, the HVR-L2 sequence with SEQ ID NO:151, and the HVR-L3 sequence with SEQ ID NO:159. 7) An antibody containing the HVR-H1 sequence with SEQ ID NO:120, the HVR-H2 sequence with SEQ ID NO:128, the HVR-H3 sequence with SEQ ID NO:136, the HVR-L1 sequence with SEQ ID NO:144, the HVR-L2 sequence with SEQ ID NO:152, and the HVR-L3 sequence with SEQ ID NO:160. 8) An antibody containing the HVR-H1 sequence with SEQ ID NO:121, the HVR-H2 sequence with SEQ ID NO:129, the HVR-H3 sequence with SEQ ID NO:137, the HVR-L1 sequence with SEQ ID NO:145, the HVR-L2 sequence with SEQ ID NO:153, and the HVR-L3 sequence with SEQ ID NO:161. 9) An antibody containing the HVR-H1 sequence with SEQ ID NO:122, the HVR-H2 sequence with SEQ ID NO:130, the HVR-H3 sequence with SEQ ID NO:138, the HVR-L1 sequence with SEQ ID NO:146, the HVR-L2 sequence with SEQ ID NO:154, and the HVR-L3 sequence with SEQ ID NO:162. 10) An antibody containing the HVR-H1 sequence with SEQ ID NO:123, the HVR-H2 sequence with SEQ ID NO:131, the HVR-H3 sequence with SEQ ID NO:139, the HVR-L1 sequence with SEQ ID NO:147, the HVR-L2 sequence with SEQ ID NO:155, and the HVR-L3 sequence with SEQ ID NO:163. 11) An antibody containing the HVR-H1 sequence with SEQ ID NO:124, the HVR-H2 sequence with SEQ ID NO:132, the HVR-H3 sequence with SEQ ID NO:140, the HVR-L1 sequence with SEQ ID NO:148, the HVR-L2 sequence with SEQ ID NO:156, and the HVR-L3 sequence with SEQ ID NO:164. 12) Antibodies containing the HVR-H1 sequence with SEQ ID NO:125, the HVR-H2 sequence with SEQ ID NO:133, the HVR-H3 sequence with SEQ ID NO:141, the HVR-L1 sequence with SEQ ID NO:149, the HVR-L2 sequence with SEQ ID NO:157, and the HVR-L3 sequence with SEQ ID NO:165, as well as 13) An antibody containing the HVR-H1 sequence with SEQ ID NO:126, the HVR-H2 sequence with SEQ ID NO:134, the HVR-H3 sequence with SEQ ID NO:142, the HVR-L1 sequence with SEQ ID NO:150, the HVR-L2 sequence with SEQ ID NO:158, and the HVR-L3 sequence with SEQ ID NO:166.

[0032] In some embodiments, the Disclosure provides isolated anti-C1s antibodies that specifically bind to epitopes within a region comprising the CUB1-EGF-CUB2 domain (also called the interaction domain or CUB domain) of complement component 1s (C1s), which consists of CUB1, EGF, and CUB2, also referred herein as the "CUB1-EGF-CUB2 domain of C1s." In some embodiments, the antibody does not bind to the CCP1-CCP2-SP domain (also called the catalytic domain or CCP-SP domain) of C1s. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is an epitope not located in the beta domain of C1s. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is an epitope located in the alpha domain or gamma domain of C1s. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is a linear epitope. In some embodiments, the epitopes to which the isolated anti-C1s antibody of this disclosure binds are epitopes within the range of amino acids 16-291, 16-172, 16-210, 16-111, 112-210, 131-172, or 16-130 of the complement C1s protein. In some embodiments, the above epitopes of C1s are epitopes of human C1s, or preferably epitopes of human C1s and cynomolgus monkey C1s.

[0033] In some embodiments, this disclosure provides isolated anti-C1r antibodies that specifically bind to an epitope within a region comprising the CUB1-EGF-CUB2 domain of complement component 1r (C1r), which consists of CUB1, EGF, and CUB2, also referred herein as the "CUB1-EGF-CUB2 domain of C1r." In some embodiments, this antibody does not bind to the CCP1-CCP2-SP domain (also called the catalytic domain) of C1r. In some examples, the epitope to which the isolated anti-C1r antibody of this disclosure binds is a linear epitope or a structural epitope. In some embodiments, the epitope of C1r is an epitope of human C1r, or preferably an epitope of human C1r and an epitope of cynomolgus monkey C1r.

[0034] 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.

[0035] In some embodiments, the isolated anti-C1r antibody of the present invention comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125 or 126, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142, wherein the antibody comprises a framework region of human or primate origin. In some embodiments, the isolated anti-C1r antibody of the present invention comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166, wherein the antibody comprises a framework region of human or primate origin.

[0036] 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 with SEQ ID NO: 19 and a VL sequence with SEQ ID NO: 26. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence with SEQ ID NO: 20 and a VL sequence with SEQ ID NO: 27. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence with SEQ ID NO: 21 and a VL sequence with SEQ ID NO: 28. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence with SEQ ID NO: 23 and a VL sequence with SEQ ID NO: 30. In a further embodiment, the anti-C1s antibody of the present invention comprises a VH sequence with SEQ ID NO: 24 and a VL sequence with SEQ ID NO: 31.

[0037] In some embodiments, the anti-C1r 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: 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 present 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 present invention comprises a VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118. In further embodiments, 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 further embodiments, 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 further embodiments, 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 with SEQ ID NO: 105 and a VL sequence with SEQ ID NO: 113. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence with SEQ ID NO: 106 and a VL sequence with SEQ ID NO: 114. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence with SEQ ID NO: 107 and a VL sequence with SEQ ID NO: 115. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence with SEQ ID NO: 108 and a VL sequence with SEQ ID NO: 116. In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence with SEQ ID NO: 109 and a VL sequence with SEQ ID NO: 117.In a further embodiment, the anti-C1r antibody of the present invention comprises a VH sequence with SEQ ID NO:110 and a VL sequence with SEQ ID NO:118.

[0038] In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a monoclonal antibody. In some embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In further embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody fragment that binds to C1s. In some specific embodiments, the anti-C1s antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is human IgG1 or humanized IgG1.

[0039] In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a monoclonal antibody. In some embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In further embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is an antibody fragment that binds to C1r. In some specific embodiments, the anti-C1r antibody of the present invention that inhibits the interaction between C1q and the C1r2s2 complex is human IgG1 or humanized IgG1.

[0040] 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 within the Fc region to promote a decrease in plasma antigen concentration and / or improve the pharmacokinetics of the antibody.

[0041] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and C1r2s2 complexes are a) The binding activity to the active Fcγ receptor is stronger than the binding activity of the Fc region of native human IgG1. b) Binding activity to inhibitory Fcγ receptors is stronger than that to active Fcγ receptors, c) The binding activity to FcRn at neutral pH is stronger than the binding activity to the Fc region of native human IgG1. It has a human Fc region having binding activity selected from the group consisting of the following.

[0042] In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex bind to at least human C1s, or preferably both cynomolgus monkey C1s and human C1s. In some embodiments, the isolated antibodies of the present invention that inhibit the interaction between C1q and the C1r2s2 complex bind to at least human C1r, or preferably both cynomolgus monkey C1r and human C1r.

[0043] The present invention also provides isolated nucleic acids encoding the anti-C1s antibody of the present invention. The present invention also provides isolated nucleic acids encoding the anti-C1r antibody of the present invention. The present invention also provides host cells containing the nucleic acids of the present invention. The present invention also provides a method for producing an antibody, comprising the step of culturing the host cells of the present invention so that the antibody is produced.

[0044] The present invention also provides a pharmaceutical formulation comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

[0045] The anti-C1s antibody of the present invention may be used for pharmaceutical purposes. The anti-C1s antibody of the present invention may be used to treat or prevent complement-mediated diseases or disorders. The anti-C1s antibody of the present invention may be used to enhance (or remove C1s from plasma) the clearance of C1s from plasma. The anti-C1s antibody of the present invention may be used to enhance (or remove C1r2s2 from plasma) the clearance of C1r2s2 from plasma. The anti-C1s antibody of the present invention may be used to enhance (or remove C1r2s2 from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing C1q from plasma) the clearance of C1q from plasma. In some cases, this antibody inhibits a component of the classical complement pathway, and in some cases, the component of the classical complement pathway is C1s.

[0046] The anti-C1r antibody of the present invention may be used for pharmaceutical purposes. The anti-C1r antibody of the present invention may be used to treat or prevent complement-mediated diseases or disorders. The anti-C1r antibody of the present invention may be used to enhance (or remove C1r from plasma) the clearance of C1r from plasma. The anti-C1r antibody of the present invention may be used to enhance (or remove C1r2s2 from plasma) the clearance of C1r2s2 from plasma. The anti-C1r antibody of the present invention may be used to enhance (or remove C1r2s2 from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing C1q from plasma) the clearance of C1q from plasma. In some cases, this antibody inhibits a component of the classical complement pathway, and in some cases, the component of the classical complement pathway is C1r.

[0047] The anti-C1s antibody of the present invention may be used in the manufacture of pharmaceuticals. In some embodiments, the pharmaceutical is for the treatment or prevention of complement-mediated diseases or disorders. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1s from plasma. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma. In this context, the level of enhancement of C1q clearance from plasma does not need to be zero. That is, the level of enhancement of C1q clearance from plasma may be zero, near zero, or very small to the point of being insignificant or technically negligible to those skilled in the art. In some cases, pharmaceuticals inhibit components of the classical complement pathway, and in some cases, the component of the classical complement pathway is C1s.

[0048] The enhancement of the C1s / C1q clearance (CL) can be measured, for example, as follows: The total concentrations of human C1s and C1q in mouse plasma are measured by LC / ESI-MS / MS. Calibration standard samples are prepared by mixing and diluting specified amounts of human C1s and C1q in mouse plasma to achieve human C1s concentrations of 0.477, 0.954, 1.91, 3.82, 7.64, 15.3, and 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 standard samples and plasma samples are mixed with 25 μL of 6.8 mol / L urea, 9.1 mmol / L dithiothreitol, and 0.4 μg / mL lysozyme (chicken egg white) in 50 mmol / L ammonium bicarbonate, and incubated at 56°C for 45 minutes. Next, 2 μL of 500 mmol / L iodoacetamide is added and incubated in the dark at 37°C for 30 minutes. Then, 160 μL of 0.5 μg / mL trypsin (Promega) in 50 mmol / L ammonium bicarbonate is added and incubated overnight at 37°C. Finally, 5 μL of 10% trifluoroacetic acid is added to inactivate the trypsin. 40 μL of the digested sample is subjected to LC / ESI-MS / MS analysis. LC / ESI-MS / MS is performed using a Xevo TQ-S triple quadrupole mass spectrometer (Waters) equipped with a 2D I-class UPLC (Waters). Human C1s-specific peptide LLEVPEGR and human C1q-specific peptide IAFSATR are monitored by Selected Reaction Monitoring (SRM). For human C1s, the SRM transition is [M+2H]2+ (m / z 456.8) ~ y6 ion (m / z 686.3), and for human C1q, it is [M+2H]2+ (m / z 383.2) ~ y5 ion (m / z 581.3). The peak area is plotted against the concentration, and a calibration curve is created by linear regression (weighted: 1 / x2). Human C1s and C1q concentrations in mouse plasma are calculated from this calibration curve using the analytical software Masslynx version 4.1 (Waters). The pharmacokinetics of total hC1s and hC1q after administration of anti-C1s antibody in mice are evaluated as follows. The in vivo pharmacokinetics of hC1s, hC1q, and anti-C1s antibodies will be evaluated after administering the antigen alone (hC1q, recombinant C1r2s2, or a mixture of hC1q and rC1r2s2) or in combination with an anti-C1s antibody to mice (CB17 / Icr-Prkdcscid / CrlCrlj: Charles River Japan). Three mice will be assigned to each treatment group. First, administer an intravenous injection of hC1q solution (0.84 mg / mL), rC1r2s2 (0.47 mg / mL), or a mixture containing hC1q and rC1r2s2 (0.84 and 0.47 mg / mL, respectively) to mice at a dose of 10 mL / kg. Immediately after administration of the antigen solution, administer an anti-C1s antibody solution (2.5 mg / mL) to the same individual using the same method. The dosage settings for C1q and rC1r2s2 are designed to achieve physiological concentrations in human plasma immediately after administration. During the study, the dose of anti-C1s antibody is adjusted to achieve excessive concentrations for both antigens, so it is estimated that almost all hC1s will be bound in the blood. Blood samples were collected 5 minutes, 30 minutes, 2 hours, 7 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. These blood samples were immediately centrifuged to separate the plasma samples. At each sample acquisition time, the plasma concentrations of hC1s and hC1q were measured by LC / ESI-MS / MS. The PK parameters of hC1s and hC1q were calculated by non-compartmental analysis (Phoenix WinNonlin version 8.0, Certara). (i) an antibody having an Fc that contains a mutation that reduces binding to both C1q and Fcγ receptors, or (ii) an antibody having an Fc that contains a mutation that reduces binding to C1q while maintaining binding to the Fcγ receptor, is administered to mice. For example, in the present invention, the Fc of "SG136" contains a mutation that reduces binding to both C1q and Fcγ receptors, and the Fc of "SG1148" contains a mutation that reduces binding to C1q while maintaining binding to the Fcγ receptor. The above mouse PK test is performed on the test antibody (e.g., CCP1-CCP2-SP conjugate or CUB1-EGF-CUB2 conjugate) to calculate the PK parameters of hC1q and hC1s. Subsequently, the C1s CL ratio (SG1148 / SG136) or the C1q CL ratio (SG1148 / SG136) of the conjugate can be evaluated. In some embodiments, the C1q CL ratio of the antibody of the present invention is ≤1.8, ≤1.7, ≤1.6, ≤1.5, ≤1.4, ≤1.3, ≤1.2, ≤1.1, or ≤1.0. The anti-C1r antibody of the present invention can be used in the manufacture of pharmaceuticals. In some embodiments, the pharmaceutical is for the treatment or prevention of complement-mediated diseases or disorders. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1r from plasma. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma. In some embodiments, the pharmaceutical is for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma. In some examples, the pharmaceutical inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1r.

[0049] The present invention also provides a method for treating or preventing an individual having a complement-mediated disease or disorder. In some embodiments, the method comprises administering an effective amount of the anti-C1s antibody of the present invention to the individual. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1s from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of the anti-C1s antibody of the present invention to enhance (or remove from plasma) the clearance of C1s from plasma. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma in an individual. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma in an individual. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing (or removing from plasma) the clearance of C1r2s2 from plasma. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma. In some examples, the antibody inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s.

[0050] The present invention also provides a method for treating or preventing a complement-mediated disease or disorder in an individual. In some embodiments, the method comprises administering an effective amount of the anti-C1r antibody of the present invention to the individual. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1r from plasma in an individual. In some embodiments, the method comprises administering to an individual an effective amount of the anti-C1r antibody of the present invention to enhance (or remove from plasma) the clearance of C1r from plasma. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma in an individual. The present invention also provides a method for enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma in an individual. In some embodiments, the method comprises administering to an individual an amount of the anti-C1r antibody of the present invention effective in enhancing (or removing from plasma) the clearance of C1r2s2 from plasma. In some embodiments, the method comprises administering to an individual an amount of the anti-C1r antibody of the present invention effective in enhancing (or removing from plasma) the clearance of C1r2s2 from plasma without enhancing (or removing from plasma) the clearance of C1q from plasma. In some examples, the antibody inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1r.

