Anti-complement c1s antibodies and uses thereof

Abstract

The present application relates to anti-complement Cls antibodies and uses thereof. The present disclosure provides antibodies that bind complement Cls protein; and nucleic acid molecules encoding such antibodies. The present disclosure also provides compositions comprising such antibodies, and methods of making and using such antibodies, nucleic acid molecules, and compositions. The present disclosure provides an isolated, humanized monoclonal antibody that inhibits cleavage of complement component G4, wherein the antibody does not inhibit cleavage of complement component C2. In some cases, the antibody inhibits a component of the classical complement pathway, in some cases the classical complement pathway component is Cls. In some cases, the antibody does not inhibit protease activity of Cls.

Description

[0001] This application is a divisional application of a divisional application filed on November 1, 2013, with Chinese application number 201810462335.3 and invention title "Anti-complement C1s antibody and its use" (original application number 201380068801.5).

[0002] Cross-references

[0003] This application claims the benefits of U.S. Provisional Patent Application No. 61 / 721,916, filed November 2, 2012; U.S. Provisional Patent Application No. 61 / 754,123, filed January 18, 2013; U.S. Provisional Patent Application No. 61 / 779,180, filed March 13, 2013; and U.S. Provisional Patent Application No. 61 / 846,402, filed July 15, 2013, all of which are incorporated herein by reference in their entirety. Invention Field

[0004] This disclosure provides antibodies that bind to complement C1s proteins; and nucleic acid molecules encoding such antibodies. This disclosure also provides compositions comprising such antibodies, and methods for preparing and using such antibodies, nucleic acid molecules, and compositions. This disclosure provides isolated, humanized monoclonal antibodies that inhibit the cleavage of complement component G4, wherein said antibody does not inhibit the cleavage of complement component C2. In some cases, said antibody inhibits a component of the classical complement pathway, and in some cases, the component of the classical complement pathway is C1s. In some cases, said antibody does not inhibit the protease activity of C1s. Background of the Invention

[0005] The complement system is a well-known effector mechanism of the immune response, providing not only protection against pathogens and other harmful agents but also recovery from injury. The complement pathway involves many proteins that are normally present in the body in an inactive form. The classical complement pathway is triggered by the activation of the first component of complement, known as the C1 complex, which consists of the C1q, C1r, and C1s proteins. After C1 binds to an immune complex or other activator, the C1s component, a diisopropyl fluorophosphate (DFP)-sensitive serine protease, cleaves complement components C4 and C2 to initiate activation of the classical complement pathway. The classical complement pathway appears to play a role in many diseases and conditions.

[0006] There is a need in the art for compounds that treat complement-mediated diseases or conditions. There is also a need for compounds that can detect or monitor such diseases or conditions. Furthermore, there is a need for methods for preparing and using such compounds and compositions thereof. Invention Overview

[0007] This disclosure provides antibodies that bind to complement C1s proteins; and nucleic acid molecules encoding such antibodies. This disclosure also provides compositions comprising such antibodies, and methods for preparing and using such antibodies, nucleic acid molecules, and compositions.

[0008] This disclosure provides isolated humanized monoclonal antibodies that inhibit the cleavage of complement component C4, wherein the antibodies do not inhibit the cleavage of complement component C2. In some cases, the antibodies inhibit components of the classical complement pathway; in some cases, the components of the classical complement pathway are C1s. In some cases, the antibodies do not inhibit the protease activity of C1s.

[0009] This disclosure provides isolated humanized monoclonal antibodies that specifically bind to epitopes within domains IV and V of complement component 1s (C1s). In some cases, the antibody inhibits the binding of C1s to complement component 4 (C4). In some cases, the antibody does not inhibit the protease activity of C1s. In some cases, the epitope bound by the isolated humanized monoclonal antibody of this disclosure is a conformational epitope.

[0010] This disclosure provides isolated humanized monoclonal antibodies that bind with high affinity to complement component C1s in the C1 complex.

[0011] This disclosure provides isolated humanized monoclonal antibodies that are specific for complement component C1s and have a concentration of less than 10 × 10⁻⁶. -9 M's IC50 inhibits complement-mediated cell lysis and / or at a rate less than 50 × 10⁻⁶. -9 M's IC50 inhibits C4 activation.

[0012] In any embodiment of this disclosure, the antibody may comprise one or more complementarity-determining regions (CDRs) of the antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 or one or more CDRs of the antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0013] In any embodiment of this disclosure, the antibody may comprise: a) a complementarity-determining region (CDR) 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:5 and SEQ ID NO:6; or b) a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0014] In any embodiment of this disclosure, the antibody may comprise: a) a light chain CDR comprising the antibody light chain variable region of amino acid sequence SEQ ID NO:7 or a heavy chain CDR comprising the antibody heavy chain variable region of amino acid sequence SEQ ID NO:8; or b) a light chain CDR comprising the antibody light chain variable region of amino acid sequence SEQ ID NO:37 or a heavy chain CDR comprising the antibody heavy chain variable region of amino acid sequence SEQ ID NO:38.

[0015] In any embodiment of this disclosure, the antibody may comprise: a) a light chain CDR comprising the antibody light chain variable region of amino acid sequence SEQ ID NO:7 and a heavy chain CDR comprising the antibody heavy chain variable region of amino acid sequence SEQ ID NO:8; or b) a light chain CDR comprising the antibody light chain variable region of amino acid sequence SEQ ID NO:37 and a heavy chain CDR comprising the antibody heavy chain variable region of amino acid sequence SEQ ID NO:38.

[0016] In any embodiment of this disclosure, the antibody may comprise heavy and light chain complementarity-determining regions (CDRs) having amino acid sequences selected from a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and b) CDRs having amino acid sequences selected from SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0017] This disclosure provides a humanized antibody that specifically binds complement component 1s (C1s), wherein the antibody competes with an antibody containing one or more CDRs of an antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 or one or more CDRs of an antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8 for binding to the epitope.

[0018] This disclosure provides humanized antibodies that specifically bind to complement component 1s (C1s), wherein the antibodies are selected from the group consisting of: a) humanized antibodies that specifically bind to epitopes within the complement C1s protein, wherein the antibodies compete with antibodies comprising a CDR 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:5, and SEQ ID NO:6 for binding to the epitopes; and b) humanized antibodies that specifically bind to epitopes within the complement C1s protein, wherein the antibodies compete with antibodies comprising a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36 for binding to the epitopes.

[0019] This disclosure provides humanized antibodies that bind to complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with antibodies comprising: a) a light chain CDR comprising an antibody light chain variable region comprising the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37; or b) a heavy chain CDR comprising an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0020] This disclosure provides humanized antibodies that bind to complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, and wherein the antibody competes with antibodies comprising: a) a light chain CDR comprising the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37 of the antibody light chain variable region; and b) a heavy chain CDR comprising the amino acid sequence SEQ ID NO:8 or SEQ ID NO:38 of the antibody heavy chain variable region. In some cases, the antibody competes with antibodies comprising heavy and light chain CDRs comprising: a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:142, SEQ ID NO:5, and SEQ ID NO:6; or b) SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0021] In any embodiment of this disclosure, the antibody may bind to human complement C1s protein. In any embodiment of this disclosure, the antibody may bind to rat complement C1s protein. In any embodiment of this disclosure, the antibody may bind to monkey complement C1s protein. In any embodiment of this disclosure, the antibody may bind to human complement C1s protein, rat complement C1s protein, and monkey complement C1s protein. In any embodiment of this disclosure, the antibody may comprise a humanized light chain framework region. For example, the humanized light chain framework region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acid substitutions shown in Table 6. In any embodiment of this disclosure, the antibody may comprise a humanized heavy chain framework region. For example, the humanized heavy chain framework region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions shown in Table 5. In any embodiment of this disclosure, the antibody may be an antigen-binding fragment that binds to complement C1s protein. In any embodiment of this disclosure, the antibody is selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies. In any embodiment of this disclosure, the antibody is selected from the group consisting of monospecific antibodies, bispecific antibodies, and multispecific antibodies. In any embodiment of this disclosure, the antibody may contain light chain and heavy chain regions present in a single polypeptide. In any embodiment of this disclosure, the antibody may contain light chain and heavy chain regions present in a single polypeptide. In any embodiment of this disclosure, the antibody may contain an Fc region. In any embodiment of this disclosure, the light chain and heavy chain CDRs are selected from the group consisting of: a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and b) SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0022] This disclosure provides an antibody that binds to complement C1s protein, wherein the antibody comprises a complementation-determining region (CDR) 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:5, and SEQ ID NO:6. In some embodiments, the antibody comprises a light chain variable region having amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, the antibody comprises a heavy chain variable region having amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the antibody comprises CDR-L1 having amino acid sequence SEQ ID NO:1, CDR-L2 having amino acid sequence SEQ ID NO:2, CDR-L3 having amino acid sequence SEQ ID NO:3, CDR-H1 having amino acid sequence SEQ ID NO:4, CDR-H2 having amino acid sequence SEQ ID NO:5, and CDR-H3 having amino acid sequence SEQ ID NO:6.

[0023] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7. In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8. In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7. In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO:8. In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8. In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0024] This disclosure provides antibodies that bind to complement C1s proteins, wherein the antibodies specifically bind to epitopes within the complement C1s proteins, and wherein the antibodies compete with antibodies comprising a light chain CDR containing an antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 and a heavy chain CDR containing an antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8 for binding to the epitopes. In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain CDR containing an antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 and a heavy chain CDR containing an antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0025] In any of the above embodiments, the anti-C1s antibody of this disclosure binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of at least one substrate cleaved by complement C1s protein. In some embodiments, the substrate is selected from the group consisting of complement C2 and complement C4.

[0026] In any of the above embodiments, the anti-C1s antibody of this disclosure may include a humanized light chain framework region. In any of the above embodiments, the anti-C1s antibody of this disclosure may include a humanized heavy chain framework region.

[0027] In any of the above embodiments, the anti-C1s antibody of this disclosure may be an Ig monomer or an antigen-binding fragment thereof that binds to complement C1s protein. In any of the above embodiments, the anti-C1s antibody of this disclosure may be an antigen-binding fragment that binds to complement C1s protein. In any of the above embodiments, the anti-C1s antibody of this disclosure is selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies. In any of the above embodiments, the anti-C1s antibody of this disclosure is selected from the group consisting of monospecific antibodies, bispecific antibodies, and multispecific antibodies.

[0028] In some embodiments, the anti-C1s antibody of this disclosure includes a light chain region and a heavy chain region present in a single polypeptide. In some embodiments, the anti-C1s antibody of this disclosure includes a light chain region and a heavy chain region present in a single polypeptide. In some embodiments, the anti-C1s antibody of this disclosure includes an Fc region.

[0029] This disclosure provides antibodies that competitively bind to epitopes bound by antibody IPN003 (also referred to herein as "IPN-M34" or "M34" or "TNT003"). This disclosure also provides antibodies comprising a variable domain of antibody IPN003. This disclosure further provides antibody IPN003.

[0030] This disclosure provides anti-C1s antibodies prepared by methods including recombinant preparation.

[0031] This disclosure provides antibodies that bind to complement C1s protein, wherein the antibodies are encapsulated in liposomes.

[0032] This disclosure provides antibodies that bind to complement C1s proteins, wherein the antibodies comprise covalently linked non-peptide synthetic polymers. In some embodiments, the synthetic polymer is a poly(ethylene glycol) polymer.

[0033] This disclosure provides antibodies that bind to complement C1s protein, wherein the antibodies are formulated together with reagents that promote crossing the blood-brain barrier.

[0034] This disclosure provides antibodies that bind to complement C1s proteins, wherein the antibodies are fused directly or via a linker to a compound that facilitates crossing the blood-brain barrier, wherein the compound is selected from the group consisting of carrier molecules, peptides, or proteins.

[0035] This disclosure provides nucleic acid molecules encoding anti-C1s antibodies of any embodiment disclosed herein. In some embodiments, this disclosure provides recombinant vectors comprising such nucleic acid molecules. In some embodiments, this disclosure provides recombinant molecules comprising such nucleic acid molecules. In some embodiments, this disclosure provides recombinant cells comprising such recombinant molecules.

[0036] This disclosure provides pharmaceutical compositions comprising anti-C1s antibodies and pharmaceutically acceptable excipients, including any of the embodiments disclosed herein. Some embodiments include a sterile container comprising such a pharmaceutical composition. In some embodiments, the container is selected from the group consisting of bottles and syringes.

[0037] This disclosure provides a method for treating an individual with complement-mediated diseases or conditions, the method comprising administering to the individual an anti-C1s antibody or a pharmaceutical composition thereof of any embodiment disclosed herein. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. In some embodiments, the administration is intravenous. In some embodiments, the administration is intrathecal. In some embodiments, the administration results in a result selected from the group consisting of: (a) reduced complement activation; (b) improved cognitive function; (c) reduced neuronal loss; (d) reduced phosphorylated Tau levels in neurons; (e) reduced glial cell activation; (f) reduced lymphocyte infiltration; (g) reduced macrophage infiltration; (h) reduced antibody deposition; (i) reduced glial cell loss; (j) reduced oligodendrocyte loss; (k) reduced dendritic cell infiltration; (l) reduced neutrophil infiltration; (m) reduced erythrocytes. (n) Decreased lysis; (o) Decreased phagocytosis by erythrocytes; (p) Decreased platelet phagocytosis; (q) Increased graft survival; (r) Decreased macrophage-mediated phagocytosis; (s) Improved vision; (t) Improved motor control; (u) Improved thrombosis; (v) Improved coagulation; (w) Improved renal function; (x) Decreased antibody-mediated complement activation; (y) Decreased autoantibody-mediated complement activation; (z) Improved anemia; (aa) Decreased demyelination; (ab) Decreased eosinophilia; (ac) Decreased deposition of C3 on erythrocytes (e.g., decreased deposition of C3b, iC3b, etc. on RBCs); (ad) Decreased deposition of C3 on platelets (e.g., decreased deposition of C3b, iC3b, etc.); (ae) decreased production of anaphylatoxins (e.g., C3a, C4a, C5a); (af) decreased autoantibody-mediated vesicle formation; (ag) decreased autoantibody-induced pruritus; (ah) decreased autoantibody-induced lupus erythematosus; (ai) decreased autoantibody-mediated skin erosion; (aj) decreased red blood cell destruction due to infusion reactions; (ak) decreased red blood cell lysis due to allogeneic antibodies; (al) decreased hemolysis due to infusion reactions. Reduced; (am) decreased platelet lysis mediated by allogeneic antibodies; (an) decreased platelet lysis due to infusion reaction; (ao) decreased mast cell activation; (ap) decreased histamine release from mast cells; (aq) decreased vascular permeability; (ar) decreased edema; (as) decreased complement deposition on graft endothelium; (at) decreased anaphylatoxin production in graft endothelium; (au) decreased separation at the dermal-epidermal junction; (av) decreased anaphylatoxin production at the dermal-epidermal junction; (aw) decreased complement activation mediated by allogeneic antibodies in graft endothelium;(ax) Reduced antibody-mediated loss at the neuromuscular junction; (ay) Reduced complement activation at the neuromuscular junction; (az) Reduced anaphylatoxin production at the neuromuscular junction; (ba) Reduced complement deposition at the neuromuscular junction; (bb) Reduced paralysis; (bc) Reduced numbness; (bd) Increased bladder control; (be) Increased defecation control; (bf) Reduced autoantibody-related mortality; and (bg) Reduced autoantibody-related morbidity. In some embodiments, the reduction in glial cell activation includes a reduction in astrocyte activation or microglia activation.

[0038] This disclosure provides a method for inhibiting complement activation in an individual with complement-mediated disease or condition, the method comprising administering to the individual an anti-C1s antibody or a pharmaceutical composition thereof of any embodiment disclosed herein. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. In some embodiments, the administration is intravenous. In some embodiments, the administration is intrathecal. In some embodiments, the administration is subcutaneous. In some embodiments, the administration results in a result selected from the group consisting of: (a) reduced complement activation; (b) improved cognitive function; (c) reduced neuronal loss; (d) reduced phosphorylated Tau levels in neurons; (e) reduced glial cell activation; (f) reduced lymphocyte infiltration; (g) reduced macrophage infiltration; (h) reduced antibody deposition; (i) reduced glial cell loss; (j) reduced oligodendrocyte loss; (k) reduced dendritic cell infiltration; (l) reduced neutrophil infiltration; (m) reduced erythrocytes. (n) Decreased lysis; (o) Decreased phagocytosis by erythrocytes; (p) Decreased platelet phagocytosis; (q) Increased graft survival; (r) Decreased macrophage-mediated phagocytosis; (s) Improved vision; (t) Improved motor control; (u) Improved thrombosis; (v) Improved coagulation; (w) Improved renal function; (x) Decreased antibody-mediated complement activation; (y) Decreased autoantibody-mediated complement activation; (z) Improved anemia; (aa) Decreased demyelination; (ab) Decreased eosinophilia; (ac) (a) Decreased deposition of C3 on erythrocytes (e.g., decreased deposition of C3b, iC3b, etc. on RBCs); (ad) Decreased deposition of C3 on platelets (e.g., decreased deposition of C3b, iC3b, etc. on platelets); (ae) Decreased production of anaphylatoxins; (af) Decreased blister formation mediated by autoantibodies; (ag) Decreased pruritus induced by autoantibodies; (ah) Decreased lupus erythematosus induced by autoantibodies; (ai) Decreased skin erosion mediated by autoantibodies; (aj) Decreased erythrocyte destruction due to infusion reactions; (ak) Decreased erythrocyte destruction due to allogeneic antibodies. (a) Decreased erythrocyte lysis; (al) Decreased hemolysis due to infusion reaction; (am) Decreased platelet lysis mediated by allogeneic antibodies; (an) Decreased platelet lysis due to infusion reaction; (ao) Decreased mast cell activation; (ap) Decreased histamine release from mast cells; (aq) Decreased vascular permeability; (ar) Decreased edema; (as) Decreased complement deposition on graft endothelium; (at) Decreased production of anaphylatoxins in graft endothelium; (au) Decreased separation of the dermal-epidermal junction; (av) Decreased production of anaphylatoxins at the dermal-epidermal junction;(aw) Decreased allogeneic antibody-mediated complement activation in graft endothelium; (ax) Reduced antibody-mediated loss at the neuromuscular junction; (ay) Decreased complement activation at the neuromuscular junction; (az) Decreased anaphylatoxin production at the neuromuscular junction; (ba) Decreased complement deposition at the neuromuscular junction; (bb) Decreased paralysis; (bc) Decreased numbness; (bd) Increased bladder control; (be) Increased defecation control; (bf) Decreased autoantibody-related mortality; and (bg) Decreased autoantibody-related morbidity. In some embodiments, the decrease in glial cell activation includes a decrease in astrocyte activation or microglia activation.

[0039] This disclosure provides for the use of any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof in treating an individual with a complement-mediated disease or condition.

[0040] This disclosure provides for the use of any embodiment of the anti-C1s antibody in the manufacture of a medicament for treating an individual with a complement-mediated disease or condition.

[0041] This disclosure provides the use of any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof in inhibiting complement C1s activity, wherein "inhibition of complement C1s activity" includes inhibiting complement activation, such as inhibiting the production of C4b2a (i.e., the complement C4b and C2a complex; also known as "C3 convertase"). In some embodiments, this disclosure provides the use of any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof in inhibiting complement activation in individuals with complement-mediated diseases or conditions.

[0042] This disclosure provides for the use of any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof in the manufacture of a medicament for inhibiting complement activation. In some embodiments, this disclosure provides for the use of any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof in the manufacture of a medicament for inhibiting complement activation in an individual with a complement-mediated disease or condition.

[0043] This disclosure provides anti-C1s antibodies or pharmaceutical compositions thereof for any implementation of medical treatment.

[0044] This disclosure provides anti-C1s antibodies or pharmaceutical compositions thereof for any implementation of treatment for individuals with complement-mediated diseases or conditions.

[0045] This disclosure provides anti-C1s antibodies or pharmaceutical compositions thereof for any embodiment of inhibiting complement activation. This disclosure also provides anti-C1s antibodies or pharmaceutical compositions thereof for any embodiment of inhibiting complement activation in individuals with complement-mediated diseases or conditions.

[0046] This disclosure provides a method for diagnosing complement-mediated diseases or conditions in an individual, the method comprising: (a) determining the amount of complement C1s protein in a biological sample obtained from the individual, wherein the determination step includes: (i) contacting the biological sample with an anti-C1s antibody of any embodiment; and (ii) quantifying the binding of the antibody to the complement C1s protein present in the biological sample; and (b) comparing the amount of complement C1s protein in the biological sample with a normal control value indicating the amount of complement C1s protein in a normal control individual, wherein a significant difference between the amount of C1s protein in the biological sample and the normal control value indicates that the individual has a complement-mediated disease or condition. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples.

[0047] This disclosure provides a method for monitoring the progression of complement-mediated disease or condition in an individual, the method comprising: (a) measuring a first amount of complement C1s protein in a biological sample obtained from the individual at a first time point; (b) measuring a second amount of complement C1s protein in a biological sample obtained from the individual at a second time point; and (c) comparing the second amount of complement C1s protein with the first amount of complement C1s protein. The measurement step includes: (i) contacting the biological sample with an anti-C1s antibody of any embodiment; and (ii) quantifying the binding of the antibody to the complement C1s protein present in the biological sample. In some embodiments, the first time point is a time point prior to the start of a treatment regimen, and the second time point is a time point after the start of a treatment regimen. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples.

[0048] This disclosure provides an in vitro method for detecting complement C1s protein in a biological sample obtained from an individual, the method comprising: (a) contacting the biological sample with an anti-C1s antibody according to any embodiment; and (b) detecting the binding of the antibody to the complement C1s protein present in the biological sample. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples. In some embodiments, the method is quantitative.

[0049] This disclosure provides a method for detecting complement C1s protein in a living individual in vivo, the method comprising: (a) administering an anti-C1s antibody of any embodiment to the individual; and (b) detecting the binding of the antibody to the complement C1s protein in the individual using an imaging method. In some embodiments, the binding is detected at a site in the individual where complement-mediated disease or symptom alteration occurs. In some embodiments, the binding is detected in the brain of the individual. In some embodiments, the antibody comprises a contrast agent suitable for use in the imaging method. In some embodiments, the imaging method is selected from the group consisting of magnetic resonance imaging, positron emission tomography, and IVIS instrumentation. In some embodiments, the method is quantitative.

[0050] In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, eye fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples.

[0051] In some embodiments, the method of this disclosure provides that: the individual is suspected of having a complement-mediated disease or condition, has been diagnosed with a complement-mediated disease or condition, or has a genetic predisposition to have a complement-mediated disease or condition.

[0052] This disclosure provides a composition comprising: (a) an anti-C1s antibody of any embodiment; and (b) a solution containing one or more reagents for preserving an organ or tissue intended for transplantation into a recipient individual. In some embodiments, the solution is an organ preservation solution or a tissue preservation solution. In some embodiments, the solution is an organ perfusion solution or a tissue perfusion solution. In some embodiments, the solution comprises: i) a salt; ii) an agent for reducing edema; iii) an oxygen free radical scavenger; and iii) an energy supply system component. In some embodiments, the composition comprises potassium lactobionate, KH₂PO₄, MgSO₄, raffinose, adenosine, glutathione, allopurinol, and hydroxyethyl starch.

[0053] This disclosure provides organ or tissue preservation solutions comprising anti-C1s antibodies or pharmaceutical compositions thereof, according to any embodiment.

[0054] This disclosure provides organ or tissue perfusion solutions comprising any embodiment of an anti-C1s antibody or a pharmaceutical composition thereof.

[0055] This disclosure provides a method for preserving an organ or tissue for transplantation, the method comprising contacting the organ or tissue with a composition comprising: (a) an anti-C1s antibody of any embodiment; and (b) an organ or tissue preservation solution of any embodiment or an organ or tissue perfusion solution of any embodiment.

