HK1261836A1

HK1261836A1 - Anti-complement c1s antibodies and uses thereof

Info

Publication number: HK1261836A1
Application number: HK19121744.7A
Authority: HK
Inventors: P‧范弗拉塞莱尔; G‧帕里; N‧E‧斯塔利亚诺; S‧帕尼克
Original assignee: 美国比奥维拉迪维股份有限公司
Priority date: 2012-11-02
Filing date: 2019-04-01
Publication date: 2020-01-03

Description

Anti-complement C1s antibodies and uses thereof

The application is a divisional application of an invention application with the application date of 2013, 11/01, the Chinese application number of 201380068801.5 and the invention name of 'anti-complement C1s antibody and application thereof'.

Cross-referencing

The present application claims the benefit of U.S. provisional patent application No.61/721,916, filed on day 11/2 of 2012, U.S. provisional patent application No.61/754,123, filed on day 18 of 1/2013, U.S. provisional patent application No.61/779,180, filed on day 13 of 2013, and U.S. provisional patent application No.61/846,402, filed on day 15 of 7/2013, which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure provides antibodies that bind complement C1s protein; and nucleic acid molecules encoding such antibodies. The disclosure also provides compositions comprising such antibodies, and methods of making and using such antibodies, nucleic acid molecules, and compositions. The present disclosure provides isolated, humanized monoclonal antibodies that inhibit the cleavage of complement component G4, wherein the antibodies do not inhibit the 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 C1 s. In some cases, the antibody does not inhibit the protease activity of C1 s.

Background

The complement system is a well-known effector mechanism of the immune response that provides not only protection against pathogens and other harmful agents, but also provides recovery from injury. The complement pathway includes many proteins that are normally found in the body in an inactive form. The classical complement pathway is triggered by activation of the first component of complement, known as the C1 complex, which consists of C1q, C1r and C1s proteins. Upon binding of C1 to immune complexes or other activators, the C1s component, Diisopropyl Fluorophosphate (DFP) -sensitive serine proteases, cleaves complement components C4 and C2 to initiate activation of the classical complement pathway. The classical complement pathway appears to play a role in a number of diseases and disorders.

There is a need in the art for compounds that treat complement-mediated diseases or disorders. There is also a need for compounds that can detect or monitor such diseases or conditions. There is also a need for methods of making and using such compounds and compositions thereof.

Summary of The Invention

The present disclosure provides antibodies that bind complement C1s protein; and nucleic acid molecules encoding such antibodies. The disclosure also provides compositions comprising such antibodies, and methods of making and using such antibodies, nucleic acid molecules, and compositions.

The present disclosure provides isolated humanized monoclonal antibodies that inhibit cleavage of complement component C4, wherein the antibodies do 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 C1 s. In some cases, the antibody does not inhibit the protease activity of C1 s.

The present disclosure provides isolated humanized monoclonal antibodies that specifically bind to an epitope within a region encompassing domains IV and V of complement component 1s (C1 s). 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 C1 s. In some cases, the epitope bound by the isolated humanized monoclonal antibody of the present disclosure is a conformational epitope.

The present disclosure provides isolated humanized monoclonal antibodies that bind with high affinity to complement component C1s in the C1 complex.

The present disclosure provides isolated humanized monoclonal antibodies specific for complement component C1s and at less than 10 x 10^-9IC50 for M inhibits complement-mediated cell lysis and/or inhibits complement-mediated cell lysis at less than 50X 10^-9IC50 for M inhibited C4 activation.

In any of the embodiments of the present disclosure, the antibody may comprise one or more Complementarity Determining Regions (CDRs) of an antibody light chain variable region comprising the amino acid sequence of SEQ ID No.7 or one or more CDRs of an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID No. 8.

In any of the embodiments of the present 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, 33, 3, 34, 35 and 36.

In any of the embodiments of the present 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 of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO 37 or a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 38.

In any of the embodiments of the present 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 of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO 37 and a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 38.

In any of the embodiments of the present disclosure, the antibody may comprise heavy and light chain Complementarity Determining Regions (CDRs) having an amino acid sequence 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) a CDR having an amino acid sequence selected from the group consisting of SEQ ID NO 32, 33, 3, 34, 35 and 36.

The present disclosure provides humanized antibodies that specifically bind complement component 1s (C1s), wherein the antibodies compete for binding to the epitope with an antibody comprising one or more CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID NO:7 or one or more CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO: 8.

The present disclosure provides humanized antibodies that specifically bind complement component 1s (C1s), wherein the antibodies are selected from the group consisting of: a) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope 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; and b) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope 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.

The present disclosure provides a humanized antibody that binds complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising: a) a light chain CDR comprising the variable region of the antibody light chain of amino acid sequence SEQ ID NO 7 or SEQ ID NO 37; or b) a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 8 or SEQ ID NO 38.

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

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

The present disclosure provides antibodies that bind complement C1s protein, wherein the antibodies comprise Complementarity Determining Regions (CDRs) 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 comprising the 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 comprising the 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.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID No. 7. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID No. 8. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 7. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No.7 and a heavy chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No. 8. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID No.7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 8.

The present disclosure provides an antibody that binds complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising the light chain CDRs of the antibody light chain variable region comprising amino acid sequence SEQ ID No.7 and the heavy chain CDRs of the antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 8. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain CDR of an antibody light chain variable region comprising amino acid sequence SEQ ID No.7 and a heavy chain CDR of an antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 8.

In any of the above embodiments, the anti-C1 s antibodies of the present disclosure bind to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure binds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure inhibits 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 any of the above embodiments, an anti-C1 s antibody of the present disclosure may comprise a humanized light chain framework region. In any of the above embodiments, an anti-C1 s antibody of the present disclosure can comprise a humanized heavy chain framework region.

In any of the above embodiments, an anti-C1 s antibody of the present disclosure can be an Ig monomer that binds to complement C1s protein or an antigen-binding fragment thereof. In any of the above embodiments, an anti-C1 s antibody of the present disclosure can be an antigen-binding fragment that binds complement C1s protein. In any of the above embodiments, the anti-C1 s antibody of the present disclosure is selected from the group consisting of an Ig monomer, an Fab fragment, F (ab')₂Fragments, 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-C1 s antibody of the present disclosure is selected from the group consisting of a monospecific antibody, a bispecific antibody, and a multispecific antibody.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain region and a heavy chain region present in separate polypeptides. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain region and a heavy chain region present in a single polypeptide. In some embodiments, an anti-C1 s antibody of the present disclosure comprises an Fc region.

The present disclosure provides antibodies that compete for binding to an epitope bound by antibody IPN003 (also referred to herein as "IPN-M34" or "M34" or "TNT 003"). The present disclosure provides antibodies comprising the variable domain of antibody IPN 003. The present disclosure provides the antibody IPN 003.

The present disclosure provides anti-C1 s antibodies made by methods including recombinant production.

The present disclosure provides antibodies that bind complement C1s protein, wherein the antibodies are encapsulated in liposomes.

The present disclosure provides antibodies that bind complement C1s protein, wherein the antibodies comprise a covalently linked non-peptide synthetic polymer. In some embodiments, the synthetic polymer is a poly (ethylene glycol) polymer.

The present disclosure provides antibodies that bind complement C1s protein, wherein the antibodies are formulated with an agent that facilitates crossing the blood brain barrier.

The present disclosure provides antibodies that bind complement C1s protein, wherein the antibodies are fused, directly or through a linker, to a compound that facilitates crossing the blood brain barrier, wherein the compound is selected from the group consisting of a carrier molecule, a peptide, or a protein.

The present disclosure provides nucleic acid molecules encoding anti-C1 s antibodies of any of the embodiments disclosed herein. In some embodiments, the present disclosure provides recombinant vectors comprising such nucleic acid molecules. In some embodiments, the present disclosure provides recombinant molecules comprising such nucleic acid molecules. In some embodiments, the present disclosure provides recombinant cells comprising such recombinant molecules.

The present disclosure provides a pharmaceutical composition comprising an anti-C1 s antibody of any embodiment disclosed herein and a pharmaceutically acceptable excipient. Some embodiments include sterile containers containing such pharmaceutical compositions. In some embodiments, the container is selected from the group consisting of a bottle and a syringe.

The present disclosure provides a method of treating an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an anti-C1 s 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 administering is intravenous administering. In some embodiments, the administering is intrathecal. In some embodiments, the administering results in a result selected from the group consisting of: (a) a decrease in complement activation; (b) improvement in cognitive function; (c) a reduction in neuronal loss; (d) a decrease in the level of phosphorylated Tau in neurons; (e) a reduction in glial cell activation; (f) a reduction in lymphocyte infiltration; (g) reduction in macrophage infiltration; (h) reduction in antibody deposition; (i) a reduction in glial cell loss; (j) a reduction in oligodendrocyte loss; (k) reduction of dendritic cell infiltration; (l) Reduction of neutrophil infiltration; (m) reduction in red blood cell lysis; (n) a reduction in phagocytosis of red blood cells; (o) a reduction in platelet phagocytosis; (p) reduction in platelet lysis; (q) an increase in graft survival; (r) a decrease in macrophage-mediated phagocytosis; (s) improvement of vision; (t) improvement in motion control; (u) improvement of thrombosis; (v) improvement of blood coagulation; (w) improvement in renal function; (x) A decrease in antibody-mediated complement activation; (y) a reduction in autoantibody-mediated complement activation; (z) amelioration of anemia; (aa) reduction of demyelination; (ab) reduction in eosinophilia; (ac) a reduction in deposition of C3 on red blood cells (e.g., a reduction in deposition of C3b, iC3b, etc. on RBCs); (ad) a reduction in the deposition of C3 on platelets (e.g., a reduction in the deposition of C3b, iC3b, etc. on platelets); (ae) reduction in production of anaphylatoxins (e.g., C3a, C4a, C5 a); (af) reduction of autoantibody-mediated blister formation; (ag) reduction of autoantibody induced pruritus; (ah) a reduction in autoantibody induced lupus erythematosus; (ai) reduction of autoantibody mediated skin erosion; (aj) a reduction in red blood cell destruction due to transfusion reactions; (ak) reduction of red blood cell lysis due to alloantibodies; (al) reduction of hemolysis due to infusion reactions; (am) reduction of allogeneic antibody-mediated platelet lysis; (an) reduction in platelet lysis due to infusion reaction; (ao) reduction in mast cell activation; (ap) reduction of histamine release from mast cells; (aq) a decrease in vascular permeability; (ar) reduction of edema; (as) reduction of complement deposition on graft endothelium; (at) a reduction in anaphylatoxin production in the graft endothelium; (au) reduction of separation of the dermal-epidermal junction; (av) a reduction in anaphylatoxin production in the dermoepidermal junction; (aw) a reduction in alloantibody-mediated complement activation in the graft endothelium; (ax) reduction of antibody-mediated loss at the neuromuscular junction; (ay) a reduction in complement activation at the neuromuscular junction; (az) a reduction in anaphylatoxin production at the neuromuscular junction; (ba) a reduction in complement deposition at the neuromuscular junction; (bb) reduction of paralysis; (bc) reduced numbness; (bd) increased bladder control; (be) increased bowel movement control; (bf) a reduction in mortality associated with autoantibodies; and (bg) a decrease in the incidence associated with autoantibodies. In some embodiments, the reduction in glial cell activation comprises a reduction in astrocyte activation or a reduction in microglial activation.

The present disclosure provides a method of inhibiting complement activation in an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an anti-C1 s 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 administering is intravenous administering. In some embodiments, the administering is intrathecal. In some embodiments, the administering is subcutaneous administering. In some embodiments, the administering results in a result selected from the group consisting of: (a) a decrease in complement activation; (b) improvement in cognitive function; (c) a reduction in neuronal loss; (d) a decrease in the level of phosphorylated Tau in neurons; (e) a reduction in glial cell activation; (f) a reduction in lymphocyte infiltration; (g) reduction in macrophage infiltration; (h) reduction in antibody deposition; (i) a reduction in glial cell loss; (j) a reduction in oligodendrocyte loss; (k) reduction of dendritic cell infiltration; (l) Reduction of neutrophil infiltration; (m) reduction in red blood cell lysis; (n) a reduction in phagocytosis of red blood cells; (o) a reduction in platelet phagocytosis; (p) reduction in platelet lysis; (q) an increase in graft survival; (r) a decrease in macrophage-mediated phagocytosis; (s) improvement of vision; (t) improvement in motion control; (u) improvement of thrombosis; (v) improvement of blood coagulation; (w) improvement in renal function; (x) A decrease in antibody-mediated complement activation; (y) a reduction in autoantibody-mediated complement activation; (z) amelioration of anemia; (aa) reduction of demyelination; (ab) reduction in eosinophilia; (ac) a reduction in deposition of C3 on red blood cells (e.g., a reduction in deposition of C3b, iC3b, etc. on RBCs); (ad) a reduction in the deposition of C3 on platelets (e.g., a reduction in the deposition of C3b, iC3b, etc. on platelets); (ae) reduction in anaphylatoxin production; (af) reduction of autoantibody-mediated blister formation; (ag) reduction of autoantibody induced pruritus; (ah) a reduction in autoantibody induced lupus erythematosus; (ai) reduction of autoantibody mediated skin erosion; (aj) a reduction in red blood cell destruction due to transfusion reactions; (ak) reduction of red blood cell lysis due to alloantibodies; (al) reduction of hemolysis due to infusion reactions; (am) reduction of allogeneic antibody-mediated platelet lysis; (an) reduction in platelet lysis due to infusion reaction; (ao) reduction in mast cell activation; (ap) reduction of histamine release from mast cells; (aq) a decrease in vascular permeability; (ar) reduction of edema; (as) reduction of complement deposition on graft endothelium; (at) a reduction in anaphylatoxin production in the graft endothelium; (au) reduction of separation of the dermal-epidermal junction; (av) a reduction in anaphylatoxin production in the dermoepidermal junction; (aw) a reduction in alloantibody-mediated complement activation in the graft endothelium; (ax) reduction of antibody-mediated loss at the neuromuscular junction; (ay) a reduction in complement activation at the neuromuscular junction; (az) a reduction in anaphylatoxin production at the neuromuscular junction; (ba) a reduction in complement deposition at the neuromuscular junction; (bb) reduction of paralysis; (bc) reduced numbness; (bd) increased bladder control; (be) increased bowel movement control; (bf) a reduction in mortality associated with autoantibodies; and (bg) a decrease in the incidence associated with autoantibodies. In some embodiments, the reduction in glial cell activation comprises a reduction in astrocyte activation or a reduction in microglial activation.

The present disclosure provides for the use of an anti-C1 s antibody of any embodiment or a pharmaceutical composition thereof in treating an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of any embodiment in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of any embodiment, or a pharmaceutical composition thereof, in inhibiting complement C1s activity, wherein "inhibiting complement C1s activity" comprises inhibiting complement activation, e.g., inhibiting the production of C4b2a (i.e., complement C4b and C2a complex; also referred to as "C3 convertase"). In some embodiments, the present disclosure provides the use of an anti-C1 s antibody of any embodiment or a pharmaceutical composition thereof in inhibiting complement activation in an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of any embodiment, or a pharmaceutical composition thereof, in the manufacture of a medicament for inhibiting complement activation. In some embodiments, the present disclosure provides a use of the anti-C1 s antibody of any embodiment or a pharmaceutical composition thereof in the manufacture of a medicament for inhibiting complement activation in an individual having a complement-mediated disease or disorder.

The present disclosure provides an anti-C1 s antibody or a pharmaceutical composition thereof for use in any embodiment in medical treatment.

The present disclosure provides an anti-C1 s antibody or pharmaceutical composition thereof of any embodiment for use in treating an individual having a complement-mediated disease or disorder.

The present disclosure provides an anti-C1 s antibody or pharmaceutical composition thereof of any embodiment for use in inhibiting complement activation. The present disclosure provides an anti-C1 s antibody or pharmaceutical composition thereof of any embodiment for use in inhibiting complement activation in an individual having a complement-mediated disease or disorder.

The present disclosure provides a method of diagnosing a complement-mediated disease or disorder in an individual, the method comprising: (a) determining the amount of complement C1s protein in a biological sample obtained from the individual, wherein the determining step comprises: (i) contacting the biological sample with an anti-C1 s antibody of any embodiment; and (ii) quantifying the binding of the antibody to complement C1s protein present in the biological sample; and (b) comparing the amount of complement C1s protein in the biological sample to a normal control value indicative of 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 disorder. 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, a solid tissue sample, a tissue culture sample, and a cell sample.

The present disclosure provides a method of monitoring the progression of a complement-mediated disease or disorder in an individual, the method comprising: (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 complement C1s protein to the first amount of complement C1s protein. The determining step comprises: (i) contacting the biological sample with an anti-C1 s antibody of any embodiment; and (ii) quantifying the binding of the antibody to complement C1s protein present in the biological sample. In some embodiments, the first time point is a time point before 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, a solid tissue sample, a tissue culture sample, and a cell sample.

The present disclosure provides an in vitro method of detecting complement C1s protein in a biological sample obtained from an individual, the method comprising: (a) contacting the biological sample with an anti-C1 s antibody of any embodiment; and (b) detecting binding of the antibody to 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, a solid tissue sample, a tissue culture sample, and a cell sample. In some embodiments, the method is quantitative.

The present disclosure provides a method of detecting complement C1s protein in vivo in a living individual, the method comprising: (a) administering to the individual an anti-C1 s antibody of any embodiment; and (b) detecting binding of the antibody to complement C1s protein in the individual using an imaging method. In some embodiments, the binding is detected at a site in the individual altered by a complement-mediated disease or disorder. 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 an imaging method. In some embodiments, the imaging method is selected from the group consisting of magnetic resonance imaging, positron emission tomography, and IVIS instrument detection. In some embodiments, the method is quantitative.

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, a solid tissue sample, a tissue culture sample, and a cell sample.

In some embodiments, the methods of the present disclosure provide: the individual is suspected of having a complement-mediated disease or disorder, has been diagnosed as having a complement-mediated disease or disorder, or has a genetic predisposition to develop a complement-mediated disease or disorder.

The present disclosure provides a composition comprising: (a) an anti-C1 s antibody of any embodiment; and (b) a solution comprising one or more agents that preserve an organ or tissue intended to be transplanted 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 perfusate or a tissue perfusate. In some embodiments, the solution comprises: i) salt; ii) an agent that reduces edema; iii) an oxygen radical scavenger; and iii) an energy supply system component. In some embodiments, the composition comprises potassium lactobionate, KH₂PO₄、MgSO₄Raffinose, adenosineGlutathione, allopurinol and hydroxyethyl starch.

The present disclosure provides an organ or tissue preservation fluid comprising the anti-C1 s antibody or pharmaceutical composition thereof of any embodiment.

The present disclosure provides an organ or tissue perfusate comprising the anti-C1 s antibody or pharmaceutical composition thereof of any embodiment.

The present disclosure provides a method for preserving an organ or tissue for transplantation, the method comprising contacting the organ or the tissue with a composition comprising: (a) an anti-C1 s 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.

The present disclosure provides an isolated organ or tissue preserved in a composition comprising: (a) an anti-C1 s 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 organ is selected from the group consisting of an eye, a heart, an intestine, a kidney, a liver, a lung, a pancreas, a stomach, and a thymus. In some embodiments, the tissue is selected from the group consisting of bone, bone marrow, cornea, heart valves, Langerhans islets (islets of Langerhans), tendons, skin, and veins.

The present disclosure provides an in vitro method for inhibiting complement activation in an organ or a tissue, the method comprising contacting the organ or the tissue with the anti-C1 s antibody of any embodiment, a solution comprising the anti-C1 s antibody of any embodiment, or a pharmaceutical composition comprising the anti-C1 s antibody of any embodiment.

Certain aspects of the invention are defined in the following numbered schemes (a-u).

[a] An antibody that binds complement C1s protein, wherein the antibody comprises 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.

[b] The antibody of claim a, wherein the antibody comprises a light chain variable region comprising the amino acid sequences SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3. The antibody of claim a, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequences SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6.

[c] The antibody of claim a, wherein the antibody 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.

[d] The antibody of claim a, wherein the antibody comprises a light chain variable region comprising an amino acid sequence 90% identical to the amino acid sequence of SEQ ID NO. 7. The antibody of claim a, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence 90% identical to the amino acid sequence of SEQ ID NO 8. The antibody of claim a, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO 7. The antibody of claim a, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 8.

[e] The antibody of claim a, wherein the antibody comprises a light chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID NO.7 and a heavy chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID NO. 8. The antibody of claim a, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO.7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 8.

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

[g] Scheme f wherein the antibody comprises the light chain CDRs of the antibody light chain variable region comprising amino acid sequence SEQ ID NO.7 and the heavy chain CDRs of the antibody heavy chain variable region comprising amino acid sequence SEQ ID NO. 8.

[h] The antibody of any one of claims a-g, wherein the antibody binds human complement C1s protein. The antibody of any one of claims a-g, wherein the antibody binds rat complement C1s protein or monkey complement C1s protein.

[i] The antibody of any of claims a-h, wherein the antibody inhibits cleavage of at least one substrate cleaved by complement C1s protein.

[j] The antibody of claim i, wherein the substrate is selected from the group consisting of complement C2 and complement C4.

[k] The antibody of any one of claims a-j, wherein the antibody comprises a humanized light chain framework region. The antibody of any one of claims a-j, wherein the antibody comprises a humanized heavy chain framework region.

[ l ] technical scheme any one of the antibodies of claims a-k, wherein the antibody is selected from the group consisting of Ig monomers that bind complement C1s protein and antigen-binding fragments thereof.

[ m ] the antibody of any one of claims a-k, wherein the antibody is an antigen-binding fragment that binds complement C1s protein.

[n]Antibodies according to any of claims a-k, wherein said antibodies are selected from the group consisting of Ig monomers, Fab fragments, F (ab')₂Fragments, Fd fragments, scFv, scAb, dAb, Fv, single domain heavy chain antibodies and single domain light chain antibodies.

[ o ] the antibody of any one of technical schemes a-k, wherein the antibody is selected from the group consisting of a monospecific antibody, a bispecific antibody and a multispecific antibody. The antibody of any one of claims a-k, wherein the antibody comprises a light chain region and a heavy chain region present in separate polypeptides. The antibody of any one of claims a-k, wherein the antibody comprises a light chain region and a heavy chain region present in a single polypeptide.

[ p ] the antibody of any one of claims a-k and o, wherein the antibody comprises an Fc region.

[ q ] the antibody of any one of claims a-p, wherein the antibody is encapsulated in a liposome.

[ r ] the antibody of any one of claims a-p, wherein the antibody comprises a covalently linked non-peptidic synthetic polymer.

[ s ] the antibody of claim r, wherein in the synthetic polymer is a poly (ethylene glycol) polymer.

[ t ] the antibody of any one of claims a-p, wherein the antibody is formulated with an agent that facilitates crossing the blood brain barrier.

[ u ] the antibody of any one of claims a-p, 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 a carrier molecule, a peptide or a protein.

The present application relates to the following embodiments.

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

2. The humanized monoclonal antibody of embodiment 1, wherein the antibody inhibits a component of the classical complement pathway.

3. The humanized monoclonal antibody of embodiment 2, wherein the classical complement pathway component is C1 s.

4. The humanized monoclonal antibody of embodiment 3, wherein the antibody does not inhibit the protease activity of C1 s.

5. An isolated humanized monoclonal antibody that specifically binds to an epitope within a region encompassing domains IV and V of complement component 1s (C1 s).

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

7. The isolated humanized monoclonal antibody of embodiment 6, wherein the antibody does not inhibit the protease activity of C1 s.

8. The isolated humanized monoclonal antibody of embodiment 5, wherein the epitope is a conformational epitope.

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

10. An isolated humanized monoclonal antibody that is specific for complement component C1s and that is less than 10 x 10^-9IC50 for M inhibits complement-mediated cell lysis and/or inhibits complement-mediated cell lysis at less than 50X 10^-9IC50 for M inhibited C4 activation.

11. The humanized monoclonal antibody of any one of embodiments 1-10, wherein the antibody comprises one or more Complementarity Determining Regions (CDRs) of an antibody light chain variable region comprising amino acid sequence SEQ ID NO:7 or one or more CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO: 8.

12. The humanized monoclonal antibody of any one of embodiments 1-10, wherein the antibody comprises:

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.

13. The humanized monoclonal antibody of any one of embodiments 1-10, wherein the antibody comprises:

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) The light chain CDR of the antibody light chain variable region comprising amino acid sequence SEQ ID NO 37 or the heavy chain CDR of the antibody heavy chain variable region comprising amino acid sequence SEQ ID NO 38.

14. The humanized monoclonal antibody of any one of embodiments 1-10, wherein the antibody comprises:

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) The light chain CDR of the antibody light chain variable region comprising amino acid sequence SEQ ID NO 37 and the heavy chain CDR of the antibody heavy chain variable region comprising amino acid sequence SEQ ID NO 38.

15. The humanized monoclonal antibody of any one of embodiments 1-10, wherein the antibody comprises heavy and light chain Complementarity Determining Regions (CDRs) having amino acid sequences selected from the group consisting of:

a) 1, 2, 3,4, 5 and 6; and

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

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

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

a) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope 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; and

b) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising CDRs 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.

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

a) a light chain CDR comprising the variable region of the antibody light chain of amino acid sequence SEQ ID NO 7 or SEQ ID NO 37; or

b) Heavy chain CDRs of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 8 or SEQ ID NO 38.

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

a) a light chain CDR comprising the variable region of the antibody light chain of amino acid sequence SEQ ID NO 7 or SEQ ID NO 37; and

20. The humanized antibody of embodiment 19, wherein the antibody competes for binding to the epitope with an antibody comprising heavy and light chain CDRs comprising:

a) 1, 2, 3, 142, 5 and 6; or

b) 32, 33, 3, 34, 35 and 36.

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

22. The antibody of any one of embodiments 1-20, wherein the antibody binds rat complement C1s protein.

23. The antibody of any one of embodiments 1-20, wherein the antibody binds monkey complement C1s protein.

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

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

26. The antibody of 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 set forth in table 8.

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

28. The antibody of embodiment 27, wherein the humanized heavy chain framework region comprises 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, or 12 amino acid substitutions shown in table 7.

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

30. The antibody of any one of embodiments 1-20, wherein the antibody is selected from the group consisting of an Ig monomer, a Fab fragment, F (ab')₂Fragment, Fd fragmentA fragment, scFv, scAb, dAb, Fv, single domain heavy chain antibody and single domain light chain antibody.

31. The antibody of any one of embodiments 1-20, wherein the antibody is selected from the group consisting of a monospecific antibody, a bispecific antibody, and a multispecific antibody.

32. The antibody of any one of embodiments 1-20, wherein the antibody comprises a light chain region and a heavy chain region present in separate polypeptides.

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

34. The antibody of any one of embodiments 1-20, wherein the antibody comprises an Fc region.

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

a) 1, 2, 3,4, 5 and 6; and

b) 32, 33, 3, 34, 35 and 36.

36. An antibody that binds complement C1s protein, wherein the antibody comprises Complementarity Determining Regions (CDRs) having an amino acid sequence selected from the group consisting of:

a) 1, 2, 3,4, 5 and 6; or

b) 32, 33, 3, 34, 35 and 36.

37. The antibody of embodiment 36, wherein the antibody comprises a light chain variable region comprising the amino acid sequences SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 or comprising the amino acid sequences SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 3.

38. The antibody of embodiment 36, wherein the antibody comprises a heavy chain variable region comprising amino acid sequences SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6 or comprising amino acid sequences SEQ ID NO 34, SEQ ID NO 35 and SEQ ID NO 36.

39. The antibody of embodiment 36, wherein the antibody comprises:

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

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.

40. The antibody of embodiment 36, wherein the antibody comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID No.7 or SEQ ID No. 37.

41. The antibody of embodiment 36, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID No. 8 or SEQ ID No. 38.

42. The antibody of embodiment 36, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID No.7 or SEQ ID No. 37.

43. The antibody of embodiment 36, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 8 or SEQ ID NO 38.

44. The antibody of embodiment 36, wherein the antibody comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to amino acid sequence SEQ ID No.7 or SEQ ID No. 37 and a heavy chain variable region comprising an amino acid sequence having at least 90% identity to amino acid sequence SEQ ID No. 8 or SEQ ID No. 38.