[0051] More specifically, the present invention provides the following: [1] An isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, wherein the antibody has a dissociation-promoting function that binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex. [2] The antibody according to [1], wherein the antibody binds to the C1qrs complex on the BIACORE chip and promotes the dissociation of C1q from the C1qrs complex, and in the BIACORE assay, after a sufficient amount of time has elapsed, the value of the resonance units (RU) in the presence of the antibody is lower than the value of the resonance units (RU) in the absence of the antibody. [3] The capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the analyte is 500 nM of the antibody injected at 10 μL / min. An antibody [2] in which, in a BIACORE assay under the following conditions, the crossover time in the BIACORE assay is within 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, or 1000 seconds after the start of antibody injection. [4] The capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and the analyte is 500 nM of the antibody injected at 10 μL / min. An antibody [2] in which, in a BIACORE assay under the following conditions, almost all C1q dissociates from the C1qrs complex within 100 seconds, 300 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection. [5] An isolated antibody that inhibits the interaction between the C1q and C1r2s2 complex, and which has neutralizing activity against at least 70% of human serum complement in an RBC assay. [6] An antibody that specifically binds to C1s or C1r, which is one of the antibodies described in [1] to [5]. [7] Isolated antibodies that inhibit the interaction between C1q and the C1r2s2 complex, which specifically bind to an epitope within the CUB1-EGF-CUB2 domain of C1s, and which, with respect to binding to the epitope, are described in 1) to 5) below: 1) An antibody containing the HVR-H1 sequence of SEQ ID NO:32, the HVR-H2 sequence of SEQ ID NO:33, the HVR-H3 sequence of SEQ ID NO:34, the HVR-L1 sequence of SEQ ID NO:35, the HVR-L2 sequence of SEQ ID NO:36, and the HVR-L3 sequence of SEQ ID NO:37. 2) An antibody containing the HVR-H1 sequence with SEQ ID NO:38, the HVR-H2 sequence with SEQ ID NO:39, the HVR-H3 sequence with SEQ ID NO:40, the HVR-L1 sequence with SEQ ID NO:41, the HVR-L2 sequence with SEQ ID NO:42, and the HVR-L3 sequence with SEQ ID NO:43. 3) An antibody containing the HVR-H1 sequence with SEQ ID NO:44, the HVR-H2 sequence with SEQ ID NO:45, the HVR-H3 sequence with SEQ ID NO:46, the HVR-L1 sequence with SEQ ID NO:47, the HVR-L2 sequence with SEQ ID NO:48, and the HVR-L3 sequence with SEQ ID NO:49. 4) Antibodies containing the HVR-H1 sequence of SEQ ID NO:50, the HVR-H2 sequence of SEQ ID NO:51, the HVR-H3 sequence of SEQ ID NO:52, the HVR-L1 sequence of SEQ ID NO:53, the HVR-L2 sequence of SEQ ID NO:54, and the HVR-L3 sequence of SEQ ID NO:55, as well as 5) An antibody containing the HVR-H1 sequence with SEQ ID NO: 56, the HVR-H2 sequence with SEQ ID NO: 57, the HVR-H3 sequence with SEQ ID NO: 58, the HVR-L1 sequence with SEQ ID NO: 59, the HVR-L2 sequence with SEQ ID NO: 60, and the HVR-L3 sequence with SEQ ID NO: 61. It competes with antibodies selected from the group consisting of, or It specifically binds to the epitope within the CUB1-EGF-CUB2 domain of C1r, and regarding binding to said epitope, the following 6)~13): 6) An antibody containing the HVR-H1 sequence with SEQ ID NO:119, the HVR-H2 sequence with SEQ ID NO:127, the HVR-H3 sequence with SEQ ID NO:135, the HVR-L1 sequence with SEQ ID NO:143, the HVR-L2 sequence with SEQ ID NO:151, and the HVR-L3 sequence with SEQ ID NO:159. 7) An antibody containing the HVR-H1 sequence with SEQ ID NO:120, the HVR-H2 sequence with SEQ ID NO:128, the HVR-H3 sequence with SEQ ID NO:136, the HVR-L1 sequence with SEQ ID NO:144, the HVR-L2 sequence with SEQ ID NO:152, and the HVR-L3 sequence with SEQ ID NO:160. 8) An antibody containing the HVR-H1 sequence with SEQ ID NO:121, the HVR-H2 sequence with SEQ ID NO:129, the HVR-H3 sequence with SEQ ID NO:137, the HVR-L1 sequence with SEQ ID NO:145, the HVR-L2 sequence with SEQ ID NO:153, and the HVR-L3 sequence with SEQ ID NO:161. 9) An antibody containing the HVR-H1 sequence with SEQ ID NO:122, the HVR-H2 sequence with SEQ ID NO:130, the HVR-H3 sequence with SEQ ID NO:138, the HVR-L1 sequence with SEQ ID NO:146, the HVR-L2 sequence with SEQ ID NO:154, and the HVR-L3 sequence with SEQ ID NO:162. 10) An antibody containing the HVR-H1 sequence with SEQ ID NO:123, the HVR-H2 sequence with SEQ ID NO:131, the HVR-H3 sequence with SEQ ID NO:139, the HVR-L1 sequence with SEQ ID NO:147, the HVR-L2 sequence with SEQ ID NO:155, and the HVR-L3 sequence with SEQ ID NO:163. 11) An antibody containing the HVR-H1 sequence with SEQ ID NO:124, the HVR-H2 sequence with SEQ ID NO:132, the HVR-H3 sequence with SEQ ID NO:140, the HVR-L1 sequence with SEQ ID NO:148, the HVR-L2 sequence with SEQ ID NO:156, and the HVR-L3 sequence with SEQ ID NO:164. 12) Antibodies containing the HVR-H1 sequence with SEQ ID NO:125, the HVR-H2 sequence with SEQ ID NO:133, the HVR-H3 sequence with SEQ ID NO:141, the HVR-L1 sequence with SEQ ID NO:149, the HVR-L2 sequence with SEQ ID NO:157, and the HVR-L3 sequence with SEQ ID NO:165, as well as 13) An antibody containing the HVR-H1 sequence with SEQ ID NO:126, the HVR-H2 sequence with SEQ ID NO:134, the HVR-H3 sequence with SEQ ID NO:142, the HVR-L1 sequence with SEQ ID NO:150, the HVR-L2 sequence with SEQ ID NO:158, and the HVR-L3 sequence with SEQ ID NO:166. An antibody that competes with an antibody selected from the group consisting of the following. [8] An isolated antibody that inhibits the interaction between the C1q and C1r2s2 complex, wherein the antigen-binding activity of the antibody is lower at pH 5.8 than at pH 7.4. [9] An antibody which specifically binds to an epitope in the CUB1-EGF-CUB2 domain of C1s or C1r, and which has antigen-binding activity lower at pH 5.8 than at pH 7.4, one of the antibodies [1] to [8].

[10] The antibody of [9] that binds to C1s or C1r with lower affinity at acidic pH than neutral pH, as described in (i) or (ii) below: (i) When measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. (ii) When measured under high calcium concentration at neutral pH and low calcium concentration at acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater.

[11] Any antibody from [8] to

[10] comprising an Fc region having at least one amino acid modification to promote a decrease in plasma antigen concentration and / or improve the pharmacokinetics of the antibody.

[12] The Fc region is a) The binding activity to the active Fcγ receptor is stronger than the binding activity to the Fc region of native human IgG1. b) Binding activity to inhibitory Fcγ receptors is stronger than binding activity to active Fcγ receptors, c) The binding activity to FcRn at neutral pH is stronger than the binding activity to the Fc region of natural human IgG1. An antibody of

[11] which is a human Fc region having binding activity selected from the group consisting of the following.

[13] An antibody that binds to either cynomolgus monkey C1s or human C1s, or to either cynomolgus monkey C1r or human C1r, as described in [1] to

[12] .

[14] A pharmaceutical preparation comprising one of the antibodies [1] to

[13] and a pharmaceutically acceptable carrier.

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

[13] to the individual. [Brief explanation of the drawing]

[0052] [Figure 1A] Figure 1A shows the binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1s protein. BIACORE® sensorgram of anti-C1s antibody against recombinant human C1s CCP1-CCP2-SP-His protein. [Figure 1B] Figure 1B shows the binding specificity of antibodies to the CUB1-EGF-CUB2 domain of the C1s protein. BIACORE® sensorgram of anti-C1s antibodies against the naturally occurring proenzyme human C1s protein. [Figure 2A]Figure 2A shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers immobilized on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs to the sensor surface. Sensorgram 2 (long dotted line) shows antibody binding to C1qrs and enhancement of C1q dissociation from C1r2s2. Sensorgram 3 (solid line) shows the baseline when only the antibody binds to C1r2s2 in the absence of C1q. For comparison of these sensorgrams, the RU values ​​at time 0 in Figure 2A are normalized (i.e., set to be identical). [Figure 2B] Figure 2B shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers immobilized on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs to the sensor surface. Sensorgram 2 (long dotted line) shows the binding of the antibody to C1qrs and the enhancement of the dissociation of C1q from C1r2s2. Sensorgram 3 (solid line) shows the baseline when only the antibody binds to C1r2s2 in the absence of C1q. For comparison of these sensorgrams, the RU values ​​at time 0 in Figure 2B are normalized (i.e., set to be identical). [Figure 2C]Figure 2C shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers immobilized on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (long dotted line) shows antibody binding to C1qrs and enhancement of the dissociation of C1q from C1r2s2. For comparison of these sensorgrams, the RU values ​​at the time of Ab injection are normalized (i.e., set to be identical) in Figure 2C. [Figure 2D] Figure 2D shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers immobilized on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (long dotted line) shows antibody binding to C1qrs and enhancement of C1q dissociation from C1r2s2. For comparison of these sensorgrams, the RU values ​​at the time of Ab injection are normalized (i.e., set to be the same) in Figure 2D. [Figure 3] Figure 3 shows antibody-mediated enhancement of the dissociation of recombinant human C1r2s2 Flag / His tetramer from biotinylated native human C1q immobilized on the surface of a BIACORE® sensor. The recombinant human C1r2s2 Flag / His tetramer was flowed to bind to the immobilized native human C1q, followed by either flowing only buffer (solid line) or flowing antibody (dotted line) to dissociate C1r2s2 and monitor the rate of C1r2s2 dissociation. [Figure 4] Figure 4 shows antibody-mediated blocking of native human C1q binding to recombinant human C1r2s2 Flag / His tetramer. Antibodies with C1q blocking function competed with C1q for binding to C1r2s2. [Figure 5] Figure 5 shows the neutralization of human serum complement activity. [Figure 6] Figure 6 shows the results of competitive epitope binning of antibodies that bind to the CUB1-EGF-CUB2 domain of C1s. [Figure 7] Figure 7 shows the pharmacokinetics of human C1s and human C1q after administration of anti-C1s antibodies in mice. [Figure 8] Figure 8 shows the time-dependent neutralization of human serum complement activity by anti-C1s antibodies. [Figure 9] Figure 9 shows antibody binding to native human proenzyme C1s in reduced and unreduced Western blot analysis. [Figure 10] Figure 10 shows antibody binding to cleaved C1s protein in reduced Western blot. [Figure 11A] Figure 11A shows the binding specificity of the antibody to the CUB1-EGF-CUB2 domain of the C1r protein. BIACORE® sensorgram of anti-C1r antibody against recombinant human C1r CCP1-CCP2-SP-FLAG protein. [Figure 11B] Figure 11B shows the binding specificity of antibodies to the CUB1-EGF-CUB2 domain of the C1r protein. BIACORE® sensorgram of anti-C1r antibodies against the native human C1r enzyme. [Figure 12A]Figure 12A shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (long dotted line) shows antibody binding to C1qrs and enhancement of C1q dissociation from C1r2s2. Sensorgram 3 (solid line) shows the baseline when only the antibody binds to C1r2s2 in the absence of C1q. For comparison of these sensorgrams, the RU values ​​at time 0 in Figure 12A are normalized (i.e., set to be identical). [Figure 12B] Figure 12B shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on the surface of a BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of three sensorgrams. Sensorgram 1 (short dotted line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (long dotted line) shows antibody binding to C1qrs and enhancement of C1q dissociation from C1r2s2. Sensorgram 3 (solid line) shows the baseline when only the antibody binds to C1r2s2 in the absence of C1q. For comparison of these sensorgrams, the RU values ​​at time 0 in Figure 12B are normalized (i.e., set to be identical). [Figure 12C]Figure 12C shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on the surface of the BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of two sensorgrams. Sensorgram 1 (solid line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (dotted line) shows antibody binding to C1qrs and enhancement of the dissociation of C1q from C1r2s2. For comparison of these sensorgrams, the RU values ​​at the time of Ab injection are normalized (i.e., set to be the same) in Figure 12C. [Figure 12D] Figure 12D shows antibody-mediated enhancement of the dissociation of native human C1q from recombinant human C1r2s2 Flag / His tetramers captured on the surface of the BIACORE® sensor. The antibody-mediated enhancement of native human C1q dissociation is shown by the superposition of two sensorgrams. Sensorgram 1 (solid line) shows the stable capture of C1qrs on the sensor surface. Sensorgram 2 (dotted line) shows antibody binding to C1qrs and enhancement of the dissociation of C1q from C1r2s2. For comparison of these sensorgrams, the RU values ​​at the time of Ab injection are normalized (i.e., set to be the same) in Figure 12D. [Figure 13] Figure 13 shows the neutralization of human serum complement activity. [Modes for carrying out the invention]

[0053] The methods and procedures described or cited herein are generally well understood, and refer to, for example, 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 Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Conventional techniques, such as those widely used by those skilled in the art, are commonly employed by those skilled in the art, as described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0054] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. 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 general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0055] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0056] In the spirit of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0057] "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd or KD). Affinity can be measured by common methods known in the art, including those described herein. Individual specific examples and exemplary embodiments for measuring binding affinity are described below. The terms "affinity," "binding affinity," "binding ability," and "binding activity" may be used interchangeably. The term "binding activity" refers to the strength of the combined non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In this specification, binding activity is not strictly limited to the activity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). When members of a binding pair can bind to each other in both monovalent and polyvalent forms, binding activity is the sum of the strengths of these bonds. The binding activity of molecule X to its partner Y can usually be expressed by the dissociation constant (KD). Alternatively, binding and dissociation rates (Kon and Koff) may be used to evaluate binding. Binding activity can be measured by common methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0058] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[0059] The term "anti-C1s antibody" or "antibody that binds to C1s" refers to an antibody that can bind to C1s with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when it targets C1s. In one embodiment, the degree of binding of the anti-C1s antibody to irrelevant non-C1s proteins is less than about 10% of the binding of the antibody to C1s when measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C1s has a dissociation constant (Kd) of 1 micromolar concentration (μM) or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-C1s antibody binds to an epitope of C1s that is conserved among C1s from different species.

[0060] The term "anti-C1r antibody" or "antibody that binds to C1r" refers to an antibody that can bind to C1r with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when it targets C1r. In one embodiment, the degree of binding of the anti-C1r antibody to irrelevant non-C1r proteins is less than about 10% of the binding of the antibody to C1r when measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to C1r has a dissociation constant (Kd) of 1 micromolar concentration (μM) or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-C1r antibody binds to an epitope of C1r that is conserved among C1r from different species.

[0061] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0062] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include 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.

[0063] An antibody that "binds to the same epitope as the reference antibody" is defined as an antibody that, in a competitive assay, inhibits the binding of the reference antibody to its own antigen by 50% or more, and conversely, the reference antibody inhibits the binding of the aforementioned antibody to its own antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0064] The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0065] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0066] As used herein, the term “cytotoxic agent” means a substance that inhibits or interferes with the function of a cell 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; chemotrepinephrine or chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleases and their fragments; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including their fragments and / or variants); and various antitumor or anticancer agents as disclosed below.

[0067] "Effector function" refers to the different biological activities that occur depending on the antibody isotype, stemming from the Fc region of the antibody. 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.

[0068] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period that is effective in achieving the desired therapeutic or prophylactic outcome.

[0069] 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 targeting that antigen and contains specific amino acids that are in direct contact with the antibody. Epitope determinants can include a group of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can possess specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on that target antigen in a complex mixture of proteins and / or macromolecules.

[0070] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both native sequence Fc regions and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (residues 446-447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0071] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0072] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0073] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0074] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0075] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0076] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0077] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0078] An "immunoconjugate" is an antibody that has been conjugated to one or more heterologous molecules (the heterologous molecules may include, but are not limited to, cytotoxic agents).

[0079] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0080] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0081] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0082] "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 the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules mounted on one or more vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0083] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0084] A "naked antibody" is an antibody that is not conjugated with a different part (e.g., a cytotoxic part) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.

[0085] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of an antibody may be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0086] The term “package insert” is used to refer to instructions for use that are typically included in the commercial packaging of therapeutic products and contain information about indications, usage, dosage, method of administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products.

[0087] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the maximum percentage sequence identity and gaps have been introduced as necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or publicly available computer software such as GENETYX® (Genetyx Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0088] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0089] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein can exert its effect, and which does not contain any additional elements that are toxic to an extent unacceptable to the subject to which the preparation is administered.

[0090] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0091] As used herein, the term “specifically binds” means the activity or characteristic of an antibody to bind to an antigen of interest at a binding level that includes background binding (i.e., nonspecific binding) but does not include significant binding (i.e., specific binding). In other words, “specifically binds” means the activity or characteristic of an antibody to bind to an antigen of interest at a binding level that includes significant binding (i.e., specific binding) in addition to or instead of background binding (i.e., nonspecific binding). Specificity can be measured by any method referred herein or known in the art. The level of nonspecific binding or background binding may be zero, near zero, or so small that it is technically negligible to a person skilled in the art. For example, if a person skilled in the art cannot detect or observe a significant (or relatively strong) signal regarding binding between an antibody and an antigen of interest in a suitable binding assay, it can be said that the antibody “does not specifically bind” to the antigen of interest. Conversely, if a person skilled in the art can detect or observe a significant (or relatively strong) signal regarding binding between an antibody and an antigen of interest in a suitable binding assay, it can be said that the antibody “specifically binds” to the antigen of interest.