[0056] This disclosure provides isolated organs or tissues preserved in a composition comprising: (a) an anti-C1s antibody of any embodiment; and (b) an organ or tissue preservation solution of any embodiment or an organ or tissue perfusion solution of any embodiment. In some embodiments, the organs are selected from the group consisting of the eye, heart, intestine, kidney, liver, lung, pancreas, stomach, and thymus. In some embodiments, the tissues are selected from the group consisting of bone, bone marrow, cornea, heart valves, Langerhans islets, tendons, skin, and veins.

[0057] This disclosure provides an in vitro method for inhibiting complement activation in an organ or tissue, the method comprising contacting the organ or tissue with an anti-C1s antibody of any embodiment, a solution containing an anti-C1s antibody of any embodiment, or a pharmaceutical composition containing an anti-C1s antibody of any embodiment.

[0058] Certain aspects of the present invention are defined in the following numbered technical solutions (au).

[0059] [a] An antibody that binds to complement C1s protein, wherein the antibody comprises a complement determination region (CDR) 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:5 and SEQ ID NO:6.

[0060] [b]An antibody of technical solution a, wherein the antibody comprises a light chain variable region containing amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. An antibody of technical solution a, wherein the antibody comprises a heavy chain variable region containing amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0061] [c]An antibody of technical solution a, wherein the antibody comprises CDR-L1 having amino acid sequence SEQ ID NO:1, CDR-L2 having amino acid sequence SEQ ID NO:2, CDR-L3 having amino acid sequence SEQ ID NO:3, CDR-H1 having amino acid sequence SEQ ID NO:4, CDR-H2 having amino acid sequence SEQ ID NO:5 and CDR-H3 having amino acid sequence SEQ ID NO:6.

[0062] [d] An antibody of technical solution a, wherein the antibody comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7. An antibody of technical solution a, wherein the antibody comprises a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8. An antibody of technical solution a, wherein the antibody comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7. An antibody of technical solution a, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0063] [e] An antibody of technical solution a, wherein the antibody comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8. An antibody of technical solution a, wherein the antibody comprises a light chain variable region containing an amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing an amino acid sequence SEQ ID NO:8.

[0064] [f] An antibody that binds to complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody containing a light chain CDR containing an antibody light chain variable region of amino acid sequence SEQ ID NO:7 and an antibody heavy chain CDR containing an antibody heavy chain variable region of amino acid sequence SEQ ID NO:8 for binding to the epitope.

[0065] [g] Technical solution f, wherein the antibody comprises a light chain CDR containing the antibody light chain variable region of amino acid sequence SEQ ID NO:7 and a heavy chain CDR containing the antibody heavy chain variable region of amino acid sequence SEQ ID NO:8.

[0066] [h] An antibody of any one of the technical solutions ag, wherein the antibody binds to human complement C1s protein. An antibody of any one of the technical solutions ag, wherein the antibody binds to rat complement C1s protein or monkey complement C1s protein.

[0067] [i] An antibody of any one of the technical solutions ah, wherein the antibody inhibits the cleavage of at least one substrate cleaved by complement C1s protein.

[0068] [j]An antibody of technical solution i, wherein the substrate is selected from the group consisting of complement C2 and complement C4.

[0069] [k] An antibody of any one of the technical solutions A1, wherein the antibody comprises a humanized light chain framework region. An antibody of any one of the technical solutions A1, wherein the antibody comprises a humanized heavy chain framework region.

[0070] [l] An antibody of any one of the technical solutions ak, wherein the antibody is selected from the group consisting of an Ig monomer that binds to complement C1s protein and its antigen-binding fragment.

[0071] [m]An antibody of any one of the technical solutions ak, wherein the antibody is an antigen-binding fragment that binds to complement C1s protein.

[0072] [n]An antibody of any one of the technical solutions ak, wherein the antibody is selected from the group consisting of Ig monomer, Fab fragment, F(ab')2 fragment, Fd fragment, scFv, scAb, dAb, Fv, single-domain heavy chain antibody and single-domain light chain antibody.

[0073] [o] An antibody of any one of the technical solutions ak, wherein the antibody is selected from the group consisting of monospecific antibodies, bispecific antibodies, and multispecific antibodies. An antibody of any one of the technical solutions ak, wherein the antibody contains a light chain region and a heavy chain region present in a single polypeptide. An antibody of any one of the technical solutions ak, wherein the antibody contains a light chain region and a heavy chain region present in a single polypeptide.

[0074] [p]An antibody of either ak or o, wherein the antibody comprises an Fc region.

[0075] [q] An antibody of any one of the technical solutions ap, wherein the antibody is encapsulated in a liposome.

[0076] [r]An antibody of any one of the technical solutions ap, wherein the antibody comprises a covalently linked non-peptide synthetic polymer.

[0077] [s]Antibody of technical solution r, wherein the synthetic polymer is a poly(ethylene glycol) polymer.

[0078] [t]An antibody in any of the technical solutions ap, wherein the antibody is formulated together with a reagent that promotes crossing of the blood-brain barrier.

[0079] [u]An antibody of any one of the technical solutions ap, wherein the antibody is fused directly or via a linker to a compound that promotes crossing the blood-brain barrier, wherein the compound is selected from the group consisting of carrier molecules, peptides or proteins.

[0080] This application relates to the following implementation scheme.

[0081] 1. An isolated humanized monoclonal antibody that inhibits the cleavage of complement component C4, wherein the antibody does not inhibit the cleavage of complement component C2.

[0082] 2. The humanized monoclonal antibody according to embodiment 1, wherein the antibody inhibits components of the classical complement pathway.

[0083] 3. The humanized monoclonal antibody according to implementation scheme 2, wherein the classical complement pathway component is C1s.

[0084] 4. The humanized monoclonal antibody according to embodiment 3, wherein the antibody does not inhibit the protease activity of C1s.

[0085] 5. An isolated humanized monoclonal antibody that specifically binds to epitopes within the regions of domains IV and V of complement component 1s (C1s).

[0086] 6. The isolated humanized monoclonal antibody according to embodiment 5, wherein the antibody inhibits the binding of C1s to complement component 4 (C4).

[0087] 7. The isolated humanized monoclonal antibody according to embodiment 6, wherein the antibody does not inhibit the protease activity of C1s.

[0088] 8. The isolated humanized monoclonal antibody according to embodiment 5, wherein the epitope is a conformational epitope.

[0089] 9. An isolated humanized monoclonal antibody that binds to complement component C1s in the C1 complex with high affinity.

[0090] 10. An isolated humanized monoclonal antibody that is specific for complement component C1s and whose concentration is less than 10 × 10⁻⁶. -9 M's IC50 inhibits complement-mediated cell lysis and / or at a rate less than 50 × 10⁻⁶. -9 M's IC50 inhibits C4 activation.

[0091] 11. The humanized monoclonal antibody according to any one of embodiments 1-10, wherein the antibody comprises one or more complementarity-determining regions (CDRs) of the antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 or one or more CDRs of the antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0092] 12. The humanized monoclonal antibody according to any one of embodiments 1-10, wherein the antibody comprises:

[0093] a) Possesses a complementarity-determining region (CDR) of 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:5, and SEQ ID NO:6; or

[0094] b) A CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0095] 13. The humanized monoclonal antibody according to any one of embodiments 1-10, wherein the antibody comprises:

[0096] a) A light chain CDR containing the variable region of the antibody light chain of SEQ ID NO:7 or a heavy chain CDR containing the variable region of the antibody heavy chain of SEQ ID NO:8; or

[0097] b) A light chain CDR containing the variable region of the antibody light chain containing the amino acid sequence SEQ ID NO:37, or a heavy chain CDR containing the variable region of the antibody heavy chain containing the amino acid sequence SEQ ID NO:38.

[0098] 14. The humanized monoclonal antibody according to any one of embodiments 1-10, wherein the antibody comprises:

[0099] a) The light chain CDR containing the variable region of the antibody light chain of SEQ ID NO:7 and the heavy chain CDR containing the variable region of the antibody heavy chain of SEQ ID NO:8; or

[0100] b) The light chain CDR containing the variable region of the antibody light chain containing the amino acid sequence SEQ ID NO:37 and the heavy chain CDR containing the variable region of the antibody heavy chain containing the amino acid sequence SEQ ID NO:38.

[0101] 15. A humanized monoclonal antibody according to any one of embodiments 1-10, wherein the antibody comprises heavy chain and light chain complementarity-determining regions (CDRs) having amino acid sequences selected from:

[0102] a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and

[0103] b) A CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0104] 16. A humanized antibody that specifically binds complement component 1s (C1s), wherein the antibody competes with an antibody comprising one or more CDRs containing an antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 or one or more CDRs containing an antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:8 for binding epitopes.

[0105] 17. A humanized antibody that specifically binds to complement component 1s (C1s), wherein the antibody is selected from the group consisting of:

[0106] a) A humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody comprising a CDR 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:5, and SEQ ID NO:6 for binding to the epitope; and

[0107] b) A humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody comprising a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36 for binding to the epitope.

[0108] 18. A humanized antibody that binds to complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody comprising:

[0109] a) A light chain CDR containing the variable region of the antibody light chain, which includes the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37; or

[0110] b) Heavy chain CDR containing the variable region of the antibody heavy chain with amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0111] 19. A humanized antibody that binds to complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody comprising:

[0112] a) A light chain CDR containing the variable region of the antibody light chain, which includes the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37; and

[0113] b) Heavy chain CDR containing the variable region of the antibody heavy chain with amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0114] 20. The humanized antibody according to embodiment 19, wherein the antibody competitively binds to the epitope with an antibody comprising heavy chain and light chain CDRs containing:

[0115] a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:142, SEQ ID NO:5 and SEQ ID NO:6; or

[0116] b) SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0117] 21. The antibody according to any one of embodiments 1-20, wherein the antibody binds to human complement C1s protein.

[0118] 22. The antibody according to any one of embodiments 1-20, wherein the antibody binds to rat complement C1s protein.

[0119] 23. The antibody according to any one of embodiments 1-20, wherein the antibody binds to monkey complement C1s protein.

[0120] 24. The antibody according to any one of embodiments 1-20, wherein the antibody binds to human complement C1s protein, rat complement C1s protein and monkey complement C1s protein.

[0121] 25. The antibody according to any one of embodiments 1-20, wherein the antibody comprises a humanized light chain framework region.

[0122] 26. The antibody according to embodiment 25, wherein the humanized light chain framework region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid substitutions shown in Table 6.

[0123] 27. The antibody according to any one of embodiments 1-20, wherein the antibody comprises a humanized heavy chain framework region.

[0124] 28. The antibody according to embodiment 27, wherein the humanized heavy chain framework region comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid substitutions as shown in Table 5.

[0125] 29. The antibody according to any one of embodiments 1-20, wherein the antibody is an antigen-binding fragment that binds to complement C1s protein.

[0126] 30. The antibody according to any one of embodiments 1-20, wherein the antibody is selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies.

[0127] 31. The antibody according to any one of embodiments 1-20, wherein the antibody is selected from the group consisting of monospecific antibodies, bispecific antibodies and multispecific antibodies.

[0128] 32. The antibody according to any one of embodiments 1-20, wherein the antibody comprises a light chain region and a heavy chain region present in a separate polypeptide.

[0129] 33. The antibody according to any one of embodiments 1-20, wherein the antibody comprises a light chain region and a heavy chain region present in a single polypeptide.

[0130] 34. The antibody according to any one of embodiments 1-20, wherein the antibody comprises an Fc region.

[0131] 35. The antibody according to any one of embodiments 1-20, wherein the light chain and heavy chain CDRs are selected from the group consisting of:

[0132] a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and

[0133] b) SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0134] 36. An antibody that binds to complement C1s protein, wherein the antibody comprises a complement-determining region (CDR) having an amino acid sequence selected from the group consisting of:

[0135] a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; or

[0136] b) SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0137] 37. The antibody according to embodiment 36, wherein the antibody comprises a light chain variable region containing amino acid sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 or containing amino acid sequences SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:3.

[0138] 38. The antibody according to embodiment 36, wherein the antibody comprises a heavy chain variable region containing amino acid sequences SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or containing amino acid sequences SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0139] 39. The antibody according to embodiment 36, wherein the antibody comprises:

[0140] i) CDR-L1 having the amino acid sequence SEQ ID NO:1, CDR-L2 having the amino acid sequence SEQ ID NO:2, CDR-L3 having the amino acid sequence SEQ ID NO:3, CDR-H1 having the amino acid sequence SEQ ID NO:4, CDR-H2 having the amino acid sequence SEQ ID NO:5, and CDR-H3 having the amino acid sequence SEQ ID NO:6; or

[0141] ii) CDR-L1 having the amino acid sequence SEQ ID NO:32, CDR-L2 having the amino acid sequence SEQ ID NO:33, CDR-L3 having the amino acid sequence SEQ ID NO:3, CDR-H1 having the amino acid sequence SEQ ID NO:34, CDR-H2 having the amino acid sequence SEQ ID NO:35, and CDR-H3 having the amino acid sequence SEQ ID NO:36.

[0142] 40. The antibody according to embodiment 36, wherein the antibody comprises a light chain variable region containing an amino acid sequence having at least 90% identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37.

[0143] 41. The antibody according to embodiment 36, wherein the antibody comprises a heavy chain variable region containing an amino acid sequence having at least 90% identity with the amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0144] 42. The antibody according to embodiment 36, wherein the antibody comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37.

[0145] 43. The antibody according to embodiment 36, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0146] 44. The antibody according to embodiment 36, wherein the antibody comprises a light chain variable region having an amino acid sequence having at least 90% identity with the amino acid sequence SEQ ID NO:7 or SEQ ID NO:37 and a heavy chain variable region having an amino acid sequence having at least 90% identity with the amino acid sequence SEQ ID NO:8 or SEQ ID NO:38.

[0147] 45. The antibody according to embodiment 36, wherein the antibody comprises:

[0148] i) Containing the light chain variable region of amino acid sequence SEQ ID NO:7 and the heavy chain variable region containing amino acid sequence SEQ ID NO:8; or

[0149] ii) The light chain variable region containing the amino acid sequence SEQ ID NO:37 and the heavy chain variable region containing the amino acid sequence SEQ ID NO:38.

[0150] 46. ​​The antibody according to embodiment 36, wherein the antibody is a monoclonal antibody.

[0151] 47. The antibody according to embodiment 36, wherein the antibody is a humanized antibody.

[0152] 48. The antibody according to embodiment 36, wherein the antibody is an antibody fragment.

[0153] 49. The antibody according to any one of embodiments 1-48, wherein the antibody is encapsulated in liposomes.

[0154] 50. The antibody according to any one of embodiments 1-48, wherein the antibody comprises a covalently linked non-peptide synthetic polymer.

[0155] 51. The antibody according to embodiment 50, wherein the synthetic polymer is a poly(ethylene glycol) polymer.

[0156] 52. The antibody according to any one of embodiments 1-48, wherein the antibody is formulated together with a reagent that promotes crossing of the blood-brain barrier.

[0157] 53. The antibody according to any one of embodiments 1-48, wherein the antibody is fused directly or via a linker to a compound that facilitates crossing the blood-brain barrier, wherein the compound is selected from the group consisting of carrier molecules, peptides, or proteins.

[0158] 54. A nucleic acid comprising a nucleotide sequence encoding an antibody according to any one of embodiments 1-48.

[0159] 55. A recombinant vector comprising the nucleic acid as described in embodiment 54.

[0160] 56. A recombinant cell comprising the nucleic acid according to embodiment 54 or the recombinant vector according to embodiment 55.

[0161] 57. A pharmaceutical composition comprising an antibody according to any one of embodiments 1-48 and a pharmaceutically acceptable excipient.

[0162] 58. A sterile container comprising the pharmaceutical composition according to embodiment 57.

[0163] 59. The container according to embodiment 58, wherein the container is selected from the group consisting of bottles and syringes.

[0164] 60. A method for inhibiting the activation of complement component C4 in an individual, the method comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0165] 61. A method for inhibiting complement C1s activity in an individual, the method comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0166] 62. A method for treating an individual with a complement-mediated disease or condition, the method comprising administering to the individual an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0167] 63. A method for inhibiting complement activation in an individual with a complement-mediated disease or condition, the method comprising administering to the individual an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0168] 64. The method according to any one of embodiments 60-63, wherein the individual is a mammal.

[0169] 65. The method according to any one of embodiments 60-63, wherein the individual is a person.

[0170] 66. The method according to any one of embodiments 60-65, wherein the administration is intravenous.

[0171] 67. The method according to any one of embodiments 60-65, wherein the application is subcutaneous.

[0172] 68. The method according to any one of embodiments 60-65, wherein the application is intrathecal.

[0173] 69. The method according to any one of embodiments 60-68, wherein the application results in a result selected from the group consisting of:

[0174] (a) Decreased complement activation;

[0175] (b) Improved cognitive function;

[0176] (c) Reduction in neuronal loss;

[0177] (d) Decreased levels of phosphorylated Tau in neurons;

[0178] (e) Decreased activation of glial cells;

[0179] (f) Decreased lymphocyte infiltration;

[0180] (g) Decreased macrophage infiltration;

[0181] (h) Reduction in antibody deposition;

[0182] (i) Reduced loss of glial cells;

[0183] (j) Reduced loss of oligodendrocytes;

[0184] (k) Decreased dendritic cell infiltration;

[0185] (l) Decreased neutrophil infiltration;

[0186] (m) Decreased erythrocyte lysis;

[0187] (n) Decreased phagocytosis by red blood cells;

[0188] (o) Decreased platelet phagocytosis;

[0189] (p) Reduced platelet lysis;

[0190] (q) Improved graft survival rate;

[0191] (r) Decreased macrophage-mediated phagocytosis;

[0192] (s) Improved vision;

[0193] (t) Improved motor control;

[0194] (u) Improvement in thrombus formation;

[0195] (v) Improvement in blood clotting;

[0196] (w) Improvement in kidney function;

[0197] (x) Decreased antibody-mediated complement activation;

[0198] (y) Decreased complement activation mediated by autoantibodies;

[0199] (z) Improvement of anemia;

[0200] (aa) Reduction of demyelination;

[0201] (ab) Decrease in eosinophilia;

[0202] (ac) Reduced deposition of C3 on erythrocytes;

[0203] (ad) Decreased deposition of C3 on platelets;

[0204] (ae) Decrease in the production of allergens;

[0205] (af) Reduced blister formation mediated by autoantibodies;

[0206] (ag) Reduction of itching induced by autoantibodies;

[0207] (ah) Reduction in autoantibody-induced lupus erythematosus;

[0208] (ai) Reduction of autoantibody-mediated skin erosion;

[0209] (aj) Reduction in red blood cell destruction due to infusion reaction;

[0210] (ak) Reduction in red blood cell lysis due to allogeneic antibodies;

[0211] (al) Reduction of hemolysis due to infusion reaction;

[0212] (am) Reduced platelet lysis mediated by allogeneic antibodies;

[0213] (an) Decreased platelet lysis due to infusion reaction;

[0214] (ao) Decreased mast cell activation;

[0215] (ap) Decreased histamine release from mast cells;

[0216] (aq) Decreased vascular permeability;

[0217] (ar) Reduction of edema;

[0218] (as) Reduced complement deposition on graft endothelium;

[0219] (at) a decrease in the production of anaphylatoxins in the graft endothelium;

[0220] (au) Reduction of separation at the dermal-epidermal junction;

[0221] (av) Reduction of allergen production at the dermal-epidermal junction;

[0222] (aw) Decreased complement activation mediated by allogeneic antibodies in graft endothelium;

[0223] (ax) Reduction of antibody-mediated loss at the neuromuscular junction;

[0224] (ay) Decreased complement activation at the neuromuscular junction;

[0225] (az) Reduced production of anaphylatoxins at the neuromuscular junction;

[0226] (ba) Decreased complement deposition at the neuromuscular junction;

[0227] (bb) Paralysis is reduced;

[0228] (bc) Reduced numbness;

[0229] (bd) Increased bladder control;

[0230] (be) Increased bowel control;

[0231] (bf) A reduction in mortality related to autoantibodies; and

[0232] (bg) Decreased incidence of autoantibody-related diseases.

[0233] 70. The method according to embodiment 69, wherein the glial cells are selected from the group consisting of astrocytes and microglia.

[0234] 71. The method according to any one of embodiments 60-68, wherein the antibody is administered in an amount providing a peak serum level of about 1 μg / ml to about 1 mg / ml.

[0235] 72. Use of the antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 57 for the treatment of an individual with a complement-mediated disease or condition.

[0236] 73. Use of the antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 57 for the manufacture of a medicament for treating an individual with a complement-mediated disease or condition.

[0237] 74. Use of the antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 57 for inhibiting complement activation in an individual with complement-mediated disease or condition.

[0238] 75. Use of the antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 57 for the manufacture of a medicament for inhibiting complement activation in an individual with a complement-mediated disease or condition.

[0239] 76. The antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 56, used in medical treatment.

[0240] 77. An antibody as claimed in any one of embodiments 1-48 or a pharmaceutical composition as described in embodiment 56, for the treatment of an individual with a complement-mediated disease or condition.

[0241] 78. An antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 56, for inhibiting complement activation in an individual with complement-mediated disease or condition.

[0242] 79. A method for diagnosing complement-mediated diseases or conditions in an individual, the method comprising:

[0243] (a) Determining the amount of complement C1s protein in a biological sample obtained from said individual, wherein said determination step includes:

[0244] (i) Contact the biological sample with the antibody according to any one of embodiments 1-48; and

[0245] (ii) Quantifying the binding of the antibody to the complement C1s protein present in the biological sample; and

[0246] (b) The amount of complement C1s protein present in the biological sample is compared with a normal control value indicating the amount of complement C1s protein in a normal control individual, wherein a significant difference between the amount of C1s protein in the biological sample and the normal control value indicates that the individual has a complement-mediated disease or condition.

[0247] 80. A method for monitoring the progression of complement-mediated disease or condition in an individual, the method comprising:

[0248] (a) Determine the first amount of complement C1s protein in a biological sample obtained from the individual at the first time point;

[0249] (b) Determining a second amount of complement C1s protein in a biological sample obtained from said individual at a second time point; and

[0250] (c) Compare the second amount of complement C1s protein with the first amount of complement C1s protein.

[0251] The determination steps mentioned above include:

[0252] (i) Contact the biological sample with the antibody according to any one of embodiments 1-48; and

[0253] (ii) Quantify the binding of the antibody to the complement C1s protein present in the biological sample.

[0254] 81. The method according to embodiment 80, wherein the first time point is a time point before the start of the treatment regimen, and wherein the second time point is a time point after the start of the treatment regimen.

[0255] 82. The method according to embodiment 81, comprising adjusting the treatment regimen based on the amount of complement C1s protein in a biological sample obtained from the individual at the second time point.

[0256] 83. An in vitro method for detecting complement C1s protein in a biological sample obtained from an individual, the method comprising:

[0257] (a) Contacting the biological sample with the antibody according to any one of embodiments 1-48; and

[0258] (b) Detect the binding of the antibody to the complement C1s protein present in the sample.

[0259] 84. The method according to any one of embodiments 79-83, wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, eye fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples.

[0260] 85. The method according to any one of embodiments 79-83, wherein the method comprises:

[0261] a) Treat the biological sample with a calcium chelating agent to form C1s monomers;

[0262] b) Contact the chelating agent-treated biological sample with an immobilized first antibody that binds to C1s but does not compete with the anti-C1s antibody according to any one of embodiments 1-48 to form an immobilized first antibody / C1s monomer complex.

[0263] c) Contacting the immobilized first antibody / C1s monomer complex with the antibody according to any one of embodiments 1-48; and

[0264] d) Detect the binding of the monoclonal anti-C1s antibody to the immobilized C1s monomer.

[0265] 86. The method according to any one of embodiments 79-83, wherein the method comprises:

[0266] a) Contact the biological sample with an immobilized first antibody that binds to C1s but does not compete with the anti-C1s antibody according to any one of embodiments 1-48 to form an immobilized first antibody / C1s complex.

