Anti-c1s antibodies and uses thereof

Abstract

The present application relates to anti-C1S antibodies and uses thereof. Provided are antibodies and antigen-binding fragments thereof that bind to C1s and inhibit C1s activity and modulate the activity of at least one component of the classical pathway (CP) of complement activation; and methods of treating complement-mediated disorders using the anti-C1s antibodies and fragments.

Description

[0001] This application is a divisional application of the invention patent application with the application date of June 28, 2022, application number "202280045741.4", and name "Anti-C1S antibodies and their uses".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 342,544, filed on May 16, 2022, and U.S. Provisional Application No. 63 / 221,131, filed on July 13, 2021, each of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] The parent application of this divisional application submitted a sequence listing text file named SEQ_LIST_PIPL_1121-102PCT.txt with a size of 279,262 bytes, created on June 28, 2022. This divisional application submitted a sequence listing in ST.26 format. Technical Field

[0006] The present disclosure relates to antibodies and antigen-binding fragments that bind to C1s protein, and the use of antibodies and antigen-binding fragments that bind to C1s protein for treating complement-mediated disorders. Background Art

[0007] The complement system is a closely regulated protein network that forms an important part of the innate and adaptive immune systems. Through multiple activation pathways and extensive complement-mediated effector functions, the complement system plays a key role in inflammation, pathogen defense and recovery after injury. Complement activation can have both beneficial and harmful effects. The defense response mediated by complement activation can eliminate pathogens. Inappropriate, undesirable, excessive or lacking complement activation is associated with disease conditions such as certain autoimmune diseases and inflammatory syndromes, which reflects the complex balance of complement between immunomodulation and inflammatory tissue damage.

[0008] The complement system can be activated through three different pathways that converge at the effector phase, where the C3 convertase activates the complement C3 protein and triggers a cascade of downstream effects. Each pathway has different triggers, initiator molecules, and enzyme cascades upstream of the production of the C3 convertase: (1) the classical pathway (CP) can be triggered when an antibody-antigen complex binds to the C1 complex and C1 is activated; (2) the alternative pathway (AP) can be triggered by cell surface components that are foreign to the host; and (3) the mannose-binding lectin pathway (LP) can be triggered when circulating lectins bind to mannose residues on the surface of microorganisms. Following the generation of the C3 convertase in the upstream cascade of each pathway, subsequent cleavage of complement proteins C3 and C5: (1) produces the anaphylatoxins C3a and C5a, which attract and activate effector immune cells to sites of antibody binding / complement activation; (2) deposits the C3b opsonin, which mediates phagocytosis and lymphocyte activation; and (3) triggers the lytic pathway to form the membrane attack complex (MAC), which disrupts the cell membrane and leads to cell destruction.

[0009] The CP is not only crucial for antibody-mediated defense against foreign pathogens, but is also known or suspected to be involved in triggering immune responses to the engagement of autoantibodies with self-antigens, i.e., autoimmune disorders. The CP is triggered by the activation of the C1 complex, which normally circulates in the plasma as an inactive complex of its subcomponent proteins, C1q, C1r, and C1s. Following recognition and binding of the antibody-antigen complex, the subcomponents of the C1 complex are sequentially activated in an enzymatic cascade, where binding of C1q to the Fc regions of two antigen-bound antibodies activates the C1r subunit, which in turn activates C1s by cleaving the C1s zymogen into active C1s having two subcomponents (A chain and B chain) connected by an interchain disulfide bond. The activated C1s B chain is a serine protease that can, in turn, cleave serum C4 and C2 to form the C3 convertase, C4b2a. The formation of the C3 convertase in the CP then triggers downstream effector processes common to all three complement pathways.

[0010] The C1s protein (complement component 1s) plays a central role in CP function, where the C1s zymogen in the inactive C1 complex must be cleaved into the active two-subunit (two-chain) C1s that can cleave and activate serum C4 in order for the downstream effector cascade to proceed. For example, in human patients, deficiency of the C1s protein can cause severe immune complex disease because immune complex deposits cannot be effectively cleared in the absence of functional C1, C2, or C4 and subsequently normal levels of C3b. Several autoimmune and / or inflammatory disorders are known or suspected to involve spontaneous activation of CP by complexes of autoantibodies (particularly complement-fixing autoantibodies) that bind to self-antigens. Known complement-mediated disorders include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (atypical HUS), hereditary angioedema (HAE), age-related macular degeneration (AMD), and autoimmune hemolytic anemias such as cold agglutinin disease (CAD). The complement system, specifically CP, is suspected of playing a role in certain autoimmune disorders, such as immune thrombocytopenic purpura (ITP), an autoimmune bleeding disorder characterized by isolated thrombocytopenia with a platelet count of <150,000 / μL, which is suspected to be caused by the development of autoantibodies targeting autoplatelet antigens. CP is also suspected of playing a role in neuromyelitis optica (NMO), an autoimmune disorder with inflammatory demyelinating lesions in the central nervous system (particularly the spinal cord and optic nerves), which is believed to be caused by the binding of anti-aquaporin-4 (AQP4) autoantibodies to astrocytes. Therapeutic approaches for the treatment of complement-mediated disorders include small molecule regulators such as protease inhibitors, although some known therapies, such as the C1 inhibitor C1 INH, are known to exert effects beyond the regulation of CP, including regulation of the lectin pathway and kinin, coagulation and fibrinolysis systems. Therapeutic approaches using antibodies to components of the CP pathway are being explored. Summary of the Invention

[0011] The present invention relates to novel antibodies and antigen-binding fragments that bind to human complement component 1s (C1s), and methods of making and using antibodies and antigen-binding fragments that bind to C1s.

[0012] The present disclosure provides anti-C1s antibodies, anti-C1s antibody fragments that bind to C1s, nucleic acids encoding the anti-C1s antibodies and anti-C1s antibody fragments, and methods for making and using the anti-C1s antibodies and anti-C1s antibody fragments. The term "anti-C1s antibody" in the present disclosure encompasses anti-C1s antibodies and antigen-binding fragments thereof that can bind to C1s, specifically anti-C1s antibodies and antigen-binding fragments thereof that specifically bind to C1s. The anti-C1s antibodies as disclosed herein are capable of binding to the zymogen human C1s (native C1s). The anti-C1s antibodies as disclosed herein are capable of binding to active human C1s. The anti-C1s antibodies as disclosed herein are capable of inhibiting C1s. The anti-C1s antibodies as disclosed herein are capable of binding to the zymogen C1s and inhibiting C1s. The anti-C1s antibodies as disclosed herein are capable of binding to active C1s and inhibiting C1s. The anti-C1s antibody disclosed herein is a recombinant anti-C1s antibody or an antigen-binding fragment thereof that is capable of specifically binding to and inhibiting human C1s.

[0013] The present disclosure provides anti-C1s antibodies for in vivo, ex vivo or in vitro therapeutic and diagnostic purposes. Anti-C1s antibodies as disclosed herein can be used to treat disorders mediated by the function of C1s. Anti-C1s antibodies as disclosed herein can be used to treat complement-mediated disorders, specifically disorders mediated by the classical pathway (CP) of complement activation. Anti-C1s antibodies as disclosed herein are capable of binding to C1s and inhibiting C1s, which has an effect on the activity of the CP pathway, which can be measured by the effect on the activity of at least one component of the CP downstream of C1s activation, wherein the component can be a molecule or biological process associated with CP. Anti-C1s antibodies as disclosed herein are capable of inhibiting the activity of the CP pathway. Anti-C1s antibodies as disclosed herein can be used to treat disorders mediated by the function of CP. Anti-C1s antibodies as disclosed herein can be used to target C1s to selectively inhibit CP in complement-mediated autoimmune disorders and / or inflammatory disorders. Anti-C1s antibodies as disclosed herein can be used to treat complement-mediated autoimmune disorders and / or inflammatory disorders, including but not limited to immune thrombocytopenic purpura (ITP) or neuromyelitis optica (NMO).

[0014] In certain embodiments, the anti-C1s antibodies disclosed herein are capable of binding to zymogen (native) C1s and inhibiting the effects of C1s activation. In certain embodiments, the anti-C1s antibodies disclosed herein are capable of binding to active C1s and inhibiting the effects of C1s activation. The anti-C1s antibodies disclosed herein are capable of inhibiting one or more effects of C1s activation on at least one downstream component. In certain embodiments, the downstream component is at least one of the cleavage of C2 and / or the cleavage of C4 and / or the formation of C3 convertase and / or serum complement-induced lysis of antibody-sensitized cells, and the binding of the anti-C1s antibody to active C1s has the effect of inhibiting the cleavage of C2 and / or the cleavage of C4 and / or the formation of C3 convertase and / or serum-induced lysis of antibody-sensitized cells.

[0015] In one aspect, anti-C1s antibodies are provided that are capable of binding to C1s and modulating at least one effect of CP activation triggered by an antibody-antigen complex. In certain embodiments, the anti-C1s antibodies disclosed herein are capable of modulating at least one effect of CP activation triggered by an antibody-antigen complex in a dose-dependent manner. In certain embodiments, the anti-C1s antibodies disclosed herein are capable of inhibiting at least one effect of CP activation triggered by an antibody-antigen complex in a dose-dependent manner. In certain embodiments, the anti-C1s antibodies disclosed herein are capable of inhibiting serum-induced lysis of antibody-sensitized cells in a dose-dependent manner.

[0016] In one aspect, anti-C1s antibodies are provided that can have the effect of modulating the activity of at least one component involved in the cleavage and / or deposition of C4 triggered by the antibody-antigen complex. In certain embodiments, the anti-C1s antibodies disclosed herein can inhibit the activity of at least one component involved in the deposition of C4 from serum triggered by IgM. In certain embodiments, the anti-C1s antibodies disclosed herein can inhibit the deposition of human C4 from human serum triggered by IgM in a dose-dependent manner. In certain embodiments, the anti-C1s antibodies disclosed herein can modulate the activity of at least one component involved in the deposition of cynomolgus monkey C4 from cynomolgus monkey serum triggered by IgM in a dose-dependent manner. In certain embodiments, the anti-C1s antibodies disclosed herein can inhibit the deposition of cynomolgus monkey C4 from cynomolgus monkey serum triggered by IgM in a dose-dependent manner.

[0017] In another aspect, anti-C1s antibodies are provided that selectively act within the CP, upstream of the production and activity of common complement system effectors. In certain embodiments, selective inhibition of the CP by the anti-C1s antibodies disclosed herein prevents C4 cleavage and the downstream effects triggered by C4 cleavage. In certain embodiments, the anti-C1s antibodies disclosed herein prevent the formation of C3 convertase via the CP pathway.

[0018] In another aspect, anti-C1s antibodies that bind to C1s zymogen and active C1s protein are provided. In certain embodiments, the anti-C1s antibodies disclosed herein bind to C1s zymogen and active C1s protein with similar affinity. In certain embodiments, the anti-C1s antibodies disclosed herein specifically bind to C1s zymogen and active C1s protein with similar high affinity, with KD values ​​in the low nanomolar to picomolar range.

[0019] In another aspect, anti-C1s antibodies are provided that show cross-reactivity with at least one non-human C1s. In certain embodiments, the anti-C1s antibodies disclosed herein may be capable of binding to human C1s and rat C1s. In certain embodiments, the anti-C1s antibodies disclosed herein may be capable of binding to human C1s and cynomolgus monkey C1s. In certain embodiments, the anti-C1s antibodies disclosed herein may be capable of binding to human C1s, rat C1s, and cynomolgus monkey C1s. In certain embodiments, the anti-C1s antibodies disclosed herein do not show detectable binding to mouse C1s.

[0020] In another aspect, anti-C1s antibodies are provided that bind to epitopes accessible on zymogen C1s and active C1s in an internal region of C1s located within a 151 amino acid region from residues Y272 to R422 (M151) in the N-terminal heavy chain (NHC), wherein the key residues include R316, K336, and a subregion G390 to R422 of human C1s (SEQ ID NO: 99).

[0021] The anti-C1s antibodies as provided herein are recombinantly expressed and are recombinant antibodies. The anti-C1s antibodies as provided herein may further be one or more of the following: a humanized antibody, a chimeric antibody, a single-chain antibody, a Fab fragment, a single-chain variable fragment (scFv), an aptamer, a single-domain antibody (VHH, nanobody, camel antibody) or any other C1s binding fragment or variant. Specifically, the anti-C1s antibodies disclosed herein are humanized antibodies. In certain embodiments, the anti-C1s antibodies disclosed herein may include a framework in which amino acids have been substituted into an existing antibody framework, specifically to affect properties such as antigen binding ability. In certain embodiments, the anti-C1s antibodies disclosed herein may include complementary determining regions (CDRs) from a source (parent) antibody that have been transplanted (fused) into a framework (specifically, a recipient human framework) from an antibody of a different type (class) and / or from an organism different from the parent antibody. In certain embodiments, the anti-C1s antibodies disclosed herein may include a framework in which amino acids have been substituted, mutated, or replaced in regions outside of the CDRs to affect properties such as antigen binding or antibody structure, for example, in the variable region framework surrounding the CDRs and / or in the constant region (particularly the Fc region). In certain embodiments, one or more of the CDRs have been substituted, mutated, or replaced. In certain embodiments, the anti-C1s antibodies disclosed herein may be humanized anti-C1s antibody variants.

[0022] In certain embodiments, the anti-C1s antibodies disclosed herein include at least one polypeptide having an amino acid sequence as shown in Table 1 ("Variable region of an anti-C1s antibody") or the Sequence Listing, or a sequence substantially identical to an amino acid sequence as shown in Table 1 or the Sequence Listing (e.g., a sequence at least about 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical). The anti-C1s antibodies disclosed herein may include at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35 SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77;SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:129; SEQ ID NO:130; SEQ ID NO:133; SEQ ID NO:134; SEQ ID NO:135; SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:139; or SEQ ID NO:140. ;

[0023] In one embodiment, the anti-C1s antibody disclosed herein comprises a heavy chain variable region (VH) polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or a sequence substantially identical to SEQ ID NO: 1; and a light chain variable region (VL) polypeptide having the amino acid sequence shown in SEQ ID NO: 6 or a sequence substantially identical to SEQ ID NO: 6. In one embodiment, the anti-C1s antibody disclosed herein comprises: a heavy chain complementary determining region 1 (HC CDR1), wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO: 2; a heavy chain complementary determining region 2 (HC CDR2), wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO: 3; a heavy chain complementary determining region 3 (HC CDR3), wherein the HC CDR3 has the amino acid sequence shown in SEQ ID NO: 4; a light chain complementary determining region 1 (LC CDR1), wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO: 7; a light chain complementary determining region 2 (LC CDR2), wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO: 8; and a light chain complementary determining region 3 (LC CDR3), wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO: 9; or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR regions. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as 2-7, comprising: an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:71; and a LC polypeptide having the amino acid sequence set forth in SEQ ID NO:73.