[0092] As used herein, the term "C1s" refers to any native C1s from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both C1s that have not undergone "full-length" processing and any form of C1s resulting from processing within cells. This term also encompasses naturally occurring variants of C1s, such as splice variants and allele 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 NO:3 and 2, respectively. As used herein, the term "C1r" refers to any native C1r from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both C1r that has not undergone "full-length" processing and any form of C1r resulting from processing within cells. This term also encompasses naturally occurring variants of C1r, such as splice variants and allele 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 NO:5 and 6, respectively.

[0093] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0094] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0095] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0096] II. Compositions and Methods In one aspect, the present invention is based in part on antibodies that inhibit the interaction between C1q and the C1r2s2 complex and the use thereof. In certain embodiments, antibodies that bind to C1s are provided. In certain embodiments, antibodies that bind to C1r are provided. The antibodies of the present invention are useful, for example, in the prevention or treatment of complement-mediated diseases or disorders.

[0097] A. Exemplary anti-complement component antibodies In one aspect, the present invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex. In another aspect, the present invention provides an isolated antibody having a dissociation-promoting function that binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex. In another aspect, the present invention provides an isolated antibody that binds to C1s. In another aspect, the present invention provides an isolated antibody that binds to C1s, the binding activity of which changes depending on the ion concentration. In a particular embodiment, the binding activity of an anti-C1s antibody changes depending on the pH, i.e., the hydrogen ion (proton) concentration. In a particular embodiment, the binding activity of an anti-C1s antibody changes depending on the calcium concentration. In a particular embodiment, the binding activity of an anti-C1s antibody changes depending on both the pH and the calcium concentration. In yet another aspect, the present invention provides an isolated antibody that binds to C1r. In another aspect, the present invention provides an isolated antibody that binds to C1r, the binding activity of which changes depending on the ion concentration. In certain embodiments, the binding activity of the anti-C1r antibody changes depending on the pH, i.e., the hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1r antibody changes depending on the calcium concentration. In certain embodiments, the binding activity of the anti-C1r antibody changes depending on both the pH and the calcium concentration.

[0098] Throughout the section "Modes for Carrying Out the Invention," the term "C1s" may be replaced with "C1r," except for descriptions relating to sequences specific to anti-C1s antibodies and sequences and domains specific to C1s proteins.

[0099] Such antibodies are expected to be particularly excellent as pharmaceuticals because they can reduce the dose and frequency of administration in patients, and consequently reduce the total dose. Anti-C1s antibodies are expected to have a superior safety profile compared to antibodies that bind to the C1qrs complex and remove it from plasma, as they only remove C1r2s2 from plasma (through binding to C1s) and not C1q from plasma. As a result, side effects associated with C1q deficiency may be avoided. In addition, antibodies with the ability to rapidly promote C1q dissociation are expected to show faster neutralization of complement activity, which may lead to a faster onset of therapeutic effect.

[0100] (BIACORE (registered trademark) / Concept of promoting dissociation) In one aspect, the isolated antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, is an antibody that binds to the C1qrs complex on a surface plasmon resonance assay chip (e.g., a BIACORE® chip) and promotes the dissociation of C1q from the C1qrs complex. In several aspects, the function of binding to the C1qrs complex and promoting the dissociation of C1q from the C1qrs complex is referred herein to as “dissociation-promoting function / activity” or “C1q dissociation-promoting function / activity”. This function / activity can be appropriately evaluated qualitatively or quantitatively using a surface plasmon resonance assay, such as the BIACORE® assay described herein. In a further aspect, the antibody of the present invention can be determined to have a dissociation-promoting function if, in a surface plasmon resonance assay, such as the BIACORE® assay, the value of the resonance units (RU) in the presence of the antibody is lower than the value of the resonance units (RU) in the absence of the antibody after a sufficient amount of time has elapsed. In the sensorgram obtained from such an assay, a “crossover point” can be identified where the curve in the presence of C1q and the absence of antibody intersects the curve in the presence of both C1q and antibody (see Examples for details). Strictly speaking, due to noise or oscillations in the latter curve when it intersects the former curve, multiple crossover points may be observed even in a single sensorgram. In such cases, any of the multiple crossover points may be selected as the “crossover point.” “Sufficient time has elapsed” means that, for the purpose of measurement, the measurement point of the unit of resonance (RU) value is well after the “crossover point.” In some embodiments, the measurement point of the unit of resonance (RU) value is at least 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection. Alternatively, the measurement time may be at least 100 seconds, 200 seconds, 300 seconds, 400 seconds, 500 seconds, 600 seconds, 700 seconds, 800 seconds, 900 seconds, 1000 seconds, 3000 seconds, 5000 seconds, 7000 seconds, or 10000 seconds after the crossover time.

[0101] In one aspect, the isolated antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, can be determined to have a dissociation-promoting function if, for example, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and a 500 nM antibody as analyte is injected at a rate of 10 microliters (μL) / min," the crossover time (in the BIACORE® assay) is within 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection.

[0102] In one aspect, the isolated antibody of the present invention, which inhibits the interaction between C1q and the C1r2s2 complex, can be determined to have a dissociation-promoting function if, for example, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and a 500 nM antibody as an analyte is injected at 10 μL / min," almost all (or all) of C1q dissociates from the C1qrs complex within 100 seconds, 300 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, 2000 seconds, 3000 seconds, 5000 seconds, 7000 seconds, or 10000 seconds after the start of antibody injection. For example, in a sensorgram obtained from such an assay, if the value (RU) in the presence of C1q and the antibody is close to or reaches the value (RU) in the presence of the antibody and in the absence of C1q, it can be determined that "almost all (or all) of C1q dissociates from the C1qrs complex." In this specification, "almost all (of C1q)" represents a percentage of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, and "all (of C1q)" represents a percentage of 100%. The proportion of C1q that dissociates can be quantitatively determined by any assay described herein. In some aspects, the present invention provides a screening method for antibodies that promote the dissociation of C1q from the C1r2s2 complex, the screening method being used to measure the “dissociation-promoting function / activity” of such antibodies. In one embodiment, the screening method includes the step of selecting an antibody that inhibits the interaction between C1q and the C1r2s2 complex, i.e., an antibody that binds to the C1qrs complex and promotes the dissociation of C1q from the C1qrs complex. Antibodies having dissociation-promoting function / activity can be appropriately selected using a surface plasmon resonance assay, such as the BIACORE® assay described herein.In some embodiments, the screening method includes the step of determining (i) the value of the resonance unit (RU) in the presence of the antibody and (ii) the value of the resonance unit (RU) in the absence of the antibody by a surface plasmon resonance assay, such as the BIACORE® assay, after a sufficient amount of time has elapsed. The screening method may include the step of comparing the value in (i) with the value in (ii). The screening method may include the step of selecting the antibody if the value in (i) is lower than the value in (ii). The screening method may include the step of identifying a “crossover point” where the curve in the presence of C1q and the absence of the antibody intersects the curve in the presence of both C1q and the antibody. As described above, multiple crossover points may be observed even in a single sensorgram, and any of the multiple crossover points may be selected as the “crossover point”. In some embodiments, the screening method may include measuring the value of the resonance unit (RU) at least 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection. Alternatively, the screening method may include measuring the value of the resonance unit (RU) at least 100 seconds, 200 seconds, 300 seconds, 400 seconds, 500 seconds, 600 seconds, 700 seconds, 800 seconds, 900 seconds, 1000 seconds, 3000 seconds, 5000 seconds, 7000 seconds, or 10000 seconds after the crossover time. In some embodiments, the screening method may include a step of selecting an antibody that inhibits the interaction between C1q and the C1r2s2 complex or has a dissociation-promoting function, for example, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and a 500 nM antibody is injected as an analyte at a rate of 10 microliters (μL) / min," the antibody's crossover time is within 60 seconds, 100 seconds, 150 seconds, 200 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, or 2000 seconds after the start of antibody injection.In some embodiments, the screening method may include a step of selecting an antibody that inhibits the interaction between C1q and the C1r2s2 complex or has a dissociation-promoting function, for example, in a BIACORE® assay under the conditions that "the capture amounts of the C1r2s2 complex and C1q are 200 resonance units (RU) and 200 resonance units (RU), respectively, and a 500 nM antibody as an analyte is injected at 10 μL / min", the step of selecting an antibody in which almost all (or all) of C1q dissociates from the C1qrs complex within 100 seconds, 300 seconds, 500 seconds, 700 seconds, 1000 seconds, 1500 seconds, 2000 seconds, 3000 seconds, 5000 seconds, 7000 seconds, or 10000 seconds after the start of antibody injection. As stated above, “(Almost all) (of C1q)” represents a percentage of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, and “(All) (of C1q)” represents 100%, and the percentage of C1q to dissociate can be quantitatively determined by any assay described herein, including the BIACORE® assay.

[0103] (BIACORE(registered trademark) / blocking concept) In one aspect, the present invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex, the antibody having a blocking function such that it binds to C1r2s2 and inhibits the binding of C1q to C1r2s2. In a further aspect, the antibodies of the present invention have a blocking ratio of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher. This blocking function / activity or blocking ratio can be determined by using the BIACORE® assay. To evaluate the level of C1q blocking, the following conditions may be used: the amount of C1r2s2 captured is targeted at 50, 100, 200, and 400 resonance units (RU). Antibody binding is saturated by injecting antibody variants of 250, 500, 1000, and 2000 nM, followed by injection of human C1q of 50, 100, and 200 nM with or without the antibody variants. The blocking ratio is calculated using the following formula: [1 - (human C1q binding response in the presence of antibody variants / human C1q binding response in the absence of antibody variants)] × 100%.

[0104] (pH dependent) In one aspect, the antibody of the present invention binds to an antigen (e.g., C1s) or to the C1r2s2 complex in a pH-dependent manner. In a preferred embodiment, the present invention provides an isolated antibody that inhibits the interaction between C1q and the C1r2s2 complex (through binding to C1s), wherein its antigen-binding activity at pH 5.8 (i.e., binding activity to C1s) is lower than that at pH 7.4. In a preferred embodiment, the antibody specifically binds to an epitope within the CUB1-EGF-CUB2 domain of C1s, and the antigen-binding activity of the antibody is lower at pH 5.8 than at pH 7.4.

[0105] In addition to binding to C1s in a pH-dependent manner, the effect of calcium on the pH-dependent affinity of antibodies to C1s may be another important property. C1s form dimers under high calcium concentrations but dissociate into monomers under low calcium concentrations. When C1s are in a dimer state, bivalent antibodies can form immune complexes by crosslinking multiple C1s molecules. This allows the antibody to bind to C1s molecules within the complex through both affinity and avidity interactions, thereby increasing the apparent affinity of the antibody. In contrast, when C1s are in a monomeric state, antibodies bind to C1s only through affinity interactions. This means that pH-dependent C1s antibodies can form immune complexes with dimeric C1s in plasma, but when C1s enter the acidic environment of endosomes, they dissociate into monomers. This breaks down the immune complex, which enhances the pH-dependent dissociation of the antibody from the antigen.

[0106] In one aspect, when the isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, the ratio of its C1s binding activity KD value at acidic pH to its C1s binding activity KD value at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In another aspect, when the isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of its C1s binding activity KD value at acidic pH to its C1s binding activity KD value at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In some embodiments, when an isolated anti-C1s antibody of the present invention is measured under low calcium concentrations at both neutral and acidic pH, the ratio of its KD value for C1s binding activity at acidic pH to its KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater, where the anti-C1s antibody binds to dimeric C1s.

[0107] While not bound by any particular theory, if 1) the epitope structure of C1s bound by the antibody of the present invention can change stereochemically due to the absence of calcium, thereby altering the antibody's affinity, or if 2) the interaction of the antibody of the present invention (affinity type or avidity type) can change depending on the state of C1s (monomer or dimer), then measurements using specific conditions (high calcium concentration at neutral pH and low calcium concentration at acidic pH) may be used to evaluate the ratio of KD values ​​(KD(acidic pH) / KD(neutral pH)).

[0108] In other words, the antibody of the present invention binds to C1s with higher affinity at neutral pH than at acidic pH, as described in (i) or (ii) below: (i) When measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. (ii) When measured under high calcium concentration at neutral pH and low calcium concentration at acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater.

[0109] More generally, although not bound by any particular theory, if 1) the epitope structure of a particular antigen bound by the antibody of the present invention may change stereochemically due to the absence of calcium, thereby altering the antibody's affinity, or if 2) the interaction of the antibody of the present invention (affinity-type or avidity-type) may change depending on the state of the antigen (monomer-type or dimer-type), then measurements using specific conditions (high calcium concentration at neutral pH and low calcium concentration at acidic pH) may be used to evaluate the ratio of KD values ​​(KD(acidic pH) / KD(neutral pH)).

[0110] Therefore, the antibody of the present invention binds to the antigen with higher affinity at neutral pH than at acidic pH, as follows: When measured under high calcium concentration at neutral pH and low calcium concentration at acidic pH, the ratio of the KD value of antigen-binding activity at acidic pH to the KD value of antigen-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more.

[0111] 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 to the original (parent) antibody. The original (parent) antibody can be any known antibody or a newly isolated antibody, as long as it specifically binds to C1s. Therefore, in one aspect, for the isolated anti-C1s antibody of the present invention, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 3 times, 3.5 times, 4 times, 5 times, 8 times, and 10 times higher than the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH to the KD value of C1s binding activity at neutral pH of the original (parent) antibody (KD(acidic pH) / KD(neutral pH)). In other words, the present invention provides isolated anti-C1s antibodies in which one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) are introduced into the parent (original) antibody, such that the ratio of (i) to (ii) below 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 of the isolated anti-C1s antibody at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)); (ii) the ratio of the KD value of the C1s binding activity of the parent (original) antibody at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)). These KD ratios can be measured under any (high or low) calcium concentration, for example, under high calcium concentrations at both neutral and acidic pH, or under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH.

[0112] In one aspect, the antibodies of the present invention have different antigen-binding activities under intracellular and extracellular conditions. Intracellular and extracellular conditions refer to different conditions between the inside and outside of a cell. Categories of conditions include, for example, ion concentrations, more specifically metal ion concentrations, hydrogen ion concentrations (pH), and calcium ion concentrations. "Intracellular conditions" preferably refer to the environment characteristic of the endosome environment, and "extracellular conditions" preferably refer to the environment characteristic of plasma. Antibodies having antigen-binding activity that changes depending on ion concentration can be obtained by screening a large number of antibodies for domains having such properties. For example, antibodies having the above properties can be obtained by producing a large number of antibodies with mutually different sequences using a hybridoma method or antibody library method and measuring their antigen-binding activity under different ion concentrations. B cell cloning is one example of a method for screening such antibodies. Furthermore, as described later, at least one characteristic amino acid residue can be identified that confers the property of antigen-binding activity that changes depending on the ion concentration to the antibody, and a library of numerous antibodies having different sequences while sharing this characteristic amino acid residue as a common structure is prepared. Such a library can be screened to effectively isolate antibodies having the above-mentioned properties.

[0113] In one aspect, the present invention provides an antibody that binds to C1s with higher affinity at neutral pH than at acidic pH. In another aspect, the present invention provides an anti-C1s antibody that exhibits pH-dependent binding to C1s. As used herein, the expression "pH-dependent binding" means "binding at acidic pH that is reduced compared to binding at neutral pH," and the two expressions may be interchangeable. For example, an anti-C1s antibody "having pH-dependent binding properties" includes an antibody that binds to C1s with higher affinity at neutral pH than at acidic pH.

[0114] In certain embodiments, when measured under high calcium concentrations at both neutral and acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In certain embodiments, the antibodies of the present invention bind to C1s at neutral pH with an affinity 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 times, or higher, than at acidic pH.

[0115] In certain embodiments, when measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater. In certain embodiments, the antibodies of the present invention bind to C1s at neutral pH with at least 2, 3, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times, or higher affinity than at acidic pH.

[0116] In the above example, for instance, the acidic pH is 5.8 and the neutral pH is 7.4, and therefore KD(acidic pH) / KD(neutral pH) is KD(pH 5.8) / KD(pH 7.4). Examples of acidic and neutral pH are described in detail later in this specification. In some embodiments, KD(acidic pH) / KD(neutral pH), for example KD(pH 5.8) / KD(pH 7.4), can range from 2 to 10,000.

[0117] When the antigen is a soluble protein, the antibody may have a longer half-life in plasma than the antigen itself and can function as a carrier for the antigen. Therefore, antibody binding to the antigen can lead to an extension of the antigen's plasma half-life (i.e., reduced clearance of the antigen from plasma). This is due to the recycling of the antigen-antibody complex by FcRn via the intracellular endosomal pathway (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties, which bind to antigens in a neutral extracellular environment but release them into the acidic endosomal compartment after entering the cell, are expected to have superior properties in terms of antigen neutralization and clearance compared to their pH-independent binding counterparts (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).