[0267] b) Contact the immobilized first antibody / C1s complex with a calcium chelating agent to form an immobilized C1s monomer;

[0268] c) Contacting the immobilized C1s monomer with the anti-C1s antibody according to any one of embodiments 1-48; and

[0269] d) Detect the binding of the antibody according to any one of embodiments 1-48 to the immobilized C1s monomer.

[0270] 87. A method for detecting complement C1s protein in vivo, the method comprising:

[0271] (a) Administering an antibody to an individual according to any one of embodiments 1-48; and

[0272] (b) Use imaging methods to detect the binding of the antibody to the complement C1s protein in the individual.

[0273] 88. The method according to embodiment 87, wherein the binding is detected at the site of complement-mediated disease or symptom alteration in the individual.

[0274] 89. The method according to embodiment 87, wherein the binding is detected in the brain of the individual.

[0275] 90. The method according to any one of embodiments 87-89, wherein the antibody comprises a contrast agent suitable for use in the imaging method.

[0276] 91. The method according to any one of embodiments 87-90, wherein the imaging method is selected from the group consisting of magnetic resonance imaging and positron emission tomography.

[0277] 92. The method according to any one of embodiments 79-91, wherein the method is quantitative.

[0278] 93. The method according to any one of embodiments 79-92, wherein the individual is suspected of having a complement-mediated disease or condition, has been diagnosed with a complement-mediated disease or condition, or has a genetic predisposition to have a complement-mediated disease or condition.

[0279] 94. A composition comprising:

[0280] (a) Anti-C1s antibody according to any one of embodiments 1-48; and

[0281] (b) A solution containing one or more reagents for preserving organs or tissues intended to be transplanted into an individual recipient.

[0282] 95. The composition according to embodiment 94, wherein the solution is an organ preservation solution or a tissue preservation solution.

[0283] 96. The composition according to embodiment 94, wherein the solution is an organ perfusion solution or a tissue perfusion solution.

[0284] 97. The composition according to embodiment 94, wherein the solution comprises: (i) a salt; (ii) an edema-reducing agent; (iii) an oxygen free radical scavenger; and (iv) an energy supply system component.

[0285] 98. The composition according to embodiment 97, wherein the composition comprises potassium lactobionate, KH2PO4, MgSO4, raffinose, adenosine, glutathione, allopurinol and hydroxyethyl starch.

[0286] 99. An organ or tissue preservation solution comprising an anti-C1s antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0287] 100. An organ or tissue perfusion solution comprising an anti-C1s antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

[0288] 101. A method of preserving an organ or tissue for transplantation, the method comprising contacting the organ or tissue with a composition according to any one of embodiments 94-98.

[0289] 102. An isolated organ or tissue preserved in a composition according to any one of embodiments 94-98.

[0290] 103. The organ according to embodiment 102, wherein the organ is selected from the group consisting of the eye, heart, intestine, kidney, liver, lung, pancreas, stomach and thymus.

[0291] 104. The tissue according to embodiment 102, wherein the tissue is selected from the group consisting of bone, bone marrow, cornea, heart valves, Langerhans islets, tendons, skin, blood, and veins.

[0292] 105. The tissue according to embodiment 104, wherein the blood tissue includes whole blood, red blood cells, white blood cells or umbilical cord blood.

[0293] 106. The tissue according to embodiment 104, wherein the blood tissue is a separated population of blood cells.

[0294] 107. An in vitro method for inhibiting complement activation in an organ or tissue, the method comprising contacting the organ or tissue with an antibody according to any one of embodiments 1-48, a pharmaceutical composition according to embodiment 57, or a solution containing an antibody according to any one of embodiments 1-48.

[0295] 108. The method according to embodiment 107, wherein the solution is an organ preservation solution or a tissue preservation solution.

[0296] 109. The method according to embodiment 107, wherein the solution is an organ perfusion solution or a tissue perfusion solution.

[0297] 110. The method according to embodiment 107, wherein the solution comprises: (i) a salt; (ii) an agent for reducing edema; (iii) an oxygen free radical scavenger; and (iv) an energy supply system component.

[0298] 111. The method according to embodiment 107, wherein the solution comprises potassium lactobionate, KH2PO4, MgSO4, raffinose, adenosine, glutathione, allopurinol and hydroxyethyl starch. Attached Figure Description

[0299] Figure 1 Describe the amino acid sequence of the Homo sapiens complement C1s protein (SEQ ID NO:9).

[0300] Figure 2 Table 2 is provided.

[0301] Figure 3 Describe the competition between IPN003 (M34) and M81 for binding with human C1s.

[0302] Figure 4 Describe the inhibition of C1s-mediated activation of human complement protein C4 by IPN003.

[0303] Figure 5 The effect of IPN003, as determined using a standard hemolysis assay, on the activation of the intact classical complement cascade is depicted.

[0304] Figure 6 and Figure 7 The inhibition of complement by IPN003 in serum from two monkey species was described.

[0305] Figure 8 Describe the specificity of IPN003 for C1s.

[0306] Figure 9 Table 4 is provided.

[0307] Figure 10 Describe the binding of IPN003 and M81 to rat C1s.

[0308] Figure 11 Describe the inhibition of human C4 cleavage mediated by IPN003 in rats.

[0309] Figure 12 The inhibition of rat C1s-mediated human C4 cleavage by IPN003 and the inhibition of human C1s-mediated human C4 cleavage by IPN003 were described.

[0310] Figure 13 To depict the effect of IPN003 on hemolysis of human erythrocytes mediated by patient serum.

[0311] Figure 14 To depict the effect of IPN003 on C3b deposition mediated by patient serum on human erythrocytes.

[0312] Figure 15Provide the amino acid sequences of the IPN003 VL and VH regions, IPN003 VL CDR and IPN003 VH CDR.

[0313] Figure 16 Depicting the amino acid sequence of humanized IPN003 VH variant 1; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 46).

[0314] Figure 17 Depicting the amino acid sequence of humanized IPN003 VH variant 2; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 47).

[0315] Figure 18 Depicting the amino acid sequence of humanized IPN003 VH variant 3; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 48).

[0316] Figure 19 Depicting the amino acid sequence of humanized IPN003 VH variant 4; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 49).

[0317] Figure 20 Depicting the amino acid sequence of humanized IPN003 Vκ variant 1; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 50).

[0318] Figure 21 Depicting the amino acid sequence of humanized IPN003 Vκ variant 2; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 51).

[0319] Figure 22 Depicting the amino acid sequence of humanized IPN003 Vκ variant 3; and the nucleotide sequence encoding said amino acid sequence (SEQ ID NO: 52).

[0320] Figure 23 Table 5 is provided, which shows the amino acid differences between the parent IPN003 VH and the exemplary VH variant; and Table 6, which shows the amino acid differences between the parent IPN003 VL and the exemplary VL variant.

[0321] Figure 24 Tables 7 and 8 are provided, which show the binding characteristics of the humanized IPN003 variant with activated C1s and pro-C1s.

[0322] Figure 25 Describing humanized variants of IPN003 for ICs that compete with IPN003 for binding C1s 50 .

[0323] Figure 26 Describe the inhibition of the classical complement pathway by humanized variants of IPN003.

[0324] Figure 27A and Figure 27B Describing the effects of three humanized IPN003 variants on the classical complement pathway ( Figure 27A ) and alternative complement pathways ( Figure 27B The effect of activation.

[0325] Figure 28 The effects of the humanized IPN003 variant on complement-mediated hemolysis and on C3b deposition on antibody-sensitized red blood cells (RBCs) were depicted.

[0326] Figure 29 Describe the inhibition of plasma-mediated hemolysis in patients with cold agglutinin disease (CAD) by IPN003 or a humanized IPN003 variant (hu-IPN003).

[0327] Figure 30 Describe the inhibition of anaphylactic toxin production by IPN003 or hu-IPN003.

[0328] Figure 31 To depict the inhibition of C3b deposition on human RBCs mediated by plasma from CAD patients by IPN003.

[0329] Figure 32 Describe the concentration-dependent inhibition of IPN003 on plasma-mediated C3b deposition in human RBCs from CAD patients.

[0330] Figure 33A and Figure 33B Describing the humanized IPN003 variant ( Figure 33A ) and for chimeric anti-C1s antibodies ( Figure 33B The proliferation response of ).

[0331] Figure 34 To depict the effect of TNT003 on complement-dependent hemolysis mediated by autoantibodies present in the plasma of patients with cold agglutinin disease (CAD).

[0332] Figure 35 To depict the effect of TNT003 on complement-dependent C3b deposition mediated by autoantibodies present in the plasma of CAD patients.

[0333] Figure 36 To depict the effect of TNT003 on complement-dependent phagocytosis mediated by autoantibodies present in the plasma of CAD patients.

[0334] Figures 37A-37CTo depict the effects of TNT003 on complement-dependent C3a, C4a, and C5a mediated by autoantibodies present in the plasma of CAD patients.

[0335] Figure 38A and Figure 38B Describe the in vitro activity of TNT003 on hemolysis and C3b deposition after administration to non-human primates.

[0336] Figure 39 Describe the in vivo effects of TNT003 on C4a after administration to non-human primates.

[0337] Figure 40 Depict the combination of TNT003 with human C1s fragments.

[0338] Figure 41 To depict the effect of mutations in D343 and D357 on the inhibition of C1s activity by TNT003.

[0339] Figure 42 The inhibitory effect of TNT003 on human C1s activity was described.

[0340] Figure 43A and Figure 43B Describe the binding of TNT003 to C1s present in the C1 complex.

[0341] Figure 44 Describe the inhibition of human C1s by TNT003 and TNT003 fragments.

[0342] Figure 45 Describe the binding of TNT003 with human C1s under non-reducing conditions.

[0343] Figure 46 The inhibition of complement C4 activation, but not complement C2 activation, by TNT003 was described.

[0344] Figure 47 The levels of C1s in plasma samples from healthy volunteers and patients with CAD were depicted.

[0345] definition

[0346] The terms “antibody” and “immunoglobulin” include any isotype of antibody or immunoglobulin that maintains specific binding to an antigen, antibody fragments including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single-domain antibodies (dAb), single-domain heavy-chain antibodies, single-domain light-chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen-binding) portion of both antibody and non-antibody proteins. Antibodies may be detectably labeled, for example, with radioisotopes, enzymes that produce detectable products, fluorescent proteins, etc. Antibodies may be further conjugated to other portions, such as members of specific binding pairs, such as biotin (a member of the biotin-antibiotin protein specific binding pair). Antibodies may also be conjugated to solid supports, including but not limited to polystyrene plates or beads. The term also covers Fab', Fv, F(ab')2, and / or other antibody fragments that maintain specific binding to an antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all generated by a single cell through repeated cell replication. That is, the cell clone produces only a single type of antibody. While monoclonal antibodies can be prepared using hybridoma techniques, other preparation methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are “Y-shaped” molecules composed of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

[0347] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin comprising immunoglobulin moieties from different sources, wherein at least one moiety contains a human-derived amino acid sequence. For example, a humanized antibody may comprise a moiety derived from a non-human source (such as mouse) with necessary specificity and a moieties derived from human-derived immunoglobulin sequences (e.g., chimeric immunoglobulins), chemically concatenated by conventional techniques (e.g., synthesis) or prepared as a continuous polypeptide using genetic engineering techniques (e.g., expressing DNA encoding the protein moieties of the chimeric antibody to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR (correlation domain) of an antibody derived from a non-human source and a framework region of a human-derived light chain and / or heavy chain (e.g., CDR-transplanted antibodies with or without framework variations). The term humanized immunoglobulin also encompasses chimeric or CDR-transplanted single-chain antibodies. See, for example, Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, EA et al., European Patent Application No. 0,519,596 A1. For information on single-chain antibodies, see also Ladner et al., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786; and Bird, RE et al., Science, 242: 423-426 (1988)).

[0348] For example, humanized immunoglobulins can be produced by using synthetic and / or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized strand. For example, nucleic acid (e.g., DNA) sequences encoding humanized variable regions can be constructed by using PCR mutagenesis to alter the DNA sequence encoding a human or humanized strand, such as DNA templates derived from previously humanized variable regions (see, for example, Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, BL, et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, AP and JS Crowe, Gene, 101: 297-302 (1991)). Variants can also be readily prepared using these or other suitable methods. For example, the variable region of the clone can be mutagenized, and the sequence encoding the variant with desired specificity can be selected (e.g., from a phage library; see, for example, Krebber et al., U.S. Patent No. 5,514,548; Hoogenboom et al., WO 93 / 06213, published April 1, 1993).

[0349] An "antibody fragment" contains a portion of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (Zapata et al., ProteinEng. 8(10): 1057-1062 (1995)); and domain antibodies (dAb; Holt et al., (2003)). Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named for its tendency to crystallize. Pepsin treatment yields the F(ab')2 fragment, which has two antigen-binding sites and remains capable of cross-linking the antigen.

[0350] "Fv" is the smallest antibody fragment containing both a complete antigen recognition site and an antigen binding site. This region consists of a dimer of a tightly bound, non-covalently associated heavy chain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define V. H -V LAntigen-binding sites on the surface of the dimer. In general, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, but the affinity is lower compared to the entire binding site.

[0351] The “Fab” fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, which includes one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name used herein for Fab' fragments in which the cysteine ​​residues of the constant domain have a free thiol group. The F(ab')2 antibody fragment was initially generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0352] The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types (called κ and λ) based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their constant domain of heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. These subclasses can be further subdivided into types, such as IgG2a and IgG2b.

[0353] A single-chain Fv, sFv, or scFv antibody fragment contains the antibody's V. H and V L Domains, wherein these domains are present within a single polypeptide chain. In some embodiments, the Fv polypeptide is further contained in V H With V L The polypeptide linkers between the domains enable sFv to form the structures required for antigen binding. For a review of sFv, see [link to review]. Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer- Verlag, New York, pp. 269-315 (1994) ).

[0354] The term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, the fragment being contained within the same polypeptide chain linked to a light chain variable domain (V). L The heavy chain variable structural domain (V) H ) (V H -V LBy using linkers that are too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain on another strand, creating two antigen-binding sites. Biantibodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., (1993). Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0355] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two reagents (e.g., antibody and antigen) and is expressed as a dissociation constant (Ka). D The term "affinity" can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1,000 times or more of the antibody's affinity for unrelated amino acid sequences. The antibody's affinity for the target protein can be, for example, from about 100 nanomolar concentrations (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar concentration (pM), or from about 100 nM to about 1 femtomolar concentration (fM) or less. As used herein, the term "affinity" refers to the resistance to the dissociation of a complex of two or more reagents upon dilution. The terms "immunoresistance" and "preferential binding" are used interchangeably herein with respect to antibody and / or antigen-binding fragments.

[0356] The term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding (including interactions such as salt bridges and water bridges). The subject anti-C1s antibody specifically binds to an epitope within the complement C1s protein. "Specific binding" refers to binding at least approximately 10... -7 M or larger, for example 5×10 -7 M, 10 -8 M, 5×10 -8 M binds with greater affinity. "Non-specific binding" refers to binding with an affinity less than approximately 10. -7 The affinity of M, for example, with 10 -6 M, 10 -5 M, 10 -4 Affinity binding of M, etc.

[0357] As used herein, the term “CDR” or “complementarity-determining region” is intended to refer to a discontinuous antigen-binding site present in the variable region of a heavy-chain and light-chain polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlap or subsets of amino acid residues when compared to one another. However, the application of any definition to mean that the CDR of an antibody or transplanted antibody or its variant is intended to be within the scope of the terminology as defined and used herein. The amino acid residues covering the CDRs defined by the references cited above are described in Table 1 below for comparison. The CDRs listed in Table 2 are based on the definition in Kabat 1991.

[0358] Table 1: CDR Definition

[0359]

[0360] 1 Residue numbering follows that of Kabat et al. Same naming convention

[0361] 2 Residue numbering follows that of Chothia et al. Same naming convention

[0362] 3 Residue numbering follows MacCallum et al., Same naming convention

[0363] As used herein, the terms "CDR-L1", "CDR-L2", and "CDR-L3" refer to the first, second, and third CDRs in the variable region of a light chain, respectively. As used herein, the terms "CDR-H1", "CDR-H2", and "CDR-H3" refer to the first, second, and third CDRs in the variable region of a heavy chain, respectively. As used herein, the terms "CDR-1", "CDR-2", and "CDR-3" refer to the first, second, and third CDRs in the variable region of any chain, respectively.

[0364] As used herein, the term "framework," when used with respect to the variable region of an antibody, is intended to refer to all amino acid residues outside the CDR region within the variable region of the antibody. The variable region frame is typically a discontinuous amino acid sequence of about 100-120 amino acids in length, but is intended to refer only to those amino acids outside the CDR. As used herein, the term "framework region" is intended to refer to the individual domains of the frame separated by the CDR.

[0365] "Isolated" antibodies are antibodies that have been identified and isolated and / or recovered from components of their native environment. Contaminant components of their native environment are substances that could interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody will be purified (1) to greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, for example, greater than 99% by weight, (2) to a degree sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues, by using a turn-cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions, using Coomassie blue or silver staining. Isolated antibodies include recombinant intracellular in situ antibodies, as at least one component of the antibody's native environment will be absent. In some cases, isolated antibodies will be prepared by at least one purification step.

[0366] In this document, the terms “polypeptide,” “peptide,” and “protein,” which are used interchangeably, refer to an amino acid in polymeric form of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. The terms include fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunotagged proteins; and so on.

[0367] As used herein, the terms “treatment,” “treating,” “treat,” etc., refer to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or as a side effect attributable to said disease. As used herein, “treatment” encompasses any treatment of diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease in subjects who may be susceptible to said disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e. causing the disease to regress.

[0368] In this document, the terms “individual,” “subject,” “host,” and “patient,” which are used interchangeably, refer to mammals, including but not limited to rodents (rats, mice), non-human primates, humans, canines, felines, and ungulates (e.g., equines, bovines, sheep, pigs, goats). These terms also encompass any animal with a complement system, such as mammals, fish, and some invertebrates. Therefore, these terms include mammals with a complement system, fish and invertebrate companion animals, agricultural animals, working animals, zoo animals, and laboratory animals.

[0369] "Therapeutic effective dose" or "effective dose" refers to the amount of anticomplement C1s antibody that is sufficient to treat a disease when administered to a mammal or other subject. The "therapeutic effective dose" will vary depending on the anticomplement C1s antibody, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0370] "Biological sample" encompasses a wide range of sample types obtained from an individual and used in diagnostic or monitoring assays. The definition covers blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after acquisition, such as by treatment with reagents, solubilization, or enrichment of certain components such as polynucleotides. The term "biological sample" covers clinical samples and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, eye discharge, synovial fluid, and blood fractions such as plasma and serum. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

[0371] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore, modifications are naturally possible. Furthermore, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0372] When a range of values ​​is provided, it should be understood that, unless the context clearly indicates otherwise, the invention covers every intermediate value (one-tenth of a unit to the lower limit) between the upper and lower limits of that range and any other stated value or intermediate value within that range. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered by the invention, subject to any specific exclusion limits within the range. When the range includes one or both limits, ranges excluding any one or both of those included limits are also included in the invention.

[0373] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used to practice or test the invention, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials relating to the cited publications.

[0374] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “described” as used herein and in the appended claims include a plural of the referred to. Thus, for example, reference to “humanized anti-complement C1s antibody” includes a plurality of such antibodies, and reference to “complement-mediated disease” includes reference to one or more complement-mediated diseases known to those skilled in the art and their equivalents, etc. Furthermore, it should be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a premise for the use of exclusive terms such as “only,” “merely,” etc., or the use of “negative” limiting terms in relation to references to claim elements.

[0375] It should be understood that certain features of the invention described for clarity in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, multiple features of the invention described for brevity in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments of the invention are specifically covered by the invention and disclosed herein, as if each and all combinations were individually and explicitly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also specifically covered by the invention and disclosed herein, as if each and all such sub-combinations were individually and explicitly disclosed herein.

[0376] The publications discussed herein are provided only with respect to their disclosure prior to the filing date of this application. Nothing herein should be construed as allowing the invention to rely on prior art without authorization. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification. Invention Details

[0377] This disclosure provides antibodies that bind to complement C1s proteins (i.e., anti-complement C1s antibodies, also referred to herein as anti-C1s antibodies and C1s antibodies) and nucleic acid molecules encoding such antibodies. This disclosure also provides compositions comprising such antibodies, and methods for preparing and using such antibodies, nucleic acid molecules, and compositions. This disclosure provides methods for treating complement-mediated diseases or conditions, including the administration of anti-C1s antibodies. This disclosure further provides in vitro and in vivo detection methods for using the anti-C1s antibodies described herein.

[0378] Anti-complement C1s antibody

[0379] This disclosure provides anti-complement C1s antibodies and pharmaceutical compositions comprising such antibodies. Complement C1s is an attractive target because it is upstream of the complement cascade and has a narrow range of substrate specificity. Furthermore, it is possible to obtain antibodies (e.g., but not limited to monoclonal antibodies) that specifically bind to the activated form of C1s.

[0380] This disclosure provides isolated antibodies that specifically bind to epitopes within the complement C1s protein. Unless otherwise stated, the complement C1s protein used herein is the activated C1s protein. In some embodiments, the isolated anti-C1s antibody of this disclosure binds to the activated C1s protein. In some embodiments, the isolated anti-C1s antibody of this disclosure binds to an inactive form of C1s. In other cases, the isolated anti-C1s antibody of this disclosure binds to both the activated C1s protein and an inactive form of C1s. In some cases, the antibody is humanized, for example, one or more framework regions of the heavy chain variable region and / or light chain variable region include sequences derived from human immunoglobulin frameworks.

[0381] This disclosure provides isolated monoclonal antibodies that inhibit C4 cleavage, wherein the isolated monoclonal antibody does not inhibit C2 cleavage. In some cases, the isolated monoclonal antibody is humanized. In some cases, the antibody inhibits a component of the classical complement pathway. In some cases, the component of the classical complement pathway inhibited by the antibody is C1s. This disclosure also provides methods for treating complement-mediated diseases or conditions, the methods comprising administering to an individual in need an effective amount of an isolated monoclonal antibody that inhibits C4 cleavage, or a pharmaceutical composition comprising the isolated monoclonal antibody, wherein the isolated monoclonal antibody does not inhibit C2 cleavage.

[0382] This disclosure provides isolated monoclonal antibodies that inhibit C4 cleavage caused by C1s, i.e., inhibit C1s-mediated C4 proteolytic cleavage. In some cases, the isolated monoclonal antibody is humanized. In some cases, the antibody inhibits C1s-mediated C4 cleavage by inhibiting the binding of C4 to C1s; for example, in some cases, the antibody inhibits C1s-mediated C4 cleavage by inhibiting the binding of C4 to the C4 binding site of C1s. Thus, in some cases, the antibody acts as a competitive inhibitor. This disclosure also provides a method of treating complement-mediated diseases or conditions, the method comprising administering to an individual in need an effective amount of isolated monoclonal antibody that inhibits C4 cleavage caused by C1s, i.e., inhibits C1s-mediated C4 proteolytic cleavage.

[0383] This disclosure provides isolated monoclonal antibodies that inhibit C4 cleavage mediated by C1s, wherein the antibody does not inhibit C1s cleavage of complement component C2; that is, the antibody inhibits C1s-mediated C4 cleavage but not C1s-mediated C2 cleavage. In some cases, the isolated monoclonal antibody is humanized. In some cases, the monoclonal antibody inhibits the binding of C4 to C1s but not the binding of C2 to C1s. This disclosure also provides a method of treating complement-mediated diseases or conditions, the method comprising administering to an individual in need an effective amount of isolated monoclonal antibody that inhibits C1s cleavage of C4, wherein the antibody does not inhibit C1s cleavage of complement component C2; that is, the antibody inhibits C1s-mediated C4 cleavage but not C1s-mediated C2 cleavage. In some embodiments of the method, the antibody is humanized.