45. The antibody of embodiment 36, wherein the antibody comprises:

i) a light chain variable region comprising the amino acid sequence of SEQ ID NO.7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 8; or

ii) a light chain variable region comprising the amino acid sequence SEQ ID NO 37 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO 38.

46. The antibody of embodiment 36, wherein the antibody is a monoclonal antibody.

47. The antibody of embodiment 36, wherein the antibody is a humanized antibody.

48. The antibody of embodiment 36, wherein the antibody is an antibody fragment.

49. The antibody of any one of embodiments 1-48, wherein said antibody is encapsulated in a liposome.

50. The antibody of any one of embodiments 1-48, wherein the antibody comprises a covalently linked non-peptidic synthetic polymer.

51. The antibody of embodiment 50, wherein in the synthetic polymer is a poly (ethylene glycol) polymer.

52. The antibody of any one of embodiments 1-48, wherein said antibody is formulated with an agent that facilitates crossing the blood brain barrier.

53. The antibody of any one of embodiments 1-48, wherein said antibody is fused, directly or through a linker, to a compound that facilitates crossing the blood brain barrier, wherein said compound is selected from the group consisting of a carrier molecule, a peptide, or a protein.

54. A nucleic acid comprising a nucleotide sequence encoding the antibody of any one of embodiments 1-48.

55. A recombinant vector comprising the nucleic acid according to embodiment 54.

56. A recombinant cell comprising the nucleic acid of embodiment 54 or the recombinant vector of embodiment 55.

57. A pharmaceutical composition comprising the antibody of any one of embodiments 1-48 and a pharmaceutically acceptable excipient.

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

59. The container of embodiment 58, wherein the container is selected from the group consisting of a bottle and a syringe.

60. A method of inhibiting 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.

61. A method of 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.

62. A method of treating an individual having a complement-mediated disease or disorder, 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.

63. A method of inhibiting complement activation in an individual having a complement-mediated disease or disorder, 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.

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

65. The method of any one of embodiments 60-63, wherein the individual is a human.

66. The method according to any one of embodiments 60-65, wherein said administering is intravenous administering.

67. The method according to any one of embodiments 60-65, wherein said administering is subcutaneous administering.

68. The method according to any one of embodiments 60-65, wherein said administering is intrathecal.

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

(a) a decrease in complement activation;

(b) improvement in cognitive function;

(c) a reduction in neuronal loss;

(d) a decrease in the level of phosphorylated Tau in neurons;

(e) a reduction in glial cell activation;

(f) a reduction in lymphocyte infiltration;

(g) reduction in macrophage infiltration;

(h) reduction in antibody deposition;

(i) a reduction in glial cell loss;

(j) a reduction in oligodendrocyte loss;

(k) reduction of dendritic cell infiltration;

(l) Reduction of neutrophil infiltration;

(m) reduction in red blood cell lysis;

(n) a reduction in phagocytosis of red blood cells;

(o) a reduction in platelet phagocytosis;

(p) reduction in platelet lysis;

(q) an increase in graft survival;

(r) a decrease in macrophage-mediated phagocytosis;

(s) improvement of vision;

(t) improvement in motion control;

(u) improvement of thrombosis;

(v) improvement of blood coagulation;

(w) improvement in renal function;

(x) A decrease in antibody-mediated complement activation;

(y) a reduction in autoantibody-mediated complement activation;

(z) amelioration of anemia;

(aa) reduction of demyelination;

(ab) reduction in eosinophilia;

(ac) reduction in deposition of C3 on red blood cells;

(ad) reduction in deposition of C3 on platelets;

(ae) reduction in anaphylatoxin production;

(af) reduction of autoantibody-mediated blister formation;

(ag) reduction of autoantibody induced pruritus;

(ah) a reduction in autoantibody induced lupus erythematosus;

(ai) reduction of autoantibody mediated skin erosion;

(aj) a reduction in red blood cell destruction due to transfusion reactions;

(ak) reduction of red blood cell lysis due to alloantibodies;

(al) reduction of hemolysis due to infusion reactions;

(am) reduction of allogeneic antibody-mediated platelet lysis;

(an) reduction in platelet lysis due to infusion reaction;

(ao) reduction in mast cell activation;

(ap) reduction of histamine release from mast cells;

(aq) a decrease in vascular permeability;

(ar) reduction of edema;

(as) reduction of complement deposition on graft endothelium;

(at) a reduction in anaphylatoxin production in the graft endothelium;

(au) reduction of separation of the dermal-epidermal junction;

(av) a reduction in anaphylatoxin production in the dermoepidermal junction;

(aw) a reduction in alloantibody-mediated complement activation in the graft endothelium;

(ax) reduction of antibody-mediated loss at the neuromuscular junction;

(ay) a reduction in complement activation at the neuromuscular junction;

(az) a reduction in anaphylatoxin production at the neuromuscular junction;

(ba) a reduction in complement deposition at the neuromuscular junction;

(bb) reduction of paralysis;

(bc) reduced numbness;

(bd) increased bladder control;

(be) increased bowel movement control;

(bf) a reduction in mortality associated with autoantibodies; and

(bg) reduction in the incidence associated with autoantibodies.

70. The method of embodiment 69, wherein said glial cells are selected from the group consisting of astrocytes and microglia.

71. The method of any one of embodiments 60-68, wherein said antibody is administered in an amount that provides a peak serum level of about 1 μ g/ml to about 1 mg/ml.

72. Use of the antibody of any one of embodiments 1-48 or the pharmaceutical composition of embodiment 57 for treating an individual having a complement-mediated disease or disorder.

73. Use of an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57 in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

74. Use of an antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57 for inhibiting complement activation in an individual having a complement-mediated disease or disorder.

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

76. An antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 56 for use in medical treatment.

77. An antibody as claimed in any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 56 for use in treating an individual having a complement-mediated disease or disorder.

78. An antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 56 for use in inhibiting complement activation in an individual having a complement-mediated disease or disorder.

79. A method of diagnosing a complement-mediated disease or disorder in an individual, the method comprising:

(a) determining the amount of complement C1s protein in a biological sample obtained from the individual, wherein the determining step comprises:

(i) contacting the biological sample with an antibody according to any one of embodiments 1-48; and

(ii) quantifying the binding of the antibody to complement C1s protein present in the biological sample; and

(b) comparing the amount of complement C1s protein present in the biological sample to a normal control value indicative of 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 disorder.

80. A method of monitoring the progression of a complement-mediated disease or disorder in an individual, the method comprising:

(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 complement C1s protein to the first amount of complement C1s protein,

wherein the determining step comprises:

(ii) quantifying the binding of the antibody to complement C1s protein present in the biological sample.

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

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

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

(a) contacting the biological sample with an antibody according to any one of embodiments 1-48; and

(b) detecting binding of the antibody to complement C1s protein present in the sample.

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, ocular fluid, synovial fluid, solid tissue samples, tissue culture samples, and cell samples.

85. The method of any one of embodiments 79-83, wherein the method comprises:

a) treating the biological sample with a calcium chelator, thereby forming a C1s monomer;

b) contacting the chelator-treated biological sample with an immobilized first antibody that binds C1s but does not compete with the anti-C1 s antibody according to any one of embodiments 1-48 for binding to C1s, thereby forming an immobilized first antibody/C1 s monomer complex;

c) contacting the immobilized first antibody/C1 s monomer complex with an antibody according to any one of embodiments 1-48; and

d) detecting binding of the monoclonal anti-C1 s antibody to the immobilized C1s monomer.

86. The method of any one of embodiments 79-83, wherein the method comprises:

a) contacting the biological sample with an immobilized first antibody that binds C1s but does not compete with the anti-C1 s antibody of any one of embodiments 1-48 for binding to C1s, thereby forming an immobilized first antibody/C1 s complex;

b) contacting the immobilized first antibody/C1 s complex with a calcium chelator, thereby forming an immobilized C1s monomer;

c) contacting the immobilized C1s monomer with an anti-C1 s antibody according to any one of embodiments 1-48; and

d) detecting binding of the antibody of any one of embodiments 1-48 to the immobilized C1s monomer.

87. A method of detecting complement C1s protein in vivo, the method comprising:

(a) administering to an individual an antibody according to any one of embodiments 1-48; and

(b) detecting binding of the antibody to complement C1s protein in the individual using an imaging method.

88. The method of embodiment 87, wherein the binding is detected at a site in the individual that is altered by a complement-mediated disease or disorder.

89. The method of embodiment 87, wherein said binding is detected in the brain of said subject.

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

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.

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

93. The method of any one of embodiments 79-92, wherein the individual is suspected of having a complement-mediated disease or disorder, has been diagnosed as having a complement-mediated disease or disorder, or has a genetic predisposition to develop a complement-mediated disease or disorder.

94. A composition, comprising:

(a) the anti-C1 s antibody according to any one of embodiments 1-48; and

(b) a solution comprising one or more agents that preserve an organ or tissue intended for transplantation into a recipient individual.

95. The composition of embodiment 94, wherein said solution is an organ preservation solution or a tissue preservation solution.

96. The composition of embodiment 94, wherein said solution is an organ perfusate or a tissue perfusate.

97. The composition of embodiment 94, wherein said solution comprises: (i) salt; (ii) an agent that reduces edema; (iii) an oxygen radical scavenger; and (iv) an energy supply system component.

98. The composition of embodiment 97, wherein the composition comprises potassium lactobionate KH₂PO₄、MgSO₄Raffinose, adenosine, glutathione, allopurinol and hydroxyethyl starch.

99. An organ or tissue preservation fluid comprising an anti-C1 s antibody according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 57.

100. An organ or tissue perfusate comprising the anti-C1 s antibody according to any one of embodiments 1-48 or the pharmaceutical composition according to embodiment 57.

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

102. An isolated organ or tissue stored in the composition of any one of embodiments 94-98.

103. The organ of embodiment 102, wherein the organ is selected from the group consisting of an eye, a heart, an intestine, a kidney, a liver, a lung, a pancreas, a stomach, and a thymus.

104. The tissue of embodiment 102, wherein the tissue is selected from the group consisting of bone, bone marrow, cornea, heart valves, islets of langerhans, tendons, skin, blood, and veins.

105. The tissue of embodiment 104, wherein the blood tissue comprises whole blood, red blood cells, white blood cells, or cord blood.

106. The tissue of embodiment 104, wherein the blood tissue is an isolated population of blood cells.

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

108. The method of embodiment 107, wherein the solution is an organ preservation solution or a tissue preservation solution.

109. The method of embodiment 107, wherein the solution is an organ perfusate or a tissue perfusate.

110. The method of embodiment 107, wherein the solution comprises: (i) salt; (ii) an agent that reduces edema; (iii) an oxygen radical scavenger; and (iv) an energy supply system component.

111. The method of embodiment 107, wherein the solution comprises potassium lactobionate KH₂PO₄、MgSO₄Raffinose, adenosine, glutathione, allopurinol and hydroxyethyl starch.

Drawings

FIG. 1 depicts the amino acid sequence of homo sapiens complement C1s protein (SEQ ID NO: 9).

Fig. 2 provides table 2.

Fig. 3 depicts competition for IPN003(M34) for M81 binding to human C1 s.

Fig. 4 depicts the inhibition of C1 s-mediated activation of human complement protein C4 by IPN 003.

Figure 5 depicts the effect of IPN003 on activation of the complete classical complement cascade as determined using a standard hemolytic assay.

Fig. 6 and 7 depict the inhibition of complement in sera from two monkey species by IPN 003.

Fig. 8 depicts the specificity of IPN003 for C1 s.

Fig. 9 provides table 4.

Fig. 10 depicts the binding of IPN003 and M81 to rat C1 s.

Fig. 11 depicts the inhibition of rat C1 s-mediated cleavage of human C4 by IPN 003.

Fig. 12 depicts the inhibition of rat C1 s-mediated human C4 cleavage by IPN003 and the inhibition of human C1 s-mediated human C4 cleavage by IPN 003.

Fig. 13 depicts the effect of IPN003 on patient serum-mediated hemolysis of human red blood cells.

Fig. 14 depicts the effect of IPN003 on patient serum-mediated C3b deposition on human red blood cells.

Fig. 15 provides the amino acid sequences of the IPN003 VL and VH regions, the IPN003 VL CDR, and the IPN003VH CDR.

Figure 16 depicts the amino acid sequence of humanized IPN003VH variant 1; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 46).

Figure 17 depicts the amino acid sequence of humanized IPN003VH variant 2; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 47).

Figure 18 depicts the amino acid sequence of humanized IPN003VH variant 3; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 48).

Figure 19 depicts the amino acid sequence of humanized IPN003VH variant 4; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 49).

Figure 20 depicts the amino acid sequence of humanized IPN003 vk variant 1; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 50).

Figure 21 depicts the amino acid sequence of humanized IPN003 vk variant 2; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 51).

Figure 22 depicts the amino acid sequence of humanized IPN003 vk variant 3; and a nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 52).

Figure 23 provides table 7, which shows the amino acid differences between the parent IPN003VH and exemplary VH variants; and table 8, which shows the amino acid differences between the parent IPN003 VL and exemplary VL variants.

Figure 24 provides tables 9 and 10, which show the binding properties of humanized IPN003 variants to activated C1s and pro-C1 s.

FIG. 25 depicts the IC of humanized variants of IPN003 for competing with IPN003 for binding to C1s₅₀。

Figure 26 depicts the inhibition of the classical complement pathway by humanized variants of IPN 003.

Fig. 27A and 27B depict the effect of 3 humanized IPN003 variants on activation of the classical complement pathway (fig. 27A) and the alternative complement pathway (fig. 27B).

Figure 28 depicts the effect of humanized IPN003 variants on complement-mediated hemolysis and on C3b deposition on antibody-sensitized Red Blood Cells (RBCs).

Fig. 29 depicts inhibition of Cold Agglutinin Disease (CAD) patient plasma-mediated hemolysis by IPN003 or humanized IPN003 variants (hu-IPN 003).

FIG. 30 depicts the inhibition of anaphylatoxin production by IPN003 or hu-IPN 003.

Fig. 31 depicts the inhibition of CAD patient plasma-mediated C3b deposition on human RBCs by IPN 003.

Fig. 32 depicts the concentration-dependent inhibition of IPN003 on CAD patient plasma-mediated C3b deposition on human RBCs.

Fig. 33A and 33B depict proliferative responses to humanized IPN003 variants (fig. 33A) and to chimeric anti-C1 s antibody (fig. 33B).

Figure 34 depicts the effect of TNT003 on complement dependent hemolysis mediated by autoantibodies present in the plasma of Cold Agglutinin Disease (CAD) patients.

Figure 35 depicts the effect of TNT003 on complement-dependent C3b deposition mediated by autoantibodies present in the plasma of CAD patients.

Figure 36 depicts the effect of TNT003 on complement-dependent phagocytosis mediated by autoantibodies present in the plasma of CAD patients.

Fig. 37A-37C depict the effect of TNT003 on complement dependent C3a, C4a, and C5a mediated by autoantibodies present in the plasma of CAD patients.

Fig. 38A and 38B depict ex vivo activity of TNT003 on hemolysis and C3B deposition following administration to a non-human primate.

Figure 39 depicts the in vivo effect of TNT003 on C4a following administration to a non-human primate.

Figure 40 depicts the binding of TNT003 to the human C1s fragment.

Figure 41 depicts the effect of mutations at D343 and D357 on TNT003 inhibition of C1s activity.

Figure 42 depicts the inhibition of human C1s activity by TNT 003.

Fig. 43A and 43B depict the binding of TNT003 to C1s present in the C1 complex.

Fig. 44 depicts the inhibition of human C1s by TNT003 and TNT003 fragments.

Figure 45 depicts TNT003 binding to human C1s under non-reducing conditions.

Figure 46 depicts the inhibition of complement C4 activation by TNT003, but not complement C2 activation.

Fig. 47 depicts C1s levels in plasma samples from healthy volunteers and from CAD patients.

Definition of

The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype that retain 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 (scabs), single domain antibodies (dabs), single domain heavy chain antibodies, single domain light chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising an antibody and an antigen-binding (also referred to herein as antigen-binding) portion of a non-antibody protein. The antibody can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or the like. The antibodies can be further conjugated to other moieties, such as specific binding pairsSuch as biotin (a member of a biotin-avidin specific binding pair), and the like. The antibody may also be bound to a solid support including, but not limited to, polystyrene plates or beads, and the like. The term also encompasses Fab ', Fv, F (ab')₂And or other antibody fragments that retain specific binding to the antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which are produced by a single cell through repeated cell replication. That is, the cloning of cells produces only a single antibody species. Although monoclonal antibodies can be prepared using hybridoma preparation techniques, other methods of preparation known to those skilled in the art (e.g., antibodies derived from antibody phage display libraries) can also be used. The antibody may be monovalent or bivalent. An antibody may be an Ig monomer, which is a "Y-shaped" molecule consisting of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

The term "humanized immunoglobulin" as used herein refers to an immunoglobulin comprising immunoglobulin parts of different origin, wherein at least one part comprises an amino acid sequence of human origin. For example, a humanized antibody can comprise portions derived from an immunoglobulin of non-human origin (such as a mouse) and portions derived from an immunoglobulin sequence of human origin (e.g., a chimeric immunoglobulin) with the requisite specificity joined together chemically by conventional techniques (e.g., synthesis) or prepared as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portion of the chimeric antibody can be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising CDRs derived from an antibody of non-human origin and framework regions derived from a light chain and/or a heavy chain of human origin (e.g., a CDR-grafted antibody with or without framework changes). The term humanized immunoglobulin also encompasses chimeric or CDR-grafted single chain antibodies. See, e.g., Cabilly et al, U.S. Pat. Nos. 4,816,567; cabilly et al, European patent No.0,125,023B 1; boss et al, U.S. Pat. No.4,816,397; boss et al, European patent No.0,120,694B 1; neuberger, M.S. et al, WO 86/01533; neuberger, m.s. et al, european patent No.0,194,276B 1; winter, U.S. Pat. No.5,225,539; winter, european patent No.0,239,400B 1; padlan, E.A. et al, European patent application No.0,519,596A 1. For single chain antibodies, see also Ladner et al, U.S. Pat. nos. 4,946,778; huston, U.S. patent No.5,476,786; and Bird, R.E. et al, Science,242:423-426 (1988)).

For example, synthetic and/or recombinant nucleic acids can be used to prepare genes (e.g., cdnas) encoding the desired humanized chains to produce humanized immunoglobulins. For example, PCR mutagenesis methods can be used to alter a DNA sequence encoding a human or humanized strand to construct a nucleic acid (e.g., DNA) sequence encoding a humanized variable region, such as a DNA template from a previously humanized variable region (see, e.g., Kamman, M., et al, Nucl. acids Res.,17:5404 (1989)); sato, K., et al, cancer research,53:851-856 (1993); daugherty, B.L. et al, Nucleic Acids Res.,19(9):2471-2476 (1991); and Lewis, A.P. and J.S.Crowe, Gene,101:297-302 (1991)). Variants can also be readily prepared using these or other suitable methods. For example, cloned variable regions can be mutagenized and sequences encoding variants with the desired specificity can be selected (e.g., from phage libraries; see, e.g., Krebber et al, U.S. Pat. No.5,514,548; Hoogenboom et al, WO 93/06213, published on 1/4/1993).

An "antibody fragment" comprises a portion of an intact antibody, e.g., an antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (Zapata et al, ProteinEng.8(10):1057-1062 (1995)); domain antibodies (dAb; Holt et al, (2003) trends Biotechnol.21: 484); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. Pepsin treatment to give F (ab')₂Fragments having two antigen binding sites and still being capable of cross-linkingAn antigen.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. 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 an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity compared to the entire binding site.

The "Fab" fragment also contains the constant domain of the light chain and the first constant domain of the heavy Chain (CH)₁). Fab fragments differ from Fab' fragments in that they contain a heavy chain CH including one or more cysteines from the antibody hinge region₁Several residues are added at the carboxy terminus of the domain. Fab '-SH is the name given herein for Fab' in which the cysteine residue of the constant domain bears a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different classes (termed kappa and lambda) depending on the amino acid sequence of its constant domain. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The subclasses can be further divided into classes such as IgG2a and IgG2 b.

"Single chain Fv" or "sFv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein these domains are present in a singleIn a polypeptide chain. In some embodiments, the Fv polypeptide is further comprised at V_HAnd V_LPolypeptide linkers between the domains that enable the sFv to form the structure required for antigen binding. For an overview of sFv see Pluckthun, the Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V) linked in the same polypeptide chain to a light chain variable domain_L) Heavy chain variable domain of (V)_H)(V_H-V_L). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies are more fully described in e.g. EP 404,097; WO 93/11161; and Hollinger et al, (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two reagents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (K)_D) And (4) showing. The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1,000-fold or more of the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a protein of interest may be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomolar (fM) or less. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoresponsiveness" and "preferential binding" are used interchangeably herein with respect to antibodies and/or antigen binding fragments.

The term "binding" means between two molecules due to, for example, covalent, electrostatic forcesHydrophobic, and ionic and/or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). The subject anti-C1 s antibodies specifically bind to an epitope within the complement C1s protein. "specifically binds" means to at least about 10^-7M or greater, e.g. 5X 10^-7M、10^-8M、5×10^-8M and greater affinity binding. By "non-specific binding" is meant binding at less than about 10^-7Affinity binding of M, e.g. at 10^-6M、10^-5M、10^-4M, etc.

As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites present within the variable regions of heavy and light chain polypeptides. CDRs have been determined by Kabat et al, J.biol.chem.252:6609-6616 (1977); kabat et al, u.s.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), wherein the definition includes overlapping or subsets of amino acid residues when compared to each other. However, the use of any definition means that the CDRs of the antibody or grafted antibody or variants thereof are intended to be within the scope of the terms as defined and used herein. Amino acid residues encompassing the CDRs as defined by each of the above-cited references are set forth in table 1 below for comparison. The CDRs listed in table 2 are defined according to Kabat 1991.

Table 1: CDR definition

	Kabat¹	Chothia²	MacCallum³
				V_HCDR-I	31-35	26-32	30-35
V_HCDR-2	50-65	53-55	47-58
				V_HCDR-3	95-102	96-101	93-101
V_LCDR-I	24-34	26-32	30-36
				V_LCDR-2	50-56	50-52	46-55
V_LCDR-3	89-97	91-96	89-96

¹Residue numbering follows the Kabat et al, supra nomenclature

²Residue numbering follows the nomenclature of Chothia et al, supra

³Residue numbering follows the nomenclature of MacCallum et al, supra

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

As used herein, the term "framework" when used in reference to an antibody variable region is intended to refer to all amino acid residues outside of the CDR regions within the variable region of an antibody. Variable region frameworks are typically discontinuous amino acid sequences of about 100-120 amino acids in length, but are intended to refer only to those amino acids outside of the CDRs. As used herein, the term "framework region" is intended to refer to the domains of the framework separated by CDRs.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 90%, greater than 95%, or greater than 98% by weight of the antibody as determined by the Lowry method, e.g., greater than 99%, by (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues, by using a rotary cup sequencer, or (3) to homogeneity, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under reducing or non-reducing conditions, using Coomassie blue (Coomassie blue) or a silver stain. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. In some cases, the isolated antibody will be prepared by at least one purification step.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acids of any length in polymeric form, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; an immunologically labeled protein; and so on.

As used herein, the term "treating" or the like refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or side effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease in a subject who may be predisposed to said disease but has not yet been diagnosed as having said disease; (b) inhibiting the disease, i.e. arresting its development; and (c) alleviating, i.e., causing regression of, the disease.

The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, a murine (rat, mouse), a non-human primate, a human, a canine, a feline, an ungulate (e.g., equine, bovine, ovine, porcine, caprine), and the like. These terms also encompass any animal having a complement system, such as mammals, fish, and some invertebrates. Thus, these terms include mammals, fish and invertebrate companion animals, farm animals, work animals, zoo animals, and laboratory animals that contain the complement system.

By "therapeutically effective amount" or "effective amount" is meant an amount of the anti-complement C1s antibody which, when administered to a mammal or other subject for treatment of a disease, is sufficient to effect treatment of such disease. The "therapeutically effective amount" will vary depending on the anti-complement C1s antibody, the disease and its severity, and the age, weight, etc., of the subject to be treated.

"biological samples" encompass a variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. Furthermore, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

When a range of values is provided, it is understood that the invention encompasses each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "humanized anti-complement C1s antibody" includes a plurality of such antibodies, and reference to a "complement-mediated disease" includes reference to one or more complement-mediated diseases and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments of the invention are specifically contemplated by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically contemplated by the present invention and disclosed herein, as if each and every such subcombination was individually and specifically disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Furthermore, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Detailed Description

The present disclosure provides antibodies that bind complement C1s protein (i.e., anti-complement C1s antibody, also referred to herein as anti-C1 s antibody and C1s antibody) and nucleic acid molecules encoding such antibodies. The disclosure also provides compositions comprising such antibodies, and methods of making and using such antibodies, nucleic acid molecules, and compositions. The present disclosure provides methods of treating a complement-mediated disease or disorder comprising administering an anti-C1 s antibody. The present disclosure further provides in vitro and in vivo detection methods using the anti-C1 s antibodies described herein.

Anti-complement C1s antibodies

The present 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 substrate specificity range. Furthermore, it is possible to obtain antibodies (such as, but not limited to, monoclonal antibodies) that specifically bind to the activated form of C1 s.

The present disclosure provides isolated antibodies that specifically bind to an epitope within a complement C1s protein. Unless otherwise indicated, complement C1s protein as used herein is an activated C1s protein. In some embodiments, an isolated anti-C1 s antibody of the present disclosure binds to activated C1s protein. In some embodiments, an isolated anti-C1 s antibody of the present disclosure binds to the inactive form of C1 s. In other cases, the isolated anti-C1 s antibodies of the present disclosure bind activated C1s protein and the inactive form of C1 s. In some cases, the antibody is humanized, e.g., one or more framework regions of the heavy chain variable region and/or the light chain variable region comprise sequences derived from a human immunoglobulin framework.

The present disclosure provides isolated monoclonal antibodies that inhibit cleavage of C4, wherein the isolated monoclonal antibodies do not inhibit cleavage of C2. 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 that is inhibited by the antibody is C1 s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual in need thereof 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.

The present disclosure provides isolated monoclonal antibodies that inhibit C4 cleavage by C1s, i.e., inhibit C1 s-mediated proteolytic cleavage of C4. In some cases, the isolated monoclonal antibody is humanized. In some cases, the antibody inhibits cleavage of C4 by C1s by inhibiting binding of C4 to C1 s; for example, in some cases, the antibody inhibits C1 s-mediated C4 cleavage by inhibiting the binding of C4 to the C4 binding site of C1 s. Thus, in some cases, the antibody acts as a competitive inhibitor. The present disclosure also provides a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual in need thereof an effective amount of an isolated monoclonal antibody that inhibits C4 cleavage by C1s, i.e., inhibits C1 s-mediated proteolytic cleavage of C4.

The present disclosure provides isolated monoclonal antibodies that inhibit cleavage of C4 by C1s, wherein the antibodies do not inhibit cleavage of complement component C2 by C1 s; that is, the antibody inhibits C1 s-mediated C4 cleavage, but does not inhibit C1 s-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 does not inhibit the binding of C2 to C1 s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual in need thereof an effective amount of an isolated monoclonal antibody that inhibits cleavage of C4 by C1s, wherein the antibody does not inhibit cleavage of complement component C2 by C1 s; that is, the antibody inhibits C1 s-mediated C4 cleavage, but does not inhibit C1 s-mediated C2 cleavage. In some embodiments of the method, the antibody is humanized.

The present disclosure provides isolated humanized monoclonal antibodies that specifically bind to an epitope within a region encompassing domains IV and V of C1 s. For example, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in FIG. 1 and set forth in SEQ ID NO. 9. In some cases, the isolated humanized monoclonal antibody specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in FIG. 1 and set forth in SEQ ID NO.9, and inhibits the binding of C4 to C1 s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual in need thereof an effective amount of an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 272-422 of the amino acid sequence depicted in figure 1 and set forth in SEQ ID No.9, and inhibits the binding of C4 to C1 s.