[0024] In one embodiment, the anti-C1s antibody disclosed herein comprises a VH polypeptide having the amino acid sequence shown in SEQ ID NO: 11 or a sequence substantially identical to SEQ ID NO: 11; and a VL polypeptide having the amino acid sequence shown in SEQ ID NO: 16 or a sequence substantially identical to SEQ ID NO: 16. In one embodiment, the anti-C1s antibody disclosed herein comprises: an HC CDR1 having the amino acid sequence set forth in SEQ ID NO: 12; an HC CDR2 having the amino acid sequence set forth in SEQ ID NO: 13; an HC CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; an LC CDR1 having the amino acid sequence set forth in SEQ ID NO: 17; an LC CDR2 having the amino acid sequence set forth in SEQ ID NO: 18; and an LC CDR3 having the amino acid sequence set forth in SEQ ID NO: 19, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as 2-8, comprising: an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 75; and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 77.

[0025] In one embodiment, an anti-Cls antibody disclosed herein comprises a VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 21 or a sequence substantially identical thereto, and a VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 26 or a sequence substantially identical thereto. In one embodiment, an anti-Cls antibody disclosed herein comprises a HC CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a HC CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, a HC CDR3 having the amino acid sequence set forth in SEQ ID NO: 24, a LC CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, a LC CDR2 having the amino acid sequence set forth in SEQ ID NO: 28, and a LC CDR3 having the amino acid sequence set forth in SEQ ID NO: 29, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-Cls antibody disclosed herein is the antibody identified herein as hz2-7 (H1L2), which comprises a HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 79, and a LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 81.

[0026] In one embodiment, the anti-C1s antibody disclosed herein comprises a VH polypeptide having the amino acid sequence shown in SEQ ID NO:31 or a sequence substantially identical to SEQ ID NO:31, and a VL polypeptide having the amino acid sequence shown in SEQ ID NO:36 or a sequence substantially identical to SEQ ID NO:36. In one embodiment, the anti-C1s antibody disclosed herein includes: HC CDR1, wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO:32; HC CDR2, wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO:33; HC CDR3, wherein the HCCDR3 has the amino acid sequence shown in SEQ ID NO:34; LC CDR1, wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO:37; LC CDR2, wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO:38; and LC CDR3, wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO:39, or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR region. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as hz2-7 (H1L2 G131A), which comprises: an HC polypeptide having the amino acid sequence shown in SEQ ID NO:83; and a LC polypeptide having the amino acid sequence shown in SEQ ID NO:85.

[0027] In one embodiment, the anti-C1s antibody disclosed herein comprises: an HC variable region polypeptide having the amino acid sequence shown in SEQ ID NO:41 or a sequence substantially identical to SEQ ID NO:41; and an LC variable region polypeptide having the amino acid sequence shown in SEQ ID NO:46 or a sequence substantially identical to SEQ ID NO:46. In one embodiment, the anti-C1s antibody disclosed herein includes: HC CDR1, wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO:42; HC CDR2, wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO:43; HC CDR3, wherein the HC CDR3 has the amino acid sequence shown in SEQ ID NO:44; LC CDR1, wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO:47; LC CDR2, wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO:48; and LC CDR3, wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO:49, or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR region. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as hz2-8 (H1L2), which comprises: an HC polypeptide having the amino acid sequence shown in SEQ ID NO:87; and a LC polypeptide having the amino acid sequence shown in SEQ ID NO:89.

[0028] In one embodiment, the anti-C1s antibody disclosed herein comprises a VH polypeptide having the amino acid sequence shown in SEQ ID NO:51 or a sequence substantially identical to SEQ ID NO:51; and a VL polypeptide having the amino acid sequence shown in SEQ ID NO:56 or a sequence substantially identical to SEQ ID NO:56. In one embodiment, the anti-C1s antibody disclosed herein includes: HC CDR1, wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO:52; HC CDR2, wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO:53; HC CDR3, wherein the HCCDR3 has the amino acid sequence shown in SEQ ID NO:54; LC CDR1, wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO:57; LC CDR2, wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO:58; and LC CDR3, wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO:59, or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR region. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as hz2-8 (H1L2 G80A / T82A), which comprises: an HC polypeptide having the amino acid sequence shown in SEQ ID NO:91; and a LC polypeptide having the amino acid sequence shown in SEQ ID NO:93.

[0029] In one embodiment, the anti-C1s antibody disclosed herein comprises a VH polypeptide having the amino acid sequence shown in SEQ ID NO:61 or a sequence substantially identical to SEQ ID NO:61, and a VL polypeptide having the amino acid sequence shown in SEQ ID NO:66 or a sequence substantially identical to SEQ ID NO:66. In one embodiment, the anti-C1s antibody disclosed herein includes: HC CDR1, wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO:62; HC CDR2, wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO:63; HC CDR3, wherein the HCCDR3 has the amino acid sequence shown in SEQ ID NO:64; LC CDR1, wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO:67; LC CDR2, wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO:68; and LC CDR3, wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO:69, or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR region. In a non-limiting embodiment, the anti-C1s antibody disclosed herein is the antibody identified herein as hz2-8 (H1L2 G80A), which comprises: an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:95; and a LC polypeptide having the amino acid sequence set forth in SEQ ID NO:97.

[0030] In another aspect, CDR consensus sequences of the anti-C1s antibodies disclosed herein are provided. Table 1.b. discloses non-limiting exemplary examples of anti-C1s antibody CDR consensus sequences. In one embodiment, an antibody is provided wherein the VH comprises: an HC CDR 1 having the amino acid sequence set forth in SEQ ID NO: 129; an HC CDR2 having the amino acid sequence set forth in SEQ ID NO: 130; and an HC CDR3 having the amino acid sequence set forth in SEQ ID NO: 131, and the VL comprises: an LC CDR 1 having the amino acid sequence set forth in SEQ ID NO: 132; an LC CDR 2 having the amino acid sequence set forth in SEQ ID NO: 133; and an LC CDR 3 having the amino acid sequence set forth in SEQ ID NO: 134. In another embodiment, an antibody is provided, wherein the VH comprises: HC CDR1, wherein the HC CDR1 has the amino acid sequence shown in SEQ ID NO: 135; HC CDR2, wherein the HC CDR2 has the amino acid sequence shown in SEQ ID NO: 136; and HC CDR3, wherein the HC CDR3 has the amino acid sequence shown in SEQ ID NO: 137, and the VL comprises: LC CDR1, wherein the LC CDR1 has the amino acid sequence shown in SEQ ID NO: 138; LC CDR2, wherein the LC CDR2 has the amino acid sequence shown in SEQ ID NO: 139; and LC CDR3, wherein the LC CDR3 has the amino acid sequence shown in SEQ ID NO: 140.

[0031] In another aspect, anti-C1s antibodies (including variants and fragments as disclosed herein) are provided, which can be used to treat conditions mediated by CP activation, including but not limited to conditions mediated by CP activation triggered by antibody-antigen complexes, and specifically conditions associated with or characterized by CP activation by autoantibodies bound to self-antigens. Methods and compositions are provided for using the anti-C1s antibodies disclosed herein for therapeutic uses, including but not limited to treating conditions mediated by CP activation, specifically conditions characterized by CP activation by autoantibodies bound to self-antigens. Methods and compositions are provided for using the anti-C1s antibodies as disclosed herein for therapeutic uses, including treating ITP and / or NMO. In certain embodiments, pharmaceutical compositions are provided comprising the anti-C1s antibodies disclosed herein and suitable carriers and / or excipients.

[0032] In another aspect, methods for treating complement-mediated disorders are provided, such methods comprising administering to a subject in need thereof an effective amount of an anti-C1s antibody disclosed herein. According to this aspect, the method for treating complement-mediated disorders comprises administering an effective amount of an anti-C1s antibody disclosed herein to have the effect of modulating or inhibiting one or more biological effects associated with CP activation triggered by the antibody-antigen complex. According to this aspect, an effective amount of an anti-C1s antibody is an amount of the antibody sufficient to cause the desired level of modulation or inhibition. In certain embodiments, the method comprises administering to a subject an effective amount of an anti-C1s antibody that results in modulation of one or more biological effects associated with CP activation by autoantibodies bound to self-antigens, including but not limited to reducing platelet lysis and / or antibody-mediated platelet removal triggered by anti-platelet antibodies.

[0033] In another aspect, methods are provided for treating a disease or condition in which undesirable, abnormal, inappropriate, or excessive CP activation is involved, such methods comprising administering to a subject in need thereof an effective amount of an anti-C1s antibody disclosed herein.

[0034] In another aspect, methods for diagnosing or screening a subject for a complement-mediated disorder are provided. The methods for diagnosing or screening a complement-mediated disorder can be practiced in vivo, ex vivo, or in vitro. In certain embodiments, the methods comprise administering an anti-C1s antibody to a subject known or suspected of having a complement-mediated autoimmune disorder, and measuring one or more biological effects or symptoms associated with the complement-mediated autoimmune disorder. In certain embodiments, the ex vivo or in vitro methods comprise administering an anti-C1s antibody to a sample taken from the subject, and measuring one or more biological effects or symptoms associated with the complement-mediated autoimmune disorder.

[0035] In another aspect, one or more isolated nucleic acid molecules are provided that encode at least a portion of at least one anti-C1s antibody disclosed herein. In certain embodiments, the isolated nucleic acid molecule encoding at least a portion of at least one of the anti-C1s antibodies disclosed herein comprises a nucleotide sequence as set forth in Table 2 herein, or a sequence substantially identical (e.g., at least about 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to a nucleotide sequence as set forth in Table 2. In certain embodiments, the isolated nucleic acid molecule encoding at least one of the heavy chain (HC) sequences of the anti-C1s antibodies disclosed herein may comprise a nucleotide sequence selected from at least one of the following: SEQ ID NO: 5 or a sequence substantially identical to SEQ ID NO: 5; SEQ ID NO: 15 or a sequence substantially identical to SEQ ID NO: 15; SEQ ID NO: 25 or a sequence substantially identical to SEQ ID NO: 25; SEQ ID NO: 35 or a sequence substantially identical to SEQ ID NO: 35; SEQ ID NO: 45 or a sequence substantially identical to SEQ ID NO: 45; SEQ ID NO: 55 or a sequence substantially identical to SEQ ID NO: 55; SEQ ID NO: 65 or a sequence substantially identical to SEQ ID NO: 65; SEQ ID NO: 72 or a sequence substantially identical to SEQ ID NO: 72; SEQ ID NO: 76 or a sequence substantially identical to SEQ ID NO: 76; SEQ ID NO: 80 or a sequence substantially identical to SEQ ID NO: 80; SEQ ID NO: 84 or a sequence substantially identical to SEQ ID NO: 84; SEQ ID NO: 88 or a sequence substantially identical to SEQ ID NO: 89. SEQ ID NO: 88 or a sequence substantially identical to SEQ ID NO: 88; SEQ ID NO: 92 or a sequence substantially identical to SEQ ID NO: 92; or SEQ ID NO: 96 or a sequence substantially identical to SEQ ID NO: 96.

[0036] In certain embodiments, an isolated nucleic acid molecule encoding at least one of the light chain (LC) sequences of the anti-Cls antibodies or antigen-binding fragments thereof disclosed herein can comprise a nucleotide sequence selected from at least one of: SEQ ID NO: 10 or a sequence substantially identical to SEQ ID NO: 10; SEQ ID NO: 20 or a sequence substantially identical to SEQ ID NO: 20; or SEQ ID NO: 30 or a sequence substantially identical to SEQ ID NO: 30; SEQ ID NO: 40 or a sequence substantially identical to SEQ ID NO: 40; SEQ ID NO: 50 or a sequence substantially identical to SEQ ID NO: 50; SEQ ID NO: 60 or a sequence substantially identical to SEQ ID NO: 60; SEQ ID NO: 70 or a sequence substantially identical to SEQ ID NO: 70; SEQ ID NO: 74 or a sequence substantially identical to SEQ ID NO: 74; SEQ ID NO: 78 or a sequence substantially identical to SEQ ID NO: 78; SEQ ID NO: 82 or a sequence substantially identical to SEQ ID NO: 82; SEQ ID NO: 86 or a sequence substantially identical to SEQ ID NO: 86; SEQ ID NO: 90 or a sequence substantially identical to SEQ ID NO: 90; SEQ ID NO: 94 or a sequence substantially identical to SEQ ID NO: 94; or SEQ ID NO: 98 or a sequence substantially identical to SEQ ID NO: 98.

[0037] In another aspect, a vector comprising one or more nucleic acid molecules encoding at least one amino acid sequence of an anti-Cls antibody disclosed herein is provided. In certain embodiments, a vector comprising one or more nucleic acid molecules encoding at least one of the sequences of a heavy chain (HC) or a light chain (LC) of an anti-Cls antibody disclosed herein is provided.

[0038] In another aspect, at least one host cell containing a vector comprising one or more nucleic acid molecules encoding an amino acid sequence of an anti-Cls antibody disclosed herein is provided. In certain embodiments, a host cell is transiently transfected with a vector comprising one or more nucleic acid molecules encoding an amino acid sequence of an anti-Cls antibody or antigen-binding fragment thereof disclosed herein, wherein the host cell is capable of supporting expression and recombinant production of the anti-Cls antibody or antigen-binding fragment thereof encoded by the vector. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 A schematic diagram showing the proposed roles of CP and anti-C1s antibodies, where Ag indicates antigen, IgG and IgM indicate types of antibodies that can bind to Ag, C1 indicates the C1 complex, where C1q, C1r, and C1s indicate subcomponent proteins of the C1 complex, anti-C1s indicates anti-C1s antibodies that bind to C1s, the labels C2a, C2b, C3, C3a, C3b, C4a, C4b, and C5 indicate components of the CP cascade arranged in a schematic diagram of the steps in the cascade, and MAC indicates the membrane attack complex.