[0118] In one aspect, the present invention provides an antibody that binds to C1s with higher affinity under high calcium concentration conditions than under low calcium concentration conditions.

[0119] In the present invention, preferred metal ions include, for example, calcium ions. Calcium ions are involved in regulating many biological phenomena, including muscle contraction (e.g., skeletal muscle, smooth muscle, and cardiac muscle); activation of leukocytes (motility, phagocytosis, etc.); activation of platelets (morphological changes, secretion, etc.); activation of lymphocytes; activation of mast cells (including histamine secretion); cellular responses mediated by catecholamine alpha receptors or acetylcholine receptors; exocytosis; release of neurotransmitters from neuronal terminals; and axonal flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chains, which have several calcium ion binding sites and are thought to have originated from a common molecular evolutionary site. Many calcium-binding motifs are also known. Such well-known motifs include, for example, the cadherin domain, the EF hand of calmodulin, the C2 domain of protein kinase C, the Gla domain of blood coagulation protein factor IX, type C lectins of asialoglycoprotein receptor and mannose-binding receptor, the A domain of the LDL receptor, annexin, the thrombospondin type 3 domain, and EGF-like domains.

[0120] In the present invention, when the metal ion is a calcium ion, it is desirable that the antigen-binding activity under low calcium ion concentration conditions be lower than the antigen-binding activity under high calcium ion concentration conditions. On the other hand, intracellular calcium ion concentration is lower than extracellular calcium ion concentration. Conversely, extracellular calcium ion concentration is higher than intracellular calcium ion concentration. In the present invention, the low calcium ion concentration is preferably 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. On the other hand, in the present invention, the high calcium ion concentration is preferably 100 μM to 10 μM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). In the present invention, it is preferable that the low calcium ion concentration is the calcium ion concentration in endosomes, and the high calcium ion concentration is the calcium ion concentration in plasma. When comparing the level of antigen binding activity under low calcium ion concentrations and high calcium ion concentrations, the binding of the antibody of the present invention is preferably stronger under high calcium ion concentrations than under low calcium ion concentrations. In other words, the antigen binding activity of the antibody of the present invention is preferably lower under low calcium ion concentrations than under high calcium ion concentrations. When the level of binding activity is expressed by the dissociation constant (KD), the value of KD(low calcium ion concentration) / KD(high calcium ion concentration) is greater than 1, preferably 2 or more, more preferably 10 or more, and even 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 may be any value such as 100, 400, 1000, or 10000, as long as it can be produced by the art. It is also possible to use the dissociation rate constant (kd) instead of KD. If it is difficult to calculate the KD value, the activity may be evaluated based on the level of binding reactivity when the analyte is run at the same concentration in BIACORE®.When an antigen is flowed onto a chip immobilized with the antigen-binding molecule of the present invention, the binding reactivity under low calcium concentrations is preferably 1 / 2 or less, more preferably 1 / 3 or less, even more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less, of the binding reactivity under high calcium concentrations. Generally, it is known that the calcium ion concentration in extracellular environments (e.g., in plasma) is high, while the calcium ion concentration inside cells (e.g., inside endosomes) is low. Therefore, in the present invention, it is preferable that the extracellular conditions are high calcium ion concentration and the intracellular conditions are low calcium ion concentration. When the antigen-binding molecule (e.g., antibody) of the present invention is given the characteristic of having lower antigen-binding activity under intracellular calcium ion concentration conditions compared to extracellular calcium ion concentration conditions, the antigen bound to the antigen-binding molecule of the present invention outside the cell dissociates from the antigen-binding molecule of the present invention inside the cell, thereby enhancing the uptake of the antigen from extracellular to intracellular. When such an antibody is administered to a living organism, it is possible to reduce the antigen concentration in plasma and reduce the physiological activity of the antigen in the living organism, making the antibody of the present invention useful. Methods for screening antigen-binding domains or antibodies that exhibit lower antigen-binding activity under low calcium ion concentration conditions compared to high calcium ion concentration conditions include, for example, the method described in WO2012 / 073992 (e.g., paragraphs 0200-0213). The method for conferring the characteristic of weakly binding to an antigen under low calcium ion concentration conditions compared to high calcium ion concentration conditions to the antigen-binding domain of the present invention is not particularly limited and may be carried out by any method. Specifically, such a method is described in Japanese Patent Application No. 2011-218006 and includes, for example, a method of substituting at least one amino acid residue in the antigen-binding domain with an amino acid residue having metal-chelating activity, and / or inserting at least one amino acid residue having metal-chelating activity into the antigen-binding domain. An antigen-binding molecule of the present invention in which at least one amino acid residue in the antigen-binding domain is substituted with an amino acid residue having metal-chelating activity, and / or in which at least one amino acid residue having metal-chelating activity is inserted into the antigen-binding domain is one preferred embodiment of the antigen-binding molecule of the present invention.

[0121] Amino acid residues having metal chelating activity preferably include, for example, serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. Furthermore, amino acid residues that change the antigen-binding activity of the antigen-binding domain depending on the calcium ion concentration preferably include, for example, amino acid residues that form calcium-binding motifs. Calcium-binding motifs are well known to those skilled in the art and have been reported in detail (e.g., Springer et al., (Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490); Moncrief et al., (J. Mol. Evol. (1990) 30, 522-562); Chauvaux et al., (Biochem. J. (1990) 265, 261-265); Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol. (1990) 2, 410-419); Schaefer et al., (Genomics (1995) 25, 638 to 643); Economou et al., (EMBO J. (1990) 9, 349-354); Wurzburg et al., (Structure. (2006) 14, 6, 1049-1058). The EF domains of troponin C, calmodulin, parvalbumin, and myosin light chain; the C2 domain of protein kinase C; the Gla domain of blood coagulation protein factor IX; the C-type lectins of asialoglycoprotein receptor and mannose-binding receptor, ASGPR, CD23, and DC-SIGN; the A domain of LDL receptor; the annexin domain; the cadherin domain; the thrombospondin type 3 domain; and the EGF-like domain are preferably used as calcium-binding motifs.

[0122] The antigen-binding domain of the present invention may include amino acid residues that change their antigen-binding activity depending on the calcium ion concentration, such as amino acid residues having metal chelating activity or amino acid residues forming calcium-binding motifs. The position of such amino acid residues within the antigen-binding domain is not particularly limited and may be any position as long as the antigen-binding activity changes depending on the calcium ion concentration. Furthermore, as long as the antigen-binding activity changes depending on the calcium ion concentration, such amino acid residues may be included individually or in combination of two or more. Suitable examples of such amino acid residues include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. If the antigen-binding domain is a variable region of the antibody, these amino acid residues may be included in the heavy chain variable region and / or the light chain variable region. In a preferred embodiment, these amino acid residues may be included in the CDR3 of the heavy chain variable region, and more preferably in positions 95, 96, 100a, and / or 101 as represented by the Kabat numbering of the CDR3 of the heavy chain variable region.

[0123] In another preferred embodiment, these amino acid residues may be contained in CDR1 of the light chain variable region, more preferably in positions 30, 31 and / or 32 as represented by the Kabat numbering of CDR1 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR2 of the light chain variable region, more preferably in position 50 as represented by the Kabat numbering of CDR2 of the light chain variable region. In yet another preferred embodiment, these amino acid residues may be contained in CDR3 of the light chain variable region, more preferably in position 92 as represented by the Kabat numbering of CDR3 of the light chain variable region.

[0124] Furthermore, the above embodiments may be combined, for example, the amino acid residue may be contained in two or three CDRs selected from CDR1, CDR2, and CDR3 of the light chain variable region, and more preferably in one or more of the 30th, 31st, 32nd, 50th, and / or 92nd positions represented by the Kabat numbering of the light chain variable region.

[0125] By creating a library of numerous antigen-binding domains that have different sequences but share a common structure of amino acid residues that change antigen-binding activity depending on the calcium ion concentration, as described above, and then screening from this library, it is possible to efficiently obtain antigen-binding domains that have binding activity to a desired antigen and whose antigen-binding activity changes depending on the calcium ion concentration.

[0126] For the purposes of this disclosure, the “affinity” of an antibody against C1s is expressed by the KD of that antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of the antibody’s binding to that antigen, the weaker its binding affinity to that particular 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 an antibody’s binding to C1s at acidic pH is at least twice as high as the KD of an antibody’s binding to C1s at neutral pH, then the antibody is considered to bind to C1s with higher affinity at neutral pH than at acidic pH. Accordingly, the present invention includes an antibody that binds to C1s at an acidic pH with a KD 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 times greater than the KD of the antibody binding to C1s at a neutral pH. In another embodiment, the KD value of the antibody at a neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10-10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It could be M or higher.

[0127] The binding properties to a particular antigen can also be expressed by the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody against a particular antigen, and is the reciprocal of seconds (i.e., sec). -1 The kd value is expressed in units of ). An increase in the kd value means that the antibody's binding to its antigen is weaker. Therefore, the present invention includes antibodies that bind to C1s at acidic pH with a higher kd value than neutral pH. The present invention includes antibodies that bind to C1s at acidic pH with a kd value 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 times, or more than the kd of the antibody's binding to C1s at neutral pH. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s or more.

[0128] In certain examples, “reduced binding at acidic pH compared to binding at neutral pH” is expressed as the ratio of the antibody’s KD value at acidic pH to the antibody’s KD value at neutral pH (or vice versa). For example, if an antibody exhibits an acid / neutral KD ratio of 2 or higher, for the purposes of the present invention, that antibody may be considered to exhibit “reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH.” In certain exemplary embodiments, the acid / neutral KD ratio in the antibody of the present 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 times, or more. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It could be M or higher.

[0129] In certain examples, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's kd value at acidic pH to its kd value at neutral pH (or vice versa). For example, if an antibody exhibits an acid / neutral kd ratio of 2 or higher, for the purposes of the present invention, that antibody may be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH." In certain exemplary embodiments, the acid / neutral kd ratio in the antibody of the present 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 times, or more. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s or more.

[0130] As used herein, the term "acidic pH" refers to a pH range of 4.0 to 6.5. The term "acidic pH" includes pH values ​​of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain contexts, "acidic pH" is 5.8 or 6.0.

[0131] As used herein, the term "neutral pH" refers to a pH range of 6.7 to approximately 10.0. The term "neutral pH" includes pH values ​​of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In certain contexts, "neutral pH" is 7.0 or 7.4.

[0132] As used herein, the expressions "under high calcium concentration conditions" or "at high calcium concentrations" mean 100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). The expressions "under high calcium concentration conditions" or "at high calcium concentrations" refer to 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 0.5 mM, 0.7 mM, 0.9 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2.0 mM, 2.2 mM, 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM Ca 2+This includes the calcium concentration value. In certain situations, "under high calcium concentration conditions" or "under high calcium concentration conditions" is 1.2 mM Ca 2+ It represents.

[0133] As used herein, the expressions "under low calcium concentration conditions" or "at low calcium concentrations" mean 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. The expressions "under low calcium concentration conditions" or "at low calcium concentrations" mean 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2.0 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM Ca 2+ This includes the calcium concentration value. In certain situations, "under low calcium concentration conditions" or "under low calcium concentration conditions" refers to 3.0 μM Ca 2+ It represents.

[0134] The KD and kd values ​​expressed herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, for example, Example 2 herein.) The KD and kd values ​​can be determined at 25°C or 37°C. This determination can be carried out in the presence of 150 mM NaCl. In some embodiments, this determination can be carried out using surface plasmon resonance technology with an immobilized antibody and an antigen as an analyte, using the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl at 37°C.

[0135] In one aspect, the present invention provides a method for enhancing the clearance of C1s from plasma in an individual. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing the clearance of C1s from plasma. The present invention also provides a method for enhancing the clearance of a C1r-C1s complex from plasma in an individual. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing the clearance of a C1r-C1s complex from plasma. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing the clearance of C1r2s2 from plasma. In some embodiments, the method comprises administering to an individual an amount of the anti-C1s antibody of the present invention effective in enhancing the clearance of C1r2s2 from plasma without enhancing the clearance of C1q from plasma.

[0136] In another aspect, the present invention provides a method for removing C1s from plasma, comprising the steps of: (a) identifying an individual from whom C1s need to be removed from the plasma; (b) providing an antibody that binds to C1s, wherein the antibody binds to C1s through its antigen-binding (C1s-binding) domain and has a KD(pH 5.8) / KD(pH 7.4) value of 2 to 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of the KD to C1s at pH 5.8 to the KD to C1s at pH 7.4, when the KD is determined using surface plasmon resonance technology; and (c) administering the antibody to the individual. In a further context, such surface plasmon resonance techniques may be used at 37°C and 150 mM NaCl. In a further context, such surface plasmon resonance techniques may be used with an immobilized antibody and an antigen as an analyte, under the conditions of "10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37°C".

[0137] In another aspect, the present invention relates to a method for removing C1s from plasma in a subject, comprising: (a) identifying a first antibody, wherein the first antibody binds to C1s through the antigen-binding domain of the first antibody; (b) identifying a second antibody, wherein the second antibody (1) binds to C1s through the antigen-binding (C1s-binding) domain of the second antibody, (2) has the same amino acid sequence as the first antibody except that at least one amino acid in the variable region of the first antibody is substituted with histidine and / or at least one histidine is inserted into the variable region of the first antibody, and (3) has a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the first antibody and is between 2 and 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of KD to C1s at pH 5.8 to KD to C1s at pH 7.4 when KD is determined using surface plasmon resonance technology, and provides a method comprising the steps of: (4) binding to C1s in plasma in vivo, (5) dissociating from bound C1s under conditions present in endosomes in vivo, and (6) being human IgG or humanized IgG; (c) identifying a subject who needs to have their plasma C1s levels reduced; and (d) administering a second antibody to the subject so that the plasma C1s levels of the subject are reduced. In a further context, such surface plasmon resonance technology may be used at 37°C and 150 mM NaCl. In a further context, such surface plasmon resonance technology may be used at 37°C and 150 mM NaCl. In a further context, such surface plasmon resonance techniques may be used by immobilizing antibodies, using antigens as analytes, and under the following conditions: "10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37°C."

[0138] In another aspect, the present invention relates to a method for removing C1s from plasma in a subject, comprising the steps of (a) identifying a first antibody, wherein the first antibody (1) binds to C1s through the antigen-binding domain of the first antibody, (2) has the same amino acid sequence as a second antibody that binds to C1s through the antigen-binding (C1s-binding) domain of the second antibody, except that at least one variable region of the first antibody has at least one more histidine residue than the corresponding variable region of the second antibody, and (3) has a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the second antibody and is between 2 and 10,000, where KD(pH 5.8) / KD(pH 7.4) is the KD for C1s at pH 5.8 and pH 7.4 when the KD is determined using surface plasmon resonance technology. The present invention provides a method comprising the steps of: (4) binding to C1s in plasma in vivo, (5) dissociating from bound C1s under conditions present in endosomes in vivo, and (6) being human IgG or humanized IgG; (b) identifying a subject for whom a reduction in plasma C1s levels is needed; and (c) administering the subject at least once with the first antibody to reduce the plasma C1s levels of the subject. In a further context, such surface plasmon resonance technology may be used at 37°C and 150 mM NaCl. In a further context, such surface plasmon resonance technology may be used at 37°C and 150 mM NaCl. In a further context, such surface plasmon resonance technology may be used with an immobilized antibody and an antigen as an analyte, and using the conditions "10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl at 37°C". In some cases, antibodies inhibit components of the classical complement pathway, and in some cases, the component of the classical complement pathway is C1s.

[0139] In one aspect, the Disclosure provides a method for modulating complement activation. In some embodiments, the method inhibits complement activation, for example, by reducing the production of C4b2a. In some embodiments, the Disclosure provides a method for modulating complement activation in an individual having a complement-mediated disease or disorder, comprising the step of administering to the individual an anti-C1s antibody of the Disclosure or a pharmaceutical composition of the Disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of the 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 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.

[0140] In certain embodiments, the anti-C1s antibody of the present invention binds to C1s derived from two or more species. In certain embodiments, the anti-C1s antibody binds to C1s derived from humans and non-human animals. In certain embodiments, the anti-C1s antibody binds to C1s derived from humans, rats, and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, and baboons).

[0141] In one aspect, the present invention provides an anti-C1s antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising amino acid sequences of SEQ ID NO: 32, 38, 44, 50, or 56; (b) HVR-H2 comprising amino acid sequences of SEQ ID NO: 33, 39, 45, 51, or 57; (c) HVR-H3 comprising amino acid sequences of SEQ ID NO: 34, 40, 46, 52, or 58; (d) HVR-L1 comprising amino acid sequences of SEQ ID NO: 35, 41, 47, 53, or 59; (e) HVR-L2 comprising amino acid sequences of SEQ ID NO: 36, 42, 48, 54, or 60; and (f) HVR-L3 comprising amino acid sequences of SEQ ID NO: 37, 43, 49, 55, or 61.