The present disclosure provides isolated humanized monoclonal antibodies that specifically bind conformational epitopes within the region encompassing domains IV and V of C1 s. For example, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in FIG. 1 and set forth in SEQ ID NO. 9. In some cases, the isolated humanized monoclonal antibody specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in fig. 1 and shown in SEQ ID No.9, and inhibits binding of C4 to C1 s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, the method comprising administering to an individual in need thereof an effective amount of an isolated humanized monoclonal antibody that specifically binds to a conformational epitope within amino acids 272-422 of the amino acid sequence depicted in figure 1 and set forth in SEQ ID No.9, and inhibits the binding of C4 to C1 s.

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

Humanization of the framework regions reduces the risk of the antibody eliciting a human anti-mouse antibody (HAMA) response in humans. Art-recognized methods of measuring immune response can be performed to monitor HAMA response in a particular patient or during clinical trials. The immunogenicity assessment can be given to patients administered the humanized antibody at the beginning of treatment and throughout the course of administration. The HAMA response is measured, for example, by detecting antibodies to the humanized therapeutic agent in a serum sample from the patient using methods known to those skilled in the art, including surface plasmon resonance technology (BIACORE) and/or solid phase enzyme-linked immunosorbent assay (ELISA) assays. In many cases, the subject humanized anti-C1 s antibodies do not substantially elicit a HAMA response in a human subject.

Certain amino acids from human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and/or binding antigen. Non-natural juxtaposition of murine CDR regions with human variable framework regions can result in non-natural conformational constraints that, unless corrected for by substitution of certain amino acid residues, would result in loss of binding affinity.

The choice 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 made starting from the resolved structure of an immunoglobulin chain or domain thereof. The chain to be modeled is compared with the chain or domain having an resolved three-dimensional structure with respect to amino acid sequence similarity, and the chain or domain showing 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, e.g., those sharing at least 60%, at least 70%, at least 80%, at least 90%, or more sequence identity are selected for modeling. The resolved starting structures were modified to allow differences between the actual amino acids in the immunoglobulin chains or domains being modeled and those in the starting structures. The modified structure is then assembled into a complex immunoglobulin. Finally, the model was refined by energy minimization and by verifying that all atoms were within the appropriate distance of each other and that bond lengths and angles were within chemically acceptable ranges.

The CDR and framework regions are as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991). Chothia et al, J.mol.biol.196:901 (1987); nature 342:878 (1989); and j.mol.biol.186:651(1989) (collectively, "Chothia") alternative structural definitions have been proposed. When framework residues as defined by Kabat, supra constitute structural loop residues as defined by Chothia, supra, amino acids present in the mouse antibody can be selected for substitution into the humanized antibody. Residues "adjacent to" a CDR region include amino acid residues in the immediate position to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., in the immediate position to a CDR as defined by Kabat or a CDR as defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs and, if selected from the acceptor, distort the donor CDRs and reduce affinity. In addition, adjacent amino acids may 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.

In some embodiments, an anti-C1 s antibody of the present disclosure (e.g., a subject antibody that specifically binds to an epitope in a complement C1s protein) comprises: a) one, two or three V comprising IPN003 antibody_LA light chain region of a CDR; and b) one, two or three V comprising an IPN003 antibody_HA heavy chain region of a CDR; wherein said V_HAnd V_LThe CDRs are as defined by Kabat (see, e.g., Table 1 above; and Kabat 1991). In some such embodiments, the anti-C1 s antibody comprises humanized V_HAnd/or V_LFramework Region (FR).

In some embodiments, an anti-C1 s antibody of the present disclosure (e.g., a subject antibody that specifically binds to an epitope in a complement C1s protein) comprises: a) one, two or three V comprising IPN003 antibody_LA light chain region of a CDR; and b) one, two or three V comprising an IPN003 antibody_HA heavy chain region of a CDR; wherein said V_HAnd V_LCDRs are as defined by Chothia (see, e.g., Table 1 above; and Chothia 1987). In some such embodiments, the anti-C1 s antibody comprises humanized V_HAnd/or V_LA framework region.

CDR amino acid sequence and V of IPN003 antibody_LAnd V_HThe amino acid sequences are provided in table 2 (fig. 2). Table 2 also provides the assigned SEQ ID NOs for each amino acid sequence.

In some embodiments, an anti-C1 s antibody of the present disclosure (e.g., a subject antibody that specifically binds to an epitope in a complement C1s protein) 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-C1 s antibody comprises a humanized V_HAnd/or V_LA framework region.

SEQ ID NO:1:SSVSSSYLHWYQ；

SEQ ID NO:2:STSNLASGVP；

SEQ ID NO:3:HQYYRLPPIT；

SEQ ID NO:4:GFTFSNYAMSWV；

SEQ ID NO:5:ISSGGSHTYY；

SEQ ID NO:6:ARLFTGYAMDY。

In some embodiments, the anti-C1 s antibodies of the present disclosure comprise 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.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO: 3.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising the amino acid sequences SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6.

In some embodiments, an anti-C1 s antibody of the present 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.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID No. 7.

SEQ ID NO:7:DIVMTQTTAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK。

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID No. 8.

SEQ ID NO:8:QVKLEESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVT。

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID No. 7.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID No. 8.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 7.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No.7 and a heavy chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No. 8.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID No.7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 8.

In some embodiments, an anti-C1 s antibody of the present disclosure specifically binds to an epitope within complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising the light chain CDRs of the antibody light chain variable region comprising amino acid sequence SEQ ID No.7 and the heavy chain CDRs of the antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 8.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain CDR of an antibody light chain variable region comprising amino acid sequence SEQ ID No.7 and a heavy chain CDR of an antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 8.

In some cases, humanized V_HFrame or V_LThe frame is a mannequin frame. The co-humanized framework may be represented in human immunoglobulin V_LOr V_HOf framework sequencesThe amino acid residues most commonly present in the selection.

Is suitable for use with V as described herein_HConsensus V for CDR use_HNon-limiting examples of framework regions include (common to subclass III):

a)V_HFR1:EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:53)；

b)V_HFR2:WVRQAPGKGLEWV(SEQ ID NO:54)；

c)V_HFR3: RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 55); and

d)V_HFR4:WGQGTLVTVSS(SEQ ID NO:56)。

in some cases, V_HFR3 comprises amino acid substitutions at positions 71, 73 and/or 78; for example, wherein RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55) wherein the underlined and bolded R is amino acid 71(Kabat numbering); RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55) wherein the underlined and bolded N is amino acid 73(Kabat numbering); and RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:55) the underlined and bolded L is amino acid 78(Kabat numbering). 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, suitable co-humanized V_HFR3 comprises the amino acid sequence: RFTISADTSKNTAYLQMNSLRAEDTAVYYC(SEQ ID NO:57)。

Is suitable for use with V as described herein_HConsensus V for CDR use_HNon-limiting examples of framework regions include (common to subclass I):

a)V_HFR1:QVQLVQSGAEVKKPGASVKVSCKAS(SEQ ID NO:58)；

b)V_HFR2:WVRQAPGQGLEWM(SEQ ID NO:59)；

c)V_HFR3: RVTITADTSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 60); and

d)V_HFR4:WGQGTLVTVSS(SEQ ID NO:56)。

is suitable for use with V as described herein_HConsensus V for CDR use_HNon-limiting examples of framework regions include (common to subclass II):

a)V_HFR1:QVQLQESGPGLVKPSQTLSLTCTVS(SEQ ID NO:61)；

b)V_HFR2:WIRQPPGKGLEWI(SEQ ID NO:62)；

c)V_HFR3: RVTISVDTSKNQFSLKLSSVTAADTAVYYC (SEQ ID NO: 63); and

d)V_HFR4:WGQGTLVTVSS(SEQ ID NO:56)。

is suitable for use with V as described herein_LConsensus V for CDR use_LNon-limiting examples of framework regions include (common to subclass I):

a)V_LFR1:DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:57)；

b)V_LFR2:WYQQKPGKAPKLLIY(SEQ ID NO:58)；

c)V_LFR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 59); and

d)V_LFR4:FGQGTKVEIK(SEQ ID NO:60)。

is suitable for use with V as described herein_LConsensus V for CDR use_LNon-limiting examples of framework regions include (common to subclass II):

a)V_LFR1:DIVMTQSPLSLPVTPGEPASISC(SEQ ID NO:64)；

b)V_LFR2:WYLQKPGQSPQLLIY(SEQ ID NO:65)；

c)V_LFR3: GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 66); and

d)V_LFR4:FGQGTKVEIK(SEQ ID NO:60)。

is suitable for use with V as described herein_LConsensus V for CDR use_LNon-limiting examples of framework regions include (common to subclass III):

a)V_LFR1:DIVMTQSPDSLAVSLGERATINC(SEQ ID NO:67)；

b)V_LFR2:WYQQKPGQPPKLLIY(SEQ ID NO:68)；

c)V_LFR3: GVPDRFSGSGSGTDFTLTISSLQAEDFAVYYC (SEQ ID NO: 69); and

d)V_LFR4:FGQGTKVEIK(SEQ ID NO:60)。

is suitable for use with V as described herein_LConsensus V for CDR use_LNon-limiting examples of framework regions include (common to subclass IV):

a)V_LFR1:DIVMTQSPDSLAVSLGERATINC(SEQ ID NO:67)；

b)V_LFR2:WYQQKPGQPPKLLIY(SEQ ID NO:68)；

c)V_LFR3: GVPDRFSGSGSGTDFTLTISSLQAEDFAVYYC (SEQ ID NO: 69); and

d)V_LFR4:FGQGTKVEIK(SEQ ID NO:60)。

in some embodiments, an anti-C1 s antibody of the disclosure binds to complement C1s protein from an individual having the complement system. In some embodiments, an anti-C1 s antibody of the present disclosure binds to complement C1s protein from a mammal, fish, or invertebrate having a complement system. In some embodiments, an anti-C1 s antibody of the disclosure binds to a mammalian complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure binds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure binds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure binds to complement C1s protein having SEQ ID No. 9. Amino acid sequence SEQ ID NO 9 represents homo sapiens complement C1s protein having the amino acid sequence shown in FIG. 1.

In some embodiments, an anti-C1 s antibody of the disclosure has a dissociation constant (K) of no greater than 2.5nM_D) Binds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 2nM_DBinds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 1nM_DBinds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.9nM, no greater than 0.8nM, no greater than 0.7nM, no greater than 0.6nM, no greater than 0.5nM, no greater than 0.4nM, no greater than 0.3nM, no greater than 0.2nM, no greater than 0.1nM_DBinds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.3nM_DBinds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.2nM_DBinds complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.1nM_DBinds complement C1s protein. Methods for measuring binding of an antibody to C1s protein can be determined by one skilled in the art.

In some embodiments, the anti-C1 s antibodies of the present disclosure have a K of no greater than 90pM, no greater than 80pM, no greater than 70pM, no greater than 60pM, no greater than 50pM, no greater than 40pM, no greater than 30pM, no greater than 20pM, no greater than 10pM, no greater than 9pM, no greater than 8pM, no greater than 7pM, no greater than 6pM, no greater than 5pM, no greater than 4pM, no greater than 3pM, no greater than 2pM, no greater than 1pM_DBinds complement C1s protein.

In some embodiments, an anti-C1 s antibody of the disclosure has a dissociation constant (K) of no greater than 2.5nM_D) Binds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 2nM_DBinds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 1nM_DBinds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.9nM, no greater than 0.8nM, no greater than 0.7nM, no greater than 0.6nM, no greater than 0.5nM, no greater than 0.4nM, no greater than 0.3nM, no greater than 0.2nM, no greater than 0.1nM_DBinds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.3nM_DBinds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.2nM_DBinds to human complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.1nM_DBinds to human complement C1s protein. Methods for measuring binding of an antibody to human C1s protein can be determined by one skilled in the art. In some embodiments, the binding assay as described in the examples is used to determine the K between an antibody and a human C1s protein_D。

In some embodiments, the anti-C1 s antibodies of the present disclosure have a K of no greater than 90pM, no greater than 80pM, no greater than 70pM, no greater than 60pM, no greater than 50pM, no greater than 40pM, no greater than 30pM, no greater than 20pM, no greater than 10pM, no greater than 9pM, no greater than 8pM, no greater than 7pM, no greater than 6pM, no greater than 5pM, no greater than 4pM, no greater than 3pM, no greater than 2pM, no greater than 1pM_DBinds to human complement C1s protein.

In some embodiments, an anti-C1 s antibody of the present disclosure that binds to human complement C1s protein also binds to complement C1s protein of another species. In some embodiments, an anti-C1 s antibody of the present 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, an anti-C1 s antibody of the present disclosure that binds to human complement C1s protein also binds to rabbit complement C1s protein, e.g., rabbit C1s protein. In some embodiments, an anti-C1 s antibody of the present 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 cynomolgus monkey (Macaca mulatta) and cynomolgus monkey (Macaca fascicularis). In some embodiments, such cross-reactivity binds to the K of the human complement C1s protein with the antibody_DK of similar magnitude_DThe antibody binds to complement C1s protein of another (non-human) species. In some embodiments, an anti-C1 s antibody of the disclosureBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure that binds to human complement C1s protein also binds to rat complement C1s protein.

In some embodiments, an anti-C1 s antibody of the disclosure has a dissociation constant (K) of no greater than 2.5nM_D) Binds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 2nM_DBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 1nM_DBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.9nM, no greater than 0.8nM, no greater than 0.7nM, no greater than 0.6nM, no greater than 0.5nM, no greater than 0.4nM, no greater than 0.3nM, no greater than 0.2nM, no greater than 0.1nM_DBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.3nM_DBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.2nM_DBinds to rat complement C1s protein. In some embodiments, an anti-C1 s antibody of the disclosure has a K of no greater than 0.1nM_DBinds to rat complement C1s protein. Methods for measuring binding of an antibody to rat C1s protein can be determined by one skilled in the art. In some embodiments, the binding assay as described in the examples is used to determine the K between an antibody and rat C1s protein_D。

In some embodiments, the anti-C1 s antibodies of the present disclosure have a K of no greater than 90pM, no greater than 80pM, no greater than 70pM, no greater than 60pM, no greater than 50pM, no greater than 40pM, no greater than 30pM, no greater than 20pM, no greater than 10pM, no greater than 9pM, no greater than 8pM, no greater than 7pM, no greater than 6pM, no greater than 5pM, no greater than 4pM, no greater than 3pM, no greater than 2pM, no greater than 1pM_DBinds to rat complement C1s protein.

In some embodiments, an anti-C1 s antibody of the disclosure binds to native complement C1s protein and denatured complement C1s protein. As used herein, "native protein" refers to a protein that folds in its naturally occurring physiological state, and thus excludes denatured proteins. Detection of binding can be performed by western blotting. In such embodiments, the anti-C1 s antibodies of the present disclosure bind to C1s protein applied to native gels and also bind to C1s protein applied to denatured (e.g., Sodium Dodecyl Sulfate (SDS)) gels. In some embodiments, the subject anti-C1 s antibodies of the present disclosure bind a linear epitope in C1 s. Methods for determining whether an antibody binds to native C1s protein or denatured C1s protein are known to those of skill in the art. In some embodiments, gel electrophoresis is used to determine whether an antibody binds native and/or denatured C1s protein.

In some embodiments, an anti-C1 s antibody of the present disclosure reduces C4b2a (i.e., complement C4b and C2a complexes; also referred to as "C3 convertases") production 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% as compared to the amount of C4b2a produced in the absence of the subject anti-C1 s antibody. Methods of measuring C4b2a production are known in the art.

In some embodiments, an anti-C1 s antibody of the present disclosure inhibits 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, an anti-C1 s antibody of the disclosure inhibits cleavage of complement C2. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of complement C4. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of complement C2 and complement C4.

In some embodiments, an anti-C1 s antibody of the present disclosure inhibits 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, an anti-C1 s antibody of the disclosure inhibits cleavage of human complement C2. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of human complement C4. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of human complement C2 and human complement C4. In some embodiments, an anti-C1 s antibody of the disclosure inhibits rat C1 s-mediated cleavage of human complement C4. In some embodiments, an anti-C1 s antibody of the present disclosure inhibits human C1 s-mediated cleavage of human complement C4.

In some embodiments, an anti-C1 s antibody of the present disclosure inhibits 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, an anti-C1 s antibody of the disclosure inhibits cleavage of rat complement C2. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of rat complement C4. In some embodiments, an anti-C1 s antibody of the disclosure inhibits cleavage of rat complement C2 and rat complement C4.

In some embodiments, the anti-C1 s antibodies of the present disclosure inhibit C1s by sterically blocking access to the C1s active site or by sterically blocking access to a substrate.

In some embodiments, an anti-C1 s antibody of the present disclosure inhibits C1 s-mediated complement C4 activation. For example, in some cases, an anti-C1 s antibody of the disclosure is administered at less than 50 x 10^-9M, less than 25X 10^-9M, less than 10X 10^-9M, less than 5X 10^- ⁹M, less than 1X 10^-9M, less than 0.5X 10^-9M, less than 0.1X 10^-9M or less than 0.1X 10^-10IC of M₅₀Inhibits C1 s-mediated complement C4 activation.

In some cases, an anti-C1 s antibody of the disclosure inhibits complement-mediated cell lysis, e.g., in an in vitro cell lysis assay. For example, in some cases, an anti-C1 s antibody of the disclosure is administered at less than 10 x 10^-9M, less than 5X 10^-9M, less than 1X 10^-9M, less than 0.5X 10^-9M, less than 0.1X 10^-9M or less than 0.1X 10^-10IC of M₅₀Inhibiting complement-mediated cell lysis.

In some embodiments, an anti-C1 s antibody of the present disclosure competes for binding to an epitope bound by IPN 003.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a variable domain of an IPN003 antibody.

In some embodiments, an anti-C1 s antibody of the present disclosure is an IPN003 antibody.

The present disclosure provides any anti-C1 s antibody of the embodiments to be humanized. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a humanized framework region. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a humanized light chain framework region. In some embodiments, an anti-C1 s antibody of the present disclosure comprises a humanized heavy chain framework region.

In some embodiments, the subject anti-C1 s antibodies comprise one or more humanized Framework Regions (FRs). In some embodiments, the subject anti-C1 s antibodies comprise a light chain variable region comprising one, two, three, or four light chain FRs that have been humanized. In some embodiments, the subject antibodies comprise a light chain variable region comprising, in order from N-terminus to C-terminus: humanized light chain FR 1; CDR-L1 as set forth herein; humanized light chain FR 2; CDR-L2 as set forth herein; humanized light chain FR 3; CDR-L3 as set forth herein; and humanized light chain FR 4. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are: 1, 2 and 3.

For example, the subject antibody may comprise a light chain variable region comprising, in order from N-terminus to C-terminus: humanized light chain FR 1; CDR-L1 comprising the amino acid sequence SEQ ID NO. 1; humanized light chain FR 2; CDR-L2 comprising the amino acid sequence SEQ ID NO. 2; humanized light chain FR 3; CDR-L3 comprising the amino acid sequence SEQ ID NO. 3; and humanized light chain FR 4.

In some embodiments, the subject anti-C1 s antibodies comprise a heavy chain variable region comprising one, two, three, or four heavy chain FRs that have been humanized. In some embodiments, the subject antibodies comprise a heavy chain variable region comprising, in order from N-terminus to C-terminus: humanized heavy chain FR 1; CDR-H1 as set forth herein; humanized heavy chain FR 2; CDR-H2 as set forth herein; humanized heavy chain FR 3; CDR-H3 as set forth herein; and humanized heavy chain FR 4.

For example, the subject antibody may comprise a heavy chain variable region comprising, in order from N-terminus to C-terminus: humanized heavy chain FR 1; CDR-H1 comprising the amino acid sequence SEQ ID NO 4; humanized heavy chain FR 2; CDR-H2 comprising the amino acid sequence SEQ ID NO. 5; humanized heavy chain FR 3; CDR-H3 comprising the amino acid sequence SEQ ID NO 6; and humanized heavy chain FR 4.

In some embodiments, an anti-C1 s antibody of the present disclosure (e.g., a subject antibody that specifically binds to an epitope in a 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, 35 and 36. In some such embodiments, the anti-C1 s antibody comprises a humanized V_HAnd/or V_LA framework region.

SEQ ID NO:32:TASSSVSSSYLH；

SEQ ID NO:33:STSNLAS；

SEQ ID NO:3:HQYYRLPPIT；

SEQ ID NO:34:NYAMS；

SEQ ID NO:35:TISSGGSHTYYLDSVKG；

SEQ ID NO:36:LFTGYAMDY。

In some embodiments, the anti-C1 s antibodies of the present disclosure comprise 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.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO: 3.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO: 36.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises CDR-L1 having amino acid sequence SEQ ID NO:32, CDR-L2 having amino acid sequence SEQ ID NO:33, CDR-L3 having amino acid sequence SEQ ID NO:3, CDR-H1 having amino acid sequence SEQ ID NO:34, CDR-H2 having amino acid sequence SEQ ID NO:35, and CDR-H3 having amino acid sequence SEQ ID NO: 36.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID No. 37.

SEQ ID NO:37:QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK。

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 38.

SEQ ID NO:38:EVMLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVTVSS。

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 37.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 38.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 37.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 38.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No. 37 and a heavy chain variable region comprising an amino acid sequence having 90% identity to amino acid sequence SEQ ID No. 38.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising an amino acid sequence having 95% identity to amino acid sequence SEQ ID No. 37 and a heavy chain variable region comprising an amino acid sequence having 95% identity to amino acid sequence SEQ ID No. 38.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID No. 37 and a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 38.

In some embodiments, an anti-C1 s antibody of the present disclosure specifically binds to an epitope within complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising the light chain CDRs of the antibody light chain variable region comprising amino acid sequence SEQ ID No. 37 and the heavy chain CDRs of the antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 38.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises the light chain CDRs of the antibody light chain variable region comprising the amino acid sequence SEQ ID NO:37 and the heavy chain CDRs of the antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO: 38.

In some embodiments, the subject anti-C1 s antibodies comprise one or more humanized Framework Regions (FRs). In some embodiments, the subject anti-C1 s antibodies comprise a light chain variable region comprising one, two, three, or four light chain FRs that have been humanized. In some embodiments, the subject antibodies comprise a light chain variable region comprising, in order from N-terminus to C-terminus: humanized light chain FR 1; CDR-L1 as set forth herein; humanized light chain FR 2; CDR-L2 as set forth herein; humanized light chain FR 3; CDR-L3 as set forth herein; and humanized light chain FR 4. In some embodiments, the respective amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are: 32, 33 and 3.

For example, the subject antibody may comprise a light chain variable region comprising, in order from N-terminus to C-terminus: humanized light chain FR 1; CDR-L1 comprising amino acid sequence SEQ ID NO 32; humanized light chain FR 2; CDR-L2 comprising the amino acid sequence SEQ ID NO. 33; humanized light chain FR 3; CDR-L3 comprising the amino acid sequence SEQ ID NO. 3; and humanized light chain FR 4.

For example, the subject antibody may comprise a heavy chain variable region comprising, in order from N-terminus to C-terminus: humanized heavy chain FR 1; CDR-H1 comprising the amino acid sequence SEQ ID NO: 34; humanized heavy chain FR 2; CDR-H2 comprising the amino acid sequence SEQ ID NO. 35; humanized heavy chain FR 3; CDR-H3 comprising amino acid sequence SEQ ID NO: 36; and humanized heavy chain FR 4.

The subject anti-C1 s antibodies can comprise a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:39 and depicted in fig. 16 (VH variant 1).

The subject anti-C1 s antibodies can comprise a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:40 and depicted in fig. 17 (VH variant 2).

The subject anti-C1 s antibodies can comprise a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:41 and depicted in fig. 18 (VH variant 3).

The subject anti-C1 s antibodies can comprise a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:42 and depicted in fig. 19 (VH variant 4).

The subject anti-C1 s antibodies can comprise a light chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO:43 and depicted in fig. 20 (VK variant 1).

The subject anti-C1 s antibodies can comprise a light chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO:44 and depicted in fig. 21 (VK variant 2).

The subject anti-C1 s antibodies can comprise a light chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO:45 and depicted in fig. 22 (VK variant 3).

The subject anti-C1 s antibodies can comprise a heavy chain variable region comprising 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, or 12 Framework (FR) amino acid substitutions relative to the FR amino acid sequence of the IPN003 parent antibody depicted in table 7 (fig. 23).

For example, the subject anti-C1 s antibodies can comprise a heavy chain variable region comprising 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 FR 1.

As another example, the subject anti-C1 s antibodies can comprise a heavy chain variable region comprising 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 FR 2.

As another example, the subject anti-C1S antibodies can comprise a heavy chain variable region comprising 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 FR 3.

As another example, the subject anti-C1S antibody can comprise a heavy chain variable region comprising an S → L substitution at amino acid position 108 of VH FR 4.

The subject anti-C1 s antibodies can comprise a light chain variable region comprising 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 Framework (FR) amino acid substitutions relative to the FR amino acid sequence of the IPN003 parent antibody depicted in table 8 (fig. 23).

For example, a subject anti-C1S antibody can comprise a light chain variable region comprising an I → T substitution at position 10 in VL FR1 and/or an M → L substitution at amino acid position 11 in VLFR1 and/or an a → L substitution at position 13 of 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 of VL FR1 and/or a T → S substitution at amino acid position 22 in VL FR 1.

As another example, the subject anti-C1S antibody can comprise a light chain variable region comprising 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 FR 2.

As another example, a subject anti-C1S antibody can comprise a light chain variable region comprising 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 FR 3.

As another example, the subject anti-C1 s antibody can comprise a light chain variable region comprising 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 FR 4.

In some cases, an anti-C1 s antibody of the present disclosure comprises:

i) VH variant 1 comprising the amino acid sequence depicted in FIG. 16 and shown in SEQ ID NO: 39; and Vk variant 1 comprising the amino acid sequence depicted in figure 20 and shown in SEQ ID No. 43;

ii) VH variant 1 comprising the amino acid sequence depicted in FIG. 16 and shown in SEQ ID NO: 39; and Vk variant 2 comprising the amino acid sequence depicted in figure 21 and shown in SEQ ID No. 44;

iii) VH variant 1 comprising the amino acid sequence depicted in FIG. 16 and shown in SEQ ID NO: 39; and Vk variant 3 comprising the amino acid sequence depicted in figure 22 and shown in SEQ ID No. 45;

iv) VH variant 2 comprising the amino acid sequence depicted in FIG. 17 and shown in SEQ ID NO: 40; and Vk variant 1 comprising the amino acid sequence depicted in figure 20 and shown in SEQ ID No. 43;

v) VH variant 2 comprising the amino acid sequence depicted in FIG. 17 and shown in SEQ ID NO: 40; and Vk variant 2 comprising the amino acid sequence depicted in figure 21 and shown in SEQ ID No. 44;

vi) VH variant 2 comprising the amino acid sequence depicted in FIG. 17 and shown in SEQ ID NO: 40; and Vk variant 3 comprising the amino acid sequence depicted in figure 22 and shown in SEQ ID No. 45;

vii) VH variant 3 comprising the amino acid sequence depicted in FIG. 18 and shown in SEQ ID NO: 41; and Vk variant 1 comprising the amino acid sequence depicted in figure 20 and shown in SEQ ID No. 43;

viii) VH variant 3 comprising the amino acid sequence depicted in FIG. 18 and shown in SEQ ID NO: 41; and Vk variant 2 comprising the amino acid sequence depicted in figure 21 and shown in SEQ ID No. 44;

ix) VH variant 3 comprising the amino acid sequence depicted in figure 18 and shown in SEQ ID NO: 41; and Vk variant 3 comprising the amino acid sequence depicted in figure 22 and shown in SEQ ID No. 45;

x) VH variant 4 comprising the amino acid sequence depicted in figure 19 and shown in SEQ ID No. 42; and Vk variant 1 comprising the amino acid sequence depicted in figure 20 and shown in SEQ ID No. 43;

xi) VH variant 4 comprising the amino acid sequence depicted in FIG. 19 and shown in SEQ ID NO: 42; and Vk variant 2 comprising the amino acid sequence depicted in figure 21 and shown in SEQ ID No. 44; or

xii) VH variant 4 comprising the amino acid sequence depicted in FIG. 19 and shown in SEQ ID NO: 42; and Vk variant 3 comprising the amino acid sequence depicted in figure 22 and shown in SEQ ID NO: 45.