[0040] Figures 2A-2D Results are shown for the ability of anti-C1s antibodies to inhibit IgM-induced C4 deposition from serum in a functional assay. Figure 2A Shown are the effects of anti-C1s antibody 2-7, anti-C1s antibody 2-8, and antibody from non-functional clone 3-30 on IgM-induced deposition of human C4 from human serum (NHS) over the indicated antibody concentration ranges. Figure 2B Shown are the effects of anti-C1s antibodies 2-7 and 2-8 on IgM-induced deposition of cynomolgus monkey C4 from cynomolgus monkey serum (CMS) over the indicated antibody concentration ranges. Figures 2A-2B In the figures, the symbols are used as follows: open circles represent the results using anti-C1s antibody 2-7; open triangles represent the results using anti-C1s antibody 2-8; and open squares represent the results using anti-C1s antibody 3-30. Figure 2C Shown are the effects of humanized anti-C1s antibody hz2-7(H1L2), humanized anti-C1s antibody hz2-8(H1L2), humanized anti-C1s antibody variant hz2-7(H1L2 G131A), humanized anti-C1s antibody variant hz2-8(H1L2 G80A), and humanized anti-C1s antibody variant hz2-8(H1L2 G80A / T82A) on IgM-induced deposition of human C4 from human serum (NHS) over the indicated antibody concentration ranges. Figure 2D Shown are the effects of humanized anti-C1s antibody hz2-7(H1L2), humanized anti-C1s antibody hz2-8(H1L2), humanized anti-C1s antibody variant hz2-7(H1L2 G131A), humanized anti-C1s antibody variant hz2-8(H1L2 G80A) and humanized anti-C1s antibody variant hz2-8(H1L2 G80A / T82A) on IgM-induced deposition of cynomolgus monkey C4 from cynomolgus monkey serum (CMS) over the indicated antibody concentration ranges. Figures 2C-2DIn the graphs, the symbols are used as follows: open circles represent results using anti-Cls antibody hz2-7 (H1L2); black filled circles represent results using anti-Cls antibody hz2-7 (H1L2 G131A); open triangles represent results using anti-Cls antibody hz2-8 (H1L2); black filled triangles represent results using anti-Cls antibody hz2-8 (H1L2 G80A); and triangles with diagonal lines represent results using anti-Cls antibody hz2-8 (H1L2 G80A / T82A).

[0041] Figures 3A-3V Results of assays to determine binding affinity and cross-reactivity of anti-Cls antibodies are shown. Figures 3A-3D Results of measuring anti-Cls antibody affinity using Bio-Layer Interferometry technology (Octet®) and determining binding of antibodies to active human Cls protein by combining the antibodies with the indicated concentrations of active human Cls protein for the indicated times using AHC biosensors are shown. Results of RED96e system assays to determine binding kinetics of active human Cls protein are shown, wherein Figure 3A Results of binding of anti-Cls antibody 2-7 to active human Cls are shown, Figure 3B Results of binding of anti-Cls antibody 2-8 to active human Cls are shown, Figure 3C Humanized anti-Cls antibody hz2-7 (H1L2) binds to active human Cls, and Figure 3D Humanized anti-Cls variant hz2-8 (H1L2 G80A / T82A) binds to active human Cls. Figure 3E Summary table showing values of KD, k on , k off , and R 2 values calculated for each indicated combination of antibody and target. Figures 3F-3G Results of measuring anti-Cls antibody binding to active and zymogen forms of Cls using ELISA are shown, wherein Figure 3F Results of binding of 2-7, 2-8, hz2-7 (H1L2), and hz2-8 (H1L2 G80A / T82A) at the indicated concentrations, and human IgG1 as a control, to active human Cls are shown, and Figure 3G Results of binding of 2-7, 2-8, hz2-7 (H1L2), and hz2-8 (H1L2 G80A / T82A) at the indicated concentrations, and human IgG1 as a control, to human Cls zymogen are shown. Figures 3H-3Q Results of measuring anti-Cls antibody affinity using Bio-Layer Interferometry technology and determining binding of antibodies to human (Hu Cls, Figures 3H-3K ), rat (Ra Cls, Figures 3L-3M) and rhesus monkey (CyC1s, Figures 3N-3Q ) recombinant C1s, wherein Figure 3H binding of 2-7 to human C1s (HuC1s) is shown, Figure 3I binding of 2-8 to HuC1s is shown, Figure 3J binding of hz2-7 (H1L2) to HuC1s is shown, Figure 3K binding of hz2-8 (H1L2 G80A / T82A) to HuC1s is shown, Figure 3L binding of 2-8 to RaC1s is shown, Figure 3M binding of hz2-8 (H1L2 G80A / T82A) to RaC1s is shown, Figure 3N binding of 2-7 to CyC1s is shown, Figure 3O binding of 2-8 to CyC1s is shown, Figure 3P binding of hz2-7 (H1L2) to CyC1s is shown, and Figure 3Q binding of hz2-8 (H1L2 G80A / T82A) to CyC1s is shown. Figure 3R is a summary table showing the values of KD, k on , k off and R 2 calculated for each indicated combination of antibody and target. Figure 3S-3V results of anti-C1s antibody cross-reactivity measurements using ELISA, wherein the graphs show binding of 2-7, 2-8, hz2-7 (H1L2), hz2-8 (H1L2 G80A / T82A) and IgG (control) at the indicated concentrations to human C1s (HuC1s, Figure 3S ), mouse C1s (MoC1s, Figure 3T ), rat C1s (RatC1s, Figure 3U ), and rhesus monkey C1s (CyC1s, Figure 3V ). In Figures 2S-2V and 3S-3V, the symbols are used as follows: open circles indicate results using anti-C1s antibody 2-7; open triangles indicate results using anti-C1s antibody 2-8; open squares indicate results using mouse IgG; black filled circles indicate results using anti-C1s antibody hz2-7 (H1L2); black filled triangles indicate results using anti-C1s antibody hz2-8 (H1L2 G80A / T82A); and black filled squares indicate results using human IgG1. Figures 3F-3G

[0042] Figures 4A-4D results of anti-C1s antibody binding to recombinant C1s full length and different truncated forms measured using ELISA are shown. Figure 4A ​Binding of 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), and IgG controls (mouse IgG, human IgG1) to full-length human C1s (HuC1s) is shown. Figure 4B Binding of 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), and IgG control (mouse IgG, human IgG1) to full-length mouse C1s (MoC1s) is shown. Figure 4C Binding of 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), and IgG controls (mouse IgG, human IgG1) to truncated human C1s NHC (HuC1s(NHC)) is shown. Figure 4D Binding of 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), and IgG controls (mouse IgG, human IgG1) to truncated human C1s M151 (HuC1s(M151)) is shown. Figures 4A-4D In the figure, the symbols are used as follows: hollow circles represent the results using anti-C1s antibody 2-7; hollow triangles represent the results using anti-C1s antibody 2-8; hollow squares represent the results using mouse IgG; black solid circles represent the results using anti-C1s antibody hz2-7 (H1L2); black solid triangles represent the results using anti-C1s antibody hz2-8 (H1L2); and black solid squares represent the results using human IgG1.

[0043] Figures 5A-5B Binding to full-length and truncated forms (including deletion mutants) of C1s is shown, along with a schematic comparison thereof. Figure 5AImages showing representative binding of anti-Cls antibody 2-8 to full-length and different truncated forms of Cls measured using Western blot. 200 ng of full-length human Cls (HuCl s), in lane 1, 200 ng of full-length mouse Cls (MoCl s), in lane 2, 200 ng of truncated human Cls M151 (HuCl s(M151)), in lane 3, and 200 ng of truncated human Cls NHC (HuCl s(NHC)), in lane 4, were resolved on SDS-PAGE under non-reducing (left blot) and reducing (right blot) conditions, followed by transfer to nitrocellulose membrane (blot). Blocked blots were incubated with antibody 2-8, and developed with HRP-labeled secondary antibody. In the blot of the gel run without reducing agent (left), lane 1 shows that antibody 2-8 binds to full-length human Cls (HuCl s, upper band) and to the auto-cleaved Cls heavy chain (lower band), lane 2 shows that antibody 2-8 does not bind to full-length mouse Cls (MoCl s) (no detectable antibody binding), lane 3 shows that antibody 2-8 binds to multiple isoforms of truncated human Cls M151 (HuCl s(M151)), and lane 4 shows that antibody 2-8 binds to truncated human Cls NHC (HuCl s(NHC)). On the blot of the gel run with reducing agent (right), lane 1 shows low levels of antibody 2-8 binding to the auto-cleaved Cls heavy chain from full-length human Cls (HuCl s), lane 2 shows that antibody 2-8 does not bind to full-length mouse Cls (MoCl s) (no detectable binding), lane 3 shows that antibody 2-8 binds to truncated human Cls M151 (HuCl s(M151)), and lane 4 shows that antibody 2-8 binds to truncated human Cls NHC (HuCl s(NHC)). Figure 5B A schematic comparison and alignment of full-length human Cls, HuCl s (e.g., as exemplified in SEQ ID NO: 99), human Cls deletion mutant 1 (M151) designated as HuCl s(M151) or M151 (e.g., as exemplified in SEQ ID NO: 107), human Cls deletion mutant 2 (NHC) designated as HuCl s(NHC) or NHC (e.g., as exemplified in SEQ ID NO: 109), and human Cls deletion mutant 3 (NHCΔ33) designated as human Cls(NHCΔ33) or NHCΔ33 (e.g., as exemplified in SEQ ID NO: 111) is shown. Figure 5B The positions of the point mutations introduced into HuCl s, M151, NHC, and NHCΔ33 in Example 6 are also shown.

[0044] Figures 6A-6UResults of binding measurements of hz2-7 (H1L2), hz2-8 (H1L2 G80A), hz2-8 (H1L2 G80A / T82A), and human IgG1 (control) to NHCΔ33 and point mutants M1-M20 of human C1s using ELISA are shown as follows: Figure 6A showing binding to NHCΔ33; Figure 6B showing binding to M1 (M277I); Figure 6C showing binding to M2 (M277I and K331Q); Figure 6D showing binding to M3 (M277I, P278S, and N329D); Figure 6E showing binding to M4 (P280A and E282K); Figure 6F showing binding to M5 (K336G); Figure 6G showing binding to M6 (P280A, E282K, and K336G); Figure 6H showing binding to M7 (D343Y and E351A); Figure 6I showing binding to M8 (E351A); Figure 6J showing binding to M9 (K336G, D343Y, and E351A); Figure 6K showing binding to M10 (D283I and P285A); Figure 6L showing binding to M11 (S349P); Figure 6M showing binding to M12 (G381E and G382E); Figure 6N showing binding to M13 (A292D); Figure 6O showing binding to M14 (R316H); Figure 6P showing binding to M15 (S360N); Figure 6Q showing binding to M16 (R368H); Figure 6R showing binding to M17 (N380H). Figure 6S showing binding to M18 (V288T); Figure 6T showing binding to M19 (Q303K); and Figure 6U showing binding to M20 (A320S). In Figures 6A to 6U the figures, the following notation is used: open circles represent results using anti-C1s antibody hz2-7 (H1L2); black solid triangles represent results using anti-C1s antibody hz2-8 (H1L2 G80A); triangles with diagonal lines represent results using anti-C1s antibody hz2-8 (H1L2 G80A / T82A); and open squares represent results using human IgG1 (HuIgG1).

[0045] Figures 7A-7BThe results show the ability of anti-C1s antibodies to inhibit complement-mediated lysis of antibody-sensitized sheep erythrocytes (EA cells). The results show the ability of anti-C1s antibodies hz2-7 (H1L2), hz2-8 (H1L2), hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) and isotype control human IgG1 to normal human serum (NHS, Figure 7A ) or cynomolgus monkey serum (CMS, Figure 7B )-induced EA cell lysis. Symbols are used as follows: open circles represent the results using the anti-C1s antibody hz2-7 (H1L2); open triangles represent the results using the anti-C1s antibody hz2-8 (H1L2); black solid triangles represent the results using the anti-C1s antibody hz2-8 (H1L2 G80A); triangles with diagonal lines represent the results using the anti-C1s antibody hz2-8 (H1L2 G80A / T82A); and open squares represent the results using human IgG1. DETAILED DESCRIPTION

[0046] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to C1s, as well as methods of making and using the novel antibodies and antigen-binding fragments thereof.

[0047] Terms / Definitions

[0048] Unless otherwise defined, the scientific and technical terms used in conjunction with the present invention should have the meanings commonly understood by those of ordinary skill in the art. Unless the context clearly dictates otherwise, the use of singular terms ("a / an" or "the" or other uses of singular terms) include plural referents, and plural terms should include the singular. Thus, for example, reference to an "antibody" includes "one or more" antibodies or "a plurality" of such antibodies. All publications mentioned herein are hereby incorporated herein by reference in their entirety.

[0049] Generally, the term and technology of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry and recombinant DNA technology known to those skilled in the art can be used for antibody disclosed herein, Fab, composition and method.Technique and procedure as herein described are usually according to conventional method well known in the art and as described in various general and more specific references, described reference is especially Sam Brook (Sambrook) et al. (1989) molecular cloning: laboratory manual (MOLECULAR CLONING:ALABORATORY MANUAL) (2nd edition, the Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)) and Ausubel (Ausubel) et al. (1994) contemporary molecular biology experimental guide (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), I-III volumes (John Wiley & Sons, NY) in New York). Unless otherwise indicated herein, otherwise according to the specification sheets of manufacturers or as generally realized in this area or as described herein, enzymatic reaction and purification technique are carried out. Conventional terms are used herein to describe techniques and methods for the preparation and formulation of drugs and treatment of subjects.

[0050] "Antibody" in the broadest sense refers to a polypeptide or combination of polypeptides that recognizes and binds an antigen through one or more immunoglobulin variable regions, which may be naturally occurring or non-naturally occurring, for example, as a result of engineering, chimerization, humanization, optimization, CDR grafting, or affinity maturation.