[0142] In one aspect, the present invention provides an anti-C1s antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. In a further embodiment, the antibody comprises HVR-H3 containing the amino acid sequence SEQ ID NO: 34, 40, 46, 52, or 58; HVR-L3 containing the amino acid sequence SEQ ID NO: 37, 43, 49, 55, or 61; and HVR-H2 containing the amino acid sequence SEQ ID NO: 33, 39, 45, 51, or 57. In a further embodiment, the antibody comprises (a) HVR-H1 containing the amino acid sequence SEQ ID NO: 32, 38, 44, 50, or 56; (b) HVR-H2 containing the amino acid sequence SEQ ID NO: 33, 39, 45, 51, or 57; and (c) HVR-H3 containing the amino acid sequence SEQ ID NO: 34, 40, 46, 52, or 58.

[0143] 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 (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61.

[0144] In another aspect, the anti-C1s antibody of the present invention comprises a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (a)(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58; and (b)(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60; and (c) SEQ ID The VL domain contains at least one, at least two, or all three VL HVR sequences selected from HVR-L3 containing amino acid sequences NO:37, 43, 49, 55, or 61.

[0145] In some embodiments, anti-C1s antibody variants are provided that are prepared by introducing amino acid modifications to an antibody containing a VH sequence with SEQ ID No: 19, 20, 21, 23, or 24 and a VL sequence with SEQ ID No: 26, 27, 28, 30, or 31.

[0146] In some embodiments, the anti-C1s antibody of the present invention contains histidine at one or more of the following positions in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97.

[0147] In some embodiments, the anti-C1s antibody of the present invention comprises at least one histidine substituted for one or more amino acid residues at a position selected from the following positions in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96, and L97.

[0148] In any of the above embodiments, the anti-C1s antibody is a humanized antibody. In one embodiment, the anti-C1s antibody comprises an HVR of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1s antibody comprises an HVR of any of the above embodiments and further comprises a VH or VL containing an FR sequence. In a further embodiment, the anti-C1s antibody of the present invention comprises the following heavy chain or light chain variable domain FR sequence.

[0149] In another aspect, the anti-C1s antibody contains 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 a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions to the reference sequence, but the anti-C1s antibody containing that sequence retains the ability to bind to C1s. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 19, 20, 21, 23, or 24. In certain embodiments, substitutions, insertions, or deletions occur in the outer region (i.e., FR) of the HVR. Optionally, the anti-C1s antibody includes a VH sequence with SEQ ID NO: 19, 20, 21, 23, or 24, including those with post-translational modifications. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 32, 38, 44, 50, or 56, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 33, 39, 45, 51, or 57, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 34, 40, 46, 52, or 58. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0150] In another aspect, an anti-C1s antibody is provided that contains 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 a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions to the reference sequence, but the anti-C1s antibody containing that sequence retains the ability to bind to C1s. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 26, 27, 28, 30, or 31. In certain embodiments, substitutions, insertions, or deletions occur in the outer region (i.e., FR) of the HVR. Optionally, the anti-C1s antibody includes a VL sequence with SEQ ID NO: 26, 27, 28, 30, or 31, including those with post-translational modifications of said sequence. In certain embodiments, said VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 35, 41, 47, 53, or 59, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 36, 42, 48, 54, or 60, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 37, 43, 49, 55, or 61. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0151] In another aspect, an anti-C1s antibody is provided comprising a VH sequence and a VL sequence of any of the above embodiments. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 19 and SEQ ID NO: 26, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 20 and SEQ ID NO: 27, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 21 and SEQ ID NO: 28, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, this antibody includes VH and VL sequences of SEQ ID NO: 23 and SEQ ID NO: 30, respectively, which include post-translational modifications of their sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, this antibody includes VH and VL sequences of SEQ ID NO: 24 and SEQ ID NO: 31, respectively, which include post-translational modifications of their sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0152] In a further aspect, the present invention provides an antibody that binds to the same epitope as the anti-C1s antibody provided herein. In a preferred aspect, this antibody specifically binds to the same epitope as the anti-C1s antibody provided herein. For example, in a particular embodiment, 1) An antibody containing the HVR-H1 sequence of SEQ ID NO:32, the HVR-H2 sequence of SEQ ID NO:33, the HVR-H3 sequence of SEQ ID NO:34, the HVR-L1 sequence of SEQ ID NO:35, the HVR-L2 sequence of SEQ ID NO:36, and the HVR-L3 sequence of SEQ ID NO:37. 2) An antibody containing the HVR-H1 sequence with SEQ ID NO:38, the HVR-H2 sequence with SEQ ID NO:39, the HVR-H3 sequence with SEQ ID NO:40, the HVR-L1 sequence with SEQ ID NO:41, the HVR-L2 sequence with SEQ ID NO:42, and the HVR-L3 sequence with SEQ ID NO:43. 3) An antibody containing the HVR-H1 sequence with SEQ ID NO:44, the HVR-H2 sequence with SEQ ID NO:45, the HVR-H3 sequence with SEQ ID NO:46, the HVR-L1 sequence with SEQ ID NO:47, the HVR-L2 sequence with SEQ ID NO:48, and the HVR-L3 sequence with SEQ ID NO:49. 4) Antibodies containing the HVR-H1 sequence of SEQ ID NO:50, the HVR-H2 sequence of SEQ ID NO:51, the HVR-H3 sequence of SEQ ID NO:52, the HVR-L1 sequence of SEQ ID NO:53, the HVR-L2 sequence of SEQ ID NO:54, and the HVR-L3 sequence of SEQ ID NO:55, as well as 5) An antibody containing the HVR-H1 sequence with SEQ ID NO: 56, the HVR-H2 sequence with SEQ ID NO: 57, the HVR-H3 sequence with SEQ ID NO: 58, the HVR-L1 sequence with SEQ ID NO: 59, the HVR-L2 sequence with SEQ ID NO: 60, and the HVR-L3 sequence with SEQ ID NO: 61. An antibody is provided that binds (specifically) to the same epitope as an antibody selected from the group consisting of the following.

[0153] In some embodiments, the isolated anti-C1s antibody of the present invention competes with antibodies selected from the group consisting of 1) to 5) below for binding to C1s. In some embodiments, the isolated anti-C1s antibody of the present invention competes with antibodies selected from the group consisting of 1) to 5) below for binding to C1s at a neutral pH: 1) An antibody containing the HVR-H1 sequence of SEQ ID NO:32, the HVR-H2 sequence of SEQ ID NO:33, the HVR-H3 sequence of SEQ ID NO:34, the HVR-L1 sequence of SEQ ID NO:35, the HVR-L2 sequence of SEQ ID NO:36, and the HVR-L3 sequence of SEQ ID NO:37. 2) An antibody containing the HVR-H1 sequence with SEQ ID NO:38, the HVR-H2 sequence with SEQ ID NO:39, the HVR-H3 sequence with SEQ ID NO:40, the HVR-L1 sequence with SEQ ID NO:41, the HVR-L2 sequence with SEQ ID NO:42, and the HVR-L3 sequence with SEQ ID NO:43. 3) An antibody containing the HVR-H1 sequence with SEQ ID NO:44, the HVR-H2 sequence with SEQ ID NO:45, the HVR-H3 sequence with SEQ ID NO:46, the HVR-L1 sequence with SEQ ID NO:47, the HVR-L2 sequence with SEQ ID NO:48, and the HVR-L3 sequence with SEQ ID NO:49. 4) Antibodies containing the HVR-H1 sequence of SEQ ID NO:50, the HVR-H2 sequence of SEQ ID NO:51, the HVR-H3 sequence of SEQ ID NO:52, the HVR-L1 sequence of SEQ ID NO:53, the HVR-L2 sequence of SEQ ID NO:54, and the HVR-L3 sequence of SEQ ID NO:55, as well as 5) An antibody containing the HVR-H1 sequence with SEQ ID NO: 56, the HVR-H2 sequence with SEQ ID NO: 57, the HVR-H3 sequence with SEQ ID NO: 58, the HVR-L1 sequence with SEQ ID NO: 59, the HVR-L2 sequence with SEQ ID NO: 60, and the HVR-L3 sequence with SEQ ID NO: 61.

[0154] In one aspect, the present invention relates to (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125 or 126, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149 or 150, (e) SEQ ID The present invention provides an anti-C1r antibody comprising at least one, two, three, four, five, or six HVRs selected from HVR-L2 containing amino acid sequences NO: 151, 152, 153, 154, 155, 156, 157, or 158, and HVR-L3 containing amino acid sequences NO: 159, 160, 161, 162, 163, 164, 165, or 166.

[0155] In one aspect, the present invention provides an anti-C1r antibody comprising at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125 or 126, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142. In another embodiment, the antibody comprises HVR-H3 containing the amino acid sequence SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142, and HVR-L3 containing the amino acid sequence SEQ ID NO: 159, 160, 161, 162, 163, 164, 165 or 166. In a further embodiment, the antibody comprises HVR-H3 containing the amino acid sequence SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142, HVR-L3 containing the amino acid sequence SEQ ID NO: 159, 160, 161, 162, 163, 164, 165 or 166, and HVR-H2 containing the amino acid sequence SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134. In a further embodiment, the antibody comprises (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125 or 126, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142.

[0156] 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 (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 containing the amino acid sequence SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 containing the amino acid sequence SEQ ID NO: 159, 160, 161, 162, 163, 164, 165, or 166.

[0157] In another aspect, the anti-C1r antibody of the present invention comprises a VH domain containing at least one, at least two or three all VH HVR sequences selected from (a)(i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125 or 126, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133 or 134, and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141 or 142, and (b)(i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149 or 150, and (ii) SEQ ID The VL domain contains at least one, at least two, or all three VL HVR sequences selected from (c)SEQ ID NO:159, 160, 161, 162, 163, 164, 165, or 166 of HVR-L2 and (c)SEQ ID NO:159, 160, 161, 162, 163, 164, 165, or 166.

[0158] In some embodiments, anti-C1r antibody variants are provided that are prepared by introducing amino acid modifications to an antibody containing a VH sequence with SEQ ID No: 103, 104, 105, 106, 107, 108, 109, or 110 and a VL sequence with SEQ ID No: 111, 112, 113, 114, 115, 116, 117, or 118.

[0159] In some embodiments, the anti-C1r antibody of the present invention is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 One or more of these contain histidine.

[0160] In some embodiments, the anti-C1r antibody of the present invention is located in the following position in the Kabat numbering system: Heavy chains: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56, L91, L92, L93, L94, L95, L95a, L96 and L97 It comprises at least one histidine substituted for one or more amino acid residues at a position selected from.

[0161] In any of the above embodiments, the anti-C1r antibody is humanized. In one embodiment, the anti-C1r antibody comprises an HVR of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1r antibody comprises an HVR of any of the above embodiments and further comprises a VH or VL containing an FR sequence. In a further embodiment, the anti-C1r antibody of the present invention comprises the following heavy chain or light chain variable domain FR sequence.

[0162] In another aspect, the anti-C1r antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-C1r antibody containing that sequence retains the ability to bind to C1r. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110. In certain embodiments, the substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., FR). Optionally, the anti-C1r antibody contains the VH sequence of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, or 110, including post-translational modifications of its sequence. In certain embodiments, VH comprises one, two, or three HVRs selected from (a) HVR-H1 having amino acid sequences of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, or 126; (b) HVR-H2 having amino acid sequences of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, or 134; and (c) HVR-H3 having amino acid sequences of SEQ ID NO: 135, 136, 137, 138, 139, 140, 141, or 142. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0163] In another aspect, an anti-C1r antibody is provided that contains a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-C1r antibody containing that sequence retains the ability to bind to C1r. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of the HVR (i.e., FR). Optionally, the anti-C1r antibody contains the VL sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, 117, or 118, including post-translational modifications of its sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 143, 144, 145, 146, 147, 148, 149, or 150; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, or 158; and (c) HVR-L3 containing 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, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0164] In another aspect, an anti-C1r antibody is provided comprising a VH sequence and a VL sequence of any of the above embodiments. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 103 and SEQ ID NO: 111, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 104 and SEQ ID NO: 112, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody comprises a VH sequence and a VL sequence of SEQ ID NO: 105 and SEQ ID NO: 113, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, this antibody includes VH and VL sequences of SEQ ID NO: 106 and SEQ ID NO: 114, respectively, including post-translational modifications of their sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, this antibody includes VH and VL sequences of SEQ ID NO: 107 and SEQ ID NO: 115, respectively, including post-translational modifications of their sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody comprises VH and VL sequences of SEQ ID NO:108 and SEQ ID NO:116, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.In one embodiment, the antibody includes VH and VL sequences of SEQ ID NO: 109 and SEQ ID NO: 117, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain. In one embodiment, the antibody includes VH and VL sequences of SEQ ID NO: 110 and SEQ ID NO: 118, respectively, including post-translational modifications of those sequences. The post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy or light chain.

[0165] In a further aspect, the present invention provides an antibody that binds to the same epitope as the anti-C1r antibody provided herein. In a preferred aspect, this antibody specifically binds to the same epitope as the anti-C1r antibody provided herein. For example, in a particular embodiment, 6) An antibody containing the HVR-H1 sequence with SEQ ID NO:119, the HVR-H2 sequence with SEQ ID NO:127, the HVR-H3 sequence with SEQ ID NO:135, the HVR-L1 sequence with SEQ ID NO:143, the HVR-L2 sequence with SEQ ID NO:151, and the HVR-L3 sequence with SEQ ID NO:159. 7) An antibody containing the HVR-H1 sequence with SEQ ID NO:120, the HVR-H2 sequence with SEQ ID NO:128, the HVR-H3 sequence with SEQ ID NO:136, the HVR-L1 sequence with SEQ ID NO:144, the HVR-L2 sequence with SEQ ID NO:152, and the HVR-L3 sequence with SEQ ID NO:160. 8) An antibody containing the HVR-H1 sequence with SEQ ID NO:121, the HVR-H2 sequence with SEQ ID NO:129, the HVR-H3 sequence with SEQ ID NO:137, the HVR-L1 sequence with SEQ ID NO:145, the HVR-L2 sequence with SEQ ID NO:153, and the HVR-L3 sequence with SEQ ID NO:161. 9) An antibody containing the HVR-H1 sequence with SEQ ID NO:122, the HVR-H2 sequence with SEQ ID NO:130, the HVR-H3 sequence with SEQ ID NO:138, the HVR-L1 sequence with SEQ ID NO:146, the HVR-L2 sequence with SEQ ID NO:154, and the HVR-L3 sequence with SEQ ID NO:162. 10) An antibody containing the HVR-H1 sequence with SEQ ID NO:123, the HVR-H2 sequence with SEQ ID NO:131, the HVR-H3 sequence with SEQ ID NO:139, the HVR-L1 sequence with SEQ ID NO:147, the HVR-L2 sequence with SEQ ID NO:155, and the HVR-L3 sequence with SEQ ID NO:163. 11) An antibody containing the HVR-H1 sequence with SEQ ID NO:124, the HVR-H2 sequence with SEQ ID NO:132, the HVR-H3 sequence with SEQ ID NO:140, the HVR-L1 sequence with SEQ ID NO:148, the HVR-L2 sequence with SEQ ID NO:156, and the HVR-L3 sequence with SEQ ID NO:164. 12) Antibodies containing the HVR-H1 sequence with SEQ ID NO:125, the HVR-H2 sequence with SEQ ID NO:133, the HVR-H3 sequence with SEQ ID NO:141, the HVR-L1 sequence with SEQ ID NO:149, the HVR-L2 sequence with SEQ ID NO:157, and the HVR-L3 sequence with SEQ ID NO:165, as well as 13) An antibody containing the HVR-H1 sequence with SEQ ID NO:126, the HVR-H2 sequence with SEQ ID NO:134, the HVR-H3 sequence with SEQ ID NO:142, the HVR-L1 sequence with SEQ ID NO:150, the HVR-L2 sequence with SEQ ID NO:158, and the HVR-L3 sequence with SEQ ID NO:166. An antibody is provided that binds (specifically) to the same epitope as an antibody selected from the group consisting of the following.