In some embodiments, an anti-C1 s antibody of the present disclosure is an Ig monomer or antigen-binding fragment thereof that binds complement C1s protein. In some embodiments, an anti-C1 s antibody of the present disclosure is an Ig monomer. In some embodiments, an anti-C1 s antibody of the present disclosure is an antigen-binding fragment that binds an Ig monomer of a complement C1s protein.

In some embodiments, an anti-C1 s antibody of the present disclosure is selected from the group consisting of an Ig monomer, an Fab fragment, F (ab')₂Fragments, Fd fragments, scFv, scAb, dAb, Fv, single domain heavy chain antibodies and single domain light chain antibodies.

In some embodiments, the anti-C1 s antibodies of the present disclosure are selected from the group consisting of monospecific antibodies, bispecific antibodies, and multispecific antibodies.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain region and a heavy chain region present in separate polypeptides.

In some embodiments, an anti-C1 s antibody of the present disclosure comprises a light chain region and a heavy chain region present in a single polypeptide.

In some embodiments, the subject antibody comprises an anti-C1 s heavy chain CDR and an anti-C1 s light chain CDR in a single polypeptide chain, e.g., in some embodiments, the subject antibody is an scFv.

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

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; connecting the subregions; 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: 32, 33, 3, 34, 35 and 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 linking sub-region may be about 5 amino acids (aa) to about 50 amino acids in length, for example about 5 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa in length.

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

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 amino acid sequences of each of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 are: 1, 2, 3, 34, 35 and 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 linking sub-region may be about 5 amino acids to about 50 amino acids in length, for example about 5 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa in length.

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

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; connecting the subregions; 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 amino acid sequences of each of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 are: 1, 2, 3,4, 5 and 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 linking sub-region may be about 5 amino acids (aa) to about 50 amino acids in length, for example about 5 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa in length.

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

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 amino acid sequences of each of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 are: 1, 2, 3,4, 5 and 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 linking sub-region may be about 5 amino acids to about 50 amino acids in length, for example about 5 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa in length.

Linkers suitable for use with the subject antibodies include "flexible linkers". If present, the linker molecule is typically of sufficient length to allow some flexible movement between the regions of linkage. In some embodiments, the linker molecule is generally about 6 to 50 atoms in length. The linker molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Other linker molecules that can bind to polypeptides can be used in accordance with the present disclosure.

Suitable linkers can be readily selected and can be 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.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-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 act as neutral tethers between components. Glycine polymers are of particular interest because glycine is close to even significantly more phi-psi space than alanine and is much less constrained than residues with longer side chains (see Scheraga, rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:12), GGSGG (SEQ ID NO:13), GSGSGSG (SEQ ID NO:14), GSGGG (SEQ ID NO:15), GGGSG (SEQ ID NO:16), GSSSG (SEQ ID NO:17), and the like. One of ordinary skill in the art will recognize that the design of the peptide conjugated to any of the elements described above may include a linker that is flexible in whole or in part, such that the linker may include a flexible linker and one or more moieties that impart a less flexible structure.

In some embodiments, the anti-C1 s antibodies of the present disclosure comprise scFv multimers. For example, in some embodiments, a subject antibody is a scFv dimer (e.g., comprising two scFv in series (scFv)₂) scFv trimer (e.g., comprising three scFv in series (scFv)₃) scFv tetramers (e.g., comprising four scFv in series (scFv)₄) Or multimers with more than four scfvs (e.g., in tandem). The scFv monomers may be connected in series via a linker that is from about 2 amino acids to about 10 amino acids (aa) in length, e.g., 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)_xWherein x is an integer from 2 to 10. Other suitable linkers are those discussed above. In some embodiments, each scFv monomer in the subject scFv multimer isHumanized as described above.

In some cases, a subject antibody comprises a constant region (e.g., an Fc region) of an immunoglobulin. The Fc region, if present, may 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 present, the antibody may contain light and heavy chain constant regions. Suitable heavy chain constant regions include the CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include antibodies with all types of constant regions including IgM, IgG, IgD, IgA, and IgE and any isotype (including IgG1, IgG2, IgG3, and IgG 4). An example of a suitable heavy chain Fc region is the human isotype IgG1 Fc. Another example of a suitable heavy chain Fc region is the human isotype IgG2 Fc. Another example of a suitable heavy chain Fc region is the human isotype IgG3 Fc. The light chain constant region can be lambda or kappa. A subject antibody (e.g., a subject humanized antibody) can comprise sequences from more than one species or isotype. The antibody may comprise tetrameric forms of two light chains and two heavy chains, single heavy chain, light chain forms, Fab ', F (ab')₂And Fv forms, or single chain antibody forms in which the heavy and light chain variable domains are linked via a spacer.

In some cases, the heavy chain region is of isotype IgG 4. In some such embodiments, the hinge region comprises the S241P substitution. See, e.g., Angal et al, (1993) mol.Immunol.30: 105. In some such embodiments, the hinge region comprises the substitution of L236E (or L235E, using EU numbering; Kabat et al (1991) Sequences of Proteins of immunological Interest, 5 th edition, U.S. Dept. health and Human Services, Bethesda, MD, NIH publication No. 91-3242). See, e.g., Reddy et al, (2000) J.Immunol.164: 1925; and Klechevsky et al, (2010) Blood 116: 1685. In some such embodiments, the hinge region comprises the S241P substitution and the L236E substitution.

The subject antibodies may comprise a free thiol (-SH) group at the carboxy terminus, wherein the free thiol group may be used to link the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, and the like.

In some embodiments, the subject antibodies comprise one or more non-naturally occurring amino acids. In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. For suitable non-naturally occurring amino acids, see, e.g., U.S. patent No.7,632,924. Including non-naturally occurring amino acids can provide for attachment to a polymer, second polypeptide, backbone, or the like. For example, a subject antibody linked to a water-soluble polymer can be prepared by reacting a water-soluble polymer comprising a carbonyl group (e.g., PEG) with an antibody, wherein the antibody comprises a non-naturally encoded amino acid comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. As another example, a subject antibody linked to a water-soluble polymer can be prepared by reacting a subject antibody comprising an alkyne-containing amino acid with a water-soluble polymer comprising an azide moiety (e.g., PEG). In some embodiments, the azide or alkyne group is linked to the PEG molecule through an amide linkage. "non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid", "non-naturally occurring amino acid" and various hyphenated and non-hyphenated forms thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that occur through modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that are not themselves naturally incorporated into a growing polypeptide chain through a translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

in some embodiments, the subject antibodies are linked (e.g., covalently linked) to polymers (e.g., polymers other than polypeptides), 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, polyalkenylene or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or isopolysaccharides, suitable polymers include, for example, ethylene-vinyl alcohol copolymers (commonly known by the generic name EVOH or by the trade name EVAL), polybutylmethacrylate, poly (hydroxyvalerate), poly (L-lactic acid), polycaprolactone, poly (lactide-co-glycolide), poly (hydroxybutyrate-co-valerate), polydioxanone, polyorthoesters, polyanhydrides, poly (glycolic acid), poly (D, L-lactic acid), poly (glycolic acid-co-trimethylene carbonate), poly (phosphourethane, poly (vinyl amino acid), cyanoacrylates, poly (trimethylene carbonate), poly (iminoiminomethyl cellulose, poly (ethylene-co-propylene-co-ethylene-vinyl acetate), poly (ethylene-co-ethylene-propylene-co-ethylene-propylene-co-ethylene-vinyl acetate; poly (ethylene-co-propylene-ethylene-co-ethylene-propylene-co-ethylene-propylene-co-ethylene-propylene-co-propylene-co-ethylene-propylene-ethylene-propylene-co-ethylene-co-propylene-co-propylene-co-ethylene-co-ethylene-propylene-co-propylene-ethylene-propylene-co-propylene-co-ethylene-propylene-co-propylene-co-propylene-co-propylene.

Suitable synthetic polymers include unsubstituted and substituted linear or branched poly (ethylene glycol), poly (propylene glycol), poly (vinyl alcohol), and derivatives thereof, for example, substituted poly (ethylene glycols), such as methoxy poly (ethylene glycol), and derivatives thereof. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen and derivatives thereof.

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

As described above, in some embodiments, the subject antibodies are covalently linked to a non-peptide synthetic polymer. In some embodiments, the subject antibody is covalently attached to a PEG polymer. In some embodiments, the subject scFv multimer is covalently linked to a PEG polymer. See, e.g., Albrecht et al, (2006) j. Methods and reagents suitable for the pegylation 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-CH)₂-CH₂)_nO-R, wherein R is hydrogen or a protecting group such as an alkyl or alkanol group, and wherein n is an integer from 1 to 1,000. When R is a protecting group, it typically has from 1 to 8 carbons.

In some embodiments, the PEG conjugated to the subject antibody is a linear chain. In some embodiments, the PEG conjugated to the subject antibody is branched. Branched PEG Derivatives such as those described in U.S. patent No.5,643,575, "star PEG" and multi-arm PEGs such as those described in Shearwater Polymers, inc. Star-shaped PEGs are described in the art, including, for example, in U.S. patent No.6,046,305.

The subject antibodies can be glycosylated, for example, the subject antibodies can comprise covalently linked carbohydrate or polysaccharide moieties. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody (for N-linked glycosylation sites) is conveniently achieved by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences. Changes (for O-linked glycosylation sites) can also be made by adding one or more serine or threonine residues to the original antibody sequence or by substitution with one or more serine or threonine residues. Similarly, removal of glycosylation sites can be achieved by amino acid changes within the native glycosylation sites of the antibody.

In some embodiments, the subject antibodies will comprise "radiopaque" markers, such as markers that can be readily visualized using, for example, x-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide, bromide, or barium salts. Other radiopaque materials are also known and include, but are not limited to, organobismuth derivatives (see, e.g., U.S. patent No.5,939,045), radiopaque polyurethanes (see, e.g., U.S. patent No.5,346,981), organobismuth complexes (see, e.g., U.S. patent No.5,256,334), radiopaque barium polymer complexes (see, e.g., U.S. patent No.4,866,132), and the like.

The subject antibodies can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the subject antibody, a synthetic polymer, a carbohydrate, etc.) using, for example, glutaraldehyde, homobifunctional or heterobifunctional crosslinkers. Glutaraldehyde crosslinks the polypeptide via its amino moiety. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (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 polypeptides to be linked. Homobifunctional NHS esters and imino esters crosslink the amines containing the polypeptides. At mildly alkaline pH, the imine reacts only with primary amines to form imidoamides, and the overall charge of the crosslinked polypeptide is unaffected. Homobifunctional thiol-reactive crosslinkers include bismaleimide hexane (BMH), 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB), and 1, 4-bis- (3',2' -pyridyldithio) propionylaminobutane (DPDPDPPB).

The heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a thiol-reactive moiety) and are crosslinked with one polypeptide via the amine or thiol-reactive moiety and then reacted with another polypeptide via a 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 reagent used to couple a carboxyl group with an amine, which produces an amide bond.

The subject antibodies can be immobilized on a solid support. Suitable supports are well known in the art and include, inter alia, 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, duracytes, wells of reaction trays (e.g., multi-well plates), plastic tubes, and the like. The solid support may comprise any of a variety of substances including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. Methods suitable for immobilizing the subject antibodies onto solid supports are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. The solid support may be soluble or insoluble, for example in aqueous solution. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

In some embodiments, the subject antibody will comprise a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable include, but are not limited to, magnetic beads (e.g., Dynabeads)^TM) Fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g.,³H、¹²⁵I、³⁵S、¹⁴c or³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and other enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

In some embodiments, the subject antibodies comprise a contrast agent or radioisotope, wherein the contrast agent or radioisotope is a contrast agent or radioactive suitable for use in an imaging procedure, such as one performed on a human. Non-limiting examples of labels include radioisotopes such as¹²³¹I (iodine)¹⁸F (fluorine),⁹⁹Tc (technetium),¹¹¹In and (indium) and⁶⁷ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotope (¹⁵³Gd) is also available and suitable for imaging procedures in non-human mammals the subject antibodies can be labeled using standard techniques for example, chloramine T or 1,3,4, 6-tetrachloro-3 α,6 α -diphenylglycoluril can be used to iodinate the subject antibodies for fluorination, during synthesis throughA fluoride ion displacement reaction to add fluorine to the subject antibody. For a review of the synthesis of proteins with such 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). The subject antibodies can also be labeled with contrast agents by standard techniques. For example, the subject antibody may be labeled with Gd by conjugating a low molecular Gd chelate such as Gd diethylenetriaminepentaacetic acid (GdDTPA) or Gd tetraazacyclododecane tetraacetic 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 labeled with Gd by, for example, conjugating a polylysine-Gd chelate to the antibody. See, e.g., Curtet et al, invest, radio, 33(10):752-761 (1998). Alternatively, the subject antibody may be labeled with Gd by incubating paramagnetic polymerized liposomes comprising Gd chelator lipids with avidin and a biotin-labeled antibody. See, e.g., Sipkins et al, Nature Med.,4:623-626 (1998).

Suitable fluorescent proteins that can be linked to the subject antibodies include, but are not limited to, green fluorescent protein from victoria multiphoton jellyfish (Aequoria victoria) or mutants or derivatives thereof, for example as described in U.S. Pat. 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, 5,874,304; for example, enhanced GFP, many of which are commercially available from, for example, Clontech, inc; a red fluorescent protein; a yellow fluorescent protein; any of a variety of fluorescent and colored proteins from the coral species (Anthozonan), as described, for example, in Matz et al, (1999) Nature Biotechnol.17: 969-973; and so on.

In some embodiments, the subject antibodies are conjugated to a therapeutic agent. Any of the subject antibodies disclosed herein can be used to form antibody-agent conjugates. The agent may be linked to the N-terminus of the light chain, the C-terminus of the light chain, the N-terminus of the heavy chain, or the C-terminus of the heavy chain. In some embodiments, the agent is attached to the hinge of the antibody or one or more other sites on the antibody. For single chain antibodies, the agent may be attached to the N-or C-terminus of the single chain antibody. The agent may be conjugated to the antibody, either 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.

In some embodiments, the subject antibody will be linked (e.g., covalently or non-covalently) to a fusion partner, e.g., a ligand; an epitope tag; a peptide; proteins other than antibodies; and so on. Suitable fusion partners include peptides and polypeptides having the following properties: confer enhanced in vivo stability (e.g., enhanced serum half-life); providing convenience of purification, e.g., (His)_ne.g., 6His, etc., providing secretion of the fusion protein from the cell, providing epitope tags, e.g., 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 a detectable signal, e.g., an enzyme that produces a detectable product (e.g., β -galactosidase, luciferase) or an auto-detectable protein, e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc., providing multimerization, e.g., a multimerization domain such as the Fc portion of an immunoglobulin, etc.

The fusion may also include an affinity domain that includes a peptide sequence that can interact with a binding partner, e.g., such as a peptide sequence immobilized on a solid support that can be used for identification or purification. A single amino acid, such as histidine, in sequence, when fused to a protein, can be used for one-step purification of the fusion protein by high affinity binding to a resin column, such as nickel sepharose. Exemplary affinity domains include His5(HHHHH) (SEQ ID NO: 21); HisX6 (HHHHHHHHHH) (SEQ ID NO: 22); c-myc (EQKLISEEDL) (SEQ ID NO: 20); flag (DYKDDDDK) (SEQ ID NO: 19); StrepTag (WSHPQFEK) (SEQ ID NO: 23); hemagglutinin, e.g., HA tags (YPYDVPDYA; SEQ ID NO: 18); glutathione-S-transferase (GST); thioredoxin; a cellulose binding domain; RYIRS (SEQ ID NO: 24); Phe-His-His-Thr (SEQ ID NO: 25); a chitin binding domain; an S-peptide; a T7 peptide; an SH2 domain; c-terminal RNA label; WEAAAREACCRECCARA (SEQ ID NO: 26); metal binding domains, e.g. zinc binding domains or calcium binding domains, such as those from calcium binding proteins, e.g. calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-regulatory protein, cone protein, VILIP, calcineurin, hippocampal calpain, frequenin, kaladain, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K and calretinin, intein, biotin, streptavidin, MyoD, leucine zipper sequence and maltose binding protein.

In some embodiments, an anti-C1 s antibody of the present disclosure is formulated with an agent that facilitates crossing the Blood Brain Barrier (BBB). In some embodiments, the antibody is fused, directly or through a linker, to a compound that facilitates crossing the BBB. Examples of such compounds include, but are not limited to, carrier molecules, peptides or proteins. In some embodiments, the subject antibodies will be fused to polypeptides that bind to endogenous BBB receptors. Linkage of the subject antibodies to polypeptides that bind to endogenous BBB receptors facilitates crossing the BBB, e.g., in the subject methods of treatment (see below) comprising administering the subject antibodies to an individual in need thereof. Suitable polypeptides that bind to an endogenous BBB receptor include antibodies, e.g., monoclonal antibodies or antigen-binding fragments thereof, that specifically bind to an endogenous BBB receptor. Suitable endogenous BBB receptors include, but are not limited to, insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and insulin-like growth factor receptor. See, e.g., U.S. patent publication No. 2009/0156498.

As an example, the subject anti-C1 s antibody can be a bispecific antibody comprising a first antigen-binding portion 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 example, in some cases, the subject anti-C1 s antibodies are bispecific antibodies comprising a first antigen-binding portion that specifically binds to an epitope in the C1s protein; and a second antigen-binding moiety that binds to transferrin receptor.

For example, an anti-C1 s antibody of the present disclosure can be fused to a peptide that facilitates crossing the BBB, the peptide having a length of about 15 amino acids to about 25 amino acids and comprising an amino acid sequence having at least about 85% amino acid sequence identity to 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, e.g., U.S. patent publication No. 2011/0288011; and 2009/0016959. Peptides that facilitate crossing the BBB can be fused to the N-terminus of the anti-C1 s light chain region, the C-terminus of the anti-C1 s light chain region, the N-terminus of the anti-C1 s heavy chain region, the C-terminus of the anti-C1 s heavy chain region, the N-terminus of the subject anti-C1 s single chain antibody, the C-terminus of the subject anti-C1 s single chain antibody, and the like.

In some embodiments, the subject antibodies comprise polyamine modifications. Polyamine modification of the subject antibodies enhances the permeability of the modified antibodies at the BBB. The subject antibodies may 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, norspermidine, homoplasminogenimine, thermoamine (thermoamine), thermopramine (caldopentamine), homophilicpentaamine (homocaltropine), and canavanine (canavalimine). Putrescine, spermidine and spermine are particularly useful. Synthetic polyamines of the empirical formula C_XH_YN_ZThe composition can be a cyclic or acyclic, branched or unbranched hydrocarbon chain having 3 to 12 carbon atoms, further comprising 1 to 6 NR or N (R)₂Moiety wherein R is H, (C)₁-C₄) Alkyl, phenyl or benzyl. The polyamine can be attached to the antibody using any standard crosslinking method.

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

Methods of producing subject antibodies

The subject antibodies can be produced by any known method, e.g., conventional synthetic methods for protein synthesis; recombinant DNA methods; and so on. In some embodiments, the subject antibodies are produced by a method selected from the group consisting of recombinant production and chemical synthesis.

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 a liquid or solid phase. Solid Phase Polypeptide Synthesis (SPPS) is an example of a suitable method for chemically synthesizing the subject antibodies, in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize the subject antibodies. The technique for solid phase synthesis is described by: barany and Merrifield, Solid-Phase Peptide Synthesis; the Peptides: Analysis, Synthesis, Biology, pp.3-284, Vol.2: specialty Methods in peptide Synthesis, part A, Merrifield et al, J.Am.chem.Soc.,85:2149-2156 (1963); stewart et al, Solid Phase Peptide Synthesis, 2 nd edition, Pierce chem.Co., Rockford, Ill. (1984); and Ganesan A.2006 Mini Rev. Med chem.6: 3-10; and Camarero JA et al, 2005 Protein PeptLett.12: 723-8. Briefly, small insoluble porous beads were treated with the functional units constructed with peptide chains above. After repeated cycles of coupling/deprotection, the free N-terminal amine of the attached solid phase is coupled with a single N-protected amino acid unit. This unit is then deprotected to expose a new N-terminal amine to which another amino acid can be attached. The peptide is still immobilized on a solid phase and undergoes a filtration process before being cleaved off.

Standard recombinant methods can be used to produce the subject antibodies. For example, nucleic acids encoding the light and heavy chain variable regions, optionally linked to a constant region, are inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is operably linked to control sequences in an expression vector that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are 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 a eukaryotic host cell (e.g., COS or CHO cells). Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the antibody.

Due to the degeneracy of the code, multiple nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence may be generated by de novo solid phase DNA synthesis or by Polymerase Chain Reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for making substitution, deletion and insertion variants of a target polypeptide DNA. See Adelman et al, DNA 2:183 (1983). Briefly, target polypeptide DNA is altered by hybridization of an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize the entire second complementary strand of the template that incorporates the oligonucleotide primer and encodes the selected change in the target polypeptide DNA.

Suitable expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selectable marker (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.

other suitable microbial hosts for use include Bacillus (Bacillus), such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella (Salmonella), Serratia (Serratia), and various Pseudomonas species (Pseudomonas), in which expression vectors can also be prepared, which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.

Other microorganisms, such as yeast, may also be used for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia (Pichia) are examples of suitable yeast host cells, where appropriate vectors have expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as necessary. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in vitro cell culture) can be used to express and produce the anti-C1 s antibodies of the present disclosure (e.g., polynucleotides encoding the subject anti-C1 s antibodies). See Winnacker, From Genes to Clones, VCH Publishers, n.y., n.y. (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 for these cells can include expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.89:49(1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J.Immunol.148:1149 (1992).

Once synthesized (chemically or recombinantly), the intact 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 columns, column chromatography, High Performance Liquid Chromatography (HPLC) Purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, n.y., (1982)). the subject antibody can be substantially pure, e.g., 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% pure, or higher, e.g., free of contaminants such as cellular debris, macromolecules other than the subject antibody, etc.

Composition comprising a metal oxide and a metal oxide

The present disclosure provides compositions comprising the subject antibodies. In addition to the subject antibodies, the subject antibody compositions can comprise one or more of the following: salts, e.g. NaCl, MgCl₂、KCl、MgSO₄Etc.; buffers, for example Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-Tris [ hydroxymethyl ] methane]Methyl-3-aminopropanesulfonic acid (TAPS); a solubilizer; detergents, e.g., non-ionic detergents such as Tween-20 and the like; a protease inhibitor; glycerol; and so on.

Nucleic acid molecules, expression vectors and host cells

The present disclosure provides nucleic acid molecules comprising a nucleotide sequence encoding the subject anti-C1 s antibody.

In some embodiments, a nucleic acid molecule of the present disclosure encodes a subject anti-C1 s 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 to the amino acid sequence set forth in seq id No. 7. In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 7.

In some embodiments, a nucleic acid molecule of the present disclosure encodes a subject anti-C1 s 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 to the amino acid sequence set forth in seq id No. 8. In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

In some embodiments, a nucleic acid molecule of the present disclosure encodes a subject anti-C1 s 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 to the amino acid sequence set forth in seq id No. 37. In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37.

In some embodiments, a nucleic acid molecule of the present disclosure encodes a subject anti-C1 s 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 to the amino acid sequence set forth in seq id No. 38. In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38.

In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:3, respectively.

In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

In some embodiments, the nucleic acid molecules of the present disclosure encode the subject anti-C1 s antibodies comprising a light chain variable region and a heavy chain variable region.

Nucleic acid molecules encoding the subject antibodies can be operably linked to one or more regulatory elements, such as promoters and enhancers, that allow for expression of the nucleotide sequence in the desired target cell (e.g., a cell genetically modified to synthesize the encoded antibody).

Suitable promoter and enhancer elements are known in the art. Promoters suitable for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the T3 promoter; the T5 promoter; a lambda P promoter; a trp promoter; a lactose operon promoter; hybrid promoters, e.g., lac/tac hybrid promoter, tac/trc hybrid promoter, trp/lac promoter, T7/lac promoter; a trc promoter; tac promoter, etc.; a gpt promoter; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. patent publication No.20040131637), the pagC promoter (Pulkkien and Miller, J.Bacteriol.,1991:173(1): 86-93; Alpuche-Aranda et al, PNAS, 1992; 89(21):10079-83),nirB promoter (Harborne et al, (1992) mol. micro.6:2805-2813), and the like (see, e.g., Dunstan et al, (1999) feed. Immun.67: 5133-5141; McKelvie et al, (2004) Vaccine 22: 3243-3255; and Chatfield et al, (1992) Biotechnol.10: 888-892); sigma70 promoter, e.g., consensus sigma70 promoter (see, e.g., GenBank accession nos. AX798980, AX798961, and AX 798183); stationary phase promoters such as dps promoter, spv promoter, etc.; a promoter derived from SPI-2 of the pathogenicity island (see, for example, WO 96/17951); the actA promoter (see, e.g., Shetron-Rama et al, (2002) feed. Immun.70: 1087-1096); the rpsM promoter (see, e.g., Valdivia and Falkow (1996). mol. Microbiol.22: 367); the tet promoter (see, e.g., Hillen, W., and Wissmann, A. (1989) in Saenger, W., and Heinemann, U. (eds.), Topics in molecular and Structural Biology, Protein-Nucleic Acid Interaction, Macmillan, London, UK, Vol.10, pp.143-162); the SP6 promoter (see, e.g., Melton et al, (1984) Nucl. acids Res.12: 7035); and so on. Strong promoters suitable for use in prokaryotes such as E.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 protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operon), the tryptophan promoter operon (the TrpR repressor protein has a conformation that binds to the operon when complexed with tryptophan; the TrpR repressor protein has a conformation that does not bind to the operon in the absence of tryptophan), and the tac promoter operon (see, e.g., deBoer et al, (1983) Proc. Natl. Acad. Sci. U.S. A.80: 21-25).

In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, and the like; or a regulatable promoter such as 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 use in pichia spp.).

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

The selection of appropriate vectors and promoters is well within the level of skill of those of ordinary skill in the art.

Nucleic acid molecules encoding the subject antibodies may be present in expression vectors and/or cloning vectors. The present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding a subject antibody in a cloning vector. The present disclosure also provides a recombinant molecule comprising a nucleic acid molecule encoding the subject antibody operably linked to appropriate regulatory sequences in an expression vector to ensure expression of the encoded antibody. When the subject antibody comprises two separate polypeptides, the nucleic acid molecules encoding the two polypeptides can 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 replication and/or maintenance of the recombinant molecule.

A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available to produce 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). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors typically have convenient restriction sites located adjacent to the promoter sequence to provide for insertion of the nucleic acid sequence encoding the heterologous protein. There may be a selectable marker effective in the expression host. Suitable expression vectors include, but are not limited to, viral vectors. Examples of viral vectors include, but are not limited to, viral vectors based on: vaccinia virus; poliovirus; adenoviruses (see, e.g., Li et al, Invest Opthalmol Vis Sci 35: 25432549,1994; Borras et al, Gene Ther 6: 515524,1999; Li and Davidson, PNAS 92: 77007704,1995; Sakamoto et al, H Gene Ther 5: 10881097,1999; WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated viruses (see, e.g., Ali et al, Hum Gene Ther 9: 8186,1998; Flannery et al, PNAS 94: 69166921,1997; Bennett et al, Invest Opthalmol Vis Sci38: 28572863,1997; Jomary et al, Gene Ther 4: 683690,1997; Rolling et al, Hum Genether 10: 641648,1999; 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); SV 40; herpes simplex virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma (Rousssarcoma) virus, Harvey Sarcoma (Harvey Sarcoma) virus, avian leukemia virus, human immunodeficiency virus (see, e.g., Miyoshi et al, PNAS 94: 1031923,1997; Takahashi et al, J Virol 73: 78127816,1999), myeloproliferative Sarcoma virus, and mammary tumor virus); and so on.

As described above, the subject nucleic acid molecules comprise a nucleotide sequence encoding an anti-C1 s antibody of the disclosure. In some embodiments, the subject nucleic acid molecules comprise nucleotide sequences encoding the heavy and light chain CDRs of the subject IPN003 antibody. In some embodiments, the subject nucleic acid molecules comprise nucleotide sequences encoding the CDRs of the heavy and light chains of the subject antibodies, wherein the CDR-encoding sequences have FR-encoding nucleotide sequences interspersed therein. In some embodiments, the FR-encoding nucleotide sequence is a human FR-encoding nucleotide sequence.