[0051] As disclosed herein, an "antibody" may be a complete (intact, full-length) antibody, a single-chain antibody, or an antigen-binding fragment having one or two chains, and may be naturally occurring or non-naturally occurring. An antibody comprises at least sufficient complementarity determining regions (CDRs) interspersed with framework regions (FRs) for the antibody to recognize and bind to an antigen. The anti-C1s antibodies disclosed herein may be, but are not limited to, at least one of: a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a Fab fragment, a single-chain variable fragment (scFv), an aptamer, a single domain antibody (VHH, nanobody, camelid antibody), a recombinant antibody, a modified antibody having a peptide / other moiety attached to the antibody and / or additional amino acids added to the N-terminus or C-terminus, or other C1s binding fragment or variant. Complete antibody, full-length antibody, intact antibody, naturally occurring antibody, or equivalent terms should be understood to refer to a polypeptide, in particular a glycoprotein, comprising in its standard form at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC comprises a heavy chain variable region (VH) and an HC constant region (CH), and each light chain comprises a light chain variable region (VL) and a LC constant region (CL). The HC variable region VH and the LC variable region VL comprise binding domains that interact with antigens. The VH and VL regions can be further subdivided into CDR regions characterized by hypervariable regions interspersed with generally more conserved FR regions. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. It will be understood that the assignment of amino acids to each domain is according to methods known in the art, in particular according to the definitions found in, inter alia, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed., NIH Publication No. 91-3242 (1991); Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the classical complement system. Generally, an antibody comprises at least heavy chain (HC) CDR1, CDR2 and CDR3 and light chain (LC) CDR1, CDR2 and CDR3 sequences, any of which may be naturally occurring or non-naturally occurring.An antibody may include fewer CDR sequences, as long as the antibody can recognize and bind to the antigen. Alternatively, a complete antibody may be a single-chain antibody, such as a heavy chain-only isotype, with antigen binding mediated by one variable domain.

[0052] The anti-C1s antibodies disclosed herein may be variants comprising at least one altered CDR or framework sequence, wherein the CDR and / or framework sequence can be optimized by mutating the nucleic acid molecules encoding such framework sequences. Variants may be constructed from HC and LC portions independently derived from different sources. Techniques for generating variants include, but are not limited to, conservative amino acid substitution, computer modeling, screening of candidate polypeptides, and codon optimization, alone or in combination, and it is understood that a skilled artisan will be able to generate antibody variants as needed. The anti-C1s antibodies disclosed herein may be fragments. The antigen-binding function of an antibody can be performed by fragments such as: a Fab fragment; a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment; a bivalent fragment comprising two Fab fragments connected at the hinge region by a disulfide bond; a Fd fragment consisting of the VH and CH1 domains; a single-chain variable fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody; a single-domain antibody (dAb) fragment consisting of the VH domain; and isolated CDRs (VHH, nanobodies) or aptamers. The antigen binding portion can be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23(9): 1126-1136). The antigen binding portion of an antibody can be grafted onto a polypeptide-based scaffold to form a monoclonal antibody (see, e.g., U.S. Patent No. 6,703,199, which describes a fibronectin polypeptide monoclonal antibody).

[0053] The term antibody encompasses polypeptides of various broad classes that can be distinguished biochemically. The "classification" of an antibody can refer to the type of constant domain or constant region that its heavy chain has. Those skilled in the art will appreciate that there are five main classes of antibodies, i.e., IgA, IgD, IgE, IgG and IgM, and some antibodies in these antibodies can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, and every kind of subclass in these subclasses is well characterized and known to confer functional specialization. Every kind of classification in these classifications and isotypes and the modified version of the isotype are easily distinguishable and within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure.

[0054] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain (HC) and / or light chain (LC) involved in forming the immunoreactive site is derived from a particular source or species, while the remainder of the HC and / or LC is derived from a different source or species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or non-human primate) and the constant region will be from a human source.

[0055] As used herein, phrase "humanized antibody " refers to an antibody (immunoglobulin) or antibody variant comprising a portion of an antibody (immunoglobulin), the portion comprising an amino acid sequence with different sources, wherein at least a portion comprises an amino acid sequence from a human source. Humanized antibodies include portions derived from non-human antibodies, typically derived from portions of mouse antibodies combined with a target of interest, wherein the portion from non-human antibodies can be a portion of the overall variable region or variable region from a "parent" non-human antibody, such as one or more CDRs from a "parent" non-human antibody. Humanized antibodies may include one or more variable regions from non-human antibodies that are connected to an immunoglobulin framework of human origin, specifically to a human variable region framework. Humanized antibodies may include CDRs from the parental non-human antibody transplanted (fused) in a framework, the framework comprising portions of the variable region derived from a human immunoglobulin framework, specifically a receptor human framework or a human shared framework. The techniques and principles for designing, making and testing humanized antibodies are known (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 1986, 321(6069):522-5; Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008, 13:1619-33). It will be appreciated that changes can be made to one or more CDR sequences and / or acceptor frameworks at various positions in order to develop humanized antibodies with improved characteristics depending on the desired use, e.g., high affinity for the target, specificity for the selected epitope, avoidance of undesirable events such as isomerization or deamidation, low clearance, low toxicity, etc. The anti-C1s antibodies disclosed herein may be humanized variants.

[0056] "Binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). "Affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, binding affinity as used herein refers to intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity can be measured by common methods known in the art, including those described herein. Approximately 50% of maximal binding (calculated EC 50 The affinity of a molecule X for its partner Y can usually be determined by the dissociation constant (Kd or KD, representing the k value measured for the interaction). off / k on "Specific binding" or "specifically binds" or similar terms refer to high affinity binding, particularly binding with a measured affinity (Kd, KD) in at least the nanomolar range. The anti-C1s antibodies of the present disclosure specifically bind to the C1s protein.

[0057] "Substantially identical" as used to refer to a sequence that is substantially identical to an identified amino acid sequence or nucleotide sequence is to be understood as a sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% identical to an amino acid sequence or nucleotide sequence, determined as the percentage of residues in the candidate amino acid or nucleic acid sequence that are identical with the residues in the identified reference sequence, after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art, for example publicly available computer software / programs such as Clustal Omega (from the European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI), available at https: / / www.ebi.ac.uk / Tools / msa / clustalo / When using such software / programs, it is preferred to use the default parameters, e.g., the default parameters for gap penalties and extension penalties.

[0058] A "subject" is a mammal, wherein mammals include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys), domesticated animals (e.g., cattle, sheep, cats, dogs, pigs, llamas, and horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human. The phrase "to a subject in need thereof" or "to a patient in need thereof" or "to a patient in need of treatment" or "to a subject in need of treatment" may include a subject who would benefit from the administration of an anti-C1s antibody disclosed herein for the treatment of a complement-mediated disorder. It should be understood that administration of an anti-C1s antibody encompasses administration to a "subject in need thereof" and may be interpreted to refer to a subject who is known or suspected of having a complement-mediated disorder such as ITP or NMO based on indicators such as symptoms, family history, or genotype. It should be further understood that an anti-C1s antibody may be administered to a subject who is not known or suspected of having a complement-mediated disorder for purposes that may include, but are not limited to, prophylactic or preventative purposes, for screening, for diagnosis, for research purposes, or to achieve a result other than treating a disorder.

[0059] An "effective amount" of an anti-C1s antibody, for example, in a pharmaceutical formulation, refers to an amount effective, at a dosage and for a period of time necessary, to achieve the desired therapeutic or prophylactic result. It should be understood that an "effective amount" is intended to refer to an amount of an anti-C1s antibody or a pharmaceutical composition comprising an anti-C1s antibody that will elicit a biological response or desired therapeutic effect in the cell, tissue, system, non-human animal subject, non-human mammalian subject, or human subject being measured. The terms "therapeutically effective amount," "pharmacologically effective amount," and "physiologically effective amount" are used interchangeably and refer to the amount of anti-C1s antibody required to provide a threshold level of active antibody in the bloodstream or target tissue. The precise amount will depend on a variety of factors, such as the specific anti-C1s antibody (active agent); the components and physical properties of the composition; the intended population of subjects / patients to be treated; considerations such as the disease state, age, sex, and weight of the subject, etc., and can be readily determined by one skilled in the art based on the information provided herein or otherwise available in the relevant literature. The terms "improve," "increase," or "decrease" as used in this context refer to a value or parameter relative to a baseline measurement, such as the measurement of the same subject before starting a treatment described herein, or the measurement of a control individual (or multiple control individuals) in the absence of a treatment described herein, or a value available in an information source such as a textbook, manual, or database.

[0060] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in a form that permits the biological activity of the active ingredient contained therein (specifically, the anti-C1s antibodies disclosed herein) to be effective. It should be understood that a pharmaceutical composition may contain more than one active ingredient, for example, more than one anti-C1s antibody or a combination of an anti-C1s antibody and another active ingredient that acts on a different target, wherein the combination may be, but is not limited to, a combination of an anti-C1s antibody and another active ingredient that has a desired effect on other complement pathways or other processes involved in inflammation, a combination of an anti-C1s antibody and a gene therapy agent, or a combination of an anti-C1s antibody and a protein directed to another target (e.g., an Fc fusion protein). A "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. It should be understood that a pharmaceutically acceptable carrier may be, but is not limited to, a buffer, an excipient, a stabilizer, an adjuvant, or a preservative.

[0061] As used herein, the term "treat" or similar terms may refer to an outcome that is believed to be beneficial for a particular subject under a defined set of circumstances. Treating a complement-mediated disorder may nonexclusively refer to any of alleviating, ameliorating, slowing, interrupting, preventing, alleviating, stopping, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, and may further encompass preventing or delaying the onset of one or more symptoms of a complement-mediated disorder, and / or reducing the severity or frequency of one or more symptoms of a complement-mediated disorder. The terms "treat" or "treatment method," or equivalents, may encompass one or more uses of the anti-C1s antibodies disclosed herein, including but not limited to therapeutic uses, prophylactic uses, preventative uses, diagnostic uses, imaging uses, and screening uses.

[0062] As used herein, "vector" refers to a nucleic acid molecule capable of propagating nucleic acids to which the vector sequences are linked in a host cell into which the vector is introduced. Vectors capable of directing the expression of nucleic acids to which they are operatively linked are referred to herein as "expression vectors."

[0063] Anti-C1S antibodies

[0064] Antibodies and antigen-binding fragments are provided that are capable of binding to Cls and modulating the activity of Cls, thereby having an effect of modulating the activity of at least one component involved in at least one complement-mediated disorder, in particular at least one disorder associated with CP activation by autoantibodies that bind to autoantigens. Antibodies and antigen-binding fragments are provided that are capable of binding to Cls and modulating the activity of Cls, thereby having an effect of modulating the activity of at least one component of CP involved in ITP and / or NMO. Anti-Cl s antibodies that are capable of binding to Cls and modulating the activity of Cls can be used in methods and compositions for treating complement-mediated disorders associated with CP activation by autoantibodies that bind to autoantigens, in particular ITP.

[0065] Once antibodies or fragments specific for Cls are provided, the desired biological activity of modulating the activity of Cls or having an effect of modulating at least one component involved in a complement-mediated disorder can be tested by several methods known to those skilled in the art.

[0066] It should be understood that "modulate" or "modulating" or similar terms, as used herein, can refer to one or more effects that may occur when the anti-C1s antibodies disclosed herein bind to their target molecule, C1s. "Modulate" and its equivalents can refer to different modes of action and effects depending on the component in question, that is, modulation can refer to neutralizing, reversing, inhibiting, blocking, reducing, antagonizing, agonizing, amplifying, enhancing, or otherwise altering the activity of at least one component involved in the complement system, specifically CP. It should be understood that "inhibit C1s" or "inhibiting C1s" or "inhibition of C1s" or similar terms, as used herein, refers to inhibiting at least one activity or function of C1s involved in the complement cascade, such that inhibition of C1s has downstream effects, such as inhibition of activation of enzymes involved in the complement cascade, association or dissociation of molecules involved in the complement cascade, production of downstream effectors that require C1s activation, or the extent of serum complement-induced lysis of antibody-sensitized cells. It should be understood that "C1s inhibition" or similar terms, as used herein, do not require demonstration of the specific activity or function of C1s that is inhibited when the C1s antibodies of the invention bind to the C1s target molecule, as measurements showing inhibition of processes or effectors known to be downstream of C1s activation can be used as surrogate measurements or indicators of C1s activity or function, and therefore as indicators of inhibition of C1s activity or function. Thus, a C1s antibody capable of binding to and inhibiting C1s is an antibody capable of, inter alia, inhibiting the activity of the classical pathway of complement activation (CP), inhibiting downstream effects of C1s activation, inhibiting IgM-induced C4 deposition from serum, inhibiting antibody-antigen complex-mediated C4 cleavage, inhibiting antibody-antigen complex-mediated C4b deposition, inhibiting the formation of C3 convertase, inhibiting downstream effects of CP activation, etc.

[0067] It is to be understood that the term "component" can refer not only to the target molecule C1s, but also to molecules, processes or pathways involved in complement-mediated effects downstream of C1s activation, in particular CP-mediated effects downstream of C1s activation. It is to be understood that by targeting C1s with an anti-C1s antibody, CP can be selectively targeted, such that CP-mediated effects can be specifically or selectively inhibited. This selective targeting effect on CP is also understood to leave the alternative and lectin pathways intact to combat infection. It is to be understood that an anti-C1s antibody as disclosed herein can be used to therapeutically target antibody-antigen complex triggered CP activation and modulate the activity of CP according to antibody-antigen complex triggered CP activation. An anti-C1s antibody as disclosed herein can be used to therapeutically target at least one component involved in a complement-mediated disorder, in particular specifically targeting at least one component involved in a CP-mediated disorder, in particular at least one component involved in a CP-mediated autoimmune disorder, in particular ITP and / or NMO. Without wishing to be bound to a particular mechanism of action, targeting C1s using an anti-C1s antibody as disclosed herein is to be understood as specifically inhibiting CP-mediated cytolysis, which is the basis for the effects leading to autoimmune pathologies such as destruction of e.g. self-platelets, red blood cells or astrocytes. In certain embodiments, an anti-C1s antibody as disclosed herein can be used to inhibit the CP pathway in a dose-dependent manner. In certain embodiments, an anti-C1s antibody as disclosed herein can be used to inhibit the effects of antibody-antigen complex triggered CP activation in a dose-dependent manner.

[0068] In certain embodiments, an anti-C1s antibody as disclosed herein can be used to therapeutically target antibody-antigen complex triggered CP activation and modulate the activity of CP downstream of antibody-antigen complex triggered CP activation. In particular, an anti-C1s antibody as disclosed herein can be used to therapeutically target antibody-antigen complex triggered CP activation and inhibit the activity of CP downstream of antibody-antigen complex triggered CP activation.

[0069] Therapeutically targeting at least one component involved in a CP-mediated disorder using an anti-C1s antibody as disclosed herein is to be understood as allowing for a precise modulation of the activity of CP and downstream effects of CP activity. In particular, therapeutically targeting at least one component involved in a CP-mediated disorder using an anti-C1s antibody as disclosed herein is to be understood as allowing for a precise inhibition of the activity of CP and downstream effects of CP activity.