[0384] This disclosure provides isolated humanized monoclonal antibodies that specifically bind to epitopes within the regions covering domains IV and V of the C1s structure. For example, this disclosure provides specific binding... Figure 1 The isolated humanized monoclonal antibody is an isolated humanized monoclonal antibody containing epitopes within amino acids 272-422 of the amino acid sequence depicted in [image / description] and shown in SEQ ID NO:9. In some cases, the isolated humanized monoclonal antibody specifically binds to [specific amino acids]. Figure 1 The epitopes within amino acids 272-422 of the amino acid sequence depicted in and shown in SEQ ID NO:9 are inhibited, and the binding of C4 to C1s is also inhibited. This disclosure also provides a method for treating complement-mediated diseases or conditions, the method comprising administering to an individual in need an effective amount of an isolated humanized monoclonal antibody, the isolated humanized monoclonal antibody specifically binding to… Figure 1 Epitopes within amino acids 272-422 of the amino acid sequence depicted in and shown in SEQ ID NO:9, and inhibit the binding of C4 to C1s.

[0385] This disclosure provides isolated humanized monoclonal antibodies that specifically bind to conformational epitopes within the regions covering domains IV and V of C1s. For example, this disclosure provides specific binding... Figure 1 The isolated humanized monoclonal antibody is a bioassay of the conformational epitopes within amino acids 272-422 of the amino acid sequence depicted in [image description] and shown in SEQ ID NO:9. In some cases, the isolated humanized monoclonal antibody specifically binds to [specific amino acids]. Figure 1 The conformational epitopes within amino acids 272-422 of the amino acid sequence depicted in and shown in SEQ ID NO:9 are inhibited, and the binding of C4 to C1s is suppressed. This disclosure also provides a method for treating complement-mediated diseases or conditions, the method comprising administering an effective amount of an isolated humanized monoclonal antibody to an individual in need, the isolated humanized monoclonal antibody specifically binding to… Figure 1 The conformational epitopes within amino acids 272-422 of the amino acid sequence depicted in and shown in SEQ ID NO:9 are inhibited, and the binding of C4 to C1s is suppressed.

[0386] This disclosure provides isolated monoclonal antibodies that bind to the complement component C1s in a C1 complex. The C1 complex consists of 6 molecules of C1q, 2 molecules of C1r, and 2 molecules of C1s. In some cases, the isolated monoclonal antibody is humanized. Therefore, in some cases, this disclosure provides isolated humanized monoclonal antibodies that bind to the complement component C1s in a C1 complex. In some cases, the antibody binds to C1s present in the C1 complex with high affinity.

[0387] Humanization of the frame region reduces the risk of the antibody inducing a human anti-mouse antibody (HAMA) response in humans. Methods recognized in the art for assessing immune responses can be used to monitor HAMA responses in specific patients or during clinical trials. Immunogenicity assessment can be performed on patients receiving the humanized antibody at the start of treatment and throughout administration. HAMA responses can be measured, for example, by detecting antibodies to the humanized therapeutic agent in serum samples from patients using methods known to those skilled in the art, including surface plasmon resonance (BIACORE) and / or solid-phase enzyme-linked immunosorbent assay (ELISA) analysis. In many cases, the subject-modified anti-C1s antibody does not substantially induce a HAMA response in human subjects.

[0388] Certain amino acids from human variable framework residues are selected for substitution based on their potential impact on CDR conformation and / or antigen binding. Unnatural juxtaposition of the murine CDR region with the human variable framework region can lead to unnatural conformational restriction, which, unless corrected by substitution of certain amino acid residues, results in a loss of binding affinity.

[0389] The selection of amino acid residues for substitution may be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are constructed starting with the resolved structure of an immunoglobulin chain or its domains. The chain to be modeled is compared with chains or domains having resolved three-dimensional structures regarding amino acid sequence similarity, and the chain or domain exhibiting the greatest sequence similarity is selected as the starting point for constructing the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling; for example, those sharing at least 60%, at least 70%, at least 80%, at least 90%, or greater sequence identity are selected. The resolved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain being modeled and those in the starting structure. The modified structure is then assembled into a complex immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable ranges.

[0390] CDRs and framework regions are defined as follows: Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Alternative structural definitions have been proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651 (1989) (collectively, “Chothia”). When framework residues, as defined by Kabat, constitute loop residues, as defined by Chothia, amino acids present in mouse antibodies can be substituted into humanized antibodies. Residues “adjacent to a CDR region” include amino acid residues located in the immediate vicinity of one or more CDRs in the primary sequence of the humanized immunoglobulin chain, such as in a CDR as defined by Kabat or as defined by Chothia (see, for example, Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDR and, if selected from the receptor, distort the donor CDR and reduce affinity. Furthermore, adjacent amino acids can interact directly with the antigen (Amit et al., Science, 233:747 (1986)) and it may be necessary to select these amino acids from the donor to maintain all antigen contacts that provide affinity in the original antibody.

[0391] In some embodiments, the anti-C1s antibody of this disclosure (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) one, two, or three V antibodies comprising IPN003 antibody. L The light chain region of the CDR; and b) containing one, two, or three V antibodies of IPN003. H The heavy chain region of the CDR; wherein the V H and V L CDRs are as defined by Kabat (see, for example, Table 1 above; and Kabat 1991). In some such embodiments, anti-C1s antibodies include humanized V... H and / or V L Frame area (FR).

[0392] In some embodiments, the anti-C1s antibody of this disclosure (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) one, two, or three V antibodies comprising IPN003 antibody. L The light chain region of the CDR; and b) containing one, two, or three V antibodies of IPN003. H The heavy chain region of the CDR; wherein the V H and V L CDRs are as defined by Chothia (see, for example, Table 1 above; and Chothia 1987). In some such embodiments, anti-C1s antibodies include humanized V... H and / or V L Frame area.

[0393] The CDR amino acid sequence and V of the IPN003 antibody L and V H The amino acid sequences are provided in Table 2 ( Figure 2 Table 2 also provides the specified SEQ ID NO for each amino acid sequence.

[0394] In some embodiments, the anti-C1s antibody of this disclosure (e.g., a subject antibody that specifically binds to epitopes in complement C1s proteins) comprises: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some such embodiments, the anti-C1s antibody comprises humanized V H and / or V L Frame area.

[0395] SEQ ID NO:1: SSVSSSYLHWYQ;

[0396] SEQ ID NO:2: STSNLASGVP;

[0397] SEQ ID NO:3: HQYYRLPPIT;

[0398] SEQ ID NO:4: GFTFSNYAMSWV;

[0399] SEQ ID NO:5: ISSGGSHTYY;

[0400] SEQ ID NO:6: ARLFTGYAMDY.

[0401] In some embodiments, the anti-C1s antibody of this disclosure comprises a CDR 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:5 and SEQ ID NO:6.

[0402] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0403] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing the amino acid sequences SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0404] In some embodiments, the anti-C1s antibody of this disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:1, CDR-L2 having the amino acid sequence SEQ ID NO:2, CDR-L3 having the amino acid sequence SEQ ID NO:3, CDR-H1 having the amino acid sequence SEQ ID NO:4, CDR-H2 having the amino acid sequence SEQ ID NO:5, and CDR-H3 having the amino acid sequence SEQ ID NO:6.

[0405] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7.

[0406] SEQ ID NO:7: DIVMTQTTAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0407] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8.

[0408] SEQ ID NO:8: QVKLEESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVT.

[0409] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7.

[0410] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8.

[0411] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7.

[0412] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0413] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:8.

[0414] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:7 and a heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0415] In some embodiments, the anti-C1s antibody of this disclosure specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody containing a light chain CDR having an antibody light chain variable region containing the amino acid sequence SEQ ID NO:7 and an antibody heavy chain variable region containing an antibody heavy chain CDR having the amino acid sequence SEQ ID NO:8 for binding to the epitope.

[0416] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain CDR containing the antibody light chain variable region of amino acid sequence SEQ ID NO:7 and a heavy chain CDR containing the antibody heavy chain variable region of amino acid sequence SEQ ID NO:8.

[0417] In some cases, humanized V H Frame or V L The framework is a shared human framework. A shared humanized framework can represent human immunoglobulin V. L or V H The selection of the framework sequence is based on the most commonly present amino acid residues.

[0418] Suitable for use with V as described herein H Co-owners V who use CDR together H Unrestricted instances of the frame region include (common to subclass III):

[0419] a)V H FR1: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:53);

[0420] b) V H FR2: WVRQAPGKGLEWV (SEQ ID NO:54);

[0421] c) V H FR3: RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55); and

[0422] d) V H FR4: WGQGTLVTVSS (SEQ ID NO:56).

[0423] In some cases, V H FR3 includes amino acid substitutions at positions 71, 73, and / or 78; for example, where RFTIS R The underlined and bold R in DNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55) is amino acid 71 (Kabat number); RFTISRD NIn SKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55), the underlined and bold N represents amino acid 73 (Kabat number); and RFTISRDNSKNT L In YLQMNSLRAEDTAVYYC (SEQ ID NO:55), the underlined and bold L represents amino acid 78 (Kabat number). For example, in some cases, amino acid 71 is A; and / or amino acid 73 is T; and / or amino acid 78 is A. As an example, in some cases, the appropriate co-humanized V H FR3 contains the amino acid sequence: RFTIS A D T SKNT A YLQMNSLRAEDTAVYYC (SEQ ID NO:57).

[0424] Suitable for use with V as described herein H Co-owners V who use CDR together H Unrestricted instances of the frame region include (common to subclass I):

[0425] a)V H FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO:58);

[0426] b) V H FR2: WVRQAPGQGLEWM (SEQ ID NO:59);

[0427] c) V H FR3: RVTITADTSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO:60); and

[0428] d) V H FR4: WGQGTLVTVSS (SEQ ID NO:56).

[0429] Suitable for use with V as described herein H Co-owners V who use CDR together H Unrestricted instances of the frame region include (common to subclass II):

[0430] a)V H FR1: QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO:61);

[0431] b) V HFR2: WIRQPPGKGLEWI (SEQ ID NO:62);

[0432] c) V H FR3: RVTISVDTSKNQFSLKLSSVTAADTAVYYC (SEQ ID NO:63); and

[0433] d) V H FR4: WGQGTLVTVSS (SEQ ID NO:56).

[0434] Suitable for use with V as described herein L Co-owners V who use CDR together L Unrestricted instances of the frame region include (common to subclass I):

[0435] a)V L FR1: DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO:57);

[0436] b) V L FR2: WYQQKPGKAPKLLIY (SEQ ID NO:58);

[0437] c) V L FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:59); and

[0438] d) V L FR4: FGQGTKVEIK (SEQ ID NO:60).

[0439] Suitable for use with V as described herein L Co-owners V who use CDR together L Unrestricted instances of the frame region include (common to subclass II):

[0440] a)V L FR1: DIVMTQSPLSLPVTPGEPASISC (SEQ ID NO:64);

[0441] b) V L FR2: WYLQKPGQSPQLLIY (SEQ ID NO:65);

[0442] c) V LFR3: GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO:66); and

[0443] d) V L FR4: FGQGTKVEIK (SEQ ID NO:60).

[0444] Suitable for use with V as described herein L Co-owners V who use CDR together L Unrestricted instances of the frame region include (common to subclass III):

[0445] a)V L FR1: DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:67);

[0446] b) V L FR2: WYQQKPGQPPKLLIY (SEQ ID NO:68);

[0447] c) V L FR3: GVPDRFSGSGSGTTDFTLTISSLQAEDFAVYYC (SEQ ID NO:69); and

[0448] d) V L FR4: FGQGTKVEIK (SEQ ID NO:60).

[0449] Suitable for use with V as described herein L Co-owners V who use CDR together L Unrestricted instances of the frame region include (common to subclass IV):

[0450] a) V L FR1: DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:67);

[0451] b) V L FR2: WYQQKPGQPPKLLIY (SEQ ID NO:68);

[0452] c) V L FR3: GVPDRFSGSGSGTTDFTLTISSLQAEDFAVYYC (SEQ ID NO:69); and

[0453] d) V LFR4: FGQGTKVEIK (SEQ ID NO:60).

[0454] In some embodiments, the anti-C1s antibody of this disclosure binds to complement C1s protein from an individual possessing a complement system. In some embodiments, the anti-C1s antibody of this disclosure binds to complement C1s protein from mammals, fish, or invertebrates possessing a complement system. In some embodiments, the anti-C1s antibody of this disclosure binds to mammalian complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure binds to complement C1s protein having SEQ ID NO:9. The amino acid sequence SEQ ID NO:9 represents Homo sapiens complement C1s protein, which has… Figure 1 The amino acid sequence shown.

[0455] In some embodiments, the anti-C1s antibody of this disclosure has a dissociation constant (Ki) of no more than 2.5 nM. D (This refers to the binding of complement C1s protein.) In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 2 nM K. D Binds to complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 1 nM. D Binds to complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is present at a concentration of no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM. D Binds to complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.3 nM. D Binds to complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.2 nM. D Binds to complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.1 nM. D Binding to complement C1s protein. The method for measuring the binding of the antibody to the C1s protein can be determined by those skilled in the art.

[0456] In some embodiments, the anti-C1s antibody of this disclosure is present in a Kc concentration of no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no more than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM, no more than 3 pM, no more than 2 pM, and no more than 1 pM. D It binds to complement C1s protein.

[0457] In some embodiments, the anti-C1s antibody of this disclosure has a dissociation constant (Ki) of no more than 2.5 nM. D This antibody binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 2 nM K. D Binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 1 nM. D It binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is present at a concentration of no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM. D Binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 0.3 nM K. D Binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 0.2 nM K. D Binds to human complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.1 nM. D The antibody binds to human complement C1s protein. The method for measuring the binding of the antibody to the human C1s protein can be determined by someone skilled in the art. In some embodiments, the binding assay as described in the examples is used to determine the K-linked antibody-human C1s protein. D .

[0458] In some embodiments, the anti-C1s antibody of this disclosure is present in a Kc concentration of no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no more than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM, no more than 3 pM, no more than 2 pM, and no more than 1 pM. D It binds to human complement C1s protein.

[0459] In some embodiments, the anti-C1s antibody of this disclosure that binds to human complement C1s protein also binds to complement C1s protein of another species. In some embodiments, the anti-C1s antibody of this disclosure that binds to human complement C1s protein also binds to rodent complement C1s protein. Examples of rodent complement C1s proteins include, but are not limited to, guinea pig C1s protein, hamster C1s protein, mouse C1s protein, and rat C1s protein. In some embodiments, the anti-C1s antibody of this disclosure that binds to human complement C1s protein also binds to rabbit complement C1s protein, such as rabbit C1s protein. In some embodiments, the anti-C1s antibody of this disclosure that binds to human complement C1s protein also binds to non-human primate complement C1s protein, wherein exemplary non-human primates include monkeys, such as rhesus monkeys. Macaca mulatta ) and crab-eating macaques ( Macaca fascicularis In some implementations, this cross-reactivity is used to bind the antibody to the K+ of the human complement C1s protein. D K of similar magnitude D The antibody binds to the complement C1s protein of another (non-human) species. In some embodiments, the anti-C1s antibody of this disclosure binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure that binds to human complement C1s protein also binds to rat complement C1s protein.

[0460] In some embodiments, the anti-C1s antibody of this disclosure has a dissociation constant (Ki) of no more than 2.5 nM. D This antibody binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 2 nM K. D Binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 1 nM. D Binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is present at a concentration of no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, or no more than 0.1 nM. D Binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is expressed at a concentration of no more than 0.3 nM K. D Binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.2 nM. D Binds to rat complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is in a K+ concentration of no more than 0.1 nM. DThe antibody binds to rat complement C1s protein. The method for measuring the binding of the antibody to rat C1s protein can be determined by those skilled in the art. In some embodiments, the binding assay as described in the examples is used to determine the K-linked antibody-rat C1s protein. D .

[0461] In some embodiments, the anti-C1s antibody of this disclosure is present in a Kc concentration of no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no more than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM, no more than 3 pM, no more than 2 pM, and no more than 1 pM. D Binds to rat complement C1s protein.

[0462] In some embodiments, the anti-C1s antibody of this disclosure binds to both native and denatured complement C1s proteins. As used herein, "native protein" means a protein folded in its naturally occurring physiological state, and therefore excludes denatured proteins. Binding can be detected by Western blotting. In such embodiments, the anti-C1s antibody of this disclosure binds to C1s proteins applied to native gels and also to C1s proteins applied to denatured (e.g., sodium dodecyl sulfate (SDS)) gels. In some embodiments, the subject matter anti-C1s antibody of this disclosure binds to linear epitopes in C1s. Methods for determining whether an antibody binds to native or denatured C1s proteins are known to those skilled in the art. In some embodiments, gel electrophoresis is used to determine whether an antibody binds to native and / or denatured C1s proteins.

[0463] In some embodiments, the anti-C1s antibody of this disclosure reduces the production of C4b2a (i.e., the complement C4b and C2a complex; also referred to as "C3 convertase") by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C4b2a produced in the absence of the subject anti-C1s antibody. Methods for measuring C4b2a production are known in the art.

[0464] In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of at least one substrate cleaved by complement C1s protein. In some embodiments, the substrate is selected from the group consisting of complement C2 and complement C4. In some embodiments, the substrate is complement C2. In some embodiments, the substrate is complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of complement C2. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of both complement C2 and complement C4.

[0465] In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of at least one substrate cleaved by human complement C1s protein. In some embodiments, the substrate is selected from the group consisting of human complement C2 and human complement C4. In some embodiments, the substrate is human complement C2. In some embodiments, the substrate is human complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of human complement C2. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of human complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of both human complement C2 and human complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits rat C1s-mediated cleavage of human complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits human C1s-mediated cleavage of human complement C4.

[0466] In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of at least one substrate cleaved by rat complement C1s protein. In some embodiments, the substrate is selected from the group consisting of rat complement C2 and rat complement C4. In some embodiments, the substrate is rat complement C2. In some embodiments, the substrate is rat complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of rat complement C2. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of rat complement C4. In some embodiments, the anti-C1s antibody of this disclosure inhibits the cleavage of both rat complement C2 and rat complement C4.

[0467] In some embodiments, the anti-C1s antibody of this disclosure inhibits C1s by spatially blocking access to the C1s active site or by spatially blocking access to the substrate.

[0468] In some embodiments, the anti-C1s antibody of this disclosure inhibits C1s-mediated complement C4 activation. For example, in some cases, the anti-C1s antibody of this disclosure is less than 50 × 10⁻⁶. -9 M, less than 25×10 -9 M, less than 10×10 -9 M, less than 5×10-9 M, less than 1×10 -9 M, less than 0.5×10 -9 M, less than 0.1×10 -9 M or less than 0.1×10 -10 M's IC 50 Inhibit C1s-mediated complement C4 activation.

[0469] In some cases, the anti-C1s antibody of this disclosure inhibits complement-mediated cell lysis, for example, in in vitro cell lysis assays. For example, in some cases, the anti-C1s antibody of this disclosure exhibits a concentration of less than 10 × 10⁻⁶. -9 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 0.5×10 -9 M, less than 0.1×10 -9 M or less than 0.1×10 -10 M's IC 50 Inhibit complement-mediated cell lysis.

[0470] In some embodiments, the anti-C1s antibody of this disclosure competitively binds to the epitope bound by IPN003.

[0471] In some embodiments, the anti-C1s antibody of this disclosure includes a variable domain of the IPN003 antibody.

[0472] In some embodiments, the anti-C1s antibody disclosed herein is an IPN003 antibody.

[0473] This disclosure provides any anti-C1s antibody with embodiments to be humanized. In some embodiments, the anti-C1s antibody of this disclosure includes a humanized framework region. In some embodiments, the anti-C1s antibody of this disclosure includes a humanized light chain framework region. In some embodiments, the anti-C1s antibody of this disclosure includes a humanized heavy chain framework region.

[0474] In some embodiments, the subject anti-C1s antibody comprises one or more humanized framework regions (FRs). In some embodiments, the subject anti-C1s antibody comprises a light chain variable region containing one, two, three, or four humanized light chain FRs. In some embodiments, the subject antibody comprises light chain variable regions containing, in order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 as described herein; humanized light chain FR2; CDR-L2 as described herein; humanized light chain FR3; CDR-L3 as described herein; and humanized light chain FR4. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0475] For example, the subject antibody may comprise a light chain variable region containing the following in order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 containing the amino acid sequence SEQ ID NO:1; humanized light chain FR2; CDR-L2 containing the amino acid sequence SEQ ID NO:2; humanized light chain FR3; CDR-L3 containing the amino acid sequence SEQ ID NO:3; and humanized light chain FR4.

[0476] In some embodiments, the subject anti-C1s antibody comprises a heavy chain variable region containing one, two, three, or four humanized heavy chain FRs. In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus, the following heavy chain variable regions: humanized heavy chain FR1; CDR-H1 as described herein; humanized heavy chain FR2; CDR-H2 as described herein; humanized heavy chain FR3; CDR-H3 as described herein; and humanized heavy chain FR4.

[0477] For example, the subject antibody may comprise heavy chain variable regions containing the following in order from N-terminus to C-terminus: humanized heavy chain FR1; CDR-H1 containing the amino acid sequence SEQ ID NO:4; humanized heavy chain FR2; CDR-H2 containing the amino acid sequence SEQ ID NO:5; humanized heavy chain FR3; CDR-H3 containing the amino acid sequence SEQ ID NO:6; and humanized heavy chain FR4.

[0478] In some embodiments, the anti-C1s antibody of this disclosure (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:3; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. In some such embodiments, the anti-C1s antibody comprises humanized V H and / or V L Frame area.

[0479] SEQ ID NO:32: TASSSVSSSYLH;

[0480] SEQ ID NO:33: STSNLAS;

[0481] SEQ ID NO:3: HQYYRLPPIT;

[0482] SEQ ID NO:34: NYAMS;

[0483] SEQ ID NO:35: TISSGGSHTYYLDSVKG;

[0484] SEQ ID NO:36: LFTGYAMDY.

[0485] In some embodiments, the anti-C1s antibody of this disclosure comprises a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0486] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequences SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:3.

[0487] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing the amino acid sequences SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0488] In some embodiments, the anti-C1s antibody of this disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:32, CDR-L2 having the amino acid sequence SEQ ID NO:33, CDR-L3 having the amino acid sequence SEQ ID NO:3, CDR-H1 having the amino acid sequence SEQ ID NO:34, CDR-H2 having the amino acid sequence SEQ ID NO:35, and CDR-H3 having the amino acid sequence SEQ ID NO:36.

[0489] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37.

[0490] SEQ ID NO:37: QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0491] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38.

[0492] SEQ ID NO:38: EVMLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVTVSS.

[0493] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:37.

[0494] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:38.

[0495] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:37.

[0496] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO:38.

[0497] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:37 and a heavy chain variable region containing an amino acid sequence having 90% identity with the amino acid sequence SEQ ID NO:38.

[0498] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 95% identity with the amino acid sequence SEQ ID NO:37 and a heavy chain variable region containing an amino acid sequence having 95% identity with the amino acid sequence SEQ ID NO:38.

[0499] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing the amino acid sequence SEQ ID NO:37 and a heavy chain variable region containing the amino acid sequence SEQ ID NO:38.

[0500] In some embodiments, the anti-C1s antibody of this disclosure specifically binds to an epitope within the complement C1s protein, wherein the antibody competes with an antibody containing a light chain CDR of an antibody light chain variable region containing the amino acid sequence SEQ ID NO:37 and a heavy chain CDR of an antibody heavy chain variable region containing the amino acid sequence SEQ ID NO:38 for binding to the epitope.

[0501] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain CDR containing the antibody light chain variable region of amino acid sequence SEQ ID NO:37 and a heavy chain CDR containing the antibody heavy chain variable region of amino acid sequence SEQ ID NO:38.

[0502] In some embodiments, the subject anti-C1s antibody comprises one or more humanized framework regions (FRs). In some embodiments, the subject anti-C1s antibody comprises a light chain variable region containing one, two, three, or four humanized light chain FRs. In some embodiments, the subject antibody comprises light chain variable regions containing, in order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 as described herein; humanized light chain FR2; CDR-L2 as described herein; humanized light chain FR3; CDR-L3 as described herein; and humanized light chain FR4. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are: SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:3.