Host cell

The present disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with the subject nucleic acid molecules. In some embodiments, the isolated genetically modified subject host cell can produce the subject antibody. Such cells are referred to as recombinant cells. The recombinant cells comprise a recombinant molecule encoding the subject antibody.

Suitable host cells include eukaryotic host cells, such as mammalian cells, insect host cells, yeast cells; and prokaryotic cells, such as bacterial cells. Introduction of the subject nucleic acids into host cells can be accomplished, for example, by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. 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), HeHLpG 2 cells, and the like. In some cases, the cell is a HEK cell. In some cases, the cell is a CHO cell, such as a CHO-K1 cell (ATCC No. CCL-61), a CHO-M cell, a CHO-DG44 cell (ATCCNO.PTA-3356), and the like. In some embodiments, the host cell is a COS cell. In some embodiments, the host cell is a 293 cell. In some embodiments, the host cell is a CHO cell.

Suitable yeast cells include, but are not limited to, Pichia pastoris (Pichia pastoris), Pichia finlandica (Pichia finlandica), Pichia trehalase (Pichia pastoris), Pichia pastoris (Pichia pastoris), Pichia salicicola (Pichia villosa), Pichia pastoris (Pichia pastoris), Pichia methanolica (Pichia methanolica), Pichia pastoris (Pichia pastoris), Pichia pastoris (Pichia sp), Saccharomyces cerevisiae, Saccharomyces polymorpha (Hansenula polymorpha), Pichia pastoris (Pichia pastoris), Aspergillus niger (Aspergillus niger, Aspergillus, Trichoderma reesei (Trichoderma reesei), Microsporum aurantium (Chrysosporium lucknowense), certain species of Neurospora (Fusarium sp.), Fusarium graminearum (Fusarium gramineum), Fusarium venenatum (Fusarium venenatum), Neurospora crassa (Neurospora crassa), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), and the like. In some embodiments, the host cell is a saccharomyces. In some embodiments, the host cell is of the genus pichia.

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, and the like. See, e.g., Carrier et al, (1992) J.Immunol.148: 1176-1181; U.S. patent nos. 6,447,784; and Sizemore et al, (1995) Science 270: 299-302. Typically, the laboratory strain is a non-pathogenic strain. In some embodiments, the host cell is e. In some embodiments, the host cell is bacillus subtilis.

Pharmaceutical composition

The present 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. By "effective amount" is meant a dose sufficient to produce a desired result, e.g., a reduction in adverse symptoms associated with a complement-mediated disease or disorder, an amelioration of symptoms of a complement-mediated disease or disorder, a slowing of the progression of a complement-mediated disease or disorder, etc. The desired result is generally at least a reduction in the symptoms of the complement-mediated disease or disorder compared to a control. In some embodiments, a subject antibody is formulated and/or modified to enable the antibody to cross the blood brain barrier. In some embodiments, the subject antibodies are delivered in a manner that avoids the blood brain barrier. In some embodiments, an anti-C1 s antibody of the present disclosure is formulated with an agent that facilitates crossing the blood brain barrier. In some embodiments, the subject antibodies are fused, directly or through a linker, to a compound that facilitates crossing the blood brain barrier.

Preparation

In the subject methods, the subject antibodies can be administered to the host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Thus, agents can be incorporated into a variety of formulations for therapeutic administration. More specifically, the subject antibodies may be formulated into pharmaceutical compositions by combination with an appropriate pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, or other pharmaceutically acceptable excipient, and may be formulated into formulations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols. In some embodiments, the pharmaceutical composition comprises the subject antibody and a pharmaceutically acceptable excipient.

In pharmaceutical dosage forms, the subject antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The following methods and excipients are exemplary only, and not in any way limiting.

For oral formulations, the subject antibodies may be used alone or in combination with suitable 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, acacia, corn starch or gelatin; disintegrants, such as corn starch, potato starch or sodium carboxymethyl cellulose; lubricants, such as talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives and flavoring agents.

The subject antibodies can be formulated as an injectable formulation 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 fatty acid glycerides, injectable organic esters (e.g., ethyl oleate), higher fatty acid esters or propylene glycol esters; and if necessary together with conventional additives such as solubilizers, isotonizing agents, suspending agents, emulsifiers, stabilizers and preservatives. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. In addition, the pharmaceutical compositions of the present disclosure may additionally comprise other agents, such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

Pharmaceutical compositions comprising the subject antibodies are prepared by mixing the subject antibodies of the desired purity with optional physiologically acceptable carriers, other excipients, stabilizers, surfactants, buffers, and/or tonicity agents. Acceptable carriers, other excipients, and/or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, 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, sorbose, raffinose, glucosamine, N-methylglucamine, galactosamine, and neuraminic acid; and/or a non-ionic surfactant such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

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

Exemplary antibody concentrations in the subject pharmaceutical compositions can range from about 1mg/mL to about 200mg/mL, or from about 50mg/mL to about 200mg/mL, or from about 150mg/mL to about 200 mg/mL.

Aqueous formulations of the antibodies may be prepared in a pH buffered solution, for example, at a pH in the range of about 4.0 to about 7.0 or about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for a pH in this range include phosphate, histidine, citrate, succinate, acetate buffers and other organic acid buffers. The buffer concentration may be from about 1mM to about 100mM, or from about 5mM to about 50mM, depending on, for example, the desired tonicity of the buffer and formulation.

A tonicity agent may be included in the antibody formulation to adjust the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may be suitable. The term "isotonic" means a solution having the same tonicity as some other solution with which it is compared, such as physiological saline solution or serum. The tonicity agent may be used in an amount of about 5mM to about 350mM, for example 100mM to 350 nM.

Surfactants may also be added to the antibody formulation to reduce aggregation of the formulated antibody and/or minimize particle formation in the formulation 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). An example of a suitable polyoxyethylene sorbitan-fatty acid ester is polysorbate 20 (under the trade mark Tween 20)^TMSold) and polysorbate 80 (under the trademark Tween 80)^TMSales). Examples of suitable polyethylene-polypropylene copolymers are by nameF68 or Poloxamer188^TMThose that are sold. An example of a suitable polyoxyethylene alkyl ether is the trademark Brij^TMThose that are sold. Exemplary concentrations of the surfactant may range from about 0.001% to about 1% w/v.

Lyophilization protecting groups may also be added to protect labile active ingredients (e.g., proteins) against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant may be included in an amount of about 10mM to 500 nM.

In some embodiments, a subject formulation includes a subject antibody, and one or more of the above-identified agents (e.g., surfactants, buffers, stabilizers, tonicity agents) and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloromethylcresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, preservatives are included in the formulations, for example, at concentrations ranging from about 0.001 to about 2% (w/v).

For example, the subject formulations may be liquid or lyophilized formulations suitable for parenteral administration, and may comprise: about 1mg/mL to about 200mg/mL of the subject antibody; from about 0.001% to about 1% of at least one surfactant; about 1mM to about 100mM of a buffering agent; optionally about 10mM to about 500mM of a stabilizer; and about 5mM to about 305mM of a tonicity agent; and has a pH of about 4.0 to about 7.0.

As another example, the subject parenteral formulation is a liquid or lyophilized formulation comprising: about 1mg/mL to about 200mg/mL of the subject antibody; 0.04% Tween20 w/v; 20mM L-histidine; and 250mM sucrose; and has a pH of 5.5.

As another example, the subject parenteral formulations comprise a lyophilized formulation comprising: 1)15mg/mL of the subject antibody; 0.04% Tween20 w/v; 20mM L-histidine; and 250mM sucrose; and has a pH of 5.5; or 2)75mg/mL of the subject antibody; 0.04% Tween20 w/v; 20mM L-histidine; and 250mM sucrose; and has a pH of 5.5; or 3)75mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM sucrose; and has a pH of 5.5; or 4)75mg/mL of the subject antibody; 0.04% Tween20 w/v; 20mM L-histidine; and 250mM trehalose; and has a pH of 5.5; or 5)75mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM trehalose; and has a pH of 5.5.

As another example, the subject parenteral formulation is a liquid formulation comprising: 1)7.5mg/mL of the subject antibody; 0.02% Tween20 w/v; 120mM L-histidine; and 250125 mM sucrose; and has a pH of 5.5; or 2)37.5mg/mL of the subject antibody; 0.02% Tween20 w/v; 10mM L-histidine; and 125mM sucrose; and has a pH of 5.5; or 3)37.5mg/mL of the subject antibody; 0.01% Tween20 w/v; 10mM L-histidine; and 125mM sucrose; and has a pH of 5.5; or 4)37.5mg/mL of the subject antibody; 0.02% Tween20 w/v; 10mM L-histidine; 125mM trehalose; and has a pH of 5.5; or 5)37.5mg/mL of the subject antibody; 0.01% Tween20 w/v; 10mM L-histidine; and 125mM trehalose; and has a pH of 5.5; or 6)5mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM trehalose; and has a pH of 5.5; or 7)75mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM mannitol; and has a pH of 5.5; or 8)75mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM Lhistidine; and 140mM sodium chloride; and has a pH of 5.5; or 9)150mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM trehalose; and has a pH of 5.5; or 10)150mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 250mM mannitol; and has a pH of 5.5; or 11)150mg/mL of the subject antibody; 0.02% Tween20 w/v; 20mM L-histidine; and 140mM sodium chloride; and has a pH of 5.5; or 12)10mg/mL of the subject antibody; 0.01% Tween20 w/v; 20mM L-histidine; and 40mM sodium chloride; and has a pH of 5.5.

The subject antibodies may be utilized in aerosol formulations for administration via inhalation. The subject antibodies can be formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservatives and isotonicity agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

In addition, the subject antibodies can be formulated as suppositories by mixing with various bases such as emulsifying bases or water-soluble bases. The subject antibodies may be administered rectally via suppositories. Suppositories may include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature but still solidify at room temperature.

Unit dosage forms for oral or rectal administration may be provided, such as syrups, elixirs and suspensions, wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may comprise the subject antibody in a composition in the form of a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of an anti-C1 s antibody of the present disclosure, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specification of the subject antibody may depend on the particular antibody used and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

Other modes of administration will also be useful in the methods of the present disclosure. For example, the subject antibodies can be formulated as suppositories, and in some cases, as aerosols and intranasal compositions. For suppositories, the vehicle composition will include conventional binders and carriers such as polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), for example about 1% to about 2%.

Intranasal formulations will generally include vehicles that do not cause irritation to the nasal mucosa, nor do they significantly interfere with ciliary function. A diluent such as water, saline solution, or other known substances may be used. Nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance absorption of the subject antibody by the nasal mucosa.

The subject antibodies may be administered in injectable formulations. Generally, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection may also be prepared. The formulation may also be emulsified or the antibody encapsulated in a liposome vehicle.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the vehicle may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents, if desired. The actual methods of making such dosage forms are known or will be apparent to those skilled in the art.See, e.g.Remington's pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17 th edition, 1985. The composition or formulation to be administered will in any case contain the subject antibody in an amount sufficient to achieve the desired state in the subject being treated.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents and the like, are readily available to the public.

In some embodiments, the subject antibodies are formulated in a controlled release formulation. Sustained release formulations can be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly D- (-) -3-hydroxybutyric acid. Possible loss of biological activity and possible change in immunogenicity of antibodies contained in sustained release formulations can be prevented by the use of appropriate additives, by controlling the moisture content and by the development of specific polymer matrix compositions.

Controlled release within the scope of the present disclosure may be considered to refer to any of a variety of extended release dosage forms. For the purposes of this disclosure, the following terms may be considered to be substantially equivalent to controlled release: continuous release, controlled release, delayed release, depot, extended release, gradual release, immediate release, long-term release, programmed release, extended release, moderate release, sustained release, depot, delayed, slow release, intermittent release, sustained release, timed coating, timed release, delayed action, extended action, layered timed action, long-term action, extended action, repeated action, slow action, sustained action, and sustained action. Further discussion of these terms can be found in Lesczek Krowczynski,Extended-Release Dosage Forms1987(CRC Press, Inc.).

Various controlled release technologies encompass a very wide range of pharmaceutical dosage forms. Controlled release techniques include, but are not limited to, physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems with rate controlling membranes, such as micro-encapsulation, macro-encapsulation, and membrane systems; reservoir systems without rate controlling membranes, such as hollow fibers, ultra microporous cellulose triacetate, and porous polymer substrates and foams; integral systems, including those systems that are physically dissolved in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, environmental agent invading and degradable), and materials that are physically dispersed in a non-porous, polymeric, or elastomeric matrix (e.g., non-erodible, environmental agent invading and degradable); a layered structure comprising a reservoir chemically similar or dissimilar to the outer control layer; and other physical methods such as osmotic pumps, or adsorption onto ion exchange resins.

Chemical systems include, but are not limited to, chemical erosion (e.g., heterogeneous or homogeneous erosion) of the polymer matrix, or biological erosion (e.g., heterogeneous or homogeneous) of the polymer matrix. Other discussions of the category of controlled release systems can be found in Agis f. kydonious,Controlled Release Technologies:Methods,Theory and Applications1980(CRC Press, Inc.).

There are a variety of controlled release pharmaceutical formulations developed for oral administration. These include, but are not limited to, osmotic pressure controlled gastrointestinal delivery systems; a hydrodynamic pressure controlled gastrointestinal delivery system; a membrane permeation control gastrointestinal delivery system comprising a microporous membrane permeation control gastrointestinal delivery device; gastric juice resistant enteric-targeted controlled release gastrointestinal delivery devices; a gel diffusion controlled gastrointestinal delivery system; and ion exchange controlled gastrointestinal delivery systems, including cationic and anionic drugs. Additional information on controlled release drug delivery systems can be found at Yie w.Novel Drug Delivery Systems,1992(MarcelDekker,Inc.).

Dosage form

The appropriate dosage can be determined by the attending physician or other qualified medical professional based on a variety of clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the particular compound to be administered, the patient's sex, time and route of administration, general health, and other drugs being administered concurrently. The subject antibody may be administered in an amount of from 1ng/kg body weight to 20mg/kg body weight, such as from 0.1mg/kg body weight to 10mg/kg body weight, such as from 0.5mg/kg body weight to 5mg/kg body weight per dose; however, doses below or above this exemplary range are contemplated, particularly in view of the foregoing factors. If the regimen is a continuous infusion, it may also be in the range of 1 μ g to 10mg per kg body weight per minute.

In some embodiments, the dose of the subject anti-C1 s antibody is in the range of 0.001 μ g to 1000 μ g; however, doses below or above this exemplary range are contemplated, particularly in view of the foregoing factors. In some embodiments, the dose can be in a range of, e.g., about 0.0001 to 100mg/kg, or about 0.01 to 5mg/kg (e.g., 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, etc.) of body weight. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight, or in the range of 1-10mg/kg, or at least 1 mg/kg. Intermediate doses within the above ranges are also contemplated within the scope of the invention.

In some embodiments, the subject anti-C1 s antibody is administered in an amount that provides a peak serum concentration of about 1 μ g/ml to about 1mg/ml, e.g., about 1 μ g/ml to about 2.5 μ g/ml, about 2.5 μ g/ml to about 5 μ g/ml, about 5 μ g/ml to about 7.5 μ g/ml, about 7.5 μ g/ml to about 10 μ g/ml, about 10 μ g/ml to about 25 μ g/ml, about 25 μ g/ml to about 50 μ g/ml, about 50 μ g/ml to about 100 μ g/ml, about 100 μ g/ml to about 250 μ g/ml, about 250 μ g/ml to about 500 μ g/ml, about 500 μ g/ml to about 750 μ g/ml, or about 750 μ g/ml to about 1000 μ g/ml. In some embodiments, the subject anti-C1 s antibody is administered in an amount that provides a peak serum concentration greater than 1mg/ml, e.g., from about 1mg/ml to about 2mg/ml, from about 2mg/ml to about 5mg/ml, or from about 5mg/ml to about 10 mg/ml.

Such doses may be administered to an individual daily, every other day, weekly, or any other schedule determined from empirical analysis. Exemplary treatments include administration in multiple doses over an extended period of, for example, at least six months. Other exemplary data regimens include administration once every two weeks or once a month or once every 3 to 6 months. Exemplary dosing schedules include 1-10mg/kg or 15mg/kg daily, 30mg/kg every other day, or 60mg/kg once a week. 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 indicated ranges. Progress can be monitored by periodic assessment.

One skilled in the art will readily appreciate that the dosage level and schedule of administration may vary with the particular antibody, the severity of the symptoms, and the subject's susceptibility to side effects. The preferred dosage and schedule of administration for a given compound is readily determined by one of skill in the art by a variety of means.

Route of administration

The subject antibodies are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intrathecal, intracranial, subcutaneous, intradermal, topical, intravenous, intraperitoneal, intraarterial (e.g., via the carotid artery), spinal or brain delivery, rectal, nasal, oral, and other enteral and parenteral routes of administration. The routes of administration can be combined, if desired, or adjusted according to the antibody and/or desired effect. The subject antibody compositions can be administered in a single dose or in multiple doses. In some embodiments, the subject antibody compositions are administered orally. In some embodiments, the subject antibody compositions are administered via the inhalation route. In some embodiments, the subject antibody compositions are administered intranasally. In some embodiments, the subject antibody compositions are administered topically. In some embodiments, the subject antibody compositions are administered transcranially. In some embodiments, the subject antibody compositions are administered intravenously. In some embodiments, the subject antibody compositions are administered intrathecally.

The antibodies of the present disclosure can be administered to a host using any of the conventional methods and routes available for delivering conventional drugs, including systemic or local routes. In general, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

Parenteral routes of administration other than administration by inhalation include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrathecal and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be performed to achieve systemic or local delivery of the subject antibodies. When systemic delivery is desired, administration typically involves invasive administration of the pharmaceutical formulation or local or mucosal administration of systemic absorption.

The subject antibodies may also be delivered to a subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., administration of suppositories).

Treatment means at least an improvement in the symptoms associated with the pathological condition afflicting the host, wherein improvement is used in a broad sense to mean at least a reduction in the magnitude of a parameter (e.g., symptom) associated with the pathological condition being treated, such as a complement-mediated disease or disorder. Thus, treatment also includes the following: wherein the pathological condition, or at least the symptoms associated therewith, is completely inhibited, e.g., prevented from occurring or ceasing (e.g., terminating), such that the host no longer suffers from the pathological condition, or at least the symptoms characteristic of the pathological condition.

In some embodiments, the subject antibodies are administered to a site in a cerebral artery or directly into brain tissue, e.g., by injection and/or delivery. The subject antibodies may also be administered directly to the target site, e.g., delivered to the target site by biolistic methods.

A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject", "individual" and "patient") can be treated according to the subject methods. Generally, these hosts are "mammals" or "mammalian", where these terms are used broadly to describe organisms within the mammalian class, including the orders carnivora (e.g., cats), grazing (e.g., cows, 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 having 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, work animal, zoo animal or laboratory animal that contains the complement system. In some embodiments, the host is a human.

Embodiments include compositions comprising a container suitable for containing a composition comprising the subject C1s antibody for administration to an individual. For example, the subject antibodies can be placed in a container suitable for containing a pharmaceutical composition. The container may be, for example, a bottle (e.g., with a closure device such as a cap), a blister pack (e.g., which may provide closure of one or more doses per blister), a vial, a flexible package (e.g., a sealed Mylar or plastic bag), an ampoule (for single doses in solution), a dropper, a syringe, a film, a tube, and the like. In some embodiments, a 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.

Kits are provided having a unit dose of the subject antibody, e.g., in an oral or injectable dose. In such kits, in addition to the container containing the unit dose, will be an information package insert describing the use and concomitant benefits of the antibody for treating the pathological condition of interest. Preferred compounds and unit doses are those described above.

Methods of treating complement-mediated diseases or disorders

The present disclosure provides methods of treating complement-mediated diseases or disorders. The methods generally comprise administering to an individual in need thereof an effective amount of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising the antibody. In some cases, administration of the subject anti-C1 s antibodies can modulate complement C1s activity in a cell, tissue, or fluid of the individual and treat the complement-mediated disease or disorder. The present disclosure provides methods of inhibiting activation of complement component C4 in an individual, comprising administering to the individual an effective amount of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising the antibody. The present disclosure provides methods of inhibiting complement C1s activity in an individual comprising administering to the individual an effective amount of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising the antibody.

In some embodiments, the methods of the present disclosure for treating an individual having a complement-mediated disease or disorder comprise administering to the individual an effective amount of an anti-C1 s antibody of the present disclosure or an effective amount of a pharmaceutical composition comprising: a) an anti-C1 s antibody of the present disclosure; and a pharmaceutically acceptable excipient suitable for administration to the subject. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration can be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, the administration is intravenous administration. In some embodiments, the administering is intrathecal. In some embodiments, the administering is subcutaneous administration.

In some embodiments, an "effective amount" of an anti-C1 s antibody of the present disclosure or of a subject pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure is an amount that, when administered to an individual in need thereof in one or more doses, reduces C4b2a production in the individual (or in a tissue or organ of the individual) 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., complement C4b and C2a complex; also referred to as "C3 convertase") produced in the individual or tissue or organ in the absence of the subject anti-C1 s antibody. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration can be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, the administration is intravenous administration. In some embodiments, the route of administration is intrathecal. In some embodiments, the route of administration is intravenous administration. In some embodiments, the route of administration is subcutaneous administration.

The present disclosure provides methods of modulating complement activation. In some embodiments, the method inhibits complement activation, e.g., reduces C4b2a production. In some embodiments, the present disclosure provides a method of modulating complement activation in an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an anti-C1 s antibody of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the pharmaceutical composition comprises an anti-C1 s antibody of the present 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 can be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, the administration is intravenous administration. In some embodiments, the administering is intrathecal.

A complement-mediated disease or disorder is a disorder characterized by an abnormal amount of complement C1s or abnormal levels of complement C1s proteolytic activity in a cell, tissue, or fluid of an individual.

In some cases, the complement-mediated disease or disorder is characterized by the presence of an elevated (above-normal) amount of C1s or an elevated level of complement C1s activity in a cell, tissue, or fluid. For example, in some cases, a complement-mediated disease or disorder is characterized by the presence of elevated amounts of C1s and/or elevated C1s activity in brain tissue and/or cerebrospinal fluid. A "higher than normal" amount of C1s in a cell, tissue, or fluid indicates that the amount of C1s in a cell, tissue, or fluid is higher than a normal control level, e.g., higher than a normal control level for an individual or population of individuals of the same age group. An "above-normal" level of C1s activity in a cell, tissue, or fluid indicates that proteolytic cleavage by C1s is above a normal control level in the cell, tissue, or fluid, e.g., above a normal control level for an individual or a population of individuals of the same age group. In some cases, an individual having a complement-mediated disease or disorder exhibits one or more other symptoms of such disease or disorder.

In other instances, the complement-mediated disease or disorder is characterized by the presence of a sub-normal amount of C1s or a lower level of complement C1s activity in a cell, tissue, or fluid. For example, in some cases, a complement-mediated disease or disorder is characterized by the presence of lower amounts of C1s and/or lower C1s activity in brain tissue and/or cerebrospinal fluid. A "less than normal" amount of C1s in a cell, tissue, or fluid indicates that the amount of C1s in a cell, tissue, or fluid is less than a normal control level, e.g., less than a normal control level for an individual or population of individuals of the same age group. A "below normal" level of C1s activity in a cell, tissue, or fluid indicates that proteolytic cleavage by C1s is below a normal control level in the cell, tissue, or fluid, e.g., below a normal control level for an individual or population of individuals of the same age group. In some cases, an individual having a complement-mediated disease or disorder exhibits one or more other symptoms of such disease or disorder.

A complement-mediated disease or disorder is one in which the amount or activity of complement C1s, for example, contributes to the disease or disorder in an individual. In some embodiments, the complement-mediated disease or disorder is selected from the group consisting of an autoimmune disease, a cancer, a hematologic disease, an infectious disease, an inflammatory disease, an ischemia-reperfusion injury, a neurodegenerative disease, a neurodegenerative disorder, an ocular disease, a renal disease, transplant rejection, a vascular disease, and a vasculitic disease. In some embodiments, the complement-mediated disease or disorder is an autoimmune disease. In some embodiments, the complement-mediated disease or disorder is cancer. In some embodiments, the complement-mediated disease or disorder is an infectious disease. In some embodiments, the complement-mediated disease or disorder is an inflammatory disease. In some embodiments, the complement-mediated disease or disorder is a hematological disease. In some embodiments, the complement-mediated disease or disorder is ischemia-reperfusion injury. In some embodiments, the complement-mediated disease or disorder is an ocular disease. In some embodiments, the complement-mediated disease or disorder is a kidney disease. In some embodiments, the complement-mediated disease or disorder is transplant rejection. In some embodiments, the complement-mediated disease or disorder is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease or disorder is a vascular disease. In some embodiments, the complement-mediated disease or disorder is an inflammatory disease disorder. In some embodiments, the complement-mediated disease or disorder is a neurodegenerative disease or disorder. In some embodiments, the complement-mediated disease is a neurodegenerative disease. In some embodiments, the complement-mediated disorder is a neurodegenerative disorder. In some embodiments, the complement-mediated disease or disorder is tauopathy.

Examples of complement-mediated diseases or disorders include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, dementia with silvery particles, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's diseaseAmyloid angiopathy of the british type, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease), Crohn's disease (Crohn's disease), cryoglobulinemia vasculitis, boxer dementia, lewy body dementia (dementia with lewy bodies, DLB), diffuse neurofibrillar with calcification, discoid lupus erythematosus, Down's syndrome (Down's disease), focal segmental glomerulosclerosis, form thought disorder, temporal dementia (FTD), barotemporal dementia with parkinsonism linked to chromosome 17, temporal lobar degeneration, getmann-scheinsler-strainer disease, gillin-gillett syndrome (gill-lance-schnei-s disease), gill-schwanner-schleme disease (schwerner-schlem-s-schlem-s), neurone disease (crohnson-schwanner-s-syndrome), neurone disease (crohnson-schwanner disease) associated with chromosome 17, morse, morbikurt, Hemolytic uremic syndrome, hereditary angioedema, hypophosphatasia (hypophosphatasis), idiopathic pneumonia syndrome, immune complex diseases, inclusion body myositis, infectious diseases (e.g., diseases caused by bacteria (e.g., Neisseria meningitidis (Neisseria meningitidis) or Streptococcus (Streptococcus)), viruses (e.g., Human Immunodeficiency Virus (HIV)) or other infectious agents), inflammatory diseases, ischemia/reperfusion injury, mild cognitive impairment, Immune Thrombocytopenic Purpura (ITP), molybdenum cofactor deficiency type a (MoCD), type I membranoproliferative glomerulonephritis (MPGN), type II membranoproliferative glomerulonephritis (MPGN) (compact deposit disease), membranous nephritis, multi-infarct dementia, lupus (e.g., Systemic Lupus Erythematosus (SLE)), glomerulonephritis, Kawasaki disease (Kawasaki disease), multifocal motor neuropathy, Multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C (Niemann-Pick disease), non-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic parkinsonism, polymyositis, prion protein cerebral amyloid angiopathy, progressive progression of cerebral amyloid angiopathyProgressive subcortical hyperplasia, progressive supranuclear palsy, psoriasis, sepsis, Shiga-toxin E coli (STEC) -HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, tangle-only dementia, graft rejection, vasculitis (e.g., ANCA-related vasculitis), wegener's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, primary mixed cryoglobulinemia, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, acquired epidermolysis bullosa, delayed hemolytic transfusion response, low complement blood disease urticaria vasculitis syndrome, pseudocrystalloid bullous keratopathy, and platelet transfusion ineffectiveness (pateletrefractorins).