[0166] In some embodiments, the isolated anti-C1r antibody of the present invention competes with antibodies selected from the group consisting of 6) to 13) below for binding to C1r. In some embodiments, the isolated anti-C1r antibody of the present invention competes with antibodies selected from the group consisting of 6) to 13) below for binding to C1r at a neutral pH: 6) An antibody containing the HVR-H1 sequence with SEQ ID NO:119, the HVR-H2 sequence with SEQ ID NO:127, the HVR-H3 sequence with SEQ ID NO:135, the HVR-L1 sequence with SEQ ID NO:143, the HVR-L2 sequence with SEQ ID NO:151, and the HVR-L3 sequence with SEQ ID NO:159. 7) An antibody containing the HVR-H1 sequence with SEQ ID NO:120, the HVR-H2 sequence with SEQ ID NO:128, the HVR-H3 sequence with SEQ ID NO:136, the HVR-L1 sequence with SEQ ID NO:144, the HVR-L2 sequence with SEQ ID NO:152, and the HVR-L3 sequence with SEQ ID NO:160. 8) An antibody containing the HVR-H1 sequence with SEQ ID NO:121, the HVR-H2 sequence with SEQ ID NO:129, the HVR-H3 sequence with SEQ ID NO:137, the HVR-L1 sequence with SEQ ID NO:145, the HVR-L2 sequence with SEQ ID NO:153, and the HVR-L3 sequence with SEQ ID NO:161. 9) An antibody containing the HVR-H1 sequence with SEQ ID NO:122, the HVR-H2 sequence with SEQ ID NO:130, the HVR-H3 sequence with SEQ ID NO:138, the HVR-L1 sequence with SEQ ID NO:146, the HVR-L2 sequence with SEQ ID NO:154, and the HVR-L3 sequence with SEQ ID NO:162. 10) An antibody containing the HVR-H1 sequence with SEQ ID NO:123, the HVR-H2 sequence with SEQ ID NO:131, the HVR-H3 sequence with SEQ ID NO:139, the HVR-L1 sequence with SEQ ID NO:147, the HVR-L2 sequence with SEQ ID NO:155, and the HVR-L3 sequence with SEQ ID NO:163. 11) An antibody containing the HVR-H1 sequence with SEQ ID NO:124, the HVR-H2 sequence with SEQ ID NO:132, the HVR-H3 sequence with SEQ ID NO:140, the HVR-L1 sequence with SEQ ID NO:148, the HVR-L2 sequence with SEQ ID NO:156, and the HVR-L3 sequence with SEQ ID NO:164. 12) Antibodies containing the HVR-H1 sequence with SEQ ID NO:125, the HVR-H2 sequence with SEQ ID NO:133, the HVR-H3 sequence with SEQ ID NO:141, the HVR-L1 sequence with SEQ ID NO:149, the HVR-L2 sequence with SEQ ID NO:157, and the HVR-L3 sequence with SEQ ID NO:165, as well as 13) An antibody containing the HVR-H1 sequence with SEQ ID NO:126, the HVR-H2 sequence with SEQ ID NO:134, the HVR-H3 sequence with SEQ ID NO:142, the HVR-L1 sequence with SEQ ID NO:150, the HVR-L2 sequence with SEQ ID NO:158, and the HVR-L3 sequence with SEQ ID NO:166.

[0167] In one aspect, the Disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding affinity that specifically binds to an epitope within the region containing the CUB1-EGF-CUB2 domain of complement component 1s (C1s), consisting of CUB1, EGF, and CUB2. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is an epitope not located in the beta domain of C1s. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is an epitope located in the alpha domain or the gamma domain of C1s. In some embodiments, the epitope to which the isolated anti-C1s antibody of the Disclosure binds is a linear epitope. In some embodiments, the epitopes to which the isolated anti-C1s antibody of this disclosure binds are epitopes within the range of amino acids 16-291 of the complement C1s protein, amino acids 16-172 of the complement C1s protein as shown in SEQ ID NO:1, amino acids 16-210 of the complement C1s protein as shown in SEQ ID NO:1, amino acids 16-111 of the complement C1s protein as shown in SEQ ID NO:1, amino acids 112-210 of the complement C1s protein as shown in SEQ ID NO:1, amino acids 131-172 of the complement C1s protein as shown in SEQ ID NO:1, or amino acids 16-130 of the complement C1s protein as shown in SEQ ID NO:1. In some embodiments, the above epitopes of C1s are epitopes of human C1s. In some embodiments, the isolated anti-C1s antibody of the present invention can bind to both activated C1s protein and inactive forms of C1s.

[0168] In some embodiments, the Disclosure provides isolated anti-C1r antibodies that specifically bind to epitopes within a region comprising the CUB1-EGF-CUB2 domain of complement component 1r (C1r), consisting of CUB1, EGF, and CUB2. In some examples, the epitopes to which the isolated anti-C1r antibodies of the Disclosure bind are linear epitopes or structural epitopes. In some embodiments, the epitopes of C1r are human C1r epitopes.

[0169] In a further aspect of the present invention, the anti-C1s antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric, humanized, or human antibody. In one embodiment, the anti-C1s antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1, IgG2, IgG3, or IgG4 antibody, or any other antibody class or isotype as defined herein.

[0170] In further developments, anti-C1s antibodies according to any of the above embodiments may incorporate any of the characteristics described in items 1 to 7 below, either alone or in combination.

[0171] 1. Antibody affinity In certain embodiments, the antibodies provided herein have concentrations 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 (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd or KD) of M).

[0172] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2-5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0173] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (GE Healthcare) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 micrograms (μg) / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 microliters (μl) / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0174] In some embodiments, the binding affinity of each histidine substitution variant of the present invention at pH 7.4 and pH 5.8 is determined at 37°C using a Biacore® T200 instrument (GE Healthcare). Recombinant protein A / G (Pierce) can be immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes can be prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4), 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8), or 5(-) buffer (20 mM ACES, 150 mM NaCl, 3 μM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody can be captured on the sensor surface by protein A / G. The target antibody capture amount is 200 resonance units (RU). Prepared 50 nM natural proenzyme human C1s (CompTech) or recombinant human C1s may be injected and subsequently dissociated. A specific example of the steps of the Biacore assay according to the present invention is as follows: The binding affinity of the C1s CUB1-EGF-CUB2 conjugate is determined at 37°C using a Biacore® T200 instrument (GE Healthcare). Recombinant protein A / G (Pierce) is immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes are prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4). Each antibody is captured on the sensor surface by protein A / G. The target antibody capture amount is 100 resonance units (RU). Natural proenzyme human C1s (Comptech A103) (50 nM monomer) or recombinant human C1s CCP1-CCP2-SP-His (100 nM monomer) is injected and then dissociated. The sensor surface is regenerated using 10 mM glycine-HCl pH 1.5 after each cycle. It can be determined that the C1s CUB1-EGF-CUB2 conjugate bound to the native proenzyme human C1s, but not to the recombinant human C1s CCP1-CCP2-SP-His, which is a cleaved protein lacking the CUB1-EGF-CUB2 domain. The antibody's C1q dissociation-promoting function is demonstrated by the C1r2s2 capture method using a BIACORE® T200 instrument (GE Healthcare) at 37°C. Anti-His antibody (GE Healthcare) is immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Each antibody, recombinant human C1r2s2 Flag / His tetramer, and native human C1q (Comptech A099) are prepared in pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). First, the recombinant human C1r2s2 Flag / His tetramer is captured on the sensor surface by the anti-His antibody ("hc1r2s2"). The target capture amount is 200 resonance units (RU). 100 nM of natural human C1q is injected to capture 200 RU ("hc1q"), followed immediately by injection of 500 nM antibody at 10 μL / min for 120 seconds. After each cycle, the sensor surface is regenerated using 10 mM glycine-HCl (pH 1.5). Antibodies with C1q dissociation-promoting function will cause the resonance units of sensorgram 2 (where C1r2s2, C1q, and antibody are present) to be lower than those of sensorgram 1 (where C1r2s2, C1q, and buffer are present, but antibody is absent) after the point at which sensorgrams 1 and 2 cross ("crossover point"). The crossover point is identified by subtracting the buffer response (sensorgram 1) from the antibody (Ab) response (sensorgram 2), and is the point at which the difference changes from positive to negative. The antibody's C1q dissociation-promoting function is demonstrated by a C1q capture method using a BIACORE® T200 instrument (GE Healthcare) at 37°C. Each antibody, recombinant human C1r2s2 Flag / His tetramer, and biotinylated native human C1q (Comptech A099) are prepared in pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). First, biotinylated native human C1q is captured on one flow cell of a CAP sensor tip (GE-Healthcare). The target capture amount is within the range of 800–1000 resonance units (RU). A 300 nM recombinant human C1r2s2 Flag / His tetramer is injected, followed by an injection of 500 nM antibody at 10 μL / min for 180 seconds. After each cycle, the sensor surface is regenerated using 8M guanidine-HCl and 1M NaOH in a 3:1 ratio. Antibodies with C1q dissociation-promoting function improve the dissociation rate of C1r2s2, i.e., the curve in the presence of the antibody is below the curve in the absence of the antibody. To evaluate the blocking of C1q binding to C1r2s2 by antibodies, a blocking assay is performed at 37°C using a BIACORE® T200 instrument (GE Healthcare). Anti-His antibodies (GE Healthcare) are immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Each antibody, recombinant human C1r2s2 Flag / His tetramer, and native human C1q are prepared in pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / mL BSA (IgG-free), 1 mg / mL CMD, 0.05% Tween 20, 0.005% NaN3, pH 7.4). First, recombinant human C1r2s2 Flag / His tetramer is captured on the sensor surface by the anti-His antibody ("hc1r2s2"). The target capture amount is 200 resonance units (RU). A 500 nM antibody variant is injected, followed by 100 nM of native human C1q ("hc1q"). After each cycle, the sensor surface is regenerated using 10 mM glycine-HCl (pH 1.5). The antibody possessing C1q blocking function is one that competes with C1q for binding to C1r2s2. In some embodiments, a further dissociation region at pH 5.8 is incorporated immediately after the dissociation region at pH 7.4, as needed. The rate of this dissociation in 5(+) buffer can be determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0175] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, see WO93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0176] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0177] A single-domain antibody is an antibody fragment containing 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 (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516 B1).

[0178] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0179] 3. Chimeric antibodies and humanized antibodies 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 in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0180] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally includes at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0181] Humanized antibodies and their preparation methods have been reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes resurfacing); Further details can be found in Dall'Acqua et al., Methods 36:43-60 (2005) (which describes FR shuffling), as well as in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (which describes a "guided selection" approach for FR shuffling).

[0182] The human framework regions that can be used for humanization are not limited to these, but include: framework regions selected using the "best fit" method (see Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684). (See also 1997 and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0183] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various methods known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0184] Human antibodies may be prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0185] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (See, for example, 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 produced 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, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005).

[0186] Human antibodies can also be generated by isolating selected Fv clone variable domain sequences from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. A method for selecting human antibodies from antibody libraries is described below.

[0187] 5. Antibodies derived from libraries The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies with desired binding properties are known in the art. Such methods are reviewed in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0188] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), recombined randomly in a phage library, and the phage library can be screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phages typically display antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), a naive repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization. Finally, naive libraries can also be made synthetically by cloning V-gene segments prior to rearrangement from stem cells and using PCR primers that encode the hypervariable CDR3 region and contain random sequences to achieve rearrangement in vitro. Patent documents describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0189] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0190] 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is to C1s and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of C1s. A bispecific antibody may be used to localize a cytotoxic agent to cells expressing C1s. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0191] Methods for producing multispecific antibodies are not limited to these, but include, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies are produced by manipulating the electrostatic steering effect to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science 229: 81 (1985)); creating antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1994)). They may also be prepared by (see reference) and by preparing a trispecific antibody as described, for example, in Tutt et al., J. Immunol. 147: 60 (1991).

[0192] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576 A1).

[0193] In this specification, an antibody or fragment also includes a “dual-acting Fab” or “DAF” which includes one antigen-binding site that binds to C1s and another different antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0194] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are also considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding ability).

[0195] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1 under the heading "Exemplary Substitutions" and are described in detail below, with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen-binding, reduced immunogenicity, or improved ADCC or CDC.

[0196] [Table 1]

[0197] Amino acids can be grouped according to their common side-chain characteristics: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: Aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: Tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitution refers to replacing one member of one class with one of another.

[0198] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants, and those selected for further testing, will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-matured antibody, which can be appropriately produced using, for example, a phage display-based affinity-mature technique (e.g., one described herein). Briefly, one or more HVR residues are mutated, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0199] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may be tested for binding affinity. Affinity maturation by construction and re-selection from secondary libraries is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is prepared. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0200] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

[0201] A useful method for identifying antibody residues or regions that can be targeted for mutational introduction is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989), Science 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues, e.g., arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants may be screened to determine whether they possess the desired properties.

[0202] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues into a sequence, but also the fusion of polypeptides ranging in length from one to over 100 residues at the amino and / or carboxyl terminals. An example of terminal insertion is an antibody with a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0203] b) Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0204] If the antibody comprises an Fc region, the carbohydrates added thereto may be modified. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are usually attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and also include fucose attached to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention may be performed to create antibody variants with certain improved properties.

[0205] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0206] Further antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0207] c) Fc region mutant (Sweeping technique) In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc region is the Fc region of human IgG1.

[0208] To facilitate a decrease in plasma antigen concentrations and / or improve the pharmacokinetics of antibodies, amino acid residues at the binding site to FcRn within the Fc region of IgG can be modified to promote their uptake into cells. When a pH-dependent antibody is modified in this way, the variant may be a “sweeping” antibody that binds more strongly to FcRn and can efficiently transport and degrade the antigen into endosomes (where pH is acidic), while being more efficiently recycled to the cell surface itself. Such a modified “sweeping” antibody may bind more strongly to FcRn on the cell surface at neutral pH and improve antigen uptake and degradation compared to the original (parent) antibody without the modification (Semin Immunopathol. 2018; 40(1): 125-140).

[0209] In some aspects, the antibody includes an Fc region having at least one amino acid modification within the Fc region to promote a decrease in plasma antigen concentration and / or improve the pharmacokinetics of the antibody.

[0210] In some embodiments, the Fc region is a human Fc region whose binding activity to the active Fcγ receptor is stronger than that of the Fc region of native human IgG1. For example, as mentioned in WO 2013 / 047752, to improve the binding activity to the active Fcγ receptor, the Fc regions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 24 7, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 2 94, 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 One or more amino acids selected from the group consisting of amino acids at positions 379, 380, 382, ​​385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 (EU numbering) may be modified to differ from the amino acids at the corresponding sites within the Fc region of the parent (original) antibody, native human IgG1.

[0211] In some embodiments, the Fc region is a human Fc region whose binding activity to the inhibitory Fcγ receptor is stronger than its binding activity to the active Fcγ receptor. For example, as mentioned in WO 2013 / 125667, in order to improve the binding activity to the inhibitory Fcγ receptor, the Fc regions 244, 245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, One or more amino acids selected from the group consisting of amino acids at positions 356, 360, 362, 375, 376, 377, 378, 380, 382, ​​385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU numbering) may be modified to differ from the amino acids at the corresponding sites within the Fc region of natural human IgG1.

[0212] In some embodiments, the Fc region is a human Fc region whose binding activity to FcRn at neutral pH is stronger than that of the Fc region of native human IgG1. For example, as mentioned in WO 2011 / 122011, to improve the binding activity to FcRn at neutral pH, the Fc regions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 3 One or more amino acids selected from the group consisting of amino acids at positions 15, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) may be modified to differ from the amino acids at the corresponding sites within the Fc region of natural human IgG1.

[0213] In certain embodiments, antibody variants possessing some, but not all, effector functions are also within consideration of the present invention, such effector functions making the antibody a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent No. 5,821,337 (see Bruggemann M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, the ACT1® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg MS et al., Blood 101:1045-1052 (2003); and Cragg MS and MJ Glennie et al., Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, Petkova SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0214] Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581) with alanine substitutions at residues 265 and 297, and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0215] Certain antibody variants exhibiting improved or decreased binding affinity to FcR have been described. (See U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0216] In certain embodiments, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in EU numbering) of the Fc region).

[0217] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or reduced) C1q binding and / or complement-dependent cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0218] Antibodies with increased half-life and improved binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994))) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve its binding affinity to FcRn. Such Fc variants include those involving 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 (for example, substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0219] d) Cysteine-modified antibody variant In certain embodiments, it would be desirable to produce a cysteine-modified antibody (e.g., "thioMAb") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in an accessible site of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. The cysteine-modified antibody may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0220] e) antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0221] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. 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 may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

[0222] B. Method and configuration of rearrangement For example, as described in U.S. Patent No. 4,816,567, antibodies can be manufactured using recombinant methods or compositions. In one embodiment, an isolated nucleic acid encoding the anti-C1s antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, SP2 / 0 cells). In one embodiment, a method for producing an anti-C1s antibody is provided, comprising culturing host cells containing the nucleic acid encoding the antibody as described above under conditions suitable for the expression of the anti-C1s antibody, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0223] For the recombinant production of anti-C1s antibodies, nucleic acids encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0224] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste into soluble fractions and further purified.