[0070] The anti-C1s antibodies disclosed herein allow for the development of treatments that can be customized for each subject (e.g., dosage, frequency of administration), wherein the treatment can be easily continued and interrupted, and combined with other therapies. In certain strategic embodiments, the anti-C1s antibodies disclosed herein can be combined with other therapies that address multiple therapeutic targets and / or address deficiencies or undesirable effects of one of the therapies in the combination therapy.

[0071] Exemplary Examples of Anti-C1s Antibodies and Their Uses

[0072] Non-limiting exemplary embodiments of the anti-C1s antibodies of the present invention are now disclosed, particularly in the Examples, Tables and Figures.

[0073] Antibodies that can specifically bind to C1s and inhibit C1s function.

[0074] The anti-C1s antibodies of the invention can be identified and characterized using a functional cascade, wherein the first step in the cascade involves screening C1s-challenged clones for antibodies capable of binding to C1s, followed by sequencing the cognate variable regions, recombinant expression of chimeric antibodies having VH and VL from the mouse clones linked to human constant regions, purification of the recombinant antibodies, and screening of the antibodies using functional assays.

[0075] As demonstrated by the exemplary embodiments disclosed in Example 2, recombinant chimeric antibodies capable of modulating C1s function, as measured by an assay for IgM-induced C4 deposition from serum, were obtained. Exemplary recombinant chimeric anti-C1s antibodies capable of inhibiting C1s function, as measured by the ability to inhibit IgM-induced human C4 deposition from human serum and the ability to inhibit IgM-induced cynomolgus monkey C4 deposition from cynomolgus monkey serum, were obtained. Exemplary recombinant chimeric anti-C1s antibodies capable of inhibiting C1s function in a dose-dependent manner were obtained.

[0076] Humanized variants

[0077] Humanized antibodies comprising CDRs derived from non-human sources grafted into a humanized antibody framework are expected to be non-immunogenic when administered to human subjects. As demonstrated by the exemplary embodiments disclosed in Example 2, humanized anti-C1s antibody variants were successfully generated, tested, optimized, and selected. Humanization of the 2-7 and 2-8 antibodies resulted in humanized anti-C1s antibody variants, of which the antibodies identified as hz2-7 (H1L2) and hz2-8 (H1L2) had the highest in vitro activity. After preliminary design and testing, variants that demonstrated the desired antigen-binding affinity and cross-reactivity were selected for further evaluation and development, including but not limited to modifications to some parent CDR sequences to avoid potential undesirable events (such as aspartate isomerization and asparagine deamidation) and selected modifications to the framework sequences.

[0078] Anti-C1s antibodies with high affinity for biologically relevant targets

[0079] Exemplary anti-C1s antibodies exhibit high affinity for biologically appropriate targets. Anti-C1s antibodies exhibit high affinity binding to both zymogen (native) and active C1s proteins. As demonstrated by exemplary affinity measurements performed using various methods as disclosed in Examples 2 and 3 and illustrated in Figures 2 and 3, antibodies 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), hz2-8(H1L2 G80A), and hz2-8(H1L2 G80A / T82A) exhibit favorable affinity properties.

[0080] Anti-C1s antibodies with cross-reactivity to non-human targets

[0081] It is desirable that therapeutically useful antibodies or antibody fragments have sufficient cross-reactivity with non-human targets (non-human homologs) from sources relevant to additional studies (e.g., preclinical efficacy studies, animal models of disease, toxicology studies, etc.) such that the antibodies or antibody fragments should recognize, for example, rat homologs and / or primate homologs, such as homologs from cynomolgus monkeys. As demonstrated by exemplary embodiments disclosed in Example 2 and shown in Figure 2 in a functional screening assay, antibodies 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), hz2-8(H1L2 G80A), and hz2-8(H1L2 G80A / T82A) inhibited IgM-induced deposition of human C4 from human serum and IgM-induced deposition of cynomolgus monkey C4 from cynomolgus monkey serum, thereby providing antibodies raised against human C1s that are functionally cross-reactive with cynomolgus monkey C1s in the cynomolgus monkey system. As demonstrated by the exemplary embodiments of binding affinity measurements disclosed in Example 3 and shown in Figure 3, antibodies 2-7, 2-8, hz2-7(H1L2), hz2-8(H1L2), and hz2-8(H1L2G80A / T82A) exhibited detectable binding and high affinity to human C1s and cynomolgus monkey C1s, while antibodies 2-8 and hz2-8(H1L2 G80A / T82A) also exhibited detectable binding and fairly high affinity to rat C1s.

[0082] Anti-C1s antibodies that selectively inhibit the classical pathway (CP)

[0083] For CP-mediated conditions, such as those triggered by complement fixing autoantibodies bound to antigens, it is desirable to selectively inhibit CP from producing common complement system effectors, specifically CP at a step upstream of the C3 convertase. Selectively inhibiting CP should be understood as inhibiting the action of CP activation triggered by the antibody-antigen complex, while leaving the alternative and lectin complement pathways intact to combat infection. For CP-mediated conditions, selectively inhibiting CP at a step upstream of the C3 convertase production should be understood as selectively inhibiting the undesirable action of CP activation associated with the condition, while leaving the alternative and lectin complement pathways substantially intact to combat infection. The anti-C1s antibodies of the present invention have a selective action within CP by inhibiting C1s (i.e., inhibiting at least one C1s activity or function), thereby acting upstream of the formation of common complement system effectors.

[0084] As demonstrated by the exemplary embodiments disclosed in Example 2, the anti-C1s antibodies disclosed herein are capable of binding to C1s and inhibiting C1s functions involved in IgM-induced C4 deposition from human serum and cynomolgus monkey serum, wherein it is understood that inhibition of C4 deposition in a test assay indicates at least one of the following: C4 cleavage does not occur; C3 convertase formation is inhibited (prevented); and / or CP activation does not produce downstream effects mediated by the common complement pathway effector C3. Specifically, the anti-C1s antibodies disclosed herein are capable of specifically binding to C1s and inhibiting at least one activity or function of C1s, such that IgM-induced C4 deposition from human serum and cynomolgus monkey serum is inhibited, C4 cleavage is inhibited, C3 convertase formation is inhibited (prevented), and CP activation is inhibited.

[0085] The exemplary embodiments disclosed in Example 7 demonstrate that the anti-C1s antibodies disclosed herein are capable of modulating C1s functions involved in the classical pathway (CP) of complement. Sheep erythrocytes coated with rabbit anti-sheep erythrocyte antiserum (hemolysin), also known as antibody-sensitized sheep erythrocytes ("EA" or "EA cells"), have traditionally been used to measure the activity of the classical pathway (CP) of complement in serum samples. Commercially available EA cells (Complement Technology, #B202) and known assay systems were used to study complement-mediated EA lysis in response to exposure to serum. Humanized anti-C1s antibodies hz2-7 (H1L2), hz2-8 (H1L2), hz2-8 (H1L2 G80A) and hz2-8 (H1L2G80A / T82A) inhibited complement-mediated EA lysis in a dose-dependent manner (Example 7, Figure 7A and 7B ), among which antibody hz2-8(H1L2) was the most potent inhibitor, hz2-8(H1L2), antibodies hz2-8(H1L2 G80A) and hz2-8(H1L2G80A / T82A were also highly potent inhibitors, and antibody hz2-7(H1L2) was less potent and showed species dependence.

[0086] The anti-C1s antibodies disclosed herein show different levels of inhibition under different conditions, for example, using different antibodies under the same assay conditions, or using the same antibody under different assay conditions, such as using sera from different species. Thus, the anti-C1s antibodies disclosed herein provide tools and approaches for studying the role of specific epitopes and binding interactions in the CP pathway.

[0087] Composition

[0088] Compositions comprising an anti-Cls antibody of the present application and a pharmaceutically acceptable carrier or excipient appropriate for the intended use of each composition are provided. Such carriers include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, excipients, stabilizers, preservatives, or combinations thereof. It will be appreciated that pharmaceutical formulations should be matched to the mode of administration.

[0089] The anti-Cls antibodies disclosed herein can be administered by any suitable means, including, but not limited to, injection or parenteral administration. Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, intraspinal (including epidural or intrathecal), intraocular, intracerebral, intracerebroventricular, intracardiac, intradermal / intracutaneous, intraarticular, intralymphatic, or intrabone administration. The anti-Cls antibodies disclosed herein can be formulated into compositions to be introduced into the circulatory system by parenteral administration, particularly by intravenous or intraarterial administration. The anti-Cls antibodies disclosed herein can be administered using a device, or as a depot, or in the form of a slow release formulation, e.g., a semipermeable matrix of a solid hydrophobic polymer containing the antibody, or a microencapsule, to allow a slow and / or measured and / or local delivery. The anti-Cls antibodies disclosed herein can be formulated and administered using a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in a coarse suspension.

[0090] Methods

[0091] Methods of treating complement-mediated disorders using an effective amount of an anti-Cls antibody disclosed herein are provided. Without wishing to be bound by a particular mechanism of action, methods of precisely targeting and inhibiting Cls using an anti-Cls antibody disclosed herein are provided, thereby allowing for the precise targeting of Cls to selectively modulate the activity of CP. Methods for precisely targeting and inhibiting Cls can be methods using routes of administration to target the location or system involved in the disorder, methods using timing of administration to target critical time periods, and combinations of these methods. Methods for treating complement-mediated disorders in which CP is activated by complement-fixing autoantibodies bound to self-antigens are provided, and in which such activation of CP results in deleterious effects on tissues bearing the self-antigens. Methods for treating disorders associated with unwanted activation of CP are provided, in which such methods use an effective amount of an anti-Cls antibody disclosed herein to selectively modulate the activity of CP processes such as C4 cleavage and deposition, resulting in unwanted attack and destruction of self-tissues. In particular, methods of treating autoimmune conditions by inhibiting downstream effects of CP activation by complexes of complement-fixing autoantibodies bound to self-antigens are provided.

[0092] Methods and compositions are provided for precisely targeting and inhibiting C1s using a route of administration to target a location or system involved in the condition. Without wishing to be bound by a particular mechanism of action, methods and compositions are provided for treating a condition characterized by unwanted, abnormal, inappropriate, or excessive CP activation in the circulatory system that can be targeted by, for example, introducing an anti-C1s antibody into the circulatory system by intravenous or intraarterial administration. Without wishing to be bound by a particular mechanism of action, methods and compositions are provided for treating a condition characterized by unwanted, abnormal, inappropriate, or excessive CP activation in the central nervous system (CNS) that can be targeted by introducing an anti-C1s antibody into one or more CNS tissues or structures by intraspinal (including epidural or intrathecal) administration, intraocular administration, intracerebral administration, or intraventricular administration. Without wishing to be bound by a particular mechanism of action, methods and compositions are provided for treating a condition characterized by unwanted, abnormal, inappropriate, or excessive CP activation in one or more joints that can be targeted by introducing an anti-C1s antibody into one or more joints by intra-articular administration.

[0093] Methods are provided for treating immune thrombocytopenic purpura (ITP) by precisely targeting C1s using an effective amount of an anti-C1s antibody disclosed herein. Compositions of anti-C1s antibodies disclosed herein can be formulated for introduction into the circulatory system, e.g., intravenous and / or intra-arterial administration, and introduced by methods that allow for targeting and inhibition of C1s in the circulatory system. Without wishing to be bound by a particular mechanism of action, methods using the anti-C1s antibodies disclosed herein can inhibit antibody-mediated platelet removal by inhibiting CP. Without wishing to be bound by a particular mechanism of action, methods using the anti-C1s antibodies disclosed herein can inhibit deposition of C4 on platelets, which can lead to undesirable platelet destruction. When the CP pathway has been activated by complement-fixing autoantibodies bound to self-antigens on platelets, the methods provided herein allow for precise targeting of C1s, wherein inhibition of C1s activity interrupts the CP pathway and prevents downstream effects, including but not limited to inhibition of IgM-induced C4 deposition from serum; inhibition of antibody-antigen complex-mediated C4 cleavage; inhibition of antibody-antigen complex-mediated C4b deposition on platelets; platelet destruction; platelet elimination; thrombocytopenia; and hemorrhagic disorders.

[0094] Methods are provided for treating immune neuromyelitis optica (NMO) by precisely targeting C1s using an effective amount of an anti-C1s antibody disclosed herein. Compositions of anti-C1s antibodies disclosed herein can be formulated for targeted introduction into the CNS, specifically for intraspinal (e.g., epidural or intrathecal) or intracerebral / intraventricular introduction, and are introduced by methods that allow targeting and inhibition of C1s in the CNS where complement-dependent cytotoxicity (CDC) may occur. Without wishing to be bound by a particular mechanism of action, methods using the anti-C1s antibodies disclosed herein can inhibit complement-dependent cytotoxicity (CDC) triggered by autoantibodies that bind to autoantigens, including but not limited to inhibition of antibody-antigen complex-mediated C4 cleavage; inhibition of antibody-antigen complex-mediated C4b deposition on astrocytes; inhibition of downstream effects of antibody-antigen complex-mediated CP activation and inhibition of downstream inflammation leading to, for example, oligodendrocyte and neuronal damage, as well as inflammatory demyelinating lesions in the CNS, specifically the spinal cord and optic nerve.

[0095] The methods and compositions provided herein allow for precise timing of treatment. Without wishing to be bound by a particular mechanism of action, the anti-C1s antibodies disclosed herein can be administered during the acute phase of a condition to inhibit CP-mediated processes during the acute phase of the condition. Without wishing to be bound by a particular mechanism of action, selective inhibition of CP-mediated processes during the acute phase of a condition using anti-C1s antibodies can interrupt the acute event and / or prevent progression of the condition, and then allow CP to resume normal activity after the anti-C1s antibody has dissociated from the C1s target.

[0096] The methods and compositions provided herein allow for precise targeting by timing and location of treatment. Without wishing to be bound by a particular mechanism of action, the timing of administration of the anti-C1s antibodies disclosed herein can occur before (e.g., in response to an indicator that an acute phase may develop) or during the acute phase of a condition to selectively inhibit CP-mediated processes during the acute phase without inhibiting the function of other complement pathways, and the route of administration can be selected to target specific tissues, systems, or structures involved in the condition. Without wishing to be bound by a particular mechanism of action, the use of anti-C1s antibodies to inhibit CP-mediated processes before or during the acute phase of a condition can prevent or interrupt the acute event without inhibiting the normal function of other complement pathways, and thereafter allow CP to return to normal activity after the anti-C1s antibody has dissociated from the C1s target.