Alzheimer's disease and some forms of frontotemporal dementia (pick's disease, sporadic frontotemporal dementia and frontotemporal dementia with parkinsonism linked to chromosome 17) are the most common forms of tauopathies. Accordingly, the present invention relates to any of the methods as described above, wherein said tauopathy is alzheimer's disease, pick's disease, sporadic frontotemporal dementia and frontotemporal dementia with parkinson's disease associated with chromosome 17. Other tauopathies include, but are not limited to, Progressive Supranuclear Palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

Neurodegenerative tauopathies include Alzheimer's disease, amyotrophic lateral sclerosis/Parkinson's disease complex, dementia with silverfish particles, English amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, frontotemporal dementia with Parkinson's disease associated with chromosome 17, frontotemporal lobal degeneration, Gerstmann-Straussler-Scheinker disease, Harlervorden-Spatz disease, somatic myositis, multiple system atrophy, myotonic dystrophy, Niemann-pick C disease, non-Guamanin motor neuron disease with neurofibrillary tangles, pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, ischemic stroke, Chronic Traumatic Encephalopathy (CTE), Traumatic Brain Injury (TBI), and stroke.

The present disclosure also provides methods of treating synucleinopathies such as Parkinson's Disease (PD); dementia with lewy bodies (DLB); multiple System Atrophy (MSA), and the like. For example, PD (PDD) associated with dementia can be treated with the subject methods.

In some embodiments, the complement-mediated disease or disorder comprises alzheimer's disease. In some embodiments, the complement-mediated disease or disorder comprises parkinson's disease. In some embodiments, the complement-mediated disease or disorder comprises transplant rejection. In some embodiments, the complement-mediated disease or disorder is antibody-mediated transplant rejection.

In some embodiments, an anti-C1 s antibody of the present disclosure prevents or delays the onset of at least one symptom of a complement-mediated disease or disorder in an individual. In some embodiments, an anti-C1 s antibody of the present disclosure reduces or eliminates at least one symptom of a complement-mediated disease or disorder in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune diseases, cancer, hematologic diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, kidney diseases, transplant rejection, ocular diseases, vascular diseases, or vasculitic disorders. The symptom may be a neurological symptom, e.g., impaired cognitive function, memory impairment, loss of motor function, etc. The condition may also be C1s protein activity in a cell, tissue, or fluid of the individual. The symptom may also be the extent of complement activation in the cells, tissues or fluids of the individual.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual can modulate complement activation in a cell, tissue, or fluid of the individual. In some embodiments, administration of the subject anti-C1 s antibody to an individual can inhibit complement activation in a cell, tissue, or fluid of the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, 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 90% or more, compared to complement activation in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, the anti-C1 s antibodies of the present disclosure reduce deposition of C3 on red blood cells; for example, in some embodiments, an anti-C1 s antibody of the present disclosure reduces deposition of C3b, iC3b, and the like on RBCs. In some embodiments, an anti-C1 s antibody of the present disclosure inhibits complement-mediated red blood cell lysis.

In some embodiments, an anti-C1 s antibody of the present disclosure reduces deposition of C3 on platelets; for example, in some embodiments, an anti-C1 s antibody of the present disclosure reduces deposition of C3b, iC3b, and the like on platelets.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure results in a result selected from the group consisting of: (a) a decrease in complement activation; (b) improvement in cognitive function; (c) a reduction in neuronal loss; (d) a decrease in the level of phosphorylated Tau protein in neurons; (e) a reduction in glial cell activation; (f) a reduction in lymphocyte infiltration; (g) reduction in macrophage infiltration; (h) reduction in antibody deposition; (i) a reduction in glial cell loss; (j) a reduction in oligodendrocyte loss; (k) reduction of dendritic cell infiltration; (l) Reduction of neutrophil infiltration; (m) reduction in red blood cell lysis; (n) a reduction in phagocytosis of red blood cells; (o) a reduction in platelet phagocytosis; (p) reduction in platelet lysis; (q) an increase in graft survival; (r) a decrease in macrophage-mediated phagocytosis; (s) improvement of vision; (t) improvement in motion control; (u) improvement of thrombosis; (v) improvement of blood coagulation; (w) improvement in renal function; (x) A decrease in antibody-mediated complement activation; (y) a reduction in autoantibody-mediated complement activation; (z) amelioration of anemia; (aa) reduction of demyelination; (ab) reduction in eosinophilia; (ac) a reduction in deposition of C3 on red blood cells (e.g., a reduction in deposition of C3b, iC3b, etc. on RBCs); and (ad) a reduction in the deposition of C3 on platelets (e.g., a reduction in the deposition of C3b, iC3b, etc. on platelets); and (ae) reduction in anaphylatoxin production; (af) reduction of autoantibody-mediated blister formation; (ag) reduction of autoantibody induced pruritus; (ah) a reduction in autoantibody induced lupus erythematosus; (ai) reduction of autoantibody mediated skin erosion; (aj) a reduction in red blood cell destruction due to transfusion reactions; (ak) reduction of red blood cell lysis due to alloantibodies; (al) reduction of hemolysis due to infusion reactions; (am) reduction of allogeneic antibody-mediated platelet lysis; (an) reduction in platelet lysis due to infusion reaction; (ao) reduction in mast cell activation; (ap) reduction of histamine release from mast cells; (aq) a decrease in vascular permeability; (ar) reduction of edema; (as) reduction of complement deposition on graft endothelium; (at) a reduction in anaphylatoxin production in the graft endothelium; (au) reduction of separation of the dermal-epidermal junction; (av) a reduction in anaphylatoxin production in the dermoepidermal junction; (aw) a reduction in alloantibody-mediated complement activation in the graft endothelium; (ax) reduction of antibody-mediated loss at the neuromuscular junction; (ay) a reduction in complement activation at the neuromuscular junction; (az) a reduction in anaphylatoxin production at the neuromuscular junction; (ba) a reduction in complement deposition at the neuromuscular junction; (bb) reduction of paralysis; (bc) reduced numbness; (bd) increased bladder control; (be) increased bowel movement control; (bf) a reduction in mortality associated with autoantibodies; and (bg) a decrease in the incidence associated with autoantibodies.

In some embodiments, when the subject anti-C1 s antibody is administered as a monotherapy or a combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, 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 results is achieved, as compared to the level or extent of the results in the individual prior to treatment with the anti-C1 s antibody: (a) complement activation; (b) decline in cognitive function; (c) neuronal loss; (d) phosphorylated Tau levels in neurons; (e) activation of glial cells; (f) lymphocyte infiltration; (g) infiltrating macrophages; (h) antibody deposition; (i) loss of glial cells; (j) oligodendrocyte loss; (k) infiltration of dendritic cells; (l) Neutrophil infiltration; (m) red blood cell lysis; (n) red blood cell phagocytosis; (o) platelet phagocytosis; (p) platelet lysis; (q) graft rejection; (r) macrophage-mediated phagocytosis; (s) loss of vision; (t) antibody-mediated complement activation; (u) autoantibody mediated complement activation; (v) demyelination; (w) eosinophilia.

In some embodiments, when the subject anti-C1 s antibody is administered as a monotherapy or a combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, 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 90% or more of one or more of the following results can be achieved as compared to the level or extent of the results in the individual prior to treatment with the anti-C1 s antibody: a) cognitive function; b) graft survival rate; c) eyesight; d) controlling the motion; e) thrombosis; f) blood coagulation; g) renal function; and h) hematocrit (red blood cell count).

In some embodiments, administration of an anti-C1 s antibody of the disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, complement activation in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to complement activation in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure increases cognitive function in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, cognitive function in the individual is increased 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% as compared to cognitive function in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure reduces the rate of decline of cognitive function in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, the rate of decline of cognitive function in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to the rate of decline of cognitive function in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces neuronal loss in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, neuronal loss in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to neuronal loss in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administering to an individual an anti-C1 s antibody of the present disclosure reduces the level of phosphorylated Tau in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, the phosphorylated Tau in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to the level of phosphorylated Tau in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces glial activation in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as a monotherapy or a combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, 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% as compared to glial cell activation in the individual prior to treatment with the anti-C1 s antibody. In some embodiments, the glial cell is an astrocyte or microglia.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces lymphocyte infiltration in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as a monotherapy or a combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, lymphocyte infiltration in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to lymphocyte infiltration in an individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces macrophage infiltration in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, macrophage infiltration in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to macrophage infiltration in an individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, antibody deposition in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to antibody deposition in the individual prior to treatment with the anti-C1 s antibody.

In some embodiments, administration of an anti-C1 s antibody of the present disclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a) production in the individual. For example, in some embodiments, when the subject anti-C1 s antibody is administered as monotherapy or as combination therapy in one or more doses to an individual having a complement-mediated disease or disorder, anaphylatoxin production in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% as compared to the level of anaphylatoxin production in the individual prior to treatment with the anti-C1 s antibody.

The present disclosure provides for the use of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient in the treatment of an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides for the use of an anti-C1 s antibody of the present disclosure in treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides for the use of a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient in the treatment of an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of the disclosure in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation. In some embodiments, the present disclosure provides the use of an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of an anti-C1 s antibody of the present disclosure for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides for the use of a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder.

The present disclosure provides for the use of an anti-C1 s antibody of the present disclosure for the manufacture of a medicament for modulating complement activation. In some embodiments, the drug inhibits complement activation. In some embodiments, the drug inhibits complement activation in an individual having a complement-mediated disease or disorder.

The present disclosure provides an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in medical treatment. In some embodiments, the present disclosure provides anti-C1 s antibodies of the present disclosure for use in medical therapy. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in medical treatment.

The present disclosure provides an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in treating an individual having a complement-mediated disease or disorder. In some embodiments, the disclosure provides an anti-C1 s antibody of the disclosure for use in treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in treating an individual having a complement-mediated disease or disorder.

The present disclosure provides an anti-C1 s antibody of the present disclosure or a pharmaceutical composition comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for use in modulating complement activation. In some embodiments, the disclosure provides anti-C1 s antibodies of the disclosure for use in modulating complement activation. In some embodiments, the present disclosure provides pharmaceutical compositions comprising an anti-C1 s antibody of the present disclosure and a pharmaceutically acceptable excipient for modulating complement activation. In some embodiments, the anti-C1 s antibody inhibits complement activation.

Combination therapy

The anti-C1 s antibodies of the present disclosure can be administered alone (e.g., in the form of a monotherapy); or administered to an individual in need thereof in combination therapy with one or more other therapeutic agents.

for the treatment of AD, suitable additional therapeutic agents include, but are not limited to, acetylcholinesterase inhibitors, including, but not limited to, Aricept (donepezil), Exelon (rivastigmine), metribuzin (metrifonate), and tacrine (Cognex), anti-a β antibodies, non-steroidal anti-inflammatory agents, including, but not limited to, ibuprofen (ibuprofen) and indomethacin (indomethacin), cyclooxygenase-2 (Cox2) inhibitors, such as celecoxib (Celebrex), and monoamine oxidase inhibitors, such as Selegilene (eldeprenyl).

Another suitable additional therapeutic agent for the treatment of AD is an agent that inhibits tau protein aggregation, for example a naphthoquinone derivative that inhibits tau aggregation, as described in U.S. patent No.7,605,179. Another suitable additional therapeutic agent is an agent that inhibits phosphorylation of tau, for example, a 3-substituted-4-pyrimidinone derivative that inhibits tau protein kinase 1, as described in U.S. Pat. No.7,572,793.

As used herein, "in combination with … …" means used as follows: wherein the first compound is administered, for example, during the entire course of administering the second compound; wherein the first compound is administered within a period of time that overlaps with the administration of the second compound, for example wherein administration of the first compound begins before administration of the second compound and administration of the first compound ends before the end of administration of the second compound; wherein administration of the second compound begins before administration of the first compound and administration of the second compound ends before administration of the first compound ends; wherein administration of the first compound begins before administration of the second compound begins and administration of the second compound ends before administration of the first compound ends; wherein administration of the second compound begins before administration of the first compound begins and administration of the first compound ends before administration of the second compound ends. Thus, "combination" may also refer to a regimen that includes the administration of two or more compounds. As used herein, "in combination with … …" also refers to the administration of two or more compounds that can be administered in the same or different formulations, by the same or different routes, and in the same or different dosage form types.

The individual to be treated

Individuals suitable for treatment with the subject anti-C1 s antibodies include individuals that have been diagnosed as having a complement-mediated disease or disorder; an individual at greater risk of developing a complement-mediated disease or disorder than the general population (e.g., an individual having a genetic predisposition to develop a complement-mediated disease or disorder); (ii) an individual with parkinson's disease (PDD) with dementia; a subject with alzheimer's disease; and so on. In some cases, the individual is an adult. In some cases, the adult is 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, or 80 years or older. For example, the adult may 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 individual is a human child. In some cases, the human child is less than 20 years old, less than 10 years old, or less than 5 years old.

In vitro testing and animal models

The present disclosure provides methods of testing the efficacy of the subject antibodies in vitro or in vivo. In vitro tests include methods of determining the binding of the subject antibody to complement C1s protein, methods of determining the ability of the subject antibody to inhibit the production of the C4b2a complex, methods of identifying the epitope or epitope characteristic of the anti-C1 s antibodies of the present disclosure. Non-human animal models to test the efficacy of the subject antibodies include experimental autoimmune encephalomyelitis (see, e.g., Weerth et al, Am J Path.163:1069-1080 (2003); Theien et al, J.Clin.invest.107:995-1006(2001)), myasthenia gravis (see, e.g., Morgan et al, Clin.Exp.Immun.146:294-302(2006)), myocardial ischemia and reperfusion (see, e.g., Busche et al, GMS Ger.Med.Sci.8: Doc20(2010)), and Streptococcus pneumoniae (see, e.g., Brown et al, Proc.Natl.Acad.Sci.99: 16969-169974)). Also suitable are non-human animal models of graft rejection (see, e.g., Racki et al, (2010) Transplantation 89: 527; and Baldwin et al, (2010) am. J. Transplantation 10: 1135). In some embodiments, the model is a murine (e.g., rat or mouse) model. Such models are known to those skilled in the art.

Detection method

The present disclosure provides in vitro methods of detecting complement C1s protein in a biological sample obtained from an individual; and methods of detecting C1s protein in vivo in a living individual. The subject in vitro detection methods can be quantitative. The C1s protein may alternatively serve as a biomarker for the progression of, or response to treatment of, a complement-mediated disease or disorder.

The complement C1s protein that is detected/quantified can be the full-length C1s protein or any fragment thereof that comprises the epitope to which the anti-C1 s antibodies of the disclosure bind.

Suitable biological samples include, but are not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, solid tissue samples, tissue culture samples, cell samples, and other biological samples known to those of skill in the art.

The in vitro methods of the present disclosure for detecting complement C1s protein in a biological sample obtained from an individual generally comprise: a) contacting the biological sample with an anti-C1 s antibody of the present disclosure; and b) detecting binding of said antibody to C1s protein present in said sample.

The detection methods of the present disclosure can be used to determine whether an individual has or is at risk for developing a complement-mediated disease or disorder. The detection methods of the present disclosure can be used to determine the stage (severity) of a complement-mediated disease or disorder. The detection methods of the present disclosure are useful for monitoring the progression of a complement-mediated disease or disorder in an individual. The detection methods of the present disclosure can be used to determine the response of an individual to a treatment regimen for treating a complement-mediated disease or disorder. The subject detection methods can be used to test biological samples obtained from individuals suspected of having a complement-mediated disease or disorder, individuals who have been diagnosed as having a complement-mediated disease or disorder, individuals who have a genetic predisposition to develop a complement-mediated disease or disorder, and the like.

The present disclosure provides methods of diagnosing a complement-mediated disease or disorder 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 to a reference value, standard value, or normal control value indicative of 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 disorder. In some embodiments, the determining step comprises contacting the biological sample with an anti-C1 s antibody of the present disclosure and quantifying binding of the antibody to complement C1s protein present in the sample.

The present disclosure provides methods of monitoring the progression of a complement-mediated disease or disorder in an individual. The methods generally include comparing the amount of complement C1s protein in a biological sample obtained from the individual at a first time point to the amount of complement C1s protein in a biological sample obtained from the individual at a second time point. A difference in the amount of complement C1s protein in the biological sample obtained from the individual at the second time point compared to the amount of complement C1s protein in the biological sample obtained from the individual at the first time point may provide an indication of: i) whether the complement-mediated disease or disorder is progressing or whether progression of the disease has slowed or stopped; and/or ii) how fast the complement-mediated disease or disorder progresses; 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 disorder. In some embodiments, the determining step comprises contacting the biological sample with an anti-C1 s antibody of the present disclosure and quantifying binding of the antibody to complement C1s protein present in the sample. In some embodiments, the step of comparing indicates whether the disease or disorder is progressing.

The present disclosure provides methods of monitoring a response to treatment of a complement-mediated disease or disorder in an individual. The methods generally include comparing the amount of complement C1s protein in a biological sample obtained from the individual at a first time point to the amount of complement C1s protein in a biological sample obtained from the individual at a second time point. A difference in the amount of complement C1s protein in the biological sample obtained from the individual at the second time point compared to the amount of complement C1s protein in the biological sample obtained from the individual at the first time point may 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 disorder. In some embodiments, the determining step comprises contacting the biological sample with an anti-C1 s antibody of the present disclosure and quantifying binding of the antibody to complement C1s protein present in the sample. In some embodiments, the step of comparing indicates whether progression of the disease is slowed or stopped.

The present disclosure provides methods for staging a complement-mediated disease or condition. For example, the subject methods can provide for the determination of the stage of alzheimer's disease. For example, the amount of complement C1s protein in a biological sample from a living individual can provide an indication as to the Braak stage of AD. Braak and Braak (1995) Neurobiol. aging 16: 271. For example, the amount of complement C1s protein in a biological sample from a living individual can provide information as to whether the individual is in the transverse internal nasal phase I-II of AD; limbic stages III-IV of AD; or indication of neocortical stages V-VI of AD.

The amount of complement C1s protein in a biological sample can be assessed by any suitable method known in the art. Suitable methods include, but are not limited to, protein ("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), Fast Protein Liquid Chromatography (FPLC), multidimensional Liquid Chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser densitometry.

The present disclosure provides methods of monitoring the progression of a complement-mediated disease or disorder in an individual, wherein the methods generally comprise: 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 to the first amount of the complement C1s protein. In some embodiments, the determining step comprises: i) contacting the biological sample with a subject anti-C1 s antibody; and ii) quantifying the binding of the antibody to complement C1s protein present in the sample. In some embodiments, the comparison indicates whether the disease has progressed.

In some cases, the first time point is a time point before a start of a treatment protocol, and the second time point is a time point after the start of the treatment protocol. Accordingly, the present disclosure provides a method of detecting a response to treatment with an agent that treats a complement-mediated disease or disorder, 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 prior to initiating treatment with an agent that treats a complement-mediated disease or disorder; b) determining a second amount of complement C1s protein in a biological sample obtained from the individual at a second time point after initiation of treatment with the agent that treats the complement-mediated disease or disorder; and C) comparing the second amount of complement C1s protein to the first amount of complement C1s protein.

The subject methods of monitoring the progression of a complement-mediated disease or disorder can also be used to monitor tauopathies or synucleinopathies such as Parkinson's Disease (PD); a method for the progression of dementia with lewy bodies (DLB) and the like. For example, the subject methods can be used to monitor the progression of PD (PDD) with dementia.

The subject methods may include the use of kits or assay devices that include the subject anti-C1 s antibodies. The present disclosure provides kits and assay devices for performing the methods as described herein. The subject kits include an anti-C1 s antibody of the present disclosure.

The anti-C1 s antibody can be immobilized on an insoluble support (e.g., a test strip, a well of a multi-well plate, a bead (e.g., a magnetic bead), etc.). Suitable supports are well known in the art and comprise, inter alia, 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 trays (e.g., multi-well plates), plastic tubes, and the like. The solid support may comprise any of a variety of materials including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. Methods suitable for immobilizing the subject antibodies on solid supports are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. The solid support may be soluble or insoluble, for example, in an aqueous solution. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

The anti-C1 s antibodies of the present disclosure may comprise a detectable label. When the antibody comprises a detectable label, the subject kits may comprise one or more reagents for producing a detectable label. The labeled antibody may comprise a label such as a chemiluminescent agent, a particulate label, a colorimetric agent, an energy transfer agent, an enzyme, a fluorescent agent, or a radioisotope. 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, texasred, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.); the radioactive label (e.g.,³H、¹²⁵I、³⁵S、¹⁴c or³²P); and enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and other enzymes that act on a substrate to produce a product that can be detected by fluorescent, colorimetric, or spectrophotometric means).

In some cases, the disclosed methods for detecting/quantifying C1s in a biological sample obtained from an individual 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 such that the polypeptides forming the C1 complex separate from each other, thereby producing a monomeric C1 complex component.

In some cases, the disclosed methods for detecting/quantifying C1s in a biological sample obtained from an individual comprise: a) contacting the biological sample with an immobilized first antibody that binds C1s, but does not compete with the subject anti-C1 s antibody for binding to C1s (e.g., a rabbit polyclonal antibody that binds C1s), thereby forming an immobilized first antibody/C1 s complex; b) contacting the immobilized first antibody/C1 s complex with a chelating agent (e.g., EDTA), thereby forming an immobilized first antibody/C1 s monomer complex; c) contacting the immobilized first antibody/C1 s monomer complex with a monoclonal anti-C1 s antibody of the present disclosure; and d) detecting binding of the monoclonal anti-C1 s antibody to the immobilized C1s monomer. In some cases, the disclosed methods for detecting/quantifying C1s in a biological sample obtained from an individual comprise: a) treating the biological sample with a chelating agent (e.g., EDTA), thereby forming C1s monomers; b) contacting the chelator-treated biological sample with an immobilized first antibody that binds C1s, but does not compete with the subject anti-C1 s antibody for binding to C1s (e.g., a rabbit polyclonal antibody that binds C1s), thereby forming an immobilized first antibody/C1 s monomer complex; c) contacting the immobilized first antibody/C1 s monomer complex with a monoclonal anti-C1 s antibody of the present disclosure; and d) detecting binding of the monoclonal anti-C1 s antibody to the immobilized C1s monomer. Detection of binding of the monoclonal anti-C1 s antibody to the immobilized C1s monomer can be accomplished in a variety of ways. For example, wherein the monoclonal anti-C1 s antibody comprises a detectable label that is detected using a method suitable for the label. Alternatively, the monoclonal anti-C1 s antibody can be detected using a detectably labeled secondary antibody that binds the monoclonal anti-C1 s antibody. The subject kits can comprise the subject monoclonal anti-C1 s antibody; and may further comprise one or more of the following: 1) chelating agents (e.g., EDTA); and 2) an anti-C1 s antibody (e.g., a polyclonal anti-C1 s antibody, such as a rabbit polyclonal antibody) that does not compete with the subject anti-C1 s antibody for binding to C1 s.

The subject kits may further comprise one or more additional components, wherein suitable additional components include: 1) a positive control; 2) buffers (e.g., binding buffers; washing the buffer solution; etc.); 3) a reagent for generating a detectable signal; and so on. Other optional components of the kit include: a protease inhibitor; a detectable label; and so on. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined in a single container as desired.

In addition to the components described above, the subject kits can include instructions for using the components of the kit to perform the subject methods. The instructions for carrying out the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic or the like. Thus, the instructions may be present in the kit in the form of a package insert, in labeling of the containers of the kit or components thereof (i.e., associated with a package or sub-package), and the like. In other embodiments, the instructions are in the form of electronically stored data files on a suitable computer readable storage medium, such as a compact disc read only memory (CD-ROM), Digital Versatile Disc (DVD), floppy disk, and the like. In other embodiments, the actual instructions are not present in the kit, but provide a means for obtaining the instructions from a remote source, such as via the internet. An example of this embodiment is a kit that includes a web site from which instructions can be viewed and/or from which instructions can be downloaded. With respect to the instructions, such means for obtaining the instructions are recorded on a suitable substrate.

The assay device may comprise the subject anti-C1 s antibody immobilized on a solid substrate. The assay device can take any of a variety of forms, such as a test strip, test paper, and the like.

In vivo imaging

As discussed above, the present disclosure provides for detection, for example, by in vivo imaging techniquesA method of detecting complement C1s protein in a living individual. For example, in one embodiment, in vivo imaging of the C1s protein may be achieved by Positron Emission Tomography (PET), single photon emission tomography (SPECT), Near Infrared (NIR) optical imaging, or Magnetic Resonance Imaging (MRI). In some embodiments, use is made ofInstruments such asSpectroscopy for in vivo imaging. The subject anti-C1 s antibodies are administered to an individual and the presence and/or amount of complement C1s protein is detected. The anti-C1 s antibody may comprise a label suitable for use in PET, SPECT, NIR, MRI or IVIS. Such labels include contrast agents or radioisotopes, wherein the contrast agents or radioisotopes are suitable for imaging (e.g., imaging procedures performed on a human as described above).

Generating reports

In some cases, a subject detection method comprises detecting complement C1s protein in a biological sample obtained from an individual; and generating a report and/or directing treatment or management of the individual from which the biological sample was obtained based on the amount of complement C1s protein detected.

The report may include one or more of the following: an indication as to whether the individual is likely to have a complement-mediated disease or disorder; an indication of the severity of the complement-mediated disease or disorder; an indication as to whether the individual exhibits a beneficial clinical response to treatment of a complement-mediated disease or disorder; and so on.

Thus, the report may include, for example, the following information: a predicted likelihood that the individual has or will develop a complement-mediated disease or disorder; a recommendation for further evaluation; recommendations for therapeutic drugs and/or other health management interventions; and so on.

For example, the methods disclosed herein may further include the step of generating or outputting a report providing the results of the subject's assessment, which report may be provided in the form of electronic media (e.g., an electronic display on a computer monitor) or tangible media (e.g., a report printed on paper or other tangible media). An assessment of the likelihood that an individual has or is at risk for developing a complement-mediated disease or disorder may be referred to as a "risk report," risk score, "or" likelihood score. The individual or entity preparing the report ("report generator") may also perform steps such as sample collection, sample processing, and the like. Alternatively, an entity other than the report generator may perform steps such as sample collection, sample processing, and the like. A risk assessment report may be provided to the user. The "user" may be a healthy professional (e.g., a clinician, laboratory technician, or physician).

Guiding health management

thus, for example, depending on the results of the subject detection methods, it may be recommended that the subject undergo therapeutic intervention (treatment) for a complement-mediated disease or condition and/or that the subject be considered for specific health management therapeutic intervention may include, for example, drug therapy for the treatment of Alzheimer's disease examples of drug therapy for the treatment of Alzheimer's disease include, but are not limited to, acetylcholinesterase inhibitors, including, but not limited to, Aricept (donepezil), Exelon (rivastigmine), mellophosphote and tacrine (Cognex), anti-A β antibodies (e.g., sorafezumab (solanezumab)), anti-C1 s antibodies, non-steroidal anti-inflammatory agents, including, but not limited to, ibuprofen and indomethacin, cyclooxygenase-2 (Cox2) inhibitors, such as celecoxib, and monoamine oxidase inhibitors, such as selegiline (Eldeprenyl or Deprenyl), the respective doses of the foregoing agents being known in the art.

Organ preservation and perfusion

The present disclosure provides compositions and methods for organ preservation and perfusion.

Composition comprising a metal oxide and a metal oxide

The present disclosure provides compositions comprising an anti-C1 s antibody of the present disclosure. Such compositions may comprise a pharmaceutically acceptable excipient.

The composition may comprise one or more agents for perfusion into an organ or tissue. The perfusion composition may be used, for example, for in situ or ex vivo perfusion of a tissue or organ. When perfusion is performed in situ, the donor individual is typically not a living and healthy individual.

The composition may comprise one or more agents that preserve an organ or tissue intended for transplantation into a recipient subject. Preservation compositions may be used, for example, for ex vivo preservation of tissues or organs.

For example, a tissue or organ obtained from or to be obtained from a donor individual is perfused with a perfusate in situ or ex vivo at or after removal from the donor individual. The tissue or organ may be stored ex vivo in a preservation solution for a period of time before being transplanted into a recipient individual. In some cases, the perfusion composition and the preservation composition are the same.

In some cases, the subject compositions are aqueous solutions comprising: (a) an anti-C1 s antibody of the present disclosure; and (b) one or more of the following: (i) salt; (ii) an agent that reduces edema; (iii) agents that scavenge free radicals ("oxygen radical inhibitors" or "oxygen radical scavengers"); and (iv) an energy supply system component. In some cases, the subject compositions are aqueous solutions comprising: (a) an anti-C1 s antibody of the present disclosure; and (b) one or more of the following: (i) sugars (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides); and (ii) an agent having pH buffering properties; and optionally, (c) one or more of: (iii) a calcium transport blocker; (iv) a thromboxane inhibitor; (v) a calcium chelating agent; and (vi) an iron chelator.