[0503] For example, the subject antibody may comprise a light chain variable region containing the following in order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 containing the amino acid sequence SEQ ID NO:32; humanized light chain FR2; CDR-L2 containing the amino acid sequence SEQ ID NO:33; humanized light chain FR3; CDR-L3 containing the amino acid sequence SEQ ID NO:3; and humanized light chain FR4.

[0504] In some embodiments, the subject anti-C1s antibody comprises a heavy chain variable region containing one, two, three, or four humanized heavy chain FRs. In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus, the following heavy chain variable regions: humanized heavy chain FR1; CDR-H1 as described herein; humanized heavy chain FR2; CDR-H2 as described herein; humanized heavy chain FR3; CDR-H3 as described herein; and humanized heavy chain FR4.

[0505] For example, the subject antibody may comprise heavy chain variable regions containing the following in order from N-terminus to C-terminus: humanized heavy chain FR1; CDR-H1 containing the amino acid sequence SEQ ID NO:34; humanized heavy chain FR2; CDR-H2 containing the amino acid sequence SEQ ID NO:35; humanized heavy chain FR3; CDR-H3 containing the amino acid sequence SEQ ID NO:36; and humanized heavy chain FR4.

[0506] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37.

[0507] In some embodiments, the anti-C1s antibody of this disclosure comprises a heavy chain variable region containing an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38.

[0508] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:39 and Figure 16 The heavy chain variable region of the amino acid sequence depicted (VH variant 1) has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence.

[0509] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:40 and Figure 17 The heavy chain variable region of the amino acid sequence depicted (VH variant 2) has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence.

[0510] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:41 and Figure 18 The heavy chain variable region of the amino acid sequence depicted (VH variant 3) has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence.

[0511] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:42 and Figure 19 The heavy chain variable region of the amino acid sequence depicted (VH variant 4) has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence.

[0512] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:43 and Figure 20 The amino acid sequence depicted (VK variant 1) has a light chain variable region with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0513] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:44 and Figure 21 The amino acid sequence depicted (VK variant 2) has a light chain variable region with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0514] The subject anti-C1s antibody may contain the same as shown in SEQ ID NO:45 and Figure 22 The amino acid sequence depicted (VK variant 3) has a light chain variable region with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0515] The subject anti-C1s antibody may include, relative to Table 5 ( Figure 23 The FR amino acid sequence of the IPN003 parent antibody described in the document contains heavy chain variable regions with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 frame (FR) amino acid substitutions.

[0516] For example, a subject anti-C1s antibody may contain a heavy chain variable region containing an M→Q substitution at amino acid position 3 of VH FR1 and / or an A→G substitution at amino acid position 10 of VH FR1 and / or a K→R substitution at amino acid position 19 of VH FR1.

[0517] As another example, a subject anti-C1s antibody may contain a heavy chain variable region containing an I→A substitution at amino acid position 40 of VH FR2 and / or an E→G substitution at amino acid position 42 of VH FR2 and / or an R→G substitution at amino acid position 44 of VH FR2.

[0518] As another example, the subject anti-C1s antibody may contain a heavy chain variable region containing an A→S substitution at amino acid position 74 of VH FR3 and / or an R→K substitution at amino acid position 75 of VH FR3 and / or a D→N substitution at amino acid position 76 of VH FR3 and / or an S→N amino acid substitution at amino acid position 82A of VH FR3 and / or an S→A amino acid substitution at amino acid position 84 of VH FR3.

[0519] As another example, a subject-specific anti-C1s antibody may contain a heavy chain variable region with an S→L substitution at amino acid position 108 of VH FR4.

[0520] The subject anti-C1s antibody may contain substances relative to those in Table 6 ( Figure 23The light chain variable region is a replacement of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 frame (FR) amino acids in the amino acid sequence of the IPN003 parent antibody described in the document.

[0521] For example, a subject anti-C1s antibody may contain a light chain variable region containing an I→T substitution at position 10 in VL FR1 and / or an M→L substitution at amino acid position 11 in VL FR1 and / or an A→L substitution at position 13 in VL FR1 and / or an L→P substitution at position 15 in VL FR1 and / or a V→A substitution at amino acid position 19 in VL FR1 and / or an M→L substitution at amino acid position 21 in VL FR1 and / or a T→S substitution at amino acid position 22 in VL FR1.

[0522] As another example, the subject anti-C1s antibody may contain a light chain variable region containing an S→K substitution at amino acid position 42 in VL FR2 and / or an S→A substitution at amino acid position 43 in VL FR2.

[0523] As another example, a subject anti-C1s antibody may contain a light chain variable region containing an A→S substitution at amino acid position 60 in VL FR3 and / or an F→D substitution at amino acid position 70 in VL FR3 and / or an S→T substitution at amino acid position 72 in VL FR3 and / or an M→L substitution at amino acid position 78 in VL FR3 and / or an E→Q substitution at amino acid position 79 in VL FR3 and / or an A→P substitution at amino acid position 80 in VL FR3 and / or a D→F substitution at amino acid position 83 in VL FR3.

[0524] As another example, a subject anti-C1s antibody may contain a light chain variable region with an A→Q substitution at amino acid position 100 in VL FR4 and / or an L→I substitution at amino acid position 106 in VL FR4.

[0525] In some cases, the anti-C1s antibodies disclosed herein comprise:

[0526] i) Includes Figure 16 VH variant 1 containing the amino acid sequence depicted in and shown in SEQ ID NO:39; and VH variant 1 containing Figure 20 Vk variant 1 of the amino acid sequence depicted in and shown in SEQ ID NO:43;

[0527] ii) Contains Figure 16 VH variant 1 containing the amino acid sequence depicted in and shown in SEQ ID NO:39; and VH variant 1 containing Figure 21 Vk variant 2 of the amino acid sequence depicted in and shown in SEQ ID NO:44;

[0528] iii) Contains Figure 16 VH variant 1 containing the amino acid sequence depicted in and shown in SEQ ID NO:39; and VH variant 1 containing Figure 22 Vk variant 3 of the amino acid sequence depicted in and shown in SEQ ID NO:45;

[0529] iv) contains Figure 17 VH variant 2, which contains the amino acid sequence depicted in [the text] and shown in SEQ ID NO:40; and [the text continues with other related information]. Figure 20 Vk variant 1 of the amino acid sequence depicted in and shown in SEQ ID NO:43;

[0530] v) contains Figure 17 VH variant 2, which contains the amino acid sequence depicted in [the text] and shown in SEQ ID NO:40; and [the text continues with other related information]. Figure 21 Vk variant 2 of the amino acid sequence depicted in and shown in SEQ ID NO:44;

[0531] vi) includes Figure 17 VH variant 2, which contains the amino acid sequence depicted in [the text] and shown in SEQ ID NO:40; and [the text continues with other related information]. Figure 22 Vk variant 3 of the amino acid sequence depicted in and shown in SEQ ID NO:45;

[0532] vii) contains Figure 18 VH variant 3 containing the amino acid sequence depicted in and shown in SEQ ID NO:41; and VH variant 3 containing Figure 20 Vk variant 1 of the amino acid sequence depicted in and shown in SEQ ID NO:43;

[0533] viii) contains Figure 18 VH variant 3 containing the amino acid sequence depicted in and shown in SEQ ID NO:41; and VH variant 3 containing Figure 21 Vk variant 2 of the amino acid sequence depicted in and shown in SEQ ID NO:44;

[0534] ix) contains Figure 18 VH variant 3 containing the amino acid sequence depicted in and shown in SEQ ID NO:41; and VH variant 3 containing Figure 22 Vk variant 3 of the amino acid sequence depicted in and shown in SEQ ID NO:45;

[0535] x) contains Figure 19VH variant 4, which contains the amino acid sequence depicted in and shown in SEQ ID NO:42; and VH variant 4 containing Figure 20 Vk variant 1 of the amino acid sequence depicted in and shown in SEQ ID NO:43;

[0536] xi) contains Figure 19 VH variant 4, which contains the amino acid sequence depicted in and shown in SEQ ID NO:42; and VH variant 4 containing Figure 21 Vk variant 2 of the amino acid sequence depicted in and shown in SEQ ID NO:44; or

[0537] xii) contains Figure 19 VH variant 4, which contains the amino acid sequence depicted in and shown in SEQ ID NO:42; and VH variant 4 containing Figure 22 Vk variant 3 of the amino acid sequence depicted in and shown in SEQ ID NO:45.

[0538] In some embodiments, the anti-C1s antibody of this disclosure is an Ig monomer or an antigen-binding fragment thereof that binds to the complement C1s protein. In some embodiments, the anti-C1s antibody of this disclosure is an Ig monomer. In some embodiments, the anti-C1s antibody of this disclosure is an antigen-binding fragment of an Ig monomer that binds to the complement C1s protein.

[0539] In some embodiments, the anti-C1s antibody of this disclosure is selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies.

[0540] In some embodiments, the anti-C1s antibody of this disclosure is selected from the group consisting of monospecific antibodies, bispecific antibodies and multispecific antibodies.

[0541] In some embodiments, the anti-C1s antibody of this disclosure comprises a light chain region and a heavy chain region present in a separate polypeptide.

[0542] In some embodiments, the anti-C1s antibody of this disclosure includes a light chain region and a heavy chain region present in a single polypeptide.

[0543] In some embodiments, the subject antibody comprises anti-C1s heavy chain CDR and anti-C1s light chain CDR in a single polypeptide chain; for example, in some embodiments, the subject antibody is scFv.

[0544] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length. In some implementations, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. For example, in some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1 comprising the amino acid sequence shown in SEQ ID NO:32; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:33; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1 comprising the amino acid sequence shown in SEQ ID NO:34; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2 comprising the amino acid sequence shown in SEQ ID NO:35; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3 comprising the amino acid sequence shown in SEQ ID NO:36; and a seventh amino acid sequence of about 5 to about 25 amino acids in length.

[0545] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a light chain FR1 region; CDR-L1; a light chain FR2 region; CDR-L2; a light chain FR3 region; CDR-L3; optionally a light chain FR4 region; a linker region; optionally a heavy chain FR1 region; CDR-H1; a heavy chain FR2 region; CDR-H2; a heavy chain FR3 region; CDR-H3; and a heavy chain FR4 region. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. In some such embodiments, one or more FR regions are humanized FR regions. In some such embodiments, each FR region is a humanized FR region. The length of the connecting subregion can be from about 5 amino acids (aa) to about 50 amino acids, for example, from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.

[0546] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length. In some implementations, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. For example, in some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1 comprising the amino acid sequence shown in SEQ ID NO:34; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2 comprising the amino acid sequence shown in SEQ ID NO:35; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3 comprising the amino acid sequence shown in SEQ ID NO:36; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1 comprising the amino acid sequence shown in SEQ ID NO:32; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:33; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length.

[0547] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a heavy chain FR1 region; CDR-H1; a heavy chain FR2 region; CDR-H2; a heavy chain FR3 region; CDR-H3; optionally a heavy chain FR4 region; a linker; optionally a light chain FR1 region; CDR-L1; a light chain FR2 region; CDR-L2; a light chain FR3 region; CDR-L3; and a light chain FR4 region. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. In some such embodiments, one or more FR regions are humanized FR regions. In some such embodiments, each FR region is a humanized FR region. The length of the connecting subregion can be from about 5 amino acids to about 50 amino acids, for example, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids.

[0548] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. For example, in some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO:1; a second amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:2; a third amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO:4; a fifth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO:5; a sixth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO:6; and a seventh amino acid sequence of about 5 to about 25 amino acids in length.

[0549] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a light chain FR1 region; CDR-L1; a light chain FR2 region; CDR-L2; a light chain FR3 region; CDR-L3; optionally a light chain FR4 region; a linker region; optionally a heavy chain FR1 region; CDR-H1; a heavy chain FR2 region; CDR-H2; a heavy chain FR3 region; CDR-H3; and a heavy chain FR4 region. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some such embodiments, one or more FR regions are humanized FR regions. In some such embodiments, each FR region is a humanized FR region. The length of the connecting subregion can be from about 5 amino acids (aa) to about 50 amino acids, for example, from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.

[0550] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; CDR-H1; a second amino acid sequence of about 5 to about 25 amino acids in length; CDR-H2; a third amino acid sequence of about 5 to about 25 amino acids in length; CDR-H3; a fourth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L1; a fifth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L2; a sixth amino acid sequence of about 5 to about 25 amino acids in length; CDR-L3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. For example, in some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO:4; a second amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO:5; a third amino acid sequence of about 5 to about 25 amino acids in length; a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO:6; a fourth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO:1; a fifth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:2; a sixth amino acid sequence of about 5 to about 25 amino acids in length; a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:3; and a seventh amino acid sequence of about 5 to about 25 amino acids in length.

[0551] In some embodiments, the subject antibody comprises, in order from N-terminus to C-terminus: a heavy chain FR1 region; CDR-H1; a heavy chain FR2 region; CDR-H2; a heavy chain FR3 region; CDR-H3; optionally a heavy chain FR4 region; a linker; optionally a light chain FR1 region; CDR-L1; a light chain FR2 region; CDR-L2; a light chain FR3 region; CDR-L3; and a light chain FR4 region. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some such embodiments, one or more FR regions are humanized FR regions. In some such embodiments, each FR region is a humanized FR region. The length of the connecting subregion can be from about 5 amino acids to about 50 amino acids, for example, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids.

[0552] Linkers suitable for use with the subject antibody include “flexible linkers.” If present, the linker molecule typically has a sufficient length to allow some flexible movement between the linked regions. In some embodiments, the linker molecule is typically about 6-50 atoms in length. The linker molecule can also be, for example, arylaceyne, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Other linker molecules capable of binding peptides can be used according to this disclosure.

[0553] Suitable linkers can be easily selected and can have any of a variety of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0554] An exemplary flexible connector includes a glycine polymer (G). n Glycine-serine polymers (including, for example, (GS)) n (GSGGS) n (SEQ ID NO:10) and (GGGS) n(SEQ ID NO:11), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because these two amino acids are relatively unstructured and therefore can serve as neutral tethers between components. Glycine polymers are of particular interest because glycine is even closer to a significantly larger phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:12), GGSGG (SEQ ID NO:13), GGSSG (SEQ ID NO:14), GGSGG (SEQ ID NO:15), GGGSG (SEQ ID NO:16), GSSSG (SEQ ID NO:17), etc. Those skilled in the art will recognize that the design of peptides conjugated to any of the above elements may include wholly or partially flexible linkers, such that the linker may include a flexible linker and one or more portions imparting a less flexible structure.

[0555] In some embodiments, the anti-C1s antibody of this disclosure comprises scFv multimers. For example, in some embodiments, the subject antibody is an scFv dimer (e.g., comprising two tandem scFvs (scFv2)), an scFv trimer (e.g., comprising three tandem scFvs (scFv3)), an scFv tetramer (e.g., comprising four tandem scFvs (scFv4)), or a multimer having more than four scFvs (e.g., tandemly). The scFv monomers may be tandemly linked via linkers of about 2 amino acids to about 10 amino acids (aa), for example, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkers include, for example, (Gly). x , where x is an integer from 2 to 10. Other suitable linkers are those discussed above. In some implementations, each scFv monomer in the topic scFv multimer is humanized as described above.

[0556] In some cases, the subject antibody contains a constant region (e.g., an Fc region) of an immunoglobulin. The Fc region, if present, can be a human Fc region or an Fc region from any animal with a complement system. In some embodiments, the Fc region, if present, is a human Fc region. If a constant region is present, the antibody may contain both light and heavy chain constant regions. Suitable heavy chain constant regions include CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and any isotype (including IgG1, IgG2, IgG3, and IgG4). An example of a suitable heavy chain Fc region is human isotype IgG1 Fc. Another example of a suitable heavy chain Fc region is human isotype IgG2 Fc. Another example of a suitable heavy chain Fc region is human isotype IgG3 Fc. The light chain constant region may be λ or κ. The subject antibody (e.g., a subject humanized antibody) may contain sequences from more than one species or isotype. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as well as as single heavy chains, light chains, Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which variable domains of the heavy and light chains are linked via spacers.

[0557] In some cases, the heavy chain region belongs to isotype IgG4. In some such embodiments, the hinge region contains an S241P substitution. See, for example, Angal et al., (1993). Mol. Immunol. 30:105. In some such implementations, the hinge area includes L236E (or L235E, using EU designation; Kabat et al., (1991)). Sequences of Proteins of Immunological Interest (5th edition, US Dept. Health and Human Services, Bethesda, MD, NIH Publication No. 91-3242) Replacement. See, for example, Reddy et al., (2000) J. Immunol. 164:1925; and Klechevsky et al., (2010) Blood 116:1685. In some such implementations, the hinge region includes S241P and L236E substitutions.

[0558] The subject antibody may contain a free thiol (-SH) group at the carboxyl terminus, wherein the free thiol group can be used to link the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a backbone, a carrier, etc.

[0559] In some embodiments, the subject antibody comprises one or more non-naturally occurring amino acids. In some embodiments, the non-naturally encoded amino acids comprise carbonyl, acetyl, aminooxy, hydrazine, acylhydrazine, aminourea, azide, or alkynyl groups. For example, see U.S. Patent No. 7,632,924 for suitable non-naturally occurring amino acids. Including non-naturally occurring amino acids can provide a link to polymers, second peptides, backbones, etc. For example, a subject antibody linked to a water-soluble polymer can be prepared by reacting an antibody containing a carbonyl group (e.g., PEG) with an antibody, wherein the antibody comprises a non-naturally encoded amino acid containing an aminooxy, hydrazine, acylhydrazine, or aminourea group. As another example, a subject antibody linked to a water-soluble polymer can be prepared by reacting a subject antibody containing an alkynyl-containing amino acid with a water-soluble polymer containing an azide moiety (e.g., PEG). In some embodiments, the azide or alkynyl group is linked to the PEG molecule via an amide bond. "Non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolidone or selenocysteine. Other terms that can be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid," "non-naturally occurring amino acid," "non-naturally occurring amino acid," and their various hyphenated and unhyphenated forms. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that appear through modifications (e.g., post-translational modifications) of naturally encoded amino acids (including, but not limited to, 20 common amino acids or pyrrolidone and selenocysteine), but are not naturally incorporated into the grown polypeptide chain by the translation complex itself. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine.

[0560] In some embodiments, the subject antibody is linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a peptide). Suitable polymers include, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally occurring polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene, polyolefin, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolymers or heteropolymers. Suitable polymers include, for example, ethylene-vinyl alcohol copolymers (usually known by the common name EVOH or the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolic acid); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphate; polyurethane polyphosphate; poly(amino acid); cyanoacrylate; poly(trimethylene carbonate); poly(imino carbonate); copoly(ether-ester) (e.g., poly(ethylene oxide)-poly(lactic acid)). (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; polysiloxanes; polyesters; polyolefins; polyisobutylene and ethylene-α-olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers such as polyvinyl chloride; polyethylene ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyvinyl aromatic compounds such as polystyrene; polyethylene esters such as polyvinyl acetate; ethylene Copolymers of alkenyl monomers with each other and with olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides, such as nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylene; polyimide; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; poly(ethylene glycol); and carboxymethyl cellulose.

[0561] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, such as substituted poly(ethylene glycol), such as methoxylated poly(ethylene glycol), and their derivatives. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen, and their derivatives.

[0562] Suitable polymers may have an average molecular weight in the range of 500 Da to 50,000 Da, for example, 5,000 Da to 40,000 Da, or 25,000 Da to 40,000 Da. For example, in some embodiments in which the subject antibody comprises a poly(ethylene glycol) (PEG) or methoxy poly(ethylene glycol) polymer, said PEG or methoxy poly(ethylene glycol) polymer may have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0563] As described above, in some embodiments, the subject antibody is covalently linked to a non-peptide synthetic polymer. In some embodiments, the subject antibody is covalently linked to a PEG polymer. In some embodiments, the subject scFv polymer is covalently linked to a PEG polymer. See, for example, Albrecht et al., (2006). J. Immunol. Methods 310:100. Methods and reagents suitable for the polyethylene glycolation of proteins are well known in the art and can be found, for example, in U.S. Patent No. 5,849,860. PEG suitable for conjugation to proteins is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2). n OR, where R is hydrogen or a protecting group such as alkyl or alkanoyl, and where n is an integer from 1 to 1,000. When R is a protecting group, it typically has 1 to 8 carbons.

[0564] In some embodiments, the PEG conjugated to the subject antibody is linear. In some embodiments, the PEG conjugated to the subject antibody is branched. Branched PEG derivatives include those described in U.S. Patent No. 5,643,575, "star-shaped PEGs," and multi-arm PEGs include those described in Shearwater Polymers, Inc.'s catalogue "Polyethylene Glycol Derivatives 1997-1998." Star-shaped PEGs are described in the art, including, for example, in U.S. Patent No. 6,046,305.

[0565] The subject antibody can be glycosylated; for example, the subject antibody may contain a covalently linked carbohydrate or polysaccharide moiety. Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the linkage of the carbohydrate moiety to a side chain of asparagine residues. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences used for the enzymatic linkage of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0566] Glycosylation sites can be conveniently added to antibodies by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding one or more serine or threonine residues to the original antibody sequence or by substituting one or more serine or threonine residues (for O-linked glycosylation sites). Similarly, glycosylation sites can be removed by altering the amino acid composition within the antibody's native glycosylation site.

[0567] In some embodiments, the subject antibody will contain a "transparent" label, such as a label that can be readily visualized using, for example, X-rays. Transparent materials are well known to those skilled in the art. The most common transparent materials include iodides, bromides, or barium salts. Other transparent materials are also known and include, but are not limited to, organobismuth derivatives (see, for example, U.S. Patent No. 5,939,045), transparent polyurethane (see, for example, U.S. Patent No. 5,346,981), organobismuth complexes (see, for example, U.S. Patent No. 5,256,334), transparent barium polymer complexes (see, for example, U.S. Patent No. 4,866,132), etc.

[0568] A subject antibody can be covalently linked to a second moiety (e.g., lipids, peptides other than the subject antibody, synthetic polymers, carbohydrates, etc.) using, for example, glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks the peptide via its amino moiety. Homobifunctional crosslinkers (e.g., homobifunctional imine esters, homobifunctional N-hydroxysuccinimide (NHS) esters, or homobifunctional thiol reactive crosslinkers) contain two or more identical reactive moieties and can be used in a single-step reaction procedure in which the crosslinker is added to a solution containing a mixture of peptides to be linked. Homobifunctional NHS esters and imine esters crosslink the amine containing the peptide. In a mildly alkaline pH, the imine ester reacts only with the primary amine to form an iminoamide, and the total charge of the crosslinked peptide is unaffected. Homogeneous bifunctional thiol reactive crosslinking agents include bismaleimide hexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-bis-(3',2'-pyridyldithio)propionylaminobutane (DPDPB).

[0569] Heterobifunctional crosslinking agents have two or more distinct reactive moieties (e.g., an amine-reactive moiety and a thiol-reactive moiety) and crosslink with one polypeptide via the amine or thiol-reactive moiety, then react with another polypeptide via the non-reactive moiety. A variety of heterobifunctional haloacetyl crosslinking agents are available, as are pyridyl disulfide crosslinking agents. Carbodiimide is a typical example of a heterobifunctional crosslinking agent used for coupling a carboxyl group to an amine (which generates an amide bond).

[0570] The subject antibody can be immobilized on a solid support. Suitable supports are well known in the art and include, in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, durates, pores of reaction discs (e.g., multi-well plates), plastic tubing, etc. The solid support can comprise any of a variety of materials, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. Suitable methods for immobilizing the subject antibody onto the solid support are well known and include, but are not limited to, ionicity, hydrophobicity, covalent interactions, etc. The solid support can be soluble or insoluble in aqueous solutions, for example. In some embodiments, suitable solid supports are typically insoluble in aqueous solutions.