[0097] The methods and compositions provided herein allow for control of the amount of therapeutic agent present at one or more time points during treatment. The anti-C1s antibodies disclosed herein can inhibit C1s activity and selectively inhibit CP pathway activity in a dose-dependent manner. Without wishing to be bound by a particular mechanism of action, the dose sufficient to ameliorate at least one biological effect or symptom of a condition ("effective amount") can depend on factors specific to the subject in need thereof, such that treatment can comprise determining the dose of anti-C1s antibody required to constitute an effective amount for the subject to be treated and administering to the subject an effective amount of the anti-C1s antibody, wherein the level of the therapeutic agent (anti-C1s antibody) is expected to decrease following treatment due to dissociation from the C1s target and clearance from the blood.

[0098] As provided herein, methods for treating complement-mediated disorders comprise administering to a subject in need thereof an effective amount of an anti-C1s antibody disclosed herein, wherein administration of the effective amount of the anti-C1s antibody ameliorates at least one biological effect or symptom associated with the disorder. Methods are provided for treating complement-mediated disorders associated with autoantibody-triggered CP activation, wherein administering to a subject in need thereof an effective amount of an anti-C1s antibody disclosed herein inhibits the CP pathway and prevents the undesirable downstream effects of CP activation.

[0099] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0100] Examples

[0101] Example 1: Antibody Generation and Identification of Antibodies Binding to C1S

[0102] The generation of novel monoclonal antibodies against C1s was performed under contract by GenScript USA, Inc. (Piscataway, NJ) using in vivo rodent immunization and single B cell cloning technology. The active form of the C1s protein (CompTech A104 C1s enzyme) was used as the antigen to immunize BALB / cJ and SJL mice (The Jackson Laboratories). Sufficient plasma titers were obtained as determined by enzyme-linked immunosorbent assay (ELISA), which triggered downstream antibody recovery and screening activities. Secondary lymphoid organs were harvested for CD138 plasma cell enrichment, which were then loaded into 14K chip (Berkeley Lights, Inc., Emeryville, CA) to screen single B cells that secrete antibodies that specifically bind to the C1s antigen. Positive single B cell clones were selected and derived for subsequent variable domain sequencing and recombinant antibody expression. Full-length recombinant chimeric antibodies were obtained by expression in ExpiCHO cells using pcDNA3.4-VH-CH (wherein VH was from a positive B cell clone (mouse) and CH was IgG1 (human)) and pcDNA3.4-VL-CL (wherein VL was from a positive B cell clone (mouse) and CL was κ (human)). The functional activity of the recombinantly expressed and purified antibodies was further screened in an in vitro cell-free system. From the obtained positive C1s-binding B cell clones, 159 clones were successfully derived for antibody sequencing, and 39 clones were sequenced using a pair of cognate heavy and light chains. All 39 anti-C1s clones were recombinantly expressed and purified, and functional screening was performed.

[0103] Example 2. Functional screening of anti-C1S antibodies

[0104] Anti-C1s antibodies from all 39 anti-C1s clones were recombinantly expressed and purified, and then subjected to a functional screening assay to identify anti-C1s antibodies capable of inhibiting IgM-induced C4 deposition. Briefly, anti-C1s antibodies were incubated with serum in IgM-coated plates under conditions suitable for IgM-induced C4 deposition from serum, and the amount of deposited C4 was subsequently detected. Based on the functional model, this functional assay serves as a surrogate assay for C1s activity, in which C4 must be cleaved by active C1s prior to C4b deposition (specifically, deposition of C4b on target cells such as platelets), such that measurement of the effect of anti-C1s antibodies on IgM-induced C4 deposition from serum is used to report the effect of anti-C1s antibodies on C1s activity. This functional assay serves as a surrogate assay for C1s activity (C1s function, C1s biological activity) in antibody-antigen complex-mediated CP activation and the downstream effects of such CP activation (see Figure 1 Anti-C1s antibodies that inhibited IgM-induced C4 deposition were selected for further study.

[0105] Functional screening was performed by first coating 96-well plates with 300 ng / well of IgM from human serum (Sigma-Aldrich I8260) overnight. The next morning, the plates were blocked with gelatin blocking buffer (1% in PBS, Alfa Aesar J62755) for 1 hour before adding various concentrations of anti-C1s antibody diluted in gelatin blocking buffer to each well. After briefly equilibrating the anti-C1s antibody by gently shaking the plate, NHS (normal human serum, CompTech NHS) was added to each well to a final concentration of 1.25% (v / v). The plates were mixed thoroughly and incubated at 37°C for 1 hour to allow C4 deposition induced by IgM. The plates were then washed thoroughly before incubating with goat anti-human C4 (CompTech A205) diluted in gelatin blocking buffer for 1 hour. After incubation, the plates were washed again and incubated for 1 hour with rabbit anti-goat IgG (H+L) secondary antibody HRP conjugated (Invitrogen 81-1620) diluted in gelatin blocking buffer. Finally, the plates were washed with PBS and developed with ELISA liquid substrate (Sigma-Aldrich T4444), followed by stopping the reaction by adding the same volume of 1 M H2SO4 ELISA liquid substrate. 450nm The bound antibody was measured by absorbance at 4°C to determine the amount of deposited C4. Figure 2A Results are shown for the top candidates, recombinant antibodies from clone 2-7 (designated anti-C1s antibody 2-7) and from clone 2-8 (designated anti-C1s antibody 2-8), as well as results for an antibody from one of the non-functional clones (3-30) that binds to C1s but does not significantly inhibit C1s function as measured in a functional assay. The half-maximal inhibitory concentration (IC50) for each antibody, which shows the ability to inhibit IgM-induced C4 deposition, was determined, with the IC50 values ​​for anti-C1s antibody 2-7 and anti-C1s antibody 2-8 being 0. 50 Values ​​ranged between 0.3 μg / ml and 0.7 μg / ml.

[0106] The functional screening assay described above was repeated using normal cynomolgus monkey serum (CMS, CompTech NCYS). Figure 2B It was shown that anti-C1s antibodies 2-7 and 2-8 also effectively inhibited IgM-induced C4 deposition in cynomolgus monkeys, with the IC of anti-C1s antibody 2-7 being 50 is approximately 0.3 μg / ml, and the IC of anti-C1s antibody 2-8 50 It is about 0.5μg / ml.

[0107] Generation and screening of humanized anti-C1s antibody variants

[0108] CDR is utilized to be transplanted onto the human antibody framework and carry out the humanization of parental antibody.First, the homology modeling of the 3-dimensional structure of parental antibody is carried out to set up the structural model of parental antibody. Based on the removal of overall sequence identity, matching VH-VL interface position, similar classification of CDR standard position (Kabat numbering) and potential N glycosylation site, the amino acid sequence of variable segment framework is identified. Humanized antibody is designed by producing a plurality of hybridization sequences that the selected part of parental antibody sequence is fused with human framework sequence. The isotype that selects to form humanized antibody is the IgG1 of heavy chain and the IgG1 κ of light chain. Use 3D model, by eye and computer modeling, these humanization sequences are systematically analyzed to isolate the sequence that is most likely to retain antigen binding. The goal is to maximize the amount of the people's sequence in the final humanized antibody while maintaining the original antibody specificity. Then, humanized variants that paired humanized VH and VL were expressed in ExpiCHO cells using the pcDNA3.4-VL-CL-IRES-VH-CH vector, where IRES is an "internal ribosome entry site" that allows simultaneous expression of light and heavy chains. The humanized variants were designed and constructed. The expressed recombinant humanized variants were then purified for affinity analysis.

[0109] As part of affinity analysis, in one round of design, generation and testing of variants, three VH variants were generated with the VH-CDRs of the parent antibody in corresponding positions in three different human IgG1 derived frameworks, and three VL (VK) variants were generated with the VL-CDRs of the parent antibody in corresponding positions in three different human IgG1κ derived frameworks. A total of nine (9) humanized variants representing each combination of VH and VL (VK) variants were prepared according to a 3VH x 3VK matrix, and the antigen binding properties (k on 、k off , KD) for evaluation.

[0110] Variants that showed the desired antigen binding affinity based on the cutoff value were selected for further evaluation and development.In some cases, the parent CDR sequences were modified to avoid potential undesirable events such as aspartic acid isomerization or asparagine deamidation.

[0111] The humanized anti-C1s antibodies hz2-7 (H1L2) and hz2-8 (H1L2) were selected from all humanized variants tested because of their excellent ability to inhibit IgM-induced C4 deposition from NHS and CMS (see Figure 2C and 2D ). IC 50 Comparison of the values ​​showed that antibody hz2-7 (H1L2) inhibited IgM-induced C4 from NHS ( Figure 2D) and CMS( Figure 2C ) deposition was not as good as that of its parental antibody 2-7 ( Figure 2A (NHS), Figure 2B (CMS)) was effective, but antibody hz2-8 (H1L2) inhibited IgM-induced C4 from NHS ( Figure 2C ) and CMS( Figure 2D ) deposition than its parent antibody 2-8 ( Figure 2A (NHS), Figure 2B (CMS)) is more effective.

[0112] Figure 2C Results are shown for the inhibition of IgM-induced C4 deposition from NHS by humanized antibodies hz2-7 (H1L2) and hz2-8 (H1L2), as well as variants hz2-7 (H1L2 G131A), hz2-8 (H1L2 G80A), and hz2-8 (H1L2 G80A / T82A). For C1s-binding antibodies capable of inhibiting IgM-induced C4 deposition from NHS, the following IC values ​​were calculated. 50 value( Figure 2C ): hz2-7 (H1L2) IC 50 The IC value of hz2-8(H1L2) is 1.1 μg / ml. 50 The IC of hz2-8(H1L2G80A) is 0.2μg / ml; 50 is 0.5 μg / ml; and the IC of hz2-8 (H1L2 G80A / T82A) 50 0.4μg / ml. Figure 2C As shown in Figure 2, the variant hz2-7 (H1L2 G131A) became functionally inactive after the introduction of the mutation G131A located in CDR3, so the IC of the functionally inactive variant hz2-7 (H1L2 G131A) was not calculated. 50 Variants hz2-8(H1L2G80A) and hz2-8(H1L2 G80A / T82A) were both functionally active, but neither was as good as hz2-8(H1L2)( Figure 2C )efficient.

[0113] Figure 2D Results are shown for the inhibition of IgM-induced C4 deposition from CMS by humanized antibodies hz2-7(H1L2) and hz2-8(H1L2), as well as variants hz2-7(H1L2G131A), hz2-8(H1L2G80A), and hz2-8(H1L2G80A / T82A). Figure 2C and Figure 2D The comparison shows that when using CMS ( Figure 2D ) instead of the NHS( Figure 2CFor C1s-binding antibodies capable of inhibiting IgM-induced C4 deposition from CMS, the following IC 50 value( Figure 2D ): hz2-7 (H1L2) IC 50 The IC value of hz2-8(H1L2) is 0.9 μg / ml. 50 The IC of hz2-8 (H1L2 G80A) is 0.2 μg / ml. 50 is 0.5 μg / ml; and the IC of hz2-8 (H1L2 G80A / T82A) 50 The IC value of the functionally inactive variant hz2-7 (H1L2 G131A) was not calculated. 50 value.

[0114] Antibody hz2-8 (H1 L2G80A / T82A), which has both the aspartate isomerization and asparagine deamidation sites removed, was selected for further development.

[0115] The following Table 1.a. shows the SEQ ID NOs assigned to the heavy chain (HC) and light chain (LC) of each full-length antibody used in the Examples, and presents the VH amino acid sequence and SEQ ID NO: and the VL amino acid sequence and SEQ ID NO: of each antibody, wherein the CDRs are indicated by underlines.

[0116]

[0117]

[0118]

[0119] Example 3. Binding affinity of anti-C1S antibodies

[0120] Anti-C1s Antibody Affinity and Binding Kinetics: Measurements Using Biolayer Interferometry

[0121] use The RED96e system (Sartorius AG) uses biolayer interferometry for anti-C1s antibody affinity measurements and binding kinetics. For the first baseline, pre-hydrated anti-human IgG Fc capture (AHC) biosensors were first equilibrated in 1x KB (kinetic buffer, 1x PBS pH 7.4 + 0.02% Tween-20 + 0.1% BSA) for 120 seconds, followed by incubation with 10 mg / ml anti-C1s antibody (2-7, Figure 3A ; 2-8, Figure 3B ;hz2-7(H1L2), Figure 3C;hz2-8(H1L2 G80A / T82A), Figure 3D ) was loaded onto the AHC biosensor for 240 seconds. A second baseline signal was then established for 120 seconds before associating with various concentrations of active human C1s (CompTech A104) for 240 to 360 seconds. Finally, the analytes were dissociated in 1x KB for 360 seconds. Anti-C1s antibodies 2-7 and 2-8 were also analyzed against human C1s zymogen (CompTech A103) (data not shown). Data analysis was performed using HT software (Sartorius). KD and k for each antibody and target combination were calculated. on 、k off and R 2 Values, such as Figure 3E As summarized in , high affinity binding was shown with KD values ​​in the low nanomolar to picomolar range. Figure 3E It was further shown that anti-C1s antibodies 2-7 and 2-8 gave identical calculated values ​​for C1s zymogen and active C1s protein, where K D The values ​​ranged from 0.14 nM to 1.2 nM.

[0122] Anti-C1s Antibody Binding Affinity: Measurement Using ELISA

[0123] ELISA was performed by first using 200 ng / well of human active C1s (CompTech A104, Figure 3F ) or human C1s zymogen (CompTech A103, Figure 3G ) protein coated 96-well plates overnight. The next morning, under gentle shaking, the plate was blocked for 1 hour with SuperBlock T20 blocking buffer (Thermo Fisher Scientific Inc., catalog number 37536). Then, various concentrations of anti-C1s antibody diluted in SuperBlock T20 blocking buffer were added to the plate and incubated for 1 hour under gentle shaking. Human IgG1 (BioXCell BP0297) was used as a negative control. Under gentle shaking, before incubation for 1 hour with HRP-conjugated secondary antibodies diluted in SuperBlock T20 blocking buffer (goat anti-human IgG antibody, HRP conjugate (Millipore AP309P)), the plate was washed. Finally, the plate was washed and developed with ELISA liquid substrate (Sigma Aldrich), followed by stopping the reaction by adding the same volume of 1M H2SO4 ELISA liquid substrate. The plate was then analyzed by OD 450nm The bound antibody was measured by absorbance at 4°C.