Suitable sugars include, but are not limited to, sucrose, raffinose, and mannitol. Suitable pH buffering agents include sodium phosphate buffer, potassium phosphate buffer, and the like; for example, Na₂PO₄、NaH₂PO₄、K₂PO₄、KH₂PO₄And the like.

Suitable oxygen radical scavengers include, but are not limited to, allopurinol and reduced glutathione. Suitable energizing system components include adenosine (or Adenosine Triphosphate (ATP)).

Examples of suitable calcium chelators include citrate and Ethylene Glycol Tetraacetic Acid (EGTA). An example of a suitable iron chelator is ethylenediaminetetraacetic acid (EDTA).

Agents that reduce edema include impermeable anions and colloidal osmotic agents.

As used herein, the term "impermeable anion" refers to a compound that counters the swelling of organs that have been exposed to cryogenic temperatures. Examples of impermeable anions include, but are not limited to, gluconate and lactobionic acid.

Agents that reduce edema include colloidal osmotic agents, for example, poly (ethylene glycol) (PEG), succinylated gelatin, Ficoll (polysaccharide), or starch products (e.g., hydroxyethyl starch).

In some cases, the subject compositions also include an amino acid, for example, glutamine, glycine, or N-acetyl cysteine.

In some cases, the subject compositions also include antimicrobial agents, e.g., antibiotics, antifungal agents, and the like.

The subject compositions may include inorganic or organic solutes. Suitable inorganic solutes are those comprising for example Na⁺、K⁺、Cl^-、OH^-、Ca²⁺、Mg²⁺Etc. of a cation andand/or an anionic electrolyte. The electrolyte may be present, for example, at the following concentrations: (i) na (Na)⁺From about 50mmol/L to about 150 mmol/L; (ii) k⁺From about 0mmol/L to about 25 mmol/L; (iii) cl^-From about 0mmol/L to about 100 mmol/L; (iv) OH group^-From about 0mmol/L to about 75 mmol/L; (v) ca²⁺From about 0mmol/L to about 2 mmol/L; (vi) mg (magnesium)²⁺From about 0mmol/L to about 10 mmol/L.

The osmolality of the subject compositions can range from about 300mosmol/l to about 450mosmol/l, for example, from about 300mosmol/l to about 325mosmol/l, from about 325mosmol/l to about 350mosmol/l, from about 350mosmol/l to about 375mosmol/l, from about 375mosmol/l to about 400mosmol/l, from about 400mosmol/l to about 425mosmol/l, or from about 425mosmol/l to about 450 mosmol/l.

The pH of the subject compounds can range from about 6.9 to about 7.8, for example, the subject compositions can have a pH of 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.

In some cases, the subject compositions are aqueous solutions comprising: (a) an anti-C1 s antibody of the present disclosure; and (b) one or more of the following: (i) hydroxyethyl starch; (ii) lactobionic acid; and (iii) raffinose.

In some cases, the subject compositions are aqueous solutions comprising: (a) an anti-C1 s antibody of the present disclosure; (b) potassium lactobionate (100 mmol); (c) KH (Perkin Elmer)₂PO₄(25mmol)；(d)MgSO₄(45 mmol); (e) raffinose (30 mmol); (f) adenosine (5 mmol); (g) glutathione (3 mmol); (h) insulin (100 units); (i) broad spectrum antibiotics such as trimethoprim (16 mg/mL); j) dexamethasone (dexamethasone) (8 mg/L); k) allopurinol (1 mM); and L) hydroxyethyl starch (e.g., hydroxyethyl starch having a molecular weight of about 200,000 daltons to about 300,000 daltons and a degree of substitution of about 0.4 to 0) (50 g/L).

In some cases, the subject compositions are aqueous solutions comprising: (a) an anti-C1 s antibody of the present disclosure; and one or more of the following: (i) hydroxyethyl starch (30g/L to 100 g/L); (ii) NaCl (85mM to 145 mM); (i)ii) KCl (3mM to 6 mM); (iv) CaCl₂(1.0mM to 1.6 mM); (v) KH (Perkin Elmer)₂PO₄(0.7mM to 1.3 mM); (vi) MgSO (MgSO)₄(0.9mM to 1.5 mM); (vii) allopurinol (0.05mM to 5.0 mM); (viii) deferoxamine (desferrioxamine) (0.02mM to 2.0 mM); (ix) glutathione (0.5mM to 10.0 mM); (x) Nicardipine (nicardipene) (0.1 μ M to 5.0 μ M); (xi) Adenosine (0.1mM to 5.0 mM); (xii) Fructose (1.0 to 50.0 mM); (xiii) Glucose (1.0 to 50.0 mM); (xiv) Insulin (5U/L to 250U/L); (xv)3- (N-morpholino) propanesulfonic acid (MOPS) (2mM to 40 mM).

In some cases, the subject compositions comprise:

(a) an anti-C1 s antibody of the present disclosure;

(b) potassium lactobionate (e.g., 100 mM);

(c)KH₂PO₄(e.g., 5 mM);

(d) raffinose (e.g., 30 mM);

(e) adenosine (e.g., 5 mM);

(f) glutathione (e.g., 3 mM);

(g) allopurinol (e.g., 1 mM); and

(h) hydroxyethyl starch (e.g., 50 g/L).

The subject anti-C1 s antibody is present in an effective amount in the subject tissue/organ preservation or perfusion fluid. An "effective amount" of a subject anti-C1 s antibody is an amount that inhibits C4b2a complex production 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 C4b2a in the absence of anti-C1 s antibody. The concentration of anti-C1 s antibody in the composition can range from about 1mg/mL to about 200mg/mL, for example, from about 1mg/mL to about 5mg/mL, from about 5mg/mL to about 10mg/mL, from about 10mg/mL to about 25mg/mL, from about 25mg/mL to about 50mg/mL, from about 50mg/mL to about 100mg/mL, or from about 100mg/mL to about 150 mg/mL.

The present disclosure provides isolated (e.g., ex vivo) organs or tissues present in preservation/perfusion fluids as described above.

Organ perfusion method and organ preservation method

The present disclosure provides tissue or organ perfusion methods and ex vivo tissue or organ preservation methods using compositions comprising anti-C1 s antibodies as described herein.

Perfusion methods generally comprise introducing a perfusate comprising an anti-C1 s antibody of the present disclosure into and/or around a donor tissue or donor organ in an amount sufficient to perfuse the tissue or organ with the perfusate, in situ or ex vivo. When perfusion is performed in situ, the donor individual is typically not a living and healthy individual. Perfusion can be achieved, for example, by introducing a perfusate of the present disclosure into a vascular bed of a tissue or organ. Perfusion may be performed to flush a tissue or organ with a perfusate, for example to at least partially replace blood present in the vasculature.

Preservation methods generally include introducing a preservation fluid comprising an anti-C1 s antibody of the present disclosure ex vivo into and/or around a donor tissue or organ in an amount sufficient to maintain the tissue or organ for later use, e.g., for use in a transplant.

The perfusion and storage methods described herein generally provide for inhibition of complement activation in a tissue or organ. Accordingly, the present disclosure provides a method for inhibiting complement activation in a tissue or organ, the method comprising introducing a perfusate or preservation fluid as described herein in situ or ex vivo into or around the tissue or organ, wherein the perfusate or preservation fluid is introduced in an amount sufficient to inhibit complement activation in the tissue or organ.

Organs and tissues that can be preserved using the subject methods include, but are not limited to, kidney, liver, pancreas, heart, lung, skin, blood tissue (including whole blood; red blood cells; white blood cells; cord blood; and the like, wherein the blood tissue can include an isolated population of blood cells (buffy coat; red blood cells; platelets; lymphocytes; T cells; B cells; or some other population), wherein the blood tissue includes a mixed population of cells), small intestine, endothelial tissue, vascular tissue (e.g., blood vessels), eye, stomach, thymus, bone marrow, cornea, heart valves, langerhans islets, or tendons. As used herein, "organ" encompasses an entire organ or a portion of an organ. As used herein, "tissue" encompasses whole tissue or a portion of tissue.

The organ or tissue may be of human origin. The organ or tissue may be of non-human animal (e.g. porcine) origin. In some cases, the tissue or organ is an allograft, i.e., the tissue or organ is allogeneic to the intended recipient. In some cases, the tissue or organ is a xenograft, i.e., the tissue or organ is from a xenogeneic source relative to the intended recipient. The organ or tissue may be obtained from a living individual, or from a recently deceased individual (e.g., where the organ or tissue is obtained from an individual within about 1 minute or hours after death of the individual).

The organ or tissue may be stored in a subject preservation or perfusion fluid at low or normal temperatures. For example, an organ or tissue may be stored in a subject preservation or perfusion fluid at a low temperature of about 1 ℃ to about 10 ℃. As another example, the organ or tissue may be stored in the subject preservation or perfusion fluid at ambient temperatures of about 12 ℃ to about 24 ℃.

The organ or tissue may be stored in the subject preservation or perfusion fluid for a period of time from about 1 minute to about 24 hours, e.g., from about 1 minute to about 15 minutes, from about 15 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, or from about 12 hours to about 24 hours. In some cases, the organ or tissue may be stored in a subject preservation or perfusion fluid for a period of time greater than 24 hours. The organ or tissue may be perfused with the subject preservation or perfusion solution for a period of time from about 1 minute to about 24 hours, e.g., from about 1 minute to about 15 minutes, from about 15 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, or from about 12 hours to about 24 hours.

Tables 2 and 3 provide a list of SEQ ID NOs disclosed in the present application. Fig. 2 provides table 2. Table 3 is provided below. It is understood that because nucleic acid sequencing techniques are not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent the apparent nucleic acid sequences of the nucleic acid molecules of the embodiments and the apparent amino acid sequences of the proteins of the embodiments, respectively.

TABLE 3 list of non-antibody amino acid sequences disclosed herein

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pairs; kb, kilobases; pl, picoliter; s or sec, seconds; min, min; h or hr, hours; aa, an amino acid; kb, kilobases; bp, base pair; nt, nucleotide; i.m., intramuscular; i.p., intraperitoneally; s.c., subcutaneous; and so on.

Example 1: preparation and characterization of anti-complement C1s IPN003 antibody

The anti-C1 s monoclonal antibody IPN003 (also referred to as "IPN-M34" or "M34") was prepared as follows: BALB/C and NZBW mice were immunized with the purified human activated C1s protein (EMD Millipore, Billerica, MA) (SEQ ID NO:9) in double-stranded form, resulting in two separate hybridoma libraries that were screened with C1s protein using techniques known to those of skill in the art (see, e.g., Galfre et al, Methods in Enzymology 73:346 (1981)). Single cell clones were generated using flow cytometry and supernatants from these individual clones were screened for activation C1s that binds to the biotin label using solution phase monoclonal antibody capture assays, such as those disclosed in Nix et al, immunologic systems, academic Approach, edited by j.p. gosling, pages 239-261, Oxford University Press (2000). 171 clones bound activated C1s with high affinity. One of the clones isolated from the NZBW mice that bound activated C1s produced an antibody called IPN003 (or IPN-M34; or M34; or TNT 003).

Amino acid sequencing of the VH and VL regions of the IPN003 anti-C1 s antibody was performed using techniques known to those skilled in the art (MCLAB, South San Francisco, CA). Specifically, cell pellets were prepared from hybridoma cell lines expressing the IPN003 monoclonal antibody, and usedRNA was extracted using a kit (Life Technologies Inc., GrandIsland, NY). The V regions were amplified by reverse transcription polymerase chain reaction (RT-PCR) using a degenerate primer pool of murine antibody signal sequences and constant region primers for IgMVH, IgGVH, Ig kappa VL and Ig lambda VL. Polymerase Chain Reaction (PCR) products from each successful amplification are purified and cloned into a ` TA ` cloning vector that acquires the sequence(s) (II)Promega, Madison, WI). Deduced amino acid sequences of VH and VL regions of IPN-M34 antibodyThe columns are provided in table 2. The CDR for IPN003 is also provided in table 2 (fig. 2).

Example 2: binding characteristics of IPN003

The binding characteristics of IPN003 were compared to those of M81. For M81, see, e.g., Matsumoto et al, (1986) j.immunol.137: 2907; matsumoto et al, (1989) J.Immunol.142: 2743; and Nakagawa et al, (1999) Ann. Rheum. Dis.58: 175.

IPN003 competes with M81 for binding to human C1 s.

To determine whether IPN003 can compete with M81 for binding to human C1s, a competition assay was performed in which biotin-labeled M81 (final concentration 0.5E-9M) was incubated with increasing concentrations of unlabeled antibody in wells coated with human C1 s. The data are shown in figure 3.

Unlabeled M81 IC at 3E-9M₅₀Competes for binding with labeled M81, while the control antibody does not. As shown in fig. 3, IPN003(M34) competed with M81 for binding to human C1 s. IPN003 is a more potent M81 binding Inhibitor (IC) than M81₅₀0.33E-9M), indicating that the epitope for IPN003 is different from, but overlapping with, the epitope recognized by M81.

IPN003 inhibited human C1s activation of human C4.

Human complement protein C4 was incubated with activated human C1s in the presence of increasing concentrations of monoclonal antibody M81 or IPN 003. As shown in fig. 4, the data show that IPN003 inhibits C1 s-mediated activation of human complement protein C4. IPN003 with 3E-9M IC₅₀Inhibit C4 activation. In contrast, M81 is a much less effective inhibitor and has an IC of 55E-9M₅₀Inhibit C4 activation.

IPN003 inhibited human complement-mediated cell lysis.

The ability of IPN003 to inhibit complement-mediated cell lysis was measured in a standard sheep red blood cell (sRBC) hemolysis assay using human serum as a source of complement protein. The data are shown in figure 5.

Control IgG had no effect on cell lysis, while IPN003 had an IC of 1.1E-9M₅₀Inhibiting cell lysis. In contrast, M81 is a much less potent inhibitor than IPN003, and has an IC of 11.3E-9M₅₀Inhibition of sRBC dissolution. Thus, the data presented in fig. 5 show that IPN003 can inhibit activation of the entire classical complement cascade using a standard hemolytic assay; and IPN003 activity was significantly greater than M81, consistent with the data presented in fig. 3 and 4.

IPN003 inhibits cell lysis mediated by cynomolgus monkey complement and by cynomolgus monkey complement.

To determine whether IPN003 can inhibit complement-mediated cell lysis in a species suitable for toxicology studies, a hemolytic assay was performed in which complement proteins were provided by sera from cynomolgus monkeys and from cynomolgus monkeys, two monkey species considered suitable for toxicology studies. The data are shown in fig. 6 and 7.

As shown in FIG. 6, the IC for IPN003 at 4.6E-9M₅₀Inhibit cell lysis mediated by serum from cynomolgus monkeys. In contrast, M81 is a much less effective inhibitor and has an IC of 189E-9M₅₀Inhibition of sRBC lysis.

As shown in FIG. 7, the IC for IPN003 at 4.5E-9M₅₀Inhibits cell lysis mediated by sera from cynomolgus monkeys. In contrast, M81 is a much less effective inhibitor and has an IC of 83E-9M₅₀Inhibition of sRBC lysis.

The data presented in fig. 6 and 7 also strongly suggest that IPN003 cross-reacts with C1s from at least two monkey species. The data also indicate that IPN003 is a more potent inhibitor of complement activation than M81.

IPN003 is specific for the C1s component of the complement cascade.

To determine whether IPN003 can bind to other components of the complement cascade, an ELISA assay was performed with target proteins immobilized on microtiter plates. The data shown in fig. 8 indicate that IPN003 is specific for C1s and does not bind other components of the complement pathway.

Table 4 (provided in fig. 9) summarizes the binding characteristics of IPN 003.

IPN003 binding to rat C1s

ELISA assays were performed with incremental dilutions of purified rat C1s and IPN003 or M81 immobilized on microtiter plates. The data presented in FIG. 10 show that IPN003 is at a K of 0.2E-9M_DRat C1s bound, whereas M81 was with a K of 0.8E-9M_DBound to rat C1 s.

Example 3: inhibition of rat C1 s-mediated cleavage of human C4 by IPN003

Human C4(0.25mg/ml) was incubated with rat C1s (0.64. mu.g/ml) with different concentrations of IPN003 at 37 ℃. Reducing the sample containing the reaction product; separating the reduced sample on a 4-12% NuPAGE gel; and the gel was stained with Coomassie blue (Coomassie blue). The band of C4a cleavage product on coomassie gel was quantified on a Licor scanner. The results are shown in FIG. 11. As shown in FIG. 11, IPN003 inhibited rat C1 s-mediated cleavage of human C4 in a concentration-dependent manner, with IC₅₀It was 1.47. mu.g/ml.

Inhibition of human C4 cleavage mediated by rat C1s and human C4 cleavage mediated by human C1s by IPN003 was compared. Mu.l rat C1s (0.64. mu.g/ml) or 5. mu.l human C1s (0.2. mu.g/ml) were added to 5. mu.l of 1mg/ml IPN003 or an irrelevant control IgG; and the mixture was kept at room temperature for 30 minutes. After incubation, 10. mu.l of 1mg/ml human C4 was added; this mixture was incubated at 37 ℃ for 80 minutes. Samples containing reaction products were analyzed by coomassie staining of the gel and by ELISA. The results are shown in FIG. 12. As shown in fig. 12, the extent to which IPN003 inhibits human C4 cleavage mediated by rat C1s is similar to the extent to which IPN003 inhibits human C1s mediated human C4 cleavage.

Example 4: inhibition of human RBC lysis and C3b deposition by IPN003 from patient serum

An assay was performed to determine whether IPN003 inhibited autoimmune hemolytic anemia (AIHA) patient serum-induced human RBC lysis and C3b deposition.

Samples containing 10. mu.l packed human RBC (hRBC) and 50. mu.l patient serum were incubated at 30 ℃ for 30 minutes. Normal human serum served as a control. After 30 minutes incubation, the samples were centrifuged and washed once. Complement-competent human serum (12.5%) with or without IPN003 (100. mu.g/ml) was then added to the sensitized hRBCs; and the samples were incubated at 37 ℃ for 1 hour. After an incubation period of one hour, the supernatant was collected and the absorbance at 540nm was measured. The results are shown in FIG. 13. The data presented in fig. 13 shows that IPN003 inhibits hemolysis of pre-incubated hrbcs in AIHA serum from patient P3-1. Complement-independent mechanisms are used to mediate background hemolysis mediated by normal human serum.

An assay was performed to determine whether IPN003 inhibited the deposition of C3b on human RBCs by serum from AIHA patients. As shown in fig. 14, IPN003 completely inhibited the deposition of C3b on hrbcs incubated with AIHA serum samples (patient P3-1).

Example 5: humanized IPN003 variants

Humanized variants of IPN003 were generated. The amino acid sequences of the heavy chain VH domains of humanized variants 1-4 and the nucleotide sequences encoding the heavy chain VH domains of the humanized variants are shown in fig. 16-19. The amino acid sequence of the light chain VL domain of humanized variants 1-3 and the nucleotide sequence encoding the light chain VL domain of the humanized variants are shown in figures 20-22. The amino acid differences relative to the amino acid sequence of IPN003 (VL SEQ ID NO: 37; VH SEQ ID NO:38) are summarized in Table 7 and Table 8 (FIG. 23).

The single letter amino acid codes are as follows (with the 3-letter amino acid codes in parentheses):

G-Glycine (Gly)

P-proline (Pro)

A-alanine (Ala)

V-valine (Val)

L-leucine (Leu)

I-isoleucine (Ile)

M-methionine (Met)

C-cysteine (Cys)

F-phenylalanine (Phe)

Y-tyrosine (Tyr)

W-Tryptophan (Trp)

H-histidine (His)

K-lysine (Lys)

R-arginine (Arg)

Q-Glutamine (Gln)

N-asparagine (Asn)

E-glutamic acid (Glu)

D-aspartic acid (Asp)

S-serine (Ser)

T-threonine (Thr)

Example 6: characterization of humanized IPN003 variants

The relative binding affinities of the various humanized IPN003 variants to activated C1s are shown in table 9, which is presented in fig. 24. The relative binding affinities of the various humanized IPN003 variants to pro-C1s are shown in table 10, which is presented in fig. 24. The humanized variant binds to active C1s with approximately 2-fold higher affinity than the parent IPN003 antibody. For comparison, K for IPN003 and human C1s_D(M) is 1.58E-9 to 2.04E-9; k_on(1/Ms) is 3.56E + 05; and areAnd K is_dis(1/s) is 5.53E-04.

Humanized variants were generated with hinge-stabilized IgG4 constant region (with S241P substitution) and with reduced effector function (with L235E substitution). All 24 combinations (VH variant 1+ Vk variant 1; VH variant 1+ Vk variant 2; VH variant 1+ Vk variant 3; VH variant 2+ Vk variant 1; VH variant 2+ Vk variant 2; VH variant 2+ Vk variant 3; VH variant 3+ Vk variant 1; VH variant 3+ Vk variant 2; VH variant 3+ Vk variant 3; VH variant 4+ Vk variant 1; VH variant 4+ Vk variant 2; VH variant 4+ Vk variant 3) were transiently expressed in HEK cells. Each humanized variant was tested for its ability to compete with IPN003 for binding to C1 s. The data are shown in figure 25.

Each humanized variant was tested in a commercial assay that measures activation of the classical Complement Pathway (CP). The results are shown in FIG. 26. The data show that all 12 humanized variants with similar IC to IPN003₅₀Inhibiting CP activation.

As shown in fig. 27A and 27B, of the 3 humanized variants tested, all variants were specific for the classical pathway. FIG. 27A shows concentration-dependent inhibition of CP by humanized variants VH2/VK3, VH3/VK1, and VH4/VK 2. IPN003 is shown for comparison. FIG. 27B shows the effect of humanized variants VH2/VK3, VH3/VK1 and VH4/VK2 on Alternative Pathway (AP).

Humanized variants were tested for Red Blood Cell (RBC) lysis inhibition, and for inhibition of C3b deposition on RBCs. The data are shown in figure 28.

Example 7: inhibition of complement dependent hemolysis

An assay was performed to determine whether IPN003 or a humanized variant of IPN003 can inhibit human RBC lysis by plasma in Cold Agglutinin Disease (CAD) patients. Assays were also performed to determine whether IPN003 or humanized variants of IPN003 could inhibit anaphylatoxin production.

The hemolysis assay was performed essentially as described in example 4. Plasma (C) of Cold Agglutinin Disease (CAD) patients at 4 deg.CAD patient 5; "CAD P5") in the presence of oxygen^-Normal human red blood cells, which allow autoantibodies in plasma to bind to RBCs, produce sensitized hrbcs. 10mM EDTA was added during sensitization to prevent complement activation. After about 1/2 hour, the plasma was washed away and 25% normal human serum containing active complement and antibodies (IPN003, humanized variant of IPN003, or control IgG2a) was added. After addition of human serum and antibody to the sensitized hRBCs, the samples were incubated at about 18 ℃ for 1 hour. After an incubation period of one hour, the supernatant was collected and the absorbance at 540nm was measured. The results are shown in FIG. 29.

As shown in fig. 29, plasma of CAD patient P5 caused hRBC lysis. IPN003 and humanized variants of IPN003 (but not control IgG2a) inhibited complement-dependent CAD patient plasma-mediated hRBC hemolysis. Complement-dependent lysis is inhibited in a concentration-dependent manner.

The CAD P5 supernatant from the hemolysis experiment described above was used to test the production of the anaphylatoxins C3a, C4a and C5 a. The data are shown in fig. 30. As shown in figure 30, IPN003 and the humanized variant of IPN003 (hu-IPN003) but not control IgG2a inhibited the production of all three anaphylatoxins with similar efficacy (competitive inhibition) and potency (about 4.5-5.5 μ g/mL) compared to hemolysis.

Example 8: inhibition of C3b deposition on red blood cells

Four CAD patient plasma samples were tested for their ability to inhibit C3b deposition on hrbcs. Incubating plasma samples from CAD patients P8, P11, P14 and P15 with IPN003 or control IgG2 a; and the percentage of C3b positive cells was determined using flow cytometry. The data are shown in fig. 31 and 32. As shown in figure 31, 100 μ g/mL IPN003 (but not control IgG2a) significantly inhibited C3b deposition on RBC surfaces. The average inhibition was about 90%. As shown in figure 32, CAD plasma-mediated deposition of C3b on hrbcs was inhibited in a concentration-dependent manner by IPN003, but not by control IgG2 a. IC of IPN003₅₀Is about 6.6. mu.g/ml.

Example 9: immunogenic potential of humanized variants of IPN003

The immunogenic potential of the humanized anti-C1 s antibody was evaluated. Using EpiScreen^TMAnd (4) measuring. See, e.g., Jones et al, (2004) j. interferon Cytokine res.24: 560; and Jones et al, (2005) J.Thromb.Haemost.3: 991. Using CD8⁺-cleared Peripheral Blood Mononuclear Cells (PBMCs) for time course T cell assays; and incorporated by multiple time points after addition of the test antibody sample³H]Thymidine to measure T cell proliferation. The proliferative responses to the humanized anti-C1 s antibody or the chimeric anti-C1 s antibody are shown in fig. 33A and fig. 33B.

As shown in fig. 33A, the fully humanized IPN003 test antibody had low immunogenic potential (below the SI threshold of 2.0). Fig. 33B shows the results for a reference chimeric antibody with IPN003 murine heavy and light chain variable regions and a human IgG4 constant region.

Example 10: effect of TNT003 on complement-dependent CAD autoantibody-mediated Activity

The effect of TNT003 on hemolysis, phagocytosis and C4a production mediated by autoantibodies present in the serum of Cold Agglutinin Disease (CAD) patients was determined.

The data are shown in fig. 34-37C.

As shown in figure 34, both TNT003 and anti-C5 mAb prevented complement-dependent CAD autoantibody-mediated hemolysis in a concentration-dependent manner. In 96-well plates, O^-Type red blood cells were incubated in the presence of plasma from CAD patients and 10mM EDTA to allow autoantibody binding (45 minutes at 4 ℃). The cells were then washed with GVB + + (GVB with Ca + + and Mg + +) buffer. GVB + + buffer: 0.1% gelatin, 5mM Veronal, 145mM NaCl, 0.025% NaN₃pH 7.3. Fresh normal cells containing 0.15mM calcium chloride and 0.5mM magnesium chloride were then added to the cellsHuman serum (25% final concentration in GVB + +) and various concentrations of TNT003 or anti-C5 mAb and incubated at 17 ℃ for 1 hour. After incubation, 50 μ L GVB + + was added to each well to stop the reaction. The 96-well plate is then centrifuged and a portion of the supernatant is collected and transferred to a new 96-well plate. The absorbance peak (540nm) was read on a microplate reader. Wells containing 10mM EDTA instead of antibody were used as control wells to determine non-complement mediated hemolysis (positive control). The absorbance value of this well was subtracted from all other wells to determine the extent of complement-dependent lysis. Similarly, control wells without antibody were used to determine maximum complement-dependent hemolysis (negative control).

As shown in figure 35, TNT003, but not anti-C5 mAb or control mouse IgG2a antibody, inhibited C3b deposition mediated by CAD autoantibodies. In 96-well plates, O^-Type red blood cells were incubated in the presence of plasma from CAD patients and 10mM EDTA or healthy normal plasma (negative control) to allow autoantibody binding (45 minutes at 4 ℃). Cells were then washed with GVB + + buffer. Fresh normal human serum (25% final concentration in GVB + +) and 100 μ g/mL of TNT003, mouse IgG2a control Ab, or anti-C5 mAb were then added to the cells and incubated at 17 ℃ for 1 hour. Cells were then washed with FACS buffer (2 ×). FACS buffer: phosphate Buffered Saline (PBS) w/o + 0.5% Bovine Serum Albumin (BSA) + 0.1% NaN₃). FACS buffers are buffers that incubate cells and antibodies prior to operation in a flow cytometer. The cells were then incubated in the presence of mouse anti-human C3b monoclonal antibody (1 hour, 4 ℃). Cells were then washed with FACS buffer (3 ×) and incubated with secondary antibody (Alexa Fluor 488-bound goat anti-mouse IgG1) for 30 minutes at room temperature. Cells were then washed with FACS buffer (3 ×) and fluorescence read at 488nm on a flow cytometer.