[0225] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0226] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0227] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0228] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Examples include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include DHFR -This includes Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0229] pH-dependent antibodies may be obtained by using screening and / or mutagenesis methods, such as those described in WO 2009 / 125825. The screening method may include any process for identifying antibodies with pH-dependent binding properties from a population of antibodies specific to a particular antigen. In certain embodiments, the screening method may include measuring one or more binding parameters (e.g., KD or kd) of individual antibodies in an initial antibody population at both acidic and neutral pH. The antibody binding parameters may be measured using, for example, surface plasmon resonance, or any other analytical method that allows for quantitative or qualitative evaluation of the antibody's binding properties to a particular antigen. In certain embodiments, the screening method may include identifying antibodies that bind to an antigen at an acidic KD / neutral KD ratio of 2 or more. Alternatively, the screening method may include identifying antibodies that bind to an antigen at an acidic kd / neutral kd ratio of 2 or more.

[0230] In another embodiment, a mutagenesis method may involve incorporating amino acid deletions, substitutions, or additions within the heavy and / or light chains of an antibody to enhance the pH-dependent binding affinity of the antibody to an antigen. In a particular embodiment, mutagenesis may occur within one or more variable domains of the antibody, for example, within one or more HVRs (e.g., CDRs). For example, mutagenesis may involve substituting an amino acid within one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In a particular embodiment, mutagenesis may involve substituting one or more amino acids within at least one HVR (e.g., CDR) of the antibody with histidine. In a particular embodiment, “enhanced pH-dependent binding” means that the mutant antibody exhibits a larger acid-KD / neutral-KD ratio or a larger acid-kD / neutral-kD ratio than the original “parent” antibody before mutagenesis (i.e., the antibody with low pH dependence). In a particular embodiment, the mutant antibody has an acid-KD / neutral-KD ratio of 2 or more. Or, the mutant antibody has an acid-kD / neutral-kD ratio of 2 or more.

[0231] Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is converted to an immunogenic protein in the immunized species, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy trypsin inhibitor, and a bifunctional substance or derivatizer, for example, maleimide benzoyl sulfosuccinimide (conjugation via cysteine ​​residue), N-hydroxysuccinimide (conjugation via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR(where R and R 1 It may be useful to conjugate them using different alkyl groups.

[0232] Animals (usually non-human mammals) are immunized to an antigen, immunogenic conjugate, or derivative by intradermal injection at multiple sites of a solution containing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) combined with three times the volume of Freund's complete adjuvant. After one month, the animals are boost-immunized with 1 / 5 to 1 / 10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, blood is collected from the animals and the serum is assayed for antibody titer. The animals are boost-immunized until the titer reaches a plateau. Preferably, the animals are boost-immunized with conjugates that are the same antigen but conjugated to a different protein and / or conjugated via a different crosslinking reagent. The conjugates can also be prepared as protein fusions in recombinant cell cultures. Coagulants such as alum are also preferably used to enhance the immune response.

[0233] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for some naturally occurring potential mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates a characteristic of the antibody that it is not a mixture of distinct antibodies.

[0234] For example, monoclonal antibodies can be produced using the hybridoma method, first described in Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as described herein to induce lymphocytes capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0235] Immunotherapeutic agents typically include antigen proteins or their fusion variants. Generally, peripheral blood lymphocytes (PBLs) are used when human-derived cells are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with immortalized cell lines using appropriate fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0236] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus produced are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the proliferation or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas would typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the proliferation of HGPRT-deficient cells.

[0237] Preferred immortalized myeloma cells are those that efficiently fuse, facilitate stable and high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Among these, mouse myeloma strains, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (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)).

[0238] The culture medium in which hybridoma cells are proliferating is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by 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 as described in Munson, Anal Biochem. 107(1):220-239 (1980).

[0239] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, these clones can be subcloned by limiting dilution and grown using standard methods (Goding, cited above). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0240] Monoclonal antibodies secreted by subclones can be appropriately isolated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0241] C. Measurement Method (Assay) The anti-C1s antibodies provided herein may be identified, screened, or elucidated for their physical / chemical properties and / or biological activity by various assay methods known in the art.

[0242] 1. Combined measurement methods and other measurement methods In one aspect, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0243] In another context, a competitive assay may be used to identify antibodies that compete with any of the anti-C1s antibodies described herein in terms of 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, if such a competitive antibody is present in excess, it will block (e.g., reduce) the binding of the 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 competitive antibody will bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-C1s antibodies described herein. Detailed exemplary methods for mapping antibody-bound epitopes are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In certain embodiments, such competitive assays can be performed under neutral pH conditions. In some embodiments, competitive assays can be performed, for example, in Octet TM This is a tandem competitive assay using a system.

[0244] In an exemplary competition assay, immobilized C1s are incubated in a solution containing a first labeled antibody that binds to C1s (e.g., one of those described herein) and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to C1s. The second antibody may be present in the hybridoma supernatant. As a control, immobilized C1s are incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to C1s, any excess unbound antibody is removed and the amount of label bound to the immobilized C1s is measured. If the amount of label bound to the immobilized C1s is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing 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).

[0245] In another context, antibodies that bind to the same epitope as the anti-C1s antibody provided herein, or that compete with the anti-C1s antibody provided herein for binding to C1s, can be identified using a sandwich assay. A sandwich assay involves using two antibodies, each of which can bind to a different immunogenic moiety or epitope of the protein to be detected. In a sandwich assay, a test sample analyte is bound to a first antibody immobilized on a solid support, followed by the binding of a second antibody to the analyte, thereby forming an insoluble ternary complex. See David & Greene, U.S. Patent No. 4,376,110. The second antibody may be labeled with a detectable moiety itself (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. An antibody that binds to C1s simultaneously with the anti-C1s antibody provided herein can be determined to be an antibody that binds to a different epitope than the anti-C1s antibody. Therefore, an antibody that does not bind to C1s simultaneously with the anti-C1s antibody provided herein may 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.

[0246] 2. Activity assay In one aspect, an assay is provided for identifying anti-C1s antibodies with biological activity. This biological activity may include inhibition of the activation of the classical pathway and the inhibition of the production of cleavage products resulting from the activation of said pathway: C2a, C2b, C3a, C3b, C4a, C4b, C5a, and C5b. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0247] In certain embodiments, the antibodies of the present invention are tested for such biological activity. In some embodiments, the antibodies of the present invention can be evaluated for their ability to inhibit complement-mediated hemolysis of sheep erythrocytes (RBCs) sensitized with an antibody against sheep erythrocyte antigen, i.e., by using an RBC assay. In some embodiments, the antibodies of the present invention can be evaluated for their ability to inhibit complement-mediated hemolysis of chicken erythrocytes (cRBCs) sensitized with an antibody against chicken erythrocyte antigen. 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 hemoglobin released by spectrophotometric analysis.

[0248] The RBC assay can be adequately performed using known methods, e.g., the method disclosed in J. Vis. Exp. 2010; (37): 1923. This document describes a method for performing a 50% hemolytic complement (CH50) assay as an RBC lysis assay. Briefly, this assay measures the activation of the classical complement pathway and detects a decrease, absence, or inactivity of any component of that pathway. It assesses the erythrolytic activity of complement components in serum. When an antibody is incubated with test serum, this pathway is activated, and hemolysis is induced. A decrease in one or more components of the classical pathway results in a decrease in the CH50 value. While the CH50 assay is not entirely identical to the assay used in the examples herein to measure the percentage of inhibition of cell lysis by complement components, the concept and basic configuration are substantially identical to those of the present invention. In one embodiment of the present invention, the RBC assay is performed as follows: Human serum is pre-incubated with the antibody of interest (e.g., 37 degrees Celsius (°C) for 3 hours). Next, this serum is added to an equal volume of sensitized sheep erythrocytes and incubated to lyse the erythrocytes (e.g., 1 hour at 37°C). Then the reaction is stopped. The mixture is centrifuged to pellet the unlysed cells, the supernatant is taken out, and hemoglobin release is analyzed using the absorbance (OD) at 415 nm minus the OD at 630 nm. To calculate the erythrocyte lysis inhibition rate (%), 0% inhibition is set as the condition with no antibody added (buffer only), and 100% inhibition is set as the condition with the addition of EDTA at a final concentration of 5 mM (see, for example, Example 7). When an antibody shows a certain erythrocyte lysis inhibition rate (%), this means that the antibody has neutralizing activity against human serum complement, for example, activity to inhibit the interaction between the C1q and C1r2s2 complex.

[0249] Thus, to evaluate the activity of inhibiting the interaction between C1q and C1r2s2 complexes, the neutralizing activity of the antibody of the present invention against human serum complement can be evaluated using an RBC assay. In one embodiment, the present invention provides an isolated antibody that inhibits the interaction between C1q and C1r2s2 complexes, having neutralizing activity against at least 70% of human serum complement in an RBC assay.

[0250] D. immunoconjugate The present invention also provides an immunoconjugate comprising one or more cytotoxic agents (e.g., chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof) or radioisotopes) conjugated with the anti-C1s antibodies of this specification.

[0251] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, the following: These include: meitansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235B1); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calicheamycin or its derivatives (see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342) (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and see U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0252] In another aspect, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to: diphtheria A chain, 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 protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene.

[0253] In another aspect, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples are 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioisotopes of Pb, and Lu. When used for detection, the radioactive conjugate may contain a radioactive atom for scintigraphy (e.g., Tc-99m or 123 I), or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI) (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron).

[0254] Antibody and cytotoxic agent conjugates can be prepared using a variety of bifunctional protein conjugates. Examples include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0255] The immunoconjugates or ADCs described herein expressly consider conjugates prepared using crosslinking 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, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0256] E. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-C1s antibodies provided herein are useful for detecting the presence of C1s in a biological sample. As used herein, the term “detection” encompasses quantitative or qualitative detection. In certain embodiments, the biological sample 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, breast milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ophthalmic lens solution, or mucus.

[0257] In one embodiment, an anti-C1s antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of C1s in a biological sample is provided. In a particular embodiment, the method comprises contacting a biological sample with the anti-C1s antibody described herein under conditions in which binding of the anti-C1s antibody to C1s is permitted, and detecting whether a complex has been 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 using the anti-C1s antibody, for example, when C1s is a biomarker for patient selection.

[0258] Examples of disorders that can be diagnosed using the antibodies of the present invention include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic granule dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behçet's disease, British-type amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemia vasculitis, boxer's dementia, Lewy body dementia (DLB), diffuse neurofibrillary tangle disease with calcification, discoid lupus erythematosus, Down syndrome, and focal segmental glomeruloscopy. Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Streussler-Scheinker syndrome, Guillain-Barré syndrome, Haller-Holden-Spats syndrome, hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g., bacteria (e.g., meningococcus or streptococcus), viruses (e.g., Diseases caused by human immunodeficiency virus (HIV) or other infectious pathogens, inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, polyinfarct dementia, lupus (systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy - Multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam type motor neuron disease presenting with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, post-encephalitis parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (E. coli).This includes, but is not limited to, *Celluloscolon* (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, graft rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, acquired epidermolysis bullosa, delayed hemolytic transfusion reaction, hypocomplementemia-induced urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractory conditions.

[0259] In certain embodiments, labeled anti-C1s antibodies are provided. Labeling includes, but is not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, high electron density labels, chemiluminescent labels, and radioactive labels) as well as moieties indirectly detectable through, for example, enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands). Exemplary labels, but are not limited to, include: radioisotopes. 32 P, 14 C, 125 I, 3 H and 131I, fluorescent phosphopoides such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, luciferases such as dansyl, umbelliferone, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), enzymes linked to oxidizing pigment precursors with hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, and similar substances.

[0260] F. Pharmaceutical preparations The pharmaceutical formulations of anti-C1s antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing an antibody of the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and Examples of pharmaceutically acceptable carriers herein include: m-cresol, etc.; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP) (e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Specific exemplary sHASEGPs and their uses (including rHuPH20) are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0261] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0262] The formulations described herein may contain more than one active ingredient if necessary for the specific indication being treated. Preferably, these ingredients have complementary activities that do not adversely affect each other. For example, it may be desirable to provide further formulations for use in combination therapy. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0263] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, a droplet formation (coacervation) technique or interfacial polymerization, or into a colloidal drug delivery system (e.g., liposomes, albumin spheres, microemulsions, nanoparticles, and nanocapsules), or into a macroemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0264] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0265] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0266] G. Therapeutic methods and therapeutic compositions Any of the anti-C1s antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-C1s antibody is provided for use as a pharmaceutical. In a further aspect, an anti-C1s antibody is provided for use in the treatment of complement-mediated diseases or disorders. In a particular embodiment, an anti-C1s antibody is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-C1s antibody for use in a method for treating an individual having a complement-mediated disease or disorder, the method comprising the step of administering an effective amount of an anti-C1s antibody to the individual. In one such embodiment, the method further comprises the step of administering an effective amount of at least one additional therapeutic agent to the individual. In any of the above embodiments, the “individual” is preferably a human.

[0267] In a further embodiment, the present invention provides an anti-C1s antibody for use in the treatment of complement-mediated diseases or disorders. In a further embodiment, the anti-C1s antibody of the present invention may be used for enhancing the clearance of C1s from plasma. In a further embodiment, the anti-C1s antibody of the present invention may be used for enhancing the clearance of C1r2s2 from plasma. In a further embodiment, the anti-C1s antibody of the present invention may be used to enhance the clearance of C1r2s2 from plasma without enhancing the clearance of C1q from plasma. In some examples, the antibody inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s. In a particular embodiment, the present invention provides an anti-C1s antibody for use in a method for treating complement-mediated diseases or disorders. In a particular embodiment, the present invention provides an anti-C1s antibody for use in a method for enhancing the clearance of C1s from plasma. In a particular embodiment, 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 enhancing the clearance of C1r2s2 from plasma without enhancing the clearance of C1q from plasma. In certain embodiments, the present invention provides an anti-C1s antibody for use in a method of inhibiting a component of the classical complement pathway, where in some examples the component of the classical complement pathway is C1s. In any of the above embodiments, “individual” is preferably human.

[0268] In one aspect, the Disclosure provides a method for modulating complement activation. In some embodiments, the method inhibits complement activation, for example, by reducing the production of C4b2a. In some embodiments, the Disclosure provides a method for modulating complement activation in an individual having a complement-mediated disease or disorder, comprising the step of administering to the individual an anti-C1s antibody of the Disclosure or a pharmaceutical composition of the Disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of the 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 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.

[0269] Complement-mediated disorders are disorders characterized by abnormal amounts of complement C1s or abnormal levels of complement C1s proteolytic activity in the cells, tissues, or fluids of an individual.

[0270] In some cases, complement-mediated diseases or disorders are characterized by the presence of increased (more than normal) amounts of C1s or elevated levels of complement C1s activity in cells, tissues, or fluids. For example, in some cases, complement-mediated diseases or disorders are characterized by the presence of increased amounts of C1s and / or elevated levels of C1s activity in brain tissue and / or cerebrospinal fluid. An "more than normal" amount of C1s in cells, tissues, or fluids indicates that the amount of C1s in those cells, tissues, or fluids is greater than the normal control level, e.g., greater than the normal control level for individuals or populations of the same age group. A "higher than normal" level of C1s activity in cells, tissues, or fluids indicates that the proteolytic cleavage mediated by C1s in those cells, tissues, or fluids is higher than the normal control level, e.g., greater than the normal control level for individuals or populations of the same age group. In some cases, individuals with complement-mediated diseases or disorders exhibit one or more further symptoms of such diseases or disorders.

[0271] In other cases, complement-mediated diseases or disorders are characterized by the presence of less-than-normal amounts of C1s or lower levels of complement C1s activity in cells, tissues, or fluids. For example, in some cases, complement-mediated diseases or disorders are characterized by the presence of less-than-normal amounts of C1s and / or C1s with lower activity in brain tissue and / or cerebrospinal fluid. A “less-than-normal” amount of C1s in cells, tissues, or fluids indicates that the amount of C1s in those cells, tissues, or fluids is lower than the normal control level, e.g., lower than the normal control level for individuals or populations of the same age group. A “lower-than-normal” level of C1s activity in cells, tissues, or fluids indicates that the proteolytic cleavage mediated by C1s in those cells, tissues, or fluids is lower than the normal control level, e.g., lower than the normal control level for individuals or populations of the same age group. In some cases, individuals with complement-mediated diseases or disorders exhibit one or more further symptoms of such diseases or disorders.