[0571] In some implementations, the subject antibody will contain a detectable marker. Suitable detectable markers include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable markers include, but are not limited to, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), and radioactive markers (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and other enzymes commonly used in enzyme-linked immunosorbent assays (ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0572] In some embodiments, the subject antibody comprises a contrast agent or a radioactive isotope, wherein the contrast agent or radioactive isotope is suitable for imaging, such as imaging procedures performed on a human. Non-limiting examples of the label include radioactive isotopes such as… 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium) and 67 Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes ( 153Gd is also available and suitable for imaging procedures in non-human mammals. Subject antibodies can be labeled using standard techniques. For example, the subject antibody can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycourea. For fluorination, fluorine is added to the subject antibody during synthesis via a fluoride ion exchange reaction. For a review of the synthesis of proteins having the aforementioned radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). Subject antibodies can also be labeled with contrast agents using standard techniques. For example, the subject antibody can be labeled with Gd by conjugating a low-molecular-weight Gd chelate such as Gd diethylenetriaminepentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic acid (GdDOTA) to the antibody. See Caravan et al., Chem. Rev. 99:2293-2352 (1999); and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). The subject antibody can be Gd-labeled, for example, by conjugating a polylysine-Gd chelate to the antibody. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, the subject antibody can be Gd-labeled by incubating a paramagnetic polymeric liposome comprising a Gd chelating lipid with an anti-biotin protein and a biotin-labeled antibody. See, for example, Sipkins et al., Nature Med., 4:623-626 (1998).

[0573] Suitable fluorescent proteins that can be linked to the subject antibody include, but are not limited to, those from the Victoria multituberculate jellyfish ( Aequoria victoria Green fluorescent protein (GFP) or its mutants or derivatives thereof, such as those described in U.S. Patent Nos. 6,066,476, 6,020,192, 5,985,577, 5,976,796, 5,968,750, 5,968,738, 5,958,713, 5,919,445, and 5,874,304; for example, enhanced GFP, many of which are commercially available from, for example, Clontech, Inc.; red fluorescent protein; yellow fluorescent protein; any of a variety of fluorescent and colored proteins from anthozoan species, such as, for example, Matz et al., (1999). Nature Biotechnol. As described in 17:969-973; etc.

[0574] In some embodiments, the subject antibody is conjugated to a therapeutic agent. Any subject antibody disclosed herein can be used to form an antibody-drug conjugate. The drug may be linked to the N-terminus of a light chain, the C-terminus of a light chain, the N-terminus of a heavy chain, or the C-terminus of a heavy chain. In some embodiments, the drug is linked to a hinge of the antibody or one or more other sites on the antibody. For single-chain antibodies, the drug may be linked to the N- or C-terminus of the single-chain antibody. The drug may be conjugated to the antibody directly or via a linker using techniques known to those skilled in the art. The linker may be cleavable or non-cleavable. Examples of such therapeutic agents (e.g., for use in therapy) are known to those skilled in the art.

[0575] In some implementations, the subject antibody is linked (e.g., covalently or non-covalently) to a fusion partner, such as a ligand; an epitope tag; a peptide; a protein other than the antibody; and so on. Suitable fusion partners include peptides and polypeptides that possess the following properties: confer enhanced in vivo stability (e.g., enhanced serum half-life); provide ease of purification, e.g., (His) n Examples of suitable proteins include 6His, etc.; providing fusion protein secretion from cells; providing epitope tags, such as GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:18), FLAG (e.g., DYKDDDDK; SEQ ID NO:19), c-myc (e.g., EQKLISEEDL; SEQ ID NO:20), etc.; providing detectable signals, such as enzymes that produce detectable products (e.g., β-galactosidase, luciferase) or self-detectable proteins, such as green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.; providing polymerization, such as polymeric domains such as the Fc portion of immunoglobulins; etc.

[0576] The fusion compound may also include an affinity domain comprising a peptide sequence that can interact with a binding chaperone, such as a peptide sequence immobilized on a solid support that can be used for identification or purification. Sequential single amino acids, such as histidines, when fused to a protein, can be used for one-step purification of the fusion protein by binding with a resin column such as nigarose via high affinity. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO:21); HisX6 (HHHHHH) (SEQ ID NO:22); c-myc (EQKLISEEDL) (SEQ ID NO:20); Flag (DYKDDDDK) (SEQ ID NO:19); StrepTag (WSHPQFEK) (SEQ ID NO:23); hemagglutinin, such as the HA tag (YPYDVPDYA; SEQ ID NO:18); glutathione S-transferase (GST); thioredoxin; cellulose-binding domain; RYIRS (SEQ ID NO:24); Phe-His-His-Thr (SEQ ID NO:25); chitin-binding domain; S-peptide; T7 peptide; SH2 domain; C-terminal RNA tag; WEAAAREACCRECCARA (SEQ ID NO:21). NO:26); Metal-binding domains, such as zinc-binding or calcium-binding domains, such as those from calcium-binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recovery protein, S-regulatory protein, cone protein, VILIP, neurotrophin, hippocampal calcineurin, frequenin, calcium tether, calpain large subunit, S100 protein, parvalbumin, calcium-binding protein D9K, calcium-binding protein D28K, and caloretinin, containing peptides, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose-binding proteins.

[0577] In some embodiments, the anti-C1s antibody of this disclosure is formulated together with an agent that promotes crossing the blood-brain barrier (BBB). In some embodiments, the antibody is fused directly or via a linker to a compound that promotes BBB crossing. Examples of such compounds include, but are not limited to, carrier molecules, peptides, or proteins. In some embodiments, the subject antibody is fused to a polypeptide that binds to an endogenous BBB receptor. The linking of the subject antibody to the polypeptide that binds to the endogenous BBB receptor promotes BBB crossing, for example, in subject therapy methods that include administering the subject antibody to an individual in need (see below). Suitable polypeptides that bind to endogenous BBB receptors include antibodies that specifically bind to endogenous BBB receptors, such as monoclonal antibodies or antigen-binding fragments thereof. Suitable endogenous BBB receptors include, but are not limited to, insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and insulin-like growth factor receptors. See, for example, U.S. Patent Publication No. 2009 / 0156498.

[0578] As an example, a subject anti-C1s antibody can be a bispecific antibody comprising a first antigen-binding moiety that specifically binds to an epitope in the complement C1s protein, and a second antigen-binding moiety that binds to an endogenous BBB receptor. For instance, in some cases, a subject anti-C1s antibody is a bispecific antibody comprising a first antigen-binding moiety that specifically binds to an epitope in the C1s protein, and a second antigen-binding moiety that binds to a transferrin receptor.

[0579] For example, the anti-C1s antibody of this disclosure can be fused to a peptide that promotes crossing the BBB, said peptide having a length of about 15 to about 25 amino acids and comprising an amino acid sequence having at least about 85% amino acid sequence identity with one of the following peptides: Angiopep-1 (TFFYGGCRGKRNNFKTEEY) (SEQ ID NO:27); Angiopep-2 (TFFYGGSRGKRNNFKTEEY) (SEQ ID NO:28); cys-Angiopep-2 (CTFFYGGSRGKRNNFKTEEY) (SEQ ID NO:29); Angiopep-2-cys (TFFYGGSRGKRNNFKTEEYC) (SEQ ID NO:30); and an aprotinin fragment (TFVYGGCRAKRNNFKS) (SEQ ID NO:31). See, for example, U.S. Patent Publications No. 2011 / 0288011; and 2009 / 0016959. Peptides that promote crossing the BBB can be fused to the N-terminus of the anti-C1s light chain region, the C-terminus of the anti-C1s light chain region, the N-terminus of the anti-C1s heavy chain region, the C-terminus of the anti-C1s heavy chain region, the N-terminus of the subject anti-C1s single-chain antibody, the C-terminus of the subject anti-C1s single-chain antibody, etc.

[0580] In some embodiments, the subject antibody comprises a polyamine modification. Polyamine modification of the subject antibody enhances the permeability of the modified antibody at the BBB. The subject antibody can be modified with naturally occurring or synthetic polyamines. See, for example, U.S. Patent No. 5,670,477. Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1,3-diaminopropane, nosemidine, homosemidine, thermomine, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines are derived from empirical formula C... X H Y N Z The composition can be a cyclic or acyclic, branched or unbranched hydrocarbon chain having 3-12 carbon atoms, further comprising 1-6 NR or N(R)2 moieties, wherein R is H, (C1-C4) alkyl, phenyl, or benzyl. Polyamines can be linked to antibodies using any standard crosslinking method.

[0581] In some embodiments, the subject antibody is modified to include a carbohydrate moiety, wherein the carbohydrate moiety is covalently linked to the antibody. In some embodiments, the subject antibody is modified to include a lipid moiety, wherein the lipid moiety is covalently linked to the antibody. Suitable lipid moieties include, for example, N-aliphatic acyl groups, such as N-lauroyl, N-oleoyl, etc.; aliphatic amines, such as dodecylamine, oleamide, etc.; C3-C16 long-chain aliphatic lipids; and so on. See, for example, U.S. Patent No. 6,638,513. In some embodiments, the subject antibody is incorporated (e.g., encapsulated) into liposomes.

[0582] Methods for generating subject antibodies

[0583] The subject antibody can be produced by any known method, such as conventional synthetic methods used for protein synthesis; recombinant DNA methods; and so on. In some embodiments, the subject antibody is produced by a method selected from the group consisting of recombinant generation and chemical synthesis.

[0584] When the subject antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, the synthesis can be carried out via liquid or solid phase. Solid-phase peptide synthesis (SPPS) is an example of a method suitable for the chemical synthesis of subject antibodies, in which the C-terminal amino acid of the sequence is linked to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize subject antibodies. Techniques for solid-phase synthesis are described in the following references: Barany and Merrifield, Solid-Phase Peptide Synthesis; The Peptides: Analysis, Synthesis, Biology, pp. 3-284, Volume 2: Special Methods in Peptide Synthesis, Part A, Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006. Mini Rev. Med Chem. 6:3-10; and Camarero JA et al., 2005 Protein Pept Lett. 12:723-8. In simple terms, small, insoluble porous beads are treated with the functional units constructed above with peptide chains. After repeated cycles of coupling / deprotection, the free N-terminal amine of the attached solid phase is coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing a new N-terminal amine that can be linked to another amino acid. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0585] Standard recombinant methods can be used to generate the subject antibody. For example, nucleic acids encoding variable regions of a light chain and a heavy chain, optionally linked to a constant region, are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. A DNA segment encoding an immunoglobulin chain is operatively linked to a control sequence in an expression vector that ensures the expression of the immunoglobulin polypeptide. The expression control sequence includes, but is not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, repressor elements, and transcription termination sequences. The expression control sequence may be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of the antibody.

[0586] Due to the degeneracy of the code, multiple nucleic acid sequences can encode the amino acid sequence of each immunoglobulin. The desired nucleic acid sequence can be generated by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of a previously prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion, and insertion variants of the target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). In simple terms, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation with a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize the entire second complementary strand of the template, which incorporates the oligonucleotide primer and encodes the selected alteration in the target polypeptide DNA.

[0587] Suitable expression vectors can typically replicate in a host organism either as a free gene or as part of the host's chromosomal DNA as a whole. Typically, expression vectors contain selection markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence.

[0588] Escherichia coli ( Escherichia coli ) is an example of a prokaryotic host cell that can be used to clone polynucleotides encoding the subject antibody. Other suitable microbial hosts include Bacilli, such as Bacillus subtilis ( Bacillus subtilis ); and other enterobacteria, such as Salmonella ( Salmonella ), Serratia ( Serratia ), and various Pseudomonas species ( Pseudomonas ) species. Expression vectors can also be prepared in these prokaryotic hosts, which will typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. Additionally, any number of well-known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or promoter systems derived from λ phage. These promoters will typically be optionally controlled along with an operon sequence and will have ribosome binding site sequences for initiating and completing transcription and translation, etc.

[0589] Other microorganisms, such as yeast, can also be used for expression. Yeast (genus *Yeast*) Saccharomyces (e.g., brewer's yeast) S. cerevisiae )) and Pichia pastoris ( Pichia A vector is an example of a suitable yeast host cell, in which a suitable vector has, where necessary, expression control sequences (e.g., promoters), origins of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters particularly include promoters from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

[0590] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell cultures) can also be used to express and produce the anti-C1s antibodies of this disclosure (e.g., polynucleotides encoding the subject anti-C1s antibodies). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors used for these cells may include expression control sequences, such as origin of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomaviruses, cytomegaloviruses, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0591] Once synthesized (chemically or recombinantly), the complete antibody, its dimer, individual light and heavy chains, or other forms of the subject antibody (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The subject antibody can be substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or higher purity, for example, free from contaminants such as cell debris, macromolecules other than the subject antibody, etc.

[0592] Composition

[0593] This disclosure provides compositions comprising the subject antibody. In addition to the subject antibody, the subject antibody composition may comprise one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizers; detergents, such as nonionic detergents like Tween-20, etc.; protease inhibitors; glycerol; etc.

[0594] Nucleic acid molecules, expression vectors, and host cells

[0595] This disclosure provides nucleic acid molecules containing nucleotide sequences encoding subject anti-C1s antibodies.

[0596] In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a light chain variable region containing the amino acid sequence shown in SEQ ID NO:7.

[0597] In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:8.

[0598] In some embodiments, the nucleic acid molecules of this disclosure encode a subject anti-C1s antibody, said antibody comprising light chain variable regions containing CDR-L1, CDR-L2, and CDR-L3, respectively, SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0599] In some embodiments, the nucleic acid molecules of this disclosure encode a subject anti-C1s antibody, said antibody comprising heavy chain variable regions containing CDR-H1, CDR-H2, and CDR-H3, respectively, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0600] In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:37. In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a light chain variable region containing the amino acid sequence shown in SEQ ID NO:37.

[0601] In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:38. In some embodiments, the nucleic acid molecule of this disclosure encodes a subject-specific anti-C1s antibody, said antibody comprising a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:38.

[0602] In some embodiments, the nucleic acid molecules of this disclosure encode a subject anti-C1s antibody, said antibody comprising light chain variable regions containing CDR-L1, CDR-L2, and CDR-L3, respectively, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:33.

[0603] In some embodiments, the nucleic acid molecules of this disclosure encode a subject anti-C1s antibody, said antibody comprising heavy chain variable regions containing CDR-H1, CDR-H2, and CDR-H3, respectively, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0604] In some embodiments, the nucleic acid molecules of this disclosure encode a subject anti-C1s antibody, said antibody comprising a light chain variable region and a heavy chain variable region.

[0605] Nucleic acid molecules encoding the subject antibody can be operatively linked to one or more regulatory elements, such as promoters and enhancers, which allow the nucleotide sequence to be expressed in the intended target cells (e.g., cells that have been genetically modified to synthesize the encoded antibody).

[0606] Suitable promoters and enhancer elements are known in the art. Promoters suitable for prokaryotic host cells include, but are not limited to, the phage T7 RNA polymerase promoter; T3 promoter; T5 promoter; λP promoter; trp promoter; lactose operon promoter; heterozygous promoters, such as lac / tac heterozygous promoters, tac / trc heterozygous promoters, trp / lac promoters, T7 / lac promoters; trc promoters; tac promoters, etc.; gpt promoters; araBAD promoters; and in vivo regulated promoters, such as... ssaG Promoters or related promoters (see, for example, U.S. Patent Publication No. 20040131637), pagC Promoters (Pulkkinen and Miller, J. Bacteriol ., 1991: 173(1): 86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), nirB Promoters (Harborne et al., (1992)) Mol. Micro 6:2805-2813), etc. (see, for example, Dunstan et al., (1999) Infect. Immun. 67:5133-5141; McKelvie et al., (2004) Vaccine 22:3243-3255; and Chatfield et al., (1992) Biotechnol. 10:888-892); Sigma 70 starters, such as common Sigma 70 starters (see, for example, GenBank login numbers AX798980, AX798961 and AX798183); fixed-phase starters, such as... dps promoter, spv Promoters, etc.; promoters derived from the pathogenic island SPI-2 (see, for example, WO96 / 17951); actA promoter (see, for example, Shetron-Rama et al., (2002)). Infect. Immun. 70:1087-1096); rpsM promoter (see, for example, Valdivia and Falkow (1996). Mol. Microbiol. 22:367); tet promoter (see, for example, Hillen, W. and Wissmann, A. (1989) in Saenger, W. and Heinemann, U. (edited)). Topics in Molecular and Structural Biology , Protein–Nucleic Acid Interaction middle. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, for example, Melton et al., (1984) Nucl. Acids Res. 12:7035); etc. Strong promoters applicable to prokaryotes such as *Escherichia coli* include, but are not limited to, Trc, Tac, T5, T7, and P. λ Non-limiting examples of operons for use in bacterial host cells include the lactose promoter operon (the LacI repressor changes conformation upon contact with lactose, thereby preventing the LacI repressor from binding to the operon), the tryptophan promoter operon (the TrpR repressor has a conformation that binds to the operon when complexed with tryptophan; in the absence of tryptophan, the TrpR repressor has a conformation that does not bind to the operon), and the tac promoter operon (see, for example, deBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0607] In some implementations, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulated promoters, such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for Pichia pastoris).

[0608] For expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoters; herpes simplex virus thymidine kinase promoters; early and late SV40 promoters; promoters present in long terminal repeat sequences from retroviruses; mouse metallothionein-I promoters; and various tissue-specific promoters known in the art.

[0609] The selection of appropriate carriers and promoters is well within the technical skill level of those skilled in the art.

[0610] Nucleic acid molecules encoding a subject antibody may be present in expression vectors and / or cloning vectors. This disclosure provides a recombinant vector containing a nucleic acid molecule encoding a subject antibody in a cloning vector. This disclosure also provides a recombinant molecule containing a nucleic acid molecule encoding a subject antibody operably linked to an expression vector to ensure expression of the encoded antibody. When the subject antibody comprises two separate polypeptides, nucleic acid molecules encoding said two polypeptides may be cloned in the same or separate vectors to form one or more recombinant molecules. Recombinant molecules may include selectable markers, origins of replication, and other features that provide for the replication and / or maintenance of the recombinant molecule.

[0611] A wide range of suitable vectors and promoters are known to those skilled in the art; many are commercially available to generate the subject recombinant molecules. For example, the following vectors are provided: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0612] Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. Selectable markers that are effective in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors. Examples of viral vectors include, but are not limited to, those based on: vaccinia virus; poliovirus; adenovirus (see, for example, Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, for example, Ali et al., HumGene Ther 9:81 86, 1998; Flannery et al., PNAS 9:81 86, 1998). 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997; Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava WO 93 / 09239; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma virus, Harvey Sarcoma virus, avian leukemia virus, human immunodeficiency virus (see, for example, Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999), myeloproliferative sarcoma virus, and mammary tumor virus); etc.

[0613] As described above, the subject nucleic acid molecule comprises a nucleotide sequence encoding the disclosed anti-C1s antibody. In some embodiments, the subject nucleic acid molecule comprises nucleotide sequences encoding the heavy and light chain CDRs of the subject IPN003 antibody. In some embodiments, the subject nucleic acid molecule comprises nucleotide sequences encoding the heavy and light chain CDRs of the subject antibody, wherein the CDR encoding sequences are interspersed with FR-encoded nucleotide sequences. In some embodiments, the FR-encoded nucleotide sequence is a human FR-encoded nucleotide sequence.

[0614] host cells

[0615] This disclosure provides isolated, genetically modified host cells (e.g., in vitro cells) that are genetically modified with a subject nucleic acid molecule. In some embodiments, the isolated, genetically modified subject host cells can produce a subject antibody. Such cells are referred to as recombinant cells. Recombinant cells contain a recombinant molecule encoding the subject antibody.

[0616] Suitable host cells include eukaryotic host cells, such as mammalian host cells, insect host cells, and yeast cells; and prokaryotic cells, such as bacterial cells. The introduction of the subject nucleic acid into the host cell can be achieved, for example, by calcium phosphate precipitation, DEAE-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0617] Suitable mammalian cell lines include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc. In some cases, the cells are HEK cells. In some embodiments, the cells are CHO cells, such as CHO-K1 cells (ATCC No. CCL-61), CHO-M cells, CHO-DG44 cells (ATCC No. PTA-3356), etc. In some embodiments, the host cells are COS cells. In some embodiments, the host cells are 293 cells. In some embodiments, the host cells are CHO cells.

[0618] Suitable yeast cells include, but are not limited to, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia oysteratiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, a specific species of *Pichia* sp., *Saccharomyces cerevisiae*, a specific species of *Saccharomyces cerevisiae*, *Hansenula polymorpha*, a specific species of *Kluyveromyces* sp., and *Kluyveromyces lactis*. The host cells include *Lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium gramineum*, *Fusarium venenatum*, *Neurospora crassa*, and *Chlamydomonas reinhardtii*. In some embodiments, the host cell is a yeast. In some embodiments, the host cell is *Pichia pastoris*.

[0619] Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of *Escherichia coli*, *Bacillus* (e.g., *Bacillus subtilis*), *Lactobacillus*, etc. See, for example, Carrier et al., (1992). J. Immunol .148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al., (1995) Science270:299-302. Typically, the laboratory strain is a non-pathogenic strain. In some embodiments, the host cell is *Escherichia coli*. In some embodiments, the host cell is *Bacillus subtilis*.

[0620] Pharmaceutical Composition

[0621] This disclosure provides compositions, including pharmaceutical compositions comprising a subject antibody. Generally, a pharmaceutical composition (also referred to herein as a formulation) comprises an effective amount of the subject antibody. "Effective amount" means a dose sufficient to produce a desired result, such as a reduction in adverse symptoms associated with complement-mediated disease or condition, improvement in symptoms of complement-mediated disease or condition, slowing the progression of complement-mediated disease or condition, etc. The desired result is typically at least a reduction in symptoms of complement-mediated disease or condition compared to a control. In some embodiments, the subject antibody is formulated and / or modified to enable the antibody to cross the blood-brain barrier. In some embodiments, the subject antibody is delivered in a manner that avoids the blood-brain barrier. In some embodiments, the anti-C1s antibody of this disclosure is formulated together with an agent that promotes blood-brain barrier crossing. In some embodiments, the subject antibody is fused directly or via a linker to a compound that promotes blood-brain barrier crossing.

[0622] preparation

[0623] In the subject approach, the subject antibody can be administered to the host using any convenient method capable of producing the desired therapeutic or diagnostic effect. Therefore, the agent can be incorporated into a variety of formulations for therapeutic administration. More specifically, the subject antibody can be formulated into a pharmaceutical composition by combining it with a suitable pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, or other pharmaceutically acceptable excipient, and can be formulated into a formulation in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, and aerosols. In some embodiments, the pharmaceutical composition comprises the subject antibody and a pharmaceutically acceptable excipient.

[0624] In pharmaceutical dosage forms, the subject antibody may be administered in its pharmaceutically acceptable salt form, or it may be used alone or in appropriate associations, as well as in combination with other pharmaceutically active compounds. The methods and excipients described below are merely exemplary and in no way limiting.

[0625] For oral formulations, the subject antibody can be used alone or in combination with appropriate additives to prepare tablets, powders, granules, or capsules, for example, in combination with: conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethyl cellulose; lubricants such as talc or magnesium stearate; and, if necessary, diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0626] The subject antibody can be formulated into an injectable preparation by dissolving, suspending, or emulsifying the antibody in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, propylene glycol, synthetic aliphatic acid glycerides, injectable organic esters (e.g., ethyl oleate), higher aliphatic esters, or propylene glycol esters; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Parenteral mediators include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous mediators include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Furthermore, the pharmaceutical compositions of this disclosure may additionally contain other agents, such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

[0627] Pharmaceutical compositions containing a subject antibody are prepared by mixing a subject antibody of desired purity with an optional physiologically acceptable carrier, other excipients, stabilizers, surfactants, buffers, and / or tonicants. The acceptable carrier, other excipients, and / or stabilizers are non-toxic to the recipient at the doses and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citrate; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); and amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, and isoleucine. Leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine and neuraminic acid; and / or nonionic surfactants, such as Tween, Brij Pluronics, Triton-X or polyethylene glycol (PEG).