[0124] Figure 3F and Figure 3G Together, the top candidates 2-7 and 2-8, as well as their humanized variants hz2-7 (H1L2) and hz2-8 (H1L2 G80A / T82A), respectively, all showed similarity to human active C1s (CompTech A104) ( Figure 3F ) and human C1s zymogen (CompTech A103) ( Figure 3G ) with strong binding, where the half-maximal effective concentration (EC 50 ) values ​​ranged from 8 ng / ml to 13 ng / ml. The EC values ​​for each antibody and target combination were 50 The determination was as follows: For antibodies 2-7, the EC of human active C1s 50 The EC of human C1s zymogen is 12 ng / ml. 50 9 ng / ml; for antibodies 2-8, the EC of human active C1s 50 The EC of human C1s zymogen is 12 ng / ml. 50 is 10 ng / ml; for variant hz2-7 (H1L2), the EC of human active C1s 50 The EC of human C1s zymogen is 13 ng / ml. 50 was 12 ng / ml; and for variant hz2-8 (H1L2 G80A / T82A), the EC of human active C1s 50 is 10 ng / ml and the EC of human C1s zymogen 50 A control assay using the same concentration of human IgG1 showed that these patterns did not reflect nonspecific binding ( Figures 3F-3G ).

[0125] Anti-C1s Antibody Cross-Reactivity: Measurement Using Biolayer Interferometry

[0126] For the first baseline, pre-hydrated anti-human IgG Fc capture (AHC) biosensors were first equilibrated in 1x KB (kinetic buffer, 1x PBS pH 7.4 + 0.02% Tween-20 + 0.1% BSA) for 120 seconds and then incubated with 10 mg / ml anti-C1s antibody (2-7, Figure 3H and 3N ; 2-8, Figure 3I 、 3L and 3O; hz2-7(H1L2), Figure 3J and 3P ;hz2-8(H1L2 G80A / T82A), Figure 3K 、 3Mand 3Q) were loaded on the AHC biosensor for 240 seconds. Then, incubation with various concentrations of human C1s (generated in-house (SEQ ID NO: 99), Figures 3H-3K ), rat C1s (generated in-house (SEQ ID NO: 105), Figures 3L-3M ) or cynomolgus monkey C1s (generated in-house (SEQ ID NO: 103), Figures 3N-3Q A second baseline signal was established for 120 seconds before the analyte was associated for 240 to 360 seconds (as shown in FIG). Finally, the analyte was dissociated in 1x KB for 360 seconds. Data analysis was performed using HT software (Sartorius). KD and k for each antibody and target combination were calculated. on 、k off and R 2 Values, such as Figure 3R The top candidates 2-7 and 2-8 and their humanized variants hz2-7 (H1L2) and hz2-8 (H1L2G80A / T82A) showed strong binding to human C1s and cynomolgus monkey C1s, respectively, but did not show strong binding to mouse C1s ( Figure 3R ) combination, where K D The anti-C1s antibodies 2-7 and its humanized variant hz2-7 (H1L2) did not show binding to rat C1s, while 2-8 and its humanized variant hz2-8 (H1L2 G80A / T82A) showed binding to rat C1s, with K D Values ​​ranged from 16nM to 17nM ( Figure 3R The binding affinity of 2-8 and hz2-8 (H1L2 G80A / T82A) to rat C1s is weaker than that to human C1s and cynomolgus monkey C1s ( Figure 3R ).

[0127] Anti-C1s Antibody Cross-Reactivity: Measurement Using ELISA

[0128] The ELISA was performed by first using 200 ng / well of human C1s (identified as full-length human C1s, HuC1s, wt human C1s) (generated in-house (SEQ ID NO: 99), Figure 3S ), mouse C1s (generated in-house (SEQ ID NO: 101), Figure 3T ), rat C1s (generated in-house (SEQ ID NO: 105), Figure 3U ) or cynomolgus monkey C1s (generated in-house (SEQ ID NO: 103), Figure 3V) were performed overnight using protein-coated 96-well plates. The next morning, the plates were blocked with SuperBlock T20 blocking buffer (Thermo Scientific 37536) for 1 hour with gentle shaking. Then, various concentrations of anti-Cls antibodies diluted in SuperBlock T20 blocking buffer were added to the plates and incubated for 1 hour with gentle shaking. Mouse IgG (Sigma Aldrich I5381) and human IgGl (BioXCell BP0297) were used as negative controls. Before incubation with HRP-conjugated secondary antibodies (goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody HRP (Jackson ImmunoResearch G-21040) or goat anti-human IgG antibody, HRP conjugate (Millipore AP309P)) diluted in SuperBlock T20 blocking buffer for 1 hour with gentle shaking, the plates were washed. Finally, the plates were washed and developed with ELISA liquid substrate (Sigma Aldrich), followed by stopping the reaction by adding the same volume of 1 M H2SO4. Bound antibody was measured by absorbance at OD 450nm Top candidates 2-7 and 2-8, and their humanized variants hz2-7 (H1L2) and hz2-8 (H1L2 G80A / T82A), respectively, all showed strong binding to human Cls Figure 3S ) and cynomolgus monkey Cls Figure 3V ), but no binding to mouse Cls Figure 3T , with EC 50 between 4 ng / ml and 15 ng / ml. Anti-Cls 2-7 and its humanized variant hz2-7 (H1L2) showed no binding to rat Cls, while 2-8 and its humanized variant hz2-8 (H1L2 G80A / T82A) showed weaker binding to rat Cls Figure 3U , with EC 50 between 63 ng / ml and 105 ng / ml.

[0129] Example 4. Binding of anti-C1S antibodies to full-length and truncated C1S measured by ELISA

[0130] The binding of anti-Cls antibodies to full-length human Cls protein, full-length mouse Cls protein, the NHC fragment of human Cls protein Figure 5B for alignment), and an intermediate fragment of human Cls located within NHC identified as “M151” or “huC1sM151” encompassing 151 amino acids at residues 272 to 422 of human Cls located upstream of the autocleavage site R422-I423 Figure 5BFor comparison purposes). Constructs were developed for the expression of full-length human C1s (HuC1s or "wt human C1s") (SEQ ID NO: 99), full-length mouse C1s (moC1s) (SEQ ID NO: 101), human C1s deletion mutant 1 (huC1sM151) (SEQ ID NO: 107), and human C1s deletion mutant 2 (huC1sNHC) corresponding to the NHC of C1s (SEQ ID NO: 109), with a C-terminal 6xHis tag on each expressed protein. All expression and purification of full-length and truncated C1s constructs (SEQ ID NOs: 100, 102, 108, 110) and the resulting proteins (SEQ ID NOs: 99, 101, 107, 109) were performed in-house.

[0131] The ELISA was performed by first coating a 96-well plate with 200 ng / well of full-length or truncated C1s protein overnight. The next morning, the plate was blocked with Superblock T20 blocking buffer (Thermo Fisher Scientific 37536) for 1 hour with gentle shaking. Then, various concentrations of anti-C1s antibody diluted in Superblock T20 blocking buffer were added to the plate and incubated for 1 hour with gentle shaking. Mouse IgG (Sigma-Aldrich I5381) and human IgG1 (BioXCell BP0297) were used as negative controls. The plate was washed with gentle shaking before incubation for 1 hour with HRP-conjugated secondary antibodies (goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody HRP (Invitrogen G-21040) or goat anti-human IgG antibody, HRP conjugate (Millipore AP309P)) diluted in Superblock T20 blocking buffer. Finally, the plates were washed and developed with ELISA liquid HRP substrate (Sigma-Aldrich), and the reaction was stopped by adding the same volume of 1 M H2SO4 as the ELISA liquid substrate. 450nm The bound antibodies were measured by measuring the absorbance at . The results are shown in Figure 4. Top candidates 2-7 and 2-8 and their humanized variants hz2-7 (H1L2) and hz2-8 (H1L2), respectively, showed binding to full-length human C1s ( Figure 4A ), but did not show strong binding to full-length mouse C1s ( Figure 4B All anti-C1s antibodies showed higher EC binding to two truncated human C1s (NHC and M151) compared to full-length C1s. 50 ( Figure 4C -D), suggesting that human C1s (M151) retains the primary binding site for anti-C1s and that the flanking regions may help maintain the correct (or preferred) conformation.

[0132] Example 5. Binding of anti-C1S antibodies to full-length and truncated C1S demonstrated by Western blotting

[0133] Western blot analysis was performed by first resolving 200 ng / lane of full-length or truncated C1s (human C1s, mouse C1s, NHC of human C1s, and M151 of human C1s) on SDS-PAGE with or without a reducing agent (Invitrogen B0004) as follows: in lane 1, 200 ng full-length human C1s (HuC1s; SEQ ID NO: 99); in lane 2, 200 ng full-length mouse C1s (MoC1s; SEQ ID NO: 101); in lane 3, 200 ng truncated human C1s M151 (HuC1s(M151); SEQ ID NO: 107); and in lane 4, 200 ng truncated human C1s NHC (HuC1s(NHC); SEQ ID NO: 109). The protein was then transferred to a nitrocellulose membrane and blocked overnight with 5% blotto (ChemCruz sc-2325) diluted in TBST (Teknova T1688). The next morning, the blocked membrane was first incubated with 1 μg / ml of anti-C1s antibody diluted in 5% blotto-TBST for 1 hour with gentle shaking. The membrane was then washed with gentle shaking and incubated with HRP-conjugated secondary antibody diluted in 5% blotto-TBST (goat anti-human IgG antibody, HRP conjugate (Millipore AP309P)) for 1 hour. Finally, the membrane was washed thoroughly and developed using SuperSignalWest Dura persistent substrate (Thermo Fisher Scientific 34075) and imaged under an azure biosystem. Representative results are shown in Figure 5A In the present study, the results of anti-C1s antibody 2-8 were presented.

[0134] Wherein the blot on the left is from a gel run under non-reducing conditions and the blot on the right is from a gel run under reducing conditions. On the blot of the gel run without a reducing agent (left), lane 1 shows antibody 2-8 binding to two (2) major bands of full-length human C1s (HuC1s), with the top band corresponding to full-length HuC1s and the bottom band corresponding to the auto-cleaved heavy chain, lane 2 shows no detectable antibody 2-8 binding to full-length mouse C1s (MoC1s), lane 3 shows antibody 2-8 binding to multiple bands of truncated human C1s M151 (HuC1s(M151)), and lane 4 shows antibody 2-8 binding to several high molecular weight bands of truncated human C1s NHC (HuC1s(NHC)). On the blot (right) of the gel run with a reducing agent, lane 1 shows low levels of antibody 2-8 binding to the band corresponding to the autocleaved heavy chain of full-length human C1s (HuC1s), lane 2 shows no detectable antibody 2-8 binding to full-length mouse C1s (MoC1s), lane 3 shows low levels of antibody 2-8 binding to a monomer of truncated human C1s M151 (HuC1s(M151)), and lane 4 shows low levels of antibody 2-8 binding to a monomer of truncated human C1s NHC (HuC1s(NHC)).

[0135] The results using anti-C1s antibodies 2-7, hz2-7(H1L2) and hz2-8(H1L2) showed that Figure 5A The binding patterns were identical to those for antibody 2-8 shown in Figure 2 (data not shown). These results show that the binding sites for anti-C1s antibody 2-7, anti-C1s antibody 2-8, anti-C1s antibody hz2-7 (H1L2), and anti-C1s antibody hz2-8 (H1L2) are located within the human C1sM151 region on the NHC.

[0136] Example 6. Epitope Mapping of Anti-C1S Antibodies Using Truncated C1S and C1S with Point Mutations

[0137] As indicated in Examples 4-5, the epitope of the anti-C1s antibodies disclosed herein is located within human C1s (M151) (SEQ ID NO: 107). Human C1s (M151) is the last 151 amino acids upstream of the autocleavage site (R422-I423) within the human C1s NHC. To further define the epitope within human C1s (M151), an additional truncated C1s mutant was designed. This mutant was designated human C1s deletion mutant 3 (NHCΔ33) (SEQ ID NO: 111) and lacks the G390-R422 segment of the human C1s NHC ( Figure 5BC1s(NHCΔ33) (SEQ ID NO: 111) was cloned and expressed with a C-terminal 6xHis tag, and its expression and purification were performed in-house. Figure 5B Shown are schematic comparisons and alignments of full-length human C1s, HuC1s (SEQ ID NO: 99), human C1s deletion mutant 1 (M151), HuC1s (M151) (SEQ ID NO: 107), human C1s deletion mutant 2 (NHC), HuC1s (NHC) (SEQ ID NO: 109), and human C1s deletion mutant 3 (NHCΔ33), human C1s (NHCΔ33) (SEQ ID NO: 111) used in these experiments.

[0138] Binding of various anti-C1s antibodies to human C1s (NHCΔ33) (SEQ ID NO: 111) was performed by ELISA as described in Example 4, and the results are shown in Figure 6A Hz2-7(H1L2) showed almost no binding to human C1s(NHCΔ33), while hz2-8(H1L2 G80A) and hz2-8(H1L2 G80A / T82A) showed strong binding to C1s(NHCΔ33), with EC 50 The values ​​were 8 ng / ml and 15 ng / ml, respectively. As expected, human IgG1 showed no binding. This different binding profile of the humanized 2-7 variant versus the humanized 2-8 variant strongly suggests that antibodies 2-7 and 2-8 bind to different epitopes within human C1s (M151), as demonstrated in Example 4. The results indicate that the epitope of hz2-7 (H1L2) is within the 33 amino acids upstream of the cleavage site (G390-R422), and the epitopes of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) are located within the remainder of M151 (Y272-A389).

[0139] To identify the epitopes of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A), various point mutations were introduced into human C1s alone or in double / triple combinations, and 20 human C1s mutants designated M1-M20 (SEQ ID NOs: 112-128 and 141-143) were obtained, as shown in Table 2.

[0140] For all point mutations, the residue at a position in human C1s was replaced by the corresponding residue in mouse C1s. Of the 22 residues mutated, 6 residues were identical between human C1s and rat C1s, 10 residues were identical between rat C1s and mouse C1s, and the remaining 6 residues were unique at the corresponding positions in human C1s, rat C1s, and mouse C1s. M1-M20 of human C1s (SEQ ID NOs: 112-128 and 141-143) were cloned with a C-terminal 6xHis tag, expressed in-house, and purified.