As shown in figure 36, TNT003 prevented complement-mediated CAD autoantibody-induced phagocytosis. To understand the effect of CAD autoantibody mediated complement deposition on RBC phagocytosis, a phagocytosis assay was developed using the THP-1 monocyte cell line. Labeling O-type human RBCs with Cell Tracker Green andsensitization with CAD autoantibodies was performed by incubation overnight (4 ℃) in the presence of CAD patient plasma and 10mM EDTA. After washing out the CAD plasma, normal human serum (NHS; final concentration 25%) and 100. mu.g/mL TNT003 or 100. mu.g/mL IgG2a control Ab (1 hour at 17 ℃) were added. The retinoic acid-treated THP-1 cells (3. mu.M for 3 days) were then placed in 96-well plates (1X 10)⁵Individual cells/well) and treated with FcX. FcX is an agent for preventing FcgR activation, a potential confounder for FcgR in phagocytosis assays that may also mediate phagocytosis; see, for example, www (. lamda.) biolegend (. lamda.) com. Then 5X 10 at 37 deg.C⁶Individual cells/well of THP-1 cells were supplemented with hrbcs to allow phagocytosis (red bars). Phagocytosis was determined by flow cytometry and expressed as a percentage of THP-1 cells containing Cell Tracker Green labeled RBCs. A portion of hrbcs were also stained with anti-C3 b Ab to quantify C3b deposition by FACS analysis as described above (blue bars).

It has been found that hrbcs coated with CAD autoantibodies but not incubated in the presence of NHS have very low levels of membrane bound C3b and are not readily phagocytosed by THP-1 cells (no NHS). Upon exposure to 25% NHS, C3b deposition increased by about 30-fold and phagocytosis increased by about 5-fold (NHS). TNT003 (rather than the IgG2a control antibody) at 100 μ g/mL inhibited C3b deposition and phagocytosis to baseline levels, providing evidence that TNT003 can prevent CAD autoantibody mediated RBC phagocytosis. The data presented are the average of two independent experiments performed with one patient sample (P18) and represent results generated from 5 different CAD patient samples.

As shown in fig. 37A and 37B, TNT003 (but not anti-C5 mAb) prevented C3a and C4a generation by CAD autoantibodies. As shown in fig. 37C, both TNT003 and anti-C5 mAb were able to inhibit CAD autoantibody mediated C5a production. Commercially available ELISA kits were used to detect and quantify C3a, C4a, and C5a from the supernatants of the above experiments. The levels of C3a, C4a, and C5a in human serum used as a source of complement were subtracted as background.

Example 11：TNT003 in vivo Effect

TNT003 was tested for in vivo effects in non-human primates. The data are shown in fig. 38A, 38B and 39.

As shown in fig. 38A and 38B, TNT 003-containing sera from cynomolgus monkeys given a single intravenous dose of TNT003(30mg/kg) could not be used to induce complement-dependent hemolysis or deposit C3B on plasma membranes of IgM-sensitized sheep red blood cells. Sera taken from monkeys at various time points before and after intravenous injection were used as a source of complement for inducing hemolysis of IgM sensitized sheep red blood cells. The final serum concentration was 1.25%. As depicted in FIG. 38A, shortly after intravenous TNT003 injection, serum samples obtained from monkeys were not used to hemolyze IgM-sensitized sheep red blood cells in all samples containing detectable TNT003 levels (up to and including 72 hours; plotted on the right Y-axis). At 96 hours, TNT003 dropped below detectable levels (as determined by ELISA capture assay; plotted on the left Y-axis), at which time the hemolytic capacity of the serum returned to pre-hemorrhagic levels. FACS assays designed to detect the presence of membrane bound C3B (fig. 38B) showed that TNT003 with serum also failed to deposit C3B on plasma membranes of IgM-sensitized sheep red blood cells. These data indicate that TNT003 is present at effective levels to inhibit the classical complement pathway in ex vivo hemolytic assays after in vivo administration.

As shown in figure 39, TNT003 inhibited C4a production in vivo in cynomolgus monkeys given a single intravenous dose of TNT003(30 mg/kg). The concentration of C4a in serum samples obtained from monkeys given TNT003 was determined using a commercially available ELISA kit (plotted on the right Y-axis). The data show that the C4a level decreased by about 90% immediately after TNT003 administration and remained low (up to and including 72 hours) in all samples containing detectable TNT 003. At 96 hours, TNT003 dropped below detectable levels (as determined by an ELISA capture assay; plotted on the left Y-axis) at which time serum C4a returned to pre-hemorrhage levels. These data provide evidence that TNT003 is active in vivo and reaches a level of inhibition of classical pathway activity in cynomolgus monkeys after 30mg/kg administration.

Example 12: TNT003 epitope mapping

To identify the minimal human C1s (hC1s) region required for TNT003 binding, full-length hC1s and N-terminal and C-short truncated hC1s were expressed in HEK293 cells. The recombinant protein was purified by affinity chromatography and analyzed by western blotting of non-reducing SDS-PAGE gels. As shown in fig. 40, a fragment consisting of amino acids 272-422 that specifically binds TNT003 (hC1s fragment 1.b) was identified. The other N-terminal and C-truncated forms of this amino acid fragment abolished TNT003 binding.

FIG. 40 is a schematic view. The smallest human C1s fragment required for TNT003 binding was identified. Fragments of full-length C1s or C1s were expressed in HEK293 cells. The proteins were purified by affinity chromatography and analyzed by western blotting on a non-reducing SDS-PAGE gel with TNT 003. TNT003 bound to the full length C1s (lane 3) and the C1s fragment containing amino acids 272-422 of the C1s a chain (fragment 1. b).

To further identify the epitope of TNT003 in full-length human C1s, alanine scanning mutagenesis was performed using standard techniques. Mutation of amino acid 357 (aspartic acid) to alanine significantly reduced TNT003 binding to human C1 s. Importantly, as shown in figure 41, this mutation had no significant effect on the catalytic activity of C1s and did not alter its ability to cleave its substrate, C4, indicating that the protein was correctly folded. However, in contrast to its effect on wild-type C1s, TNT003 failed to inhibit the activity of this mutant C1s even at high concentrations. Taken together, these data indicate that the epitope of TNT003 contains aspartic acid 357 and that this amino acid is critical for the binding and inhibitory activity of the antibody.

FIG. 41: the specific amino acid (aspartic acid 357) required for TNT003 binding to human C1s was identified. Alanine scanning mutagenesis of human C1s was performed using standard techniques. Each C1s mutant protein was expressed in HEK293 cells and purified by affinity chromatography. To measure enzyme activity, each mutant C1s protein was incubated with purified human C4 in the presence or absence of TNT003 at 37 ℃ for 1 hour. The reaction was analyzed by SDS-PAGE to identify the cleavage product of C4 (C4a and C4 b). Mutation of aspartate 357 abrogates the ability of TNT003 to inhibit C1s activity. In contrast, mutation of aspartate 343 to alanine had no effect on the C1s enzyme activity or the inhibitory ability of TNT 003.

Example 13: affinity for TNT003

TNT003 is a potent inhibitor of C1s activity in both purification systems and in functional assays (e.g., red blood cell hemolysis). TNT003 activity was compared to other C1s antibodies in a plate-based C4 deposition assay (fig. 42). TNT003 is a more effective inhibitor of C1s activity than other anti-C1 s antibodies (TNT004, TNT005, TNT 006; anti-C1 s antibody that inhibits C1s protease activity), even though other anti-C1 s antibodies bind C1s with higher affinity. These data indicate that the efficacy of TNT003 is mediated in part by its affinity for C1s within the C1 complex, and that a single TNT003 molecule can contact (and thus inhibit) two C1s molecules simultaneously.

FIG. 42: TNT003 is a potent inhibitor of human C1s activity. Wells of a 96-well plate were coated with human IgM antibody and non-specific binding was blocked using gelatin. Human serum (final concentration 1.25%) was added to wells with or without increasing concentrations of antibody and incubated at 37 ℃ for 1 hour. The amount of C4 deposited on the plate was measured using a biotin-labeled anti-human C4 antibody and streptavidin-HRP conjugate.

To demonstrate that TNT003 can bind to the C1 complex, biotin-labeled TNT003 was incubated with purified C1 complex and the reaction was fractionated on a Sepharose Superdex-200 column. As shown in figure 43B, C1 bound TNT003 to form complexes in which the molecular weight was consistent with a single TNT003 antibody bound to each C1 complex.

Fig. 43A and 43B: TNT003 bound to the C1 complex. TNT003 was labeled with biotin and incubated with purified human C1 complex. The reaction was fractionated on a Sepharose Superdex-200 column and each fraction was analyzed for the presence of TNT003 by western blotting. As shown in fig. 43A, TNT003 eluted only in a unimodal form. In contrast, a second peak was observed after incubation with the purified C1 complex, consistent with the formation of the TNT003: C1 complex (fig. 43B).

To further characterize the inhibition mechanism, TNT 003F (ab')₂Fragments and TNT003 Fab fragments inhibit the activity of C1 s. As shown in FIG. 44, TNT003 and TNT 003F (ab')₂The fragment inhibited C1s activity with equal activity. In contrast, TNT003 Fab fragments are much less effective inhibitors of C1s activity. Taken together, these data indicate that the potent activity of TNT003 is at least partially compatible with antibodies (and F (ab')₂Fragments) and the ability of the antibody to inhibit C1s has a significant avidity component.

FIG. 44: TNT003 and TNT003 Fab'2 fragments are more potent inhibitors of C1s activity than TNT003 Fab. Wells of a 96-well plate were coated with human IgM antibody and non-specific binding was blocked using gelatin. To the presence or absence of increasing concentrations of TNT003 or F (ab') of TNT003₂And Fab fragments were added to the wells with human serum (final concentration 1.25%) and incubated at 37 ℃ for 1 hour. The amount of C4 deposited on the plate was measured using a biotin-labeled anti-human C4 antibody and streptavidin-HRP conjugate.

Example 14: TNT003 binding characteristics

To determine whether TNT003 can bind C1s under reducing and non-reducing conditions, western blotting on SDS-PAGE gels was performed with reducing and non-reducing activated human C1 s. As shown in figure 45, TNT003 only bound C1s under non-reducing conditions, consistent with antibodies that bound to conformationally specific epitopes within C1 s.

FIG. 45: TNT003 specifically binds to human C1s under non-reducing conditions. Activated human C1s was fractionated by SDS-PAGE under reducing and non-reducing conditions and western blotted with TNT 003. TNT003 only binds C1s under non-reducing conditions, indicating that it binds a conformation-dependent epitope.

To determine whether TNT003 inhibited complement C2 or complement C4 activation, purified C1s was incubated with C2 or C4 in the presence or absence of TNT 003. As shown in fig. 46, TNT003 specifically inhibited C1s activation of C4 but not C1s activation of C2, consistent with: TNT003 is a competitive inhibitor of C4 binding to C1s, but not an inhibitor of the C1s serine protease domain.

FIG. 46: TNT003 inhibited the activation of complement C4 by C1s but not complement C2. Purified human C1s was incubated with human complement C2 or human complement C4 for 3 hours at 37 ℃. The reaction products were separated on SDS-PAGE gels. TNT003 specifically inhibits the activation of human C1s to human complement C4 but not complement C2.

Example 15: c1s determination

C1s levels were measured in patient samples by ELISA. High binding plates (Costar, 3590) were coated with 100. mu.l of 5. mu.g/ml rabbit polyclonal C1s antibody (Abcam, ab87986) overnight in 1 XDPBS (Life Technologies, 14190-144). Blocking was performed by adding 1X pbs containing 1% gelatin (Sigma-Aldrich, G2500) for 3 hours. The plates were then stored at 4 ℃. Plates were incubated at 37 ℃ for 15 minutes prior to use. After washing the plates with buffer a (1X pbs containing 0.01% Tween20 and 20mM EDTA), serial dilutions of human serum in buffer a or purified C1s in buffer a were added to the wells. After 1 hour incubation at ambient temperature, wells were washed three times with buffer a. Each well was incubated with 100. mu.l of 1. mu.g/ml biotin-labeled TNT003 (diluted in buffer A) for 1 hour, followed by three washes with buffer A. The wells were further incubated with 5000-fold dilutions of streptavidin-conjugated Horse Radish Peroxidase (HRP) (southern Biotech, 7100-05) (diluted in buffer A) for 10 minutes. After four washes with buffer a, enzymatic color development was achieved using TMB (Thermo Scientific, 34029) and the reaction was stopped using 1M sulfuric acid. The absorbance at 450nm was measured.

The results are shown in FIG. 47. Fig. 47 compares the C1s concentration in plasma samples of healthy volunteers (green; n-13) versus CAD patients (grey; n-27). It has been found that on average the concentration of C1s is comparable between healthy individuals and CAD patients. These data provide the rationale for using similar dose levels and dosing regimens for C1s inhibitor in healthy or CAD patients in phase I trials to achieve target coverage.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Sequence listing

<110> P Vanfalaselai (VAN VLASSELAER, PETER)

G pali (Parry, Graham)

N.E. Stagliano (Nancy E)

S panick (Panicker, Sandip)

<120> anti-complement C1s antibodies and uses thereof

<130>TNRX-801WO

<150>US 61/721,916

<151>2012-11-02

<150>US 61/754,123

<151>2013-01-18

<150>US 61/779,180

<151>2013-03-13

<150>US 61/846,402

<151>2013-07-15

<160>69

<170> PatentIn 3.5 edition

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Asp Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Arg Leu Pro

85 90 95

Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

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Glu Val Met Leu Val Glu Ser Gly Gly Ala Leu Val Lys Pro Gly Gly

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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

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Ala Met Ser Trp Val Arg Gln Ile Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asp Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

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Glu Pro Thr Met Tyr Gly Glu Ile Leu Ser Pro Asn Tyr Pro Gln Ala

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Tyr Pro Ser Glu Val Glu Lys Ser Trp Asp Ile Glu Val Pro Glu Gly

20 25 30

Tyr Gly Ile His Leu Tyr Phe Thr His Leu Asp Ile Glu Leu Ser Glu

35 40 45

Asn Cys Ala Tyr Asp Ser Val Gln Ile Ile Ser Gly Asp Thr Glu Glu

50 55 60

Gly Arg Leu Cys Gly Gln Arg Ser Ser Asn Asn Pro His Ser Pro Ile

6570 75 80

Val Glu Glu Phe Gln Val Pro Tyr Asn Lys Leu Gln Val Ile Phe Lys

85 90 95

Ser Asp Phe Ser Asn Glu Glu Arg Phe Thr Gly Phe Ala Ala Tyr Tyr

100 105 110

Val Ala Thr Asp Ile Asn Glu Cys Thr Asp Phe Val Asp Val Pro Cys

115 120 125

Ser His Phe Cys Asn Asn Phe Ile Gly Gly Tyr Phe Cys Ser Cys Pro

130 135 140

Pro Glu Tyr Phe Leu His Asp Asp Met Lys Asn Cys Gly Val Asn Cys

145 150 155 160

Ser Gly Asp Val Phe Thr Ala Leu Ile Gly Glu Ile Ala Ser Pro Asn

165 170 175

Tyr Pro Lys Pro Tyr Pro Glu Asn Ser Arg Cys Glu Tyr Gln Ile Arg

180 185 190

Leu Glu Lys Gly Phe Gln Val Val Val Thr Leu Arg Arg Glu Asp Phe

195 200 205

Asp Val Glu Ala Ala Asp Ser Ala Gly Asn Cys Leu Asp Ser Leu Val

210 215 220

Phe Val Ala Gly Asp Arg Gln Phe Gly Pro Tyr Cys Gly His Gly Phe

225 230 235 240

Pro Gly Pro Leu Asn Ile Glu Thr Lys Ser Asn Ala Leu Asp Ile Ile

245 250 255

Phe Gln Thr Asp Leu Thr Gly Gln Lys Lys Gly Trp Lys Leu Arg Tyr

260 265 270

His Gly Asp Pro Met Pro Cys Pro Lys Glu Asp Thr Pro Asn Ser Val

275 280 285

Trp Glu Pro Ala Lys Ala Lys Tyr Val Phe Arg Asp Val Val Gln Ile

290 295 300

Thr Cys Leu Asp Gly Phe Glu Val Val Glu Gly Arg Val Gly Ala Thr

305 310 315 320

Ser Phe Tyr Ser Thr Cys Gln Ser Asn Gly Lys Trp Ser Asn Ser Lys

325 330 335

Leu Lys Cys Gln Pro Val Asp Cys Gly Ile Pro Glu Ser Ile Glu Asn

340 345 350

GlyLys Val Glu Asp Pro Glu Ser Thr Leu Phe Gly Ser Val Ile Arg

355 360 365

Tyr Thr Cys Glu Glu Pro Tyr Tyr Tyr Met Glu Asn Gly Gly Gly Gly

370 375 380

Glu Tyr His Cys Ala Gly Asn Gly Ser Trp Val Asn Glu Val Leu Gly

385 390 395 400

Pro Glu Leu Pro Lys Cys Val Pro Val Cys Gly Val Pro Arg Glu Pro

405 410 415

Phe Glu Glu Lys Gln Arg Ile Ile Gly Gly Ser Asp Ala Asp Ile Lys

420 425 430

Asn Phe Pro Trp Gln Val Phe Phe Asp Asn Pro Trp Ala Gly Gly Ala

435 440 445

Leu Ile Asn Glu Tyr Trp Val Leu Thr Ala Ala His Val Val Glu Gly

450 455 460

Asn Arg Glu Pro Thr Met Tyr Val Gly Ser Thr Ser Val Gln Thr Ser

465 470 475 480

Arg Leu Ala Lys Ser Lys Met Leu Thr Pro Glu His Val Phe Ile His

485 490 495

Pro Gly Trp Lys Leu Leu Glu Val Pro Glu Gly Arg Thr Asn Phe Asp

500 505 510

Asn Asp Ile Ala Leu Val Arg Leu Lys Asp Pro Val Lys Met Gly Pro

515 520 525

Thr Val Ser Pro Ile Cys Leu Pro Gly Thr Ser Ser Asp Tyr Asn Leu

530 535 540

Met Asp Gly Asp Leu Gly Leu Ile Ser Gly Trp Gly Arg Thr Glu Lys

545 550 555 560

Arg Asp Arg Ala Val Arg Leu Lys Ala Ala Arg Leu Pro Val Ala Pro

565 570 575

Leu Arg Lys Cys Lys Glu Val Lys Val Glu Lys Pro Thr Ala Asp Ala

580 585 590

Glu Ala Tyr Val Phe Thr Pro Asn Met Ile Cys Ala Gly Gly Glu Lys

595 600 605

Gly Met Asp Ser Cys Lys Gly Asp Ser Gly Gly Ala Phe Ala Val Gln

610 615 620

Asp Pro Asn Asp Lys Thr Lys Phe Tyr Ala Ala Gly Leu Val Ser Trp

625630 635 640

Gly Pro Gln Cys Gly Thr Tyr Gly Leu Tyr Thr Arg Val Lys Asn Tyr

645 650 655

Val Asp Trp Ile Met Lys Thr Met Gln Glu Asn Ser Thr Pro Arg Glu

660 665 670

Asp

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Gly Gly Gly Ser

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Gly Gly Ser Gly

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Gly Gly Ser Gly Gly

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Gly Ser Gly Ser Gly

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Gly Ser Gly Gly Gly

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Gly Gly Gly Ser Gly

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Gly Ser Ser Ser Gly

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Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

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Asp Tyr Lys Asp Asp Asp Asp Lys

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Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

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His His His His His

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Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys Thr

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Cys Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys

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Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

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Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Asp Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Arg Leu Pro

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Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

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Glu Val Met Leu Val Glu Ser Gly Gly Ala Leu Val Lys Pro Gly Gly

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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

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Ala Met Ser Trp Val Arg Gln Ile Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asp Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

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Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

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Ser Val Thr Val Ser Ser

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Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

8590 95

Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210>40

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>40

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr

6570 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>41

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>41

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210>42

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser His Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Phe Thr Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>43

<211>109

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>43

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Met Ser Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Phe Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Ala Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Arg Leu Pro

85 90 95

Pro Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>44

<211>109

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>44

Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 510 15

Glu Arg Ala Thr Met Ser Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Arg Leu Pro

85 90 95

Pro Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>45

<211>109

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis of amino acid sequence

<400>45

Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Arg Leu Pro

85 90 95

Pro Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>46

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>46

gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt cactttcagt aattatgcca tgtcttgggt tcgccaggct 120

ccagggaagg ggctggagtg ggtcgcaacc attagtagtg gtggtagtca cacctattat 180

ttagacagtg tgaagggtcg attcaccatc tccagagaca attccaagga caccctgtac 240

ctgcaaatga gcagtctgag ggctgaggac acggccctgt attattgtgc aagactgttt 300

accggctatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc ctca 354

<210>47

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>47

gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt cactttcagt aattatgcca tgtcttgggt tcgccaggct 120

ccagggaagg ggctggagtg ggtcgcaacc attagtagtg gtggtagtca cacctattat 180

ttagacagtg tgaagggtcg attcaccatc tccagagaca attccaagga caccctgtac 240

ctgcaaatga acagtctgag ggctgaggac acggccctgt attattgtgc aagactgttt 300

accggctatg ctatggacta ttggggtcaa ggaaccctgg tcaccgtctc ctca 354

<210>48

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>48

gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt cactttcagt aattatgcca tgtcttgggt tcgccaggct 120

ccagggaagg ggctggagtg ggtcgcaacc attagtagtg gtggtagtca cacctattat 180

ttagacagtg tgaagggtcg attcaccatc tccagagaca attccaagga caccctgtac 240

ctgcaaatga gcagtctgag ggctgaggac acggccctgt attattgtgc aagactgttt 300

accggctatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc ctca 354

<210>49

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>49

gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt cactttcagt aattatgccatgtcttgggt tcgccaggct 120

ccagggaagg ggctggagtg ggtcgcaacc attagtagtg gtggtagtca cacctattat 180

ttagacagtg tgaagggtcg attcaccatc tccagagaca attccaagaa caccctgtac 240

ctgcaaatga acagtctgag ggctgaggac acggccctgt attattgtgc aagactgttt 300

accggctatg ctatggacta ttggggtcaa ggaaccctgg tcaccgtctc ctca 354

<210>50

<211>327

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>50

caaattgttc tcacccagtc tccagcaatc ctgtctctgt ctccagggga acgggccacc 60

atgtcctgca cagccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120

ccaggaaagg cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

tctcgcttca gtggcagtgg gtctgggacc ttttacactc tcacaatcag cagcctgcag 240

gctgaagatt ttgccactta ttactgccac cagtattatc gtttaccacc catcacgttc 300

ggtcagggga ccaagctgga gatcaaa 327

<210>51

<211>327

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>51

caaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga acgggccacc 60

atgtcctgca cagccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120

ccaggaaagg cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

tctcgcttca gtggcagtgg gtctgggacc gattacactc tcacaatcag cagcctgcag 240

cctgaagatt ttgccactta ttactgccac cagtattatc gtttaccacc catcacgttc 300

ggtcagggga ccaagctgga gatcaaa 327

<210>52

<211>327

<212>DNA

<213> Artificial sequence

<220>

<223>Synthetic nucleic acid sequence

<400>52

caaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga acgggccacc 60

ctgtcctgca cagccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120

ccaggaaagg cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

tctcgcttca gtggcagtgg gtctgggacc gattacactc tcacaatcagcagcctgcag 240

cctgaagatt ttgccactta ttactgccac cagtattatc gtttaccacc catcacgttc 300

ggtcagggga ccaagctgga gatcaaa 327

<210>53

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>54

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>54

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

1 5 10

<210>55

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>55

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

20 25 30

<210>56

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>56

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>57

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>57

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

20 25 30

<210>58

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210>59

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>59

Trp Val Arg Gln Ala Pro Gly Gln Gly LeuGlu Trp Met

1 5 10

<210>60

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>60

Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

20 25 30

<210>61

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>61

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser

20 25

<210>62

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>62

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

1 5 10

<210>63

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>63

Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

20 25 30

<210>64

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>64

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys

20

<210>65

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>65

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

1 5 10 15

<210>66

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>66

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 1015

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210>67

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>67

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys

20

<210>68

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>68

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>69

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>69

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

Claims

1. An antibody which is:

(1) an isolated humanized monoclonal antibody that inhibits cleavage of complement component C4, wherein the antibody does not inhibit cleavage of complement component C2;

(2) an isolated humanized monoclonal antibody that specifically binds to an epitope within a region encompassing domains IV and V of complement component 1s (C1 s);

(3) an isolated humanized monoclonal antibody that binds with high affinity to complement component C1s in the C1 complex;

(4) seed separation deviceAn isolated humanized monoclonal antibody that is specific for complement component C1s and that is less than 10 x 10^-9IC50 for M inhibits complement-mediated cell lysis and/or inhibits complement-mediated cell lysis at less than 50X 10^-9IC50 for M inhibits C4 activation;

(5) a humanized antibody that specifically binds complement component 1s (C1s), wherein the antibody competes for binding to an epitope with an antibody comprising one or more CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID No.7 or one or more CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID No. 8;

(6) a humanized antibody that specifically binds complement component 1s (C1s), wherein the antibody is selected from the group consisting of:

b) a humanized antibody that specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope 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;

(7) a humanized antibody that binds complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising:

b) The heavy chain CDR of the antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 8 or SEQ ID NO 38;

(8) a humanized antibody that binds complement C1s protein, wherein the antibody specifically binds to an epitope within the complement C1s protein, wherein the antibody competes for binding to the epitope with an antibody comprising:

b) the heavy chain CDR of the antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO 8 or SEQ ID NO 38; or

(9) An antibody that binds complement C1s protein, wherein the antibody comprises Complementarity Determining Regions (CDRs) having an amino acid sequence selected from the group consisting of:

a) 1, 2, 3,4, 5 and 6; or

b) 32, 33, 3, 34, 35 and 36.

2. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable excipient.

3. A method, which is:

(1) a method of inhibiting complement component C4 activation in an individual, the method comprising administering to the individual an effective amount of an antibody of claim 1 or a pharmaceutical composition of claim 2;

(2) a method of inhibiting complement C1s activity in an individual, the method comprising administering to the individual an effective amount of an antibody of claim 1 or a pharmaceutical composition of claim 2;

(3) a method of treating an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an antibody of claim 1 or a pharmaceutical composition of claim 2;

(4) a method of inhibiting complement activation in an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an antibody of claim 1 or a pharmaceutical composition of claim 2;

(5) a method of diagnosing a complement-mediated disease or disorder in an individual, the method comprising:

(i) contacting the biological sample with an antibody of claim 1; and

(b) comparing the amount of complement C1s protein present in the biological sample to a normal control value indicative of 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 disorder;

(6) a method of monitoring the progression of a complement-mediated disease or disorder in an individual, the method comprising:

wherein the determining step comprises:

(i) contacting the biological sample with an antibody of claim 1; and

(ii) quantifying the binding of the antibody to complement C1s protein present in the biological sample;

(7) an in vitro method of detecting complement C1s protein in a biological sample obtained from an individual, the method comprising:

(a) contacting the biological sample with an antibody of claim 1; and

(b) detecting binding of the antibody to complement C1s protein present in the sample; or

(8) A method of detecting complement C1s protein in vivo, the method comprising:

(a) administering to an individual an antibody according to claim 1; and

4. A composition, comprising:

(a) the anti-C1 s antibody of claim 1; and

5. An organ or tissue preservation fluid comprising the anti-C1 s antibody according to claim 1 or the pharmaceutical composition according to claim 2.

6. An organ or tissue perfusate comprising the anti-C1 s antibody of claim 1 or the pharmaceutical composition of claim 2.

7. A method of preserving an organ or tissue for transplantation, the method comprising contacting the organ or the tissue with the composition of claim 4.

8. An isolated organ or tissue preserved in the composition of claim 4.

9. An in vitro method of inhibiting complement activation in an organ or tissue, the method comprising contacting the organ or the tissue with the antibody of claim 1, the pharmaceutical composition of claim 2, or with a solution comprising the antibody of claim 1.