[0628] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, wherein the lyophilized formulation is reconstituted with a sterile solution prior to administration. The standard procedure for reconstituted lyophilized compositions is to add back a certain volume of pure water (usually equivalent to the volume removed during lyophilization); however, solutions containing antibacterial agents may be used to prepare pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0629] Exemplary antibody concentrations in the subject pharmaceutical composition may range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0630] Aqueous formulations of antibodies can be prepared in pH buffer solutions, for example, in the pH range of about 4.0 to about 7.0 or about 5.0 to about 6.0, or optionally about 5.5. Examples of buffers suitable for this pH range include phosphate, histidine, citrate, succinate, acetate buffers, and other organic acid buffers. Buffer concentrations can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on, for example, the desired torsional strength of the buffer and the formulation.

[0631] Tonic agents can be included in antibody formulations to adjust the tonicity of the formulation. Exemplary tonic agents include sodium chloride, potassium chloride, glycerol, and any components from the group consisting of amino acids, sugars, and combinations thereof. In some embodiments, aqueous formulations are isotonic, but hypertonic or hypotonic solutions may be suitable. The term "isotonic" means a solution that has the same tonicity as some other solution compared to it, such as physiological saline or serum. Tonic agents can be used in amounts from about 5 mM to about 350 mM, for example, from 100 mM to 350 nM.

[0632] Surfactants can also be added to antibody formulations to reduce the aggregation of the formulated antibodies and / or minimize particle formation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Pluronicer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0633] Lyophilization protectants can also be added to protect unstable active ingredients (such as proteins) from instability conditions during the lyophilization process. Known lyophilization protectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The amount of lyophilization protectant can range from about 10 mM to 500 nM.

[0634] In some embodiments, the subject formulation includes a subject antibody and one or more of the reagents identified above (e.g., surfactants, buffers, stabilizers, tension agents) and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlorocresol, methylparaben or propylparaben, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation, for example, at a concentration in the range of about 0.001 to about 2% (w / v).

[0635] For example, the subject formulation may be a liquid or lyophilized formulation suitable for parenteral administration and may contain: a subject antibody of about 1 mg / mL to about 200 mg / mL; at least one surfactant of about 0.001% to about 1%; a buffer of about 1 mM to about 100 mM; optionally a stabilizer of about 10 mM to about 500 mM; and a tonic agent of about 5 mM to about 305 mM; and have a pH of about 4.0 to about 7.0.

[0636] As another example, the subject parenteral formulation is a liquid or lyophilized formulation containing: about 1 mg / mL to about 200 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5.

[0637] As another example, the parenteral formulation comprises a lyophilized preparation containing: 1) 15 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and having a pH of 5.5; or 2) 75 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and having a pH of 5.5; or 3) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and having a pH of 5.5; or 4) 75 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and having a pH of 5.5; or 5) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and having a pH of 5.5; Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

[0638] As another example, the parenteral formulation of the subject is a liquid formulation comprising: 1) 7.5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 120 mM L-histidine; and 250 mM sucrose; and having a pH of 5.5; or 2) 37.5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; and having a pH of 5.5; or 3) 37.5 mg / mL of the subject antibody; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; and having a pH of 5.5; or 4) 37.5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; 125 mM trehalose; and having a pH of 5.5; or 5) 37.5 5 mg / mL of the main antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM trehalose; with a pH of 5.5; or 7) 75 mg / mL of the main antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; with a pH of 5.5; or 8) 75 mg / mL of the main antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; with a pH of 5.5; or 9) 150 mg / mL of the main antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 125 mM trehalose; with a pH of 5.5. L-histidine; and 250 mM trehalose; and a pH of 5.5; or 10) 150 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol; and a pH of 5.5; or 11) 150 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; and a pH of 5.5; or 12) 10 mg / mL of the subject antibody; 0.01% Tween 20 w / v; 20 mM L-histidine; and 40 mM sodium chloride; and a pH of 5.5.

[0639] The subject antibody can be utilized in aerosol formulations for inhalation. The subject antibody can be formulated in pressurized, acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc. Aerosol formulations, such as nasal sprays, comprise purified aqueous or other solutions of active agents, preservatives, and isotonic agents. These formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0640] Furthermore, the subject antibody can be formulated into suppositories by mixing with various matrices such as emulsion matrices or water-soluble matrices. The subject antibody can be administered rectally via suppositories. Suppositories may include media such as cocoa butter, carbon wax, and polyethylene glycol, which melt at body temperature but remain solidified at room temperature.

[0641] Unit dosage forms, such as syrups, elixirs, and suspensions, for oral or rectal administration are available, wherein each dose unit, such as a teaspoon, tablespoon, tablet, or suppository, contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the subject antibody in a solution of the composition in sterile water, saline, or another pharmaceutically acceptable carrier.

[0642] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for use in human and animal subjects, calculated in an amount sufficient to combine with a pharmaceutically acceptable diluent, carrier, or mediator to produce the desired effect, each unit containing a predetermined amount of the anti-C1s antibody of this disclosure. The specifications of the subject antibody may depend on the specific antibody used and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

[0643] Other modes of administration will also be used in the methods of this disclosure. For example, the subject antibody can be formulated as a suppository, and in some cases as an aerosol and intranasal composition. For suppositories, the carrier composition will include conventional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories can be formed from a mixture containing about 0.5% to about 10% (w / w), for example about 1% to about 2% of the active ingredient.

[0644] Intranasal preparations typically include a medium that neither irritates the nasal mucosa nor significantly interferes with ciliary function. Diluents such as water, saline solution, or other known substances may be used. Nasal preparations may also contain preservatives, such as, but not limited to, chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance the absorption of the subject antibody by the nasal mucosa.

[0645] The subject antibody can be administered in injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions; they can also be prepared in a solid form suitable for dissolution or suspension in a liquid medium prior to injection. Formulations can also be emulsified or the antibody encapsulated in a liposome medium.

[0646] Suitable excipient mediators include, for example, water, saline solution, dextrose, glycerol, ethanol, and combinations thereof. Additionally, if desired, the mediator may contain small amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffers. Practical methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will in any event contain an amount of the subject antibody sufficient to achieve the desired state in the treated subject.

[0647] Pharmaceutically acceptable excipients, such as mediators, adjuvants, carriers, or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tension modifiers, stabilizers, and wetting agents, are readily available to the public.

[0648] In some embodiments, the subject antibody is formulated in a controlled-release formulation. Sustained-release formulations can be prepared using methods well known in the art. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody, wherein the matrix is ​​in the form of a shaped article such as a film or microcapsule. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, hydrogels, polylactide, degradable lactic-glycolic acid copolymers, and poly(-)-3-hydroxybutyric acid. Possible loss of bioactivity and possible changes in the immunogenicity of the antibody contained in the sustained-release formulation can be prevented by using appropriate additives, by controlling the moisture content, and by developing specific polymer matrix compositions.

[0649] Controlled release within the scope of this disclosure can be considered to refer to any of a variety of extended-release dosage forms. For the purposes of this disclosure, the following terms can be considered substantially equivalent to controlled release: continuous release, controlled release, delayed release, reservoir, extended release, stepwise release, immediate release, long-term release, programmed release, extended release, moderate release, sustained release, reservoir, delayed, slow release, interval release, continuous release, timed coating, timed release, delayed action, prolonged action, stratified timed action, long-term action, prolonged action, repeated action, slow action, sustained action, and sustained action. Further discussion of these terms can be found in Lesczek Krowczynski, Extended-Release Dosage Forms ,1987 (CRC Press, Inc.)

[0650] Controlled release technologies cover a very wide range of drug dosage forms. These technologies include, but are not limited to, physical and chemical systems.

[0651] Physical systems include, but are not limited to, reservoir systems with rate-controlled membranes, such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlled membranes, such as hollow fiber, ultraporous cellulose triacetate, and porous polymer substrates and foams; monolithic systems, including those physically dissolved in nonporous, polymeric, or elastomeric matrices (e.g., non-erosive, erosive, environmentally infiltrated, and degradable), and materials physically dispersed in nonporous, polymeric, or elastomeric matrices (e.g., non-erosive, erosive, environmentally infiltrated, and degradable); laminated structures, including reservoirs that are chemically similar to or dissimilar to an external control layer; and other physical methods, such as osmotic pumps, or adsorption onto ion exchange resins.

[0652] Chemical systems include, but are not limited to, chemical erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion) or biological erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion). Further discussion of the categories of controlled-release systems can be found in Agis F. Kydonieus. Controlled Release Technologies: Methods, Theory and Applications , 1980 (CRC Press, Inc.)

[0653] Various controlled-release drug delivery systems have been developed for oral administration. These include, but are not limited to, osmotic pressure-controlled gastrointestinal delivery systems; hydrodynamic pressure-controlled gastrointestinal delivery systems; membrane osmotic control gastrointestinal delivery systems, including microporous membrane osmotic control gastrointestinal delivery devices; gastric juice-resistant intestinal-targeted controlled-release gastrointestinal delivery devices; gel diffusion-controlled gastrointestinal delivery systems; and ion exchange-controlled gastrointestinal delivery systems, including cationic and anionic drugs. Further information on controlled-release drug delivery systems can be found in Yie W. Chien, Novel Drug Delivery Systems , 1992 (Marcel Dekker, Inc.).

[0654] dose

[0655] The attending physician or other qualified medical personnel may determine the appropriate dosage based on a variety of clinical factors. As is well known in the medical field, the dosage for any patient depends on many factors, including the patient's body size, body surface area, age, the specific compound to be administered, the patient's sex, time and route of administration, general health, and other concurrent medications. The subject antibody may be administered in doses ranging from 1 ng / kg body weight to 20 mg / kg body weight, for example, from 0.1 mg / kg body weight to 10 mg / kg body weight, for example, from 0.5 mg / kg body weight to 5 mg / kg body weight; however, doses below or above this exemplary range are contemplated, especially taking into account the foregoing factors. If the regimen is a continuous infusion, it may also be administered in doses ranging from 1 μg / kg body weight per minute to 10 mg / min.

[0656] In some embodiments, the dose of the subject anti-C1s antibody is in the range of 0.001 μg to 1000 μg; however, doses below or above this exemplary range are contemplated, especially considering the foregoing factors. In some embodiments, the dose may be in the range of, for example, about 0.0001 to 100 mg / kg, or about 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.) body weight. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight, or in the range of 1-10 mg / kg, or at least 1 mg / kg. Intermediate doses within the above ranges are also contemplated within the scope of the invention.

[0657] In some embodiments, the subject anti-C1s antibody is administered in an amount providing a peak serum concentration of about 1 μg / ml to about 1 mg / ml, such as about 1 μg / ml to about 2.5 μg / ml, about 2.5 μg / ml to about 5 μg / ml, about 5 μg / ml to about 7.5 μg / ml, about 7.5 μg / ml to about 10 μg / ml, about 10 μg / ml to about 25 μg / ml, about 25 μg / ml to about 50 μg / ml, about 50 μg / ml to about 100 μg / ml, about 100 μg / ml to about 250 μg / ml, about 250 μg / ml to about 500 μg / ml, about 500 μg / ml to about 750 μg / ml, or about 750 μg / ml to about 1000 μg / ml. In some embodiments, the subject anti-C1s antibody is administered in an amount that provides a peak serum concentration greater than 1 mg / ml, for example about 1 mg / ml to about 2 mg / ml, about 2 mg / ml to about 5 mg / ml, or about 5 mg / ml to about 10 mg / ml.

[0658] Such doses may be administered to an individual daily, every other day, weekly, or at any other schedule determined based on empirical analysis. Exemplary treatments include administration at multiple doses over an extended period, for example, at least six months. Other exemplary data regimens include administration every two weeks, monthly, or every 3 to 6 months. Exemplary dosing schedules include continuous administration of 1-10 mg / kg or 15 mg / kg daily, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the range shown. Progress can be monitored through periodic assessment.

[0659] Those skilled in the art will readily understand that dosage levels and administration schedules can vary depending on the specific antibody, the severity of symptoms, and the subject's susceptibility to side effects. The preferred dosage and administration schedule for a given compound can be readily determined by those skilled in the art through various means.

[0660] Application route

[0661] The subject antibody shall be administered to an individual using any available methods and routes suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes.

[0662] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intrathecal, intracranial, subcutaneous, intradermal, local, intravenous, intraperitoneal, intraarterial (e.g., via the carotid artery), spinal or brain delivery, rectal, nasal, oral, and other enteral and parenteral routes. Routes of administration can be combined, if desired, or modified according to the antibody and / or desired action. The subject antibody composition can be administered in a single dose or multiple doses. In some embodiments, the subject antibody composition is administered orally. In some embodiments, the subject antibody composition is administered via inhalation. In some embodiments, the subject antibody composition is administered intranasally. In some embodiments, the subject antibody composition is administered locally. In some embodiments, the subject antibody composition is administered intracranially. In some embodiments, the subject antibody composition is administered intravenously. In some embodiments, the subject antibody composition is administered intrathecally.

[0663] The antibodies disclosed herein can be administered to the host using any available conventional methods and routes of delivery of conventional drugs, including systemic or local routes. Generally, the routes of administration covered by this invention include, but are not limited to, enteric, parenteral, or inhalation routes.

[0664] Parenteral administration routes, other than inhalation, include, but are not limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrathecal, and intravenous routes—that is, any route of administration other than through the digestive tract. Parenteral administration can be performed to achieve systemic or local delivery of the subject antibody. When systemic delivery is required, administration typically involves invasive administration of the drug formulation or local or mucosal administration for systemic absorption.

[0665] The subject antibody can also be delivered to subjects via enteral administration. Enteral administration routes include, but are not limited to, oral and rectal administration (e.g., suppository administration).

[0666] Treatment means at least improving the symptoms associated with the pathological condition troubling the host, where improvement is used in a broad sense to mean at least reducing the magnitude of parameters (e.g., symptoms) associated with the treated pathological condition, such as complement-mediated disease or symptom. Therefore, treatment also includes situations where the pathological condition or at least the associated symptoms are completely suppressed, e.g., prevented from occurring or stopped (e.g., terminated), so that the host no longer suffers from said pathological condition, or at least the characteristic symptoms of said pathological condition.

[0667] In some embodiments, the subject antibody is administered to a site in a cerebral artery, for example, by injection and / or delivery, or directly to brain tissue. The subject antibody can also be administered directly to a target site, for example, via a biogun delivery method.

[0668] Multiple hosts (where the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") may be treated according to the subject-matter approach. Generally, these hosts are "mammals" or "of mammals," where these terms are widely used to describe organisms within the class Mammalia, including Carnivora (e.g., cats), Herbivora (e.g., cattle, horses, and sheep), Omnivora (e.g., dogs, goats, and pigs), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host is an individual with a complement system, such as a mammal, fish, or invertebrate. In some embodiments, the host is a mammal, fish, or invertebrate companion animal, agricultural animal, working animal, zoo animal, or laboratory animal containing a complement system. In some embodiments, the host is a human.

[0669] Implementations include compositions comprising containers adapted to contain a composition containing a subject C1s antibody for administration to an individual. For example, the subject antibody may be placed in a container adapted to contain the pharmaceutical composition. The container may be, for example, a bottle (e.g., having a closure device, such as a cap), a blister pack (e.g., which can provide closure for one or more doses per blister), a vial, flexible packaging (e.g., a sealed Mylar or plastic bag), an ampoule (for a single dose in solution form), a dropper, a syringe, a film, a tube, etc. In some embodiments, the container (such as a sterile container) contains the subject pharmaceutical composition. In some embodiments, the container is a bottle or a syringe. In some embodiments, the container is a bottle. In some embodiments, the container is a syringe.

[0670] A kit is provided containing a unit dose of the subject antibody, for example, in an oral or injectable dose. In addition to the container containing the unit dose, such a kit will include an informational insert describing the use and associated benefits of the antibody treatment for the pathological condition of interest. Preferred compounds and unit doses are those described above.

[0671] Methods for treating complement-mediated diseases or conditions

[0672] This disclosure provides methods for treating complement-mediated diseases or conditions. The methods generally include administering an effective amount of the disclosed anti-C1s antibody or a pharmaceutical composition containing said antibody to an individual in need. In some cases, administration of the subject anti-C1s antibody can modulate complement C1s activity in the cells, tissues, or fluids of an individual and treat the complement-mediated disease or condition. This disclosure provides methods for inhibiting complement component C4 activation in an individual, the methods including administering an effective amount of the disclosed anti-C1s antibody or a pharmaceutical composition containing said antibody to the individual. This disclosure provides methods for inhibiting complement C1s activity in an individual, the methods including administering an effective amount of the disclosed anti-C1s antibody or a pharmaceutical composition containing said antibody to the individual.

[0673] In some embodiments, a method of the present disclosure for treating an individual with a complement-mediated disease or condition includes administering to the individual an effective amount of the disclosed anti-C1s antibody or an effective amount of a pharmaceutical composition comprising: a) the disclosed anti-C1s antibody; and a pharmaceutically acceptable excipient suitable for administration to the individual. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration may be performed by any means known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, administration is intrathecal. In some embodiments, administration is subcutaneous.

[0674] In some embodiments, the “effective amount” of the anti-C1s antibody of this disclosure or the “effective amount” of the subject pharmaceutical composition comprising the anti-C1s antibody of this disclosure is an amount that, when administered to an individual in need in one or more doses, reduces the production of C4b2a in the individual (or in the individual’s tissues or organs) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C4b2a (i.e., the complement C4b and C2a complex; also referred to as “C3 convertase”) produced in the absence of the subject anti-C1s antibody, by at least 100%. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration may be performed by any means known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, administration is intrathecal. In some embodiments, administration is intravenous. In some implementation methods, the route of administration is subcutaneous.

[0675] This disclosure provides methods for regulating complement activation. In some embodiments, the method inhibits complement activation, for example, by reducing C4b2a production. In some embodiments, this disclosure provides methods for regulating complement activation in an individual with a complement-mediated disease or condition, the method comprising administering to the individual an anti-C1s antibody of this disclosure or a pharmaceutical composition of this disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of this disclosure. In some embodiments, the method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration may be performed by any means known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, administration is intrathecal.

[0676] Complement-mediated diseases or conditions are characterized by abnormal amounts of complement C1s or abnormal levels of complement C1s proteolytic activity in an individual's cells, tissues, or fluids.

[0677] In some cases, complement-mediated diseases or conditions are characterized by elevated (higher than normal) levels of C1s or elevated levels of complement C1s activity in cells, tissues, or fluids. For example, in some cases, complement-mediated diseases or conditions are characterized by elevated levels of C1s and / or elevated C1s activity in brain tissue and / or cerebrospinal fluid. "Higher than normal" levels of C1s in cells, tissues, or fluids indicate that the amount of C1s in cells, tissues, or fluids is higher than normal control levels, for example, higher than normal control levels in an individual or a group of individuals of the same age. "Higher than normal" levels of C1s activity in cells, tissues, or fluids indicate that proteolytic cleavage by C1s in cells, tissues, or fluids is higher than normal control levels, for example, higher than normal control levels in an individual or a group of individuals of the same age. In some cases, individuals with complement-mediated diseases or conditions exhibit one or more other symptoms of that disease or condition.

[0678] In other cases, complement-mediated diseases or conditions are characterized by the presence of lower than normal amounts of C1s or lower levels of complement C1s activity in cells, tissues, or fluids. For example, in some cases, complement-mediated diseases or conditions are characterized by the presence of lower amounts of C1s and / or lower C1s activity in brain tissue and / or cerebrospinal fluid. "Lower than normal" amounts of C1s in cells, tissues, or fluids indicate that the amount of C1s in cells, tissues, or fluids is lower than normal control levels, for example, lower than normal control levels for an individual or an individual or population of the same age. "Lower than normal" levels of C1s activity in cells, tissues, or fluids indicate that proteolytic cleavage by C1s in cells, tissues, or fluids is lower than normal control levels, for example, lower than normal control levels for an individual or an individual or population of the same age. In some cases, individuals with complement-mediated diseases or conditions exhibit one or more other symptoms of that disease or condition.

[0679] Complement-mediated diseases or conditions are those in which the amount or activity of complement C1s causes, for example, a disease or condition in an individual. In some embodiments, the complement-mediated diseases or conditions are selected from the group consisting of autoimmune diseases, cancer, hematological diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative conditions, eye diseases, kidney diseases, transplant rejection, vascular diseases, and vasculitis. In some embodiments, the complement-mediated disease or condition is an autoimmune disease. In some embodiments, the complement-mediated disease or condition is cancer. In some embodiments, the complement-mediated disease or condition is an infectious disease. In some embodiments, the complement-mediated disease or condition is an inflammatory disease. In some embodiments, the complement-mediated disease or condition is a hematological disease. In some embodiments, the complement-mediated disease or condition is ischemia-reperfusion injury. In some embodiments, the complement-mediated disease or condition is an eye disease. In some embodiments, the complement-mediated disease or condition is a kidney disease. In some embodiments, the complement-mediated disease or condition is transplant rejection. In some embodiments, the complement-mediated disease or condition is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease or condition is a vascular disease. In some embodiments, the complement-mediated disease or condition is a vasculitis. In some embodiments, the complement-mediated disease or condition is a neurodegenerative disease or condition. In some embodiments, the complement-mediated disease is a neurodegenerative disease. In some embodiments, the complement-mediated condition is a neurodegenerative disease. In some embodiments, the complement-mediated disease or condition is tau proteinosis.

[0680] Examples of complement-mediated diseases or conditions include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), allergic reactions, auricolic granulation dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemia vasculitis, boxer's dementia, and Lewy body dementia. LewyBodies (DLB), diffuse neurofibrillary tangles with calcification, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thinking disorder, frontotemporal dementia (FTD), frontotemporal dementia with Parkinson's disease associated with chromosome 17, frontotemporal degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatase syndrome, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infectious diseases (e.g., caused by bacteria, such as Neisseria meningitidis). Neisseria meningitidis ) or Streptococcus ( StreptococcusDiseases caused by viruses (e.g., human immunodeficiency virus (HIV)) or other infectious agents, inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency A (MoCD), type I membranoproliferative glomerulonephritis (MPGN), type II membranoproliferative glomerulonephritis (MPGN) (dense deposit disease), membranous nephritis, multiple infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuronopathy, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease. Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, post-encephalitis Parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin Escherichia coli (Shiga-toxin E. coli) Coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, tangles-only dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, primary mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, acquired bullous epidermolysis bullosa, delayed hemolytic infusion reaction, hypocomplementemia, urticarial vasculitis syndrome, pseudophaloid bullous keratopathy, and platelet refusal.

[0681] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with chromosomal 17-related Parkinson's disease) are the most common forms of tau protein disorders. Accordingly, the present invention relates to any of the methods described above, wherein the tau protein disorder is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with chromosomal 17-related Parkinson's disease. Other tau protein disorders include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

[0682] Neurodegenerative tau protein disorders include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinson's disease-dementia complex, aerobatic granulation dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, boxer's dementia, diffuse neurofibrillary tangles with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with Parkinson's disease associated with chromosome 17, frontotemporal degeneration, Gerstmann-Straussler-Scheinker disease, and more. Hallewarden-Scholes disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, post-encephalitis Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangles-only dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0683] This disclosure also provides methods for treating synucleinopathies such as Parkinson's disease (PD); Lewy body dementia (DLB); and multiple system atrophy (MSA). For example, PD with dementia (PDD) can be treated using the methods described herein.

[0684] In some embodiments, complement-mediated diseases or conditions include Alzheimer's disease. In some embodiments, complement-mediated diseases or conditions include Parkinson's disease. In some embodiments, complement-mediated diseases or conditions include transplant rejection. In some embodiments, complement-mediated diseases or conditions are antibody-mediated transplant rejection.

[0685] In some embodiments, the anti-C1s antibody of this disclosure prevents or delays the onset of at least one symptom of a complement-mediated disease or condition in an individual. In some embodiments, the anti-C1s antibody of this disclosure reduces or eliminates at least one symptom of a complement-mediated disease or condition in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, hematological disorders, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative conditions, kidney diseases, transplant rejection, eye diseases, vascular diseases, or vasculitis. Symptoms can be neurological symptoms, such as cognitive impairment, memory impairment, loss of motor function, etc. Symptoms can also be the activity of the C1s protein in an individual's cells, tissues, or fluids. Symptoms can also be the degree of complement activation in an individual's cells, tissues, or fluids.