[0272] Complement-mediated diseases or disorders are diseases or disorders in which the amount or activity of complement C1s causes disease or disorder in an individual. In some embodiments, complement-mediated diseases or disorders are selected from the group consisting of autoimmune diseases, cancer, hematological diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, ocular diseases, kidney diseases, graft rejection, vascular diseases, and vasculitis. In some embodiments, complement-mediated diseases or disorders are autoimmune diseases. In some embodiments, complement-mediated diseases or disorders are cancer. In some embodiments, complement-mediated diseases or disorders are infectious diseases. In some embodiments, complement-mediated diseases or disorders are inflammatory diseases. In some embodiments, complement-mediated diseases or disorders are hematological diseases. In some embodiments, complement-mediated diseases or disorders are ischemia-reperfusion diseases. In some embodiments, complement-mediated diseases or disorders are ocular diseases. In some embodiments, complement-mediated diseases or disorders are kidney diseases. In some aspects, a complement-mediated disease or disorder is graft rejection. In some aspects, a complement-mediated disease or disorder is antibody-induced graft rejection. In some aspects, a complement-mediated disease or disorder is a vascular disease. In some aspects, a complement-mediated disease or disorder is a vasculitic disease. In some aspects, a complement-mediated disease or disorder is a neurodegenerative disease or disorder. In some aspects, a complement-mediated disease is a neurodegenerative disease. In some aspects, a complement-mediated disorder is a neurodegenerative disorder. In some aspects, a complement-mediated disease or disorder is a tauropathy.

[0273] Examples of complement-mediated diseases or disorders include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, and Barraquer-Simons syndrome. Syndrome, Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemia, boxer's dementia, Lewy body dementia (DLB), diffuse neurofibrillary tangle disease with calcification, discoid lupus erythematosus, Down syndrome, focal segmental glomeruloscopy Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Streussler-Scheinker syndrome, Guillain-Barré syndrome, Haller-Holden-Spats syndrome, hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g., bacteria (e.g., meningococcus or streptococcus), viruses (e.g., Diseases caused by human immunodeficiency virus (HIV) or other infectious pathogens, inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, polyinfarct dementia, lupus (systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy - Multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam type motor neuron disease presenting with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, post-encephalitis parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (E. coli).This includes, but is not limited to, *Celluloscolon* (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, graft rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, acquired epidermolysis bullosa, delayed hemolytic transfusion reaction, hypocomplementemia-induced urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion refractory conditions.

[0274] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia linked to chromosome 17 with parkinsonism) are the most common forms of tauropathy. Therefore, this invention relates to any of the above-mentioned ways in which tauropathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia linked to chromosome 17 with parkinsonism. Other tauropathy includes, but is not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

[0275] Neurodegenerative tauropathy includes Alzheimer's disease, amyotrophic lateral sclerosis / Parkinsonian dementia complex, argyrophilic grain dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, boxer's dementia, diffuse neurofibrillary tangle disease with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, and Gerstmann-Streussler-Scheinker syndrome. The group includes Haller-Holden-Spats syndrome, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam type motor neuron disease presenting with neurofibrillary tangles, Pick's disease, post-encephalitis-Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, neurofibrillary dementia, polyinfarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0276] This disclosure also provides methods for treating synucleinopathies, such as Parkinson's disease (PD); Lewy body dementia (DLB); and multiple system atrophy (MSA). For example, PD with dementia (PDD) may be treated using the methods of this disclosure.

[0277] In some embodiments, complement-mediated diseases or disorders include Alzheimer's disease. In some embodiments, complement-mediated diseases or disorders include Parkinson's disease. In some embodiments, complement-mediated diseases or disorders include graft rejection. In some embodiments, complement-mediated diseases or disorders include antibody-mediated graft rejection.

[0278] In some embodiments, the anti-C1s antibodies of this disclosure prevent or delay the onset of at least one symptom of complement-mediated disease or disorder in an individual. In some embodiments, the anti-C1s antibodies of this disclosure mitigate or eliminate at least one symptom of complement-mediated disease or disorder in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, hematological disorders, infectious diseases, inflammatory diseases, ischemia-reperfusion diseases, neurodegenerative diseases, neurodegenerative disorders, kidney diseases, graft rejection, eye diseases, vascular diseases, or vasculitis. Symptoms may also be neurological symptoms, such as cognitive impairment, memory impairment, or motor function loss. Symptoms may also be the activity of the C1s protein in the cells, tissues, or fluids of an individual. Symptoms may also be the degree of complement activation in the cells, tissues, or fluids of an individual.

[0279] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual modulates complement activation in the individual's cells, tissues, or body fluids. In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual inhibits complement activation in the individual's cells, tissues, or body fluids. For example, in some embodiments, when the anti-C1s antibody of this disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, it inhibits complement activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to complement activation in the individual before treatment with the anti-C1s antibody.

[0280] In some embodiments, the anti-C1s antibody of the present disclosure reduces C3 deposition on erythrocytes, for example, in some embodiments, the anti-C1s antibody of the present disclosure reduces the deposition of C3b, iC3b, etc., on RBCs. In some embodiments, the anti-C1s antibody of the present disclosure inhibits complement-mediated erythrocyte lysis.

[0281] In some embodiments, the anti-C1s antibody of the present disclosure reduces the deposition of C3 into platelets. For example, in some embodiments, the anti-C1s antibody of the present disclosure reduces the deposition of C3b, iC3b, etc., into platelets.

[0282] In some embodiments, administration of the anti-C1s antibody of this disclosure results in outcomes selected from the following group: (a) reduced complement activation, (b) improved cognitive function, (c) reduced neuronal loss, (d) reduced phosphorylated Tau levels in neurons, (e) reduced glial cell activation, (f) reduced lymphocyte infiltration, (g) reduced macrophage infiltration, (h) reduced antibody deposition, (i) reduced glial cell loss, (j) reduced oligodendrocyte loss, (k) reduced dendritic cell infiltration, (l) reduced neutrophil infiltration, (m) reduced erythrolysis, (n) reduced red blood (o) Reduced cytophagocytosis, (p) Reduced platelet phagocytosis, (q) Improved graft survival, (r) Reduced macrophage-mediated phagocytosis, (s) Improved visual acuity, (t) Improved motor control, (u) Improved thrombus formation, (v) Improved coagulation, (w) Improved renal function, (x) Reduced antibody-mediated complement activation, (y) Reduced autoantibody-mediated complement activation, (z) Improved anemia, (aa) Reduced demyelination, (ab) Reduced eosinophilia, (ac) Reduced C3 deposition in erythrocytes (e.g., reduced deposition of C3b, iC3b, etc. in RBCs), (ad) Reduced C3 deposition in platelets (e.g., reduced deposition of C3b, iC3b, etc. in platelets), and (ae) Reduced anaphylatoxin production, (af) Reduced blister formation mediated by autoantibodies, (ag) Reduced itching induced by autoantibodies, (ah) Reduced lupus erythematosus induced by autoantibodies, (ai) Reduced skin erosion mediated by autoantibodies, (aj) Reduced erythrocyte destruction due to transfusion reactions, (ak) Reduced erythrocyte lysis due to alloantibodies, (al) Reduced hemolysis due to transfusion reactions, (am) Alloantibodies (a) Reduction of platelet lysis, (a) Reduction of platelet lysis due to transfusion reaction, (ao) Reduction of mast cell activation, (ap) Reduction of mast cell histamine release, (aq) Reduction of vascular permeability, (ar) Reduction of edema, (as) Reduction of complement deposition in graft endothelium, (at) Reduction of anaphylatoxin production in graft endothelium, (au) Reduction of dermal-epidermal junction separation, (av) Reduction of anaphylatoxin production at the dermal-epidermal junction, (aw) Reduction of alloantibody-mediated complement activation in graft endothelium, (ax) Reduction of antibody-mediated neuromuscular junction loss,(ay) Reduced complement activation at the neuromuscular junction, (az) Reduced anaphylatoxin production at the neuromuscular junction, (ba) Reduced complement deposition at the neuromuscular junction, (bb) Reduced paralysis, (be) Reduced numbness, (bd) Improved bladder control, (be) Improved bowel management, (bf) Reduced autoantibody-related deaths, and (bg) Reduced autoantibody-related pathological conditions.

[0283] In some embodiments, when the anti-C1s antibody of this disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual with complement-mediated disease or disorder, the following outcomes may occur: (a) complement activation; (b) cognitive decline; (c) neuronal loss; (d) phosphorylated Tau levels in neurons; (e) glial cell activation; (f) lymphocyte infiltration; (g) macrophage infiltration; (h) antibody deposition; (i) glial cell loss; (j) oligodendrocyte loss; (k) dendritic cell infiltration; (l) neutrophil infiltration; (m) erythrolysis; (n) erythrocyte phagocytosis; (o) platelet phagocytosis; (p) platelet lysis; It is effective in reducing one or more of the following by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level or degree of the outcome in the individual before treatment with anti-C1s antibody.

[0284] In some embodiments, the anti-C1s antibodies of this disclosure, when administered in one or more doses as monotherapy or in combination therapy to individuals with complement-mediated disease or disorder, are effective in improving one or more of the following outcomes: a) cognitive function; b) graft survival; c) visual acuity; d) motor control; e) thrombus formation; f) coagulation; g) renal function; and h) hematocrit (red blood cell count) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level or degree of the outcome in the individual before treatment with the anti-C1s antibodies.

[0285] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses to an individual with a complement-mediated disease or disorder, either as monotherapy or in combination therapy, it reduces complement activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to complement activation in the individual before treatment with the anti-C1s antibody.

[0286] In some embodiments, administration of the anti-C1s antibody of the Disclosure improves cognitive function in an individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, it improves cognitive function in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to cognitive function in the individual before treatment with the anti-C1s antibody.

[0287] In some embodiments, administration of the anti-C1s antibody of the Disclosure reduces the rate of cognitive decline in an individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, it reduces the rate of cognitive decline in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the rate of cognitive decline in the individual before treatment with the anti-C1s antibody.

[0288] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces neuronal loss in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual having a complement-mediated disease or disorder, it reduces neuronal loss in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to neuronal loss in the individual before treatment with the anti-C1s antibody.

[0289] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces the phosphorylated Tau level in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual having a complement-mediated disease or disorder, it reduces the phosphorylated Tau level in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the phosphorylated Tau level in the individual before treatment with the anti-C1s antibody.

[0290] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces glial cell activation in the individual. For example, in some embodiments, when the anti-C1s antibody of this disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual having a complement-mediated disease or disorder, it reduces glial activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to glial cell activation in the individual before treatment with the anti-C1s antibody. In some embodiments, the glial cells are astrocytes or microglia.

[0291] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces lymphocyte infiltration in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual having a complement-mediated disease or disorder, it reduces lymphocyte infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to lymphocyte infiltration in the individual before treatment with the anti-C1s antibody.

[0292] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces macrophage infiltration in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses as monotherapy or in combination therapy to an individual having a complement-mediated disease or disorder, it reduces macrophage infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to macrophage infiltration in the individual before treatment with the anti-C1s antibody.

[0293] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses to an individual having a complement-mediated disease or disorder, either as monotherapy or in combination therapy, it reduces antibody deposition in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to antibody deposition in the individual before treatment with the anti-C1s antibody.

[0294] In some embodiments, administration of the anti-C1s antibody of the Disclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a) production in the individual. For example, in some embodiments, when the anti-C1s antibody of the Disclosure is administered in one or more doses to an individual having a complement-mediated disease or disorder, either as monotherapy or in combination therapy, it reduces anaphylatoxin production in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the anaphylatoxin production level in the individual before treatment with the anti-C1s antibody.

[0295] In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure or a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for treating individuals with complement-mediated diseases or disorders. In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure for treating individuals with complement-mediated diseases or disorders. In some embodiments, the Disclosure provides the use of a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for treating individuals with complement-mediated diseases or disorders.

[0296] In some embodiments, the present disclosure provides the use of the anti-C1s antibody of the present disclosure in the manufacture of a pharmaceutical product for treating an individual having a complement-mediated disease or disorder.

[0297] In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure or a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation. In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure or a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the Disclosure provides the use of a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder.

[0298] In some embodiments, the Disclosure provides the use of the anti-C1s antibody of the Disclosure in the manufacture of a pharmaceutical product for modulating complement activation. In some embodiments, the pharmaceutical product inhibits complement activation. In some embodiments, the pharmaceutical product inhibits complement activation in an individual having a complement-mediated disease or disorder.

[0299] In some embodiments, the Disclosure provides a pharmaceutical composition for use in pharmacotherapy comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient. In some embodiments, the Disclosure provides an anti-C1s antibody of the Disclosure for use in pharmacotherapy. In some embodiments, the Disclosure provides a pharmaceutical composition for use in pharmacotherapy comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient.

[0300] In some embodiments, the Disclosure provides an anti-C1s antibody of the Disclosure or a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder. In some embodiments, the Disclosure provides an anti-C1s antibody of the Disclosure for treating an individual having a complement-mediated disease or disorder. In some embodiments, the Disclosure provides a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder.

[0301] In some embodiments, the Disclosure provides a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure or the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the Disclosure provides an anti-C1s antibody of the Disclosure for modulating complement activation. In some embodiments, the Disclosure provides a pharmaceutical composition comprising the anti-C1s antibody of the Disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the anti-C1s antibody inhibits complement activation.

[0302] In a further aspect, the present invention provides the use of anti-C1s antibodies in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of complement-mediated diseases or disorders. In a further embodiment, the pharmaceutical is for use in a method for treating complement-mediated diseases or disorders, comprising the step of administering an effective amount of the pharmaceutical to an individual having a complement-mediated disease or disorder. In one such embodiment, the method further comprises the step of administering an effective amount of at least one further therapeutic agent (e.g., one described below) to the individual. In a further embodiment, the pharmaceutical is for use to enhance the clearance of C1s from plasma (or remove C1s from plasma). In a further embodiment, the pharmaceutical is for use to enhance the clearance of C1r2s2 from plasma (or remove C1r2s2 from plasma). In a further embodiment, the pharmaceutical is for use to enhance the clearance of C1r2s2 from plasma (or remove C1r2s2 from plasma) without enhancing the clearance of C1q from plasma (or removing C1q from plasma). In a further embodiment, the pharmaceutical is intended for use in inhibiting components of the classical complement pathway, in some examples, the components of the classical complement pathway being C1s.

[0303] In a further embodiment, the pharmaceutical is intended for use in a method for treating an individual having a complement-mediated disease or disorder, comprising the step of administering an effective amount of the pharmaceutical to the individual. In any of the above embodiments, “individual” may be a human.

[0304] In a further aspect, the present invention provides a method for treating complement-mediated diseases or disorders. In one embodiment, the method comprises administering an effective amount of anti-C1s antibody to an individual having such a complement-mediated disease or disorder. In one such embodiment, the method further comprises administering an effective amount of at least one further therapeutic agent (described below) to the individual. In any of the above embodiments, “individual” may be a human.

[0305] In a further aspect, the present invention provides a method for enhancing the clearance of C1s from plasma (or removing C1s from plasma) in an individual. In a further aspect, the present invention provides a method for enhancing the clearance of C1r2s2 from plasma (or removing C1r2s2 from plasma) in an individual. In a further embodiment, the present invention provides a method for enhancing the clearance of C1r2s2 from plasma (or removing C1r2s2 from plasma) without enhancing the clearance of C1q from plasma (or removing C1q from plasma) in an individual. In some examples, the present invention provides a method for inhibiting a component of the classical complement pathway in an individual, and in some examples, the component of the classical complement pathway is C1s. In one embodiment, “individual” is a human.

[0306] In a further aspect, the present invention provides a pharmaceutical formulation comprising one of the anti-C1s antibodies provided herein for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation comprises one of the anti-C1s antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises one of the anti-C1s antibodies provided herein and at least one further therapeutic agent (e.g., one described below).

[0307] The antibodies of the present invention can be used in therapy either alone or in combination with other agents. For example, the antibodies of the present invention may be administered concurrently with at least one additional therapeutic agent.

[0308] The combination therapies described above include combination administration (two or more therapeutic agents contained in the same or separate formulations) and individual administration, in which case the antibody of the present invention may be administered prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent. In one embodiment, the administration of the anti-C1s antibody and the administration of the additional therapeutic agent are performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The antibody of the present invention can be used in combination with radiotherapy.

[0309] The antibodies (and any additional therapeutic agents) of the present invention may be administered by any preferred means, including parenteral administration, intrapulmonary administration, and nasal administration, and, if desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be made by any preferred route, such as by injection, including intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various administration schedules, including single doses, repeated doses over various time points, bolus administration, and pulse infusion, are within consideration herein, but are not limited to these.

[0310] The antibodies of the present invention are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered from this perspective include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to healthcare professionals. The antibodies are formulated, optionally but not necessarily, with one or more agents already in use to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are typically used in the same doses and routes of administration as described herein, or at about 1 to 99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

[0311] For the prevention or treatment of a disease, the appropriate dose of the antibody of the present invention (when used alone or with one or more other additional therapeutic agents) will depend on the type o...

Claims

[Claim 1] The invention described herein.

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