[0141] Table 2: Point mutations in human C1s and the corresponding residues in rat and mouse C1s

[0142]

[0143] Binding of anti-C1s antibodies to mutants M1-M20 of human C1s was measured by ELISA as described in Example 4, and the results are shown in Figure 6B-6U All anti-C1s humanized variants, including hz2-7(H1L2), hz2-8(H1L2 G80A), and hz2-8(H1L2G80A / T82A), maintained strong binding properties to most (16 of 20) C1s mutants, including M1( Figure 6B )、M2( Figure 6C )、M3( Figure 6D )、M4( Figure 6E )、M7( Figure 6H )、M8( Figure 6I )、M10( Figure 6K )、M11( Figure 6L )、M12( Figure 6M )、M13( Figure 6N )、M15( Figure 6P )、M16( Figure 6Q )、M17( Figure 6R )、M18( Figure 6S )、M19( Figure 6T ) and M20( Figure 6U) and human IgG1 did not show binding to any of the mutants. These results strongly suggest that 20 of the 22 point mutations (i.e., M277I, P278S, P280A, E282K, D283I, P285A, V288T, A292D, Q303K, A320S, N329D, K331Q, D343Y, S349P, E351A, S360N, R368H, N380H, G381E, and G382E) have no effect on the binding of hz2-7 (H1L2), hz2-8 (H1L2 G80A), and hz2-8 (H1L2 G80A / T82A) to C1s. Thus, the 20 residues are not critical binding sites for any of the anti-C1s antibodies examined.

[0144] As shown in Figure 6A , the binding site of hz2-7 (H1L2) is located within the last 33 amino acids of C1s NHC (G390-R422). M1-M20 of human C1s are not expected to affect binding of hz2-7 (H1L2) because none of the point mutations are located within this region. Surprisingly, M14 of human C1s (SEQ ID NO: 125) containing only the single point mutation R316H completely abolished binding of hz2-7 (H1L2) while having no effect on the binding of hz2-8 (H1L2 G80A) or hz2-8 (H1L2 G80A / T82A) ( Figure 6O ). Human IgG1 did not show binding to M14 as expected ( Figure 6O ). These results strongly suggest that, in addition to G390-R422, R316 is another critical residue involved in the binding of hz2-7 (H1L2), further confirming the previous indication that the binding site of hz2-7 (H1L2) is different from the binding site of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A).

[0145] In addition, as shown in Figure 6J , mutant M9 (SEQ ID NO: 120) encompassing point mutations K336G, D343Y, and E351A completely abolished the binding of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) while hz2-7 (H1L2) remained bound with high potency. As previously demonstrated, D343Y and E351A are not important for the binding of any of the anti-C1s antibodies examined ( Figures 6H-6I). Therefore, K336 is (or may be) a key residue for hz2-8(H1L2G80A) and hz2-8(H1L2G80A / T82A) binding. In addition to M9, the K336G point mutation also exists in M5 and M6 of human C1s, either alone (M5) or in combination with other non-essential residues (M6). Figures 6F-6G As shown in , the single mutation K336G resulted in a significant decrease in the binding of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A), but had no effect on hz2-7 (H1L2), confirming that K336 is a key residue for the binding of hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) to C1s, constituting a unique epitope for the 2-8 antibody.

[0146] In summary, the epitope of hz2-7 (H1L2) includes R316 and G390-R422, and the key residue for hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) binding to C1s is K336. The results indicate that the epitopes of antibodies 2-7 and 2-8 are different, but all appear within the middle 151 amino acid region of human C1s (M151).

[0147] Example 7. Inhibition of complement-mediated lysis of antibody-sensitized sheep erythrocytes by anti-C1S antibodies

[0148] Sheep erythrocytes coated with rabbit anti-sheep erythrocyte antiserum (hemolysin), also known as antibody-sensitized sheep erythrocytes (EA), have traditionally been used to measure the activity of the classical complement pathway in serum samples, where these assays allow the determination of the CH50 titer of the serum. The inhibition of complement-mediated EA lysis by anti-C1s antibodies was assessed using commercially available EA (Complement Technologies, #B202). Briefly, humanized anti-C1s variants including hz2-7(H1L2), hz2-8(H1L2), hz2-8(H1L2G80A), and hz2-8(H1L2 G80A / T82A) were first serially diluted in GVB++ buffer (Complement Technologies, catalog #B100, manufacturer's ingredient list: 0.1% gelatin, 5mM veronal, 145mM NaCl, 0.025% NaN3, 0.15mM calcium chloride, 0.5mM magnesium chloride, pH 7.3). The diluted anti-C1s antibodies were then mixed with normal human serum (NHS, Figure 7A ) or cynomolgus monkey serum (CMS, Figure 7B ) were mixed to a final serum concentration of 1%. The antibody and serum mixture was allowed to equilibrate at room temperature before adding EA cells. For each reaction, approximately 0.5-1 x 10 8EA cells. To determine the 0% lysis background control value, EA cells were mixed with buffer only ("no serum control"). To determine the 100% lysis control value, GVB++ buffer was replaced with water. EA cells were mixed gently with antibody and serum, and then the mixture was incubated at 37°C for 1 hour under periodic mixing. After incubation, the remaining EA cells were spun down, and the absorbance of the supernatant was determined by measuring OD 540nm To calculate the percentage lysis that occurred under a particular incubation condition, first the 0% lysis background value was subtracted from each absorbance reading, and then the results were normalized to the 100% lysis control (minus the 0% lysis background) to determine the percentage lysis that occurred.

[0149] Results of anti-Cls antibody inhibition of human complement are shown in Figure 7A Antibody hz2-8 (H1L2) was the most potent inhibitor of complement-mediated EA lysis, with an IC 50 of 0.2 μg / ml. Antibodies hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A), which are sequence-optimized variants of hz2-8 (H1L2), also effectively inhibited human complement-mediated EA lysis, with similar IC 50 values of between 0.4 μg / ml and 0.5 μg / ml. Hz2-7 (H1L2) was the weakest inhibitor in this case, with an IC 50 of 4.1 μg / ml, and maximum inhibition reached only about 50% at the highest concentration tested (4 μg / ml). Results of cynomolgus monkey complement inhibition shown in Figure 7B were similar to the human serum results, with hz2-8 (H1L2) being the most potent inhibitor of cynomolgus monkey complement-mediated EA lysis, with an IC 50 of 0.1 μg / ml, and its variants hz2-8 (H1L2 G80A) and hz2-8 (H1L2 G80A / T82A) also maintained high inhibition potency, with similar IC 50 values of between 0.3 μg / ml and 0.4 μg / ml. Hz2-7 (H1L2) inhibited cynomolgus monkey complement more effectively than human complement, with an IC 50 of 0.8 μg / ml, and inhibition reached maximum Figure 7B These results strongly support the role of various anti-Cls antibodies in inhibiting the classical complement pathway, consistent with the findings from Example 2.

[0150] Although the present invention has been described with reference to certain specific embodiments thereof, it will be understood by those skilled in the art that changes and modifications may be made to the specifically described embodiments without departing from the principles of the invention, which is intended to be limited only by the scope of the appended claims.

Claims

1. A recombinant anti-C1s antibody or antigen-binding fragment thereof, which can specifically bind to and inhibit human C1s, the recombinant anti-C1s antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising three heavy chain complementarity determining regions (HC CDRs), the VL comprising three light chain complementarity determining regions (LC CDRs), wherein: (a) the VH comprises: an HC CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, an HC CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and an HC CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, and the VL comprises: an LC CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, an LC CDR2 consisting of the amino acid sequence of SEQ ID NO: 18, and an LC CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; (b) the VH comprises: an HC CDR1 consisting of the amino acid sequence of SEQ ID NO:42, an HC CDR2 consisting of the amino acid sequence of SEQ ID NO:43, and an HC CDR3 consisting of the amino acid sequence of SEQ ID NO:44, and the VL comprises: an LC CDR1 consisting of the amino acid sequence of SEQ ID NO:47, an LC CDR2 consisting of the amino acid sequence of SEQ ID NO:48, and an LC CDR3 consisting of the amino acid sequence of SEQ ID NO:49; (c) the VH comprises: an HC CDR1 consisting of the amino acid sequence of SEQ ID NO: 62, an HC CDR2 consisting of the amino acid sequence of SEQ ID NO: 63, and an HC CDR3 consisting of the amino acid sequence of SEQ ID NO: 64, and the VL comprises: an LC CDR1 consisting of the amino acid sequence of SEQ ID NO: 67, an LC CDR2 consisting of the amino acid sequence of SEQ ID NO: 68, and an LC CDR3 consisting of the amino acid sequence of SEQ ID NO: 69; or (d) the VH comprises: an HC CDR1 consisting of the amino acid sequence shown in SEQ ID NO:52, an HC CDR2 consisting of the amino acid sequence shown in SEQ ID NO:53, and an HC CDR3 consisting of the amino acid sequence shown in SEQ ID NO:54, and the VL comprises: an LC CDR1 consisting of the amino acid sequence shown in SEQ ID NO:57, an LC CDR2 consisting of the amino acid sequence shown in SEQ ID NO:58, and an LC CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

59. 2 . The anti-C1s antibody of claim 1 , wherein the antibody is capable of binding to residue K336 of human C1s (SEQ ID NO: 99). 3 . The anti-C1s antibody according to claim 1 , wherein the anti-C1s antibody is capable of inhibiting the activity of the classical pathway (CP) of complement activation.

4. The anti-C1s antibody of claim 3, wherein the anti-C1s antibody is capable of performing at least one of the following: inhibiting downstream effects of C1s activation, inhibiting IgM-induced C4 deposition from serum, inhibiting antibody-antigen complex-mediated C4 cleavage, inhibiting antibody-antigen complex-mediated C4b deposition, inhibiting the formation of C3 convertase, and inhibiting downstream effects of CP activation.

5. The anti-C1s antibody of claim 1, wherein the antibody shows cross-reactivity with at least one non-human C1s. The anti-C1s antibody of claim 5 , wherein the non-human C1s is cynomolgus monkey C1s. The anti-C1s antibody according to claim 6 , wherein the anti-C1s antibody shows cross-reactivity with rat C1s.

8. The anti-C1s antibody of claim 1, wherein the antibody is at least one of a chimeric antibody and an antigen-binding fragment thereof. 9 . The anti-C1s antibody of claim 1 , wherein the antibody is at least one of a humanized antibody and an antigen-binding fragment thereof.

10. A recombinant anti-C1s antibody or an antigen-binding fragment thereof, which is capable of specifically binding to and inhibiting human C1s, wherein the anti-C1s antibody is one of the following: a. an antibody, comprising: an HC polypeptide consisting of the amino acid sequence of SEQ ID NO: 75; and an LC polypeptide consisting of the amino acid sequence of SEQ ID NO: 77; b. an antibody comprising: an HC polypeptide consisting of the amino acid sequence of SEQ ID NO: 87; and an LC polypeptide consisting of the amino acid sequence of SEQ ID NO: 89; c. an antibody comprising: an HC polypeptide consisting of the amino acid sequence of SEQ ID NO: 91; and an LC polypeptide consisting of the amino acid sequence of SEQ ID NO: 93; or d. An antibody comprising: an HC polypeptide consisting of the amino acid sequence of SEQ ID NO: 95; and an LC polypeptide consisting of the amino acid sequence of SEQ ID NO:

97.

11. A recombinant anti-C1s antibody or an antigen-binding fragment thereof, which is capable of specifically binding to and inhibiting human C1s, wherein the anti-C1s antibody is one of the following: a. Antibodies, the antibodies comprising: a VH consisting of the amino acid sequence of SEQ ID NO: 11; and a VL consisting of the amino acid sequence of SEQ ID NO: 16; b. an antibody comprising: a VH consisting of the amino acid sequence of SEQ ID NO: 41; and a VL consisting of the amino acid sequence of SEQ ID NO: 46; c. an antibody comprising: a VH consisting of the amino acid sequence of SEQ ID NO: 51; and a VL consisting of the amino acid sequence of SEQ ID NO: 56; or d. an antibody comprising: a VH consisting of the amino acid sequence of SEQ ID NO: 61; and a VL consisting of the amino acid sequence of SEQ ID NO:

66.

12. The anti-C1s antibody of claim 1, wherein the antibody comprises HC CDR1 of SEQ ID NO:52, HC CDR2 of SEQ ID NO:53, HC CDR3 of SEQ ID NO:54, LC CDR1 of SEQ ID NO:57, LC CDR2 of SEQ ID NO:58, and LC CDR3 of SEQ ID NO:

59.

13. Use of the anti-C1s antibody of claim 1 in the preparation of a medicament for treating a complement-mediated disorder in a subject in need thereof, wherein the medicament inhibits the activity of the CP.

14. The use according to claim 13, wherein the complement-mediated disorder is an autoimmune disorder characterized by binding of complement-fixing antibodies to at least one autoantigen.

15. The use according to claim 14, wherein the autoimmune disorder is selected from one of immune thrombocytopenic purpura (ITP) and neuromyelitis optica (NMO).

16. The use of claim 13, wherein the drug inhibits at least one of C1s activation, antibody-antigen complex-mediated C4 cleavage, and formation of common complement pathway effectors.

17. The use of claim 13, wherein the medicament is prepared for administration during the acute phase of the complement-mediated disorder. 18 . A pharmaceutical composition comprising the anti-C1s antibody according to claim 1 and a pharmaceutically acceptable carrier and / or excipient.

19. An isolated nucleic acid molecule encoding at least a portion of the anti-C1s antibody of claim 1, wherein the nucleic acid comprises at least one nucleotide sequence selected from the group consisting of: a. a heavy chain (HC) nucleotide sequence, the HC nucleotide sequence being selected from the group consisting of a nucleotide sequence consisting of the sequence of SEQ ID NO: 15, a nucleotide sequence consisting of the sequence of SEQ ID NO: 45, a nucleotide sequence consisting of the sequence of SEQ ID NO: 55, and a nucleotide sequence consisting of the sequence of SEQ ID NO: 65, and b. A light chain (LC) nucleotide sequence, wherein the LC nucleotide sequence is selected from the group consisting of a nucleotide sequence consisting of a sequence of SEQ ID NO: 20, a nucleotide sequence consisting of a sequence of SEQ ID NO: 50, a nucleotide sequence consisting of a sequence of SEQ ID NO: 60, and a nucleotide sequence consisting of a sequence of SEQ ID NO:

70.

20. A vector comprising the nucleic acid molecule according to claim 19. A host cell comprising the vector according to claim 20 .

Images