[0686] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual modulates complement activation in the individual's cells, tissues, or fluids. In some embodiments, administration of the subject anti-C1s antibody to an individual inhibits complement activation in the individual's cells, tissues, or fluids. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, complement activation in the individual is inhibited by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to complement activation in the individual before treatment with the anti-C1s antibody.

[0687] In some embodiments, the anti-C1s antibody of this disclosure reduces C3 deposition on erythrocytes; for example, in some embodiments, the anti-C1s antibody of this disclosure reduces C3b, iC3b, etc., deposition on RBCs. In some embodiments, the anti-C1s antibody of this disclosure inhibits complement-mediated erythrocyte lysis.

[0688] In some embodiments, the anti-C1s antibody of this disclosure reduces the deposition of C3 on platelets; for example, in some embodiments, the anti-C1s antibody of this disclosure reduces the deposition of C3b, iC3b, etc. on platelets.

[0689] In some embodiments, administration of the anti-C1s antibody of this disclosure results in outcomes selected from the group consisting of: (a) reduced complement activation; (b) improved cognitive function; (c) reduced neuronal loss; (d) reduced levels of phosphorylated Tau protein in neurons; (e) reduced glial cell activation; (f) reduced lymphocyte infiltration; (g) reduced macrophage infiltration; (h) reduced antibody deposition; (i) reduced glial cell loss; (j) reduced oligodendrocyte loss; (k) reduced dendritic cell infiltration; (l) reduced neutrophil infiltration; (m) (n) Decreased erythrocyte lysis; (o) Decreased erythrocyte phagocytosis; (p) Decreased platelet phagocytosis; (q) Increased graft survival; (r) Decreased macrophage-mediated phagocytosis; (s) Improved vision; (t) Improved motor control; (u) Improved thrombosis; (v) Improved coagulation; (w) Improved renal function; (x) Decreased antibody-mediated complement activation; (y) Decreased autoantibody-mediated complement activation; (z) Improved anemia; (aa) Decreased demyelination; (ab) Decreased eosinophilia; (ac) Decreased C3 deposition on erythrocytes (e.g., decreased deposition of C3b, iC3b, etc. on RBCs); and (ad) Decreased deposition of C3 on platelets (e.g., decreased deposition of C3b, iC3b, etc.); and (ae) decreased production of anaphylatoxins; (af) decreased autoantibody-mediated blister formation; (ag) decreased autoantibody-induced pruritus; (ah) decreased autoantibody-induced lupus erythematosus; (ai) decreased autoantibody-mediated skin erosion; (aj) decreased red blood cell destruction due to infusion reactions; (ak) decreased red blood cell lysis due to allogeneic antibodies; (al) decreased hemolysis due to infusion reactions; (am) decreased platelet lysis mediated by allogeneic antibodies; (an) decreased platelet lysis due to infusion reactions; (ao) decreased mast cell activation; (ap) decreased mast cell histamine release; (aq) blood Decreased vascular permeability; (ar) reduced edema; (as) reduced complement deposition on graft endothelium; (at) reduced anaphylatoxin production in graft endothelium; (au) reduced separation at the dermo-epidermal junction; (av) reduced anaphylatoxin production at the dermo-epidermal junction; (aw) reduced allogeneic antibody-mediated complement activation in graft endothelium; (ax) reduced antibody-mediated loss at the neuromuscular junction; (ay) reduced complement activation at the neuromuscular junction; (az) reduced anaphylatoxin production at the neuromuscular junction; (ba) reduced complement deposition at the neuromuscular junction; (bb) reduced paralysis; (bc) reduced numbness; (bd) increased bladder control; (be) increased defecation control; (bf) reduced autoantibody-related mortality.and (bg) a decrease in the incidence of autoantibodies.

[0690] In some implementations, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, a reduction of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% of one or more of the following outcomes can be achieved compared to the level or extent of outcomes in the individual before treatment with the anti-C1s antibody: (a) complement activation; (b) cognitive decline; (c) neuronal loss; (d) neuronal Phosphorylated Tau levels; (e) glial cell activation; (f) lymphocyte infiltration; (g) macrophage infiltration; (h) antibody deposition; (i) glial cell loss; (j) oligodendrocyte loss; (k) dendritic cell infiltration; (l) neutrophil infiltration; (m) erythrocyte lysis; (n) erythrocyte phagocytosis; (o) platelet phagocytosis; (p) platelet lysis; (q) graft rejection; (r) macrophage-mediated phagocytosis; (s) visual impairment; (t) antibody-mediated complement activation; (u) autoantibody-mediated complement activation; (v) demyelination; (w) eosinophilia.

[0691] In some implementations, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, an improvement of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% of one or more of the following outcomes can be achieved compared to the level or extent of outcomes in the individual before treatment with the anti-C1s antibody: a) cognitive function; b) graft survival; c) visual acuity; d) motor control; e) thrombosis; f) coagulation; g) renal function; and h) hematocrit (red blood cell count).

[0692] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces complement activation in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or more in the form of a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, complement activation in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to complement activation in said individual before treatment with the anti-C1s antibody.

[0693] In some embodiments, administration of the anti-C1s antibody of this disclosure improves cognitive function in the individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, cognitive function in the individual is improved by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to cognitive function before treatment with the anti-C1s antibody.

[0694] In some embodiments, administration of the anti-C1s antibody of this disclosure reduces the rate of cognitive decline in the individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, the rate of cognitive decline in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the rate of cognitive decline in the individual before treatment with the anti-C1s antibody.

[0695] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces neuronal loss in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, the neuronal loss in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the neuronal loss in said individual before treatment with the anti-C1s antibody.

[0696] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces the level of phosphorylated Tau in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or more in the form of a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, the phosphorylated Tau in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level of phosphorylated Tau in said individual before treatment with the anti-C1s antibody.

[0697] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces glial cell activation in that individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, glial cell activation in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to glial cell activation before treatment with the anti-C1s antibody. In some embodiments, the glial cells are astrocytes or microglia.

[0698] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces lymphocyte infiltration in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, lymphocyte infiltration in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to lymphocyte infiltration in the individual before treatment with the anti-C1s antibody.

[0699] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces macrophage infiltration in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, macrophage infiltration in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to macrophage infiltration in the individual before treatment with the anti-C1s antibody.

[0700] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces antibody deposition in said individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, antibody deposition in said individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to antibody deposition in the individual before treatment with the anti-C1s antibody.

[0701] In some embodiments, administration of the anti-C1s antibody of this disclosure to an individual reduces the production of anaphylatoxins (e.g., C3a, C4a, C5a) in the individual. For example, in some embodiments, when the subject anti-C1s antibody is administered once or multiple times as a monotherapy or combination therapy to an individual with a complement-mediated disease or condition, the production of anaphylatoxins in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level of anaphylatoxin production in the individual before treatment with the anti-C1s antibody.

[0702] This disclosure provides the use of the anti-C1s antibody of this disclosure or a pharmaceutical composition comprising the anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient in the treatment of an individual with a complement-mediated disease or condition. In some embodiments, this disclosure provides the use of the anti-C1s antibody of this disclosure in the treatment of an individual with a complement-mediated disease or condition. In some embodiments, this disclosure provides the use of a pharmaceutical composition comprising the anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient in the treatment of an individual with a complement-mediated disease or condition.

[0703] This disclosure provides the use of the anti-C1s antibody of this disclosure in the manufacture of a medicament for treating an individual with a complement-mediated disease or condition.

[0704] This disclosure provides the use of the anti-C1s antibody of this disclosure or a pharmaceutical composition comprising the anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation. In some embodiments, this disclosure provides the use of the anti-C1s antibody of this disclosure or a pharmaceutical composition comprising the anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual with a complement-mediated disease or condition. In some embodiments, this disclosure provides the use of the anti-C1s antibody of this disclosure for inhibiting complement activation in an individual with a complement-mediated disease or condition. In some embodiments, this disclosure provides the use of a pharmaceutical composition comprising the anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual with a complement-mediated disease or condition.

[0705] This disclosure provides the use of the anti-C1s antibody of this disclosure in the manufacture of a medicament for regulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in individuals with complement-mediated diseases or conditions.

[0706] This disclosure provides an anti-C1s antibody of the present disclosure for use in medical treatment, or a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, this disclosure provides an anti-C1s antibody of the present disclosure for use in medical treatment. In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in medical treatment.

[0707] This disclosure provides an anti-C1s antibody of this disclosure or a pharmaceutical composition comprising an anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for treating individuals with complement-mediated diseases or conditions. In some embodiments, this disclosure provides an anti-C1s antibody of this disclosure for treating individuals with complement-mediated diseases or conditions. In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for treating individuals with complement-mediated diseases or conditions.

[0708] This disclosure provides an anti-C1s antibody of this disclosure for modulating complement activation, or a pharmaceutical composition comprising an anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient. In some embodiments, this disclosure provides an anti-C1s antibody of this disclosure for modulating complement activation. In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-C1s antibody of this disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the anti-C1s antibody inhibits complement activation.

[0709] Combination therapy

[0710] The anti-C1s antibody disclosed herein can be administered to individuals in need, either alone (e.g., as a monotherapy) or in combination with one or more other therapeutic agents.

[0711] For the treatment of Alzheimer's disease (AD), suitable other therapeutic agents include, but are not limited to, acetylcholinesterase inhibitors, including but not limited to Aricept (donepezil), Exelon (rivastigmine), metrifonate, and tacrine (Cognex); anti-Aβ antibodies; nonsteroidal anti-inflammatory agents, including but not limited to ibuprofen and indomethacin; cyclooxygenase-2 (Cox2) inhibitors, such as celecoxib (Celebrex); and monoamine oxidase inhibitors, such as selegilene (Eldepryl or Deprenyl). The dosages of each of the above agents are known in the art.

[0712] Another suitable therapeutic agent for treating AD is an agent that inhibits tau protein aggregation, such as a naphthoquinone derivative that inhibits tau aggregation, as described in U.S. Patent No. 7,605,179. Another suitable therapeutic agent is an agent that inhibits tau phosphorylation, such as a 3-substituted-4-pyrimidinone derivative that inhibits tau protein kinase 1, as described in U.S. Patent No. 7,572,793.

[0713] As used herein, “combined with” means, for example, the application of the first compound during the entire course of application of the second compound; the application of the first compound during a period overlapping with the application of the second compound, for example, the application of the first compound begins before the application of the second compound and ends before the application of the second compound; the application of the second compound begins before the application of the first compound and ends before the application of the first compound; the application of the first compound begins before the application of the second compound and ends before the application of the first compound; the application of the second compound begins before the application of the first compound and ends before the application of the second compound; the application of the second compound begins before the application of the first compound and ends before the application of the second compound. Therefore, “combined” can also refer to a scheme involving the application of two or more compounds. As used herein, “combined with” also refers to the application of two or more compounds, which may be the same or different formulations, administered via the same or different routes, and in the same or different dosage forms.

[0714] Individuals awaiting treatment

[0715] Individuals suitable for treatment with the subject anti-C1s antibody include those already diagnosed with a complement-mediated disease or condition; those at greater risk of developing a complement-mediated disease or condition compared to the general population (e.g., those with a genetic predisposition to a complement-mediated disease or condition); those with Parkinson's disease with dementia (PDD); those with Alzheimer's disease; and so on. In some cases, the individuals are adults. In some cases, the adults are 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or 80 years of age or older. For example, the adults can be 20 to 30 years old, 30 to 40 years old, 40 to 50 years old, 50 to 60 years old, 60 to 70 years old, or over 70 years old. In some cases, the individuals are children. In some cases, the children are under 20 years of age, under 10 years of age, or under 5 years of age.

[0716] In vitro testing and animal models

[0717] This disclosure provides methods for testing the efficacy of a subject antibody in vitro or in vivo. In vitro testing includes methods for determining the binding of the subject antibody to complement C1s protein, methods for determining the ability of the subject antibody to inhibit the production of the C4b2a complex, and methods for identifying the epitope or epitope characteristics to which the anti-C1s antibody of this disclosure binds. Non-human animal models used to test the efficacy of the subject antibodies include experimental autoimmune encephalomyelitis (see, for example, Weerth et al., Am J Path. 163:1069-1080 (2003); Theien et al., J. Clin. Invest. 107:995-1006 (2001)), myasthenia gravis (see, for example, Morgan et al., Clin. Exp. Immun. 146:294-302 (2006)), myocardial ischemia and reperfusion (see, for example, Busche et al., GMS Ger. Med. Sci. 8:Doc20 (2010)), and Streptococcus pneumoniae (see, for example, Brown et al., Proc. Natl. Acad. Sci. 99:16969-16974)). Additionally, non-human animal models of transplant rejection are suitable (see, for example, Racki et al., (2010)). Transplantation 89:527; and Baldwin et al., (2010) Am. J. Transplantation 10:1135). In some embodiments, the model is a rodent model (e.g., a rat or mouse). Such models are known to those skilled in the art.

[0718] Detection methods

[0719] This disclosure provides in vitro methods for detecting complement C1s protein in biological samples obtained from individuals; and in vivo methods for detecting C1s protein in living individuals. The in vitro detection methods may be quantitative. C1s protein may therefore serve as a biomarker for the progression of complement-mediated diseases or conditions, or for the response to treatment of complement-mediated diseases or conditions.

[0720] The complement C1s protein to be detected / quantified can be the full-length C1s protein or any fragment thereof containing the epitope bound by the anti-C1s antibody of this disclosure.

[0721] Suitable biological samples include, but are not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, eye discharge, synovial fluid, solid tissue samples, tissue culture samples, cell samples, and other biological samples known to those skilled in the art.

[0722] The in vitro method of the present disclosure for detecting complement C1s protein in biological samples obtained from an individual generally includes: a) contacting the biological sample with the anti-C1s antibody of the present disclosure; and b) detecting the binding of the antibody to the C1s protein present in the sample.

[0723] The detection methods disclosed herein can be used to determine whether an individual has a complement-mediated disease or condition, or is at risk of developing a complement-mediated disease or condition. The detection methods disclosed herein can be used to determine the stage (severity) of a complement-mediated disease or condition. The detection methods disclosed herein can be used to monitor the progression of a complement-mediated disease or condition in an individual. The detection methods disclosed herein can be used to determine an individual's response to a treatment regimen for a complement-mediated disease or condition. The subject detection methods can be used to test biological samples obtained from individuals suspected of having a complement-mediated disease or condition, individuals already diagnosed with a complement-mediated disease or condition, individuals with a genetic predisposition to developing a complement-mediated disease or condition, etc.

[0724] This disclosure provides a method for diagnosing complement-mediated diseases or conditions in an individual. The method generally includes: (a) determining the amount of complement C1s protein in a biological sample obtained from the individual; and (b) comparing the amount of complement C1s protein in the biological sample with a reference value, standard value, or normal control value indicating the amount of complement C1s protein in a normal control subject. A significant difference between the amount of C1s protein in the biological sample and the normal control value indicates that the individual has a complement-mediated disease or condition. In some embodiments, the determination step includes contacting the biological sample with an anti-C1s antibody of this disclosure and quantifying the binding of the antibody to the complement C1s protein present in the sample.

[0725] This disclosure provides a method for monitoring the progression of complement-mediated disease or condition in an individual. The method generally includes comparing the amount of complement C1s protein in a biological sample obtained from the individual at a first time point with the amount of complement C1s protein in a biological sample obtained from the individual at a second time point. The difference between the amount of complement C1s protein in the biological sample obtained from the individual at the second time point and the amount of complement C1s protein in the biological sample obtained from the individual at the first time point can provide an indication of: i) whether the complement-mediated disease or condition is progressing or whether the progression of the disease has slowed or stopped; and / or ii) how rapidly the complement-mediated disease or condition is progressing; and / or iii) whether the individual exhibits a beneficial clinical response to treatment with a drug or other treatment regimen for the complement-mediated disease or condition. In some embodiments, the assay step includes contacting the biological sample with an anti-C1s antibody of this disclosure and quantifying the binding of the antibody to the complement C1s protein present in the sample. In some embodiments, the comparison step indicates whether the disease or condition is progressing.

[0726] This disclosure provides a method for monitoring the response of an individual to treatment for a complement-mediated disease or condition. The method generally includes comparing the amount of complement C1s protein in a biological sample obtained from the individual at a first time point with the amount of complement C1s protein in a biological sample obtained from the individual at a second time point. The difference between the amount of complement C1s protein in the biological sample obtained from the individual at the second time point and the amount of complement C1s protein in the biological sample obtained from the individual at the first time point can provide an indication of whether the individual exhibits a beneficial clinical response to treatment with a drug or other treatment regimen for the complement-mediated disease or condition. In some embodiments, the assay step includes contacting the biological sample with an anti-C1s antibody of this disclosure and quantifying the binding of the antibody to the complement C1s protein present in the sample. In some embodiments, the comparison step indicates whether the progression of the disease has slowed or stopped.

[0727] This disclosure provides methods for staging complement-mediated diseases or conditions. For example, the subject method can provide for the determination of stages in Alzheimer's disease. For example, the amount of complement C1s protein in a biological sample from a living individual can provide an indication of the Braak stage of AD. Braak and Braak (1995) Neurobiol. Aging 16:271. For example, the amount of complement C1s protein in biological samples from living individuals can provide an indication of whether the individual is in the transverse nasal region stage I-II of AD; the limbic system stage III-IV of AD; or the neocortical stage V-VI of AD.

[0728] The amount of complement C1s protein in biological samples can be assessed by any suitable method known in the art. Suitable methods include, but are not limited to, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), surface-enhanced laser desorption / ionization time-of-flight (SELDI-TOF), high-performance liquid chromatography (HPLC), rapid protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC), and subsequent tandem mass spectrometry (MS / MS), and laser density determination.

[0729] This disclosure provides a method for monitoring the progression of complement-mediated disease or condition in an individual, wherein the method generally includes: a) determining a first amount of complement C1s protein in a biological sample obtained from the individual at a first time point; b) determining a second amount of complement C1s protein in a biological sample obtained from the individual at a second time point; and c) comparing the second amount of the second complement C1s protein with the first amount of the complement C1s protein. In some embodiments, the determination step includes: i) contacting the biological sample with a subject anti-C1s antibody; and ii) quantifying the binding of the antibody to the complement C1s protein present in the sample. In some embodiments, the comparison indicates whether the disease has progressed.

[0730] In some cases, the first time point is a time point before the start of the treatment regimen, and the second time point is a time point after the start of the treatment regimen. Therefore, this disclosure provides a method for detecting a response to treatment with an agent for treating a complement-mediated disease or condition, wherein the method comprises: a) determining a first amount of complement C1s protein in a biological sample obtained from the individual at a first time point before the start of treatment with the agent for treating a complement-mediated disease or condition; b) determining a second amount of complement C1s protein in a biological sample obtained from the individual at a second time point after the start of treatment with the agent for treating a complement-mediated disease or condition; and c) comparing the second amount of complement C1s protein with the first amount of complement C1s protein.

[0731] Thematic approaches for monitoring the progression of complement-mediated diseases or conditions can also be used to monitor the progression of tau protein diseases or synuclein diseases such as Parkinson's disease (PD); Lewy body dementia (DLB), etc. For example, thematic approaches can be used to monitor the progression of PD with dementia (PDD).

[0732] The subject method may include using a kit or assay device that includes the subject anti-C1s antibody. This disclosure provides kits and assay devices for performing the methods described herein. The subject kit includes the anti-C1s antibody of this disclosure.

[0733] Anti-C1s antibodies can be immobilized on insoluble supports (e.g., test strips, wells of multi-well plates, beads (e.g., magnetic beads), etc.). Suitable supports are well known in the art and particularly include commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction discs (e.g., multi-well plates), plastic tubes, etc. Solid supports can comprise any of a variety of substances, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. Suitable methods for immobilizing the subject antibody on a solid support are well known and include, but are not limited to, ionicity, hydrophobicity, covalent interactions, etc. Solid supports can be soluble or insoluble, for example, in aqueous solutions. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

[0734] The anti-C1s antibody disclosed herein may contain a detectable label. When the antibody contains a detectable label, the subject kit may include one or more reagents for generating the detectable label. The labeled antibody may contain labels such as chemiluminescent agents, microparticle labels, colorimetric agents, energy transfer agents, enzymes, fluorescent agents, or radioisotopes. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable detectable labels include, but are not limited to, fluorescent labels (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.); radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32 P); and enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and other enzymes that act on substrates to produce products that can be detected by fluorescence, colorimetry, or spectrophotometry).

[0735] In some cases, the methods of this disclosure for detecting / quantifying C1s in biological samples obtained from individuals include treating the sample with a chelating agent such as a calcium chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)). The chelating agent disrupts the C1 complex, causing the polypeptides that form the C1 complex to separate from each other, thereby producing monomeric C1 complex components.

[0736] In some cases, the present disclosure method for detecting / quantifying C1s in biological samples obtained from an individual includes: a) contacting the biological sample with an immobilized first antibody (e.g., a rabbit polyclonal antibody that binds C1s but does not compete with the subject anti-C1s antibody for C1s binding, thereby forming an immobilized first antibody / C1s complex; b) contacting the immobilized first antibody / C1s complex with a chelating agent (e.g., EDTA), thereby forming an immobilized first antibody / C1s monomer complex; c) contacting the immobilized first antibody / C1s monomer complex with a monoclonal anti-C1s antibody of the present disclosure; and d) detecting the binding of the monoclonal anti-C1s antibody to the immobilized C1s monomer. In some cases, the present disclosure method for detecting / quantifying C1s in biological samples obtained from an individual includes: a) treating the biological sample with a chelating agent (e.g., EDTA) to form a C1s monomer; b) contacting the chelated biological sample with an immobilized first antibody (e.g., a rabbit polyclonal antibody that binds C1s but does not co...

Claims

1. A method for generating a humanized antibody that binds to complement C1s protein, comprising: Culture recombinant cells, said recombinant cells comprising nucleic acids or nucleic acid groups encoding said humanized antibody, wherein said humanized antibody comprises: The complementarity-determining regions CDR-1, CDR-2, and CDR-3 of the antibody light chain variable region, wherein the amino acid sequence of the antibody light chain variable region is the amino acid sequence of SEQ ID NO:7; and The antibody heavy chain variable region includes CDR-1, CDR-2, and CDR-3 regions, wherein the amino acid sequence of the antibody heavy chain variable region is the amino acid sequence SEQ ID NO:8, wherein the nucleic acid or nucleic acid group is expressed in cells and produces the humanized antibody.

2. The method of claim 1, wherein, The humanized antibody includes a humanized light chain framework region and a humanized heavy chain framework region.

3. The method of claim 1, wherein, The humanized antibody is selected from the group consisting of the Fab fragment, the F(ab')2 fragment, scFv, and Fv.

4. The method according to claim 1, wherein, The humanized antibody includes the heavy chain constant region of isotype IgG1, IgG2, IgG3 or IgG4.

5. The method according to claim 1, wherein, The humanized antibody includes the heavy chain constant region of isotype IgG4.

6. The method according to claim 5, wherein, The humanized antibody comprises S241P replacement via Kabat numbering and L235E replacement via EU numbering.

7. The method according to claim 1, wherein, The humanized antibody comprises a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:45; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:45; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:44; or The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:

45.

8. The method according to claim 1, wherein, The humanized antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO:44, and the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO:

42.

9. The method according to claim 1, wherein, The method further includes: The humanized antibody was recovered.

10. The method according to any one of claims 1 to 9, wherein, The cells are mammalian cells, insect cells, yeast cells, or prokaryotic cells.

11. The method according to claim 10, wherein, The cells in question are mammalian cells.

12. The method according to claim 11, wherein the mammalian cells are selected from HeLa cells, CHO cells, 293 cells, Vero cells, NIH 3T3 cells, Huh-7 cells, BHK cells, PC12 cells, COS cells, RAT1 cells, mouse L cells, human embryonic kidney HEK cells, and HLHepG2 cells.

13. The method of claim 12, wherein the mammalian cell is a CHO cell.

14. The method of claim 11, wherein the mammalian cell is a COS-7 cell.

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